The distribution of the GABAAβ2,β3 subunit receptor in the cat superior colliculus using antibody immunocytochemistry

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Neuroscience Vol. 79, No. 4, pp. 1121–1135, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(96)00667-7

THE DISTRIBUTION OF THE GABAA â2,â3 SUBUNIT RECEPTOR IN THE CAT SUPERIOR COLLICULUS USING ANTIBODY IMMUNOCYTOCHEMISTRY R. R. MIZE* and G. D. BUTLER Departments of Anatomy and Ophthalmology and the Neuroscience Center, Louisiana State University Medical Center, New Orleans, LA, U.S.A. Abstract––GABA-containing synaptic terminals in the cat superior colliculus include two varieties of presynaptic dendrite and at least one type of axon terminal with flattened vesicles. These anatomically distinct synaptic profiles probably also mediate different types of inhibition. Whether they are associated with different types of GABA receptor is unknown and one objective of the present paper. We used the antibody mAb 62-3G1 directed against the â2,â3 subunits of the GABAA receptor complex to determine whether the distribution of this receptor subunit is specific to one or more types of GABA-containing synapse. At the light microscope level, â2,â3 immunoreactivity was densely distributed within the neuropil of the zonal and superficial gray layers, and more lightly within the optic, intermediate, and deep gray layers. No cell bodies were labelled by the antibody in the zonal and superficial gray layers, but numerous cells contained internalized cytoplasmic immunoreactivity in the optic, intermediate gray, and deeper layers. At the ultrastructural level, synaptic sites opposite axon terminals that contained flattened synaptic vesicles (F profiles) were often â2,â3 immunoreactive, while postsynaptic sites opposite presynaptic dendrites (PSD profiles) were never immunoreactive. The label at F profiles usually filled the synaptic cleft and coated the postsynaptic plasma membrane. Some membrane-associated label was also found at non-synaptic sites. We conclude that this receptor subunit is selectively associated with flattened vesicle axon terminals and not with presynaptic dendrites, a result which supports evidence that those terminal types mediate different types of inhibition. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: GABA, visual pathways, presynaptic dendrites, flattened synaptic vesicles, receptor localization, inhibition.

The neurotransmitter GABA plays an important role in inhibitory processing in the superior colliculus (SC).2,52 Iontophoretic application of GABA ligands inhibits the responses of some SC neurons.31,71,72 GABA also plays a role in the generation of saccadic eye movements.25–27 Some of these effects are mediated by intrinsic GABAergic neurons. Between 25– 40% of neurons in the cat SC contain GABA.37,38 These neurons are densely concentrated within the superficial layers and less densely within the deeper layers of most mammalian species.28,37,44,45,49 These cells comprise three distinct morphological types of GABAergic neuron in SC: horizontal cells and two classes of granule cell.39,43 Three different types of synaptic profile have also been shown to contain GABA.37,39,40,44,45 F profiles are synaptic boutons that contain dense clusters of synaptic vesicles, many of which are clearly flattened. P1 profiles are presynaptic dendrites containing loose accumulations of *To whom correspondence should be addressed. Abbreviations: GBar, GABAA receptor subunit antibody ; IGL, intermediate gray layer; LGN, lateral geniculate nucleus; OL, optic layer; PBS, phosphate-buffered saline; PSD, presynaptic dendrite; SC, superior colliculus; SGL, superficial gray layer; SN, substantia nigra; ZL, zonal layer.

pleomorphic synaptic vesicles that are often a component of synaptic triads involving retinal terminals. P2 profiles are larger calibre dendrites that have small, punctate clusters of synaptic vesicles, often at regular intervals along the dendritic trunk.44,45 All three types are densely labelled by GABA antibodies and presumably form inhibitory synapses.40 At least two of these types, F and P1 profiles, are thought to mediate different types of inhibition.39 Although these profile types have been characterized at the ultrastructural level in cat,37 monkey,44 and rabbit,45 nothing is known of their relationship to specific types of GABA receptor. Given their differences in morphology and function, it is possible that these synapse types are selectively associated with the GABAA vs GABAB receptor or with different subunits of these receptors. Physiological studies have shown that inhibition in SC can be mediated by both GABAA and GABAB receptors.32,52,56 Both GABAA and GABAB receptors have also been localized in SC using ligand receptor autoradiography. [3H]muscimol, a selective GABAA agonist, binds with high affinity in the zonal and superficial gray layers and with lower affinity in the deeper layers.41,55,65 In agreement with these reports, ligands specific to the GABAA/benzodiazepine receptor

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complex are also most densely concentrated in the superficial gray layer.57,60,81,83 GABAB receptors are also more densely distributed within the superficial layers of SC but are present in the deep layers as well.8–10,19 In the present study, we have examined the distribution of the GABAA receptor in the cat SC using the antibody mAb 62-3G1 which recognizes the â2 and â3 subunits of this receptor complex.13,74,75 This approach has allowed us to determine whether this antibody labels only selected synapse types or all synapse types containing GABA.

EXPERIMENTAL PROCEDURES

Ten domestic cats (2.7–3.7 kg) were used to localize the GABAA receptor antibody within the SC using antibody immunocytochemistry. Animals were anaesthetized with a mixture of ketamine hydrochloride and xylazine (35 mg/kg) and ventilated with 95% O2 and 5% CO2. After injection with 1 ml of 1% sodium nitrite and 1000 units of sodium heparin, each cat was perfused transcardially with a rinse solution of phosphate-buffered saline (PBS) followed by a fixative solution containing 4% paraformaldehyde and 0.1– 0.2% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4) with 0.002% CaC12. After fixation, the brains were removed and immersed in the same fixative for 4–24 h followed by overnight storage in an 8% dextrose solution in 0.1 M phosphate buffer (pH 7.4). The midbrain was then sectioned coronally at 50 µm thickness with a Vibratome. Every effort was made to eliminate animal suffering and to reduce the number of animals. Guidelines published by the National Institutes of Health were followed. The antibody used was the 62-3G1 monoclonal antibody74 that is thought to recognize the â2 and â3 subunits of the GABAA receptor. The antibody was provided by Angel de Blas. Sections were incubated in 1% sodium borohydride for 30 min, blocked in 0.1 M lysine, 4% normal horse serum (Vector, Burlingame, CA) and 0.1% bovine serum albumin in phosphate buffer for 1 h, and incubated in primary antiserum for 16–72 h at a dilution of 1:100. In some experiments 0.1–0.25% Triton X-100 was used. After exposure to primary antibody, sections were incubated in a biotinylated horse anti-mouse IgG (Vector, Burlingame, CA; 1:100 dilution) for 1 h, then incubated in an avidin– biotin–horseradish peroxidase complex (Vector, Burlingame, CA; ABC Elite, 1:50) in PBS for 1 h. PBS rinses were used between the appropriate steps. Peroxidase reaction product was demonstrated histochemically by immersion in 0.05% 3,3’-diaminobenzidine tetrahydrochloride and 0.003% hydrogen peroxide in phosphate buffer for 30 s to 14 min. Following the immunohistochemical procedures, some sections were mounted on microscope slides coated with gelatin–chrome-alum, dehydrated through alcohols and xylene and coverslipped. For electron microscopy, sections were rinsed in 0.1 M sodium cacodylate buffer and postfixed in 1% osmium for 30 min and rinsed in buffer. Sections were en bloc stained with 4% uranyl acetate in 100% ethanol and dehydrated through alcohols and propylene oxide, embedded in Medcast-Araldite and mounted flat onto blank embedding capsules. After curing, thick sections of 1–2 µm were cut and stained with Toluidine Blue. These were examined with a light microscope to locate the immunoreactive regions accurately in the superficial gray layer (SGL) and intermediate gray layer (IGL) of the cat SC. Mesas were then cut from these areas. Ultrathin sections were cut with an ultramicrotome and collected on Formvar-coated single-slot grids.

Sections were stained with uranyl acetate and lead solutions using an LKB grid stainer and then examined and photographed using a JEOL 1200 electron microscope. RESULTS

Light microscope distribution GABAA receptor subunit antibody (GBar) labelling was found throughout the cat SC (Fig. 1A–C). Fine reaction product was uniformly distributed within the zonal (ZL) and SGLs (Fig. 1A). Somewhat coarser, less uniformly distributed reaction product was seen within the optic (OL; Fig. 1B) and IGLs (Fig. 1C). Cell bodies in the ZL and the SGL were unlabelled, which produced a halo-like appearance against the dense labelling of the surrounding neuropil (Fig. 1A, asterisk). Some cell bodies in the OL and IGL were labelled (Fig. 1B–D). This internalized cytoplasmic labelling was especially common in medium- and large-sized neurons in the IGL (Fig. 1C,D). In some light microscope semithin sections, GBar reaction product was densely coated at the plasma membrane surfaces of some cells. This labelling was particularly apparent on large cell bodies and proximal dendrites in the IGL (Fig. 1E,F). Electron microscopy reveals that this GBar labelling is membrane-associated and adjacent to flattened vesicle axon terminals that contact these cells. It is therefore likely that this GBar membrane labelling is bound to postsynaptic receptors opposite GABAcontaining axon terminals. Electron microscope distribution At the electron microscope level, membraneassociated labelling was also found in all layers of SC examined, including the ZL, SGL, OL, and IGL. Internalized cytoplasmic labelling was usually not visible in electron microscope material, although some immunoreactivity was seen in the cytoplasm of a few cells in the IGL (see below). Much of the membrane-associated GBar was found at sites between presynaptic axon terminals and postsynaptic dendrites and cell bodies (Figs. 2, 3). The label was often dense and filled the extracellular space between pre- and postsynaptic profiles (arrows, Fig. 2A,C,D; Fig. 3A–C). In regions where the label was less dense, the reaction product was usually confined to the postsynaptic membrane (arrowheads; Fig. 3B,D). Thus, GBar selectively labels the postsynaptic membranes of some synapses in cat SC. Because both the synaptic cleft and postsynaptic membranes of many synapses were densely labelled by antibody, we could not always determine whether the synaptic contacts were symmetric or asymmetric. However, many synapses with wide synaptic clefts and thick postsynaptic membranes were clearly not labelled by GBar. By contrast, dense reaction

GABAA receptor in superior colliculus

Fig. 1. GBar immunoreactivity in the cat superior colliculus. (A–C) GBar uniformly labels the neuropil of the superficial gray layer (SGL) and both the neuropil and selected cell bodies in the optic layer (OL) and intermediate gray layer (IGL). Note that dense immunoreactivity fills the cytoplasm, but not the nucleus, of some cells. The asterisk in A marks an unlabelled neuron. (D) Higher magnification micrograph of GBar-labelled neuron in the IGL. (E,F) Membrane-associated label on the plasma membrane of two cells in the IGL. Note that there is no apparent cytoplasmic labelling in these cells. A–D are from 50 µm Vibratome sections. E,F are from 2 µm-thick plastic sections counterstained with Toluidine Blue. Scale bars: A–C=30 µm; D–F=20 µm.

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product was frequently seen at synaptic sites characterized by narrow synaptic clefts and thinner postsynaptic densities (Figs 2D, 3B,C). We conclude from this result that most if not all synaptic densities labelled by GBar are of the symmetric variety. The presynaptic profiles found at sites containing GBar most often contained dense clusters of small synaptic vesicles, many with flattened morphologies (Figs 2, 3). These presynaptic profiles had characteristics of axon terminals, including a bulbous boutonlike shape (Figs 2, 3), densely-packed synaptic vesicles (Figs 2A,B,D, 3C), and a glial process surrounding portions of the bouton not in synaptic contact with other profiles (asterisks, Fig. 2A,C,D) These boutons formed synaptic contacts with both large dendritic shafts (Figs 2A,B, 3C) and cell bodies (Figs 2D, 3B,D, 5A–C; Fig. 6). Labelling associated with synaptic contacts onto small dendrites (Fig. 2C) and spine-like processes (Fig. 3A,D) was less common. Superficial vs deep layer labelling GBar labeling differed in the superficial and deep layers. In the superficial SC (ZL, SGL), labelling was found at synaptic sites between flattened vesicle axon terminals and several types of postsynaptic profiles, including cell bodies (Fig. 3A,B), dendrites (Fig. 3C), and spines (Fig. 3A,D). However, GBar reaction product was rarely found in regions of the SGL that contained retinal terminals or presynaptic dendrites (PSDs). PSDs contain limited, usually sparsely distributed pleomorphic synaptic vesicles and in favourable sections receive synaptic input from presynaptic axon terminals, often of retinal origin. PSDs also form synaptic contacts with other postsynaptic profiles, usually conventional dendrites (Fig. 4A–D). PSDs are most frequently found in the upper SGL. Despite extensive sampling of this region of SC, we never found label associated with postsynaptic densities that were opposite PSDs (Fig. 4A–D). As label could be seen at other sites in the same region as PSDs (Fig. 4, arrowheads), the absence of GBar at membrane sites opposite PSDs was apparently not due to inadequate penetration of the antibody. This absence of labelling was particularly convincing when we identified PSDs that were making synaptic contact with conventional dendrites (Fig. 4A–D, arrows). In these cases it was apparent that the postsynaptic densities were not immunoreactive even though antibody labelling was readily visible at other sites in the vicinity. In fact, we occasionally found dendrites that were contacted by PSDs that did have â2,â3 immunoreactivity at non-synaptic sites but not at the synaptic contact site with the PSD. We conclude from this evidence that GBar is not associated with synapses formed by PSDs, even when it is present at other sites on the same dendrite. Within the deep layers, â2,â3 immunoreactivity was also seen at synapses between flattened vesicle

axon terminals and various postsynaptic profiles, including cell bodies (Fig. 2D), large dendrites (Fig. 2A,B) and occasionally dendritic spines (Fig. 2B,D). Membrane-associated label was especially prominent on the very large neurons within the IGL. In the best labelled material, immunoreactivity was found along large segments of membrane on both the cell bodies and dendrites of these neurons (Figs 5, 6). In most cases, this label was confined to synaptic sites between axon terminals and the soma or dendrites of the postsynaptic cell (Fig. 5; Fig. 6, arrowheads). In fortuitous examples, the membrane labelling was confined to postsynaptic sites where the presynaptic terminal had detached from the postsynaptic membrane (Fig. 5; Fig. 6, asterisks). This phenomenon is especially evident in Fig. 6 where a number of postsynaptic densities are heavily labelled by GBar at sites where the presynaptic terminal is missing (Fig. 6, asterisks). This figure also illustrates the bulb-like morphology of flattened vesicle axon terminals contacting this cell (Fig. 6, inset). These observations provide additional evidence that the GBar antibody recognizes the plasma membrane of postsynaptic densities opposite F profiles contacting these neurons. Non-synaptic labelling Particulate labelling was also visible in association with ribosomes in the cytoplasm of some neurons (Fig. 5C, small arrows). This labelling is probably homologous to the internalized cytoplasmic labelling seen with the light microscope. Although much of the antibody binding seen on plasma membranes was at synaptic sites, membrane labelling was clearly also present at non-synaptic sites (Fig. 7A–D, arrowheads). Some of this labelling was along the plasma membranes of presynaptic terminals (Fig. 7A), some at non-synaptic membrane sites between two closelyapposed synaptic terminals (Fig. 7B), some along the outer membranes of presynaptic terminals (Fig. 7C), and some at synaptic sites with presynaptic terminals that contained round synaptic vesicles (Fig. 7D, asterisk). Labelling at this location, however, was extremely rare. Whether this non-synaptic plasma membrane labelling represents active receptor sites is uncertain (see Discussion). DISCUSSION

GABAA receptor distribution in superior colliculus The gradient of labelling density that we found using the â2,â3 antibody parallels that found in studies using ligand binding to the GABAA receptor. GBar labelling in our study was very fine but densely packed within the neuropil of the ZL and SGL. GBar label was coarse in the deeper layers and not as densely distributed. This same dorsal to ventral gradient is found using ligand binding autoradiography. [3H]muscimol, a GABAA receptor agonist, binds

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Fig. 2. (A–D) Membrane-associated GBar labelling in the optic and intermediate gray layers of the cat superior colliculus. Most label is found within synaptic clefts (arrows) between axon terminals with flattened synaptic vesicles and postsynaptic profiles. In some cases, the label can be localized to the postsynaptic density (B,D, arrowheads). Glial processes surrounding portions of the bouton are indicated by asterisks in A,C,D. cb, cell body; d, dendrite; f, flattened vesicle axon teminals. Scale bar=0.5 µm.

densely in the SGL and less densely in the deep layers of rat SC.55,65 [3H]GABA binding produces a similar pattern after blocking GABAB sites with baclofen.10

In addition, the specific benzodiazepine receptor ligand [3H]flunitrazepan has been shown to label the superficial layers of the rat SC more densely than

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Fig. 3. (A–D) Membrane-associated GBar labelling in the superficial gray layer of the cat superior colliculus. Label is primarily found within synaptic clefts (arrows) between axon terminals with flattened vesicles and postsynaptic profiles. Label can be localized to the postsynaptic density (arrowhead, C). Other label is seen along membranes at non-synaptic sites (large arrow, A,C). cb, cell body; d, dendrite; f, flattened vesicle axon terminals; s, spine-like processes. Scale bar=0.5 µm.

other layers81–83 as does the benzodiazepine agonist, Ro-15-4513,57,59 and the benzodiazepine antagonists [3H]clonazepam and Ro-15-1788.60 Finally an anti-

body directed against the á1 subunit of the GABAA receptor/benzodiazepine complex densely labels the SGL of the rat SC.58 There is thus consistent

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Fig. 4. (A,D) Presynaptic dendrites (PSDs) within the upper superficial gray layer of the cat superior colliculus. PSDs contain loose clusters of pleomorphic synaptic vesicles. They also form synaptic contacts with postsynaptic profiles, usually conventional dendrites (D). Note the absence of GBar label at the postsynaptic densities opposite PSD profiles (arrows). GBar immunoreactivity is seen at other sites (arrowheads). Scale bar=0.5 µm.

evidence that GABAA receptors are most densely distributed within the superficial layers of the mammalian SC and less densely in the deeper layers.

The gradient of â2,â3 immunoreactivity through the depth of the SC matches very closely the gradient in the density of interneurons and their terminals.

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Fig. 5. Large neuron in the intermediate gray layer of the cat superior colliculus. (A) GBar label is found along the plasma membrane of both the cell body and dendrite of this neuron. This label is seen adjacent to synaptic terminals (arrowheads) and also at sites with no apparent presynaptic terminals (asterisks). Virtually all of the terminals contain flattened synaptic vesicles. Star indicates terminal illustrated in B,C. (B,C) Higher magnification views of GBar label associated with axon terminals containing flattened vesicles (arrowheads). Note that some sites of membrane label are adjacent to vacant extracellular space where axon terminals may have been damaged during fixation or processing. Some particulate label is also present within the cytoplasm of this cell (small arrows). Scale bars: A=2 µm; B=1 µm; C=0.5 µm.

Fig. 6. Another large neuron in the intermediate gray layer of the cat superior colliculus. This cell is surrounded by axon terminals with flattened synaptic vesicles (arrowheads), all of which have GBar label within the synaptic cleft. Other sites (asterisks) of GBar are adjacent to vacant regions where axon terminals were probably damaged during fixation of processing. Insets show the labelling at synaptic clefts as well as the morphology of flattened vesicle axon terminals marked by black boxes. Scale bars: Cell=2 µm; Insets=0.5 µm.

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Fig. 7. Membrane-associated GBar labelling in the cat superior colliculus. Labelling is found at both synaptic (arrows) and non-synaptic sites (arrowheads), including along the plasma membrane of large dendrites (A), between two synaptic terminals (B), along the membrane of a presynaptic terminal (C), and within the synaptic cleft of a synaptic profile with round vesicles (D) (asterisk). Scale bar=0.5 µm.

GABA neuron density is over three times higher in the SGL than in the OL and IGL and over four times higher than in the deepest layers of the cat SC.37 If receptor density matches synapse density, then higher densities of GABA receptors would be expected in

the superficial layers if a large number of GABA synapses arise from intrinsic GABAergic neurons. This seems likely because the only known extrinsic GABAergic projection to the superficial SC comes from the pretectum.1,51 Thus, the bulk of â2,â3

GABAA receptor in superior colliculus

subunit receptor labelling in the SGL is almost certainly opposite synapses that arise from intrinsic GABA containing interneurons found in this layer. By contrast, receptor labelling in the deeper layers is primarily opposite GABA-containing axon terminals that are likely of extrinsic origin. These arise from a number of sources, including the substantia nigra, zona incerta, and collicular commissure.1,3,4,7,15,76 GABAA receptor localization at F-type inhibitory synapses Our finding that the GABAA â2,â3 subunits are expressed at postsynaptic densities opposite F-type axon terminals but not opposite PSDs is an important result. Both F and PSD synaptic terminals contain GABA,37,39,40,42,44 but they arise from different cell types and form synapses that differ in ultrastructure. They also likely mediate different types of synaptic inhibition.40 In the cat SC, F terminals are densely distributed upon the somas and proximal dendrites of deep layer neurons, especially the large predorsal bundle neurons in the IGL. In many cases, these F profiles virtually surround the cell bodies of these neurons and must produce the strong inhibition that blocks conduction of action potentials in these cells. Much of this input arises from the substantia nigra (SN).3,4,7,11,15,20,23,76 The SN pathway is known to tonically inhibit pre-saccadic neurons in the IGL of the monkey SC.25,26,27 This pathway is blocked by bicuculline, a selective GABAA antagonist,25–27 which is consistent with our finding that â2,â3 GABAA receptor subunits lie opposite these terminals. The density of these terminals on postsynaptic cells, the density of synaptic vesicles within the terminals, and the length of their synaptic appositions are features well-suited to mediate the potent tonic inhibition reported by Wurtz and colleagues.25–27 F-type synapses are also present in the superficial layers of SC.37,43 In these layers they are thought to arise from at least one class of inhibitory interneuron that has a locally ramifying axon and is thought to produce feedback inhibition onto projection cells following activation by retinal input.40 PSDs in SC, which were never found to be associated with the â2,â3 GABAA receptor subunit antibody, are heterogeneous in origin and morphology. P1 PSDs probably arise from another class of intrinsic GABAergic neuron37,43,44 and they are the intermediate elements in serial synapses and synaptic triads between retinal terminals and conventional dendrites.37,43,44 This type of synaptic arrangement is thought to mediate feedforward inhibition in both SC and lateral geniculate nucleus (LGN).40,50,73 A second type of PSD, called a P2 profile,40 has discrete clusters of vesicles at synaptic sites within large calibre dendrites thought to arise from horizontal cells. The function of these P2 synapses is not known.40 Despite the diversity in the morphology

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and cellular origin of these two PSD types, neither type was ever found opposite neuronal profiles labelled by the â2,â3 antibody. GABAA receptor immunoreactivity is also specific to F-type terminals in the LGN.66,68 Both á and â GABAA subunit receptor antibodies label postsynaptic membranes opposite F1 axon terminals but not PSDs despite the fact that both profile types are GABA immunoreactive.16,47,73 Like those in SC, F1 axon terminals in the LGN arise from axons of both intrinsic and extrinsic origin,16,47,73 while PSDs in LGN are thought to be the dendrites of one class of GABAergic interneuron.22,73,77 Physiological evidence demonstrates that PSDs mediate feedforward while F1s mediate feedbackward inhibition in the LGN,50,73 just as is thought to occur in the SC.40 Thus, in both structures there is solid morphological evidence that the GABAA receptor is specific to F-type axon terminals that mediate a specific type of inhibition. The functional significance of this selectivity is uncertain. One possible explanation for the absence of GABAA receptor labelling opposite PSDs is that PSDs are selectively related to the GABAB receptor. This is an attractive hypothesis because it could in part explain why the synaptic morphologies of the two GABA synapse types differ. Unfortunately, physiological evidence does not support this idea. Both GABAA- and GABAB-induced inhibitory postsynaptic potentials can be recorded from a large number of relay cells in both SC32,52,56 and the dorsal layers of the LGN.12,66,67 In addition, both geniculate X and Y relay cells generate both GABAA and GABAB responses,67 despite ultrastructural evidence that Y cells receive many F1 synapses and few PSD synapses while X cells receive mostly PSD synapses.63,77 If GABAA receptors were selectively associated with F1 profiles then one would expect that Y cells that receive mostly F1 inputs would have only GABAA responses while X cells receiving mostly PSD input would have only GABAB responses. This is not the case. There is apparently no correlation between the type of GABAergic synapse contacting these neurons and the presence or absence of GABAA vs GABAB responses. The second possibility is that different subunits of the functional GABAA receptor are expressed at different synapses and that the â2,â3 subunits are not expressed at every GABAA receptor. The GABAA receptor is a ligand-gated receptor that can contain different combinations of the five basic subunit groups (alpha, beta, gamma, delta, and pi).18,24,29,30,53,54,61,62,79 Considerable evidence has shown that the á, â, and ä subunits are usually required to form a functional receptor.53,78 For the most part, only recombinant receptors that contain á, â, and ã subunits produce GABA-sensitive Clchannels that bind benzodiazepines.33,34,48,64 Although variations in subunit composition can affect receptor sensitivity and binding affinities to various

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ligands,33,34,35,46 immunocytochemistry studies have shown that the á1, â2,â3, and ã2 subunit antibodies are usually concentrated in the same cell types.17 Finally, the á, â2,â3, and ã2 subunits are frequently found to co-exist in purified receptor preparations.5,6 These data all suggest that the functional receptor is less heterogeneous in structure/distribution than is subunit transcript expression and that á, â, and ã subunits are required at most functional GABAA receptor sites. The absence of â2,â3 subunit labelling opposite PSDs thus suggests to us that PSDs are probably not associated with any form of the GABAA receptor but could well be specific to the GABAB receptor. GABAA internalized and non-synaptic labelling The presence of internalized receptor antibody within the cytoplasm of neurons has been a common finding in other immunocytochemistry studies.6,13,60,69,70,80 Cytoplasmic labelling probably reflects the presence of the receptor protein within the cell body. Receptor synthesis, turnover, and degradation all occur internally. Antibody labelling within cells in our study was probably specific to cytoplasmic receptor protein as it was found only in mediumand large-sized neurons in the optic and deeper layers which also contained substantial amounts of antibody labelling along their plasma membranes. The low levels of cytoplasmic labelling that we found are probably due to the fact that the epitope of the de Blas antibody is relatively specific to membraneincorporated receptor. The mAb 62-3G1 antibody recognizes an epitope at the amino terminus of the â2,â3 subunit which is thought to be localized on the extracellular side of the membrane.14,21 The antibody would thus be expected to primarily label the plasma membrane components of the postsynaptic density, a locus also suggested by our electron microscopy studies. We also found antibody labelling along nonsynaptic plasma membranes. This labelling could be an artifact, an amino epitope of a non-functional

protein, or a functional membrane receptor located at a non-synaptic site. The 62-3G1 antibody is directed against the GABAA receptor-benzodiazepine complex purified on the benzodiazepine molecule R07-1986/1 by affinity chromatography.74 Immunoblot tests have demonstrated that the 62-3G1 antibody recognizes peptides Mr of 55–57.5000 mol. wt in bovine brain which immunoprecipitates at both muscimol and flunitrazepam binding sites.74 Recognition of these binding sites suggests that this antibody may be localized at low affinity GABA receptors that are at non-synaptic sites. The nature of non-synaptic labelling in SC nevertheless requires additional study. It is also of interest that the insertion of the receptor protein into membranes appears to be selective to specific regions of the cell body or dendrites of labelled cells. We never observed F and PSD terminals contacting the same dendrite in this study, but serial reconstruction studies suggest that these two terminal types do occasionally contact the same cell but along different regions of their dendrites.43,44 Thus the â2,â3 subunit of the the GABAA receptor probably is selective in its insertion into the membranes of a neuron’s cell body or dendrites. CONCLUSIONS

In conclusion, we have shown that the â2,â3 subunits of the GABAA receptor are selectively associated with F-type synaptic terminals but not with GABAergic presynaptic dendrites. Whether GABAcontaining presynaptic dendrites are related specifically to the GABAB receptor remains to be determined. GABAB site recognition awaits development of effective antibodies for localizing this receptor.36 Acknowledgements—We thank Dr Angel de Blas for generously providing us with the mAb 62-3G1 antibody. Kevin S. Mardis and Stephanie G. Jackson typed the manuscript. This research was supported by USPHS Grant NIH EY02973 (R.R.M.) from the National Eye Institute.

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