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Physiology and Pathophysiology of the Calcium Store in the Endoplasmic Reticulum of Neurons

The University of Manchester, Faculty of Biological Sciences, Manchester, United Kingdom

ABSTRACT

I. ENDOPLASMIC RETICULUM AND CELLULAR PHYSIOLOGY: HISTORICAL REMARKS

II. ORGANIZATION OF THE ENDOPLASMIC RETICULUM

A. The Neuronal ER as a Continuous Endomembrane Structure

B. The ER as a Universal Signaling Organelle

C. The ER as an Excitable Organelle

1. Ca2+ release channels

2. SERCA pumps

3. ER Ca2+ buffers

4. The ER as an excitable medium

III. ENDOPLASMIC RETICULUM CALCIUM RELEASE IN NERVE CELLS

A. The Discovery of Ca2+ Release From the ER

B. Ca2+-Induced Ca2+ Release

1. Caffeine-induced Ca2+ release

2. The ''quantal'' nature of caffeine-induced Ca2+ release

3. Cytosolic Ca2+ regulates caffeine-induced Ca2+ release

4. Physiological CICR

A) THE ER AS A SOURCE OF AND SINK FOR CYTOSOLIC CA2+.

B) EVIDENCE FOR PHYSIOLOGICAL CICR IN NEURONS.

C) NEURONAL CICR IS GRADED BY CA2+ ENTRY.

5. Depolarization-induced CICR in nerve cells

6. Endogenous CICR modulators

A) CYCLIC ADP RIBOSE.

B) NICOTINIC ACID ADENINE DINUCLEOTIDE PHOSPHATE.

C) CALEXCITIN.

D) VANILLOID RECEPTORS.

E) PROTONS.

F) GLUTAMATE.

G) NEUROTROPHINS.

H) TNF-{alpha}/SPHINGOSINE-1-PHOSPHATE.

I) NO.

C. IICR in Nerve Cells

1. Direct evidence for neuronal IICR

2. Metabotropic receptors and neuronal IICR

3. CICR-assisted IICR

4. Endogenous IICR modulators

A) PROTEIN KINASES AND CALCINEURIN.

B) IMMUNOPHILIN FK506 BINDING PROTEIN 12.

C) CALMODULIN.

D) CALDENDRIN.

D. Do InsP3Rs and RyRs Share a Common Ca2+ Pool?

E. Luminal Ca2+ as a Regulator of Ca2+ Release

F. Refilling the Ca2+ Store and Store-Operated Ca2+ Entry

G. Pharmacology of Ca2+ Release

1. Ryanodine receptors/CICR

A) RYANODINE.

B) RUTHENIUM RED.

C) DANTROLENE.

D) GENERAL ANESTHETICS.

E) LOCAL ANESTHETICS.

F) DIHYDROPYRIDINES.

G) HEPARIN.

H) OTHER PHARMACOLOGICAL CICR REGULATORS.

2. Pharmacology of IICR

A) HEPARIN.

B) CAFFEINE.

C) 2-APB.

D) XESTOSPONGINES.

E) ADENOPHOSTINES.

IV. ENDOPLASMIC RETICULUM CALCIUM SIGNALING AND REGULATION OF NEURONAL FUNCTIONS

A. Neuronal Excitability

B. Neurotransmitter Release

C. Synaptic Plasticity

1. Short-term synaptic plasticity

2. Long-term synaptic plasticity

A) SYNAPTIC PLASTICITY IN THE HIPPOCAMPUS: A ROLE FOR CICR.

B) SYNAPTIC PLASTICITY IN THE CEREBELLUM: A ROLE FOR IICR.

C) SYNAPTIC MODIFICATION.

D) MEMORY PROCESSES.

D. Gene Expression, ER Ca2+ Waves, and ER Ca2+ Tunneling

E. Neuronal Growth and Morphological Plasticity

F. Neurohormone Release

G. Circadian Rhythms

V. ENDOPLASMIC RETICULUM CALCIUM HOMEOSTASIS AND NEURONAL PATHOPHYSIOLOGY

A. ER Stress Response and Neurodegeneration

B. ER Stores and Ischemia

C. ER Stores and Human Immunodeficiency Virus

D. ER Stores and Diabetes

E. ER Stores and Gangliosidoses (Gaucher’s and Sandhoff’s Disease)

F. ER Stores and Alzheimer’s Disease

G. ER Stores and Ageing

H. Spinocerebellar Ataxia Type 1

I. ER and Neuronal Trauma

J. ER and Parkinson’s Disease

K. ER and Epileptiform Activity

L. ER and Huntington’s Disease

VI. CONCLUDING REMARKS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. ENDOPLASMIC RETICULUM AND CELLULAR PHYSIOLOGY: HISTORICAL REMARKS

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In 1674 Antonius van Leeuwenhoek, after discovering the cross-striations of muscle fibers using one of his ingenious light microscopes, reflected "... who can tell, whether each of these filaments may not be enclosed in its proper membrane and contain within it an incredible number of still smaller filaments." (236). One century later, Luigi Galvani discovered "animal electricity" (178). To explain his observations, Galvani suggested that nerve and muscle are capable of generating electricity by accumulating positive and negative charges on two opposite surfaces. Moreover, the electrical flow necessary to produce voltage changes was explained by Galvani in terms of specific fluid-filled pores between the internal and external surfaces, which were the harbingers of ion channels.

The 19th century witnessed the emergence of the cellular theory. Furthermore, it became apparent that the cell is a complex structure containing numerous organelles. Great histologists, such as Rudolf Virchow, Gustav Retzius, Santiago Ramon y Cajal, and Jan Purkinje, to name but a few, developed various techniques to look inside the cells, and it was Camillo Golgi who, using his "black (silver chromate) reaction" technique in 1898 discovered the "fine and elegant reticulum hidden in the cell body" of owl cerebellar Purkinje neurons (187; see also Refs. 37, 384), the structure known to us as the "Golgi complex." The first detailed description of the endoplasmic reticulum was made by Golgi’s pupil, Emilio Veratti, who, by using black reaction technique on muscle fibers, was the first to discover a "true reticular apparatus constituted of filaments" (385, 668, 669; Fig. 1). This finding remained almost unnoticed for 50 years, and only when the endoplasmic reticulum (ER) was rediscovered by Keith Porter and George Palade, who used electron microscopy (481), were Veratti’s achievements generally recognized.

View larger version (82K): [in this window] [in a new window]   FIG. 1. Early drawings of intracellular reticular structures. A: original drawings of Camillo Golgi. The drawings present dorsal root ganglion neurons of an adult horse (Figs. 1–3), bovine fetus (Fig. 4), a neonatal cat (Fig. 6), and a young dog (Fig. 8). Black reticular structures ("internal reticular apparatus") are visualized by metallic impregnation (188). B: original drawing of Emilio Veratti. Longitudinal section of a limb muscle of the water beetle Hydrophilus piceus (668). The black reticular structure represents sarcoplasmic reticulum. [A from Bentivoglio et al. (37); B from Mazzarello et al. (385).]

The idea of an intracellular Ca²⁺ store and the importance of regulated intracellular Ca²⁺ release for muscle contraction was formulated by Heilbrunn, who was also the first to test these concepts experimentally (210, 211). His theory was not readily accepted, and for many years K⁺ were considered to be the regulators of contraction and relaxation, a theory very much supported by Szent-Gyorgyi (619). Only after the free intracellular Ca²⁺ in skeletal muscle was measured directly by murexide (263) or aequorin (15, 533) could internal Ca²⁺ release be visualized and the intracellular Ca²⁺ store became the legitimate member of a Ca²⁺ signaling cascade. In the meantime, the concept of intracellular Ca²⁺ pumps associated with the endomembrane was developed by W. H. Hasselbach, Setsuro Ebashi, and Anne-Marie Weber (see Ref. 159), explaining how the ER could accumulate Ca²⁺. At the same time, the first indications of ER-residing regulated ion channels, which could provide a means of Ca²⁺ release, appeared (158).

At the beginning of the 1970s the crucial role of the ER (in its sarcoplasmic reticulum disguise) in regulation of contraction became generally accepted and the concepts of Ca²⁺-induced Ca²⁺ release and depolarization-induced Ca²⁺ release firmly established (see Refs. 140, 160 for review). At the same time, the receptor-activated, second messenger-induced intracellular Ca²⁺ release was discovered (81, 457), and a possible role for inositol phospholipids in signal transduction was suggested (410). Another decade, however, passed before the seminal experiments of Hanspeter Streb, Robin Irvine, Irene Schulz, and Michael Berridge (607) showed that this alternative pathway for intracellular Ca²⁺ release is mediated by the second messenger inositol 1,4,5-trisphosphate (InsP₃). The intracellular Ca²⁺ release channels, residing within the endomembrane, were soon molecularly characterized, revealing two main families: the Ca²⁺-gated and InsP₃-gated channels (505, 613). Thus the general concept of intracellular Ca²⁺ stores was established, and the stage was set for identification of their functional role in various cell types.

The ability of nerve cells to actively buffer cytosolic Ca²⁺ was initially associated with Ca²⁺ accumulation by mitochondria, the latter utilizing H⁺ transport to develop an electrochemical gradient favoring Ca²⁺ entry (334, 544). The functional importance of the neuronal ER as a dynamic Ca²⁺ pool was first appreciated after the demonstration that neuronal microsomal fractions can accumulate Ca²⁺ via an ATP-dependent mechanism (649). Almost simultaneously it was also discovered that various neuronal preparations, such as the squid axon (64), extruded axoplasm (21), and permeabilized synaptosomes (51, 52), are able to sequester Ca²⁺ even when mitochondrial uptake was obliterated (see also Ref. 416 for review). Finally, this nonmitochondrial Ca²⁺ uptake was unequivocally associated with the ER (212–214, 682), and the latter became firmly established as a dynamic intracellular Ca²⁺ store, deeply involved in neuronal signaling.

	II. ORGANIZATION OF THE ENDOPLASMIC RETICULUM

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A. The Neuronal ER as a Continuous Endomembrane Structure

The endoplasmic reticulum is arguably the largest single intracellular organelle, which appears as a three-dimensional network formed by an endomembrane and organized in a complex system of microtubules and cisternae. In neurons, the ER extends from the nucleus and the soma to the dendritic arborization and, through the axon, to presynaptic terminals. Classical histology subdivides the ER into smooth ER, rough ER, and the nuclear envelope (32). The continuity of the endomembrane that forms these three structures was initially deduced from microscopic observations (130, 131, 371, 682). Further insights into the continuity of the ER were gained by fluorescent microscopy on cells injected with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC₁₆), which diffuses within strictly continuous lipid membranes. When injected into cells, DiIC₁₆ labeled the intracellular membrane network endowed with ER markers (632, 633). Injection of DiIC₁₆ into rat cerebellar Purkinje neurons in acutely isolated brain slices revealed a continuous membrane network extending throughout all parts of the cell (Fig. 2, A and B). The kinetics of dye spread was consistent with intramembrane diffusion; moreover, the ER labeling occurred even when slices were fixed 2 min after dye injection, thus excluding the possibility of active transport (634). Very similar ER labeling was observed in cultured hippocampal neurons (634). The concept of a continuous ER was further substantiated by experiments on nonneuronal cells. Initially, the continuity of the intra-ER aqueous space was directly demonstrated in pancreatic acinar cells, where soluble molecules, such as Ca²⁺ and fluorescent dyes, were shown to move throughout the ER lumen with remarkable ease (491, 509). Later on, it was found that much larger molecules of green fluorescence protein (GFP) targeted against the ER and expressed in Chinese hamster ovary (CHO) cells, were also able to freely diffuse within the ER lumen. Repeated local laser illumination of a spot 2 µm in diameter eventually bleached all GFP present in the cell, thus demonstrating the continuity of the ER lumen (115). Similarly, in RBL cells transfected with even larger ER-targeted elastase-GFP fusion protein (molecular weight 60 kDa), fluorescence recovery after photobleaching revealed rapid (diffusion coefficient 0.5 µm²/s) movements of this, rather hefty, protein within the entire ER lumen (610).

View larger version (70K): [in this window] [in a new window]   FIG. 2. Morphological appearance of neuronal endoplasmic reticulum (ER). A: the continuity of ER in Purkinje neurons from acutely isolated cerebellar slices. The lipophilic dye DiIC16 (absorption maximum 549 nm, emission 565 nm) was injected into the soma of two Purkinje neurons (PNs) in acutely isolated slice, and the resulting spread of fluorescence was imaged by a confocal microscope 4 h after dye injection. To obtain the image of the entire cellular network, a Z-series scan was performed, and the images were superimposed. B: spread of DiIC16 in living and fixed PNs. Images show the spread of the dye at 30 min (a) and 2 h (b) after the dye injection in a living cell; c and d show the spread of DiIC16 in PN fixed 2 min after dye injection and imaged 15 min (c) and 14 h (d) after injection. Bars for A and B, 10 µm. C: imaging of the ER in isolated sensory neuron. The same DRG neuron cultured for 24 h after isolation was stained with ER-Tracker (100 nM, 10 min at 24°C) and BODIPY FL-X fluorescent ryanodine (1 µM, 5 min). The fluorescence was excited at 357 nm (ER-Tracker) and at 488 nm (BODIPY FL-X ryanodine), and emitted light was collected at 530 ± 15 nm. [A and B from Terasaki et al. (634), copyright 1994 National Academy of Sciences, USA; C from Solovyova et al. (603), with permission from Nature Publishing Group.]

In axons, the ER mostly has a tubular structure and extends into the presynaptic terminals, where it often closely enwraps the mitochondria (48, 391, 693). The ER also spreads throughout the dendrites, terminating in the spine apparatus and thereby connects the latter with the entire ER lumen (604).

An important feature of the ER is its morphological plasticity. The ER network constantly remodels itself through multiple forms of tubular movements. These movements occur quite rapidly; new structures emerge within tens of seconds (57). Even large formations such as crystalline cisternal organelles are not static and may swiftly surface as part of a neuronal adaptive response, for example, against hypoxia (625).

The topographical heterogeneity of the ER is matched by its functional diversity. Protein synthesis within the rough ER occurs in specific compartments, with different mRNAs being specifically selected and targeted towards distinct regions of the reticulum (209, 525, 682). Sometimes mRNA is engaged in a long-distance travel to end up in the ER in dendrites, where the actual synthesis of a locally demanded protein (e.g., InsP₃ receptors, Ref. 606) takes place (180, 566). Here, the ER closely interacts with another reticular structure, the Golgi apparatus, the outposts of which are present in dendrites and are involved in final trafficking of membrane proteins and proteins destined for secretion (237, 238). Although the precise mechanisms of the intra-ER mRNA sorting remain unclear, they appear to be an important part of proteins targeting cascades. The synthesis of phospholipids and cholesterol similarly appears to be spatially heterogeneous, with different steps of the process occurring in different ER compartments (32). The enormous functional heterogeneity of the ER is nonetheless integrated into a coherent system by the continuous lumen.

B. The ER as a Universal Signaling Organelle

The functions of the ER are many. First and foremost, the ER is the site of synthesis and maturation of proteins. Protein synthesis is accomplished in the rough ER, while posttranslational processing of proteins is governed by an extended family of ER-resident chaperones, which form complexes with newly synthesized proteins, assist their folding into the final tertiary structure and prevent them from aggregation. If the process of folding somehow fails, the chaperones remain assembled with misfolded proteins, thereby preventing them from proceeding through the ER exit sites and towards the Golgi complex. Whenever the concentration of unfolded proteins rises dangerously high, the ER develops a specialized reaction known as ER stress, as a result of which transcription-affecting signals are sent to the nucleus, which, in turn, adjusts gene expression according to environmental requirements. In addition to the synthesis of proteins, the ER is the primary site of formation of phospholipids, glucosylphosphatidylinositols, and leukotrienes. The ER may also serve as a graveyard for various unwanted molecules and toxins. Owing to its continuous lumen, the ER serves as a highway allowing haulage of transport RNAs, secretory products, numerous structural proteins and, as we shall see later, ions between different parts of polarized cells. The ER is also intimately involved in rapid cellular signaling, because it is a dynamic store of Ca²⁺ that regulates the cytosolic Ca²⁺ concentration and generates Ca²⁺ fluxes between the cytosol and the ER lumen in response to extracellular stimulation. Finally, the ER coordinates all these diverse processes with the physiological state of the cell. Therefore, the ER may be defined as a multifunctional organelle able to detect and integrate incoming signals and generate output signals in response to environmental changes (40, 57). For a comprehensive overview of ER functions, see References 65, 257, 364, 388, 392, 399–402, 479, 487, 508, 509, 591, 677.

The intimate mechanisms of ER integration remain largely undiscovered, yet a central role for Ca²⁺ is emerging. First, Ca²⁺ is a key input and output signal of the ER. Indeed, Ca²⁺ concentration increases in the cytosol affect its concentration in the ER, and in turn, the ER Ca²⁺ release and uptake influence the cytosolic Ca ²⁺concentration. Second, a number of intra-ER chaperones, such as calreticulin, calnexin, grp78/BiP, endoplasmin (or glucose-regulated protein, grp94), are Ca²⁺ binding proteins, and changes in free Ca²⁺ concentration in the lumen of the ER ([Ca²⁺]_L) profoundly affect their functional activity (106, 409). Therefore, fluctuations in the ER Ca²⁺ content may provide the link between rapid signaling and long-lasting cellular adaptive responses.

C. The ER as an Excitable Organelle

The basic physiology of the ER calcium store has been extensively characterized in a variety of excitable and nonexcitable cells (for key reviews, see Refs. 38–42, 56, 75, 76, 147, 177, 308, 309, 381, 507, 508, 539, 670–672, 712). The ER acts as a dynamic Ca²⁺ store due to concerted activity of Ca²⁺ channels and transporters residing in the endomembrane, and intraluminal Ca²⁺-binding proteins, which serve as a high-capacity Ca²⁺ buffering system. Ca²⁺ efflux from the ER is executed by two families of Ca²⁺ channels, the Ca²⁺-gated Ca²⁺ channels, generally referred to as the ryanodine receptors (RyRs), and the InsP₃-gated channels, commonly known as InsP₃ receptors (InsP₃Rs). Ca²⁺ accumulation into the ER lumen results from the activity of Ca²⁺ pumps of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) family. The molecular physiology of ER channels and SERCA pumps has been comprehensively reviewed recently (RyRs, Refs. 59, 148, 161, 396, 545, 695; InsP₃Rs, Refs. 44, 411, 412, 631; SERCA pumps, Refs. 121, 134, 587, 618, 699); hence, I shall limit myself to a brief discussion of their key features in the context of neuronal signaling.

1. Ca²⁺ release channels

The Ca²⁺ release channels, the RyRs and the InsP₃Rs, are large tetrameric channel proteins that share a rather peculiar four-leaf clover-like structure when observed by electron microscopy. The RyRs were the first to receive experimental attention due to their vital role in excitation-contraction coupling in muscle. The RyR family comprises three major receptor subtypes, which were historically classified as "skeletal muscle," "heart," and "brain" types, currently referred to as RyR1, RyR2, and RyR3, respectively. The historic classification had a certain physiological sense, as the RyR1 type, dominant in skeletal muscle, possesses a rather unique activation mechanism; it can be activated solely by membrane depolarization by virtue of a direct link with the plasmalemmal Ca²⁺ channels through a huge cytosolic "foot" extension. All three RyRs can be activated by Ca²⁺ from the cytosolic side; the rank order of their sensitivity to cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) being RyR1 > RyR2 > RyR3 (93, 435). On the basis of studies on reconstituted channels in lipid bilayer membranes, the RyRs can be biophysically described as not perfectly selective Ca²⁺ channels (P_Ca/P_K 6–7) with fast activation kinetics ( of activation 0.5–1 ms) and a large conductance (100–500 pS, depending of the nature of ion carrier) (148). Yet, taking into account the imperfect selectivity toward monovalent cations and the virtual absence of selectivity between divalents, the physiological unitary Ca²⁺ current through RyRs can be as small as 0.5–0.9 pA at 2 mM intraluminal Ca²⁺ concentration (397).

All three types of RyRs have been detected in neurons; however, expression of the RyR2 isoform dominates. Interestingly, although the RyR3 was initially called the "brain" subtype, its total expression in the central nervous system does not exceed 2% of all RyRs (434). The RyR1 is heavily expressed in cerebellar Purkinje neurons, whereas RyR3 is found in hippocampal structures, the corpus striatum, and the diencephalon (170, 185, 434, 474, 574).

However, these are only general trends; in reality, many neurons coexpress either two or all three types of RyR isoforms. RyRs are found in high densities in neuronal somatas; in axons of hippocampal (574), cerebellar (475), and cortical (723) neurons; in cerebellar mossy fibers (475); and in presynaptic terminals (355). RyRs are also widely expressed in the dendritic tree; their expression is particularly high in dendritic spines of hippocampal CA1 neurons, where they represent the dominant Ca²⁺ release channel (574). Incidentally, RyRs were visualized (with fluorescent ryanodine) in the Golgi complex of sympathetic neurons, although the functional relevance of this discovery remains unclear (98).

The expression of RyRs changes considerably during development; for instance, throughout the embryonic stage, RyR1 mRNA levels are highest in the rostral cortical plate, whereas RyR3 mRNA was most prominent in the caudal cortical plate and hippocampus. Low levels of RyR2 mRNA were detected in the diencephalon and the brain stem. However, from postnatal day 7 onward, RyR2 mRNA became the major isoform in many brain regions, while RyR1 mRNA became prominent in the dentate gyrus and in the Purkinje cell layer. Postnatal downregulation in the caudal cerebral cortex restricted RyR3 mRNA expression to the hippocampus, particularly the CA1 region (427).

In vivo imaging of RyRs can be achieved using fluorescent BODIPY-ryanodine (e.g., Refs. 389, 603; Fig. 2C). Because ryanodine binding to the RyR is use dependent, the intensity of BODIPY-ryanodine staining may reflect the overall density of open channels. With the use of this technique, a redistribution of active RyRs from soma to dendrites was observed in developing hippocampal neurons (611); moreover, L-type Ca²⁺ channel-RyR coupling was identified at early developmental stages (611), reflecting a developmental switch between RyR1 and RyR2 expression in nerve cells (427).

Similar to RyRs, the InsP₃ receptor family comprises three homotetrameric isoforms, InsP₃R1, InsP₃R2, and InsP₃R3, although further diversity may arise from alternative splicing of InsP₃R1 (up to 6 variants; Ref. 463) and heteromeric expression (399). The most important difference between isoforms lies in their different sensitivity to [Ca²⁺]_i: the InsP₃R1 has the "classical" bell-shaped dependence on [Ca²⁺]_i with a maximal activation at 300–400 nM (45); the InsP₃R2 and InsP₃R3 are also stimulated by [Ca²⁺]_i increase, but are not readily inhibited by larger [Ca²⁺]_i rises (198).

In the central nervous system, the InsP₃Rs were initially discovered in the cerebellum (172, 613), but thereafter all three isoforms were identified throughtout the brain, with particularly strong expression in Purkinje neurons and in somatas of CA1 cells (170, 171, 574). The InsP₃R1 is the most abundant isoform in the brain, whereas InsP₃R2 dominates in the spinal cord and is also highly expressed in glial cells. The InsP₃R3 are expressed to a much lesser extent but are nonetheless clearly detectable in several brain regions, such as the cerebellar granule layer and the medulla (575). There is a prominent heterogeneity in the intracellular distribution of InsP₃Rs. For example, InsP₃R1 is found in dendrites, dendritic spines, cell bodies, axons, and axonal terminals of cerebellar Purkinje cells, but is mostly confined to somatic regions and proximal dendrites in other neurons (122, 551, 575). The InsP₃R3 is very much concentrated in the neuropil and neuronal terminals (575).

2. SERCA pumps

The SERCA pump belongs to the family of P-type Ca²⁺-ATPases and includes three gene products, SERCA1, SERCA2, and SERCA3; alternative splicing produces two distinct isoforms of SERCA2, namely, 2a and 2b. In central neurons, the SERCA2b is ubiquitously expressed, whereas SERCA2a and SERCA3 are found almost exclusively in cerebellar Purkinje neurons (18). The physiological role of SERCA3 remains quite mysterious, and genetic deletion of this Ca²⁺ pump does not result in any obvious phenotypic change, save minor deficits in relaxation of vascular and tracheal smooth muscles (351).

An important property of the SERCA2b pump that is relevant for ER Ca²⁺ homeostasis is the tight regulation of its activity by the free Ca²⁺ concentration in the ER lumen, mediated via the Ca⁺-binding proteins calreticulin (264) and ERp57 (341). Calreticulin senses the actual level of [Ca²⁺]_L and, by directly interacting with SERCA2b, activates Ca²⁺ pumping whenever [Ca²⁺]_L decreases. The ERp57 regulates Ca²⁺ uptake by modulating the redox state of SERCA2b thiols in a Ca²⁺-dependent manner.

The SERCA pumps have a distinct pharmacology; they are blocked irreversibly by thapsigargin (TG) in submicromolar concentrations (K_D 20 nM) and reversibly by cyclopiazonic acid (CPA) at concentrations of 20–50 µM. Inhibition of the SERCA pumps results in a relatively slow emptying of releasable Ca²⁺ from the ER, with Ca²⁺ leaving the ER through poorly understood pathways (72). It must also be noted that TG is not perfectly specific for SERCAs; it also inhibits plasmalemmal voltage-gated Ca²⁺ channels of L/N-types in DRG neurons at concentrations of 0.2–2 µM (582). Likewise, micromolar concentrations of TG substantially decreased T and L Ca²⁺ currents in adrenal glomerulosa cells (546). The concentrations of TG that inhibit Ca²⁺ channels are comparable to those used in the majority of experiments in which it is intended to block the Ca²⁺ accumulation by the ER. A certain care, therefore, should be taken when interpreting data arising from such experiments.

3. ER Ca²⁺ buffers

ER Ca²⁺ buffering is accomplished by several Ca²⁺-binding proteins; in neurons, the most abundant is calreticulin, which accounts for nearly one-half of total ER Ca²⁺ binding. Calreticulin in the neuronal ER is endowed with 20–50 low-affinity Ca²⁺ binding sites (K_D 1 mM) and therefore can bind a huge amount of Ca²⁺. As mentioned above, calreticulin acts not only as a Ca²⁺ buffer, but also as an important chaperone and regulator of SERCA pumps. Calsequestrin, the major ER Ca²⁺ buffer in skeletal myocytes, is also present in nerve cells, and like calreticulin may bind up to 50 Ca²⁺ with low affinity. Several other low-affinity Ca²⁺-binding proteins, such as endoplasmin (grp94), BiP/grp78, and proteins of the CREC family (reticulocalbin, calumenin, Cab55, etc.) also participate in intra-ER Ca²⁺ buffering (235, 399, 401, 409).

The most important property of the ER Ca²⁺ buffers is their low affinity to Ca²⁺, which allows the maintenance of high intra-ER free Ca²⁺ levels. There is a fundamental difference in Ca²⁺ handling in the cytosol and within the ER lumen. The cytoplasmic Ca²⁺ binding proteins are characterized by a very high (10–100 nM) affinity to Ca²⁺; therefore, they limit Ca²⁺ diffusion and favor localization of cytosolic Ca²⁺ signals. In contrast, the low Ca²⁺ affinity of the ER buffers allows Ca²⁺ diffusion throughout the lumen (423).

4. The ER as an excitable medium

The extensive complement of endomembrane Ca²⁺ channels and transporters, together with intra-ER Ca²⁺ storage proteins, gives rise to the excitable properties of the ER. Ca²⁺ accumulation in the ER lumen creates a large electrochemical driving force favoring Ca²⁺ movement from the ER lumen into the cytosol; hence, opening of Ca²⁺ channels in the ER membrane causes fast efflux of Ca²⁺ from the ER. In turn, the ER channels are sensitive to signals originating at the plasmalemma and so can be activated by both electrical and chemical stimulation of the cell. Furthermore, the ER channels are subject to positive autofeedback due to their sensitivity to cytosolic Ca²⁺, leading to a regenerative opening of RyRs and/or InsP₃Rs beyond a certain threshold of [Ca²⁺]_i. This regenerative process underlies propagating waves of channel activation along the ER membrane, very similar to the propagating wave of opening and closing of Na⁺ and K⁺ channels that generate plasmalemmal action potentials. Neuronal function is thus an interplay of two excitable membranes, the outer and the inner, which are coupled through a multitude of reciprocal signaling cascades (39). In contrast to plasmalemma, which is mostly specialized for integrating extracellular information, the ER uses Ca²⁺ to couple plasmalemmal and cytosolic events with intraluminal formation and transport of proteins, as well as with gene expression within the nucleus. The periodic fluctuations of the ER Ca²⁺ concentration are instrumental for this intracellular integration, because [Ca²⁺]_L regulates the excitability of the ER membrane by controlling ER Ca²⁺ release channels and SERCA pumps, and governs intra-ER enzymatic cascades.

	III. ENDOPLASMIC RETICULUM CALCIUM RELEASE IN NERVE CELLS

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A. The Discovery of Ca²⁺ Release From the ER

Historically, the first observations of the release of Ca²⁺ from an intracellular pool were very much indebted to the previous discovery that caffeine can activate RyRs in muscle cells (688). The pioneering observation indicating that caffeine affects neuronal [Ca²⁺]_i was made by K. Kuba and S. Nishi in 1976 (319). When monitoring membrane potential of neurons in isolated sympathetic ganglia of the bullfrog, Kuba and Nishi found that superfusion of the ganglia with an extracellular solution supplemented with 1–6 mM caffeine resulted in rhythmic hyperpolarizations. Further on, they found that the caffeine-induced effects on membrane potential (V_m) were mediated through a Ca²⁺-dependent K⁺ conductance, because intracellular injection of EDTA inhibited the action of caffeine. Several years later, Kenji Kuba made the logical suggestion that "rhythmic increases in the G_K under the effect of caffeine are due to oscillations of the intracellular Ca²⁺ concentration and that there may be Ca storage sites in the bullfrog sympathetic ganglion cell which are comparable to the sarcoplasmic reticulum in the skeletal muscle fiber" (317). Using K⁺ permeability as a readout of [Ca²⁺]_i, several groups confirmed these observations (219, 318, 431, 557). The advent of luminescent and fluorescent Ca²⁺ probes allowed direct monitoring of [Ca²⁺]_i, which became instrumental for detailed investigations of ER Ca²⁺ release in nerve cells.

B. Ca²⁺-Induced Ca²⁺ Release

1. Caffeine-induced Ca²⁺ release

As already mentioned, the methylxantine caffeine was the first specific agent permitting identification of ER Ca²⁺ release, and it has remained the probe of choice (Table 1). Probably the first direct indication that another methylxantine, theophylline, triggers [Ca²⁺]_i rises in vertebrate nerve cells was obtained in bullfrog sympathetic neurons injected with the bioluminescent Ca²⁺ probe arsenazo III (597). Yet, the first proper description of caffeine-induced Ca²⁺ release was published in 1984, when Ian Neering and Robert McBurney presented the results of their experiments on DRG neurons, which were isolated from newborn rats and injected with another luminescent Ca²⁺ probe, aequorin (452). When 10 mM caffeine was applied to DRG neurons, they responded by a large light transient, reflecting an elevation of [Ca²⁺]_i (Fig. 3A). In fact, the caffeine-induced [Ca²⁺]_i elevation was three to five times larger than the [Ca²⁺]_i increase evoked by action potentials (the latter artificially prolonged by addition of 20 mM TEA). The caffeine-induced [Ca²⁺]_i response was preserved in an extracellular solution containing 5 mM Co²⁺, suggesting that it was independent of plasmalemmal Ca²⁺ entry. Furthermore, Neering and McBurney (452) showed that caffeine depletes the Ca²⁺ store, which, however, can be rapidly replenished by stimulating the neuron with several action potentials (APs) (Fig. 3B). Further progress in studying caffeine-induced Ca²⁺ release was very much assisted by the introduction of fluorescent Ca²⁺ probes (653, 654) as well as by the widespread use of the patch-clamp technique (200). When combined, these techniques allowed simultaneous monitoring of membrane currents and [Ca²⁺]_i dynamics. In 1988, caffeine-induced Ca²⁺ release was identified in fura 2-loaded sympathetic neurons of the frog (348, 349) and in sympathetic (636), sensory (637), and central (638) neurons of the rat. Within the next several years caffeine-induced Ca²⁺ release was discovered in all types of neurons studied regardless of the preparation; the phenomenon was found in freshly dissociated neurons, neurons in primary cultures, and neurons in situ in brain slices (see Table 1).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Identification of caffeine-stimulated ER Ca2+ release in nerve cell. A: effect of caffeine on membrane potential (Vm) and [Ca2+]i (proportional to changes in Ilight) in a single DRG neuron. The resting Vm was –55 mV. Extracellular solution contained 20 mM TEA. Peak of caffeine-induced [Ca2+]i rise was 10 µM. B: caffeine depletes the ER calcium store. Similarly to A, the [Ca2+]i and Vm were recorded from a single DRG neuron. First application of caffeine triggered [Ca2+]i rise; however, when caffeine was applied 50 s later, no changes in [Ca2+]i were observed. After the cell was stimulated by three action potentials (AP), caffeine was once more able to produce [Ca2+]i elevation. The time elapsed after the beginning of the experiment is indicated on the graph. [From Neering and McBurney (452), with permission from Nature Publishing Group.]

The properties of caffeine-induced Ca²⁺ release are generally similar between different neurons (Table 1). Caffeine induces a [Ca²⁺]_i rise without a requirement for extracellular calcium, and the [Ca²⁺]_i elevation is not associated with plasmalemmal Ca²⁺ movements. Caffeine-evoked [Ca²⁺]_i transients are sensitive to pharmacological modulators interacting with RyRs or with SERCA pumps; specifically, these [Ca²⁺]_i responses are blocked by ryanodine, dantrolene, ruthenium red, and procaine and disappear after inhibition of ER Ca²⁺ uptake with TG or CPA (Table 1). Caffeine responses are found in all parts of neurons, from soma to dendrites and presynaptic terminals. Caffeine-induced Ca²⁺ release that is sensitive to TG and ryanodine has even been observed in individual spines of cultured hippocampal neurons, which are rich in ryanodine receptors (303).

Incubation of neurons isolated from bullfrog sympathetic ganglia with 10 mM caffeine frequently induces [Ca²⁺]_i oscillations (e.g., Refs. 164, 166, 462), which are often quite diverse in different neurons, ranging from high-amplitude regular oscillatory activity to low-amplitude decaying [Ca²⁺]_i fluctuations (111). This diversity of oscillatory patterns reflects complex mechanisms responsible for their generation, which involve ER (Ca²⁺-induced Ca²⁺ release and SERCA-dependent Ca²⁺ uptake, etc.) as well as non-ER (mitochondrial Ca²⁺ accumulation, plasmalemmal Ca²⁺ extrusion) cascades.

When using caffeine in nerve cell preparations, a certain caution is needed, because caffeine (and other methylxanthines) directly affects potassium channels (660) and effectively inhibits delayed rectifier and A-type potassium currents in vertebrate neurons (529). In certain types of neurons, caffeine may also activate plasmalemmal Ca²⁺ influx, for example, as demonstrated in acutely dissociated rabbit nodose ganglion neurons (229) and leech mechanosensitive neurons (565). The K_D values for the activation of ER Ca²⁺ release and plasmalemmal Ca²⁺ influx by caffeine were close (1.5 and 0.6 mM, respectively); this needs to be taken into account when interpreting caffeine-induced [Ca²⁺]_i responses as a sole consequence of ER Ca²⁺ release.

2. The "quantal" nature of caffeine-induced Ca²⁺ release

Caffeine releases Ca²⁺ from neuronal ER in a rather peculiar manner, which has been termed "quantal" release. When the cell is challenged with a low concentration of caffeine (2–5 mM), a transient Ca²⁺ response is triggered, and even if caffeine remains in the system, the [Ca²⁺]_i recovers back toward the prestimulated level. Applications of caffeine in submaximal concentrations fail to empty the store completely; subsequent application of a higher caffeine concentration induces a further [Ca²⁺]_i elevation. Interestingly, it appears that a submaximal caffeine concentration can empty the store "compartment" sensitive to this concentration: when 2 mM of caffeine is applied repetitively, the second application no longer produces a [Ca²⁺]_i response (Fig. 4). This peculiar quantal release was found when studying [Ca²⁺]_i dynamics in cultured chromaffin cells (92), in rat (661) and mouse (584) DRG neurons, and in myenteric neurons (286). A similar quantal release was also found when monitoring [Ca²⁺]_L in chromaffin cells expressing ER-targeted aequorin (9; see Fig. 4A). These observations can be explained by assuming either that different neuronal Ca²⁺ release channels have different sensitivities to caffeine, or that the ER is composed of several compartments, which are discharged by different concentrations of caffeine (92). Further experiments showed, however, that the quantal caffeine-induced release is controlled by intraluminal Ca²⁺ concentration, and overcharging the stores with Ca²⁺ following massive depolarization-induced Ca²⁺ entry significantly enhances the sensitivity of ER Ca²⁺ release to caffeine (Fig. 4D, Refs. 300, 584). In the context of ER Ca²⁺ release, [Ca²⁺]_L-dependent regulation of RyRs appears to be a general phenomenon, as the same sort of regulation is well established for another Ca²⁺ release channel, the InsP₃R. Similar to RyRs, elevation of luminal Ca²⁺ increases the sensitivity of InsP₃R to InsP₃ (419, 465). Changes in "luminal" [Ca²⁺] in a planar lipid system directly influenced the purified InsP₃R by affecting their inactivation (643).

View larger version (25K): [in this window] [in a new window]   FIG. 4. "Quantal" release of Ca2+ by caffeine. A: "quantal" caffeine-induced release of ER Ca2+ as measured by [Ca2+]L recordings in bovine chromaffin cells expressing ER-targeted low-affinity aequorin. The ER was refilled by incubation with medium containing 1 mM Ca2+, and the increasing concentrations of caffeine were applied as indicated. B and C: examples of [Ca2+]i recordings from acutely isolated mouse DRG neurons in response to different concentrations of caffeine. Note that (C) the second application of 2 mM caffeine fails to induce [Ca2+]i increase, although 5 mM of caffeine applied shortly after triggers Ca2+ release. D: increase in intraluminal Ca2+ content increases the sensitivity of ER Ca2+ release to caffeine. The graph shows the concentration dependence of caffeine-induced [Ca2+]i responses. The amplitudes of caffeine-induced [Ca2+]i transients measured at rest () and after conditioning depolarization (; the example of [Ca2+]i trace is shown in inset) are plotted against caffeine concentration. Data are pooled from 24 mouse DRG neurons; bars denote ±SD. [A from Alonso et al. (9) by copyright permission from The Rockefeller University Press. B–D from Shmigol et al. (584), copyright 1996 with permission from Elsevier.]

The nature of interaction of caffeine with RyRs depends on the concentration of the former: direct electrophysiological investigations of isolated ryanodine receptor channels have demonstrated that low (<2 mM) concentrations of caffeine sensitize the channel to extraluminal (i.e., cytoplasmic) calcium, shifting RyR activation towards submicromolar Ca²⁺ concentrations. In the presence of low millimolar caffeine concentrations, the RyR channel open probability (P_o) was significantly increased mostly due to a shortening of the lifetime of the closed state. At higher concentrations (>5 mM), caffeine opens RyRs even at picomolar [Ca²⁺]_i and dramatically increases the open time of the channel (e.g., Refs. 221, 547, 593).

These biophysical properties of the caffeine-RyR interactions are also reflected by the [Ca²⁺]_i dependence of caffeine action on nerve cells. The amplitude of the [Ca²⁺]_i elevation induced by 10 mM caffeine increased sharply when the [Ca²⁺]_i preceding the application of the drug was elevated by a conditioning KCl depolarization of rat sympathetic neurons (221). By changing the duration of the KCl application between 0.3 and 7 s, the conditioning [Ca²⁺]_i rise could be graded, therefore making it possible to characterize the relation between [Ca²⁺]_i and the amplitude of caffeine-induced Ca²⁺ release (Fig. 5A). This relation appeared bell-shaped: an increase of [Ca²⁺]_i from the resting level of 50–70 to 100–300 nM markedly potentiated caffeine-induced [Ca²⁺]_i response, but a further increase of [Ca²⁺]_i to >400 nM led to an inhibition of the caffeine-evoked [Ca²⁺]_i elevation. A similar effect of [Ca²⁺]_i on the response to a submaximal (2 mM) caffeine challenge was observed in rat sensory neurons (Fig. 5B), where an increase in [Ca²⁺]_i preceding caffeine application greatly potentiated Ca²⁺ release (584). Moreover, when precaffeine [Ca²⁺]_i reached a level of 350 nM, 2 mM caffeine induced a full-blown Ca²⁺ release that almost completely emptied the ER.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Effects of [Ca2+]i on caffeine-induced Ca2+ release in peripheral neurons. A: the [Ca2+]i recordings were performed on cultured rat sympathetic neurons. Left: examples of [Ca2+]i transients illustrating the effects of applying a conditioning pulse of high KCl of increasing durations (as indicated under the traces) just before a caffeine application of 5 s. The interval between the end of KCl pulse and the caffeine application remained constant at 1.5 s. Right: amplitudes of caffeine ([Ca2+]caff) measured from the records shown on the left and plotted against the "pedestal" [Ca2+]i (P), reached immediately before caffeine administration. B: two [Ca2+]i recordings taken from the same freshly isolated mouse DRG neuron. Note that increase in [Ca2+]i significantly potentiated the amplitude of 2 mM caffeine-induced response (left trace); further increase in [Ca2+]i (right trace) triggered even larger response to 2 mM caffeine, which emptied the ER, as judged by very small response to subsequent application of 20 mM caffeine. Timing of caffeine and KCl applications is indicated on the graph. [A from Hernandez-Cruz et al. (221), with permission from Blackwell Publishers Ltd. B from Shmigol et al. (584), copyright 1996 with permission from Elsevier.]

View larger version (23K): [in this window] [in a new window]   FIG. 6. Effects of [Ca2+]i on caffeine-induced Ca2+ release in Purkinje neurons. A: pseudocolor fluorescence images of [Ca2+]i illustrating the [Ca2+]i dependence of caffeine-induced Ca2+ release in various cellular compartments. The gain of intensified CCD camera was set at values that optimized detection from dendrites, but partially saturated the fluorescence signal from the soma (darkened area in the central part of the soma). B: [Ca2+]i recordings from the soma and proximal and distal dendrites of Purkinje neuron shown in A. The regions from which the mean [Ca2+]i values were calculated are indicated by rectangles on images shown in A. Arrows marked with a, b, and c indicate the time at which the digital fluorescence images in A, labeled a, b, and c, were taken. The baseline [Ca2+]i was changed by applying 6 depolarizing pulses (from –60 to 0 mV for 500 ms) immediately before the application of caffeine. [From Kano et al. (269), with permission from Blackwell Publishers Ltd.]

A) THE ER AS A SOURCE OF AND SINK FOR CYTOSOLIC CA²⁺.  Although the ability of the ER to buffer [Ca²⁺]_i and at the same time to release Ca²⁺ into the cytosol was recognized already by the mid 1980s (213, 214, 386), the concept of the ER Ca²⁺ store acting as a "Ca²⁺ sink and Ca²⁺ source" was for the first time clearly formulated by David Friel and Richard Tsien in 1992 (165). They backed up this concept with a rather thorough analysis of changes in depolarization-induced Ca²⁺ transients occurring upon various manipulations of the ER Ca²⁺ store. First, they convincingly demonstrated that ER Ca²⁺ release participates in the delivery of Ca²⁺ to the cytosol during cell depolarization, i.e., that CICR indeed acts as an amplifier of Ca²⁺ signals resulting from plasmalemmal Ca²⁺ entry. They employed a protocol, which later became a generally accepted test for CICR. This protocol compared depolarization-induced [Ca²⁺]_i transients in control conditions and in conditions when 1) ER is recovering following treatment with a high (10–20 mM) concentration of caffeine, 2) RyRs are sensitized by a low (1 mM) concentration of caffeine, and 3) RyRs are activated by 1 µM ryanodine and the stores are permanently depleted. In perfect concordance with the idea of the ER having the dual role of [Ca²⁺]_i buffer and Ca²⁺ source, these manipulations affected both the rising phase and the recovery of [Ca²⁺]_i elevations in response to membrane depolarization with 30–50 mM KCl (Figs. 7 and 8). A complete depletion of the stores decreased the amplitude and rate of rise of KCl-induced [Ca²⁺]_i transients, and accelerated their recovery by 1) removing the CICR contribution and 2) upregulating Ca²⁺ uptake into the depleted ER. Sensitization of RyRs by 1 mM caffeine increased the rate of rise and the amplitude of the depolarization-induced [Ca²⁺]_i transient, and moderately accelerated the recovery of the [Ca²⁺]_i elevation by 1) enhancing the CICR contribution and 2) augmenting Ca²⁺ uptake because increased CICR depleted the store to a higher extent compared with control. Finally, permanent emptying of the ER by 1 µM ryanodine decelerated the KCl-induced [Ca²⁺]_i transient and slowed down the recovery by 1) removing the CICR contribution and 2) occluding the ER Ca²⁺ uptake, because the store remained permanently leaky due to constantly open RyRs.

View larger version (24K): [in this window] [in a new window]   FIG. 7. ER Ca2+ store as a source of and sink for Ca2+. Top panel represents [Ca2+]i traces evoked by 30 mM KCl in control conditions and upon pharmacologically modulated ER Ca2+ store (b–e) as explained by drawings below. Arrows indicate direction and relative magnitude of the net Ca2+ flux across the surface membrane and between the cytosol and the store, during the onset and recovery phases of the response. Under control conditions (a), depolarization stimulates Ca2+ entry and a rise in [Ca2+]i, which promotes Ca2+ release from the store (onset). Repolarization permits the Ca2+ contents of both the cytosol and the store to relax towards their prestimulation levels (recovery); since the store released Ca2+ during the onset, it must accumulate Ca2+ during the recovery. b: When continuously present, caffeine (1 mM) enhances Ca2+-induced Ca2+ release (CICR) from the store (onset) so that [Ca2+]i increases more rapidly than it does under control conditions. To restore its initial Ca2+ content, the store also accumulates Ca2+ more avidly during the recovery; as a result, [Ca2+]i declines more rapidly following repolarization. If Ca2+ entry is induced while the store is filling as in c, net Ca2+ uptake by the store slows the rate at which [Ca2+]i rises during the onset. However, if [Ca2+]i is elevated long enough for the Ca2+ content of both the cytosol and the store to approximate the steady-state values achieved under control conditions (a), then [Ca2+]i will recover just as it does in the control (compare a and c, recovery). By increasing passive Ca2+ exchange between the store and cytosol, ryanodine prevents net Ca2+ accumulation by the store (d and e). Without the added effect of CICR, [Ca2+]i rises more slowly in response to stimulated Ca2+ entry (compare a and d, onset) and falls more slowly during recovery (compare a and d) than it does under control conditions. Since the store remains discharged in the presence of ryanodine, caffeine has no further effect. Therefore, the postcaffeine response in the presence of ryanodine (e) is essentially the same as the response elicited in the presence of ryanodine without caffeine pretreatment (d). [From Friel and Tsien (165), with permission from Blackwell Publishers Ltd.]

View larger version (21K): [in this window] [in a new window]   FIG. 8. Evidence for CICR in neurons: pharmacological modulation of depolarization- and synaptically induced [Ca2+]i elevation by caffeine and ryanodine. A: depletion of ER attenuates depolarization-induced [Ca2+]i response in indo 1-AM-loaded cultured rat DRG neurons. The [Ca2+]i trace shows high-K+-induced [Ca2+]i transients before and after store depletion by long application of 10 mM caffeine. The inset shows initial phase of depolarization-induced [Ca2+]i response before and immediately after caffeine application. The amplitude and the rate of rise of KCl-evoked [Ca2+]i transients were 603 nM and 202 nM/s before and 393 nM and 122 nM/s after depletion of the Ca2+ store. B: CICR accounts for major part of [Ca2+]i elevation following titanic stimulation of synaptic inputs to CA1 neurons in hippocampal slices. Incubation of slice with 10 µM ryanodine or 10 µM thapsigargin markedly suppresses [Ca2+]i elevation without affecting excitatory postsynaptic currents. [A modified from Usachev et al. (661); B modified from Alford et al. (6).]

B) EVIDENCE FOR PHYSIOLOGICAL CICR IN NEURONS.  With the use of the paradigm described above, the contribution of CICR to the [Ca²⁺]_i elevation triggered by plasmalemmal Ca²⁺ entry has been demonstrated for many types of neurons, in both the peripheral and the central nervous system (Table 2). In addition to the protocols used by Friel and Tsien, several other experimental approaches were developed, e.g., CICR can be completely blocked by high concentrations of ryanodine, or the ER store can be depleted using the SERCA pump blockers TG or CPA. The changes observed in the depolarization-induced [Ca²⁺]_i transients caused by these interventions are very much as expected from the sink and source theory: inhibition of CICR by ryanodine slows down the rate of rise and decreases the amplitude of KCl-induced [Ca²⁺]_i transients, whereas inhibition of ER Ca²⁺ uptake has the same effect on the onset of [Ca²⁺]_i signals in response to depolarization and significantly decelerates their recovery.

It is worth pointing out that the functionality of the CICR pathway may critically depend on the experimental conditions. For instance, in Purkinje neurons, the parameters of ER Ca²⁺ release are greatly affected by cell culture. Photorelease of InsP₃ or administration of caffeine induced only a tiny [Ca²⁺]_i response in a small fraction of cultured Purkinje neurons, whereas the same manipulations resulted in large and frequent [Ca²⁺]_i responses in freshly dissociated neurons (697) (however a caveat: the cultures were prepared from the embryonic cerebellum, E16-E18, whereas neurons were acutely isolated from mice at postnatal days 10–16). Conversely, no CICR in response to a single AP or trains of APs (tetanic stimulation) was observed in bullfrog sympathetic neurons, when studied in whole ganglia (462), but a prominent CICR in response to Ca²⁺ entry was invariably observed when the same cells were kept in culture conditions (240).

In certain cell types, physiological CICR exhibits a remarkable spatial heterogeneity. In leech Retzius neurons, for instance, depletion of the ER by 10 µM CPA reduced AP-induced [Ca²⁺]_i transients in dendrites, but not in axons (33). In frog sympathetic ganglion neurons, either within the whole ganglia or in culture, a single AP was able to trigger several local CICR events concentrated in the ER-rich perinuclear region or around axon hillocks, as was revealed by fast fluo 4 confocal imaging (112).

At the subcellular level, activation of CICR is represented by local release events, resulting from spontaneous activation of RyRs and subsequent recruitment of RyR clusters. These elementary [Ca²⁺]_i signals [observed in nerve growth factor-differentiated PC12 cells and in cultured hippocampal neurons (298, 398)] are analogs to "sparks" in muscle cell preparations. Neuronal [Ca²⁺]_i sparks occur spontaneously, and their frequency increases upon addition of low concentrations of caffeine or following loading of the ER store by conditioning depolarization-induced Ca²⁺ entry. In contrast to other preparations, neuronal release sites appear to contain both InsP₃Rs and RyRs, since metabotropic stimulation affected elementary Ca²⁺ release frequency at the same sites as caffeine (298). Spontaneous [Ca²⁺]_i sparks were also found in presynaptic boutons of the lizard neuromuscular junction (398) and in cerebellar basket neurons (105, 355).

More accurate characterization of CICR in nerve cells requires a direct comparison between plasmalemmal Ca²⁺ entry and [Ca²⁺]_i elevation. This necessitates simultaneous monitoring of Ca²⁺ currents (under voltage-clamp) and [Ca²⁺]_i; the amount of Ca²⁺ entering the cell can be precisely calculated by integrating Ca²⁺ current (I_Ca) over time, and hence the [Ca²⁺]_i can be directly related to I_Ca/t. Such an experiment was performed for the first time in rat sympathetic neurons (636), revealing a fairly linear relation between Ca²⁺ entry and [Ca²⁺]_i at short test pulses and its saturation at long test pulses (range of I_Ca durations varied between 10 and 640 ms). When the same experiment was repeated in the presence of 10 mM caffeine and 10 µM ryanodine, suppression of [Ca²⁺]_i was detected; however, this phenomenon was not very robust, leading the authors to conclude that CICR involvement in amplification of voltage-gated [Ca²⁺]_i transients is rather modest, if it exists at all. Several years later, Shao-Yng Hua, Mitsuo Nohmi, and Kenji Kuba (240) added a degree of sophistication to this protocol by inventing a "unitary Ca²⁺ transient" (UT), which legitimized the [Ca²⁺]_i/I_Ca/t equation being in essence a measure of how many nanomolar Ca²⁺ will appear in the cytoplasm per nanoCoulombs of Ca²⁺-carried current entering the cell. If the transmembrane calcium current would be the only source for the [Ca²⁺]_i increase, the UT should be constant irrespective of the duration or amplitude of I_Ca. An increase in UT thus reflects either the occurrence of CICR that delivers additional Ca²⁺, or saturation of cytoplasmic Ca²⁺ buffers and/or fast Ca²⁺ extrusion systems. Several groups, starting with Hua et al. have measured UT or else quantified the relation between Ca²⁺ entry and [Ca²⁺]_i in peripheral (240, 406, 585, 673) and central (68, 353) neurons, and all groups obtained virtually identical results (Table 2). When Ca²⁺ entry was graded by lengthening the depolarization step, the UT would eventually begin to increase, indicating a supralinear relation between Ca²⁺ entry and [Ca²⁺]_i (Fig. 9); the UT was similarly increased when Ca²⁺ entry was graded by increases in extracellular free Ca²⁺ concentration ([Ca²⁺]_o) or by varying the depolarization amplitude (Figs. 9 and 10). Pharmacological inhibition of RyRs, or emptying the ER by preincubation with 20 mM caffeine, would depress UT and remove the supralinear relation between [Ca²⁺]_i and Ca²⁺ entry. Conversely, sensitization of RyRs by 1 mM caffeine results in an increase of UT (i.e., in increase of [Ca²⁺]_i in response to the same I_Ca; Fig. 10). These data strongly imply the existence of physiological CICR in nerve cells.

View larger version (25K): [in this window] [in a new window]   FIG. 9. Evidence for CICR in neurons: modulation of unitary Ca2+ transients by graded Ca2+ entry. A and B: nonlinear relations between Ca2+ entry and [Ca2+]i in Purkinje neurons in cerebellar slice. The amount of Ca2+ influx was graded by changing either the duration of Ca2+ current (ICa; A) or the amplitude of test potentials evoking ICa (from –70 to –40 mV to 0 mV, B). Plots show peak [Ca2+]i values as a function of ICa integral. The insets show selected ICa (in response to depolarizations of different durations or different amplitudes) and corresponding [Ca2+]i traces. C: similarly to A, the UT was measured from single DRG neurons. Ca2+ entry was graded by varying the duration of ICa (evoked by test depolarization from –60 to 0 mV, duration 20–500 ms). Note the clear supralinear increase in [Ca2+]i at ICa longer than 200 ms. This supralinearity was completely obliterated when the experiments were repeated in the presence of 20 mM caffeine, which depleted the ER. [A and B modified from Llano et al. (353); C from Verkhratsky and Shmigol (673), copyright 1996 with permission from Elsevier.]

View larger version (22K): [in this window] [in a new window]   FIG. 10. Evidence for CICR in neurons: effects of increase in Ca2+ influx and pharmacological modulators. A: elevation of extracellular Ca2+ (from 2 to 8 mM) and hence increase in ICa (evoked by cell depolarization from –60 to 0 mV) leads to an increase in urinary Ca2+ transient (UT) in a single DRG neuron. B: incubation of DRG neuron with 1 mM caffeine results in dramatic increase of [Ca2+]i and UT in response to ICa; after store depletion in the presence of 20 mM caffeine, both UT and [Ca2+]i are markedly depressed. Throughout the experiment ICa did not change significantly. C: effects of pharmacological inhibition of ryanodine receptors (RyRs) by ryanodine on depolarization-induced [Ca2+]i transients. Ryanodine (10 µM) was added into the intrapipette solution, and [Ca2+]i and ICa were recorded simultaneously from DRG neuron. First depolarization (from –60 to 0 mV, 500 ms) delivered 5 min after the beginning of intracellular perfusion triggered large [Ca2+]i transient (left trace). Subsequent application of 20 mM caffeine (middle trace) failed to induce [Ca2+]i elevation. The second depolarization (right trace) resulted in ICa very similar to the first, yet [Ca2+]i was reduced by 80%, indicating CICR eradication by ryanodine. [A and C modified from Shmigol et al. (585); B modified from Verkhratsky and Shmigol (673).]

View larger version (23K): [in this window] [in a new window]   FIG. 11. CICR as seen from the ER lumen. A: CICR-induced changes in [Ca2+]L measured in bovine chromaffin cells transfected by ER-targeted aequorin. a: Activation of CICR by the Ca2+ entry elicited by high-K+-induced cell depolarization. The ER was refilled by perfusing medium containing 1 mM Ca2+. Then, standard medium containing 70 mM KCl was applied as indicated. b: Potentiation of CICR by 1 mM caffeine. B: direct visualization of CICR in DRG neurons. Simultaneous recordings of [Ca2+]i (normalized fluo 3 fluorescent intensity, black trace) and [Ca2+]L (ratiometric recordings with Mag-fura 2, gray trace) dynamics recorded from the same neuron are presented. The cell was challenged subsequently by an application of caffeine (20 mM) and by a 3-s step depolarization from –70 to 0 mV (the corresponding Ca2+ current is shown in the inset on the top of the fluo 3 trace). Note that depolarization triggers an increase in cytoplasmic Ca2+ and a decrease in [Ca2+]L. C: pharmacological manipulation with CICR-induced changes in [Ca2+]L. a: Low (1 mM) caffeine concentration enhances the CICR. The [Ca2+]i (black traces) and [Ca2+]L (gray traces) were measured from the same DRG neuron. The neuron was stimulated by 3-s depolarizations in control conditions and in the presence of 1 mM caffeine (the instants of depolarizations are indicated by arrows). Note the significant rise in the amplitude of the [Ca2+]L transient in the presence of caffeine. The corresponding ICa are shown on the bottom panel at a higher time resolution. b: Ryanodine completely inhibits both caffeine- and Ca2+-induced Ca2+ release. The top panel shows control [Ca2+]i and [Ca2+]L traces (depicted in black and gray, respectively) in response to 20 mM caffeine and 3-s depolarization. The lower panel demonstrates the same experiment performed on another neuron from the same culture after 10-min incubation with 50 µM ryanodine. ICa are shown in the insets. [A modified from Alonso et al. (9); B and C modified from Solovyova et al. (603).]

View larger version (19K): [in this window] [in a new window]   FIG. 12. CICR is graded by Ca2+ entry. A: changes in [Ca2+]L (top panel, gray traces) and [Ca2+]i (bottom panel, black traces) in response to step depolarizations from –70 to 0 mV of increasing duration. B: voltage protocol and Ca2+ currents from the experiment shown in A. C: the relationship between the amplitude of the [Ca2+]L decrease ([Ca2+]L) and the charge carried by Ca2+ current (ICa/t) derived from the traces shown in A and B. D: the ratio between the charge carried by ICa and the amplitude of [Ca2+]L decrease plotted against duration of corresponding Ca2+ currents. Data are means ± SD derived from 5 independent experiments similar to those shown in A. [From Solovyova et al. (603), with permission from Nature Publishing Group.]

Nonetheless, in certain conditions, and in certain cells, neuronal CICR may acquire a regenerative character, when after reaching a threshold the release develops in an all-or-none fashion. A particularly strong CICR in response to a single AP was observed in nodose neurons, where Ca²⁺ entry produced by a single AP was amplified 5- to 10-fold by CICR, suggesting a regenerative process. In fact, the inhibition of CICR in these cells resulted in complete disappearance of [Ca²⁺]_i elevations in response to less than eight APs (103). In sensory neurons regenerative CICR was also reported, although it required pharmacological sensitization of RyRs. In the presence of 5 mM caffeine, suprathreshold [Ca²⁺]_i elevations produced by Ca²⁺ entry through plasmalemmal channels invariably triggered all-or-none CICR (663). Conversely, when the same protocol was applied to hippocampal neurons in slices, it did not reveal any regenerative CICR (446).

In fact, CICR is tightly regulated by the amount of trigger Ca²⁺ entering the cell and the rate of rise of [Ca²⁺]_i in the close neighborhood of RyRs (222). In the presence of caffeine, the rise of [Ca²⁺] in the vicinity of RyRs is fast enough to trigger a regenerative response, which, by recruiting adjacent RyRs (already sensitized by caffeine), fully depletes the store. In the absence of caffeine, [Ca²⁺]_i rises are slower and inactivation of RyRs prevents the occurrence of all-or-none CICR. One also has to remember the [Ca²⁺]_i-sequestering ability of the ER, which balances the release, and sometimes may even overcome the latter. Indeed, as was elegantly shown by David Friel and colleagues (4, 234), the ER may switch between net Ca²⁺ release and net Ca²⁺ accumulation depending on the amount of Ca²⁺ entering the cell.

The mechanism that allows smoothly graded CICR in nerve cells is not entirely clear, although the very same phenomenon has been known for a long time from experiments on cardiomyocytes (149). Several theories (e.g., RyR adaptation, Ca²⁺-dependent inactivation of RyRs, [Ca²⁺]_L depletion, [Ca²⁺]_L regulation of RyRs, etc.) have been proposed to explain the inhibition of autocatalytic CICR (149, 326, 595); most likely a combination of many if not all of these factors is responsible for Ca²⁺ entry-dependent grading of Ca²⁺ release.

5. Depolarization-induced CICR in nerve cells

Although many neurons express the "skeletal" RyR1, identification of direct depolarization-induced Ca²⁺ release, which does not involve plasmalemmal Ca²⁺ entry, has long been missing. Some recently published data indicate that direct coupling between the plasmalemmal and ER Ca²⁺ channels in neurons may indeed exist. At the molecular level, for instance, direct coupling between the Ca²⁺ channels Ca_v1.2 or Ca_v1.3 and RyR1 was identified in solubilized rat brain membrane preparations (433). Some indirect evidence favoring the expression of depolarization-induced CICR in axons was presented by Ouardouz et al. (473), who demonstrated that inhibition of the voltage sensor of Ca²⁺ channel provides the same degree of protection against ischemic axonal damage as inhibition of RyRs. These authors also observed coimmunoprecipitation (suggestive of a direct physical link) of RyR1 and Ca_v1.2 and of RyR2 and Ca_v1.3, both in the whole brain and in the dorsal spinal cord column (such combinations are quite different from skeletal muscle where RyR1 is linked to Ca_v1.1). Finally, elementary Ca²⁺ release events, which occurred in the absence of Ca²⁺ entry but were potentiated by depolarization, were identified in isolated nerve terminals of magnocellular hypothalamic neurons (116). The localized [Ca²⁺]_i transients that resulted from these elementary Ca²⁺ releases were named Ca²⁺ scintillas (a spark or a glimmer in latin). They were not affected by the removal of extracellular Ca²⁺, they were sensitive to ryanodine, and their frequency increased upon depolarization.

6. Endogenous CICR modulators

A) CYCLIC ADP RIBOSE.  The effects of cyclic ADP ribose (cADPR) on Ca²⁺ homeostasis were initially discovered in sea urchin eggs microsomes where this pyridine nucleotide applied in low micromolar concentrations was found to trigger Ca²⁺ release (99, 333). The primary target of cADPR is the ryanodine receptor type 2, and cADPR triggers opening of RyR2 in various nonexcitable cells (176, 177). In neuron-related cells or in neuron-derived cell lines, the injection of cADPR was reported to induce Ca²⁺ release. Such direct cADPR-triggered CICR was observed in permeabilized bovine chromaffin cells (430), and PC12 pheochromocytoma cells (100), as well as in neuroblastoma x fibroblast hybrid cells injected with cADPR (251). An increase in [Ca²⁺]_i due to Ca²⁺ release was also observed in buccal Aplysia neurons injected with cADPR (432); moreover, cADPR also potentiated the release of ACh from cholinergic synapses belonging to these neurons. Apart from affecting the ER, cADPR may exert several other effects not connected with RyRs at all, e.g., cADPR was reported to interact with M-type potassium channels by affecting the ACh-induced inhibition of I_K(M) (61). It has to be noted also that cADPR competes with ATP for the same binding site on the RyR2 (592).

In mammalian neurons, cADPR usually does not induce Ca²⁺ release by itself, yet it potentiates CICR in response to plasmalemmal Ca²⁺ entry. In the majority of neurophysiological experiments, administration of cADPR into the nerve cells was not accompanied by acute changes in [Ca²⁺]_i. Injection of cADPR via patch pipette into NG108–15 neuroblastoma cells did not affect resting [Ca²⁺]_i, but greatly potentiated depolarization-induced [Ca²⁺]_i elevation, the effect being, at least in part, blocked by ryanodine. Apart from facilitating CICR, in these experiments cADPR also potentiated Ca²⁺ entry via L-type channels (204). In another set of experiments on the same neuroblastoma NG108–15, the inclusion of 10 µM cADPR into the pipette solution potentiated [Ca²⁺]_i transients evoked by Ca²⁺ currents and resulted in a strong nonlinearity between Ca²⁺ entry and [Ca²⁺]_i transient amplitude (thus indicating potentiated CICR, Fig. 13A, Ref. 137). Similarly, intracellular administration of cADPR facilitated depolarization-induced [Ca²⁺]_i transients in CA1 hippocampal pyramidal cells (612) and in thalamocortical neurons (68).

View larger version (31K): [in this window] [in a new window]   FIG. 13. cADPR-dependent potentiation of CICR. A, a: whole cell voltage-clamp electrophysiological recording of a cell showing ICa traces evoked upon depolarization of the membrane from –70 to –10 mV. Below the current records are the simultaneously measured [Ca2+]i traces shown at a different time scale. The colors of the [Ca2+]i traces correspond to the colors of the current traces. The arrow shows the time the voltage step was applied. The pipette used to record this cell contained 10 µM cADPR that potentiated the measured [Ca2+]i rises when longer voltage steps, leading to prolonged inward currents (red, green, and yellow), triggered an enhanced Ca2+ release. By measuring the area beneath the current trace, pA multiplied by seconds, the charge carried by ICa was derived. b: The unit transient ([Ca2+]i/ICa/t) calculated for this cell at each duration voltage step (from three separate voltage step applications) is plotted against the duration of the step. Also shown, in black, are values from a representative control cell. c: Pseudocolor images of a control cell and a cell treated with 10 µM cADPR. Images are sequential left to right and taken at 1.4-s intervals; the white arrow represents the time at which the voltage step was applied. The charge entry was similar in both cells. The pseudocolor images show that the edges of the cells were always the first regions to increase, presumably as Ca2+ entered the cell following the depolarization. In the cADPR-treated cell there was a significant rise of [Ca2+]i in the center of the cell after the influx, whereas in the control cell the rise in [Ca2+]i in the center was less apparent and more confined to the edges. B: results of a similar experiment performed on isolated DRG neurons perfused with intrapipette solution supplemented with 10 µM cADPR. The examples of ICa and corresponding [Ca2+]i transients with and without 100 µM ryanodine in the bath are shown in a. The dependence of UT from pulse duration measured for control cells (without cADPR in the pipette), for cells intracellularly exposed to cADPR and for the same cells treated with 100 µM ryanodine is shown on b. [A modified from Empson and Galione (137); B from Kostyuk and Verkhratsky (309), with the actual experiments performed by Shmigol, Eisner, and Verkhratsky.]

To act as a second messenger, production of cADPR must be controlled by relevant neurotransmitter-activated signaling cascades. Indeed, the activity of ADP-ribosyl cyclase may be regulated by metabotropic receptors, such as muscarinic cholinoreceptors (MChRs) or even glutamate receptors. Coupling between MChR and ADP-ribosyl cyclase was identified in the NG108–15 neuroblastoma, indicating that ACh may regulate cADPR production (225). The neurotransmitter-regulated cADPR-dependent Ca²⁺ release pathway was also implicated in glutamate-induced [Ca²⁺]_i signaling in midbrain dopamine neurons: local activation of mGluRs triggered propagating Ca²⁺ waves that were mediated by both InsP₃Rs and RyRs, and were blocked by concerted action of heparin and the cADPR antagonist 8-NH₂-cADPR. Quite naturally, the idea of mGluRs controlling both phospholipase C (PLC) and ADP-ribosyl cyclase was born (429), although more direct data supporting such a suggestion would be very welcome.

In addition, the activity of ADP ribosyl cyclase may be controlled by the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase cascade (224); NO and cGMP do enhance the formation of cADPR in homogenates of sea urchin eggs (176). Photorelease of cGMP from a caged precursor delivered into cultured DRG neurons via intracellular dialysis triggered a delayed Ca²⁺-dependent inward current in 50% of all neurons tested (514). This was interpreted as an indication of cADPR-induced Ca²⁺ release resulting from the activation of ADP ribosyl cyclase caused by cGMP-dependent phosphorylation. In support of this suggestion, the inclusion of the cADPR antagonist 8-NH₂-cADPR into the pipette solution inhibited the effects of cGMP. Finally, there is some evidence that cADPR may be involved in assisting CICR necessary for LTP generation in Schaffer collaterals-CA1 neuron synapses in rat hippocampus (531).

All in all, cADPR remains a strong candidate as an endogenous modulator of CICR. In particular, neurotransmitter-regulated synthesis of cADPR in synaptic regions may be very important for local modulation of [Ca²⁺]_i signals and hence relevant for synaptic plasticity.

B) NICOTINIC ACID ADENINE DINUCLEOTIDE PHOSPHATE.  Another nucleotide involved in the regulation of ER Ca²⁺ release in many types of nonexcitable cells is nicotinic acid adenine dinucleotide phosphate (NAADP). This molecule is believed to trigger Ca²⁺ release through specific, but so far unidentified, channels, the NAADPRs (75, 77, 96, 177, 374), although NAADP may also directly activate RyRs (182). In the neural tissue, the NAADP-induced Ca²⁺ release was described in brain microsomes (20) and in neurons from buccal ganglion of Aplysia californica (86). The existence and importance of NAADP-dependent signaling cascade in vertebrate neurons remains unknown.

C) CALEXCITIN.  Calexcitin (CE), a Ca²⁺ and GTP-binding protein, is a recently discovered endogenous modulator of neuronal RyRs and CICR. This protein was initially isolated from neurons of conditionally trained Hermissenda (451). CE binds Ca²⁺ by virtue of an EF Ca²⁺-binding domain and undergoes considerable and reversible conformational changes when [Ca²⁺] fluctuates between 0.1 and 10 µM (14). The physiological effects of CE are exerted through modulation of K⁺ channels and RyRs. CE binds to RyRs in a Ca²⁺-dependent manner (7, 453) and facilitates Ca²⁺ release; injection of CE into hippocampal neurons resulted in a slow-developing [Ca²⁺]_i elevation, which originated from the ER (454). CE also stimulated Ca²⁺ release from squid optic lobe microsomes; this release was blocked by dantrolene and an anti-RyR antibody (454). CE is known to be phosphorylated upon memory consolidation, which raises the interesting possibility that RyRs are involved in this process.

D) VANILLOID RECEPTORS.  The effects of capsaicin on nerve cells are executed through the TRPV1/VR1 vanilloid receptor, which belongs to a broad family of TRP channels (35, 49). In cultured DRG neurons, capsaicin increased [Ca²⁺]_i in a dose-dependent manner with ED₅₀ 13.5 µM. This [Ca²⁺]_i elevation was mimicked by the capsaicin analog resiniferatoxin (100 nM) and blocked by the VR1 receptor antagonist capsazepine (10 mM). Capsaicin-induced [Ca²⁺]_i signaling did not require extracellular Ca²⁺ and disappeared after the ER was depleted by incubating cells with a mixture of 10 mM caffeine and 12 µM ryanodine (144). The [Ca²⁺]_i transients evoked by capsaicin were also inhibited by 10 µM dantrolene or by 10 µM ruthenium red (144), as well as by 2 µM TG (271). Taken together, these data indicate that capsaicin stimulates CICR in sensory neurons.

A rather unusual suggestion of direct involvement of TRPV1/VR1 in Ca²⁺ release from the ER was made recently by Liu et al. (352), who confirmed that capsaicin, when applied in Ca²⁺-free media, produces [Ca²⁺]_i elevation in small DRG neurons. In contrast to previous findings, however, this [Ca²⁺]_i signaling pathway was not affected by TG but was sensitive to the TRPV1/VR1 receptor antagonist capsazepine. Morphological examination revealed a substantial overlap between polyclonal antibody (TRITC labeled) TRPV1/VR1 staining of small DRG neurons and staining of the same cells with ER-tracker or ER marker DiOC₅ (352). This overlap in staining was considered to indicate specific localization of TRPV1/VR1 in the ER membrane. These data prompted the intriguing speculation that TRPV1/VR1s may act as ER-resident Ca²⁺ release channel. These authors also proposed that the vanilloid receptors act as store-operated channels (but this is disputed by Karai and co-workers, Ref. 271).

E) PROTONS.  "It is a truth universally acknowledged that all intracellular reactions are sensitive to pH" wrote Roger Thomas (641),¹ and RyR function as well as CICR in various cell types are sensitive to the concentration of protons (e.g., Refs. 128, 395). In neurons, acidosis inhibits CICR and increases ER Ca²⁺ content (641), whereas alkaline shifts stimulate the release of Ca²⁺ and/or inhibit reuptake of Ca²⁺ into the ER (696).

F) GLUTAMATE.  It is generally accepted that the action of extracellular glutamate on ER Ca²⁺ release is accomplished via activation of group I metabotropic glutamate receptors (mGluRs) coupled, through G protein pathways, to PLC and InsP₃ metabolism. Data accumulated during the past decade, however, indicate the existence of an alternative route that involves direct interactions of mGluRs and RyRs (145). Initial evidence in support of this pathway was obtained in cell-attached patch-clamp experiments on cultured cerebellar granule cells (90). This study demonstrated mGluR-dependent activation of L-type VGCCs, which could not be mimicked by intracellular dialysis with InsP₃, was not blocked by heparin, but was completely obliterated by 1 µM ryanodine and simulated by caffeine. Subsequently, it was found that ryanodine effectively (90%) inhibited 1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-induced [Ca²⁺]_i elevation, while it was much less potent against [Ca²⁺]_i signals triggered by the other metabotropic agonists carbachol and histamine (119). These data led to an interesting idea about direct functional coupling between mGluRs and RyRs, thought to be mediated by Homer proteins (145). This assembly may be even more complex and may involve voltage-gated Ca²⁺ channels and Ca²⁺-dependent potassium channels. Although extremely stimulating, this hypothesis still requires additional experimentation.

Further evidence for glutamate-mediated regulation of CICR was obtained on neurons from the avian nucleus magnocellularis, in which activation of mGluRs inhibited both CICR and InsP₃-induced Ca²⁺ release (IICR). It is hypothesized that this inhibition provides a mechanism by which glutamate protects highly active neurons from calcium overload (275).

G) NEUROTROPHINS.  Neurotrophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3/4/5 (NT-3, -4, and -5), operate through an extended family of TrkA, -B, and -C receptors, tumor necrosis factor (TNF) receptors, and p75 and Fas receptors (89). Among these receptors TrkA and p75 are abundantly expressed in the brain, and their activation is important for neuronal physiological activity; for example, BDNF-dependent neuronal excitation, synaptic plasticity, and release of neurotransmitters have been reported (265, 340, 359, 685, 701). Several of these effects involve intracellular Ca²⁺ release mediated by RyRs. For instance, NGF induces release of glutamate from cultured cerebellar neurons through activation of p75 receptors and ryanodine-sensitive Ca²⁺ mobilization (464). Similar activation of glutamate release, sensitive to dantrolene and to store depletion by caffeine, was observed in cortical synaptosomes; in this case, NGF acted through TrkA receptors, and glutamate release was sensitive to heparin (527).

Another neurotrophin, NT-3, significantly potentiated the spontaneous neurotransmitter release in the neuromuscular junction; this action required both Ca²⁺ release via RyR/InsP₃Rs and the activation of Ca²⁺/calmodulin-dependent kinase II (208). In pyramidal neurons from the visual cortex, NT-4 potentiated depolarization-induced [Ca²⁺]_i elevations partly though enhancement of CICR (278).

Alternatively, neurotrophins may regulate CICR by affecting the expression of RyRs. For instance, NGF treatment of cultured adrenal chromaffin cells significantly elevated the expression of RyR2 and dramatically (>1,000% over 10 days) increased the amplitude of caffeine-induced [Ca²⁺]_i responses (260). In cerebellar granule cells, the addition of 10 ng/ml BDNF from the first day in culture augmented the contribution of CICR to depolarization-induced [Ca²⁺]_i transients by a yet unknown mechanism (408).

H) TNF-/SPHINGOSINE-1-PHOSPHATE.  Treatment of cultured rat DRG neurons with TNF- induced a Ca²⁺-dependent current in 70% of cells and triggered [Ca²⁺]_i increase in 35% of cells tested; both the current and [Ca²⁺]_i elevation were inhibited by TG (10–50 µM) and ryanodine (10 µM). The action of TNF- was mimicked by the uncaging of sphingosine or by addition of 0.4 U/ml sphingomyelinase (515). In parallel, two subtypes of TNF- receptors, TNFR1 and TNFR2, were identified in DRG neurons by immunohistochemical and RT-PCR techniques. Whether the activation of TNFRs represents a novel pathway for Ca²⁺ release mediated by sphingosine-1-phosphate through separate ER channels or through modulation of RyRs remains rather unclear (712).

I) NO.  NO, which acts as a rapidly diffusible gaseous messenger in many tissues including the brain, seems to be another endogenous regulator of RyRs (143). NO-induced activation of RyR-mediated Ca²⁺ release was observed in hippocampal CA1 neurons; this Ca²⁺ release was important for both CREB phosphorylation and LTP induction (360). NO-induced activation of Ca²⁺ release from a ryanodine-sensitive store was also implicated in hypoxic [Ca²⁺]_i rises in brain slices (394). Similarly, NO-mediated ER Ca²⁺ release via RyRs is considered to be an important pathological step in interleukin (IL)-1-induced pyrogenic effects (485).

C. IICR in Nerve Cells

1. Direct evidence for neuronal IICR

The initial suggestion of InsP₃-induced release of Ca²⁺ in neurons arose from electrophysiological studies, which demonstrated that injection of InsP₃ into various neurons or neuronlike cells [e.g., neuroblastoma x glioma cells (223), rat hippocampal neurons (387), rat dorsal raphe neurons (162), etc.] resulted in activation of Ca²⁺-dependent K⁺ currents. This suggestion was immediately confirmed by measuring [Ca²⁺]_i (Fig. 14A, Ref. 151), which demonstrated directly that intracellular administration of InsP₃ induces a [Ca²⁺]_i elevation due to Ca²⁺ release from the intracellular compartments (Table 3).

View larger version (37K): [in this window] [in a new window]   FIG. 14. Inositol trisphosphate (InsP3)-induced Ca2+ release in neurons in response to direct activation of InsP3 receptors. A: one of the first recordings of InsP3-induced Ca2+ release (IICR) in nerve cell. The Aplysia californica neuron was loaded with fura 2 and injected with InsP3 (0.8 mM InsP3 in the electrode). Pseudocolor fluorescence images of [Ca2+]i show intracellular calcium dynamics at different times during and after InsP3 injection. B: low sensitivity of neuronal IICR. Left panel shows [Ca2+]i traces recorded from Purkinje neuron in cerebellar slice after photorelease of different InsP3 concentrations. On the right panel, the amplitudes of normalized peak [Ca2+]i responses are plotted against InsP3 concentrations attained during photorelease. [A modified from Fink et al. (151); B modified from Khodakhah and Ogden (284).]

The reasons for low sensitivity of IICR in nerve cells are not quite clear. The effective InsP₃ concentrations in the cytosol can be substantially reduced by an extensive InsP₃ buffering, or else InsP₃ can be quickly degraded by enzymatic systems. These two possibilities, however, are questioned by the experiments on permeabilized cells, where cytoplasm is essentially washed out together with the InsP₃ buffers and InsP₃-degrading enzymes. Alternatively, the InsP₃ binding to the receptor may be inhibited by endogenous inositol trisphosphate receptor inhibitor (IRI), recently discovered in the cerebellum (686). The low sensitivity of neuronal IICR could be important for the maintenance of synaptic specificity by localization of InsP₃-induced signaling.

2. Metabotropic receptors and neuronal IICR

Physiological activation of IICR requires stimulation of plasmalemmal receptors coupled to PLC, which in turn hydrolyzes phosphatidylinositol 4,5-bisphosphate and gives birth to InsP₃. These plasmalemmal receptors, which usually share a common seven-transmembrane domains structure, are coupled to G proteins and are generally known as metabotropic receptors. Neurons express an impressive complement of metabotropic receptors, many of which are coupled to the InsP₃ signaling cascade. Numerous observations have shown that activation of many different types of neuronal metabotropic receptors trigger IICR (Table 4).

View larger version (41K): [in this window] [in a new window]   FIG. 15. Synaptically evoked localized IICR in Purkinje neuron. A: local dendritic [Ca2+]i signals mediated by repetitive parallel fiber stimulation. a: Confocal image of Purkinje neuron voltage-clamped and loaded with Oregon-green BAPTA-1. The dendritic regions 1 and 2 correspond to sites of stimulation (position of stimulation pipettes are depicted by dotted lines). b: These regions at higher magnification and also shows the kinetics of [Ca2+]i responses to repetitive parallel fiber stimulation (5 stimuli, 50 Hz). B: dependence of synaptic [Ca2+]i responses on ER Ca2+ release. a: Almost complete elimination of the delayed phase of [Ca2+]i transient by intracellularly applied heparin (4 mg/ml). b: Same effect of ER depletion by incubation of slices with cyclopiazonic acid (CPA; 20 µM). [Modified from Takechi et al. (624).]

3. CICR-assisted IICR

An important property of InsP₃ receptors and IICR is their regulation by [Ca²⁺]_i. An increase in the latter facilitates IICR and may trigger Ca²⁺ release through InsP₃Rs in the presence of a constant level of the second messenger. The synergism between [Ca²⁺]_i and InsP₃ may act as a coincidence detector allowing the system to identify the simultaneous activation of both signaling cascades; furthermore, pairing of InsP₃ and [Ca²⁺]_i elevations may be important in converting local signaling events into propagating waves of ER membrane excitation.

Indeed, IICR was found responsible for generation of propagating Ca²⁺ waves triggered by synaptic excitation of hippocampal pyramidal neurons. These Ca²⁺ waves were found almost exclusively in the thick apical shafts of CA1 neurons (445). The generation of the Ca²⁺ wave has a threshold nature, which most likely reflects InsP₃ accumulation (717) and amplification of the release by CICR through InsP₃Rs. This amplification can be seen directly. Pairing of mGluR stimulation (either by synaptic stimulation or by application of a metabotropic receptors agonist) with plasmalemmal Ca²⁺ entry, activated by back-propagating action potentials, greatly enhanced [Ca²⁺]_i signals (Fig. 16, Refs. 444, 446). In fact, the degree of mutual potentiation was quite remarkable: a backpropagated AP by itself resulted in a fast low-amplitude [Ca²⁺]_i increase, and the application of mGluR antagonists alone did almost nothing to [Ca²⁺]_i, yet when an AP was generated in the presence of continuous mGluR stimulation, a large (up to 40 µM) [Ca²⁺]_i increase was produced (446). Similarly, [Ca²⁺]_i waves could be induced in response to electrical stimulation of mossy fibers, which form synaptic input to CA3 hippocampal neurons. These waves were inhibited by 1 µM heparin and were potentiated by pairing mossy fiber stimulation with AP-driven Ca²⁺ influx through VGCC (270). Comparable synaptically activated propagating [Ca²⁺]_i waves resulting from IICR were also observed in neocortical pyramidal neurons from layers II/III and V. These [Ca²⁺]_i waves were dependent on stimulation of metabotropic glutamate receptors and were blocked not only by intracellularly applied heparin, but also by 5–20 µM ryanodine, but not by ruthenium red (331). These results may indicate either an amplifying role of RyR-mediated CICR or depletion of a single store shared by RyRs and InsP₃Rs.

View larger version (40K): [in this window] [in a new window]   FIG. 16. Ca2+-assisted InsP3-induced Ca2+ release. A: backpropagated action potentials cause large [Ca2+]i elevation and propagating [Ca2+]i wave in the presence of the mGluR agonist 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD). In control conditions (first trace from the left), a pair of spikes caused rapid, almost simultaneous, increases in [Ca2+]i at all locations in the cell, as shown by [Ca2+]i responses measured from green and red boxes on the cell image shown on the right. When the slice was incubated with 30 µM t-ACPD, the same pair of spikes caused the same synchronous increases in [Ca2+]i, followed by larger propagating [Ca2+]i signal (second trace). Five spikes (third trace) caused a similar secondary response that initiated earlier and more synchronously at different dendritic locations. B: synaptic stimulation followed by action potentials causes release of Ca2+ in the apical dendrites. Left panel shows bis-fura 2-filled pyramidal neuron, with regions of interest and electrode positions marked. AP-5 (100 mM) and CNQX (10 mM) were added to the bath. Scale bar, 50 µm. a: Synaptic stimulation at 100 Hz for 1 s caused no detectable change in fluorescence at any somatic or dendritic location. The membrane potential hyperpolarized because inhibition was not blocked. b: Synaptic stimulation (same stimulation parameters) followed immediately by action potentials, evoked by 1-ms intrasomatic pulses at 30-ms intervals, caused fast fluorescence increases at all locations and slower, larger increases, predominantly in the proximal apical dendrites. c: A train of 10 action potentials without synaptic stimulation evoked fluorescence increases that peaked at the time of the last spike at all locations.[A from Nakamura et al. (446), copyright 2000 by the Society for Neuroscience. B from Nakamura et al. (444), copyright 1999 with permission from Elsevier.]

4. Endogenous IICR modulators

A) PROTEIN KINASES AND CALCINEURIN.  Endogenous regulation of InsP₃Rs is mainly achieved through phosphorylation/dephosphoryaltion of the receptor molecule. Phosphorylation of the InsP₃R, which results in enhanced channel activity, is catalyzed by protein kinases A, C, and G (412, 413) and by tyrosine kinase (258). The latter is also coupled to InsP₃ metabolism and therefore may affect the IICR via several routes. Dephosphorylation of InsP₃R and hence reduction of the channel activity is catalyzed by the Ca²⁺/calmodulin-dependent phosphatase calcineurin (73), which also regulates the expression of InsP₃R at the transcriptional level, because pharmacological blockade of calcineurin abolished the expression of InsP₃Rs in cerebellar granule neurons (181).

B) IMMUNOPHILIN FK506 BINDING PROTEIN 12.  Immunophilin FK506 binding protein 12 (FKBP12), which is abundantly expressed in brain tissue, was shown to be associated with InsP₃R1. This complex is disrupted upon the addition of the immunodepressants FK506 or rapamycin. Dissociation of InsP₃R1-FKBP12 causes an increase of Ca²⁺ flux through the channel, and this can be reversed by adding FKBP12 (74). It seems that the latter serves as a physiological regulator of the conductive properties of InsP₃Rs. In biophysical experiments on purified InsP₃R1s in planar lipid bilayers, the addition of exogenous recombinant FKBP12 substantially increased the open time of the channels (113).

C) CALMODULIN.  The InsP₃R type 1, but not type 3, possesses a high-affinity binding site for calmodulin, occupation of which inhibits the channel. Inhibition of calmodulin by calmodulin inhibitory peptide sensitizes IICR in Xenopus oocytes (376).

D) CALDENDRIN.  The InsP₃Rs are also regulated by the cytoplasmic Ca²⁺-binding proteins caldendrins, which, depending on their structure, may either activate or inhibit IICR. A splice variant of caldendrin was found to bind specifically to all three InsP₃R subtypes. This binding activated InsP₃Rs, suggesting the possibility of IICR initiation even in the absence of the natural agonist (707). At the same time a long-splice variant of caldendrin, when expressed in PC12 or HeLa cells, inhibited IICR triggered by activation of histamine and purinoreceptors (206).

D. Do InsP₃Rs and RyRs Share a Common Ca²⁺ Pool?

Although morphological and functional evidence (see sect. II) clearly defined the ER as a single continuous space, the analysis of Ca²⁺ release divided the field into supporters of a single functional Ca²⁺ pool available for both CICR and IICR, and supporters of at least two separate pools, selectively sensitive to either InsP₃ or Ca²⁺/caffeine (see Table 6). The experimental strategy employed by both camps is similar: the cells are treated with maximal concentrations of CICR agonists and thereafter the ability of IICR-stimulated agents to produce Ca²⁺ release is tested, and vice versa. Provided the cell has a single Ca²⁺ pool, each agonist should be able to deplete it completely, thus rendering subsequent stimulations with other agents ineffective. Alternatively, if the cell expresses more than one functional pool, each agonist would discharge a compartment with appropriate complement of receptors. The story is old and at the moment opposing factions are far from a consensus. As far as neurons are concerned, it looks as if more data are in favor of a single continuous ER Ca²⁺ store. Furthermore, some studies supporting separate Ca²⁺ pools mention peculiar unidirectional relations between RyR-bearing and InsP₃Rs bearing stores: either ryanodine occluded IICR but not vice versa (541), or else stimulation of IICR depletes caffeine-sensitive pool but stimulation of RyRs does not affect the InsP₃-sensitive one (528). Moreover, in most cases, the separate pools were observed in cell lines or in embryonic cultures, whereas the existence of a single Ca²⁺ store is supported by experiments on primary cultures and brain slices, the latter preparations being rather more physiological (Fig. 17A). When directly investigating intra-ER Ca²⁺ dynamics in sensory neurons, a very clear and full overlap between InsP₃/ryanodine/caffeine/Ca²⁺ and TG/CPA pools was observed (Fig. 17B, Ref. 602).

View larger version (23K): [in this window] [in a new window]   FIG. 17. Neuronal ER as a single functional Ca2+ store shared by RyRs and InsP3Rs. A: ryanodine abolishes InsP3-induced [Ca2+]i elevation in Purkinje neuron studies in cerebellar slices. Left panel shows three [Ca2+]i responses to photorelease of various concentrations of InsP3 (as indicated over the recordings; instances of ultraviolet flash are indicated by solid circles). Right panel shows complete inhibition of InsP3-induced Ca2+ mobilization in a neuron that was internally dialyzed with 50 µM ryanodine. Slow equilibration of ryanodine through the Purkinje neuron most likely resulted in depletion of the store. B: complete overlap of InsP3/caffeine/ryanodine and TG/CPA-sensitive Ca2+ store in single DR neurons as judged by [Ca2+]L recordings. a–d: [Ca2+]L recordings taken from different neurons that were challenged with ER depleting/Ca2+-release channels activating agents in a sequences indicated on the graph. [A from Khodakhah and Armstrong (283), with permission from the Biophysical Society; B from Solovyova and Verkhratsky (602), with permission from Springer-Verlag.]

The level of free Ca²⁺ concentration within the ER lumen is critically important for regulation of Ca²⁺ release as it creates the driving force for Ca²⁺ exit and regulates the functional availability of Ca²⁺ release channels. That [Ca²⁺]_L in nerve cells is a labile parameter became obvious from the very beginning of experimental investigations of the ER Ca²⁺ store. The first attempts to induce Ca²⁺ release in single central neurons, either by caffeine or by metabotropic agonists, demonstrated that application of stimulating agents to resting cells triggered very tiny [Ca²⁺]_i increases in a small proportion of neurons. However, when caffeine or a metabotropic agonist was applied shortly after the cell was depolarized, and thus had experienced a large Ca²⁺ influx into the cytosol, a considerable [Ca²⁺]_i elevation due to Ca²⁺ release was apparent in a large proportion of neurons (249, 250, 375, 437, 526, 581). A small depolarization (i.e., with 10 mM KCl or with low concentrations of NMDA) which led to a steady-state [Ca²⁺]_i increase had the same effect (249).

The stimulating effect of the conditioning depolarization was transient because the amplitude of Ca²⁺ release decreased progressively when increasing the time gap between the depolarization and the application of a Ca²⁺-release stimulator (Fig. 18). Quite logically, this peculiar behavior was explained in terms of a low releasable Ca²⁺ content of the ER in resting central neurons. The empty Ca²⁺ store favors Ca²⁺ accumulation and therefore Ca²⁺ entering the cell following depolarization is taken up by the ER, thus increasing the Ca²⁺ content within the store and permitting Ca²⁺ release. An extreme situation, in which the store is empty or almost empty at rest, is usually observed in cultured cells. Application of caffeine to resting neurons in acute hippocampal slices (179) or in acutely isolated thalamocortical neurons (68) routinely evokes [Ca²⁺]_i responses; similarly stimulation of IICR triggered [Ca²⁺]_i elevation in Purkinje neurons (624) and in pyramidal cortical neurons (609) in situ. Nonetheless, the conditioning depolarization dramatically (by 500%) increased the amplitude of a subsequent caffeine-induced [Ca²⁺]_i transient in central neurons studied in acute slices (179); and similarly to what was found in cultured neurons this potentiation was short-lived (Fig. 18B). In peripheral neurons, the effect of conditioning depolarization also existed, although it was much less dramatic; conditioning depolarization increased the amplitude of subsequent Ca²⁺ release by 10–30% (583, 661). The nature of this rather prominent difference between central and peripheral neurons is unclear; similarly unclear are the mechanisms responsible for the loss of surplus Ca²⁺ in the ER after the store has been charged.

View larger version (25K): [in this window] [in a new window]   FIG. 18. Transient overcharging of ER Ca2+ stores in central neurons. A: left panel shows representative [Ca2+]i recordings from cultured hippocampal neurons challenged with 20 mM caffeine and 50 mM KCl as indicated on the graph. Note that initial application of caffeine to the resting cell fails to trigger [Ca2+]i elevation, yet caffeine applied immediately after the end of KCl depolarization produces substantial [Ca2+]i response. Increase in the time gap between conditioning KCl depolarization and application of caffeine leads to progressive decrease in the amplitude of caffeine-induced [Ca2+]i elevation (right panel shows a summary of similar experiments performed on different types of cultured central neurons), indicating that ER loses surplus Ca2+. The columns on the graph are means ± SD for the amplitudes of caffeine-induced [Ca2+]i elevation; caffeine was applied 30, 210, and 600 s after the end of KCl depolarization. B: experiment very similar to that described in A except it was performed on CA1 pyramidal neurons in acutely isolated hippocampal slices. Left panel shows examples of [Ca2+]i recordings demonstrating the changes in caffeine-induced [Ca2+]i response depending on conditioning KCl (40 mM) challenge. On the right, the bar histogram depicting the relation between the size of caffeine-induced [Ca2+]i transient versus the time elapsed after a KCl-induced [Ca2+]i elevation (t). The t was measured between the peaks of KCl-mediated and caffeine-mediated [Ca2+]i transients. Each bar represents a mean of 6–11 data points. The amplitudes of caffeine-induced [Ca2+]i transients evoked after KCl depolarization were normalized with respect to the control (i.e., before KCl) [Ca2+]i transient. [A from Shmigol et al. (581), with permission from Blackwell Publishers Ltd.; B from Garaschuk et al. (179), with permission from Springer-Verlag.]

The transient Ca²⁺ loading of the Ca²⁺ store in central neurons certainly has very important implications. Indeed, a transient increase in [Ca²⁺]_L and the consequent enhancement of Ca²⁺ release would provide the ER with a coincidence detection mechanism and with (at least short-term) memory. Further understanding of the mechanisms controlling changes in the ER Ca²⁺ concentration ultimately requires direct monitoring of the free Ca²⁺ dynamics in the ER lumen.

Several experimental approaches permitting real time measurements of [Ca²⁺]_L have been developed during the last two decades (Table 7). These techniques employ either ER-targeted Ca²⁺-sensing luminescent or fluorescent proteins (10, 120) or conventional fluorescent Ca²⁺ probes with a sufficiently low Ca²⁺ affinity (232, 492, 601). The [Ca²⁺]_L measurements in neurons began only recently (Table 7; Fig. 19), and our knowledge about ER Ca²⁺ dynamics in nerve cells remains very incomplete. The quantitative [Ca²⁺]_L measurements performed in PC12 cells and isolated sensory neurons showed a high resting [Ca²⁺]_L ranging between 100 and 800 µM and demonstrated that activation of Ca²⁺ release channels by Ca²⁺, caffeine, ryanodine, and InsP₃ results in a rapid decrease of [Ca²⁺]_L reflecting Ca²⁺ release. Direct [Ca²⁺]_L measurements in physiologically intact central neurons have not yet been performed.

View larger version (30K): [in this window] [in a new window]   FIG. 19. Examples of [Ca2+]L recordings. A: an example of [Ca2+]L recording in bovine chromaffin cells transfected by ER-targeted aequorin showing complete overlap of InsP3/ryanodine and TG-sensitive store. Left trace shows effects of caffeine (50 mM) and histamine (10 µM) on [Ca2+]L after the ER was refilled by incubation with medium containing 1 mM Ca2+. On the right, the effects of caffeine, histamine, and bradykinin (1 µM) on [Ca2+]L in control conditions and after pretreatment with either ryanodine (10 µM) or thapsigargin (1 µM) are demonstrated. B: example of [Ca2+]L recording using fluo 3FF. The cells were loaded with fluo 3FF-AM, and no attempts to washout the cytosolic portion of the dye were made, which may account for a poor dynamic range of measurements (maximal response 0.1 F/F). Cells were treated with 10 mM caffeine, 50 mM KCl, or 1 mM chloro-m-cresol as indicated on the graph. Caffeine triggered [Ca2+]L response where recovery was as fast (left trace) or even faster (right trace) that the initial [Ca2+]L decrease. C: simultaneous visualization of [Ca2+]i and [Ca2+]L dynamics in DRG neurons. On the left, a series of selected images of the Mag-fura 2 ratio (Ex. 340/380 nm, top panel) taken simultaneously with images of fluo 3 fluorescent intensity (Ex. 488 nm) from the DRG neuron exposed to 20 mM caffeine is shown. Right panel shows calibrated recordings of [Ca2+]L and normalized fluo 3 fluorescent intensity (reflecting changes in [Ca2+]i) taken from the cell shown on the left. Caffeine was applied as indicated on the graph; the positions of the images presented on the left panel are shown near the [Ca2+]L trace. [A from Alonso et al. (9) by copyright permission from The Rockfeller University Press; B from Cseresnyes et al. (110), copyright 1997 with permission from Elsevier; C from Solovyova et al. (603), with permission from Nature Publishing Group.]

Control of [Ca²⁺]_L and refilling of the Ca²⁺ store after depletion is accomplished by SERCA pumps. Numerous experiments (see Tables 1– 5) have demonstrated that inhibition of the latter prevents Ca²⁺ accumulation and results in progressive depletion of the Ca²⁺ store due to unopposed leakage of Ca²⁺ from the ER lumen. Occasional reports mention the possible existence of a TG-resistant Ca²⁺ store in nerve cells, and a TG-resistant Ca²⁺ transport was found in brain microsomes and in certain cell lines (687). The relevance of this type of Ca²⁺ pool for neuronal physiology remains unclear.

Intraluminal Ca²⁺ recordings revealed an important role of the [Ca²⁺]_L level in the regulation of the velocity of ER Ca²⁺ uptake. Decreases in [Ca²⁺]_L significantly (5–7 times) increased the speed of Ca²⁺ uptake; replenishment of the ER slowed the rate of Ca²⁺ uptake (603). Most importantly, at least in sensory neurons, the influence of an increased [Ca²⁺]_i on the Ca²⁺ uptake velocity seems to be negligible (Fig. 20). To quantify Ca²⁺ uptake velocity, Ca²⁺ release was stimulated by brief application of caffeine, and the recovery kinetics of [Ca²⁺]_L were monitored during the washout period. After [Ca²⁺]_L reached the prestimulation level, the SERCA pumps were inhibited by TG, which resulted in a relatively slow decrease in [Ca²⁺]_L due to Ca²⁺ leakage. The rate of leakage declined in proportion to the decrease in [Ca²⁺]_L, presumably reflecting a falling electrochemical driving force for Ca²⁺. It turned out that neither the rate of Ca²⁺ uptake nor the velocity of Ca²⁺ leakage was affected when a BAPTA/Ca²⁺ (10 mM/2 mM) buffer was washed into the cytosol (Fig. 20, A and B). In the latter condition [Ca²⁺]_i is clamped, yet plenty of Ca²⁺ is available for ER accumulation. Therefore, in peripheral neurons store refilling does not require an increase in [Ca²⁺]_i, albeit it requires Ca²⁺ availability in the cytosol. This seems to be a general mechanism for many cell types, and the very similar dependence of ER Ca²⁺ uptake on [Ca²⁺]_L and independence on [Ca²⁺]_i rises, was shown for pancreatic acinar cells (425; see Fig. 20C) and BHK-21 fibroblasts (231).

View larger version (27K): [in this window] [in a new window]   FIG. 20. [Ca2+]L regulates the rate of Ca2+ uptake onto the ER. A: top panel shows simultaneous [Ca2+]L (black trace) and [Ca2+]i (gray trace) recordings from single DRG neurons treated with 20 mM caffeine and 5 µM TG as indicated on the graph. Bottom panel shows the relationship between Ca2+ uptake/Ca2+ leakage rates and [Ca2+]L. The intrapipette solution did not contain any Ca2+ buffers, except 0.1 mM Ca2+ indicator fluo 4. B: the same experiment was repeated on another cell, when intrapipette solution was supplemented with 10 mM BAPTA/2 mM Ca2+ buffer, which clamped the [Ca2+]i at 90 nM (note absence of [Ca2+]i changes on gray trace). The Ca2+ uptake/Ca2+ leakage rates shown on the bottom panel of the graph do not differ significantly from those on A, demonstrating thus that an increase in [Ca2+]i does not affect Ca2+ movement into and out of the ER. C: an example of similar relations between [Ca2+]L and uptake rate obtained in pancreatic acinar cell. The Ca2+ reuptake into intracellular stores following ACh-evoked Ca2+ release was not prevented by clamping the cytoplasmic Ca2+ concentration at the normal resting level. In this experiment, the patch pipette solution was heavily buffered by a BAPTA/Ca2+ mixture, clamping [Ca2+]i at a concentration of 90 nM. Left panel shows the ACh-evoked loss of Ca2+ from the ER store and the subsequent rise of [Ca2+]L after removal of the agonist. After a period of Ca2+ reaccumulation, thapsigargin is added to arrest the ER Ca2+ ATPase, and a slow reduction in [Ca2+]L follows. Right panel shows the relationship between apparent calcium transport rates and [Ca2+]L derived from the data shown on the left. Note similar behaviors of ER Ca2+ uptake rate in acinar cell and in DRG neuron (A and B). [A from Solovyova et al. (603), with permissiion from Nature Publishing Group; C from Mogami et al. (425), with permission from Nature Publishing Group; B represents unpublished data from Solovyova and Verkhratsky.]

All in all, the regulation of [Ca²⁺]_L in peripheral neurons seems to be quite straightforward; these stores are full in the resting conditions, and an efficient [Ca²⁺]_L dependent regulation of SERCA pumping keeps [Ca²⁺]_L under tight control. This control is assisted by a [Ca²⁺]_L-dependent leakage such that every increase in [Ca²⁺]_L immediately increases the Ca²⁺ leak and decreases SERCA activity. Store depletion leads to the opposite effect, i.e., a decrease in Ca²⁺ leak and rapid upregulation of SERCA pumping velocity. Intuitively, the mechanism controlling [Ca²⁺]_L in central neurons must be very different as it should permit a high flexibility of the [Ca²⁺]_L regulation to tolerate the relatively low resting ER Ca²⁺ content. Nevertheless, this low resting [Ca²⁺]_L seems to prime SERCAs for rapid accumulation of the bulk of the Ca²⁺ entering the cytosol upon depolarization or synaptic stimulation. This also assumes the high sensitivity of the Ca²⁺ uptake mechanism to increases in [Ca²⁺]_i, which represents another clear difference between central and peripheral nerve cells.

The problem of store refilling is also inseparable from the question of neuronal expression of the store-operated Ca²⁺ entry pathway. This pathway, discovered by Jim Putney (522), is a general feature of nonexcitable cells, which lack other means of Ca²⁺ entry. A direct relation between ER depletion and SOC activation is firmly established (458, 488, 490, 523). In nerve cells, the replenishment of the Ca²⁺ store after depletion by either several consecutive applications of caffeine/metabotropic agonists or prolonged incubation of cells with Ca²⁺ release stimulating agents, requires plasmalemmal Ca²⁺ influx (179, 661). Does this Ca²⁺ influx employ a classical store-operated mechanism? Or, does the Ca²⁺ enter the cell via numerous Ca²⁺-permeable channels present in the neuronal plasmalemma? This remains a debatable matter. From the general logic of neuronal physiology, the SOC pathway can hardly be justified. Indeed, neurons possess plenty of Ca²⁺ channels, and moreover, these channels are repeatedly activated, as neurons rarely enjoy complete idleness. Furthermore, activation of SOC, according to several reports, has a threshold nature, i.e., it requires a very substantial depletion of the store (202, 489, 490). This is very difficult to reconcile with the observations of empty stores in central neurons; if SOC exists it surely would increase [Ca²⁺]_i up to the point when the stores become replenished.

Nonetheless, several groups have suggested that neurons possess a store-operated Ca²⁺ influx pathway very similar to that of nonexcitable cells (Table 8, Ref. 524), yet the data gathered are not entirely convincing. Quite often SOC expression is judged upon pharmacological sensitivity of [Ca²⁺]_i transients produced by Ca²⁺ readmission after store depletion in Ca²⁺-free media. Because the pharmacology of store-operated channels is not very well defined, this interpretation is open to criticism. The existence of SOC in central neurons, for example, was postulated by Baba et al. (17) based on [Ca²⁺]_i imaging of primary cultured pyramidal and granule neurons isolated from Ammon’s horn and dentate gyrus, respectively. After these neurons were incubated with 1 µM TG for 5 min in Ca²⁺-free solution, readmission of external Ca²⁺ triggered [Ca²⁺]_i elevation, which was inhibited by 100 µM La²⁺, 3 µM SKF96365 or 30 µM 2-aminoethoxydiphenyl borate (2-APB). No attempts, however, were made to activate SOC by more physiological depletion of the ER store such as administration of caffeine or stimulation of metabotropic receptors, yet a conclusion about SOC activation by synaptic transmission (based on partial and rather slight inhibition of [Ca²⁺]_i transients evoked by tetanic field stimulation of cultured neurons by 2-APB or SKF96365 was announced. As 2-APB and SKF96365also attenuated LTP recorded in hippocampal slices, another fundamental conclusion of SOC involvement in synaptic plasticity was made by the same authors (17). Interestingly, La²⁺, 2-APB, and SKF96365also inhibited the plateau phase of NMDA-induced [Ca²⁺]_i elevation, which may reflect their poor selectivity, rather than activation of SOC upon stimulation of ionotropic glutamate receptors.

Perhaps the most convincing case for SOC expression in neurons was presented by Usachev and Thayer, who investigated the process of store replenishment in rat cultured DRG neurons. They demonstrated that Ca²⁺ entry, essential for the refilling of ER stores after they have been discharged with caffeine, can be blocked by 2 mM Ni²⁺, and the refilling of the store is potentiated when the cells are hyperpolarized from –55 mV to –80 mV. Furthermore, Usachev and Thayer (664) substantiated their claim for SOC in nerve cells by demonstrating that ER store depletion potentiates Mn²⁺ entry into neurons. Still, these data may be explained by a very different mechanism, i.e., Ca²⁺ entry through T-type Ca²⁺ channels, which have a "window" current region around –75 to –80 mV, where they may provide for unstimulated Ca²⁺ influx. Calcium entry through conventional Ca²⁺ channels following ER depletion was, for instance, observed in cultured guinea pig myenteric neurons, in which the treatment with TG (1 µM) induced long-lasting irregular [Ca²⁺]_i oscillations arising solely from Ca²⁺ influx, yet these oscillations were completely blocked by -conotoxin MVIIA, suggesting that these neurons may use N-type Ca²⁺ channels for store refilling (665). In the same neurons, application of ATP triggered a biphasic (peak – plateau) [Ca²⁺]_i response; the plateau required extracellular Ca²⁺ but was blocked by 10 µM nifedipine (287). Likewise, the plasmalemmal voltage-gated Ca²⁺ channels were responsible for ER store refilling in cultured cerebellar granule cells (249) as well as in cultured motoneurons from larval lampreys (280).

G. Pharmacology of Ca²⁺ Release

1. Ryanodine receptors/CICR

Mechanisms of pharmacological modulation of RyRs have received much attention, and the properties of drugs that selectively modulate RyRs have been summarized in numerous reviews (e.g., Refs. 135, 216, 217, 259, 308, 310, 483, 614, 703, 720). Therefore, I shall limit myself to a brief discussion of some of these tools, which are instrumental for studying CICR in nerve cells.

A) RYANODINE.  Ryanodine was probably the first and the foremost pharmacological agent used for probing for Ca²⁺ stores and Ca²⁺ release. Its action on skeletal and cardiac muscle had been known for the last 35 years (259), and its ability to specifically bind to Ca²⁺-gated Ca²⁺ release channels led to naming the latter "ryanodine receptor" (505) a little confusing (as no ryanodine was ever in attendance in living tissues, save in roots and stem of the plant Ryania speciosa, which is the natural source of ryanodine), but generally accepted. Ryanodine exerts multiple actions on the RyRs, depending on its concentration. At very low concentrations (5–40 nM), ryanodine was reported to increase the frequency of RyR channel opening, without affecting its conductance (67). At low micromolar (1 to 5 µM) concentrations, ryanodine promotes RyR channel opening in a subconducting (40–60% compared with a normal) state, which is also characterized by a dramatic (20 times) increase in the channel open time. At higher concentrations (50 to >100 µM) ryanodine completely blocks the channel (67, 440). These multiple actions most likely reflect the existence of high- and low-affinity binding sites for ryanodine within the RyR. Notably, ryanodine binding to the low-affinity site is almost irreversible. An important feature of ryanodine is its "use dependence" (440), i.e., preferential binding of the drug to the open channel. In practical terms, this became apparent as a facilitation of ryanodine action by repetitive activation of RyRs (either by caffeine or by [Ca²⁺]_i increase), a phenomenon that is often observed in nerve cells (29, 92, 269, 308, 636, 661). Ryanodine is still the most specific pharmacological tool used for probing CICR in neurons (see Tables 1 and 2).

B) RUTHENIUM RED.  A polycationic dye, ruthenium red, is widely used as CICR blocker in nerve cells (see Tables 1 and 2). Its effective concentrations for CICR inhibition in muscle preparations range between 1 and 20 µM (720). At the single-channel level, ruthenium red effectively reduces P_o by prolonging the closed state of the RyR. Unfortunately, as an inhibitor ruthenium red is rather nonspecific as it also inhibits VGCC (97) epithelial Ca²⁺ channels ECaC1 (459), TRPV channels (194), GABA release from hippocampal synaptic terminals (568), and mitochondrial calcium uptake (534). Moreover, when applied to permeabilized chicken atria, ruthenium red prevented contractile responses induced by InsP₃ and potentiated responses to caffeine (676), which makes its employment as a specific CICR inhibitor even more dubious.

C) DANTROLENE.  Dantrolene was originally synthesized in 1967 (598) as a potent muscle relaxant, and it is currently the only available drug for the specific treatment of malignant hyperthermia, a life-threatening complication of general anesthesia (313). Incidentally, dantrolene may also help against ecstasy intoxication (28). The action of dantrolene on the ER consists of reducing the P_o of RyRs, thereby inhibiting CICR; the effective concentrations of the drug lie between 10 and 90 µM (469). Dantrolene is widely used as a CICR inhibitor in neurons (Tables 1 and 2), although there are some indications that dantrolene is effective only against RyR1 and does not inhibit RyR2 (167).

D) GENERAL ANESTHETICS.  Halothane and enflurane enhance Ca²⁺ release in cardiac preparation at low micromolar (i.e., in subtherapeutic) concentrations and increase P_o of RyR channels (720). In the neural cholinergic cell line SN56, halothane triggered Ca²⁺ release from the ryanodine-, dantrolene- and CPA-sensitive calcium store (189). Considering that volatile anesthetics affect synaptic transmission (94, 95, 293) and neuronal excitability (414), the possibility that they act by modulating CICR should not be forgotten. In addition, however, halothane also inhibits plasmalemmal Ca²⁺-ATPase (157, 683), so the resulting effects on intracellular Ca²⁺ dynamics and Ca²⁺-dependent processes may be rather complex and difficult to interpret.

E) LOCAL ANESTHETICS.  Most of the local anesthetics, such as procaine, tetracaine, lidocaine, prilocaine, the quaternary amines QX 572 and QX 314, and benzocaine, inhibit caffeine-induced Ca²⁺ release and CICR in millimolar concentrations (720), and this action was also observed in nerve cells (308, 661).

F) DIHYDROPYRIDINES.  Nifedipine, a well-known voltage-gated channel antagonist, was reported to increase the frequency of miniature end-plate potentials in synaptic terminals studied in the muscular junction of neonatal (up to 3 wk) rats. Effects of nifedipine persisted in Ca²⁺-free media and were inhibited by TG (2 µM) and ryanodine (10 µM), which allowed the authors to suggest possible stimulatory action of the drug on RyRs (512).

G) HEPARIN.  Heparin, which is customarily used as a selective inhibitor of IICR, does affect RyRs at the very same concentrations that are effective against InsP₃Rs. The effects of heparin on RyRs were initially suggested when it was found that heparin stimulates Ca²⁺ release from skeletal muscle SR vesicles (404, 538). Further experiments on purified RyR channels demonstrated that heparin increases channel openings with EC₅₀ values of 0.23 µg/ml (compared with 1–4 mg/ml concentrations usually employed for IICR inhibition) in a Ca²⁺-dependent manner (46, 135).

H) OTHER PHARMACOLOGICAL CICR REGULATORS.  The interaction with RyRs and modulation of CICR have been reported for many other pharmacological agents, which generally are not considered to possess such ability. For example, cardiac glycosides and, in particular, digoxin in therapeutic concentrations increase the rate of CICR in cardiac preparations through sensitizing RyRs to [Ca²⁺]_i and increasing channel P_o (390). Suramin, widely used as a purinoreceptor blocker, induces CICR and increases P_o of RyRs (594). Another agent, believed to activate CICR and the RyR3 channel, in particular, is chloro-m-cresol, which triggers Ca²⁺ release in skeletal muscle (719), and in glial cells (383). So far, however, no systematic attempts to use chloro-m-cresol in nerve cells were made. Another class of drugs often employed in experiments with cultured neurons, the aminoglycoside antibiotics neomycin and gentamycin, are potent inhibitors of CICR with effective concentrations in a range between 50 nM and 10 µM (720).

2. Pharmacology of IICR

IICR pharmacology is less well defined than the pharmacology of CICR. From the very beginning of InsP₃R studies it became apparent that a number of quite different pharmacological agents, including heparin, p-chloromercuribenzoic acid, cinnarisine, flunarisine, local anesthetics, such as tetracaine and lidocaine, and potassium channel blockers such as TEA, can effectively inhibit InsP₃-iduced Ca²⁺ release from microsomal ER preparations (see, e.g., Refs. 482, 484). Further complexity was added when it became clear that caffeine, which was believed to selectively interact with RyRs, inhibits InsP₃Rs (135, 680), and heparin, the most widely used InsP₃R blocker, activates RyRs (46, 135). As if this was not enough, caffeine also effectively inhibits InsP₃ production (645). All in all, the ideal inhibitor of IICR, which would penetrate biological membranes and demonstrate high selectivity against InsP₃R, remains in the realm of dreams.

A) HEPARIN.  Heparin was shown to bind to InsP₃Rs (613) and block the activity of InsP₃-gated channels incorporated into lipid bilayers (43). Heparin was also shown to inhibit IICR in many types of nonexcitable and excitable cells, and it is constantly used as a pharmacological probe for neuronal IICR (see Tables 3–5). A big disadvantage of heparin is its membrane impermeability; hence, it must be delivered into the cell interior either by injection or via intracellular dialysis. Although heparin activates RyRs in artificial membranes, I failed to find a single report that would describe heparin-induced Ca²⁺ release in nerve cells. If that would be the case, however, heparin may still inhibit IICR by depleting ER via activated RyRs, although the interpretation of such data can be difficult.

B) CAFFEINE.  The inhibitory action of caffeine on IICR was first discovered by Ole Petersen and co-authors (680) who studied agonist-mediated Ca²⁺ signaling in pancreatic acinar cells. Later on, the inhibition of IICR by millimolar concentrations of caffeine was found in Xenopus oocytes (493), cerebellar microsomes (66), and permeabilized smooth muscle cells (228). Direct electrophysiological investigations of purified InsP₃Rs supported these findings by showing inhibition of unitary InsP₃R current. The K_D of caffeine action on InsP₃R currents was 1.6 mM, and at 10 mM caffeine, a complete block of InsP₃R currents was attained (228).

C) 2-APB.  The ability of 2-APB to inhibit IICR was initially found in cerebellar microsomes stimulated with 100 µM InsP₃; 2-APB blocked Ca²⁺ release with an IC₅₀ value of 40 µM. Incubation with 2-APB also inhibited agonist-stimulated IICR in human platelets and neutrophils (373) and in HeLa cells (503). However, further investigations demonstrated that 2-APB lacks selectivity as it affects not only IICR but also Ca²⁺ pumps and store-operated Ca²⁺ entry in nonexcitable cells (55, 503).

D) XESTOSPONGINES.  Some hope for a selective, membrane-permeable InsP₃R antagonist appeared in 1997 when Isaac Pessah’s group purified and characterized a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge Xestospongia species (174). Several of these agents, dubbed Xestospongines (Xes), namely, XeA, C, and D, were reported to be powerful membrane-permeable blockers of IICR when tested on ER vesicles isolated from the cerebellum; of these, XeC was the most potent inhibitor with a K_D of 360 nM. Further experiments have demonstrated that XeC blocks [Ca²⁺]_i transients mediated by metabotropic pathways in the PC12 cell line and in primary rat cortical astrocytes. Incubation of cells with 5–20 µM XeC effectively abolished [Ca²⁺]_i responses to bradykinin and carbachol (174). Yet more detailed investigations performed later somewhat dampened the optimism for the action of xestospongines as selective membrane-permeable blockers of InsP₃Rs because it emerged that XeC acts as an effective blocker of SERCA pumps and thus empties the ER of Ca²⁺ (84, 123, 600). It is noteworthy that an indication that XeC interacts with SERCA pumps was already present in the initial paper by Gafni et al. (174); Figure 4 of this study clearly shows that incubation of PC12 cells with XeC decreases the amplitude of consequent TG-induced [Ca²⁺]_i rise (which was interpreted by authors as an inhibition of leak pathway, yet it may reflect depletion of the ER as a result of SERCA blockade).

E) ADENOPHOSTINES.  The adenophostines A and B were isolated from metabolites of fungi Penicillium brevicompactum (622) and found to be extremely potent (10–100 times more potent than InsP₃) agonists of InsP₃Rs (694). They were tested in turtle olfactory sensory neurons (272) and in single InsP₃Rs isolated from fish olfactory cilia (70). In both cases adenophostines acted as powerful agonists of InsP₃Rs inducing channel opening (70) and promoted IICR as judged by the appearance of InsP₃/[Ca²⁺]_i-dependent currents.

	IV. ENDOPLASMIC RETICULUM CALCIUM SIGNALING AND REGULATION OF NEURONAL FUNCTIONS

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A. Neuronal Excitability

Calcium release from the ER regulates neuronal excitability through several types of plasmalemmal Ca²⁺-activated channels, responsible for K⁺, Cl^–, and nonselective currents (see Tables 9 and 10). In addition, Ca²⁺ release may (at least theoretically) suppress VGCC by increased Ca²⁺-dependent inactivation. The best-characterized effect of CICR is activation of K(Ca) channels, which control postspike afterhyperpolarization (AHP). The AHP comprises three distinct components (554), the fast AHP (mediated by BK channels), the AHP (mediated by SK channels), and the (ultra)slow AHP (mechanism not yet identified). Activation of the fast AHP is mediated solely by plasmalemmal Ca²⁺ entry, while both AHP and slow AHP are sensitive to CICR inhibitors. The contribution of CICR to the AHP is quite substantial: up to 50% of the AHP current is activated by Ca²⁺ released from the ER. The mechanism of CICR-mediated regulation of the (ultra)slow AHP remains entirely unknown. In vagal afferent neurons, Ca²⁺ release activates I_AHP only when CICR is triggered by Ca²⁺ entry through N-type VGCC, which may reflect a very special colocalization of N-type Ca²⁺ channels, RyRs, and Ca²⁺-dependent K⁺ channels (107). A similar functional colocalization of RyRs and voltage-gated N-type Ca²⁺ channels was also demonstrated in bullfrog sympathetic neurons, where Ca²⁺ entry through N-type VGCC triggers regenerative CICR near the plasma membrane (2). In these cells, RyRs also seem to be functionally coupled to BK channels, thus providing a feedback between ER Ca²⁺ release, membrane excitability, and AP frequency. Finally, the same functional interactions between N-type Ca²⁺ channels and RyRs were confirmed in PC12 cells, where CICR is triggered by Ca²⁺ influx through N-type but not through L-type Ca²⁺ channels (657).

B. Neurotransmitter Release

The crucial importance of Ca²⁺ for neurotransmission was initially demonstrated by F. S. Locke for the neuromuscular junction (356), and the concept of Ca²⁺-dependent neurotransmitter release was coined by Bernard Katz and colleagues (118, 146). According to this theory, regulated exocytosis, of which neurotransmitter release represents a particular case, requires activation of numerous Ca²⁺-regulated transduction cascades that eventually result in the fusion of vesicles with the plasmalemma and release of vesicle contents into the extracellular space (in the case of neurotransmission, the synaptic cleft). Activation of exocytotic cascades requires a large (up to 100 µM) local [Ca²⁺]_i concentration, and the role of plasmalemmal Ca²⁺ entry in neurotransmitter release is well established (36). The possible role of Ca²⁺ originating from the ER store is much less understood. Certainly, exocytosis in several types of nonexcitable and excitable cells almost exclusively depends on ER Ca²⁺ release [good examples are pancreatic acinar cells (506) and pituitary gonadotrophs (652)]. In a nerve-related cell preparation, neuroblastoma cocultured with rat skeletal muscle cells, bradykinin-induced IICR elevated [Ca²⁺]_i and triggered acetylcholine release (468). However, whether ER Ca²⁺ release by itself can trigger exocytosis in presynaptic terminals in vivo remains an unanswered, albeit challenging, question.

Numerous morphological studies confirmed that functional ER is present in presynaptic terminals [e.g., Refs. 50, 82, 203, 391; see also an excellent review by Ron Bouchard et al. (59)], and moreover, ER structures are located in close proximity (as near as 40–400 nm) to the presynaptic active zone. As demonstrated already in 1986 by Westrum and Grey (693), the ER within the presynaptic terminal appears to be in close association with microtubules and synaptic vesicles and, moreover, ER portions impinge directly on the presynaptic membrane forming "tubular-fibrillar" extensions. Such close apposition of the ER and the presynaptic membrane may perfectly well allow Ca²⁺ released from the store to participate in creation of [Ca²⁺]_i microdomains relevant for exocytosis. Yet the role of ER Ca²⁺ release in regulation of neurotransmitter release remains controversial, with arguments supporting (59) or denying (721) its importance.

Indeed, there are not many indications that ER Ca²⁺ release in the absence of plasmalemmal Ca²⁺ entry can induce massive exocytosis in synaptic terminals, although numerous data (see Tables 9 and 10) demonstrate that both CICR and IICR may modulate/amplify neurotransmitter release from presynaptic compartments.

Probably the first to contemplate the importance of presynaptic Ca²⁺ release in the regulation of neurotransmission were Erulkar and Rahamimoff (141), who arrived at this idea based on analysis of tetanic stimulation-induced changes in the frequency of the miniature end-plate potentials at varying Ca²⁺ electrochemical gradients. Further experimentation, aimed at investigating the effects of pharmacological manipulations with ER Ca²⁺ handling on either spontaneous or evoked postsynaptic currents, demonstrated that both Ca²⁺ release from and Ca²⁺ uptake into the ER may significantly modulate exocytosis of neurotransmitter in synaptic terminals. It appeared that spontaneous RyR-mediated presynaptic [Ca²⁺]_i transients may trigger neurotransmitter release in resting conditions. This conclusion was based on experiments demonstrating that the frequency or amplitude of miniature postsynaptic currents (mIPSCs as well as mEPSCs) is sensitive to inhibition of RyRs (by ryanodine) or SERCA pumps (by TG and CPA). This phenomenon was observed in cerebellar neurons (26, 355) as well as in hippocampal pyramidal (139, 563) and cortical (589) neurons. In parallel, Ca²⁺ imaging of presynaptic terminals indeed revealed the spontaneous [Ca²⁺]_i release events (139, 355). Moreover, in the presynaptic terminals of basket cells, the amplitude of [Ca²⁺]_i signals resulting from spontaneous Ca²⁺ release was comparable with [Ca²⁺]_i rises produced by single AP (105).

At the same time, CICR triggered by depolarization-induced Ca²⁺ entry was detected in single presynaptic terminals in intact bullfrog sympathetic ganglia, where it accounted for 46% of the total [Ca²⁺]_i elevation induced by electrical stimulation of the nerve (502) and in the presynaptic mossy fiber terminals, where it contributed as much as 50% of the [Ca²⁺]_i elevation induced by a train of action potentials (344). In hair cells (which are presynaptic to primary afferent neurons), direct activation of RyR-mediated Ca²⁺ release by caffeine induced a [Ca²⁺]_i elevation and an increase in membrane capacitance that may indicate activation of exocytosis (337). Finally, pharmacological inhibition of ER reduced not only spontaneous but also evoked GABA-mediated IPSCs in basket cell-Purkinje neuron synapses in the cerebellum (175), whereas potentiation of CICR by intracellular addition of cADPR augmented ACh release from terminals of Aplysia buccal neurons (432). Likewise, CICR activated by Ca²⁺ entry via VGCC enhanced neurotransmitter release in the frog neuromuscular junction (450). In this preparation, a close apposition of RyRs and plasmalemmal Ca²⁺ channels was postulated, and it was also suggested that Ca²⁺ entry primes and activates CICR, which in turn contributes to neurotransmitter release (449).

The intimate mechanism of CICR-dependent potentiation of evoked neurotransmitter release may, however, be more complicated than simple amplification of Ca²⁺ entry and therefore an increase in [Ca²⁺]_i domains near the active zone. The simplest hypothesis argues that CICR events trigger neurotransmitter release and moreover favor the multiquantal release from the same active zone (105, 355). However, analysis of ryanodine effects on the amplitude and frequency of evoked IPSCs in basket cell-Purkinje neuron synapse, led Galante and Marty (175) to suggest that Ca²⁺ released by CICR arrives at the active zone too late to affect exocytosis, but it does sensitize the exocytotic machinery to a subsequent depolarization.

Sometimes the effective modulation of neurotransmitter release requires synergism between IICR and CICR. When recording simultaneously from presynaptic axons and postsynaptic motoneurons in a Lamprey spinal cord preparation, Cochilla and Alford (102) observed potentiation of synaptic transmission by Ca²⁺ release from the ER; both IICR and CICR were necessary as this potentiation could be blocked by pharmacological inhibition of mGluRs as well as by 20 µM ryanodine.

Incidentally, the CICR responsible for spontaneous neurotransmitter release may also be activated by Ca²⁺ entry through presynaptic Ca²⁺-permeable ionotropic receptors. The frequency and amplitude of spontaneous mEPSCs in CA3 hippocampal neurons were greatly enhanced following activation of presynaptic nicotinic cholinoreceptors (NChRs). Subsequent analysis revealed that CICR triggered in the presynaptic terminal by Ca²⁺ entry through Ca²⁺-permeable NChRs was instrumental in the action of nicotine on mEPSCs. Pharmacological inhibition of presynaptic CICR completely abolished NChR-dependent modulation of mEPSCs (573). Most importantly, CICR in the presynaptic terminal remodeled neurotransmitter release quite substantially, by a shift towards a multiquantal mode, or else in synchronizing release across several active zones, as judged by a dramatic (3 times) increase in mean mEPSC amplitude and appearance of a relatively high number of extralarge mEPSCs reaching 200 pA (as compared with a mean mEPSC amplitude of 20 pA in control conditions) upon activation of NChRs. This remodeling has important functional consequences as nicotine-induced facilitation of neurotransmitter release was able to trigger a burst of action potentials in the postsynaptic cell, thus indicating the existence of synaptic transmission without AP-induced depolarization of the presynaptic membrane (573). Local CICR in response to Ca²⁺ entry through NChRs was also found in nerve terminals in vas deferens; this CICR facilitated neurotransmitter release, which resulted in a 70% increase in the amplitude of excitatory junction potentials (63).

Very much in line with the concept of Ca²⁺ store/sink duality, the presynaptic ER may not only amplify, but also limit neurotransmitter release, by buffering Ca²⁺ in the terminal, thus reducing the lifetime of elevated [Ca²⁺]_i microdomains. This, for example, happens in the frog neuromuscular junction, where inhibition of SERCAs facilitates synaptic transmission (83). In the rat neuromuscular junction, CICR also limits the transmission, albeit in a completely different way, by limiting Ca²⁺ entry through VGCC as a result of increased Ca²⁺-induced inactivation of the latter (567).

However comprehensively the data discussed above favor the role of presynaptic Ca²⁺ release in the regulation of neurotransmission, it may not be regarded as an ubiquitous mechanism always present in all synapses. For example, Carter et al. (80) while investigating transmission in the hippocampus (associational-commissural synapses and mossy fiber synapses in CA1 and CA3 neurons) and cerebellum (parallel fiber-Purkinje neuron synapse) did not observe any involvement of CICR in the regulation of neurotransmitter release. Moreover, they failed to detect either caffeine-induced Ca²⁺ release or CICR in cerebellar parallel fibers. Likewise, a study of giant EPSCs [which were quite similar to extralarge ACh-mediated mEPSCs observed by Sharma and Vijayaraghavan (573)] in the mossy fiber-CA3 synapse did not show dramatic effects of either TG/CPA or ryanodine/caffeine on occurrence of giant EPSPs (215). Furthermore, the inhibition of Ca²⁺ release affected the parameters of normal EPSPs rather slightly, if at all (e.g., 10 µM ryanodine changed EPSC frequency to 126 ± 42% of the control; Ref. 215). No significant CICR (as probed by 3 mM caffeine, 10µM ryanodine, or 1 µM TG) was found during [Ca²⁺]_i recordings from the single synaptic boutons of rat sympathetic preganglionic terminals (347) and from sympathetic varicosities (as probed by 10 µM ryanodine; Ref. 62). CICR was also not found in presynaptic terminals of goldfish retinal bipolar cells (296). Or was it? The same group published results supporting the importance of intracellular Ca²⁺ release for amplification of intraterminal [Ca²⁺]_i transients and for facilitation of transmitter release from the same goldfish retinal bipolar cells (295); these two contradictory papers are separated by exactly 6 wk and were published in the same journal. These discrepancies most likely reflect the complexity of the system responsible for synaptic transmission. In particular, they may arise from differential expression of ER in various terminals, from differences in ER functional conditions (e.g., the degree of Ca²⁺ loading), and, naturally, from the experimental difficulties, as all these measurements are technically quite demanding and are performed at the very edge of the sensitivity of existing equipment. One also has to consider that incubation of whole brain slices with rather powerful pharmacological tools such as TG or ryanodine may trigger obscure indirect effects, thus complicating data interpretation.

C. Synaptic Plasticity

The living brain constantly remodels and modifies its cellular networks. Throughout life, synapses weaken and strengthen or else form and die. These processes underlie the adaptation of the brain to the constantly changing environment and represent what we know as learning and memory. Modification of synaptic transmission efficacy is an important part of this adaptation, which is manifested in short- and long-term synaptic plasticity. The electrophysiological correlates of long-term synaptic plasticity are long-term potentiation (LTP) or depression (LTD) of synaptic potentials in response to either high-frequency stimulation of single synaptic inputs or to simultaneous activation of several distinct synaptic inputs.

LTP, discovered by Tim Bliss and Terje Lomo in hippocampal neurons (53), was also the first form of synaptic plasticity, which highlighted the importance of cytoplasmic Ca²⁺ signals, as intracellular injection of the Ca²⁺ chelator EGTA effectively obliterated the potentiation of synaptic strength (363). Very soon thereafter, the importance of the cytoplasmic Ca²⁺ rise was also appreciated for cerebellar LTD (136, 301). More recent experiments also clearly demonstrate that the ER Ca²⁺ store plays an important role in the generation of [Ca²⁺]_i elevations relevant to the induction of synaptic plasticity (154, 542, 552).

1. Short-term synaptic plasticity

Short-term synaptic plasticity is a form of presynaptic strengthening of neurotransmitter release, and as such, it ultimately depends on presynaptic Ca²⁺ signaling as discussed above. The involvement of CICR in a form of short-term plasticity, known as paired-pulse facilitation (PPF), was recently proposed for CA3 hippocampal neurons studied in organotypic slices (139). In contrast, a detailed investigation of PPF in excitatory synapses in the hippocampus and cerebellum failed to reveal any contribution of ER Ca²⁺ release to this phenomenon (80).

2. Long-term synaptic plasticity

Historically, LTP and LTD were mostly investigated in the hippocampus and cerebellum, respectively. Incidentally, as far as ER Ca²⁺ release is concerned, the mechanisms of hippocampal and cerebellar plasticity clearly differ in that cerebellar LTD involves IICR, whereas CICR is important for hippocampal LTP.

A) SYNAPTIC PLASTICITY IN THE HIPPOCAMPUS: A ROLE FOR CICR.  Much of our understanding of hippocampal synaptic plasticity comes from investigations of synapses formed by CA3 neurons through Schaffer collaterals on CA1 pyramidal cells. A peculiarity of this synapse is that it possesses both LTP and LTD depending on the stimulation protocol. LTP is usually induced by high frequency (100 Hz; 100 stimuli or so) stimulation, whereas LTD requires prolonged low-frequency stimulation of Schaffer collaterals (many 100s of stimuli at 1–2 Hz) (154). Both LTP and LTD critically require a [Ca²⁺]_i increase. The precise mechanisms of differentiating between the different types of synaptic input in favor of potentiation versus depression are unclear, yet the signals arising from the ER may hold the key to further understanding.

The first and foremost task in the elucidation of the role of the ER in synaptic plasticity is to show that synaptic stimulation can result in Ca²⁺ release in dendritic spines and proximal dendrites, i.e., where exactly do the signaling events responsible for LTP/LTD occur. Hippocampal neurons have a rather peculiar distribution of the ER and Ca²⁺ release channels. First, in vivo the ER is not present in every spine. Only 25–50% of the small spines contain ER. In contrast, the latter is present in 80–90% of the big mushroom-like spines (604). Second, hippocampal pyramidal neurons have a peculiar distribution of Ca²⁺ release channels (617). Only RyRs (particularly RyR3) are present in the spines, while both RyRs and InsP₃Rs reside in the dendritic ER [although prominent species-dependent differences exist, i.e., RyR expression in the rabbit hippocampus appears to be almost negligible compared with the rat (558)].

Such a distribution of Ca²⁺ release channels suggests strong CICR in those CA1 neuron spines, which possess a functional ER. Because the distribution/localization of the latter may change depending on the experimental conditions (i.e., the animal’s age), conflicting results could be anticipated safely, and, sure enough, the controversy did not wait long to emerge. To begin with, a robust caffeine-induced Ca²⁺ release, sensitive to TG and ryanodine, and CICR in response to glutamate application, were observed in spines from cultured hippocampal neurons (303). This observation was confirmed by [Ca²⁺]_i imaging on hippocampal neurons in organotypic slices (138), which revealed that CICR is responsible for by far the largest (80–90%) part of the [Ca²⁺]_i elevation triggered by stimulation of NMDA receptors (Fig. 21A). Yet, when confocal Ca²⁺ imaging was applied to hippocampal neurons in acute slices, no signs of significant CICR following NMDA-mediated Ca²⁺ influx were detected (312), and the NMDA-induced Ca²⁺ flux was found to be almost solely responsible for the [Ca²⁺]_i elevation (Fig. 21B). All in all, the question about functional CICR in spines remains unresolved, which most likely reflects the complexity and heterogeneity of the preparations. Yet, physiological data support the notion of at least some involvement of CICR in LTP in SC-CA1 synapses, since emptying the ER with TG (1 µM) or CPA (1 µM), or blocking CICR with 10 µM ryanodine, inhibited the induction but did not affect the maintenance of LTD in acute hippocampal slices (530).

View larger version (29K): [in this window] [in a new window]   FIG. 21. CICR in spines of CA1 hippocampal neurons: evidence pro and contra. A: recordings of [Ca2+]i in a single spine of hippocampal CA1 neuron filled with Oregon green BAPTA-1 through the tip of sharp microelectrode. Top row shows [Ca2+]i traces; the row on the bottom demonstrates voltage recordings. In control conditions, synaptic stimulation triggers [Ca2+]i elevation and excitatory postsynaptic potentials (EPSPs); incubation of slice with 15 µM CPA completely abolished [Ca2+]i increase without any apparent effects on EPSPs. One hour of washout with normal saline restores the parameters of [Ca2+]i responses. B: similar recordings demonstrating a minor role played by CICR in synaptically induced [Ca2+]i signals in hippocampal slices. a: EPSP-associated spine [Ca2+]i signal (color-coded image and top trace) was reduced by 30% in the presence of ryanodine (25 µM, bottom trace). b: Time course of the effect of ryanodine (indicated by the bar) on the peak amplitudes of [Ca2+]i transients. Same experiment as in A. Single-shock stimuli were repeatedly delivered every 4 min; each data point represents a single trial. The dashed lines represent the values during control conditions (1.0) and in the presence of ryanodine (0.72). The numbers point to the individual recordings displayed in A. c: Application of CPA (30 µM, indicated by the bar) reduced the amplitude of synaptic Ca2+ signals in spines by 36%. d: Bar graph summarizing the effects of ryanodine (20 or 25 µM) and CPA (30 µM). Data points were normalized to control conditions. The first two bars represent the effect on synaptically evoked [Ca2+]i signal in spines (n = 7 and 9 cells for ryanodine and CPA, respectively). The last two bars show that both drugs effectively abolish dendritic [Ca2+]i responses evoked by local application of caffeine (20–40 mM, n = 5 for ryanodine and for CPA). Data points were normalized to the mean control value. Experiments were done at 30°C. [A from Emptage et al. (138) copyright 1999 with permission from Elsevier; B from Kovalchuk et al. (312) copyright 2000 by the Society for Neuroscience.]

Some indications of the importance of CICR in generation of LTP/LTD are coming from other neurons (CA3 hippocampal cells, cortical neurons), although they often involve both pre- and postsynaptic mechanisms (see Table 9). IICR seems to play a more important role in CA3 neurons, as deletion of InsP₃Rs facilitated LTP and completely inhibited LTD in mossy fiber-CA3 neuron synapses (254).

B) SYNAPTIC PLASTICITY IN THE CEREBELLUM: A ROLE FOR IICR.  The role for IICR in LTD generation in the cerebellum is rather well established. The LTD of parallel fibers, which form their synapses on dendrites of Purkinje neurons, occurs as a result of simultaneous activation of parallel and climbing fibers. Stimulation of climbing fibers can be fully substituted by depolarization of the neuronal soma and [Ca²⁺]_i elevation (301). Whatever protocol of LTD induction is used, [Ca²⁺]_i elevation in the PN is necessary for LTD development. In fact, stimulation of climbing fibers or somatic depolarization may be replaced by an artificial increase in [Ca²⁺]_i due to photorelease of caged Ca²⁺ (338) (incidentally, stimulation of parallel fibers can be fully substituted by NO generation, Ref. 338).

Genetic manipulation of components of the mGluR-IICR signaling cascade evidently demonstrated the crucial importance of this pathway for cerebellar LTD. Deletion of mGluR1 resulted in the appearance of a clear cerebellar phenotype, with ataxia, motor discoordination, and impaired LTD (1, 104). Both LTD and locomotive deficits can be restored by expression of mGluR1 under the control of a Purkinje neuron-specific promotor (243).

Almost simultaneously, the involvement of IICR in cerebellar LTD was suggested by physiological experimentation. Direct activation of IICR by photorelease of caged InsP₃ in Purkinje neurons produced LTD when coincident with somatic depolarization, thus fully substituting for the stimulation of parallel fibers (282). Furthermore, as shown by Finch and Augustine (150), local InsP₃ uncaging and subsequent IICR were sufficient to induce local LTD in synapses close to the uncaging foci.

It has to be noted, though, that IICR appeared not to contribute to LTD in cultured or freshly isolated Purkinje neurons, as no difference was found in [Ca²⁺]_i transients induced by depolarization and combination of depolarization and glutamate application. Moreover, LTD was insensitive to XeC, and diacylglycerol was able to substitute for mGluR activation in the induction of LTD (448). Yet, these results most likely reflect certain changes in signaling pathways introduced by tissue culture conditions or the isolation procedure.

Further evidence favoring the key role of IICR in the generation of cerebellar LTD was obtained from mice with genetically deleted InsP₃R1s, in which LTD completely vanished (Fig. 22A) (247). Interestingly, InsP₃R1^–/– animals never survived beyond the 23rd day. The same authors also reported that a similar disappearance of LTD can also be achieved by perfusing neurons with specific antibodies raised against InsP₃R1. Not only the expression but also the localization of InsP₃R1s matters, as was demonstrated on animals mutated for myosin Va. This mutation causes the ER not to enter PN spines, which therefore effectively eliminates IICR specifically from this compartment. The LTD in mutant mice and rats was completely lost (Fig. 22B); nonetheless, it could be rescued by Ca²⁺ uncaging within the spine (421).

View larger version (24K): [in this window] [in a new window]   FIG. 22. InsP3-induced Ca2+ release is critical for cerebellar long-term depression (LTD). A: loss of LTD in InsP3R1–/– Purkinje cells. a: Pairing depolarization and PF stimulation (1 Hz, 240 times) induced long-lasting depression of the PF-EPSP amplitude in control experiments using a wild-type (WT) InsP3R1+/+ cerebellar slice. b: LTD was lost in an InsP3R1–/– cerebellar slice. Insets show an average of 10 consecutive sweeps at time points indicated. c: Averaged time course of normalized EPSP amplitude. Results are presented as means ± SE. B: LTD is absent in dilute-opithotonus (dop) mutant rats (117), which lack ER Ca2+ stores in Purkinje cell dendritic spines. Top panel shows traces of PF-EPSPs measured in control (left) and mutant (right) Purkinje cell before (pre) and 25 min after conjunctive PF and CF stimulation. Bottom panel shows the time course of changes in EPSP amplitudes after PF-CF stimulation (incidence of stimulation is indicated on the graph) for control (left) and mutant (right) rat. LTD is completely absent in mutant animal. [A from Inoue et al. (247) copyright 1998 by the Society for Neuroscience; B from Miyata et al. (421) copyright 2000 with permission from Elsevier.]

D) MEMORY PROCESSES.  Not much is known about the role of Ca²⁺ release in consolidative memory processes. There are some indications that synchronous oscillations of membrane potential in the neocortex, which were suggested to be important for cognitive brain function (218), are governed by CICR triggered by Ca²⁺ entering through NMDA receptors (710). A certain importance of ER Ca²⁺ release may be also deduced from the observation that a significant increase in RyR2 expression, at both the mRNA and protein level, was found in the hippocampus of rats subjected to intensive water maze training (716).

D. Gene Expression, ER Ca²⁺ Waves, and ER Ca²⁺ Tunneling

The importance of cytoplasmic and nuclear Ca²⁺ signals in the regulation of gene expression is well established, and a variety of Ca²⁺-dependent cascades controlling transcription have been characterized (19, 91, 152, 183, 184, 692), which shall not be discussed here. However, the problem of how Ca²⁺ reaches the nucleus during physiological activity of the geometrically complex and highly polarized nerve cell remains unsolved. It is well established that cytosolic Ca²⁺ buffering hampers Ca²⁺ diffusion from often distant entry sites towards the cell interior. The ER can be instrumental in conveying Ca²⁺ over long distances and therefore providing nuclear systems with appropriate Ca²⁺ signals. Intracellular propagation of Ca²⁺ signals can be achieved by virtue of endomembrane excitability. Either RyR-mediated CICR or CICR-assisted Ca²⁺ release from InsP₃Rs or their combination may create propagating Ca²⁺ waves transforming near-plasmalemmal [Ca²⁺]_i elevations into nuclear Ca²⁺ rises. Indeed, such propagating [Ca²⁺]_i waves have been observed experimentally. For instance, CICR maintains spreading Ca²⁺ waves between the plasmalemma and the nucleus of the frog sympathetic ganglion neurons. Moreover, the speed of this wave remains constant all the way to the nucleus, suggestive of an active propagation, whereas at the moment the Ca²⁺ signal reaches the nucleus, its spread becomes diffusion governed (112, 389). Similarly, InsP₃Rs-dependent propagating Ca²⁺ waves have been described in a variety of central neurons (see Ref. 25 for review). Most interestingly, the Ca²⁺ wave can become amplified when approaching the center of the cell. For example, pairing of muscarinic stimulation of CA1 hippocampal neurons with trains of action potentials resulted in a focal Ca²⁺ elevation, which triggered a propagating Ca²⁺ wave, and the [Ca²⁺]_i increase resulting from this wave was largest in the soma and the nuclear region (518). This amplification critically depended on ER Ca²⁺ release, as emptying the ER by caffeine completely prevented wave propagation.

Yet, the ER may offer an alternative route for long-range Ca²⁺ propagation, which relies on the much faster Ca²⁺ diffusion within the ER lumen compared with the cytoplasm. This pathway, known as the intraluminal Ca²⁺ tunnel, has been described in pancreatic acinar cells, where Ca²⁺ was shown to rapidly equilibrate throughout the whole of the ER (491), and moreover, intra-ER Ca²⁺ diffusion can effectively replenish the depleted store in conditions of highly localized Ca²⁺ entry (424). As discussed above, synaptic stimulation can result in a very substantial accumulation of Ca²⁺ in the dendritic ER. This local increase of luminal Ca²⁺ may, by Ca²⁺ tunneling, rapidly reach the nucleus even from remote cellular processes. Because the ER is connected with the nuclear envelope (or rather the nuclear envelope is part of the ER), Ca²⁺ can be released directly into the nucleus via Ca²⁺ release channels residing in the envelope membrane (182, 642). Furthermore, sustaining Ca²⁺ waves in the neuronal soma would also require Ca²⁺ transport through the ER lumen. As mentioned above, the ER in central neurons may not be full in resting conditions, and therefore, initial loading of the store is necessary for the maintenance of a propagating Ca²⁺ wave. The existence of neuronal ER Ca²⁺ tunneling has not been demonstrated directly, although recent experiments performed by Hilmar Bading and co-workers (201) may provide some evidence for such a pathway. They investigated CREB-mediated gene expression in cultured hippocampal neurons treated with bicuculline. This treatment facilitates spontaneous firing in peripheral dendrites associated with substantial peripheral Ca²⁺ influx and [Ca²⁺]_i elevation in dendrites, which then had to be conveyed to the nucleus to activate CREB. Bading and co-workers (201) found that inhibition of ER Ca²⁺ accumulation by TG completely prevented the signal from reaching the nucleus. In the described experimental conditions, Ca²⁺ entry into the ER must occur in dendrites, precisely where [Ca²⁺]_i is elevated, and therefore, the possibility of Ca²⁺ diffusing through the ER lumen to the nucleus appears probable. Clearly many more experiments are needed to test for ER Ca²⁺ tunneling in nerve cells.

E. Neuronal Growth and Morphological Plasticity

Neurite outgrowth is governed by [Ca²⁺]_i fluctuations within the growth cone, where both the level of [Ca²⁺]_i and patterns of [Ca²⁺]_i fluctuations regulate the velocity of cone extension (273). For example, complex [Ca²⁺]_i transients are produced in growth cones during their migration in the embryonic spinal cord. The larger the frequency and magnitude of these [Ca²⁺]_i spikes, the slower the outgrowth. Inhibition of [Ca²⁺]_i elevation accelerates the rate of neurite extension, whereas artificial increase of [Ca²⁺]_i by Ca²⁺ photorelease inhibits axonal extension (190).

The [Ca²⁺]_i signals relevant for neurite outgrowth are produced by both Ca²⁺ entry and Ca²⁺ release, and the particular importance of CICR was appreciated quite early (297, 330). Recent analysis of growth cone movements of Xenopus neurons treated with netrin-1, an established regulator of neurite outgrowth, has further substantiated the importance of CICR for growth cone extension (233). Focal administration of netrin-1 to the neuronal culture created a local concentration gradient, which, in turn, forced the neurites to turn towards the source of netrin-1. This turning movement was modified by pharmacological inhibition of either plasmalemmal Ca²⁺ entry (by 50 µM Cd²⁺) or Ca²⁺ release from the ER (by 10 µM BHQ or 1 µM TG). When the cultures were treated with 100 µM ryanodine, however, the administration of netrin-1 produced repulsion of the cone. Most interestingly, applications of low concentrations of ryanodine (100 nM around the growth cone) caused a [Ca²⁺]_i elevation within the cone and turning of the latter towards the source of the drug (233). Thus, by regulating the pattern of local [Ca²⁺]_i signaling in the growth cone, CICR determines the direction and rate of axon extension/repulsion. A somewhat similar role of CICR was observed in the embryonic chick retina, where local CICR stabilized dendrite formation during development. Inhibition of this local Ca²⁺ release induced rapid retraction of dendrites (358).

In contrast, in embryonic chick DRG neurons, the regulation of growth cone motility seems to be regulated by InsP₃Rs. Growth cones of these neurons are particularly rich in InsP₃R1. Depletion of the ER store with TG (10 µm) arrested the extension of the cones, and inhibition of IICR with intracellularly injected heparin induced neurite retraction (626).

ER Ca²⁺ release controls not only the extension of growth cones, but also the extension of dendritic spines. Caffeine-activated release of Ca²⁺ from the ER triggers rapid elongation of spines in cultured embryonic hippocampal neurons. Spine elongation was already obvious 30 min after a short application of 5–10 mM caffeine, and after 90 min, the spine length had increased by 0.35 µm (33%; Ref. 304). Short puffs of caffeine even triggered a de novo formation of spines, although this was not very frequent (new spines appeared in 4 of 22 preparations). The effects of caffeine were a specific consequence of ER Ca²⁺ release, as 10 µM ryanodine obliterated them completely (304).

In summary, ER Ca²⁺ release acts as a powerful regulator of the motility of growth cones and dendrites, most likely through interaction with enzymes controlling polymerization/depolymerization of the cytoskeleton.

F. Neurohormone Release

An interesting hypothesis postulating that the ER Ca²⁺ store may regulate dendritic (i.e., extraterminal) release of oxytocin was developed as a result of in vivo experiments, which measured oxytocin release in the supraoptic nucleus region of anesthetized rats in response to local dialysis of ER-specific agents. TG, but not caffeine or ryanodine, induced release of the neurohormone and greatly potentiated depolarization-induced oxytocin secretion. Interestingly, this potentiation was not observed shortly after (5 min) application of TG, but a large potentiation was detected 30–90 min later (361). The authors explained their results in terms of TG-induced sustained Ca²⁺ release, which, in fact, does not seem a plausible explanation. First, [Ca²⁺]_i rises caused by SERCA inhibition are short-lived, and second, depletion of the ER store renders additional Ca²⁺ release from this compartment impossible. If anything, post-TG potentiation of depolarization-induced oxytocin release may be explained by larger [Ca²⁺]_i rises in the presence of TG due to exclusion of ER Ca²⁺ buffering, or else due to activation of an alternative Ca²⁺ entry pathway.

IICR triggered by stimulation of P2Y purinoreceptors stimulated the release of a pain-related neuromodulator, calcitonin gene-related peptide (CGRP), from small (presumed nociceptive) cultured DRG neurons (559).

Finally, the release of BDNF from electrically active neurons required activation of Ca²⁺ influx through N-type Ca²⁺ channels and CICR, as BDNF secretion was inhibited by -conotoxin GVIA, TG, and dantrolene (22).

G. Circadian Rhythms

Ca²⁺ release via ryanodine receptors is intimately involved in the control of circadian rhythms in the suprachiasmatic nucleus (SCN). Transfection of organotypic SCN slices with cytosol- and nucleus-targeted chameleon YC2.1 (422), by employing gene gun technology, allowed long-term monitoring of intracellular Ca²⁺ in neurons that maintain circadian rhythms (244). These long-lasting recordings revealed a circadian rhythm for cytosolic but not nuclear [Ca²⁺], and the former was fully synchronized with the electrical circadian rhythm measured by a multiple electrode array. Treatment with ryanodine (100 µM) depressed both the [Ca²⁺]_i and electrical rhythms, although TG (1 µM) was ineffective (244). Hence, the precise role played by the ER store remains to be investigated in much greater detail.

Most interestingly, the expression of RyR2 in SCN neurons is also governed by the circadian rhythm, with the maximal level in the second half of the day phase. This rhythm of RyR2 expression was preserved even when the animals were constantly kept in dim light, suggesting that it has a fundamental importance in setting the circadian clock (125). A role for InsP₃Rs in regulation of circadian rhythms has also been suggested, although the only evidence for this is the inhibitory action of 2-APB (199), which may not be associated with suppression of IICR at all due to the lack of specificity.

	V. ENDOPLASMIC RETICULUM CALCIUM HOMEOSTASIS AND NEURONAL PATHOPHYSIOLOGY

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A. ER Stress Response and Neurodegeneration

Since ER is responsible for transcription, posttranslational protein modification, and selective transport of proteins to different destinies, it has developed a highly sophisticated system for controlling the quality of the final protein products. Any imbalance of protein handling triggers a specific reaction generally defined as the ER stress response. This response is triggered either by accumulation of unfolded proteins (the "unfolded protein response," UPR) or by overexpression of particular proteins ("the ER overload response," EOR). The ER stress results in activation of several transcription factors (most notably IRE1 and its receptor PERK) that upregulate the expression of chaperones and reduce ER protein load by inhibiting overall protein synthesis. In addition, the ER triggers the activation of NF-B and the transcription factor C/ERP-homologous protein, which in turn control synthesis of proinflammatory and hemopoetic proteins and act as inhibitors of growth as well as promoters of apoptosis (see Refs. 12, 155, 364, 477, 478, 500). If the ER stress persists, it may result in the release of necrotic factors, which trigger cell death. As already discussed above, the activity of many intra-ER enzymatic cascades ultimately depends on the intraluminal Ca²⁺ concentration, and a strong link between ER Ca²⁺ homeostasis, ER stress, and neurodegeneration therefore has been postulated (495–498). This hypothesis is supported by several lines of evidence, which show that 1) depletion of ER stores induces upregulation of ER stress markers, 2) depletion of ER stores causes neuronal cell death, 3) exposure of neuronal cells to various insults upregulates ER stress markers, and 4) inhibition of ER Ca²⁺ release protects against neurotoxicity. Several examples of such data are discussed below.

The depletion of ER Ca²⁺ stores in primary cultured rat embryonic cortical neurons by incubation with TG (1 µM), CPA (30 µM), or caffeine (10 mM) reduced protein synthesis to 10–70% of the control and caused complete disaggregation of polysomes (129, 499). A significant decrease in protein synthesis was also observed in cultured embryonic rat cortical neurons treated with 100 nM ryanodine, a concentration which depletes the ER store by opening RyRs (5). At the same time, ER depletion following 30-min incubation with 1 µM TG led to an almost 200-fold increase in the expression of ER stress markers grp78, grp94, gadd34, and gadd153 and in a 6-fold increase of SERCA2b and BCl-2 (403). Therefore, severe disruption of ER Ca²⁺ homeostasis triggers ER stress and has a clear neurotoxic effect.

ER stress is also induced by exposure of nerve cells to various neurotoxic conditions. For example, application of excitotoxic concentrations of glutamate as well as oxidative insults elevated the level of grp78 in rat cultured hippocampal neurons (713). This increase in grp78 expression was neuroprotective, as treatment of cultures with grp78 antisense substantially increased neuronal cell death. Hypoxia/reperfusion increased the expression of another chaperon, grp94, in neuroblastoma cells. The increased level of grp94 was also cytoprotective, as treatment of neuroblastoma cells with grp94 antisense exacerbated cell death (24). Similarly, CA1 hippocampal neurons overexpressing grp94 were resistant to ischemic damage in gerbils subjected to transient carotid artery occlusion (24).

Finally, disturbances of ER Ca²⁺ handling cause neuronal death. Treatment of cultured cerebellar granule neurons with the neurotoxin mastoparan triggered massive IICR, which in turn initiated apoptosis and cell death. Inhibition of PLC (and hence InsP₃ production) had a neuroprotective effect (346). Depletion of Ca²⁺ stores with TG (300 nM), CPA (300 µM), and BHQ (300 µM) resulted in 50–60% neuroblastoma cell death within 48 h and in an almost complete demise of the culture within 72 h (455). Interestingly, loading the cells with BAPTA-AM did not protect against TG toxicity, although it prevented TG-induced [Ca²⁺]_i increases, thus demonstrating that the ER depletion, rather than [Ca²⁺]_i disturbances, were responsible for neurotoxicity (455). Experiments on the same cell line demonstrated that toxicity induced by oxygen-glucose deprivation is also mediated through Ca²⁺ depletion of the ER (684). In Caenorhabditis elegans, necrotic neuronal death can be prevented by loss of function mutations in Ca²⁺ release channels; at the same time, treatment with TG invariably exacerbated necrosis (e.g., Ref. 702).

The ER stress response may regulate neuronal death through another pathway, which involves caspase-12. This enzyme resides within the ER lumen and is activated upon various insults initiating ER stress (577). Most notably, ER stress triggered by disruption of ER Ca²⁺ homeostasis also activated caspase-12 cascade (442). This pathway is endowed with a degree of specificity as cortical neurons isolated from caspase-12-deficient mice were resistant to apoptosis induced by amyloid- proteins, but not to apoptosis induced by staurosporine or trophic factor deprivation (442). Caspase-induced cell death in embryonic hippocampal neurons similarly depended on ER Ca²⁺ signaling and required the activation of ryanodine receptors (239).

Overall, it is quite clear that nerve cells do develop ER stress upon severe disruption of ER Ca²⁺ homeostasis. Yet, the maneuvers employed for damaging ER Ca²⁺ handling, such as the irreversible block of SERCA pumps by TG, are rather brutal and are very far from the conditions that neurons may encounter even in a diseased state. Clearly more experimentation is needed to precisely characterize the link between [Ca²⁺]_L and ER stress. Another very important point lies in the determination of harmful levels of [Ca²⁺]_L. Indeed, it is evident that dramatic falls in intra-ER free Ca²⁺ are detrimental for the cell; however, increased [Ca²⁺]_L can also be toxic (147, 511). It seems that a certain normal "window" of [Ca²⁺]_L exists, and when ER Ca²⁺ rises or falls beyond a particular limit, grave consequences follow. Lastly, changes in ER Ca²⁺ concentration may cause severe fragmentation of the ER (610, 634), which may also have pathological consequences.

B. ER Stores and Ischemia

As a rule, glucose/oxygen deprivation triggers a substantial elevation of [Ca²⁺]_i in nerve cells (521, 630, 708), part of which is due to ER Ca²⁺ release (569, 644). In striatal slices exposed to hypoxic conditions, Ca²⁺ release from the ER (partially mediated by NO) was responsible for a large part of the ⁴⁵Ca²⁺ liberation (394). When cultured hippocampal neurons were subjected to an anoxic environment, mimicked by incubation with NaCN, a large [Ca²⁺]_i increase was induced. This [Ca²⁺]_i elevation was blocked by dantrolene and ruthenium red, therefore implying an important role of CICR (132) The NaCN-induced [Ca²⁺]_i increase considerably exceeded the [Ca²⁺]_i elevation in response to glutamate, although the action of the latter was grossly amplified in conditions of metabolic stress.

Similarly, energy deprivation (induced by perfusion with an oxygen/glucose free solution) produced a large [Ca²⁺]_i elevation in hippocampal neurons in slices ([Ca²⁺]_i increased up to 28 µM), and this increase was invariably followed by cell edema and death. Similar massive (up to 30 µM) increases in [Ca²⁺]_i following an interruption in blood flow were observed in hippocampal neurons impaled with ion-sensitive electrodes (588). Depletion of the ER stores by preincubation of hippocampal slices from young Wistar rats with TG (1 µM, 30 min) dramatically (5 times) reduced the [Ca²⁺]_i elevation triggered by energy deprivation (192). Promotion of ER Ca²⁺ release (by intracellular injection of InsP₃ into gebril hippocampal neurons) greatly facilitated the irreversible depolarization induced by ischemia (288). Dantrolene significantly reduced neuronal death in the CA1 area of the hippocampus in adult rats, subjected to global cerebral ischemia, but this effect could be partly attributed to reduced release of glutamate (447). Interestingly, however, the glutamate release was diminished to an equal extent by dantrolene as well as by DMSO (used as a solvent for dantrolene); DMSO alone also protected neurons against ischemia, yet dantrolene was twice as potent. Inhibition of ER Ca²⁺ release also prevented the liberation of neurotoxic free fatty acids from the ischemic cortex (510).

Ca²⁺ release from the ER also appears instrumental in ischemic damage of white matter. Destruction of axons upon ischemic stress is mediated through an increase in cytosolic Ca²⁺. Experiments on rat dorsal columns showed that this [Ca²⁺]_i elevation persisted after removal of Ca²⁺ from the perfusate; similarly, ischemic injury of axons persisted in Ca²⁺-free conditions. At the same time, the axonal [Ca²⁺]_i increases were sensitive to ER depletion by TG or by inhibition of CICR by ryanodine (473). Most intriguingly, the CICR in axons may be of the "skeletal muscle" variety, i.e., involving direct coupling between plasmalemmal Ca²⁺ channels and RyRs. This suggestion is based on the fact that blockage of the Ca²⁺ channel voltage sensor (by dihydropyridines), but not of the Ca²⁺ channel pore (by Cd²⁺), was hugely protective against ischemia. At the same time, simultaneous inhibition of RyRs (by ryanodine) and of Ca²⁺ channels by nimodipine was not additive, thus favoring interdependence of two molecules. In concordance with this suggestion, replacement of extracellular Na⁺ with impermeable cations, limiting depolarization, protected axons against ischemia, whereas substitution of Na⁺ with Li⁺ (which is carried through Na⁺ channels and hence allows depolarization) was not effective (473). Together, these findings provide very important information on mechanisms of ischemic damage of white matter, which seem to be different from those in gray matter. White matter damage is accomplished by intracellular Ca²⁺ release, whereas injury of gray matter very much depends on plasmalemmal Ca²⁺ entry.

The link between ischemia-induced changes in ER Ca²⁺ homeostasis and intra-ER chaperone activity has also been described recently, when the ischemia-induced ER chaperone orp150 (oxygen-regulated protein of 150 kDa) was isolated and characterized. Orp150 is strongly upregulated in astrocytes following ischemic stress, which may explain their exceptional resistance to ischemic conditions. Overexpression of orp150 in cultured rat cortical neurons protected them against ischemia, and vice versa, inhibition of orp150 synthesis in cultured astrocytes increased their vulnerability to hypoxic shock (627). Transgenic mice expressing orp150 under control of a platelet-derived growth factor B-chin promoter displayed high levels of orp150 in the cortex, hippocampus, and cerebellum and were much more resistant to global cerebral ischemia compared with wild-type controls (627). Interestingly, in cultured neurons with an increased level of orp150, glutamate-induced [Ca²⁺]_i loads were substantially reduced, implying that this chaperone may also stabilize cellular Ca²⁺ homeostasis, most likely by limiting Ca²⁺ release from the ER, which accompanies excessive glutamate stimulation (294).

C. ER Stores and Human Immunodeficiency Virus

The human immunodeficiency virus (HIV) envelope glycoprotein GP120 was shown to induce large [Ca²⁺]_i increases in cultured hippocampal neurons and promote their degeneration. Both the [Ca²⁺]_i elevation and the neurodegeneration can be prevented by the CICR blocker dantrolene, or by ER depletion produced by preincubation of cells with caffeine, carbachol, or TG (393).

D. ER Stores and Diabetes

The involvement of the ER Ca²⁺ store in diabetic peripheral neuropathies can be suggested on the basis of a rather limited number of observations demonstrating a reduction in the size of caffeine-induced Ca²⁺ release in peripheral neurons isolated from animals with experimental (usually streptozotocin induced) diabetes type I. Such a decrease was found in diabetic DRG neurons (242, 306, 316), and the reduced [Ca²⁺]_i responses to caffeine were especially prominent in cells isolated from lumbar ganglia (242), which are the first to be affected by diabetic neuropathies. A very substantial decrease in caffeine-induced [Ca²⁺]_i transients (from 270 nM in control animals to 30 nM in diabetic ones) was also observed in dorsal horn neurons from streptozotocin-induced diabetic rats (316, 679). In in vivo experiments, ryanodine and TG significantly modified the formalin-induced nociceptive response in streptozotocin-diabetic mice (266) providing further evidence that the ER Ca²⁺ store is involved in the pathogenesis of diabetic neuropathies.

E. ER Stores and Gangliosidoses (Gaucher’s and Sandhoff’s Disease)

Mutations of the human gene encoding glucocerebrosidase result in a reduction of activity of this enzyme and accumulation of glycosylceramide, which, in turn, promotes neurodegeneration in the central nervous system. Clinically this type of neurodegenerative disorder is known as Gaucher’s disease (27). When the activity of glucocerebrosidase in cultured hippocampal neurons was blocked by incubation with conduritol-B-epoxide, accumulation of glycosylceramide occurred, thus mimicking the pathological conditions associated with Gaucher’s disease. This condition was accompanied by a significant increase in ER tubular elements and by a great increase in the release of Ca²⁺ from the ER in response to both metabotropic stimulation and caffeine (302). Furthermore, hippocampal neurons with inhibited glucocerebrosidase were significantly more vulnerable to excitotoxic shock (exposure to high glutamate concentrations), and this increased vulnerability was blocked by ryanodine (302). Therefore, remodeling of the ER Ca²⁺ homeostasis which results in an increase of ER Ca²⁺ content may be regarded as a key element of neurodegeneration associated with Gaucher’s disease.

Another type of gangliosidoses, when gangliosides accumulate in cells due to a defect in lysosomal proteins catalyzing their degradation, the GM2 gangliosidoses, or Sandhoff’s disease, also affects the ER, as the rate of SERCA-driven Ca²⁺ uptake was found to be considerably lower in the Sandhoff mouse model (501).

F. ER Stores and Alzheimer’s Disease

Alzheimer’s disease (AD) is a form of senile dementia characterized by the advent of plaques in the brain matter (54, 153) and appearance of fibrillous structures within the neuronal cytoplasm (11). It receives much attention because of its social impact affecting the progressively ageing mankind. The importance of cellular Ca²⁺ homeostasis in the pathogenesis of AD was perceived at the beginning of 1980s, when Zaven Khachaturian promulgated his "Ca²⁺ hypothesis of AD and neuronal ageing" (281). This hypothesis postulated that long-lasting subtle changes in [Ca²⁺]_i homeostasis would eventually result in the neurodegeneration so characteristic of AD.

Investigations in the last decade confirmed this early reflection by clearly demonstrating that disruption of cellular Ca²⁺ homeostasis is a prominent feature of AD pathophysiology. Furthermore, these investigations identified ER Ca²⁺ dysregulation as a key step in the development of AD.

As already mentioned, the histopathology of AD is represented by plaques formed by aggregates of amyloid- peptide (A) and by the appearance of intracellular filamentous structures of a macrotubule-associated protein tau (378). This reflects several complex alterations of enzymatic systems controlling the synthesis and processing of A. The familial form of AD (FAD) is caused by mutations of genes encoding the A precursor protein (APP, chromosome 21) and presenilins 1 (PS1, chromosome 14) and 2 (PS2, chromosome 1). Mutations in PS1 are responsible for the majority of FAD cases. The central question for the pathophysiology of AD is related to events proximal to the malformation of A, as they could represent potential therapeutic targets.

Disrupted Ca²⁺ homeostasis has been identified as a feature of AD. Increased levels of free and bound Ca²⁺ have been detected in neurons containing neurofibrillary tangles (439). Likewise, neurons possessing tangles showed increased activity of various Ca²⁺-dependent proteases (461). Early studies on fibroblasts isolated from AD patients also demonstrated impairment of Ca²⁺ mobilization in response to metabotropic stimulation (186, 252). Direct administration of A to nerve cells caused elevation in resting [Ca²⁺]_i and greatly diminished the ability of cells to cope with Ca²⁺ loads (207, 367, 379; see also Ref. 377 for review). Most importantly however, cellular Ca²⁺ dishomeostasis occurs at the initial stages of AD (142), and studies on transgenic mice with APP or PS mutations found that impaired Ca²⁺ handling occurs long before any histopathological changes (195, 323). The mechanisms of AD-related disruption of [Ca²⁺]_i homeostasis can be multifacetous and involve plasmalemmal systems as well as intracellular organelles. The effects of A and its fragments, for instance, are reported to influence plasmalemmal Ca²⁺ transport by forming Ca²⁺-permeable pores (47, 279), although acute application of A additionally augments intracellular Ca²⁺ release (207, 299). Several recently conducted studies suggest that disrupted ER Ca²⁺ homeostasis may be regarded as a specific feature of the AD-related neuronal Ca²⁺ dysregulation (see Table 11 and Refs. 323, 378, 380, 608, 709).

Presenilins are highly expressed in neurons and reside mostly in the ER membrane. Not much is known about the involvement of presenilins in ER Ca²⁺ handling, although it has been shown that PS1 can bind to RyRs (88) and interact with sorcin (476), calsenilin (69), and calpain (372), i.e., proteins known to regulate Ca²⁺ release channels. Nonetheless, every alteration of PS, be it in the form of its genetic deletion or of expression of mutant (FAD associated) PS, results in impairment of ER function. As a rule, the latter is manifested as an increased amplitude of the Ca²⁺ response to metabotropic agonists, caffeine, or the SERCA inhibitors TG and CPA (see Table 11). This increase in [Ca²⁺]_i transients is generally regarded as a consequence of an elevated [Ca²⁺]_L. The changes in ER Ca²⁺ content are not confined to neurons, but are also observed in various peripheral cells such as, e.g., fibroblasts (336).

Manipulations of presenilin expression affect several Ca²⁺-dependent functions as well as neuronal vulnerability to various types of insults. In transgenic mice expressing mutant PS1, neurons in hippocampal slices show an increased sensitivity to kainate excitotoxicity and demonstrate facilitated LTP induction (564). Both effects are attributable to an increased Ca²⁺ release from the ER. Similarly, expression of mutant PS1 increased the size of infarction following middle cerebral artery occlusion and reperfusion in vivo and increased cell death in cortical neurons in vitro, when exposed to hypoxia and glucose deprivation (382). Likewise, cultured hippocampal neurons from the same animals displayed increased vulnerability to glutamate excitotoxic shock (195). Inhibition of ER stores by dantrolene protected PS1 mutant-bearing neurons (382). Higher susceptibility of PS1 mutant neurons to various insults correlated with failures of the Ca²⁺ homeostatic machinery. Recordings of [Ca²⁺]_i in PS1 mutant neurons revealed larger [Ca²⁺]_i elevations induced by hypoxia, glutamate, and intracellular delivery of InsP₃ (195, 382, 608). Modulation of PS functions by overexpressing calsenilin, a cytoplasmic protein interacting with PS1 and PS2, enhanced apoptosis and increased the amount of TG-released Ca²⁺, once more pointing to a possible increase in [Ca²⁺]_L (345).

The PS-mediated alterations of ER function were accompanied by biochemical changes characteristic of the ER stress response. Specifically, expression of mutant PS1 in hippocampal neurons was associated with an abnormal activation of ER resident caspase-12 (87), which, as has been mentioned previously, mediates ER stress-related apoptosis. Expression of an aberrant spliced form of PS2 (PS2V) altered UPR and increased production of A40 and A42 (561). Furthermore, neurons overexpressing the mutant (but not the wild-type) form of PS1 were more susceptible to spontaneous apoptosis and apoptosis induced by ER stress, through decreased UPR (635).

An extremely important achievement in studying the cellular mechanisms of AD occurred recently after several genetic AD models were created. Conceptually these models aimed at the insertion of various forms of APP, which replicated A deposition and plaque formation, or inclusion of mutant PS genes, which produced numerous AD-specific neuropathological changes. The recently discovered tau gene mutations (191), associated with frontotemporal dementia, have provided an additional tool for genetic modeling. A big step forward was made by Frank LaFerla, Mark Mattson, and co-workers who succeeded in creating a triple transgenic mouse bearing presenilin 1 (PS1m146V), amyloid precursor protein (APPSwe), and tau (tauP301L) transgenes (466). These animals possessed several hallmarks of AD such as progressively developing plaques and tangles as well as deficiencies in LTP, which preceded the appearance of the histological markers of AD. The functional changes were clearly age dependent, as both the amplitude of field-stimulation evoked EPSPs and LTP were significantly impaired in 6-mo-old triple transgenic animals, whereas at 1 mo there was no difference between the transgenic mice and wild-type controls (466). However, a detailed analysis of the ER signaling changes associated with this model has yet to be performed.

Although there is no doubt about the AD-related impairment of ER Ca²⁺ homeostasis, the important question of whether ER changes precede A formation or conversely A malsynthesis causes ER dysfunction remains open.

G. ER Stores and Ageing

The process of physiological ageing (646), which is not associated with any prominent neuronal loss, yet is accompanied by cognitive decline, involves changes in neuronal Ca²⁺ homeostasis, which comprise alterations of Ca²⁺ influx, mitochondrial and cytoplasmic Ca²⁺ buffering, and Ca²⁺ extrusion (108, 156, 289, 307, 329, 516, 639, 640, 647, 675, 700). The amplitudes of caffeine-induced [Ca²⁺]_i responses are moderately decreased in aged DRG, neocortical, and cerebellar granule cells (290, 291, 674) and in acutely dissociated rat basal forebrain neurons (436). This may reflect the decreased [Ca²⁺]_L, and indeed, the studies mentioned above found a decreased Ca²⁺ sequestering capacity of the ER in aged nerve cells. In contrast, old cultured hippocampal neurons (101) demonstrated prolonged CICR in response to glutamate exposure, although the size of the caffeine-releasable pool was similar in young and old cultures. Experiments in hippocampal slices isolated from old (22–24 mo) rats found that increased ER Ca²⁺ release affects LTP induction in the aged hippocampus (321). All in all, however, the age-dependent changes in ER Ca²⁺ homeostasis are rather mild when compared with the ER alterations in AD. Moreover, no obvious increase in the ER Ca²⁺ content has been discovered in old neurons, which may reflect a fundamental difference between the physiological processes of brain ageing and age-dependent neurodegeneration.