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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 sp