Hypoxia-induced activation of N-methyl-D-aspartate receptors causes retinal ganglion cell death in the neonatal retina

It is well established that hypoxia causes excess accumulation of glutamate in developing neural tissues. This study aimed to elucidate the mechanism by which glutamate can cause retinal ganglion cell (RGC) death through the N-methyl-D-aspartate (NMDA) receptors (NR) in the developing retina. One-day-old Wistar rats were exposed to hypoxia for 2 hours and then killed at different time points. Normal age-matched rats were used as controls. NR1, NR2A-D, and NR3A messenger RNA and protein expression showed significant increases over control values, notably at early time points (3 hours to 7 days) after the hypoxic exposure, and immunoexpression of NR1, NR2A-D and NR3A on retinal ganglion cells (RGCs) was enhanced in hypoxic rats and this was confirmed in cultured hypoxic RGCs. Ca(2+) influx in cultured RGCs was increased after hypoxic exposure, and the intracellular Ca(2+) concentration was suppressed by MK-801. Mitochondrial permeability transition pore opening, mitochondrial/cytosolic cytochrome c, and cytosolic caspase-3 expression levels were significantly increased in the hypoxic RGCs. These increases were reversed by MK-801, suggesting that the NMDA receptor subunits in the retina respond rapidly to the hypoxia-induced glutamate overload that leads to the cascade of events that result in RGC death.     

Postsynaptic IP3 receptor-mediated Ca2+ release modulates synaptic transmission in hippocampal neurons

Ca(2+)-dependent mechanisms are important in regulating synaptic transmission. The results herein indicate that whole-cell perfusion of inositol 1,4,5-trisphosphate receptor (IP(3)R) agonists greatly enhanced excitatory postsynaptic current (EPSC) amplitudes in postsynaptic hippocampal CA1 neurons. IP(3)R agonist-mediated increases in synaptic transmission changed during development and paralleled age-dependent increases in hippocampal type-1 IP(3)Rs. IP(3)R agonist-mediated increases in EPSC amplitudes were inhibited by postsynaptic perfusion of inhibitors of Ca(2+)/calmodulin, PKC and Ca(2+)/calmodulin-dependent protein kinase II. Postsynaptic perfusion of inhibitors of smooth endoplasmic reticulum (SER) Ca(2+)-ATPases, which deplete intracellular Ca(2+) stores, also enhanced EPSC amplitudes. Postsynaptic perfusion of the IP(3)R agonist adenophostin (AdA) during subthreshold stimulation appeared to convert silent to active synapses; synaptic transmission at these active synapses was completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Postsynaptic IP(3)R-mediated Ca(2+) release also produced a significant increase in spontaneous EPSC frequency. These results indicate that Ca(2+) release from intracellular stores play a key role in regulating the function of postsynaptic AMPARs.     

Modulation of ultra-low-molecular-weight heparin on [Ca²⁺]i in nervous cells

Heparin is an effective competitive antagonist of inositol 1,4,5-trisphosphate receptors (IP(3)Rs). It binds to IP(3)Rs and affects calcium homeostasis. Ultra-low-molecular-weight heparin (ULMWH) is heparin's derivative, the present study was designed to test the effects of ULMWH on intracellular calcium concentration ([Ca(2+)]i) in primary cultured neurons. [Ca(2+)]i was measured by Multilabel Counter Victor-1420 using Fura-2/AM as the calcium fluorescent probe. The results indicated that ULMWH decreased the resting [Ca(2+)]i with or without extracellular Ca(2+). They had no effects on high K(+)-induced elevation of intracellular Ca(2+) level indicating that ULMWH had no effect on external Ca(2+) influx mediated by voltage-dependent calcium channels. However, they partially reduced the increase in [Ca(2+)]i induced by glutamate. Furthermore, ULMWH significantly inhibited the inositol 1,4,5-trisphosphate (IP(3))-induced increase in [Ca(2+)]i both in cellular and subcellular level. These results suggest that ULMWH may reduce [Ca(2+)]i in neurons through suppressing Ca(2+) release from IP(3)-sensitive stores.     

Protective effect of nilvadipine against glutamate neurotoxicity in purified retinal ganglion cells

We determined the effect of nilvadipine, a dihydropyridine-type calcium channel blocker, in preventing glutamate neurotoxicity in purified retinal ganglion cells (RGCs). RGCs were purified from dissociated rat retinal cells (postnatal days 6-8), using a modified two-step panning method, and cultured in serum-free medium containing neurotrophic factors and forskolin. RGC survival after exposure to glutamate (25 microM) with nilvadipine or other calcium channel blockers was measured by calcein-acetoxymethyl ester staining after 3 days in culture. Changes in the level of intracellular Ca(2+) ([Ca(2+)](i)) were measured with fura-2 fluorescence. Induction of apoptosis was evaluated using the TDT-dUTP terminal nick-end labeling technique. The neurotoxic effects of low doses of glutamate were blocked by a specific alpha-amino-3-dihydro-5-methylisoxazole-4-propionate-kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (20 microM). Simultaneous application of nilvadipine (1-100 nM) with glutamate protected against glutamate neurotoxicity in a dose-dependent manner. Calcium-imaging experiments showed that the glutamate-evoked [Ca(2+)](i) increase was significantly blocked by nilvadipine (P<0.001), but not nifedipine and diltiazem, in about 50% of RGCs. In addition, the application of nilvadipine significantly reduced glutamate-induced apoptosis (P<0.001). These findings suggest that nilvadipine may partly inhibit glutamate-induced apoptotic cell death by blocking calcium influx via voltage-dependent calcium channels in purified RGCs.     

Signaling proteins in the axoglial apparatus of sciatic nerve nodes of Ranvier

During action potential conduction, the axonal specializations at the node, together with the adjacent paranodal terminations of the myelin sheath, interact with glial processes that invest the nodal gap. The nature of the mutual signals between axons and myelinating glia, however, are not well understood. Here we have characterized the distribution of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in the axoglial apparatus by immunohistochemistry, using known myelin domain-specific markers. While IP(3)R1 is not expressed in the Schwann cells or the axon, IP(3)R2 and IP(3)R3 are expressed in distinct cellular domains, suggesting distinct signaling roles for the two receptors. IP(3)R3 is the most predominant isoform in Schwann cells, and is expressed in particularly dense patches in the paranodal region. In addition to IP(3)Rs, two other members of the metabotropic Ca(2+) signaling pathway, G(alpha)q, and P(2)Y1 type of purinoceptors were also found in Schwann cells. Their pattern of expression matches the expression of their signaling partners, the IP(3)Rs. One interesting finding to emerge from this study is the expression of connexin 32 (Cx32) in close proximity with IP(3)R3. Although IP(3)R3 and Cx32 are not colocalized, their expression in the same membrane areas raises the question whether Schwann cell Ca(2+) signals either control the function of the gap junctions, or whether the gap junctional channels serve as conduits for rapid radial spread of Ca(2+) signals initiated during action potential propagation.     

Skeletal muscle IP3R1 receptors amplify physiological and pathological synaptic calcium signals

Ca(2+) release from internal stores is critical for mediating both normal and pathological intracellular Ca(2+) signaling. Recent studies suggest that the inositol 1,4,5-triphosphate (IP(3)) receptor mediates Ca(2+) release from internal stores upon cholinergic activation of the neuromuscular junction (NMJ) in both physiological and pathological conditions. Here, we report that the type I IP(3) receptor (IP(3)R(1))-mediated Ca(2+) release plays a crucial role in synaptic gene expression, development, and neuromuscular transmission, as well as mediating degeneration during excessive cholinergic activation. We found that IP(3)R(1)-mediated Ca(2+) release plays a key role in early development of the NMJ, homeostatic regulation of neuromuscular transmission, and synaptic gene expression. Reducing IP(3)R(1)-mediated Ca(2+) release via siRNA knockdown or IP(3)R blockers in C2C12 cells decreased calpain activity and prevented agonist-induced acetylcholine receptor (AChR) cluster dispersal. In fully developed NMJ in adult muscle, IP(3)R(1) knockdown or blockade effectively increased synaptic strength at presynaptic and postsynaptic sites by increasing both quantal release and expression of AChR subunits and other NMJ-specific genes in a pattern resembling muscle denervation. Moreover, in two mouse models of cholinergic overactivity and NMJ Ca(2+) overload, anti-cholinesterase toxicity and the slow-channel myasthenic syndrome (SCS), IP(3)R(1) knockdown eliminated NMJ Ca(2+) overload, pathological activation of calpain and caspase proteases, and markers of DNA damage at subsynaptic nuclei, and improved both neuromuscular transmission and clinical measures of motor function. Thus, blockade or genetic silencing of muscle IP(3)R(1) may be an effective and well tolerated therapeutic strategy in SCS and other conditions of excitotoxicity or Ca(2+) overload.     

Slow transport rates of cytoskeletal proteins change during regeneration of axotomized retinal neurons in adult rats

To investigate cytoskeletal changes associated with axonal regrowth from damaged nerve cells in the mammalian CNS, we examined the slow transport of axonal proteins during the regeneration of adult rat retinal ganglion cell (RGC) axons. Although normally such RGC axons do not regrow after injury in the CNS, they can extend several centimeters when their nonneuronal environment is changed by replacing the optic nerve (ON) with a grafted segment of peripheral nerve (PN). Proteins transported in axons of RGCs from intact control and PN-grafted animals were labeled by an intraocular injection of 35S-methionine and examined 4-60 days later by SDS PAGE. During RGC regeneration into PN grafts, the transport rate of tubulin and neurofilament increased twofold, whereas that of actin decreased to nearly one third of its normal rate. Thus, in these regenerating RGC axons, all three major cytoskeletal proteins were largely transported within a single rate component rather than in the two separate components (SCa and SCb) normally observed in the intact ON. Furthermore, the 200 kDa neurofilament protein (NF-H) was persistently detected in Western blots during periods of active regeneration, a finding that contrasts with the late appearance of the NF-H during the developmental growth of retinal axons. The changes in slow transport observed during RGC regeneration in adult rats may reflect growth-associated responses of mature CNS neurons during periods of active axonal extension.     

Expression of voltage-dependent potassium channels in the developing visual system of Xenopus laevis

Accumulating evidence suggests that voltage-dependent potassium (Kv) channels have important and varied roles in the development of neuronal and non-neuronal cell types. They have been implicated in processes such as proliferation, cell adhesion, migration, neurite outgrowth, and axon guidance. In this study, we used antibodies against several electrically active Kv channel alpha-subunits (Kv1-4) to describe the spatial and temporal expression patterns of Kv channel subunits in Xenopus laevis retinal ganglion cell (RGC) somata, axons, and growth cones. We found that RGCs express Kv1.3-, Kv1.5-, Kv3.4-, and Kv4.2-like subunits. Each subunit displayed unique cellular and subcellular distributions. Moreover, the expression patterns changed considerably over the major period of Xenopus retinal cell genesis and differentiation. Weak or no immunoreactivity was observed with antibodies against Kv1.1, Kv1.2, Kv1.4, Kv1.6, and Kv3.2 subunits in RGCs or other retinal cell types. In support of our previous pharmacologic evidence implicating Kv channels in RGC axon outgrowth, we found that Kv1.5-, Kv3.4-, and Kv4.2-like proteins, but not Kv1.3-like subunits, are abundantly expressed in RGC growth cones.     

The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina

There are few neurochemical markers that reliably identify retinal ganglion cells (RGCs), which are a heterogeneous population of cells that integrate and transmit the visual signal from the retina to the central visual nuclei. We have developed and characterized a new set of affinity‐purified guinea pig and rabbit antibodies against RNA‐binding protein with multiple splicing (RBPMS). On western blots these antibodies recognize a single band at ?24 kDa, corresponding to RBPMS, and they strongly label RGC and displaced RGC (dRGC) somata in mouse, rat, guinea pig, rabbit, and monkey retina. RBPMS‐immunoreactive cells and RGCs identified by other techniques have a similar range of somal diameters and areas. The density of RBPMS cells in mouse and rat retina is comparable to earlier semiquantitative estimates of RGCs. RBPMS is mainly expressed in medium and large DAPI‐, DRAQ5‐, NeuroTrace‐ and NeuN‐stained cells in the ganglion cell layer (GCL), and RBPMS is not expressed in syntaxin (HPC‐1)‐immunoreactive cells in the inner nuclear layer (INL) and GCL, consistent with their identity as RGCs, and not displaced amacrine cells. In mouse and rat retina, most RBPMS cells are lost following optic nerve crush or transection at 3 weeks, and all Brn3a‐, SMI‐32‐, and melanopsin‐immunoreactive RGCs also express RBPMS immunoreactivity. RBPMS immunoreactivity is localized to cyan fluorescent protein (CFP)‐fluorescent RGCs in the B6.Cg‐Tg(Thy1‐CFP)23Jrs/J mouse line. These findings show that antibodies against RBPMS are robust reagents that exclusively identify RGCs and dRGCs in multiple mammalian species, and they will be especially useful for quantification of RGCs. J. Comp. Neurol. 522:1411–1443, 2014. © 2013 Wiley Periodicals, Inc.     

Involvement of endoplasmic reticulum Ca2+ release through ryanodine and inositol 1,4,5-triphosphate receptors in the neurotoxic effects induced by the amyloid-beta peptide

Studies with in-vitro-cultured neurons treated with amyloid-beta (A beta) peptides demonstrated neuronal loss by apoptosis that is due, at least in part, to the perturbation of intracellular Ca(2+) homeostasis. In addition, it was shown that an endoplasmic reticulum (ER)-specific apoptotic pathway mediated by caspase-12, which is activated upon the perturbation of ER Ca(2+) homeostasis, may contribute to A beta toxicity. To elucidate the involvement of deregulation of ER Ca(2+) homeostasis in neuronal death induced by A beta peptides, we have performed a comparative study using the synthetic peptides A beta(25-35) or A beta(1-40) and thapsigargin, a selective inhibitor of Ca(2+) uptake into the ER. Incubation of cortical neurons with thapsigargin (2.5 microM) increased the intracellular Ca(2+) levels and activated caspase-3, leading to a significant increase in the number of apoptotic cells. Similarly, upon incubation of cortical cultures with the A beta peptides (A beta(25-35), 25 microM; A beta(1-40), 0.5 microM), we observed a significant increase in [Ca(2+)](i), in caspase-3-like activity, and in number of neurons exhibiting apoptotic morphology. The role of ER Ca(2+) release through ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (IP(3)R) in A beta neurotoxicity has been also investigated. Dantrolene and xestospongin C, inhibitors of ER Ca(2+) release through RyR or IP(3)R, were able to prevent the increase in [Ca(2+)](i) and the activation of caspase-3 and to protect partially against apoptosis induced by treatment with A beta(25-35) or A beta(1-40). In conclusion, our results demonstrate that the release of Ca(2+) from the ER, mediated by both RyR and IP(3)R, is involved in A beta toxicity and can contribute, together with the activation of other intracellular neurotoxic mechanisms, to A beta-induced neuronal death. This study suggests that A beta accumulation may have a key role in the pathogenesis of AD as a result of deregulation of ER Ca(2+) homeostasis.     

Expression of the candidate circadian photopigment melanopsin (Opn4) in the mouse retinal pigment epithelium

A number of responses to light, including circadian entrainment and pupillary constriction, are preserved in mammals that lack rod and cone photoreceptors. Recent studies have demonstrated that a subset of retinal ganglion cells (RGCs) are intrinsically photosensitive, and that these RGCs project to regions of the brain associated with the regulation of the circadian clock and pupil constriction. The photopigment gene(s) that mediate these effects of irradiance remain unidentified, although melanopsin (Opn4) has emerged as a strong candidate. For example, Opn4 is expressed within intrinsically photosensitive RGCs, and Opn4 knock-out mice show attenuated circadian and pupillary responses to light. In this study we provide the first clear evidence that Opn4 expression is not confined to these photosensitive RGCs, but is also expressed in the retinal pigment epithelium (RPE), a tissue with no known photosensensory role. We can preclude retinal contamination of RPE extracts as levels of Opn4 expression were higher in the RPE than in the retina, and the expression of rod opsin and Thy1 (a marker of the RGC layer) were barely detectable in RPE extracts. Our results raise questions about the presumed function of melanopsin, and highlight the need for biochemical studies on this protein.     

Multiple calcium-activated neutral proteinases (CANP) in mouse retinal ganglion cell neurons: specificities for endogenous neuronal substrates and comparison to purified brain CANP

Calcium-activated neutral proteinases (CANPs) and their specificities for axonally transported proteins were studied within intact axons of mouse retinal ganglion cell (RGC) neurons in vitro. Two CANP activities with markedly different properties were identified. CANP B, at endogenous calcium levels, selectively cleaved the 145,000 Da (145 kDa) neurofilament protein subunit to yield 143 and 140 kDa neurofilament proteins that are also major constituents of the axonal cytoskeleton. This process represents a posttranslational modification of the neurofilament protein subunit rather than the initial step in its degradation (Nixon et al., 1982, 1983). A second calcium-activated neutral proteinase activity, CANP A, appeared only when calcium levels in the incubating medium were 100 microM or higher. CANP A degraded most proteins in RGC axons but acted considerably more rapidly on high-molecular-weight species. In particular, a 290-320 kDa protein in the Group IV (SCb) phase of axoplasmic transport was degraded 3 X faster than other major axonal proteins, including neurofilament proteins and fodrin. When maximally expressed, CANP A activity represented an enormous proteolytic potential in RGC axons--more than 50% of the total axonal content of proteins larger than 60 kDa could be hydrolyzed within 5 min. The calcium requirements, inhibitor profile, and substrate specificity of CANP A were similar to those of mCANP, the major CANP of mouse brain purified to homogeneity, suggesting that these enzymes may be the same or highly related proteins. The existence in a single neuron type of two CANP activities with markedly different substrate specificities and enzymatic properties emphasizes the possible functional diversity of calcium-activated neutral proteinases in neurons. These functions include the posttranslational modification, as well as degradation of neuronal proteins.     

Effects of ryanodine receptor activation on neurotransmitter release and neuronal cell death following kainic acid-induced status epilepticus

Dynamic changes in intracellular free Ca(2+) concentration play a crucial role in various neural functions. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and the ryanodine (Ry) receptor (RyR) are involved in Ca(2+)-induced Ca(2+)-release (CICR). Recent studies have shown that type 3 IP3R is highly expressed in rat hippocampal neurons after kainic acid (KA)-induced seizures and that dantrolene, a RyR antagonist, reduces KA-induced neuronal cell death. We investigated the RyR-associated effects of CICR agents on basal and K(+)-evoked releases of GABA and glutamate in rat hippocampus and the changes in expression of mRNA for RyRs in mouse brain after KA-induced seizures. The stimulatory effect of Ry on releases of GABA and glutamate was concentration-dependent in a biphasic manner. The inflection point in concentration-response curves for Ry on GABA release was lower than that for glutamate in both basal and K(+)-evoked conditions, suggesting that hyperactivation of RyR-associated CICR produces the imbalance between GABAergic and glutamatergic transmission. Following KA-induced seizures, transient up-regulation of brain-type RyR mRNA was observed in the hippocampal CA3 region and striatum, and signals for c-Fos mRNA increased transiently in the hippocampus, dentate gyrus and deeper layers of the neocortex. Thereafter, some dead neurons with single-stranded DNA (ssDNA) immunoreactive fragmented nuclei appeared in these areas. These findings suggest that intracellular Ca(2+) release via the RyR might be one of the mechanisms involved in KA-induced neuronal cell death.     

Oxidative stress and Ca2+ influx upregulate calpain and induce apoptosis in PC12 cells

Calpain, a Ca2+-dependent cysteine protease, has previously been implicated in apoptosis or programmed cell death (PCD) in immune cells. Although oxidative stress and intracellular free Ca2+ are involved in neurodegenerative diseases, the mechanism of neuronal cell death in the central nervous system (CNS) due to these agents has not yet been defined. To explore a possible role for calpain in neuronal PCD under oxidative stress and Ca2+ influx, we examined the effects of H2O2 and A23187 on PC12 cells. Treatments caused PCD (light microscopy and TUNEL assay) with altered mRNA expression (RT-PCR) of bax (pro-apoptotic) and bcl-2 (anti-apoptotic) genes, resulting in a high bax/bcl-2 ratio. Control cells expressed 1.3-fold more microcalpain (requiring microM Ca2+) than mcalpain (requiring mM Ca2+). Expression of mcalpain was significantly increased following exposure to oxidative stress and Ca2+ influx. The mRNA levels of calpastatin (endogenous calpain inhibitor) and beta-actin (house-keeping) genes were not changed. Western analysis indicated degradation of 68 kDa neurofilament protein (NFP), a calpain substrate. Pretreatment of cells with MDL28170 (a cell permeable and selective inhibitor of calpain) prevented increase in bax/bcl-2 ratio, upregulation of calpain, degradation of 68 kDa NFP, and occurrence of PCD. These results suggest a role for calpain in PCD of PC12 cells due to oxidative stress and Ca2+ influx.     

Developmental changes in human cerebellum: Expression of intracellular calcium receptors,calcium-binding proteins,and phosphorylated and nonphosphorylated neurofilament protein

Few recent data are available on the development of the precise projection maps of the cerebellar cortex in humans. To address this topic, we studied temporal and spatial distribution of several antigens involved in calcium (Ca)-dependent processes: the intracellular Ca receptors, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and ryanodine receptor (RyR); the Ca-binding proteins, calbindin D-28k (CB), parvalbumin (PV), and synaptophysin; and phosphorylated (SMI 31) and nonphosphorylated (SMI 32) forms of neurofilament protein. All antigens were studied in the human cerebellum during intrauterine development. The results of this study show that immunocytochemical markers appeared in the following sequence: CB and both forms of neurofilament protein were observed at 4–5 gestational weeks (g.w.), PV appeared in the external granular layer and in a few Purkinje cells at 11 g.w., a diffuse immunostaining for IP3R1 and synaptophysin were observed at 13 g.w., whereas RyR was observed at 17–18 g.w. From 24 g.w. on, Purkinje cells expressed all four examined markers of intracellular Ca signaling as well as two forms of neurofilament protein. At the same time, compartmentation of the Purkinje cell layer was detected with three intracellular Ca-signaling molecules (IP3R1, CB, and PV) and with SMI 32. These results indicate that the developmentally regulated expression of antigens studied here may play a role in establishing a highly regular organization of terminal fields in the human cerebellar cortex. Moreover, the initial expression of these antigens is correlated temporally with other developmental processes in the cerebellum, such as cellular maturation, revealed by the immunoreaction to cytoskeletal protein, and synaptogenesis, revealed by immunoreaction to synaptophysin. J. Comp. Neurol. 396:442–460, 1998. © 1998 Wiley-Liss, Inc.     

Central retinal area is not the site where ganglion cells are generated first

The development of retinal ganglion cells (RGC) was studied in the chick from stage 18 to adulthood. Our main objectives were to identify the retinal site where the first RGCs differentiate, to locate this site relative to the optically defined central retinal area, and to map the spatial arrangement of the RGC field at different stages in development. The eyes of the experimental animals were fixed and serially sectioned. The borders of RGC fields were determined from the presence of either ganglion cell perikarya or ganglion cell axons. In seven cases between stages 21 and 26, the borders of the RGC fields were confirmed electron microscopically. The serial sections together with the RGC fields were then reconstructed in three dimensions. The reconstructed retinae were projected onto a plane by using the radially equidistant polar azimuthal projection. First, RGCs appear dorsal to the apex of the optic fissure. Ganglion cell development then initially spreads out symmetrically with respect to the optic fissure. However, from stage 29 on, the nasal half of the retina expands much more than the temporal half. This asymmetrical growth entails that the optic fissure is eventually located in the temporal half of the retina in the mature animal. The RGC fields of the embryonic stages were superimposed on the retina of a visually active animal according to their real size and position. It turned out that the central retinal area was at least 2 mm away from the site where the first RGCs were generated. It is not before stage 28 that the prospective central retinal area is included into the expanding ganglion cell field. The fact that RGCs at the central retinal area are generated 2.5 days later than first RGCs near the apex of the optic fissure has important implications for the formation of the retinotectal projection. © 1993 Wiley-Liss, Inc.     

Peak density and distribution of ganglion cells in the retinae of microchiropteran bats: implications for visual acuity

We have estimated the total number, distribution and peak density of retinal ganglion cells (RGCs) in retinal wholemounts of several species of microchiropteran (echolocating) bats. The estimates are based on counts of Nissl-stained, presumed RGCs. The total number of presumed RGCs varies among the species: from about 4,500 in Rhinolophus rouxi to about 120,000 in Macroderma gigas. In addition, in two species (Nyctophilus gouldi and M. gigas), the estimates are based on counts of positively identified RGCs retrogradely labelled with the enzyme horseradish peroxidase injected into the retinorecipient nuclei. In these two species, the numbers and distributions of retrogradely labelled RGCs and Nissl-stained presumed RGCs are very similar. In all six species studied, the peak-density regions of presumed (or positively identified) RGCs are located in the inferotemporal retinae, and the RGC isodensity lines tend to be horizontally elongated. However, the RGC densities in the high-density regions are only 2-4 times greater than those in the low-density regions in the superior retinae. The somal sizes of RGCs vary from 5 to 16 micron in diameter and are unimodally distributed. There is no indication of the existence of distinct morphological classes of RGCs. The axial lengths of microchiropteran eyes vary from 1.8 mm in R. rouxi to 7.0 mm in M. gigas. For all species the posterior nodal distance (PND) was assumed to be 0.52 of the axial length of the eye. This assumption is based on the analysis of published data concerning schematic eyes of nocturnal vertebrates. These derived values of the PNDs allowed us to calculate the retinal magnification factors and the number of RGCs per degree of visual angle. From these, the upper limits of visual acuity were derived on the basis of the assumptions of the sampling theorem. The estimated upper limits of visual acuity of the six species of echolocating bats vary from about 0.35 cycles/degree in R. rouxi to about 2 cycles/degree in M. gigas. This range is quite similar to the range of visual acuities in murid rodents.     

Calcium transient activity in cultured murine neural crest cells is regulated at the IP(3) receptor

In a previous study we have shown that cultured neural crest cells exhibit spontaneous calcium transients and that these events are required for neurogenesis. In this study, we examine the mechanism that generates these calcium transients. Extracellular Ca(2+) modulates calcium transient activity. Lanthanum (La(3+)), a general calcium channel antagonist and zero extracellular Ca(2+), reduces the percentage of cells exhibiting calcium transients (26.2 and 40. 5%, respectively) and decreases calcium spiking frequency (4.5 to 1. 0 and 2.5 to 1.0 spikes/30 min, respectively). Intracellular calcium stores also contribute to the generation of calcium transients. Depleting the calcium stores of the endoplasmic reticulum (ER) reduces the percentage of active cells (15.7%) and calcium spiking frequency (2.8 to 1.5 spikes/30 min). Ryanodine (100 microM), which blocks calcium release regulated by the ryanodine receptor (RyR), had no effect on calcium transient activity. Blocking inositol 1,4, 5-triphosphate receptor (IP(3)R)-dependent calcium release, with elevated extracellular Mg(2+) (20 mM), abolished calcium transient activity. Mg(2+) did not block caffeine-sensitive calcium release (RyR-dependent) or voltage dependent calcium channels. Mg(2+) also suppressed thimerosal-induced calcium oscillations (IP(3)R-dependent). Small increases in the intracellular calcium concentration ([Ca(2+)](i)), increases the percentage of active cells and the calcium spiking frequency, while larger increases in [Ca(2+)](i) block the transients. Reducing intracellular IP(3) levels reduces the percentage of active cells and the calcium spiking frequency. We conclude that the mechanism for generating spontaneous calcium transients in cultured neural crest cells fits the model for IP(3)R-dependent calcium excitability of the ER.     

Astrocytes in adult rat brain express type 2 inositol 1,4,5-trisphosphate receptors

Astrocytes respond to neuronal activity by propagating Ca(2+) waves elicited through the inositol 1,4,5-trisphosphate pathway. We have previously shown that wave propagation is supported by specialized Ca(2+) release sites, where a number of proteins, including inositol 1,4,5-trisphosphate receptors (IP(3)R), occur together in patches. The specific IP(3)R isoform expressed by astrocytes in situ in rat brain is unknown. In the present report, we use isoform-specific antibodies to localize immunohistochemically the IP(3)R subtype expressed in astrocytes in rat brain sections. Astrocytes were identified using antibodies against the astrocyte-specific markers, S-100 beta, or GFAP. Dual indirect immunohistochemistry showed that astrocytes in all regions of adult rat brain express only IP(3)R2. High-resolution analysis showed that hippocampal astrocytes are endowed with a highly branched network of processes that bear fine hair-like extensions containing punctate patches of IP(3)R2 staining in intimate contact with synapses. Such an organization is reminiscent of signaling microdomains found in cultured glial cells. Similarly, Bergmann glial cell processes in the cerebellum also contained fine hair-like processes containing IP(3)R2 staining. The IP(3)R2-containing fine terminal branches of astrocyte processes in both brain regions were found juxtaposed to presynaptic terminals containing synaptophysin as well as PSD 95-containing postsynaptic densities. Corpus callosum astrocytes had an elongated morphology with IP(3)R2 studded processes extending along fiber tracts. Our data suggest that PLC-mediated Ca(2+) signaling in astrocytes in rat brain occurs predominantly through IP(3)R2 ion channels. Furthermore, the anatomical arrangement of the terminal astrocytic branches containing IP(3)R2 ensheathing synapses is ideal for supporting glial monitoring of neuronal activity.