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PubMed2006

Electrical coupling, receptive fields, and relative rod/cone inputs of horizontal cells in the tiger salamander retina.

Light responses, dendritic/axonal morphology, receptive field diameters, patterns of dye coupling, and relative rod/cone inputs of various types of horizontal cells (HCs) were studied using intracellular recording and Lucifer yellow/neurobiotin dye injection methods in the flatmount tiger salamander retina. Three physiologically and morphologically distinct types of HC entities were identified. 1) The A-type HCs are somas that do not bear axons, with average (+/-SE) soma diameters of 20.01 +/- 0.59 microm, relatively sparse and thick dendrites, and they resemble the A-type HC in mammals. The average receptive field diameter of these cells is 529.6 +/- 10.87 microm and they receive inputs predominantly from cones. 2) The B-type HCs are broad-field somas that bear thin and long axons, with average soma diameters of 17.67 +/- 0.38 microm, thinner dendrites of higher density, and they resemble the B-type HC in mammals. The average receptive field diameter of these cells is 1,633.55 +/- 37.34 microm and they receive mixed inputs from rods and cones. 3) The B-type HC axon terminals are broad-field, coarse axon terminal processes and they resemble the B-type HC axon terminal in rabbits. The average receptive field diameter of these axon terminals is 1,291.67 +/- 24.02 microm and they receive mixed inputs from rods and cones. All these types of HC are dye-coupled with adjacent HCs of the same type. Additionally, B-type HCs and axon terminals are dye-coupled with subpopulations of bipolar cells whose axon terminals ramify in the proximal half of the inner plexiform layer, raising the possibility that these HCs may send feedforward antagonistic surround responses to depolarizing bipolar cells through electrical synapses.

Ambystoma↗
PubMed2003

Differential synaptic organization of GABAergic bipolar cells and non-GABAergic (glutamatergic) bipolar cells in the tiger salamander retina.

The synaptic organizations of gamma-aminobutyric acid-immunoreactive (GABA-IR, GABAergic) and non-GABA-IR (non-IR, glutamatergic) bipolar cells in salamander retina were compared by postembedding immunoelectron microscopy. A total of 238 presynaptic bipolar cell synapses were studied; 61 were GABA-IR and 177 were non-IR. Both groups were similar in that (1). they made asymmetrical ribbon synapses as well as asymmetrical non-ribbon synapses; (2). they made ribbon synapses at dyads, triads, and monads; and (3). the vast majority of ribbon synapses ( approximately 90%) were with dyads. The differences were that synapses of GABA-IR bipolar cells had a higher proportion of (1). direct contact with ganglion cells, (2). non-ribbon synapses, (3). output to GABA-IR amacrine cells, and (4). output in sublamina a. Overall, the output of GABA-IR ribbons was equally split between amacrine and ganglion cell processes, whereas for non-IR ribbons, it was approximately 2:1 in favor of amacrine cells. The ribbon:non-ribbon synapse ratio was approximately 1.2:1 (33:28) for GABA-IR but approximately 2:1 (118:59) for non-IR terminals. Thus, GABA-IR bipolar cells made more direct contacts with ganglion cells and used a higher proportion of non-ribbon synapses. GABA-IR dyads were more likely to contact GABA-IR amacrine profiles (52% vs. 38%). Finally, GABA-IR ribbon synapses were more common in sublamina a than sublamina b (2:1), whereas non-IR synapses were equally present in sublaminas a and b. This differential targeting of ganglion cells and amacrine cells in the OFF vs. ON layers indicates a difference in the role of bipolar cells in the generation of receptive field properties, depending on whether or not they use GABA as well as glutamate for their transmitter.

Animals↗
PubMed1977

Telencephalic efferents of the tiger salamander Ambystoma tigrinum tigrinum (Green).

The efferent projections of the telencephalon in the tiger salamander were examined by the Nauta and Fink-Heimer methods following unilateral hemispherectomies, rostral hemispheric ablations and pallial lesions. The cerebral hemisphere connects with most areas of the contralateral hemisphere via the pallial, anterior and habenular commissures. The descending fibers travel in the medial and lateral forebrain bundles and in the tracts comprising the stria medullaris. Degenerating fibers and terminals were present throughout the diencephalon but were more abundant ipsilaterally. Fibers reach the pretectum and optic tectum via dorsal and ventral pathways. There is a heavy projection to the midbrain tegmentum and a sparse projection to the tectum via the ipsilateral lateral forebrain bundle. This tract continues into the medulla oblongata and the cervical spinal cord. Rostral and dorsal hemispheric ablations revealed that the majority of fibers forming the olfacto-peduncular tract originate in the ventral, rostral one-third of the hemisphere. It was also determined that the majority of the descending efferent fibers located in the lateral forebrain bundle originate from the caudal lateral hemispheric wall, and that these fibers form connections characteristic of mammalian corticofugal and striatofugal systems. The cytoarchitecture and connections of the caudal lateral hemispheric wall suggest that it is homologous to parts of motor isocortex and amygdala of amniotes.

Ambystoma↗
PubMed1985

The structure of the brainstem and cervical spinal cord in lungless salamanders (family plethodontidae) and its relation to feeding.

We present an HRP study of the sensory tracts and motor nuclei associated with feeding (especially use of the tongue) in plethodontid salamanders (mainly Batrachoseps attenuatus, Bolitoglossa subpalmata, Desmognathus ochrophaeus, Eurycea bislineata, and Plethodon jordani). The nerves studied are VII (ramus hyomandibularis only), IX, X, XI, the first spinal nerve (hypoglossus), and the second spinal nerve. Two types of sensory projections are universally found in the brainstem: superficial somatosensory projections of VII, IX, and X, and deeper visceral sensory projections of IX and X to the fasciculus soltarius. The first spinal nerve and the spinal accessory nerve (XI) have no sensory projections, but the second spinal nerve has typical projections along the dorsal funiculus of the spinal cord. The motor nuclei of VII ramus hyomandibularis, IX, and X form a combined nucleus situated at the level of the IX/X root complex. The nucleus of the first spinal nerve is well separated from the combined nucleus and is situated rostral and caudal to the obex. The rostral part of the motor nucleus of the second spinal modestly overlaps that of the first. The motor nucleus of the spinal accessory nerve is more or less restricted to the region of the second spinal nerve. Its fibers leave the brain through the last root of the IX/X complex and the related ganglion. Bolitoglossine and nonbolitoglossine differ in the architecture of the spinal nuclei. Two distinct types of motor neurons occur in spinal nuclei of nonbolitoglossine species--some of those with tongue projection--but only one type is found among the tongue-projecting bolitoglossine group.

Animals↗
PubMed1985

Localization of enkephalin-like immunoreactive amacrine cells in the larval tiger salamander retina: a light and electron microscopic study.

Immunohistochemistry was utilized to examine the light and electron microscopic localization of enkephalin-like (enk) immunoreactive amacrine cells in the larval tiger salamander retina. The vast majority of enk-immunoreactive cells were typical amacrine cells whose round or oval cell bodies (14-16 microns) were situated in the innermost cell row of the inner nuclear layer. A relatively small number of enk-stained oval cell bodies (14-22 microns) were located in the ganglion cell layer and were designated as those of displaced amacrine cells. Enkephalin immunostaining was observed in the inner plexiform layer as a fine plexus in sublamina 1 and as a dense network of fibers in sublamina 5. In both the center and periphery of the retina the density of typical enk-amacrine cells was determined to be 250 +/- 16.36 cells per mm2 surface area of the retina. At the ultrastructural level typical enk-stained amacrine cells possessed a round, indented nuclear membrane. Enk-immunoreactive processes sometimes contained dense-core vesicles (60-115 nm) in addition to a rather homogeneous population of small, round, agranular synaptic vesicles (25-35 nm). In sublamina 1 the processes of enk-amacrine cells were presynaptic to amacrine and bipolar cells. They also contacted processes devoid of synaptic vesicles which possibly arise from ganglion cells. As the postsynaptic element in sublamina 1, they received synaptic input from amacrine cells. In sublamina 5 the processes of enk-amacrine cells were presynaptic to amacrine cells, bipolar cells, and the somas of cells situated in the ganglion cell layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Ambystoma↗
PubMed1986

Neuropeptide-like immunoreactive cells in the retina of the larval tiger salamander: attention to the symmetry of dendritic projections.

Light microscopic immunocytochemistry was used to study the morphology of cells that showed immunoreactivity (IR) to antisera against substance P (SP), glucagon (GLU), met enkephalin (ENK), and somatostatin (SS) in the retina of the larval tiger salamander. Both vertical sections and retinal whole mounts were studied. All four antisera labeled amacrine cells in the inner nuclear layer and cells in the ganglion cell layer (GCL). GLU-IR cells had processes stratified throughout the inner plexiform layer (IPL), whereas the other three types had bistratified projections in laminae 1 and 5, with lamina 5 being broader and more dense. Two types of SS-IR amacrine cell were observed. As seen in retinal whole mount, most GLU-IR and ENK-IR amacrine cells had processes that were symmetrically distributed about the soma, whereas processes of SS- and SP-IR amacrine cells were markedly asymmetrical. The dendritic fields of SP-IR amacrine cells were selectively oriented toward the periphery of the retina in the nasal, temporal, and dorsal areas. Immunoreactive cells in the GCL had fine projections into the IPL and in addition gave rise to two large oriented processes proximal to the soma that projected in opposite directions for 100-200 microns. The oriented processes often showed further branching; they appeared to be along the radiated lines from the optic disc but did not enter it. The implications of the selectively oriented processes of SP-IR amacrine cells and cells in the GCL are discussed.

Ambystoma↗
PubMed1986

Morphology of physiologically identified bipolar cells in the retina of the tiger salamander, Ambystoma tigrinum.

Intracellular recordings of the light responses of bipolar cells were made in the isolated perfused retina of the larval tiger salamander by microelectrodes filled with a 1% solution of the enzyme horseradish peroxidase (HRP). Two classes of bipolar cell were identified in terms of their responses to luminous spots and annuli centered upon their receptive fields: on-center cells, which depolarized in response to a centered spot and hyperpolarized in response to a concentric annulus, and off-center cells, which responded with the opposite polarities. Physiologically identified cells were labelled with HRP by iontophoretic injection and prepared for light microscopy. Examples of each of the three classes of bipolar cell described in Cajal's study of the amphibian retina were found amongst the labelled cells. The only morphological feature found to correlate with the physiological classification was the stratum at which the axon of the cell arborized in the inner plexiform layer. The axons of on-center cells arborized in the more proximal sublamina b, while those of off-center cells arborized in the more distal sublamina a. This is consistent with earlier findings in the retinae of the cat and the carp.

Ambystoma↗
PubMed1988

Localization of tyrosine-hydroxylase-like-immunoreactive amacrine cells in the larval tiger salamander retina.

Immunocytochemistry was used to localize the populations of tyrosine-hydroxylase-like (TH)-immunoreactive cells in the tiger salamander retina. Ninety percent of these cells possessed somas that were situated in the innermost cell row of the inner nuclear layer and were classified as amacrine cells. Ten percent of TH-immunoreactive somas were located in the ganglion cell layer and were tentatively designated as those of displaced amacrine cells. The processes of TH-immunoreactive cells ramified most heavily in sublayer 1 of the inner plexiform layer, while a relatively small number of TH-labelled processes distributed in sublayers 3 and 5. Less than 1% of TH-immunoreactive cells in the amacrine cell layer exhibited a short process of somal origin that extended distally toward the outer plexiform layer. However, these processes did not cross the whole of the inner nuclear layer, and no immunolabelling was observed in the outer plexiform layer. An examination of retinal whole-mounts revealed that TH-immunoreactive amacrine and displaced amacrine cells were distributed throughout the center and periphery of the retina. The density of TH-immunolabelled amacrine cells was calculated to be 49 +/- 13 (mean +/- standard error) cells per mm2. The vast majority of TH-immunoreactive amacrine and displaced amacrine cells exhibited a stellate appearance and gave rise to three or more primary dendrites. A few TH-amacrine and displaced amacrine cells possessed two primary dendrites that emerged from opposite sides of their somas. The processes of TH-immunoreactive cells were generally poorly branched and varicose with terminal branches sometimes appearing thin and beaded. Because some TH-immunolabelled processes were very long, there was considerable overlap between the dendritic fields of neighboring TH-cells. Lastly, individual TH-immunoreactive amacrine and displaced amacrine cells were often observed in whole-mounts to provide processes that ramified at more than one level of the inner plexiform layer.

Ambystoma↗
PubMed1988

Light microscopic localization of putative glycinergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographical methods.

Putative glycinergic neurons in the larval tiger salamander retina were localized by a comparative analysis of high affinity 3H-glycine uptake and glycine-like immunoreactivity (Gly-IR) at the light microscopic level. Commonly labeled neurons include at least three types of amacrine cell (Type IAd, Type IAb, Type IIAd; distinguished by soma location and dendritic ramification), cell bodies in the ganglion cell layer (GCL), and rarely observed Type II (inner) bipolar cells. With the increased resolution provided by Gly-IR, we identified a Type IAa amacrine cell, two types of Type IIAd amacrine cells, and Gly-IR interplexiform cells. Gly-IR axons in longitudinal sections of the optic nerve indicate the presence of Gly-IR ganglion cells. The percentage of labeled somas in the inner nuclear layer (INL) compared to all cells in each layer was similar for the two methods: 30-40% in INL 2 (middle layer of somas), 30-40% in INL 3 (inner layer of somas), and about 5% in the GCL. Labeled processes were found throughout the full thickness of the inner plexiform layer (IPL), but with a much denser band in the proximal half (sublamina b). The only major difference between the two methods (3H-glycine uptake vs. Gly-IR) was that Type I (outer) bipolar cells were labeled only by 3H-glycine uptake; these cells were more lightly labeled with silver grains than cell bodies in either INL 2 or INL 3. Postembed labeling of 1 micron Durcupan plastic sections for Gly-IR showed the same pattern, but with much higher resolution, as obtained with 10 micron cryostat sections. This study indicates extensive colocalization of labeling by both probes in INL 2, INL 3, the IPL, and the GCL. We conclude that Gly-IR can serve as a valid and reliable marker for glycine-containing neurons in this retina and suggest that glycine serves as a transmitter for several morphologically distinct types of amacrine cell, an interplexiform cell, and perhaps a small percentage of Type II bipolar cells and ganglion cells.

Animals↗
PubMed1988

Localization of putative GABAergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographic methods.

Putative GABAergic neurons in the larval tiger salamander retina were localized by a comparative analysis of glutamate decarboxylase immunoreactivity (GAD-IR), GABA-like immunoreactivity (GABA-IR), and high-affinity 3H-GABA uptake at the light microscopical level. Preliminary data showed that all GAD-IR neurons were double labeled for GABA-IR. However, because the weak somatic labeling with GAD-IR, we could not determine if the converse were true. Neurons commonly labeled with GABA-IR and 3H-GABA uptake include horizontal cells, type I (outer) and type II (inner) bipolar cells, type I (inner) and type II (outer) amacrine cells, and cell bodies in the ganglion cell layer (GCL). In addition, interplexiform cells were identified with GABA-IR. The presence of GABA-IR ganglion cells was indicated by GABA-IR fibers in the optic fiber layer and optic nerve as well as by a GABA-IR cell in the GCL that included a labeled axon. The percentage of labeled somas in the inner nuclear layer (INL) compared to all cells in each layer was similar for the two methods: 30% in INL 1 (outer layer of somas), 15% in INL 2 (middle layer), 43-52% in INL 3 (inner layer), and about 21-26% in the GCL. Labeled processes were found in three bands in the inner plexiform layer, with the densest band located in the most proximal part. Postembedding labeling of 1-micron Durcupan resin sections for GABA-IR showed the same general pattern as obtained with 10-microns cryostat sections, with additional staining, however, of type II (inner) bipolar cell Landolt's clubs. Extensive colocalization of labeling was indicated, and we conclude that GABA-IR can serve as a valid and reliable marker for GABA-containing neurons in this retina and suggest that GABA serves as a transmitter for horizontal cells, several types of amacrine cell, a type of interplexiform cell, and perhaps a small percentage of type I and type II bipolar cells and ganglion cells.

Animals↗
PubMed1989

Localization of serotoninlike-immunoreactive amacrine cells in the larval tiger salamander retina.

Light microscopic immunocytochemistry was used to study the populations of serotoninlike-immunoreactive cells in the larval tiger salamander retina. Of 1,135 serotonin-immunostained cells observed in transverse cryosections, 87% were identified as amacrine cells, whereas 13% were tentatively designated as displaced amacrine cells. The somas of the vast majority of serotonin-amacrine cells were situated in the innermost cell row of the inner nuclear layer. Only a few serotonin-immunostained amacrine cell somas were observed in the second row of cells from the inner nuclear layer. Serotonin-immunoreactive processes generally appeared as a diffuse plexus distributed evenly throughout all levels of the inner plexiform layer. As determined in whole-mount preparations, serotonin-amacrine cells were divisible into two populations on the basis of the diameters of their somas. Large cells (45%) ranged from 16 to 19 microns in diameter with the vast majority measuring 17-18 microns. Smaller and sometimes less intensely stained cells ranged from 14 to 16 microns in diameter with the large majority measuring 15 microns. The diameters of serotonin-displaced amacrine cells ranged from 19 to 22 microns with the large majority measuring 20 microns in diameter. An examination of whole-mount retinas revealed that serotonin-immunoreactive amacrine and displaced amacrine cells were distributed throughout the center and the periphery of the retina. The density of serotonin-amacrine cells (large and small combined) was calculated to be 173 +/- 4.5 (mean +/- standard error) cells per mm2.

Animals↗
PubMed1990

Morphology and distribution of the glossopharyngeal nerve afferent and efferent neurons in the Mexican salamander, axolotl: a cobaltic-lysine study.

Cobaltic-lysine complex was used to label the afferent and efferent components of the glossopharyngeal nerve in the ganglion and brainstem of the Mexican salamander, axolotl (Ambystoma mexicanum). The distribution of afferent cell bodies in the combined glossopharyngeal-vagus ganglion (the IX-X ganglion) was reconstructed from serial sections, and the sizes of the cell bodies were measured. The central projection of afferents and the location of efferent cell bodies were determined by the tracer. The afferent cell bodies in the ganglion were medium-sized (ca. 25 microns). Cell bodies with a single process were seen. The ganglion was not clearly divided into superior and inferior ganglia, as is observed in mammals and frogs, but comprised a single ganglion. Labelled cells were diffusely distributed in the rostral part of the IX-X ganglion. A few labelled cells also were seen in the caudal part, where the vagus nerve fibers and cell bodies were mainly distributed. Double labellings of the glossopharyngeal and vagus nerves with HRP and cobaltic-lysine demonstrated that the ganglion cells of each nerve are not clearly separated in the IX-X ganglion. In the brainstem, the majority of afferent fibers formed thick ascending and descending limbs in the solitary fasciculus. The remaining afferent fibers formed a thin bundle in the spinal tract of the trigeminal nerve, which had a short ascending limb and a long descending limb. These two bundles had terminal areas in the ipsilateral brainstem: in the dorsal gray matter for the solitary fasciculus and in the lateral funiculus for the spinal tract of the trigeminal nerve, respectively. The cell bodies of the efferent neurons possessed developed dendritic arborizations in the ventrolateral white matter, and formed a longitudinal cell column in the ventrolateral margin of the gray matter. Thus, the glossopharyngeal nerve system in the axolotl assumes a primordial form in its ganglions, but its topographical organization in the brainstem is basically similar to that in anurans.

Afferent Pathways↗
PubMed1992

Distribution of immunoreactivity for gamma-aminobutyric acid in the salamander olfactory bulb.

Intrinsic neurons provide inhibitory synaptic input to mitral (and tufted) output cells within several laminae of the olfactory bulb. In rodents, the two main types of intrinsic neurons are granule and periglomerular cells, both of which contain gamma-aminobutyric acid (GABA). In the present study, immunocytochemical techniques were used to determine whether intrinsic neurons in the salamander olfactory bulb might also contain GABA. With the aid of two antisera to different GABA-conjugates, immunoreactivity for GABA was localized within the olfactory bulb laminae. In the glomerular layer, periglomerular cells, which were strongly immunoreactive, were concentrated in clusters along the border with the olfactory nerve layer. Dendrites of the cells encircled nearby glomeruli and were presumably a primary source of intraglomerular processes that were also stained. In the subglomerular region and external plexiform layer, relatively few immunoreactive cells were observed, most of which appeared to be periglomerular and tufted cell types with glomerular dendrites. Throughout the external plexiform and mitral cell layers, however, a dense matrix of spiny processes and puncta was stained, outlining large, unstained dendrites derived from the large, unstained cell bodies of mitral cells. The spiny processes and puncta appeared to be derived from granule cells, which were the most abundant immunoreactive cells in the bulb. Granule cell bodies filled the granule cell layer. In tissue fixed with 0.1-0.2% glutaraldehyde, staining in the olfactory bulb laminae was blocked by preadsorption of the two antisera with glutaraldehyde-conjugated GABA-bovine serum albumin. The staining therefore appeared to be specific for fixed GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Ambystoma↗
PubMed1995

Nitric oxide synthase in tiger salamander retina.

Previous studies have indicated that nitric oxide, a labile freely diffusible biological messenger synthesized by nitric oxide synthase, may modulate light transduction and signal transmission in the retina. In the present work, the large size of retinal cells in tiger salamander (Ambystoma tigrinum) allowed the utilization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and nitric oxide synthase immunocytochemistry to delineate the cell-specific intracellular localization of nitric oxide synthase. NADPH-diaphorase activity was highly concentrated in the outer retina, in rod and cone inner segment ellipsoids, and between and adjacent to the photoreceptor cell bodies in the outer nuclear layer. Examination of enzymatically isolated retinal cells indicated that outer nuclear layer NADPH-diaphorase activity was localized to the distal processes of the retinal glial (Müller) cells and to putative bipolar cell Landolt clubs. Less intense NADPH-diaphorase activity was seen in the photoreceptor inner segment myoid region, in a small number of inner nuclear layer cells, in cap-like configurations at the distal poles of cells in the ganglion cell layer and surrounding ganglion cell layer somata, and in punctate form within both plexiform layers, the pigment epithelium, and the optic nerve. Nitric oxide synthase-like immunoreactivity was similarly localized, but was also concentrated along a thin sublamina centered within the inner plexiform layer. The potential for nitric oxide generation at multiple retinal sites suggests that this molecule may play a number of roles in the processing of visual information in the retina.

Animals↗
PubMed1977

Position discrimination in the salamander, Ambystoma tigrinum.

Twenty larval tiger salamanders (Ambystoma tigrinum) were collected from a playa lake in West Texas and maintained in the laboratory for 7 days prior to the beginning of position training. Ten subjects in the larval state were trained on a position problem in a T-maze for 5 trials per day to a criterion of 80% correct responses, using a self-correction procedure. Following metamorphosis to the adult stage, these subjects were tested for retention. The remaining 10 subjects were allowed to metamorphose prior to training on the position problem. The results indicate reliably faster learning in the adult (terrestrial) subjects, and reliable retention of the response following metamorphosis. These results, taken in conjunction with informal observation of the behavior of the subjects in the experimental environment, suggest that the performance of the adult subjects was a result of their better perceptual apparatus.

Animals↗
PubMed2003

Mitochondrial localization of glucocortocoid receptor in glial (Müller) cells in the salamander retina.

Glucocorticoid hormones regulate the transcription of nuclear genes by way of their receptors. In addition, these hormones modulate mitochondrial gene transcription by mechanisms that remain poorly understood. Using immunofluorescence labeling in isolated Müller and photoreceptor cells and in intact salamander retina, we found that the glucocorticoid receptor (GR) is localized in both cell types. Confocal laser scanning microscopy and double staining with cytochrome oxidase (COX) showed that GR is localized in the mitochondria of Müller cells, but not in the mitochondria of photoreceptors. GR also colocalizes with glutamine synthetase (GS) in the cytoplasm of Müller cells. GR is also localized in the microvilli of the distal process of Müller cells and in the synaptic terminal of photoreceptors. Pre-incubation of Müller cells with 1 microM dexamethasone (DEX) for 7 h led to greater than 50% inhibition of the glutamate-induced increase in mitochondrial NADH. This late effect of glucocorticoids on glutamate metabolism could be ascribed, in part, to a direct action of steroid hormones on mitochondrial metabolism.

Ambystoma↗
PubMed2005

High-voltage-activated calcium channels in Muller cells acutely isolated from tiger salamander retina.

Muller cells mediate retinal function by stabilizing the ionic environment and signal glial network activity via calcium waves. Using whole-cell patch clamp recording, we describe a high-voltage-activated, slowly inactivating Ca channel current in isolated salamander Muller cells that has unusual pharmacological properties. The Ca channel current has an activation midpoint of approximately -8 mV and an inactivation midpoint of approximately -26 mV in 10 mM Ba2+. The time constant for inactivation is approximately 380 ms at potentials positive to zero. The current is blocked by Cd2+ with an EC50 of <100 nM. nisoldipine (10 microM) blocks approximately 50%, while nifedipine (1 microM), diltiazem (20 microM), and verapamil (50 microM) each block one-third of the current. In contrast to its typical actions, BayK 8644 blocks the current by approximately 25%. Blockers of other Ca channel subtypes were also tested: omega-agatoxin IVA (200 nM) blocked only 13% of the Ca channel current, while omega-conotoxin GVIA (1 microM) blocked 84% of the current. Immnohistochemistry supported the presence of alpha1A, alpha1B, alpha1C, and alpha1D Ca channel subunits. Mapping of dihydropyridine-binding sites with DM-BODIPY revealed a distribution of channels over the entire membrane of the Muller cell with a higher density at the apical region. Overall, these observations suggest either the presence of a mix of L- and N-type Ca channels or a single, unconventional HVA Ca channel subtype sharing L- and N-type Ca channel characteristics.

Ambystoma↗
PubMed2006

Calcium-activated chloride channels in müller cells acutely isolated from tiger salamander retina.

Ca(2+)-activated chloride channels were identified with whole-cell patch-clamp recording techniques in salamander retinal Müller cells. Cl(Ca) channels were activated by membrane depolarizations that elicited Ca2+ influx or the application of the Ca2+ ionophore, ionomycin. The Ca channel blocker, Cd2+, abolished the Cl(Ca) channel tail currents. Increasing the duration of the depolarizing pulse resulted in enhancement of the Cl(Ca) channel tail current. Repetitive depolarizations with rapid pulses to +20 mV produced a buildup of I(Cl(Ca)), which reversed at 0 mV in symmetrical [Cl-] and at -40 mV when intracellular [Cl-] was reduced to 10% of the external concentration. I(Cl(Ca)) was blocked by the Cl channel blocker niflumic acid, while niflumic acid had no effect on voltage-gated Ca channels. These results offer the first demonstration of Cl(Ca) channels in a nonastrocytic glial cell and expand our understanding of the functional capacities of retinal glial cells.

Ambystoma↗