GABA-like Immunoreactivity Research Articles

Co-localization of tyrosine hydroxylase and GABA immunoreactivities in human cortical neurons

Samples of human cerebral cortex were stained immunocytochemically for tyrosine hydroxylase (TH) and γ-aminobutyric acid (GABA). TH-positive neurons were in small number and predominated in the deep infragranular layers V-VI contrasting with numerous GABA-positive neurons scattered in all layers. Co-localization of TH- and GABA-like immunoreactivities in a single cell was studied by the double immunolabeling technique with the elution-restaining procedure. Only 50% of the TH-positive neurons also expressed GABA-like immunoreactivities. The two markers were detectable in the somata and not in the processes of the cells. The double-labeled cells were mainly fusiform and medium-sized and were observed in layer VI. These observations suggest that the TH-positive cells form a mixed neuronal population, only a part of which corresponds to the GABAergic class of intrinsic interneurons.

Light and electron microscopic localization of GABA-like immunoreactivity in myenteric plexus of chicken.

Whole-mounts of 1-day-old chicken midgut were incubated with an antiserum against GABA-glutaraldehyde-BSA conjugate. The immunoreaction was visualized by using the peroxidase-antiperoxidase method, and processed for consecutive light and electronmicroscopic observation. GABA was selectively localized in some of the varicose and nonvaricose nerve fibres of the myenteric plexus. The varicose fibres formed dense networks within the myenteric ganglia, some of which--mainly in duodenum--also contained immunopositive nerve cell bodies. Some of the varicose fibres projected out from the myenteric plexus into the circular muscle layer. At the electronmicroscopic level, labelled axon terminals formed synaptic contact with unlabelled perikarya and vica versa. At the same time, no labelled terminals were found on immunostained cells. In a few cases, axon terminals with GABA positivity were situated close to the smooth muscle cells in the circular muscle layer, suggesting a prejunctional GABA effect on the neighbouring nerve terminals on the release of their transmitters.

GABAergic neurons in the rat hippocampal formation: ultrastructure and synaptic relationships with catecholaminergic terminals

Numerous studies indicate that gamma-aminobutyric acid (GABA) can either hyperpolarize or depolarize hippocampal pyramidal and granule cells. While the inhibitory action of GABA may occur directly on these cells, the excitatory action may be mediated by interactions of GABAergic neurons with each other or with catecholaminergic afferents. We sought to examine the cellular basis for these interactions and their relative frequency. Thus, the ultrastructural morphology of GABAergic neurons and their relation to terminals exhibiting immunoreactivity for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) were examined in the rat hippocampal formation using combined immunoautoradiographic and peroxidase-antiperoxidase labeling methods. By light microscopy, GABAergic perikarya and processes codistributed most noticeably with TH-containing processes in the hilus of the dentate gyrus (DG) and in strata lucidum, radiatum, and lacunosum-moleculare of the CA3 region of the hippocampus. Thus, these regions were examined further by electron microscopy. In the ultrastructural analysis, GABA-like immunoreactivity (GABA-LI) was detected in neuronal perikarya, dendrites, axons, and axon terminals. The GABA-containing perikarya were large, ovoid (20-40 microns in diameter), and contained abundant cytoplasm and an indented nucleus with one nucleolus. Synaptic junctions on the perikarya and dendrites with GABA-LI were both symmetric and asymmetric. Approximately equal numbers of TH-labeled terminals (19% of 133 in DG; 39% of 26 in CA3) and GABA-containing terminals (19% DG, 15% CA3) formed synapses with GABA-labeled perikarya. The remainder of the presynaptic terminals (62% DG, 46% CA3) were unlabeled, i.e., contained unidentified transmitters. Terminals with GABA-LI (0.5-1.6 microns) contained numerous small clear vesicles and from 0 to 2 large dense-core vesicles. The types of associations formed by terminals with GABA-LI were remarkably similar in the DG and hippocampus proper despite differences in intrinsic cell type and function. Terminals with GABA-LI formed associations with unlabeled perikarya and dendrites (24% of 151 in DG, 25% of 75 in CA3) and synapses with GABA-containing perikarya and dendrites (18% DG, 5% CA3). Additionally, GABAergic terminals converged upon the same perikarya or dendrite as a TH-containing terminal (15% DG, 21% CA3) and were in direct apposition to TH-labeled terminals (19% DG, 20% CA3). The remaining GABAergic terminals (24% DG, 28% CA3) were without any apparent synaptic relations. In both the DG and CA3, the junctions formed by GABAergic terminals were symmetric. Terminals showing colocalization of GABA-LI and TH-I were also detected although rarely.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical evidence for the presence of histamine and GABA in photoreceptors of the barnacle (Balanus nubilus)

Biochemical evidence indicates that GABA and histamine may both be synthesized by barnacle photoreceptors (Koike & Tsuda, 1980; Timpe & Stuart, 1984; Callaway & Stuart, 1989b). We used antisera against GABA- and histamine-protein conjugates to determine whether the photoreceptors contain either or both of these antigens. Both antisera labeled all of the photoreceptors in each of the three ocelli. Histamine-like immunoreactivity was found throughout each photoreceptor cell but was most intense at their presynaptic terminals. Histamine-like immunoreactivity was blocked by preincubation of the antibody either with histamine or with a histamine-protein conjugate. GABA-like immunoreactivity was found in all parts of the photoreceptors including the cell body, axon, rhabdomeric dendrites, and presynaptic terminals. GABA-protein conjugates blocked the GABA-like labeling of the photoreceptors, while protein conjugates with histamine, L-glutamate, L-glutamine, beta-alanine, and taurine did not. Histamine-like immunoreactivity in the supraesophageal ganglion was confined to the photoreceptor terminals and a second, loose plexus of endings in the main neuropil. GABA-like immunoreactivity, in contrast, was found in approximately twenty-five pairs of neurons of this ganglion. In the cirral nerves, which are expected to contain inhibitory motoneurons, unidentified axons also labeled with the GABA antiserum.

GABA-like immunoreactivity of neurons in the chicken diencephalon and mesencephalon.

The chick brain is a useful model system for studying the ontogeny and phylogeny of neural circuitry, especially that of the visual system. In this study the distribution of cells and processes showing GABA-like immunoreactivity (GABA+) in the diencephalon and mesencephalon of the posthatch chick was determined immunohistochemically with a polyclonal antibody to GABA and compared with the results of similar studies in mammals. Most of the small GABA+ cells were found in the chick visual centers such as the nucleus lateralis anterior, suprachiasmatic nucleus, ventral lateral geniculate, optic tract, dorsolateralis anterior pars lateralis, lentiformis mesencephali, ectomammillary nucleus, area pretectalis, and the optic tectum. Large GABA+ cells were found in the following nuclei: reticularis superior, posteroventralis thalami, subpretectalis, isthmi pars magnocellularis, interstitio-pretectosubpretectalis, mesencephalicus lateralis pars dorsalis. These large cell-containing nuclei receive projections from visual or auditory centers. GABA+ axons were found throughout the diencephalon and mesencephalon but were especially prominent in the ansa lenticularis, fasciculus medialis longitudinalis, and optic tract. The distribution of GABA+ cells in the chick is more widespread than in rodents and exhibits an increased association with the visual centers suggesting a correlation with the specialized visual requirements of the bird.

The morphology and topographic distribution of substance- P-like immunoreactive amacrine cells in the cat retina

In cat retinal wholemounts, substance-P-like immunoreactivity (SP-IR) was localized in a distinct population of amacrines whose cell bodies were normally placed in the ganglion cell layer. Although displaced amacrines accounted for 80-95% of the SP-IR amacrines in peripheral retina, this proportion decreased considerably within the area centralis, accounting for 50-80% of the labelled cells at maximum density. The SP-IR cells in both the inner nuclear and ganglion cell layers gave rise to well-defined varicose dendrites of uniform appearance that stratified around 60% depth (S3/S4) of the inner plexiform layer. In addition, sparse fine dendrites in stratum 1 (S1) could sometimes be traced to inner nuclear cells and occasionally to displaced amacrines. The combined SP-IR cell density ranged from less than 50 cells mm-2 in the far periphery to more than 500 cells mm-2 in the area centralis; the maximum density showed little individual variation despite wide differences in the proportion of displaced cells. The 39,000 SP-IR amacrines in a mapped retina had a triangular topographic distribution, with intermediate isodensity lines extending vertically in superior retina and horizontally along both arms of the visual streak. Colocalization experiments established that all SP-IR cells in cat retina showed GABA-like immunoreactivity, and that the SP-IR amacrines were quite distinct from the cholinergic amacrines identified by choline acetyltransferase immunohistochemistry.

Distribution of GABAergic neurons and terminals in the auditory system of the barn owl.

Antisera to GAD (glutamic acid decarboxylase) and GABA were used to determine the distribution of GABAergic cells and terminals in the brainstem and midbrain auditory nuclei of the barn owl. The owl processes time and intensity components of the auditory signal in separate pathways, and each pathway has a distinctive pattern of GAD- and GABA-like immunoreactivity. In the time pathway, all the cells of the cochlear nucleus magnocellularis and nucleus laminaris receive perisomatic GABAergic terminals, and small numbers of GABAergic neurons surround both nuclei. The ventral nucleus of the lateral lemniscus (anterior division) contains both immunoreactive terminals and some GABAergic neurons. In the intensity pathway, dense immunoreactive terminals are distributed throughout the cochlear nucleus angularis, which also contains a small number of GABAergic neurons. The superior olive contains two GABAergic cell types and immunoreactive terminals distributed throughout the neuropil. All the neurons of the nucleus of the lateral lemniscus (ventral part) appear to be GABAergic, and this nucleus also contains a moderate number of immunoreactive terminals. Immunoreactive terminals are distributed throughout the neuropil of the ventral nucleus of the lateral lemniscus (posterior division), whereas multipolar and small fusiform GABAergic neurons predominate in the dorsal regions of the nucleus. The time and intensity pathways combine in the inferior colliculus. The central nucleus of the inferior colliculus contains a larger number of fusiform and stellate GABAergic neurons and a dense plexus of immunoreactive terminals, whereas the external nucleus contains slightly fewer immunoreactive cells and terminals. The superficial nucleus contains dense, fine immunoreactive terminals and a small number of GABAergic neurons.

GABA-like immunoreactivity in the cochlea of the developing mouse.

GABA-like immunoreactivity was studied in surface preparations of cochleas from postnatal developing mice, and GAD-like immunoreactivity was studied in the adult. GABA-positive fibres are already present at birth; they innervate both the inner and outer hair cells and some spiral ganglion cells. The GABA-positive fibres that enter via the intraganglionic bundle send collaterals to the spiral ganglion and to the hair cell region. Fibres that enter along the central processes of spiral neurons end predominantly among the spiral ganglion cells. A few spiral neurons display pericellular rings of GABA-positive boutons at birth. In older animals, the endings occur on a small number of spiral ganglion cells either as rings or as brush formations. The early GABA-positive fibres reach the inner hair cells around the second day and the outer hair cells (of the upper turns only) around the seventh day. In 12-day animals, tunnel fibres arborize in the outer hair cell region; their collaterals make contacts with the outer hair cells within four to eight cell-wide segments, distributing the endings high, up to the reticular plate. In older animals, fibres (both GABA-and GAD-positive) may innervate single vertical rows of outer hair cells. In the maturing and the adult cochlea, the GABA-positive component of the inner spiral bundle is conspicuous and extends along the entire cochlear length, but the innervation of the outer hair cells comprises only the mid and apical turns. GABA-positive nerve cells occur among the small vestibular neurons, occasionally among the cells of eighth nerve nucleus and only exceptionally in the spiral ganglion. In the adult animal, GAD-positive cells, although uncommon, were observed among the spiral neurons. In the developing animal, GABA-positive fibres give rise to transitory formations: (1) a convoluted plexus running beneath and among the radial bundles and (2) a sparse plexus, continuous with the inner spiral bundle and running in the upper plane of the inner spiral sulcus. GABA-like immunoreactivity was also observed in neuronal growth cones and in some fibres running along blood vessels. In conclusion, GABA immunoreactive fibres appear to reach the cochlea by two routes: via the intraganglionic bundle and to a much lesser extent via the central bundles of the spiral ganglion. The fibres innervate sensory cells and also some spiral neurons. The occasional presence of GABA-positive neurons in the vestibular ganglion, in the VIIIth nerve nucleus, and exceptionally among the spiral neurons raises the possibility of a local GABAergic circuitry within the inner ear.

GABA-like activity in in vitro slices of the rat visual cortex: immunocytochemistry and electrophysiology

Using antisera raised against γ-aminobutyric acid (GABA), GABA-like immunoreactivity was determined in in vitro slices fixed after various incubation times. Synaptic efficacy of GABAergic innervation was concurrently assessed in adjacent slices by intracellular recordings. We show that GABA-like immunoreactivity dramatically decreases already after one hour of incubation and remains low throughout the following hours. Nevertheless robust GABA-mediated synaptic potentials are recordable for more than 8 h.

Localization of GABA- and GAD-like immunoreactivity in the turtle retina

gamma-aminobutyric acid (GABA) has been reported to be an important neurotransmitter in the retinas of many species. This immunocytochemical study detailed the localization of antigens resembling GABA and glutamic acid decarboxylase (GAD, an enzyme involved in the synthesis of GABA), in retinal neurons in the turtle, Pseudemys scripta elegans. GABA-like immunoreactivity was present within somata in the inner and outer regions of the inner nuclear layer, within somata in the ganglion cell layer, and in processes in the outer plexiform layer, inner plexiform layer, and ganglion cell axon layer. GAD-like immunoreactivity was found in somata in the inner and outer regions of the inner nuclear layer and in processes in the inner and outer plexiform layers. Cell counts indicated more somata with GABA-like than GAD-like immunoreactivity in the inner nuclear layer. Double-label studies showed that every somata in the inner nuclear layer which had GAD-like immunoreactivity also had GABA-like immunoreactivity, but that many somata had only GABA-like immunoreactivity. The stratification of immunoreactivity within the inner plexiform layer was analyzed using a scanning densitometer. We described the strate within the inner plexiform layer such that S0 represented the inner nuclear layer/inner plexiform layer border and S100 represented the inner plexiform layer/ganglion cell layer border. Analysis of GAD-like labeling yielded seven distinct strata with peak densities at positions S8, S19, S28, S42, S59, S75, and S93. GABA-like labeling provided five distinct strata with peak densities at positions S17, S28, S67, S84, and S95. The strata with peaks of GABA-like immunoreactivity at S17 and S28 were in statistically identical locations to corresponding strata with GAD-like immunoreactivity. The strata with GABA-like immunoreactivity at S67, S84, and S95 did not have statistically identical peaks of correlated GAD-like immunoreactivity, although there were corresponding strata with GAD-like immunoreactivity nearby. Antiserum directed against GABA failed to produce labeled strata at positions corresponding to the strata with GAD-like immunoreactivity at S8 and S42. In summary, our results indicated that the antisera we used, which were directed against GABA and GAD, produced significantly different labeling in the inner nuclear layer, inner plexiform layer, and the ganglion cell body and axon layers of the turtle retina. Until the physiological significance of these differences is resolved, studies employing these markers to investigate the function of GABA in the turtle retina should be interpreted with caution.

Generation of monoclonal antibodies against a nematode peptide extract: another approach for identifying unknown neuropeptides.

Monoclonal antibodies that cross-react with Ascaris neural antigens were generated in mice immunized with a conjugate made with keyhole limpet hemocyanin (KLH) linked to a crude peptide extract from Caenorhabditis elegans. The response to KLH was suppressed by injection of cyclophosphamide 3 days after immunization with a gamma-aminobutyric acid (GABA)-KLH conjugate. Screening of hybridomas was carried out by enzyme-linked immunosorbent assay and whole mount immunocytochemistry. Two similar clones produced antibodies that recognized a small subset of Ascaris neurons. This result suggests that the monoclonal antibody technique might be useful for identifying new neuropeptides since the antibodies can be used for localization of the neuropeptidelike substances and, potentially, for immunoaffinity chromatography. As a by-product of this experiment, monoclonal antibodies that recognize GABA-like immunoreactivity in whole mounts and plastic sections were also obtained.

Localization of GABA-like immunoreactivity in the ectostriatum of domestic chicks: GABA immunocytochemistry combined with Golgi impregnation.

The distribution of GABA-like immunoreactivity (GABA-LI) in the ectostriatal core (Ec) of domestic chicks (one to two days old) was investigated using (1) preembedding GABA immunocytochemistry and (2) Golgi impregnation and gold-toning combined with postembedding GABA immunocytochemistry. Two major classes of neurons which display GABA-LI were identified in chick Ec. Firstly, large GABA immunopositive cells which comprise at least two further subtypes: an ovoid or polygonal form of 14-18 microns diameter with no apparent polarity of dendrites and a smaller cell (10-14 microns) with ovoid or basket-shaped soma and often more polarized dendritic ramification. In both subtypes the dendritic surface is smooth or sparsely spiny. Secondly, a small GABA immunopositive cell which is characterized by a round cell body of 5-8 microns diameter and thin and sparsely ramifying dendrites of smooth surface or with irregular protrusions. Based upon comprehensive descriptions of ectostriatal cytoarchitectonics (Tömböl et al., 1988c), and synaptology (Watanabe et al., 1985), we argue that the GABA-immunopositive cell types of chick Ec are likely to represent inhibitory interneurons comparable with GABAergic inhibitory cell types described in mammalian visual cortex.

Coexistence of GABA and peptide immunoreactivity in non-pyramidal neurons of the basolateral amygdala

Colocalization of γ-aminobutyric acid (GABA) immunoreactivity with somatostatin (SOM), neuropeptide Y (NPY), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP) immunoreactivity was demonstrated in non-pyramidal neurons of the basolateral amygdala using a two-color immunoperoxidase procedure. Approximately 80–90% of SOM- and NPY-positive neurons in the basolateral amygdala were also immunoreactive for GABA. Virtually all large CCK-positive neurons also exhibited GABA-like immunoreactivity. About one-half of VIP-positive neurons and small CCK-positive cells were also immunoreactive for GABA.

Distribution of GABA immunoreactivity in the cat retina: A light- and electron-microscopic study

The distribution of GABA-like immunoreactivity in the cat retina was studied through the use of preembedding immunocytochemistry for light microscopy and by postembedding immunogold techniques for electron microscopy. Staining was observed in both inner and outer plexiform layers. Approximately 30% of the somata in the amacrine portion of the inner nuclear layer were immunoreactive and included amacrine and interplexiform cells. Horizontal cells and a subpopulation of cone bipolar cells were also stained. In the ganglion cell layer, staining was observed in both small- and medium-sized neurons. GABA-labeled amacrine cells were presynaptic to somata of amacrine cells and to dendrites of amacrine, bipolar, and ganglion cells. Bipolar cells were a major target, receiving more than 60% of all labeled synapses in the inner plexiform layer. Many of these contacts were reciprocal synapses. These findings support a major role for GABA-labeled amacrines in providing feedback inhibition to bipolar cells in the inner retina.

GABA-like immunoreactivity suggests an inhibitory function of the thoracic low-frequency neuron (TN1) in acridid grasshoppers

GABA-like immunoreactivity suggests an inhibitory function of the thoracic low-frequency neuron (TN1) in acridid grasshoppers

Quantitative analysis of the number and distribution of neurons richly innervated by GABA-immunoreactive axons in the rat superior cervical ganglion.

The superior cervical ganglion of rats contains a considerable number of nerve fibers with GABA-like immunoreactivity which show a nonuniform distribution within the ganglion. The topography of these fibers has been analyzed by using antibodies raised against GABA-BSA-glutaraldehyde complexes. GABA-positive axons and axon varicosities accumulated around a subpopulation of principal ganglion cells forming basketlike patterns. These neurons richly innervated by GABA-positive axons (RIG-neurons) in turn were aggregated in patches with strong immunoreactivity. The size and packing density of the patches containing RIG-neurons and GABA-positive axons approaching them had rostral-to-caudal and medial-to-lateral gradients. Similar patterns were found in right and left ganglia. In five ganglia, a quantitative analysis revealed on average 1,344 RIG-neurons per ganglion representing about 5% of the total neuron population, with small variations (standard deviation 122) despite the highly variable shape of the ganglia. The distribution of RIG-neurons resembles that of neurons sending their axons into the internal carotid nerve. To check this possible correlation, HRP was injected into the eye and applied to the transected external carotid nerve. Double staining for the retrogradely transported peroxidase and GABA immunohistochemistry revealed that RIG-neurons formed a small subpopulation of retrogradely labelled neurons in both experiments. This suggests that RIG-neurons innervate various target organs. This conclusion is in agreement with the observation that RIG-neurons also exist in other sympathetic ganglia. Data presented suggest that sympathetic ganglion cells can be classified on the basis of non-uniform innervation patterns formed by axons that use different neurotransmitters.

Diagonal band projection towards the hypothalamic supraoptic nucleus: light and electron microscopic observations in the rat.

Supraoptic nucleus (SON) neurons receive a prominent gamma-amino-butyric acid (GABA) input. This study evaluated the hypothesis, partly on the basis of recent electrophysiological data, that this innervation might arise from GABAergic neurons located in the ventral diagonal band of Broca area. For retrograde transport studies, pentobarbital-anesthetized male Long-Evans rats received 0.03-0.20-microliter injections of a suspension of rhodamine tagged latex microspheres into the SON. In two cases where such injections were confined to the SON, less than 60 retrogradely labeled neurons were detected in the ipsilateral diagonal band. In three animals where injections extended into the perinuclear zone around the SON, more than 2,000 retrogradely labeled cells were counted in the ipsilateral diagonal band. For anterograde transport studies, another group of animals received either 30% horseradish peroxidase (HRP) in 0.5% poly-L-ornithine (0.05-0.10 microliter injections) or Phaseolus vulgarus (iontophoresed from a 2% solution) into the diagonal band. After survivals of 18-24 hours (HRP) or 5 days (PHAL-L) labeled axon terminals invested the perinuclear zone above the SON. The presence of just a single fiber within the nucleus indicated a minor projection to the SON itself. The HRP-injected material was processed for ultrastructural examination and revealed dense HRP-labeled axon terminals in this perinuclear zone, most often (98%) forming axodendritic appositions. A postembedding colloidal gold technique to visualize GABA-synthesizing terminals revealed that fewer than 5% of these perinuclear HRP-labeled terminals also exhibited GABA-like immunoreactivity. Within the SON, where GABAergic axon terminals are abundant, few (less than 5%) GABAergic terminals contained HRP.(ABSTRACT TRUNCATED AT 250 WORDS)

GABA-like immunoreactivity in the cat retina: light microscopy.

Semithin sections of the cat retina were stained with antibodies against GABA conjugated to bovine serum albumin with glutaraldehyde. Labelled cells were visualized by means of the peroxidase-antiperoxidase method. In the outer plexiform layer both A- and B-type horizontal cells and the B-type axon terminal system expressed GABA-like immunoreactivity. Approximately 25-30% of all amacrine cells and the whole inner plexiform layer were heavily labelled. Two types of putative GABA-ergic interplexiform cells could be distinguished. One of them also expressed tyrosine-hydroxylase-like immunoreactivity. A few bipolar cells were also GABA-immunolabelled. GABA-like immunoreactivity and 3H-muscimol uptake were colocalized in 90% of the amacrine cells labelled. However, horizontal cells did not accumulate 3H-muscimol.

GABA-like immunoreactivity in the cat retina: electron microscopy.

The synaptic organization of the cat retina was studied with antibodies against the GABA-GA (glutaraldehyde)-BSA (bovine serum albumin) complex. The postembedding technique combined with immunogold labelling ensured ultrastructural preservation and made identification of synapses possible. The most common putative GABA-ergic synapses in the inner plexiform layer were amacrine-to-bipolar-cell synapses followed by amacrine-to-ganglion-cell and amacrine-to-amacrine-cell synapses. GABA-immunoreactive amacrine cells received most of their synaptic input from bipolar cells followed by other amacrine cells. Synapses between two labelled amacrine cells were common. Rod bipolar cells were the predominant input source and also the preferred output target of GABA-labelled amacrine cells. OFF- and ON-ganglion cells received putative GABA-ergic synapses at their dendrites in laminas a and b, respectively, and also at their somata. In the outer plexiform layer, synapses of interplexiform cells onto bipolar cell dendrites expressed GABA-like immunoreactivity. In both the cone pedicles and the rod spherules, GABA-like immunoreactivity was observed in horizontal cell processes.

Histochemical demonstration of GABA-like immunoreactivity in cobalt labeled neuron individuals in the insect olfactory pathway.

Individual olfactory neurons in the antennal lobes of Periplaneta americana were investigated for their morphological and neurochemical properties by means of intracellular cobalt injection and indirect peroxidase-antiperoxidase immunohistochemistry. GABA-like immunoreactivity was demonstrated in many local interneurons but not in uniglomerular projection neurons.