Parvalbumin and calbindin D-28K in the human entorhinal cortex. An immunohistochemical study.

Research is here reported on the distribution of immunoreactivities of the calcium-binding proteins parvalbumin and calbindin D-28K in the entorhinal cortex of normal human brains. Topographically, parvalbumin immunoreactive neurons were only seen in the lateral portion of the rostral entorhinal cortex, in continuity with the adjacent perirhinal cortex. The intermediate and caudal portions gave positive results along the mediolateral extension of the entorhinal cortex. The laminar distribution of parvalbumin immunoreactive neurons was similar throughout the entorhinal cortex. Heavy immunostaining, largely coincident with cell islands, was observed in cells and fibers in layer II, being densest in the deep half of layer III and more sparsely distributed in layers V and VI. Calbindin D-28K immunoreactivity was found throughout the entorhinal cortex. In contrast to parvalbumin immunoreactivity, calbindin D-28K was present from layer I up to upper layer III, the neurons being most numerous in the cell islands of layer II. These results show that rostromedial portions of the human entorhinal cortex contain calbindin immunoreactivity, but not parvalbumin, while the lateral, intermediate and caudal portions of the entorhinal cortex contain both calcium-binding proteins. As it is known that these two proteins belong to a subset of GABAergic neurons, we suggest that a topographical diversity in some of the cells may be responsible for inhibitory effects in the human entorhinal cortex. This proposed diversity might be relevant to the processing of information that the entorhinal cortex conveys to the dentate gyrus and receives from various components of the hippocampus, the subicular complex and other cortical and subcortical sources.     

Light microscope study of the coexistence of GABA-like and glycine-like immunoreactivities in the spinal cord of the rat

The distributions of GABA-like and glycine-like immunoreactivities in the rat spinal cord were compared by using postembedding immunohistochemistry on semithin sections. In laminae I, II, and III, the proportions of GABA immunoreactive cells were 28%, 31%, and 46%, respectively, whereas for glycine immunoreactive cells the proportions were 9%, 14%, and 30%. Nearly all of the glycine immunoreactive cells in this area were also immunoreactive with the anti-GABA antiserum. In lamina II, some Golgi-stained islet cells were glycine immunoreactive, whereas others were not. Immunoreactive cell bodies were also present in the remainder of the grey matter. Some of these reacted with anti-GABA or antiglycine antiserum; others showed immunoreactivity with both antisera. Immunoreactive axons were found in the ventral and lateral funiculi of the white matter. Many large axons reacted with antiglycine antiserum, whereas GABA-immunoreactive axons were mostly of small diameter. Some large and small axons showed both types of immunoreactivity. These results suggest that the inhibitory neurotransmitters GABA and glycine coexist within cell bodies and axons in the rat spinal cord.     

Immunoreactivity for Taurine Characterizes Subsets of Glia,GABAergic and non-GABAergic Neurons in the Neo- and Archicortex of the Rat,Cat and Rhesus Monkey: Comparison with Immunoreactivity for Homocysteic Acid

The cerebral cortex is an area rich in taurine (2-aminoethanesulphonic acid), but only limited information exists regarding its cellular distribution. We therefore examined taurine-like immunoreactivity in the cerebral cortex of the rat, cat and macaque monkey using antiserum directed against glutaraldehyde-conjugated taurine. Immunostaining was assessed at the light and electron microscopic level, and patterns obtained in light microscopic studies were compared to those produced with antiserum to gamma-aminobutyric acid (GABA) and homocysteic acid (HCA). In all three species, strong taurine-like immunoreactive perivascular endothelial cells, pericytes and oligodendrocytes were found. These cells were located throughout the neuropil, which itself showed a low level of immunoreactivity. In rats and cats, a small number of weakly taurine-enriched neurons were observed, particularly in superficial layers. In all cortical areas of the macaque, however, glial staining was matched by strong, selective staining of subpopulations of cortical neurons which were distributed in a bilaminar pattern involving layers II/III and VI. In addition, in primary visual cortex, area 17, immunopositive neurons were also present in sublayer IVCbeta, while in the hippocampus strongly taurine-positive neurons were most conspicuous in the granule cell layer of the dentate gyrus. In all regions, strongly taurine-positive neurons constituted only a subpopulation of the neurons occupying a given layer. Examination of adjacent sections for GABA immunoreactivity showed that the most strongly taurine-positive neurons in layers II/III were immunoreactive for GABA. The cells located in layers IVCbeta and VI, and the granule cells of the dentate gyrus, however, were GABA-negative. The morphological features of these latter groups suggested that the antiserum to taurine identifies subsets of spiny stellate, small pyramidal and dentate granule cells. None of these neurons showed immunoreactivity with antiserum to HCA in the primate; HCA-positive glia were found along the pial and white matter boundaries of the cortex, and showed no overlap with strongly taurine-positive glial elements. Although a transmitter role for taurine may be unlikely, particularly in view of its enrichment in subpopulations of both inhibitory and excitatory cells, the capacity of taurine to influence membrane-associated functions in excitable tissues, and its selective distribution demonstrated here, provides the potential for a contribution to communication between cortical cells.     

Evidence for coexistence of GABA and dopamine in neurons of the rat olfactory bulb

Immunoreactivities for gamma-aminobutyric acid (GABA) and the dopamine-synthesizing enzyme tyrosine hydroxylase (TH) were localized ultrastructurally and colocalized at the light microscopic level in neurons of the rat main olfactory bulb. By means of a simultaneous indirect immunofluorescence technique, GABA and TH immunoreactivities were found to coexist in a large number of neurons in the glomerular and external plexiform layers. Virtually all the TH-immunoreactive periglomerular neurons also contained GABA immunoreactivity (GABA-I) while there was an additional number of GABA-immunoreactive periglomerular cells (27%) which did not contain TH immunoreactivity (TH-I). In contrast, the numerous tufted-type neurons in the glomerular and superficial external plexiform layers which contained TH-I did not contain GABA-I. In the external plexiform layer (EPL), 41% of the immunoreactive neurons contained GABA-I alone, 24% contained TH-I alone, and 35% contained both. EPL neurons containing GABA-I only or both GABA-I and TH-I never exhibited tufted cell morphological characteristics and were generally of the short-axon type. Electron microscopic examination of GABA-I and TH-I elements in the glomerular layer detected morphologically similar periglomerular perikarya and intraglomerular processes immunoreactive for each substance and other neurons and processes of the same type containing neither GABA-I or TH-I. These data indicate that the classical neurotransmitters GABA and dopamine coexist in large numbers of neurons in the rat main olfactory bulb including characteristic periglomerular cells and certain other local-circuit neuronal types.     

Evidence for commissurally projecting parvalbumin-immunoreactive basket cells in the dentate gyrus of the rat.

The fluorescent retrograde tracer, fluorogold, was used to identify commissurally projecting neurons in the hippocampus and dentate gyrus. After injection of fluorogold into the hippocampus, the contralateral hippocampus was evaluated for fluorogold-immunoreactive or fluorescent neurons. In addition to observing labeled hilar neurons and CA3 pyramidal cells that previously have been reported to send commissurally projecting axons to the contralateral hippocampus, the authors unexpectedly found a population of fluorogold-labeled cells in the granule cell layer with the morphology and location of GABA-immunoreactive basket cells. Immunocytochemical staining revealed that all fluorogold-labeled cells of the granule cell layer were immunoreactive for parvalbumin. However, not all parvalbumin cells, shown previously to be a subset of GABA neurons, were fluorogold-labeled. The association between fluorogold transport and parvalbumin immunoreactivity was unique for these cells of the granule cell layer. In the adjacent hilus, relatively few of the many fluorogold-labeled cells were parvalbumin- or GABA-immunoreactive. These results (1) identify a population of presumed inhibitory neurons that apparently form commissural projections; (2) document that all of these cells contain the calcium-binding protein parvalbumin; and (3) indicate that the vast majority of commissurally projecting hilar neurons are neither parvalbumin- nor GABA-immunoreactive.     

Vesicular gamma-aminobutyric acid transporter expression in amacrine and horizontal cells

The vesicular gamma-aminobutyric acid (GABA) transporter (VGAT), which transports the inhibitory amino acid transmitters GABA and glycine, is localized to synaptic vesicles in axon terminals. The localization of VGAT immunoreactivity to mouse and rat retina was evaluated with light and electron microscopy by using well-characterized VGAT antibodies. Specific VGAT immunoreactivity was localized to numerous varicose processes in all laminae of the inner plexiform layer (IPL) and to the outer plexiform layer (OPL). Amacrine cell somata characterized by weak VGAT immunoreactivity in the cytoplasm were located in the ganglion cell layer and proximal inner nuclear layer (INL) adjacent to the IPL. In rat retina, VGAT-immunoreactive cell bodies also contained GABA, glycine, or parvalbumin (PV) immunoreactivity, suggesting vesicular uptake of GABA or glycine by these cells. A few varicose VGAT-immunoreactive processes entered the OPL from the IPL. VGAT immunoreactivity in the OPL was predominantly localized to horizontal cell processes. VGAT and calcium binding protein-28K immunoreactivities (CaBP; a marker for horizontal cells) were colocalized in processes and terminals distributed to the OPL. Furthermore, VGAT immunoreactivity overlapped or was immediately adjacent to postsynaptic density-95 (PSD-95) immunoreactivity, which is prominent in photoreceptor terminals. Preembedding immunoelectron microscopy of mouse and rat retinae showed that VGAT immunoreactivity was localized to horizontal cell processes and their terminals. Immunoreactivity was distributed throughout the cytoplasm of the horizontal cell processes. Taken together, these findings demonstrate VGAT immunoreactivity in both amacrine and horizontal cell processes, suggesting these cells contain vesicles that accumulate GABA and glycine, possibly for vesicular release.     

Immunohistochemical evidence that Met-enkephalin and GABA coexist in some neurones in rat dorsal horn

A pre-embedding immunohistochemical method to detect Met-enkephalin was combined with postembedding immunohistochemistry with GABA and glycine antisera, in order to determine whether or not Met-enkephalin coexisted with either of these inhibitory transmitters in neuronal cell bodies within the superficial dorsal horn of the rat. The distribution of immunostaining with the three antisera was similar to that which has been described previously. Of 74 enkephalin-immunoreactive neurones in laminae II and III, 51 were immunoreactive with the GABA antiserum and 23 were not. All of the neurones which were not GABA-immunoreactive were located in lamina II. None of the enkephalin-immunoreactive cells showed glycine-like immunoreactivity. These results suggest that enkephalin is present both in GABAergic neurones and in neurones which do not contain GABA within the rat superficial dorsal horn. It is likely that the population of neurones immunoreactive with both enkephalin and GABA antisera includes lamina II islet cells and that the population which were enkephalin-immunoreactive but not GABA-immunoreactive includes stalked cells. In addition, this latter group may correspond to those cells which possess both enkephalin- and substance P-like immunoreactivity and which have been described previously in this area     

Calcium-binding proteins as markers for subpopulations of GABAergic neurons in monkey striate cortex

Recent studies have shown that the presence of immunoreactivity for parvalbumin (PV-IR) and calbindin-D 28k (Cal-IR) can be used as markers for certain types of gamma-aminobutyric acid (GABA) immunoreactive interneurons in monkey cerebral cortex. Little quantitative information is available regarding the features that distinguish these two subpopulations, however. Therefore, in this study we localized PV-IR and Cal-IR neurons in Macaca monkey striate cortex and analyzed quantitatively their laminar distribution, cell morphology, and co-localization with GABA by double-labeling immunocytochemistry. PV-IR was found in nonpyramidal cells in all layers of the cortex, although PV-IR cells in layer 1 were rare. In contrast, Cal-IR was found mainly in nonpyramidal cells in two bands corresponding to layers 2-3 and 5-6. We found very few double-labeled PV-IR/Cal-IR cells but confirmed that almost all PV-IR and Cal-IR cells are GABAergic. Overall, 74% of GABA neurons in striate cortex displayed PV-IR compared to only 12% that displayed Cal-IR and 14% that were GABA-IR only. Quantitative analysis indicated that the relative proportion of GABA cells that displayed PV-IR or Cal-IR showed conspicuous laminar differences, which were often complementary. Cell size measurements indicated that PV-IR/GABA cells in layers 2-3 and 5-6 were significantly larger than Cal-IR/GABA cells. Analysis of the size, shape, and orientation of stained cell bodies and proximal dendrites further demonstrated that each subpopulation contained several different types of smooth stellate cells, suggesting that Cal-IR and PV-IR are found in functionally and morphologically heterogeneous subpopulations of GABA neurons. There was a thick bundle of PV-IR axons in the white matter underlying the striate but not prestriate cortex. PV-IR punctate labeling matched the cytochrome oxidase staining pattern in layers 4A and 4C, suggesting that PV-IR is present in geniculocortical afferents as well as intrinsic neurons. Cal-IR neuropil staining was high in layers 1, 2, 4B, and 5, where cytochrome oxidase staining is relatively low. We did not find a preferential localization of either PV-IR or Cal-IR cell bodies in any cytochrome oxidase compartments in layers 2-3 of the cortex. These findings indicate that PV and Cal are distributed into different neuronal circuits.     

GABA-like immunoreactivity in the squirrel monkey organ of corti

The distribution of gamma-aminobutyric acid (GABA)-like immunoreactivity in the squirrel monkey organ of Corti was determined using an antiserum against GABA conjugated to bovine serum albumin. Immunoreactive labeling was seen in the region of the inner spiral bundle, the synaptic region below inner hair cells, in terminals contacting the basal part of outer hair cells, and in tunnel spiral fibers. Examples of each of these immunoreactive components could be observed in all cochlear turns. In the region of inner hair cells, immunoreactive labeling took the form of numerous small puncta randomly distributed below the base of the cells. In the region of outer hair cells, large globular immunoreactive structures reminiscent of terminal endings at the subnuclear level were observed. Since similar structures were seen at the base of outer hair cells in other cochleas processed for AChE, we conclude that GABA-like immunoreactivity was contained in efferent terminals which synapse on outer hair cells. These results strengthen previous evidence for the presence of GABA in the olivocochlear system of the mammalian cochlea.     

Connectivity of glycine immunoreactive amacrine cells in the cat retina

The synaptic relationships of glycine immunoreactive amacrine cells in the cat retina were studied through the use of postembedding immunogold techniques. Glycine immunoreactive amacrine cells were found to synapse extensively with other amacrines and ganglion cells, particularly in strata 1-3 of the inner plexiform layer. This contrasts with GABA immunoreactive amacrine cells which provide major input to bipolar cells in strata 3-5. Glycine containing amacrine terminals exhibited diversity with respect to the morphology of their synaptic vesicles. The three types of terminals which could be distinguished were characterized by small pleomorphic (32-35 nm), medium-sized flattened (38-45 nm), or larger rounded (48-55 nm) vesicles. Comparison of retinal sections processed for glycine immunoreactivity with adjacent sections stained for GABA reactivity revealed a colocalization of glycine and GABA in 3% of the cells in the amacrine layer and approximately 40% of the cells in the ganglion cell layer. The amacrine terminals in which glycine and GABA were colocalized typically contained the small pleomorphic type of vesicles.     

The role of calcium-binding proteins in selective motoneuron vulnerability in amyotrophic lateral sclerosis

The factors contributing to selective motoneuron loss in amyotrophic lateral sclerosis (ALS) remain undefined. To investigate whether calcium-binding proteins contribute to selective motoneuron vulnerability in ALS, we compared calbindin-D_28K and parvalbumin immunoreactivity in motoneuron populations in human ALS, and in a ventral spinal cord hybrid cell line selectively vulnerable to the cytotoxic effects of ALS IgG. In human autopsy specimens, immunoreactive calbindin-D_28K and parvalbumin were absent in motoneuron populations lost early in ALS (i.e., cortical and spinal motoneurons, lower cranial nerve motoneurons), while motoneurons damaged late or infrequently in the disease (i.e., Onuf's nucleus motoneurons, oculomotor, trochlear, and abducens nerve neurons) expressed markedly higher levels of immunoreactive calbindin-D_28K and/or parvalbumin. Motoneuron–neuroblastoma VSC 4.1 hybrid cells lost immunoreactive calbindin-D_28K and parvalbumin following dibutyryl-cyclic AMP–induced differentiation and were killed by IgG from ALS patients. Undifferentiated calbindin/parvalbumin-reactive VSC 4.1 cells were not killed, nor were other cell lines expressing high levels of calbindin-D_28K and parvalbumin immunoreactivity (substantia nigra–neuroblastoma hybrid cells and N18TG2 neuroblastoma parent cells). These studies suggest that decreased calbindin-D_28K and parvalbumin immunoreactivity may help explain the selective vulnerability of motoneurons in ALS.     

Heterogeneity of layer II neurons in human entorhinal cortex.

Abnormalities in the layer II neurons of human entorhinal cortex have been implicated in the pathophysiology of Alzheimer's disease and schizophrenia. The reported abnormalities are not homogeneously distributed throughout the entorhinal cortex, suggesting that layer II of entorhinal cortex may contain different subpopulations of neurons, each with a different susceptibility to pathological mechanisms. In order to investigate the possible heterogeneity of neurons in layer II of human entorhinal cortex, we first identified distinct subdivisions of human entorhinal cortex by adapting the cytoarchitectonic criteria for subdivisions of monkey entorhinal cortex described by Amaral et al. (J Comp Neurol 264:326, 1987). The morphology and regional distribution of distinct subpopulations of human layer II neurons were determined through the use of immunohistochemical techniques. Multipolar, stellate, and modified pyramidal neurons in the characteristic cell clusters or islands of layer II were immunoreactive for nonphosphorylated neurofilament proteins. The intensity of immunoreactivity for the nonphosphorylated neurofilament proteins gradually increased along the rostrocaudal axis of entorhinal cortex and was primarily due to a similar gradient in the density of labeled neurons per island. The calcium-binding protein calbindin D-28K was found in both pyramidal and nonpyramidal neurons in layers II and superficial III. The distribution of calbindin-immunoreactive neurons also depended upon the region of entorhinal cortex. In rostral entorhinal cortex, labeled neurons were scattered throughout the superficial layers, whereas in caudal entorhinal cortex, distinctive patches of small calbindin-immunoreactive neurons were found among the layer II islands. Another calcium-binding protein, parvalbumin, was present in nonpyramidal neurons in layers II and III that were distinct from those containing calbindin. The regional distribution of parvalbumin-positive neurons was very similar to that of the neurofilament immunoreactive neurons; in rostral entorhinal cortex very few parvalbumin-labeled neurons were present but their frequency gradually increased in the caudal direction. In addition, punctate parvalbumin immunoreactivity was frequently encountered in the location of the nonphosphorylated neurofilament protein-positive layer II islands. These findings demonstrate that layer II of human entorhinal cortex contains distinct subpopulations of neurons, that the relative density of each subpopulation differs across cytoarchitectonic regions, and that the patterns of distribution of these subpopulations are in some cases similar and in other cases complementary. This heterogeneity in the organization of layer II of human entorhinal cortex has important implications for the study of some neuropsychiatric disorders.     

Progression of temporal lobe epilepsy in the rat is associated with immunocytochemical changes in inhibitory interneurons in specific regions of the hippocampal formation

Immunocytochemical markers of specific rat hippocampal interneuron subpopulations, including the calcium binding proteins parvalbumin (PV), and calretinin (CR) were examined in relation to the evolution of spontaneous seizures after electrically induced status epilepticus (SE). PV/CR/NeuN immunoreactive neurons were counted in the hippocampal formation at different time intervals after SE and related to spontaneous hippocampal discharge activity. Decreased PV immunoreactivity was observed within 1 day after SE in the hilus, pre- and parasubiculum, and in the entorhinal cortex layers II and V/VI. In layer III, the density of detectable PV immunoreactive neurons did not decrease significantly, whereas the number of surrounding principal neurons was extensively decreased within a week in most post-SE rats, and after 3–4.5 months in all rats that had developed a progressive evolution of seizures. CR immunoreactive neuron number decreased in all hippocampal subregions except for the stratum lacunosum-moleculare and the EC layer II, in which the density did not decrease significantly. The apparent decrease in the number of PV and CR immunoreactive hilar neurons was correlated with the duration of the SE and was most extensive in rats with a progressive form of epilepsy. The loss of CR and PV expression or the loss of CR- and PV-containing neurons in specific regions of the hippocampal formation may play a role in the progressive nature of epilepsy possibly via increasing the entorhinal–hippocampal activity.     

High-Resolution Light and Electron Microscopic Immunocytochemistry of Colocalized GABA and Calbindin D-28k in Somata and Double Bouquet Cell Axons of Monkey Somatosensory Cortex

A simple method for high-resolution immunocytochemical colocalization of different antigens in semithin sections 1 - 3 μm thick was used to study the colocalization of the calcium binding protein calbindin D-28k (calbindin) with γ-aminobutyric acid (GABA) in double bouquet cells of monkey (Macaca fuscata) somatosensory cortex. Double bouquet cells were first visualized in vibratome sections by pre-embedding immunocytochemical staining for calbindin. Sections containing calbindin-immunoreactive somata and double bouquet cell axons were then osmicated, embedded in Araldite, resectioned at 1–3μm and stained for GABA by postembedding immunocytochemistry after elution of the bound anti-calbindin antibodies. Other semithin sections adjacent to those eluted and still containing calbindin immunoreactive somata and processes were resectioned at 60–70 nm for electron microscopy and stained immunocytochemically for GABA by the postembedding immunogold procedure. Calbindin-positive cells are most numerous in layer II and upper layer III, where they outnumber those in all other layers combined. In layers II and upper III, -30% of the stained cells are pyramidal and do not colocalize GABA. Only approximately two-thirds of the calbindin-stained nonpyramidal cells in these layers colocalize GABA, but among these virtually all the calbindin-positive double bouquet cells and their axons are GABA-immunoreactive. In deeper layers all calbindin-positive cells are nonpyramidal and all colocalize GABA. At the electron microscopic level, however, significant numbers of calbindin-positive axon terminals making symmetrical synapses are not GABA-immunoreactive. These results suggest the calbindin cells of monkey somatosensory cortex are a heterogeneous population that includes GABAergic and non-GABAergic cell types.     

Chemical Compartmentation and Relationships between Calcium-binding Protein Immunoreactivity and Layer-specific Cortical and Caudate-projecting Cells in the Anterior Intralaminar Nuclei of the Cat

Neurons projecting to the parietal cortex or striatum and neurons showing immunoreactivity for the calcium-binding proteins parvalbumin and 28_KD-calbindin were examined in the anterior intralaminar nuclei (IL) of the cat. Retrograde tracing from deep or superficial parietal cortical layers or from the caudate nucleus was coupled with immunohistochemistry to determine which of these proteins were expressed in the projection neurons. It was found that IL neurons project to deep as well as to superficial layers of the parietal cortex, that IL-cortical neurons could be differentiated into two populations according to their cortical projection pattern and their soma size, and that IL neurons projecting to the parietal cortex or to the striatum express 28_KD calbindin immunoreactivity but not parvalbumin immunoreactivity. The distribution of immunoreactivity to 28_KD calbindin and parvalbumin in the neuropil showed a consistent complementary distribution pattern in the IL. The compartments based on differential parvalbumin and 28_KD calbindin expression may indicate the presence of functionally segregated units in IL.     

Chemical organization of the macaque monkey olfactory bulb: II. Calretinin, calbindin D-28k, parvalbumin, and neurocalcin immunoreactivity

The distribution and morphologic features of calcium-binding protein- (calbindin D-28k, calretinin, neurocalcin, and parvalbumin) immunoreactive elements were studied in the macaque monkey olfactory bulb by using specific antibodies and the avidin-biotin-immunoperoxidase method. A characteristic laminar pattern of stained elements was observed for each marker. Scarce superficial short-axon cells and superficial stellate cells demonstrated calbindin D-28k immunoreactivity in the outer layers, whereas a moderate number of calbindin D-28k-immunoreactive granule cells and scarce deep short-axon cells were observed in the inner layers. Calretinin-staining demonstrated abundant periglomerular cells and granule cells and a scarce number of other interneuronal populations. Most neurocalcin-immunopositive elements were external and medial tufted cells and periglomerular cells, although other scarcer interneuronal populations were also immunostained. A few superficial and deep short-axon cells as well as small interneurons in the external plexiform layer were the only elements immunoreactive to parvalbumin. The distribution of the immunoreactive elements in the olfactory bulb of the macaque monkey showed a high similarity to that reported in the human, whereas it demonstrated a different and simpler pattern to what has been reported in the olfactory bulb of macrosmatic animals. It suggests more homogeneous calcium-mediated cell responses after stimulation that could be correlated to the lower capability to modulate olfactory signals in microsmatic animals. In addition, these results indicate that experimental models in rodents do not provide an accurate estimation of calcium-binding protein-immunoreactive neuronal populations in the primate olfactory system.     

Regional and cellular localisation of GABA(A) receptor subunits in the human basal ganglia: An autoradiographic and immunohistochemical study.

The regional and cellular localisation of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the human basal ganglia using receptor autoradiography and immunohistochemical staining for five GABA(A) receptor subunits (alpha(1), alpha(2), alpha(3), beta(2, 3), and gamma(2)) and other neurochemical markers. The results demonstrated that GABA(A) receptors in the striatum showed considerable subunit heterogeneity in their regional distribution and cellular localisation. High densities of GABA(A) receptors in the striosome compartment contained the alpha(2), alpha(3), beta(2, 3), and gamma(2) subunits, and lower densities of receptors in the matrix compartment contained the alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. Also, six different types of neurons were identified in the striatum on the basis of GABA(A) receptor subunit configuration, cellular and dendritic morphology, and chemical neuroanatomy. Three types of alpha(1) subunit immunoreactive neurons were identified: type 1, the most numerous (60%), were medium-sized aspiny neurons that were immunoreactive for parvalbumin and alpha(1), beta(2,3), and gamma(2) subunits; type 2 (38%) were medium-sized to large aspiny neurons immunoreactive for calretinin and alpha(1), alpha(3), beta(2,3), and gamma(2) subunits; and type 3 (2%) were large sparsely spiny neurons immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits. Type 4 neurons were calbindin-positive and immunoreactive for alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. The remaining neurons were immunoreactive for choline acetyltransferase (ChAT) and alpha(3) subunit (type 5) or were neuropeptide Y-positive with no GABA(A) receptor subunit immunoreactivity (type 6). The globus pallidus contained three types of neurons: types 1 and 2 were large neurons and were immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and for parvalbumin alone (type 1) or for both parvalbumin and calretinin (type 2); type 3 neurons were medium-sized and immunoreactive for calretinin and alpha(1), beta(2, 3), and gamma(2) subunits. These results show that the subunit composition of GABA(A) receptors displays considerable regional and cellular variation in the human striatum but are more homogeneous in the globus pallidus.     

GABA-like immunoreactivity in the chick vestibular end organs

gamma-Aminobutyric acid (GABA)-like immunoreactivity in the chick vestibular endorgans was examined using an antiserum against GABA coupled with glutaraldehyde to bovine serum albumin. GABA-like immunoreactivity was confined to the cytoplasm of the hair cells in both cristae and maculae. GABA-like immunoreactive cells were evenly distributed throughout the sensory epithelia, and no difference existed between type I and type II hair cells. The results provide evidence that GABA-like immunoreactivity is localized to sensory cells and raises the possibility that GABA may serve as an afferent neurotransmitter in the chick vestibular end organs.     

Postnatal development of parvalbumin and calbindin D-28k immunoreactivities in the canine anterior cingulate cortex: transient expression in layer V pyramidal cells

We have examined the ontogeny of parvalbumin (PV) and calbindin D-28k (CB) immunoreactivities in the canine anterior cingulate cortex (ACC) from the day of birth (P0) through P180. At P7, PV immunoreactivity first appeared in layer VI multipolar cells. The PV immunoreactivity in GABAergic nonpyramidal cells appeared to follow an inside-out gradient of radial emergence. Although immunoreaction was limited mainly to the developing nonpyramidal cells, pyramid-like PV immunoreactive cells were transitorily observed in layer V from P14 to P90. The developmental pattern of CB immunoreactivity differed from that of PV immunoreactivity. CB immunoreactivity first developed in layer V pyramidal cells from P0, which continued through P90. CB immunoreactive nonpyramidal cells were located in the infragranular layers and white matter at P0 and maturated in both the supragranular and infragranular layers without clear inside-out gradient.This developmental study revealed the comparable belated expression of PV immunoreactivity and the transient expression of both calcium-binding proteins in layer V pyramidal cells. These results suggest that the transient expression of calcium-binding proteins in layer V pyramidal cells might be related to the critical period of early postnatal development.     

Immunocytochemical localization of the amino acid neurotransmitters in the chicken retina

Postembedding immunocytochemistry was used to determine the cellular localization of the amino acid neurotransmitters glutamate, aspartate, gamma-aminobutyric acid (GABA), and glycine in the avian retina. The through retinal pathway was glutamatergic, with all photoreceptors, bipolar cells, and ganglion cells being immunoreactive for glutamate. Bipolar cells displayed the highest level of glutamate immunoreactivity, with the cell bodies terminating just below the middle of the inner nuclear layer. All lateral elements, horizontal cells, amacrine cells, and interplexiform cells were immunoreactive for glycine or GABA. The GABAergic neurons consisted of two classes of horizontal cells and amacrine cells located in the lower part of the inner nuclear layer. GABA was also localized in displaced amacrine cells in the ganglion cell layer, and a population of ganglion cells that co-localize glutamate and GABA. Both the horizontal cells and GABAergic amacrine cells had high levels of glutamate immunoreactivity, which probably reflects a metabolic pool. At least two types of horizontal cells in the avian retina could be discriminated on the basis of the presence of aspartate immunoreactivity in the H2 horizontal cells. Glycine was contained in a subclass of amacrine cells, with their cell bodies located between the bipolar cells and GABAergic amacrine cells, two subclasses of bipolar cells, displaced amacrine cells in the ganglion cell layer, and ganglion cells that colocalize glutamate and glycine. Glycinergic amacrine cells had low levels of glutamate. We have also identified a new class of glycinergic interplexiform cell, with its stellate cell body located in the middle of the inner nuclear layer among the cell bodies of bipolar cells. Neurochemical signatures obtained by analyzing data from serial sections allowed the classification of subclasses of horizontal cells, bipolar cells, amacrine cells, and ganglion cells. © 1993 Wiley-Liss, Inc.