Immunocytochemical localization of polyamines in the tiger salamander retina

The polyamines spermine and spermidine are present in neural tissue, but their functions there are not well understood. Recent work suggests that the NMDA subtype of glutamate receptors, other glutamate receptor subtypes, and certain K(+)-channels, are neural targets for polyamines. To better understand the neuron-specific roles of polyamines, we have developed antibodies that interact with spermine and spermidine in aldehyde-fixed tissue and used these antibodies in immunocytochemical studies to determine the cellular localization of these polyamines in the tiger salamander retina. The affinity-purified, polyclonal antibodies were highly specific for spermine and spermidine, exhibiting < 1% cross reactivity with putrescine, and virtually no cross-reactivity with GABA, arginine, lysine, or glutaraldehyde. Polyamine labeling was most abundant in cells in the inner half of the inner nuclear layer and in the ganglion cell layer. Some cells in the outer half of the inner nuclear layer are labeled, and there was some labeling in both synaptic layers. Double-labeling experiments indicated (1) all GABAergic amacrine cells were polyamine-positive; and (2) all ganglion cells (identified by back-filling after microinjections of rhodamine in the optic nerve) were polyamine-positive. These results are consistent with a role for polyamines as modulators of NMDA receptor function and channel function in the inner retina.     

Immunocytochemical localization of GABA and glycine in amacrine and displaced amacrine cells of macaque monkey retina

Recent studies have varied widely in the percentages of GABA- and glycine-immunoreactive (GABA+, GLY+) amacrines reported for primate retina. We compared the distributions of GABA+ and GLY+ amacrines and displaced amacrines at seven locations along the horizontal meridian of macaque retina using postembedding immunogold labeling with silver intensification. The percentage of GABA+ amacrine profiles was higher in central retina (50-55%) than peripheral retina (30-40%), whereas the percentage of GLY+ amacrine profiles did not vary much with eccentricity (52-57%). GABA and glycine were colocalized in 5-20% of amacrines, depending on the eccentricity, whereas 5-30% of amacrines were not immunoreactive for either neurotransmitter. GABA+ amacrines were slightly larger than GLY+ amacrines or Müller cells. In the ganglion cell layer, 5-20% of neurons were labeled for either GABA or glycine and were identified as displaced amacrines. Of these, 53% were GABA+ only, 11% were GLY+ only, and 37% were double-labeled. A few large, very lightly labeled GABA+ cells were identified as ganglion cells. Other features that varied with eccentricity included the linear density of GABA+ and GLY+ amacrines, and the ratio of amacrines to Müller cells.     

Immunocytochemical and histochemical localization of nitric oxide synthase in the turtle retina

Recent interest in nitric oxide and its relationship to cGMP has produced many attempts to anatomically localize the enzyme synthesizing nitric oxide, nitric oxide synthase. In the retina, numerous previous studies have used the NADPH-diaphorase enzyme activity of nitric oxide synthase as a histochemical method to localize nitric oxide synthase. However, all NADPH-diaphorase activity is not necessarily nitric oxide synthase, because several enzymes have similar biochemical activity. Additionally, various histochemical methods have been used to demonstrate NADPH-diaphorase activity, which makes comparisons between studies difficult. The purpose of this study was twofold. First, we wanted to examine the histochemical labeling of NADPH-diaphorase in the turtle retina to allow comparisons to previous studies. Second, we wanted to compare the histochemical localization of NADPH-diaphorase activity to the immunocytochemical localization of nitric oxide synthase in the turtle retina. Our histochemical localization of NADPH-diaphorase activity and our localization of nitric oxide synthase-like immunoreactivity in the turtle retina both produced similar results. Both the histochemistry and immunocytochemistry consistently labeled photoreceptor inner segments, at least three amacrine cell types, and processes in the inner plexiform layer. In optimized double-labeled preparations, all cells with NADPH-diaphorase activity were also positive for nitric oxide synthase-like immunoreactivity, although some somata in the ganglion cell layer only had nitric oxide synthase-like immunoreactivity. The immunocytochemical localization of nitric oxide synthase in photoreceptors, amacrine cells, and putative ganglion cells indicates that nitric oxide may function at several levels of visual processing in the turtle retina.     

Dysplastic nitrergic neurons in the rectum of a patient with rectal ectasia

We present a histochemical study of resected colon from a 13-year-old boy diagnosed with rectal ectasia. Laminar preparations and sectioned tissue of rectum were assayed histochemically for nitric oxide (NO) synthase activity by reducing nitro blue tetrazolium salt in the presence of the cofactor NADPH. Tissue preparations displayed intensely labelled neurons and fibers throughout the gut wall. Laminar preparations of Meissner's plexus showed a hyperplasia of ganglia and NO-related neurons throughout the length of the resected rectum compared with normal bowel. Sectioned tissue of the Auerbach's plexus demonstrated a normal number of ganglia and NO-related neurons. As well, the ectatic bowel showed a proliferation of nerve fibers in keeping with the degree of circular smooth muscle hypertrophy. This proliferation may represent a reactive phenomenon secondary to the functional obstruction. The NO histochemical technique may form the basis of further investigations in defining the cause of this functional obstruction.     

Microtubule transport from the cell body into the axons of growing neurons

The present studies test the hypothesis that microtubules (MTs) are transported from the cell body into the axons of growing neurons. Dissociated sympathetic neurons were cultured using conditions that allow us to control the initiation of axon outgrowth. Neurons were injected with biotin-labeled tubulin (Bt-tub) and then stimulated to extend axons. The newly formed axons were then examined using immunofluorescence procedures for MTs with or without Bt-tub. Because the Bt-tub is fully assembly competent, all MTs that assemble after injection will contain Bt-tub. However, MTs that exist in the neuron at the time of injection and persist during the subsequent incubation will not contain Bt-tub. Because the neurons were injected before extending axons, MTs without Bt-tub are initially localized to the cell body. We specifically determined whether these MTs appeared in the newly formed axon. Such a result can only be explained by the transport of these MTs from their initial location in the cell body into the axon. The newly formed axons of many neurons contained MTs both with and without Bt-tub. MTs without Bt-tub were detected all along the axon and in some neurons represented a substantial portion of the total polymer in the proximal and middle regions of the axon. These results show that MTs are transported from the cell body into growing axons and that this transport is robust, delivering MTs to all regions of the newly formed axon.     

Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina

Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2+, Brain Res. 664 (1994) 252-256; G.D. Zeevalk, W.J. Nicklas, Action of the anti-ischemic agent ifenprodil on N-methyl-d-aspartate and kainate-mediated excitotoxicity, Brain Res. 522 (1990) 135-139; R. Huba, H.D. Hofmann, Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures, Vis. Neurosci. 6 (1991) 303-314; M. Yamashita, R. Huba, H.D. Hofmann, Early in vitro development of voltage- and transmitter-gated currents in GABAergic amacrine cells, Dev. Brain Res. 82 (1994) 95-102; R. Ientile, S. Pedale, V. Picciurro, V. Macaione, C. Fabiano, S. Macaione, Nitric oxide mediates NMDA-evoked [3H]GABA release from chick retina cells, FEBS Lett. 417 (1997) 345-348; R.C. Kubrusly, M.C. deMello, F.G. deMello, Aspartate as a selective NMDA agonist in cultured cells from the avian retina, Neurochem. Intl. 32 (1998) 47-52] or reduction of GABA in vivo [N.N. Osborn, A.J. Herrera, The effect of experimental ischaemia and excitatory amino acid agonist on the GABA and serotonin immunoreactivities in the rabbit retina, Neurosci. 59 (1994) 1071-1081]. Since the majority of GABAergic synapses in the inner retina are onto both rod and cone bipolar axon terminals [R.G. Pourcho, M.T. Owzcarzak, Distribution of GABA immunoreactivity in the cat retina: A light and electron-microscopic study, Vis. Neurosci. 2 (1989) 425-435], we hypothesize that the NMDA-receptor plays a crucial role in providing feedback inhibition onto rod and cone bipolar cells.     

Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina

Synapse-associated proteins are the scaffold for the selective aggregation of ion channels at synapses; they provide the link to cytoskeletal elements and possibly are involved with the regulation of synaptic efficacy by electrical activity. The localization of the postsynaptic density protein PSD-95 was studied in different mammalian retinae (rat, monkey, and tree shrew) by using immunocytochemical methods. Immunofluorescence for PSD-95 was most prominent in the outer plexiform layer (OPL). The axon terminals of rods and cones, the rod spherules and cone pedicles, were strongly labeled. Electron microscopy, using preembedding immunocytochemistry, showed PSD-95 localized presynaptically within the photoreceptor terminals. Distinct PSD-95 labeling was also present in the inner plexiform layer (IPL). It had a punctate appearance suggesting the synaptic clustering of PSD-95 in the IPL. Electron microscopy showed that PSD-95 was concentrated in processes that were postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for PSD-95. Double-labeling experiments were performed for PSD-95 and for SAP 102 or PSD-93, respectively, two other members of the family of synapse-associated proteins. All three were found to be colocalized in the synaptic hot spots in the IPL. In the OPL, however, PSD-95 and PSD-93 were found presynaptically, whereas SAP 102 was located postsynaptically at photoreceptor synapses. Double-labeling experiments also were performed for PSD-95 and for the NR1 subunit of the NMDA receptor. They were found to be colocalized in synaptic hot spots in the IPL.     

Amacrine cells in Necturus retina: evidence for independent gamma-aminobutyric acid- and glycine-releasing neurons

About one-half of on-off ganglion cells have inhibitory postsynaptic potentials (IPSP's) which are blocked by strychnine, while the remainder have IPSP's which are blocked by picrotoxin or bicuculline. These antagonists do not abolish light activity of the presynaptic inhibitory neuron, the amacrine cell. The existence of separate gamma-aminobutyric acid- and glycine-releasing amacrine cells is implied by these results.     

Adeno-associated virus vector-mediated transgene integration into neurons and other nondividing cell targets

The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration in several types of nondividing cells, including neurons. This novel technique will be highly useful for further delineating the mechanisms underlying AAV-mediated integration, including issues of frequency, site preference, and DNA rearrangement in human as well as animal cells. Results from these studies should be beneficial for the development of the next generation of gene delivery vectors.     

Unique retina cell phenotypes revealed by immunological analysis of recoverin expression in rat retina cells

OBJECTIVE: This study was undertaken to determine the impact of previous cardiac surgery on the presentation, management, and outcome of late dissection of the ascending aorta. PATIENTS AND METHODS: From 1976 to 1998, type A dissection developed in 56 patients with a history of previous cardiac surgery. Interval from first operation to type A dissection was 49 +/- 47 months (0.3-180 months). Previous operations were coronary artery bypass grafting (n = 40), aortic valve replacement (n = 8), and other (n = 8). RESULTS: Type A dissection was acute in 34 patients and chronic in 22. In acute dissection, aortic insufficiency occurred in 50%, malperfusion in 12%, and rupture in 18%; 2 patients (6%) were in hemodynamically unstable condition because of rupture. Of patients with previous coronary bypass grafting, 98% had preoperative coronary angiography. Type A dissection was treated by supracoronary tube graft (84%), Bentall procedure (14%), or local repair (2%). Strategies for managing previous coronary bypass grafting included reimplantation of proximal anastomoses with a button of native aorta (29 patients), interposition graft to pre-existing saphenous vein grafts (9 patients), and new saphenous vein grafts (20 patients). Eight hospital deaths occurred (14%). CONCLUSIONS: We conclude that (1) patients having type A dissection late after cardiac surgery infrequently have cardiac tamponade and hemodynamic collapse; (2) patients with previous coronary bypass grafting require coronary angiography, because operative management must account for pre-existing coronary artery disease; and (3) operative mortality is low, and this may be attributable to preoperative hemodynamic stability, delineation of coronary anatomy in those with previous coronary bypass grafting, and operative treatment of coronary artery disease.     

An analysis of the cross-correlation between ganglion cells in the retina of goldfish

Induction of autoantibodies to serotonin and dopamine in blood serum was demonstrated in a new rat model of experimental depression-like syndrome induced by intraperitoneal injection of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 20 mg/kg daily for 12 days). The level and frequency of detection of antibodies to serotonin within 2 and 3 weeks after MPTP withdrawal did not differ, and the level and frequency of detection of antibodies to dopamine were significantly reduced within 3 weeks as compared with 2 weeks after the MPTP withdrawal. In is suggested that disturbances in neuroimmune interactions play an important part in development of depressive states.     

Immunocytochemical localization of the NPY/PYY Y1 receptor in enteric neurons, endothelial cells, and endocrine-like cells of the rat intestinal tract

Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides that are considered to mediate inhibitory actions on gastrointestinal motility, secretion, and blood flow. Several receptor subtypes for these peptides have been identified and the Y1, Y2, Y4/PP1, Y5, and Y5/PP2/Y2b receptors have been cloned. In this article we report the immunocytochemical localization of the Y1 receptor to myenteric and submucosal nerve cell bodies, endothelial cells, and scattered endocrine-like cells of rat intestinal tract. Moreover, double immunofluorescence demonstrates that subpopulations of the Y1 receptor-positive nerve cell bodies are immunopositive for NPY, vasoactive intestinal polypeptide, and nitric oxide synthase. In part, such co-localizations were made possible by use of peroxidase-mediated deposition of tyramide, which permitted use of antisera derived from the same species. Our observations suggest the existence of multiple neuronal, endothelial, and endocrine target sites for NPY and PYY and that some of the actions of these regulatory peptides can be mediated by vasoactive intestinal peptide and nitric oxide synthase.     

Immunocytochemical demonstration of reduced glutathione in neurons of rat forebrain

Histochemical studies show reduced glutathione (GSH) in neuroglia, whereas immunocytochemistry of glutaraldehyde-fixed tissue reveals GSH also in neurons. Using an antibody suitable for formaldehyde-fixed tissue, we find GSH staining in the cytoplasm of neurons throughout the brain. Staining was prominent in large pyramidal neurons of cerebral cortex, in basal ganglia, and in reticular and ventrobasal thalamic nuclei.     

P19 cells differentiate into glutamatergic and glutamate-responsive neurons in vitro

The neurotransmitter L-glutamate has been associated with a number of developmental events within the central nervous system including synaptogenesis and the refinement of topographically ordered neural maps. As a model for studying such events at the molecular level, we have examined the expression of glutamate and glutamate receptors in neurons that develop from P19 cells in response to retinoids. We report here that many P19-derived neurons do contain glutamate in secretory vesicles and that this glutamate appears to function as a neurotransmitter. The neurotransmitter GABA is also present in these cultures and both glutamate and GABA appeared to co-localize in some neuronal processes. Both neurotransmitters were released from the neurons in response to membrane depolarization. These neurons also express various glutamate receptor subunits including GluR1, GluR4 and NMDAR1 as detected by immunological methods. Using whole-cell patch-clamping, we have recorded spontaneous postsynaptic potentials which increase in both amplitude and frequency with time in culture and which are sensitive to the glutamate antagonist kynurenic acid Thus, P19-derived neurons mature in culture and form electrically active neural networks involving glutamate and glutamate receptors.     

Immunocytochemical localization and description of neurons expressing serotonin2 receptors in the rat brain

Serotonin2 receptors have been implicated in a variety of behavioral and physiological processes, as well as a number of neuropsychiatric disorders. To specify the brain regions and specific cell types possessing serotonin2 receptors, we conducted an immunocytochemical study of the rat brain using a polyclonal serotonin2 receptor antibody. Perfusion-fixed rat brain sections were processed for immunocytochemistry and reactivity was visualized using an immunoperoxidase reaction. Numerous small, round neurons were heavily labeled in the granular and periglomerular regions of the olfactory bulb. Heavy labeling of medium-sized multipolar and bipolar neurons was also seen in olfactory regions of the ventral forebrain, including the anterior olfactory nucleus and olfactory tubercle. Other regions of the basal forebrain exhibiting high levels of immunoreactivity were the nucleus accumbens, ventral pallidum, Islands of Calleja, fundus striatum and endopyriform nucleus. Immunoreactive neurons were also seen in the lateral amygdala. A dense band of small, round cells was stained in layer 2 of pyriform cortex. In neocortex, a very sparse and even distribution of bipolar and multipolar neurons was seen throughout layers II-VI. A much more faintly labeled population of oval cells was observed in the deep layer of retrosplenial and posterior cingulate cortex, and in the granular layer of somatosensory frontoparietal cortex. A moderate number of medium bipolar and multipolar cells were scattered throughout the neostriatum, and a moderate number of pyramidal and pyramidal-like cells were seen in the CA fields of the hippocampus. Diencephalic areas showing immunolabeling included the medial habenula and anterior pretectal nucleus, with less labeling in the ventral lateral geniculate. In the hindbrain, two dense populations of large multipolar cells were heavily labeled in the pedunculopontine and laterodorsal tegmental nuclei, with lesser labeling in the periaqueductal gray, superior colliculus, spinal trigeminal nucleus and nucleus of the solitary tract. Based on the distribution, localization and morphology of immunoreactive neurons in these regions, we hypothesize that subpopulations of serotonin2 containing cells may be GABAergic interneurons or cholinergic neurons. Further, the observed distribution suggests that the physiological effects of serotonin acting through serotonin2 receptors are mediated by a relatively small number of cells in the brain. These observations may have strong functional implications for the pharmacological treatment of certain neuropsychiatric disorders.     

Immunocytochemical localization of aromatase-containing neurons in the rat brain during pre- and postnatal development

This article discusses the existence of both spouse abuse and child abuse within families. Recent research suggests that practitioners have often missed the coexistence of these problems within their caseloads. Practice implications for both domestic violence service providers and child welfare professionals are outlined. Recommendations for changes in assessment procedures, treatment planning, and implementation are made.     

Excitatory synaptic transmission in the inner retina: paired recordings of bipolar cells and neurons of the ganglion cell layer

Properties of glutamatergic synaptic transmission were investigated by simultaneously voltage-clamping a pair of connected bipolar cells and cells in the ganglion cell layer (GLCs) in the newt retinal slice preparation. Activation of the Ca2+ current in a single bipolar cell was essential for evoking the glutamatergic postsynaptic current in the GLC. Depolarization for as short as 15 msec activated both NMDA and non-NMDA receptors. On the other hand, analysis of the spontaneous glutamatergic synaptic currents of GLCs revealed that these currents consisted of mainly non-NMDA receptor activation with little contribution from NMDA receptors. This suggests that non-NMDA receptors of GLCs are clustered in postsynaptic membrane regions immediately beneath the release sites of bipolar cells and that NMDA receptors have lower accessibility to the released transmitter than non-NMDA receptors. Glutamate that is spilled over from the release sites may activate the NMDA receptors. When a prolonged depolarizing pulse was applied to a bipolar cell, the response induced by non-NMDA receptors was limited greatly by their fast desensitization, whereas NMDA receptors were able to produce a maintained response. The relationship between the pulse duration applied to the bipolar cell and the integrated charge of the response evoked in the GLC was almost linear. Therefore, we propose that both non-NMDA and NMDA receptors cooperate to transfer the graded photoresponses of bipolar cells proportionally to GLCs.     

Immunocytochemical localisation of parietal cells and G cells in the developing human stomach

Previous studies on the distribution of parietal cells and G cells in normal adult stomachs have shown that in about 20% of specimens parietal cells extended to the pylorus. This study aimed to examine the distribution of parietal cells and G cells in the body and antrum of the developing human stomach in relation to anatomical landmarks, using histological and immunocytochemical methods. In all 15 fetal stomachs examined, parietal cells extended to the pylorus and expressed intrinsic factor and hydrogen-potassium-ATPase activity from week 13 of gestation. By contrast, in only one of the five infant stomachs did parietal cells extend to the pylorus: this is identical to the distribution in the adult. G cells developed in the antrum from 18 weeks' gestation and their distribution did not differ between the fetal and infant stomachs. These findings indicate that parietal cells disappear from the antrum of the stomach in the third trimester of pregnancy, but this process fails to occur in approximately 20% of the population.     

A quantitative analysis of cells in the ganglion cell layer of the chick retina

This study investigated the organization of cells in the ganglion cell layer (GCL) using Nissl staining, retrograde cell degeneration with axotomy of the optic nerve, and retrograde cell labeling by injections of horseradish peroxidase (HRP) into the optic nerve of chicks (posthatching day 1 and 8, P-1 and P-8). The total number of cells in the GCL was 6.1 x 10(6) (P-1) and 4.9 x 10(6) (P-8), and the cell density was 14,300 cells/mm2 (P-1) and 10,400 cells/ mm2 (P-8) on average. Two high-density areas, the central area (CA) and the dorsal area (DA), were observed in the central and dorsal retinas in both P-1 (22,000 cells/mm2 in CA, 19,000 cells/mm2 in DA) and P-8 chicks (19,000 cells/mm2 in CA, 12,800 cells/mm2 in DA). The cell densities in the temporal periphery (TP) and the nasal (NP) peripheral retinas were 7,800 cells/mm2 and 12,500 cells/mm2, respectively, in P-1 and 5,000 cells/ mm2 and 8,000 cells/mm2, respectively, in P-8 chicks. The cell density in the temporal periphery was 35% (P-8) lower than in the nasal periphery in both P-1 and P-8 chicks. Thirty percent (1.9 x 10(6) cells in P-1) of the total cells in the GCL were resistant to axotomy of the optic nerve. The distribution of the axotomy-resistant cells showed two high-density areas in the central and dorsal retinas, corresponding to the CA (5,800 cells/mm2) and the DA (3,200 cells/mm2). These cells also exhibited a center-peripheral increase (2,200 cells/mm2 in the TP) in P-1 chicks, but the high-density area was not found in the dorsal retina of P-8 chicks. From these data and the HRP study, the number of presumptive ganglion cells in P-8 chicks was estimated to be 4 x 10(6) (8,600 cells/mm2 on average), and the density in each area was 13,500 (CA), 10,200 (DA), and 4,300 (TP) cells/mm2. The peripheral/ center ratios of the density of ganglion cells were significantly different along the nasotemporal and dorsoventral axes. The density of ganglion cells decreased more rapidly toward the temporal periphery (TP/CA ratio: 0.47 in P-1 and 0.32 in P-8) than toward the nasal periphery (NP/CA ratio: 0.67 in P-1 and 0.52 in P-8). In contrast, there was no significant difference in the peripheral/center ratios between the dorsal retina (DP/CA ratio: 0.6 in P-1 and 0.56 in P-8) and ventral retina (VP/CA ratio: 0.58 in P-1 and 0.51 in P-8). A small peak in the density of the presumptive ganglion cells was detected in the dorsal retina of both P-1 chicks (10,800 cells/mm2) and P-8 chicks (10,200 cells/mm2). The HRP-labeled cells were small in the CA (M +/- SD: 35.7 +/- 9.1 microm2) and DA (40.0 +/- 11.3 microm2), and their sizes increased toward the periphery (63.4 +/- 29.7 microm2 in the TP) accompanied by a decrease in the cell density. However, the axotomy-resistant cells did not significantly increase in size toward the peripheral retina (12.2 +/- 2.2 microm2 in the CA, 15.2 +/- 3.2 microm2 in the DA, 15.1 +/- 3.8 microm2 in the TP). The characteristic distribution of ganglion cells could be related to visual behavior based upon the specialization of avian visual fields.     

Modulation of GABAC response by Ca2+ and other divalent cations in horizontal cells of the catfish retina

GABAC responses were recorded in cultured cone-driven horizontal cells from the catfish retina using the patch clamp technique. At a holding potential of -49 mV, a bicuculline-resistant inward current (IGABA) was observed when 10 microM GABA was applied. The amplitude of IGABA increased as the extracellular Ca2+ ([Ca2+]o) was increased. Concentration-response curves of IGABA at 2.5 and 10 mM -Ca2+-o had similar EC50 (3.0 and 3.1 microM) and Hill coefficients (1.54 and 1. 24). However, the maximal response estimated at 10 mM [Ca2+]o was larger than the maximal response at 2.5 mM [Ca2+]o. Increasing Ca influx through voltage-gated Ca channels and the resulting rise in the intracellular Ca2+ concentration had no effects on IGABA. However, IGABA was inhibited by extracellular divalent cations, with the following order of the inhibitory potency: Zn2+ > Ni2+ > Cd2+ > Co2+. The inhibitory action of Zn2+ on the [Ca2+]o-dependent IGABA increase was noncompetitive. The action of [Ca2+]o on IGABA was mimicked by Ba2+ or Sr2+. These results demonstrate that the extracellular domain of GABAC receptors has two functionally distinct binding sites represented by Ca2+ (facilitation) and Zn2+ (inhibition). Since [Ca2+]o and [Zn2+]o change into the opposite direction by light, it seems likely that they modify cooperatively the efficacy of the positive feedback consisting of the GABAC receptor.