Biomedical subjects

S Yazulla

Publications and source records attributed to S Yazulla.

At least 37 records · Page 2Linked to original sources

PubMed1993

GABA and glycine in retinal amacrine cells: combined Golgi impregnation and immunocytochemistry.

Golgi-impregnated amacrine cells in the all-cone lizard retina (Anolis carolinensis) were characterized on the bases of dendritic and somatic criteria. Four major cell categories, comprising 23 types were identified: three non-stratified, 13 monostratified, five bistratified, and two tristratified types. Four of the cell types comprised two to four subtypes based on stratification of their dendrites within the inner plexiform layer (IPL). Golgi impregnation strongly favoured monostratified amacrine cells with cell bodies at the proximal margin of the inner nuclear layer. The neurotransmitter content of each of the 23 amacrine cell types was examined by combined Golgi-immunocytochemistry after morphological classification. Putative neurotransmitters examined included gamma-aminobutyric acid (GABA), glycine (GLY) and aspartate (ASP). Seventeen cell types showed GABA-immunoreactivity (IR), three cell types showed GLY-IR, and four cell types showed neither GABA-IR nor GLY-IR. No cell types showed ASP-IR. Each cell type had a characteristic neurochemical signature, with the exception of one monostratified cell type that showed three different neurochemical signatures. Postembedding immunocytochemistry on conventionally processed retinas confirmed the localization of glutamic acid decarboxylase, the synthetic enzyme for GABA, to cells similar to several of the GABA-IR Golgi-stained types. Postembedding immunocytochemistry for tyrosine hydroxylase (the synthetic enzyme for catecholamines) and GABA on serial sections demonstrated colocalization of GABA and a catecholamine, probably dopamine, in a bistratified amacrine cell type. We conclude that GABA-IR amacrine cell types are more numerous and morphologically heterogeneous than GLY-IR amacrine cells. The morphological heterogeneity and, with one exception, exclusivity of GABA-IR and GLY-IR amacrine cell types indicate that both neurotransmitters play a variety and different functional roles in the lizard inner retina.

Animals↗

PubMed1993

Goldfish bipolar cells and axon terminal patterns: a Golgi study.

The morphology and axon terminal arrangement of Golgi stained goldfish bipolar cells were examined to understand better the organization of bipolar cells in the inner plexiform layer (IPL) of the retina. Fifteen morphological bipolar cell types were identified, representing two major cell classes: mixed input cells that receive input from rod and cone photoreceptors, and cone bipolar cells that receive input from cones only. Mixed input bipolar cells comprised six types, including two new types, characterized by large somas and terminals. The terminals of mixed input bipolar cells terminated strictly within sublamina a or b of the IPL. Cone bipolar cells comprised nine subtypes, including seven new types, characterized by small somas and from one to four small terminal bulbs along the length of the axon, each having a characteristic termination depth in the IPL. The cone bipolar cell system had a complex multilaminar organization of terminals in the IPL, but maintained a high degree of anatomical symmetry about sublamina a and b. Cone bipolar cells could be divided into three groups: cells terminating within sublamina a and having an anatomically symmetrical counterpart terminating in sublamina b; cells with anatomically similar terminals in both sublamina a and b; and cells having no anatomically symmetrical counterpart or having anatomically dissimilar terminals in sublamina a and b. Based on bipolar cell terminal arrangement, we suggest that each bipolar cell type probably has a unique set of synaptic targets in the IPL, and that several bipolar cell types may be involved in functionally equivalent circuits at more than one level in the IPL.

Animals↗

PubMed1993

Immunofluorescent identification of endogenous neurotransmitter content in Golgi-impregnated neurons.

A combined Golgi-impregnation/immunocytochemistry procedure was developed to identify the endogenous neurotransmitter content of morphologically characterized neurons. Golgi-impregnated retinal amacrine cells in the lizard Anolis carolinensis were characterized morphologically in thick resin sections. Cells of interest were remounted, resectioned at 1 micron thickness and subjected to a postembedding immunofluorescence procedure to visualize the amino acid neurotransmitters gamma-aminobutyric acid (GABA) or glycine. Double-labeled cells were identified by opaque Golgi deposits in the cytoplasm under bright-field illumination and nuclear immunofluorescence under ultraviolet illumination. Twenty-seven Golgi-impregnated amacrine cells, exhibiting morphological features of GABA-immunoreactive (GABA-IR) cells, were tested for GABA-IR; 21 showed double labeling. Glycine-IR amacrine cells also were identified using the Golgi/immunocytochemistry procedure. This double-labeling procedure allows rapid assessment of endogenous neurotransmitter content in large samples of morphologically characterized neurons.

Animals↗

PubMed1992

Light-dependent plasticity of the synaptic terminals of Mb bipolar cells in goldfish retina.

We recently described spine-like protrusions (spinules) from the synaptic terminals of mixed rod-cone (Mb) bipolar cells of goldfish retina that invaginated about 5% of the presynaptic amacrine cell processes (Yazulla and Studholme, J Comp Neurol 310:11, 1991). In view of reports of a light/dark dependent-plasticity on the formation of dendritic spinules on goldfish horizontal cells (Raynauld et al., Science 204:1436, 1979; Wagner, J Neurocytol 9:573, 1980), we investigated the possibility that Mb terminal spinules also might show light/dark-dependent plasticity. Retinas were obtained at noon time from light-adapted and 3-hour dark-adapted goldfish and processed for electron microscopy using conventional histological procedures and by preembedding immunocytochemistry to detect protein kinase C immunoreactivity. Two effects of light adaptation on Mb terminal morphology were observed. First, the surface of Mb terminals was significantly more irregular after dark adaptation than light adaptation. With dark adaptation, Mb terminals appeared "amoeboid," with numerous cytoplasmic extensions between the presynaptic processes. Second, spinules were sevenfold more frequent after dark adaptation than light adaptation (8% vs. 1.2% of the presynaptic processes were invaginated by spinules). We suggest that the increased frequency of spinules during dark adaptation is related to an enhancement of synaptic transmission from a minor amacrine cell input when the major input from GABAergic amacrine cells is reduced. Also, the irregular surface of dark-adapted Mb terminals may be related to the reduction of synaptic input during dark adaptation.

Animals↗

PubMed1992

Localization of GABAA receptor subtypes in the tiger salamander retina.

Dry autoradiography was used to determine the distribution of GABAA binding sites in tiger salamander retina. High-affinity binding of [3H]-flunitrazepam [( 3H]-FNZ) was used to localize benzodiazepine receptors (BZR) and [3H]-muscimol was used to localize the GABAA recognition site. Specific [3H]-FNZ binding was present only in the inner retina, primarily in the inner plexiform layer (IPL). Co-incubation with GABA enhanced [3H]-FNZ binding by 20-50%. [3H]-muscimol binding was found throughout the IPL and in the outer plexiform layer (OPL). Mouse monoclonal antibodies 62-3G1 and BD-17, that recognize the GABAA beta 2, beta 3 polypeptides, and BD-24, that recognizes the GABAA alpha 1 polypeptide, did not label either the OPL or IPL, despite numerous variations in the fixation and immunoprocessing methods. GABAA receptor location, as revealed by [3H]-muscimol binding, matches the distribution of presumed GABAergic terminals in the OPL and IPL. We suggest that there are at least two subtypes of GABAA receptor in the tiger salamander retina: one type is present only in the inner retina, primarily in the IPL and is functionally coupled to BZRs; the other type is located in the OPL and is not coupled to the BZRs. Furthermore, GABAA receptors in the tiger salamander retina appear to be of a different epitope than GABAA receptors in numerous other preparations that are recognized by mAbs 62-3G1, BD-17, and BD-24.

Ambystoma↗

PubMed1991

Amacrine cells in the tiger salamander retina: morphology, physiology, and neurotransmitter identification.

Amacrine cells of the vertebrate retina comprise multiple neurochemical types. Yet details of their electrophysiological and morphology properties as they relate to neurotransmitter content are limited. This issue of relating light responsiveness, dendritic projection, and neurotransmitter content has been addressed in the retinal slice preparation of the tiger salamander. Amacrine cells were whole-cell clamped and stained with Lucifer yellow (LY), then processed to determine their immunoreactivity (IR) to GABA, glycine, dopamine or tyrosine hydroxylase (TOH), and glucagon antisera. Widefield, ON-OFF amacrine cells were glycine-IR. The processes of these cells extended laterally in the inner plexiform layer (IPL) from 250-600 microns. They were either multistratified in the IPL or monostratified near the IPL midline. Three multistratified ON-OFF narrowfield glycine-IR cells also were found. Four types of ON amacrine cells were found to be GABA-IR; all types had their processes concentrated in the proximal IPL (sublamina b). Type I cells were narrowfield (approximately 100 microns) with a compact projection. Type II cells were widefield (220-300 microns) with a sparse projection. Type III cells had an asymmetrical projection and varicose processes. Type IV cells were pyriform and monostratified in sublamina b. One narrowfield ON-OFF amacrine cell, with processes broadly distributed in the middle of the IPL, was GABA-IR. This cell appeared similar to an ON-OFF cell that was glycine-IR and may comprise a type in which GABA and glycine colocalize. Another class of amacrine cell, with processes forming a major plexus along the distal border of the IPL and a lesser plexus in the proximal IPL, produced slow responses at light ON and OFF; these cells were dopamine/TOH-IR. A narrowfield class of transient ON-OFF amacrine cell, with processes ramifying throughout both sublaminae a and b of the IPL, were glucagon-IR; these cells appeared to be dye-coupled at the soma. We have shown that, with respect to GABA, glycine, dopamine, and glucagon, salamander amacrine cells fall into rather discrete groups on the basis of ramification patterns in the IPL and responses to photic stimulation. The physiological, structural, and neurochemical diversity of amacrine cells is indicative of multiple and complex roles in retinal processing.

Ambystoma↗

PubMed1991

Glycinergic interplexiform cells make synaptic contact with amacrine cell bodies in goldfish retina.

Recent works utilizing glycine-immunoreactivity (IR) and combined Golgi impregnation and 3H-glycine uptake autoradiography indicate that glycinergic interplexiform cells (IPC) may synapse upon cell bodies in the inner nuclear and ganglion cell layers in fish retina. This possibility was investigated with immunocytochemical techniques using presynaptic and postsynaptic markers for glycinergic neurons: a monoclonal antibody (mAb 7A) against the 93 kDa subunit of the strychnine-sensitive glycine receptor and a polyclonal antiserum against a glycine/BSA conjugate. Synaptic contacts onto the lateral and proximal surfaces of amacrine cell bodies and onto the distal surface of cells in the ganglion cell layer were identified with both probes. The contacts were rare with one contacted amacrine cell/section of 500 linear micron. Serial 1-micron sections were processed alternately for glycine and GABA antisera using postembedding techniques at the light microscopic level. Glycine-IR processes + boutons were apposed to GABA-IR cell bodies in 16 of 17 examples, indicating that the dendro-somatic contacts were onto GABA-immunoreactive amacrine cell bodies. In context of other published morphological data, we suggest that the dendro-somatic synapses were derived from glycinergic IPCs. Glycinergic IPCs receive input from GABAergic horizontal cells and, via a shunt conductance produced by the dendro-somatic contacts, may be involved in controlling the sensitivity, temporal, or spatial properties of amacrine cell responses to large field illumination.

Animals↗

PubMed1991

Glycine-receptor immunoreactivity in retinal bipolar cells is postsynaptic to glycinergic and GABAergic amacrine cell synapses.

Glycinergic innervation of the synaptic terminals of mixed rod-cone bipolar cells in the goldfish retina was investigated by electron microscopical immunocytochemistry with presynaptic and postsynaptic markers for glycinergic neurons: a monoclonal antibody (mAb 7A) against the 93 kDa subunit of the strychnine-sensitive glycine receptor and polyclonal antisera against a glycine/BSA conjugate. Conventional "glycinergic" synaptic contacts, made by amacrine cell processes, accounted for 7-10% of the input to the bipolar cell terminals, whether determined by glycine receptor immunoreactivity (GlyR-IR) or glycine-IR. In addition to the conventional synapses, the large bipolar cell terminals in the proximal inner plexiform layer (type Mb) gave rise to spinules (spine-like protrusions) that invaginated into presynaptic amacrine cell processes. Although 85% of the spinules were GlyR-IR, no spinules were postsynaptic to glycine-IR processes; yet 86% of the spinules were postsynaptic to GAD-IR processes, suggesting that the GlyR-IR spinules were postsynaptic to GABAergic terminals. Furthermore, a single amacrine cell process could make two synapses with an Mb terminal: a GlyR-IR contact onto a spinule and a conventional synapse that was not GlyR-IR. We suggest that glycinergic innervation of bipolar cell terminals involves conventional glycinergic synapses as well as an unconventional situation in which GABA and glycine may interact in as yet undetermined manner, perhaps by potentiation.

Animals↗

PubMed1991

The mismatch problem for GABAergic amacrine cells in goldfish retina: resolution and other issues.

GABAergic neurons in the vertebrate retina have received intensive study. Yet there are several notable examples of a "mismatch" among the cytochemical markers used to identify GABAergic neurons. The mismatch between [3H]GABA uptake autoradiography and all other indicators of GABAergic neurons as they pertain to amacrine cells in goldfish retina is examined in this overview. The discrepancies can be accounted for largely by barriers to diffusion presented by significant GABA uptake sinks at the inner and outer margins of the retina and by the differential subcellular distribution of the various markers for GABAergic neurons. Also, conditions producing a redistribution of [3H]-GABA and endogenous GABA stores within the retina are described and discussed.

4-Aminobutyrate Transaminase↗

PubMed1990

Glycinergic contacts in the outer plexiform layer of the Xenopus laevis retina characterized by antibodies to glycine, GABA and glycine receptors.

Electrophysiological experiments have predicted a direct synaptic input from glycinergic interplexiform cells (IPCs) to GABAergic horizontal cells in the Xenopus retina. However, previous ultrastructural studies failed to demonstrate this input. Here, we used three immunocytochemical approaches to investigate this issue. First, double-label postembedding immunocytochemistry with GABA- and glycine-like immunoreactivity (GABA-LI and glycine-LI) was used to study possible interactions of the glycinergic IPC with GABAergic horizontal cells. Processes postsynaptic to glycine-LI IPC terminals in the outer plexiform layer (OPL) fell into two groups, small microtubule-filled processes and larger electron-lucent processes with sparse microtubules and occasional mitochondria. In no case did we find glycine-LI synapses onto GABA-LI cells or processes. Second, pre-embedding immunocytochemistry was used to label GABA-LI cells and processes in the OPL. GABA-LI was sparse in horizontal cell axons and more intense in horizontal cell somas and in small processes. In agreement with our first set of experiments, GABA-LI profiles did not receive input from conventional synapses. Third, we localized glycine-receptor-like immunoreactivity (GlyR-LI) to several types of apparent synapses in the OPL. As expected, it was found at IPC synapses. Unexpectedly, GlyR-LI was also subsynaptic at photoreceptor synapses onto second order neurons, both at ribbon and basal junction type synapses. At least some of the GlyR-LI photoreceptor synapses were from cones. Also, GlyR-LI was apposed to photoreceptors and to unidentified small diameter processes, where no other indication of synaptic input was evident. Because glycine-LI is not found in photoreceptors, we suggest that glycine receptors at photoreceptor synapses are stimulated by glycine that diffuses from other sites, possibly from IPCs. This interpretation is consistent with available physiological studies of glycinergic effects in this retina.

Animals↗

PubMed1990

Multiple subtypes of glycine-immunoreactive neurons in the goldfish retina: single- and double-label studies.

The glycinergic system in goldfish retina was studied by immunocytochemical localization of glycine antiserum at the light-microscopical level. Numerous amacrine cells, a type of interplexiform cell, interstitial cell, and displaced amacrine cell were glycine-immunoreactive (IR). Amacrine cells, accounting for 97% of the glycine-IR neurons, were of four types based solely on their level of dendritic stratification: stratified amacrine cells of the first, third, and fifth sublayers and bistratified amacrine cells of the first and fifth sublayers. Double-labeling experiments were carried out to determine possible co-localization of glycine-IR with GABA-IR, serotonin-IR, substance P-IR and somatostatin-IR. No evidence for co-localization of glycine-IR with these other transmitter substances was found, despite reports of co-localization of these substances in retinas of other species. Glycinergic neurons in goldfish retina appear to consist of a heterogeneous population of at least seven morphologically distinct subtypes that are also neurochemically distinct in regard to GABA, serotonin, substance P, and somatostatin. Since dendritic stratification in the inner plexiform layer is correlated with ON-, OFF-response types, we suggest that the subtypes of glycine-IR amacrine cells play different roles in the encoding of visual information.

Animals↗

PubMed1989

Immunocytochemical evidence that vigabatrin in rats causes GABA accumulation in glial cells of the retina.

Vigabatrin (gamma-vinyl-GABA, GVG) is an irreversible inhibitor of GABA-aminotransferase (GABA-T) that is under clinical trial as an antiepileptic drug. Rats were injected (i.p.) with GVG and killed 18 h later. GVG administration reduced retinal GABA-T activity to undetectable levels and increased the GABA content 5-fold. Immunocytochemistry using a GABA antiserum clearly revealed the presence of GABA-IR in the glial Muller cells of retinas from GVG-treated rats but not from controls. This experiment indicates that the administration of drugs which inhibit GABA-T may cause the accumulation of GABA in retinal cells that do not normally possess enough endogenous GABA to be detected by immunocytochemistry.

Aminocaproates↗

PubMed1989

Immunocytochemical localization of GABAA receptors in goldfish and chicken retinas.

A monoclonal antibody (mAb 62-3G1) to the GABAA receptor/benzodiazepine receptor/Cl- channel complex from bovine brain was used with light and electron microscopy in goldfish retina and light microscopy in chicken retina to localize GABAA receptor immunoreactivity (GABAr-IR). GABAr-IR was found in the outer plexiform layer (OPL) in both species, in three broad bands in the inner plexiform layer (IPL) of goldfish, and in seven major bands of the chicken IPL. A small percentage of amacrine cell bodies (composing at least three types) were stained in chicken. In goldfish OPL, GABAr-IR was localized intracellularly and along the plasma membrane of cone pedicles, whereas rod spherules were lightly stained, but always only intracellularly. In chicken, all three sublayers of the OPL were GABAr-IR. The presence of GABAr-IR on photoreceptor terminals is consistent with data indicating feedback from GABAergic horizontal cells to cones. In the goldfish IPL, GABAr-IR was localized to postsynaptic sites of amacrine cell synapses; intracellular staining of processes in the IPL also was observed in presumed "GABAergic" targets. A comparison of GABAr-IR with the distributions of 3H-muscimol uptake/binding, glutamate decarboxylase-IR, GABA-IR, and 3H-GABA uptake in the IPL showed either a reasonable correspondence or mismatch, depending on the marker, species, and lamina within the IPL. The distribution of GABAr-IR in the retina corresponded better with the 3H-muscimol than with 3H-benzodiazepine binding patterns yet overall was in excellent agreement with many other physiological and anatomical indicators of GABAergic function. We suggest that intracellular GABAr-IR represents the biosynthetic and/or degradative pathway of the receptor and we conclude that mAb 62-3G1 is a valid marker of GABAA receptors in these retinas and will serve as a useful probe with which to address the issue of mismatches between the localization of GABAA receptors and indicators of presynaptic GABAergic terminals.

Animals↗

PubMed1988

Colocalization of GABA and glycine immunoreactivities in a subset of retinal neurons in tiger salamander.

Alternate serial 1 micron Durcupan resin sections of tiger salamander retina were stained with antisera against GABA and glycine using postembed immunocytochemical techniques. Although the vast majority of neurons were labeled by either GABA or glycine antiserum, a small percentage of presumed amacrine cells in the inner nuclear layer and cells in the ganglion cell layer were clearly labeled by both antisera, indicative of colocalization of endogenous GABA and glycine. Although there is a greater than 90% chance that a labeled cell will be clearly labeled for either GABA or glycine immunoreactivity, the possibility for cotransmission of two inhibitory transmitters must be considered for a small percentage of these retinal neurons.

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↗

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

Light adaptation of rod and cone luminosity horizontal cells of the retina of the goldfish.

Intracellular recordings from rod horizontal cells and luminosity external horizontal cells of the goldfish retina were obtained, and the process of light adaptation induced by steady, full-field background illumination was investigated. Rod horizontal cells had remarkably steep response vs intensity (rvi) functions when dark-adapted. Background illumination reduced the sensitivity of these cells primarily due to response compression, with intense backgrounds resulting in eventual response saturation. Increment threshold functions for these cells were non-linear, and increment saturation was evident when 500-nm backgrounds exceeded 10.5 log photons s-1 cm-2. Cone luminosity cells displayed broad response operating functions when dark-adapted. Light adaptation resulted in substantial narrowing of the rvi function, as well as a shift in the operating function to greater intensities. Response compression played only a minor role in the loss of sensitivity of these cells.

Animals↗

PubMed1988

Localization of GABA and glycine in goldfish retina by electron microscopic postembedding immunocytochemistry: improved visualization of synaptic structures with LR white resin.

A post-embedding, electron microscopic immunocytochemistry technique, modified from existing protocols, was used to examine the labelling patterns of GABA immunoreactivity and glycine immunoreactivity in goldfish retina. Retinae were fixed in mixed aldehyde solution, dehydrated in ethanol, stained en bloc with uranyl acetate and phosphotungstic acid and embedded in LR White resin. Substances were localized in thin sections by floating grids first on a drop of primary antiserum and then on a colloidal gold-IgG conjugate. Finally, grids were exposed to osmium vapour. The localization of GABA immunoreactivity matched that of [3H]-GABA uptake or glutamate decarboxylase immunoreactivity as described previously. In the outer retina, GABA immunoreactivity was found in the cell bodies and axon terminals of H1 horizontal cells and their dendrites opposite cone photoreceptor terminals. Selected amacrine cell bodies were labelled, as were many processes, both synaptic and non-synaptic, throughout the inner plexiform layer, including most amacrine cell processes contacting the synaptic terminals of type Mb bipolar cells. Numerous amacrine cells, their processes in the inner and outer plexiform layers, and photoreceptor terminals contained glycine immunoreactivity in a distribution similar to that shown by [3H]-glycine uptake. Despite the absence of osmium in the primary or secondary fixative, our protocol results in excellent visibility of synaptic structures and detectability of the colloidal gold immunolabel. Also, it does not cause extraction of the HRP/DAB reaction product and is therefore suitable for double-label analysis of neurons labelled with horseradish peroxidase.

Acrylic Resins↗