Stephen Yazulla

Non-correspondence of [3H]GABA uptake and GAD localization in goldfish amacrine cells*

Ultrastructural analysis of the inner synaptic layer of goldfish retina, using a double-label technique, showed that [3H]GABA uptake and glutamic acid decarboxylase (GAD)-immunoreactivity (IR) occurred in different neuronal processes in most cases. Many [3H]GABA-accumulating processes were found surrounding GAD-IR processes, though not necessarily in a postsynaptic relationship. Co-localization of [3H]GABA uptake and GAD-IR occurred only when one GAD-IR process was juxtaposed to another GAD-IR process. This study suggests that [3H]GABA uptake may be a poor marker for GABA releasing neurons.

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.

GABAergic input to the synaptic terminals of mb1 bipolar cells in the goldfish retina

An EM-autoradiographical/immunocytochemical technique was used to study amacrine cell synapses onto mb1 bipolar cell terminals in goldfish retina. Tissue was double labeled for [3H]GABA uptake and glutamate decarboxylase (GAD) immunolocalization. Nearly 90% of the amacrine cell synaptic processes onto both proximal and distal halves of mb1 terminals were labeled with either [3H]GABA or GAD-immunoreactivity (IR). Proximal half: 73% of the amacrine synapses were labeled with [3H]GABA uptake and 82% with GAD-IR; 88% of [3H]GABA labeled contacts were double labeled. Distal half: 17% of the amacrine synapses were labeled with [3H]GABA uptake and 67% with GAD-IR; 63% of [3H]GABA labeled contacts were double labeled. After consideration of the possible sources of [3H]GABA labeled synapses onto mb1 terminals, we concluded that the synaptic terminals of pyriform Ab amacrine cells double label for [3H]GABA and GAD-IR despite our previous report that Ab cell bodies do not stain for anti-catfish brain GAD antiserum. We suggest that Ab cells contain isoenzymes of GAD which differ in subcellular distribution, thereby accounting for the differential staining of the cell bodies and dendrites obtained with the GAD antiserum we used.

Radioautographic localization of125I α-bungarotoxin binding sites in the retinas of goldfish and turtle

There is substantial evidence for the action of acetylcholine (ACh) as a neurotransmitter in the vertebrate retina. Chotineqic mimics and antagonists alter the response properties of optic nerve fibers in rabbit (Ames and Pollen, 19691, cat (Straschill, 1968) and the ERG of frog (Val’tsev. 1966). The hydrolytic enzyme acetylcholinesterase (AChE) is found in the retinas of a wide variety of vertebrates (Francis. 1953; Nichols and Koelie, 1968; Dickson, Fiumerfelt. Hollenberg and Gwyn, 1971). In mammalian and avian retinas AChE is confined largely to the inner plexiform layer (IPL) and amacrine cells (Nichols and KoeIle. 1968). However, in the newt. an amphibian, AChE was found in the outer plexiform layer (OPL) where it was associated with horizontal and bipolar ceII processes (Dickson et al.. 1971). Since the mere presence of AChE is not conclusive proof that a neuron utilizes ACh as a transmitter, additional histological markers are needed if cholinergic transmission is to be demonstrated more convincingly and localized more accurately in the retina. Such an approach has become possible with the discovery that r-bungarotoxin (r-BTX), the principal component of the venom from the banded krait, Btmynrus mdticinctus, binds to nicotinic receptors with high affinity (Chang and Lee, 1963) With radioactively-labeled &TX and the related r-toxin from fVaja nigricollis, the ACh receptor sites at neuroeffector junctions of electric organ (Bourgeois, Ryter, Menez, Fromageot, Boquet and Changeux, 1972) and muscle (Porter, Chiu, Wieckowski and Barnard, 1973; Fertuck and Salpeter, 1974) were localized and quantified. Also, toxin binding sites were described in the chick optic tectum, rat hippocampus, and other structures of the central nervous system (Polz-Tejera, Schmidt and Karten, 1975). It seems reasonable to expect that if ACh is a retinal transmitter, the location ofnicotinic receptors sites could be found by radioautography utilizing labeied Z-BTX.

Two types of receptors for α-bungarotoxin in the synaptic layers of the pigeon retina

Summary Pigeon retinae were analyzed for binding [ 125 I]α-bungarotoxin (αBgt) by radioautographic and biochemical methods. Toxin binding, localized to the outer and inner plexiform layers (OPL and IPL), was inhibited by micromolar concentrations of native αBgt and d -tubocurarine and by 1 m M acetyl- and butyrylcholine in both synaptic layers. Nicotine, at comparable concentrations affected only the IPL. In vitro drug competition experiments showed that the pigeon retina contains two types of receptors for αBgt which differ in sensitivity to inhibition by nicotine by 4 orders of magnitude. The results suggest that: (1) the receptor for αBgt in the IPL is a nicotinic receptor, (2) the receptor for αBgt in the OPL may be involved in an unusual cholinergic system, and (3) ability to bind αBgt is not a sufficient criterion for identifying nicotinic-cholinergic receptors.

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

SummaryA 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, staineden 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.

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.

Postsynaptic localization of gamma-aminobutyric acid transporters and receptors in the outer plexiform layer of the goldfish retina: An ultrastructural study.

The γ‐aminobutyric acid (GABA)‐ergic system in the outer plexiform layer (OPL) of the goldfish retina was studied via light and electron immunohistochemistry. The subcellular distributions of immunoreactivity (‐IR) of plasma membrane GABA transporters GAT2 and GAT3, the α1 and α3 subunits of the ionotropic GABAA receptor, and the ρ1 subunit of the ionotropic GABAC receptor were determined. The localization of the GAT2‐IR and GAT3‐IR to horizontal cell dendrites at the base of the cone synaptic complex was the main characteristic at the ultrastructural level. Very rarely, GAT2‐IR and GAT3‐IR were found in horizontal cell dendrites innervating rod spherules. α1‐IR and α3‐IR were seen in wide bands in the OPL, whereas ρ1‐IR appeared as a narrow band in the OPL. Most α1‐IR was intracellular in rod and cone terminals. Membrane‐associated α1‐IR was observed in cone pedicles but not in rod spherules; postsynaptic elements were also labeled. α3‐IR was concentrated in the lateral elements of horizontal cell dendrites in cone pedicles. In contrast, ρ1‐IR was found mainly on the spinules of the horizontal cell dendrites in cone pedicles. In addition, in another type of cone pedicle, ρ1‐IR was found at the position of OFF‐bipolar cell dendrites. α3‐IR and ρ1‐IR were rarely found in horizontal cell dendrites innervating rods. We suggest that two GABAergic pathways exist in the outer retina— first, a GABAergic positive loop with GABA receptors mainly on the horizontal cell dendrites and spinules and, second, a GABAergic feedback pathway involving GABA receptors on cone pedicles and GABA transporters on horizontal cells and that this pathway presumably modulates feedback strength from horizontal cells to cones. J. Comp. Neurol. 474:58–74, 2004.

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.

3H-adenosine uptake selectively labels rod horizontal cells in goldfish retina.

There are four types of horizontal cell in the goldfish retina, three cone- and one rod-type. The neurotransmitter of only one type, the H1 (cone) horizontal cell, has been identified as GABA. 3H-adenosine uptake was examined as a possible marker for the other classes of horizontal cell. Isolated goldfish retinae were incubated in 3H-adenosine (10-40 microCi) in HEPES-buffered saline for 30 min, then fixed, embedded in plastic, and processed for light-microscopic autoradiography (ARG). For double-label immuno/ARG studies, 1-micron-thick sections were processed for GABA postembed immunocytochemistry, then for ARG. 3H-adenosine uptake was localized to cone photoreceptors, presumed precursor cells in the proximal outer nuclear layer, and to a single, continuous row of horizontal cell bodies in the inner nuclear layer. No uptake was localized to the region of horizontal cell axon terminals. 3H-adenosine uptake did not colocalize with GABA-IR in H1 horizontal cells, but it did colocalize with adenosine deaminase immunoreactivity. It is concluded that 3H-adenosine uptake selectively labels rod horizontal cells in the goldfish retina based on position and staining pattern, which are similar to rod horizontal cells stained by Golgi or HRP injection methods. The use of 3H-adenosine uptake may provide a useful tool to study other properties of rod horizontal cells (i.e. development) as well as provide clues as to the transmitter used by these interneurons.