Subpopulations of GABAergic neurons containing parvalbumin, calbindin D28k, and cholecystokinin in the rat hippocampus.

The possible coexistence of calbindin D28k with parvalbumin and of calbindin D28k with cholecystokinin was studied in nonpyramidal cells of the rat dorsal hippocampal formation. Neighbouring Vibratome sections were immunostained either for calbindin D28k and parvalbumin or for calbindin D28k and cholecystokinin. The cells, halved during sectioning, were identified in both sections immunostained for different antigens. The coexistence of calbindin D28k and parvalbumin in the same neuron was rare throughout the hippocampal formation with the exception of stratum oriens of the CA1 region, where 9.6% of the parvalbumin-immunoreactive cells also contained calbindin D28k. In stratum radiatum of the CA3 region, calbindin D28k and cholecystokinin coexisted in 12.5% and 21.2% of the calbindin D28k and cholecystokinin-immunoreactive cells, respectively. In other regions of the hippocampal formation, the two markers coexisted in less than 5% of the cells of either type. The present results demonstrate that calbindin D28k-, parvalbumin- and cholecystokinin-containing nonpyramidal cells represent largely nonoverlapping cell populations and may thus be involved in different inhibitory circuits.     

Transient Colocalization of Parvalbumin and Calbindin D28k in the Postnatal Cerebral Cortex: Evidence for a Phenotypic Shift in Developing Nonpyramidal Neurons

In the adult rat cerebral cortex the calcium-binding proteins parvalbumin and calbindin D28k are present in essentially non-overlapping populations of GABAergic interneurons. These proteins follow different developmental patterns in the cortex: calbindin D28k-immunoreactive nonpyramidal neurons are abundant until the second postnatal week and decrease markedly thereafter; it is at this time that parvalbumin immunoreactivity develops in cortical nonpyramidal neurons. To determine whether parvalbumin-immunoreactive neurons derive from calbindin D28k-positive cells we used double-immunofluorescence studies for both calcium-binding proteins, together with combined immunocytochemistry for calbindin D28k and in situ hybridization for parvalbumin mRNA during postnatal development. Double-labelled cells were found in all cortical layers between P9 and P21, coinciding with the onset of parvalbumin expression. The percentage of colocalization of the two calcium-binding proteins depended on the age and layer examined. Colocalization reached a peak (80–100%) during the second postnatal week in layers II–IV and VI and decreased thereafter to adult levels by the end of the third postnatal week. Double-labelled neurons were rare in layer V at all ages studied. The present results indicate a phenotypic shift during the development of some cortical interneurons that halts the expression of calbindin D28k while parvalbumin expression starts. These findings agree with lineage analyses reporting that different types of nonpyramidal neuron arise from a common progenitor.     

The coexistence of TrkA with putative transmitter agents and calcium-binding proteins in the vagal and glossopharyngeal sensory neurons of the adult rat

The presence of the neurotrophin receptor, TrkA, in neurochemically identified vagal and glossopharyngeal sensory neurons of the adult rat was examined. TrkA was colocalized with calcitonin gene-related peptide (CGRP), parvalbumin, or calbindin D-28k in neurons of the nodose, petrosal and/or jugular ganglia. In contrast, no TrkA-immunoreactive (ir) neurons in these ganglia colocalized tyrosine hydroxylase-ir. About one-half of the TrkA-ir neurons in the jugular and petrosal ganglia contained CGRP-ir, whereas only a few of the numerous TrkA-ir neurons in the nodose ganglion contained CGRP-ir. Although 43% of the TrkA-ir neurons in the nodose ganglion contained calbindin D-28k-ir, few or no TrkA-ir neurons in the petrosal or jugular ganglia were also labeled for either calcium-binding protein. These data show distinct colocalizations of TrkA with specific neurochemicals in vagal and glossopharyngeal sensory neurons, and suggest that nerve growth factor (NGF), the neurotrophin ligand for TrkA, plays a role in functions of specific neurochemically defined subpopulations of mature vagal and glossopharyngeal sensory neurons.     

Parvalbumin and calbindin D-28 k immunoreactivity in dorsal root ganglia in acquired immunodeficiency syndrome

Various degrees of neuronal degeneration have been found in lumbosacral dorsal root ganglia of patients with acquired immunodeficiency syndrome (AIDS). To characterize the subpopulations of primary sensory neurons affected in AIDS. we immunostained dorsal root ganglion tissues from 11 AIDS patients and six controls using antibodies to the calcium binding proteins, parvalbumin and calbindin D-28 k. In controls, the proportion of neurons containing parvalbumin and calbindin was 18.0% and 22.4%, respectively. The majority of parvalbumin-positive neurons, which are thought to be proprioceptive neurons, were of medium to large size, while calbindin was found in both large- and small-sized neurons. The density of parvalbumin-immunoreactive neurons was reduced by 7.3% in AIDS patients, but the density of calbindin-immunoreactive neurons was preserved. Furthermore, in AIDS cases, the number of parvalbumin-positive neurons was reduced more in dorsal root ganglia in which human immunodeficiency virus (HIV) antigen was detected than in HIV-negative ganglia. These results suggest that specific subpopulations of sensory neurons positive for parvalbumin may be differentially affected over the course of AIDS, and that this could be related to peripheral neuropathy which frequently occurs in the late stages of AIDS.     

Calbindin D-28k and parvalbumin immunoreactivity in the frontal cortex in patients with frontal lobe dementia of non-Alzheimer type associated with amyotrophic lateral sclerosis.

The morphology and distribution of local-circuit neurons (interneurons) were examined, by calbindin D-28k and parvalbumin immunocytochemistry, in the frontal cortex (area 8) in two patients with frontal lobe dementia of non-Alzheimer type associated with classical amyotrophic lateral sclerosis (ALS), and in seven normal cases. The density of calbindin D-28k immunoreactive cells was dramatically reduced in ALS patients, but the density of parvalbumin-immunoreactive neurons was preserved. Decreased density of calbindin D-28k-immunoreactive neurons, which are mainly located in the upper cortical layers, may interfere with the normal processing of cortico-cortical connections, whereas integrity of parvalbumin-immunoreactive cells may be associated with the preservation of the major inhibitory intracortical circuits in patients with frontal lobe dementia.     

Calretinin-containing neurons which co-express parvalbumin and calbindin D-28k in the rat spinal and cranial sensory ganglia; triple immunofluorescence study

The co-expression of calretinin with parvalbumin and calbindin D-28k was examined in the rat cranial and spinal sensory ganglia by triple immunofluorescence method. In the trigeminal and nodose ganglia, 9% and 5% of calretinin-immunoreactive neurons, respectively, also contained both parvalbumin- and calbindin D-28k immunoreactivity. These neurons had large cell bodies. In the trigeminal ganglion, they were restricted to the caudal portion. Such neurons were evenly distributed throughout the nodose ganglion. The co-expression could not be detected in the dorsal root, jugular or petrosal ganglia. Nerve fibers which co-expressed all the three calcium-binding proteins were observed in the inferior alveolar nerve but not the infraorbital nerve or palate. In the periodontal ligament, these nerve fibers formed Ruffini-like endings. These findings suggest that (1) the co-expression in trigeminal neurons is intimately related to their peripheral receptive fields; (2) the three calcium-binding proteins (calretinin, parvalbumin, calbindin D-28k) co-expressed in the trigeminal neurons may have mechanoreceptive function in the periodontal ligament.     

Differential expression of calbindin D28k, calretinin and parvalbumin in the cerebellum of pups of ethanol-treated female rats

Three calcium-binding proteins (CaBPs), calbindin D28k, calretinin and parvalbumin, were immunohistochemically examined in the cerebellum of ten-day-old rat pups of ethanol-treated dams. Dams were treated with ethanol during pregnancy and/or lactation. In the cerebellar cortex of the pups from control groups, Purkinje cells with their processes and Golgi cells were positive for calbindin D28k, whereas interneurons (Lugaro, Golgi and unipolar brush cells) and sometimes Purkinje cells were positive for calretinin. Parvalbumin immunoreactivity was observed in Golgi and basket cells, stellate cells and in some Purkinje cells. The number of positive cells and staining intensity for calbindin D28k and parvalbumin decreased in all experimental groups, whereas the immunoreaction for calretinin was visible only in interneurons and was more intense in experimental than in control groups. Calbindin D28k immunoreactivity in experimental groups was detected in some Purkinje cells and rarely in Golgi cells. The localization of very intense calretinin expression was visible mainly in unipolar brush cells. A parvalbumin-positive reaction was detected in single Purkinje cells and sometimes in basket cells. The results of the present study showed that immunoreactivity of the three calcium-binding proteins was found in the cells of the cerebellum of the ten-day-old pups from the control groups. In experimental groups of females treated with ethanol during pregnancy and/or lactation, we observed the most significant decrease in both the intensity and the number of immunoreactions of calbindin D28k and parvalbumin, but the intensity of the immunoreaction for calretinin was increased for interneurons. Ischaemic damage to Purkinje cells and loss of interneurons and Purkinje cells were also noted in these groups. A possible correlation between the duration of ethanol intoxication, expression of calcium-binding proteins and pathological changes of cells in the cerebellar cortex of the pups of ethanol-treated dams is discussed.     

Coexistence of calbindin D-28k and NADPH-diaphorase in vagal and glossopharyngeal sensory neurons of the rat

The presence and coexistence of calbindin D-28k-immunoreactivity (ir) and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase activity (a marker of neurons that are presumed to convert L-arginine to L-citrulline and nitric oxide) were examined in the glossopharyngeal and vagal sensory ganglia (jugular, petrosal and nodose ganglia) of the rat. Calbindin D-28k-ir nerve cells were found in moderate and large numbers in the petrosal and nodose ganglia, respectively. Some calbindin D-28k-ir nerve cells were also observed in the jugular ganglion. NADPH-diaphorase positive nerve cells were localized to the jugular and nodose ganglia and were rare in the petrosal ganglion. A considerable portion (33–51%) of the NADPH-diaphorase positive neurons in these ganglia colocalized calbindin D-28k-ir. The presence and colocalization of calbindin D-28k-ir and NADPH-diaphorase activity in neurotransmitter-identified subpopulations of visceral sensory neurons were also studied. In all three ganglia, calcitonin gene-related peptide (CGRP)-ir was present in many NADPH-diaphorase positive neurons, a subset of which also contained calbindin D-28k-ir. In the nodose ganglion, many (42%) of tyrosine hydroxylase (TH)-ir neurons also contained NADPH diaphorase activity but did not contain calbindin D-28k-ir. These data are consistent with a potential co-operative role for calbindin D-28k and NADPH-diaphorase in the functions of a subpopulation of vagal and glossopharyngeal sensory neurons.     

Spatial distributions of chemically identified intrinsic neurons in relation to patch and matrix compartments of rat neostriatum

The spatial distributions and dendritic branching patterns of chemically identified subpopulations of striatal intrinsic neurons, defined by immunoreactivity for choline acetyltransferase (ChAT), neuropeptide Y or parvalbumin, were studied in relation to patch and matrix compartments of rat neostriatum. ChAT-immunoreactive cells and fibers showed an uneven pattern of distribution in the striatum. ChAT immunoreactivity was higher in the dorsolateral part and lower in the ventromedial part of the striatum. This regional gradient pattern is the inverse of the overall pattern of calbindin D_28k immunoreactivity. However, in small regions close to the lateral ventricle and globus pallidus, areas containing fewer ChAT-immunoreactive cells and fibers coincided with those containing low calbindin D_28k immunoreactivity. Neuropeptide Y immunoreactivity was uniform in the neostriatum. Certain neuropeptide Y cells (about 20%) were also immunoreactive for calbindin D_28k, indicating that at least a small population of calbindin D_28k-immunoreactive cells are medium aspiny cells. Parvalbumin immunoreactivity was not uniform in the striatum. A higher density of parvalbumin immunoreactivity was found in the neuropil in lateral and caudal parts than in the medial part. Small regions with weaker parvalbumin-immunoreactive neuropil partially corresponded to calbindin D_28k poor patches. Larger cells immunoreactive for parvalbumin were preferentially located in lateral and caudal parts of the striatum. Cells immunoreactive for ChAT, neuropeptide Y or parvalbumin showed basically similar distribution patterns in relation to the patch and matrix compartments. Most stained cells were located in the matrix, but some were located at the borders of patches and a few were inside patches. Most primary dendrites of stained cells in the matrix or patches remained confined to these compartments, but cells on the borders invariably extended dendrites into both compartments. The striatal intrinsic neurons form chemically differentiated neuronal circuits within the matrix, and the patches and those whose dendrites cross the borders may contribute to associational interconnections between the two compartments, unlike the spiny projection neurons whose dendrites are confined to one or the other compartment. © Wiley-Liss, Inc.     

ASIC3-immunoreactive neurons in the rat vagal and glossopharyngeal sensory ganglia

ASIC3-immunoreactivity (ir) was examined in the rat vagal and glossopharyngeal sensory ganglia. In the jugular, petrosal and nodose ganglia, 24.8%, 30.8% and 20.6% of sensory neurons, respectively, were immunoreactive for ASIC3. These neurons were observed throughout the ganglia. A double immunofluorescence method demonstrated that many ASIC3-immunoreactive (ir) neurons co-expressed calcitonin gene-related peptide (CGRP)- or vanilloid receptor subtype 1 (VRL-1)-ir in the jugular (CGRP, 77.8%; VRL-1, 28.0%) and petrosal ganglia (CGRP, 61.7%; VRL-1, 21.5%). In the nodose ganglion, however, such neurons were relatively rare (CGRP, 6.3%; VRL-1, 0.4%). ASIC3-ir neurons were mostly devoid of tyrosine hydroxylase in these ganglia. However, some ASIC3-ir neurons co-expressed calbindin D-28k in the petrosal (5.5%) and nodose ganglia (3.8%). These findings may suggest that ASIC3-containing neurons have a wide variety of sensory modalities in the vagal and glossopharyngeal sensory ganglia.     

Cortical neurons expressing calcium binding proteins are spared in dementia with Lewy bodies

The consistent regional and laminar distribution of cortical Lewy bodies (LB) in brains of patients with dementia with Lewy bodies (DLB) suggests that only a certain subpopulation of neurons develops these alpha-synuclein-immunoreactive cytoplasmic inclusions. This study examined whether four non-overlapping neuronal subpopulations, defined by the expression of non-phosphorylated neurofilaments (SMI-32) and several calcium binding proteins (parvalbumin, calretinin and calbindin D28k), are vulnerable to LB formation. We performed peroxidase immunostaining to examine the distribution and to quantitate each neuronal subpopulation within the superior temporal sulcus (STS) area, and double-label immunohistochemistry to test for colocalization of alpha-synuclein and each neuronal marker in the STS and the entorhinal cortex. There were no significant differences between DLB brains and controls in the proportional quantity or laminar distribution of each neuronal subpopulation. Parvalbumin-immunoreactive neurons represented around 7%, calbindin D28k 8%, calretinin 10%, and SMI-32 about 20% of the total neuronal population in the STS cortex. Neurons expressing parvalbumin and SMI-32 showed a widespread distribution across layers II to VI. Neurons expressing calretinin were present in superficial layers (II to IV), and calbindin D28k-immunoreactive neurons were mostly distributed within granular layers II and IV. None of the LB observed in the STS or the entorhinal cortex were located in neurons expressing calcium binding proteins; 25% of the LB were contained in SMI-32 immunoreactive neurons. In conclusion, cortical neurons expressing calcium binding proteins are spared in DLB, while SMI-32-positive neurons are affected in proportion to their density in the cortex. However, the majority of cortical LB develop in neurons not identified by any of these markers.     

GABAergic Interneurons are the Major Postsynaptic Targets of Median Raphe Afferents in the Rat Dentate Gyrus

The termination pattern of median raphe axons was studied in the rat dentate gyrus using Phaseolus vulgaris leucoagglutinin as an anterograde tracer, in combination with postembedding immunostaining for γ-amino-butyric acid (GABA), and pre-embedding immunostaining for calbindin D28k, parvalbumin and GABA. Postembedding immunogold staining for GABA revealed that the majority (73.7%) of anterogradely labelled median raphe boutons make synaptic contacts with GABA-immunoreactive postsynaptic targets, mainly with dendritic shafts and perikarya. Pre-embedding immunocytochemical double staining for the anterograde tracer and GABA confirmed the electron microscopic results and showed that varicose median raphe axons establish multiple contacts with fusiform interneurons in the hilus and different types of basket cells in the granule cell layer. Some of the innervated cells were shown to contain calbindin D28k, whereas GABAergic interneurons containing another calcium-binding protein, parvalbumin, were never seen to receive multiple contacts from axons of raphe origin. Our results suggest that serotonergic median raphe fibres influence the firing of dentate granule cells via local inhibitory interneurons. The mechanism of using these interneurons with extensive local connections as monosynaptic targets may explain the great efficacy of this pathway in the control of hippocampal electrical activity.     

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.     

Effect of mCOUP-TF1 deficiency on the glossopharyngeal and vagal sensory ganglia

Immunohistochemistry for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase and calbindin D-28k was performed on the glossopharyngeal and vagal ganglia in mCOUP-TFI knockout mice to know the effect of its deficiency on different types of primary sensory neurons. In wild type and heterozygous mice, the glossopharyngeal and vagal ganglia contained abundant CGRP-, tyrosine hydroxylase- and calbindin D-28k-immunoreactive (IR) neurons. In the ganglia of mCOUP-TFI knockout mice, a 38% decrease of CGRP-IR neurons was detected. However, the number of tyrosine hydroxylase- or calbindin D-28k-neurons was not altered by the mCOUP-TFI deficiency. In the tongue of knockout mice, the number of CGRP-IR nerve fibers decreased compared to wild-type and heterozygous mice. The development of CGRP-IR petrosal neurons, which supply innervation of the tongue, may depend on mCOUP-TFI.     

Calbindin D-28K- and parvalbumin-reacting neurons in the hypothalamic magnocellular neurosecretory nuclei of the rat.

The distribution of calbindin D-28K- and parvalbumin-reacting neurons in the hypothalamic magnocellular neurosecretory nuclei of the rat was studied using the avidin-biotin-immunoperoxidase method and highly specific monoclonal antibodies. Incubation with anticalbindin D-28K-antiserum revealed immunoreactive neurons in the following nuclei: supraoptic, paraventricular (both in the magnocellular and parvicellular regions), circularis, fornicals and medial forebrain bundle. Incubation with parvalbumin antiserum displayed immunoreactive neurons only in the circularis nucleus. Additionally, it was possible to observe scattered calbindin and parvalbumin immunoreactive neurons (which do not form part of the nuclei considered) located in the hypothalamic area between the supraoptic and the paraventricular nuclei, especially for the calbindin D-28K antiserum.     

Estrogen receptor alpha containing neurons in the monkey forebrain: lack of association with calcium binding proteins and choline acetyltransferase

The present study used single and dual immunohistochemistry to determine the topography and chemical phenotype of ERalpha containing neurons within the monkey forebrain utilizing antibodies directed against the full-length human ERalpha (NCL-ER-6F11), calcium-binding proteins calbindin-D(28k), and parvalbumin as well as choline acetyltransferase (ChAT). Our findings demonstrate for the first time ERalpha immunoreactive (-ir) cells in the monkey cerebral cortex (layers I-II) and in the claustrum. In addition, ERalpha-ir cells were seen in the septum, basal forebrain, amygdala and hypothalamus. Double-labeled cells for ERalpha and calbindin-D(28k) were seen only in the ventrolateral part of the ventromedial hypothalamic nucleus. In contrast, the co-localization of ERalpha and parvalbumin or ChAT was not seen in any of the areas of the monkey forebrain examined. These observations suggest that estrogens, at least in part, via ERalpha regulate calbindin-D(28k) hypothalamic but not parvalbumin or ChAT containing neurons in select monkey forebrain regions.     

Colocalization of neuropeptides with calcium-binding proteins in the claustral interneurons during postnatal development of the rat

The claustrum is a relatively large telencephalic structure, situated close to the border of the neo- and allocortical regions. Its neuronal population consists of glutamatergic, projecting neurons and GABA-ergic interneurons, characterized by occurrence of numerous additional biochemical markers. The postnatal development of these latter neurons has not been extensively studied. Revealing the characteristic patterns of colocalizations between selected markers may shed some light on their function and origin.We investigated the colocalization patterns between three neuropeptides: neuropeptide Y, somatostatin, vasoactive intestinal polypeptide and three calcium-binding proteins: calbindin D28k, calretinin, parvalbumin in the interneurons of the rat claustrum during a four-month postnatal period (P0–P120; P: postnatal day).Our studies revealed the following types of colocalizations: neuropeptide Y with calbindin D28k, calretinin or parvalbumin; somatostatin with calbindin D28k; vasoactive intestinal polypeptide with calretinin. Only vasoactive intestinal polypeptide- and calretinin-containing, double-labeled neurons were present at the day of birth, whereas the other double-labeled neurons appeared at later stages of development. The ratios of colocalizing neurons to single-labeled neurons in each type of colocalization were differentiated and reached the highest value (51%) for vasoactive intestinal polypeptide- and calretinin-double-labeled neurons.In conclusion, the claustral interneurons represent differentiated population in respect to the occurrence of neuropeptides and calcium-binding proteins. The expression of studied substances is changing during the postnatal period.     

Parvalbumin and calbindin D-28k immunoreactivity in transgenic mice with a G93A mutant SOD1 gene

Immunohistochemical study was performed to examine if calcium-binding proteins are involved in the degeneration of motor neurons in the brain stems and the spinal cords of transgenic mice carrying a G93A mutant human SOD1 gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the spinal cord of the controls, the density of parvalbumin-immunoreactive neurons was highest in the large anterior horn neurons and lower in the posterior horn neurons in the spinal cord. On the other hand, calbindin D-28k immunoreactivity was much less apparent than that observed with parvalbumin antisera. Rexed's lamina II was densely immunostained for calbindin D-28k, whereas, in the anterior horn, calbindin-D-28k-positive small neurons were barely dispersed in a scattered pattern. In transgenic mice, parvalbumin-positive anterior horn neurons were severely reduced, even at the presymptomatic stage, whereas calbindin-positive neurons were largely preserved. At the symptomatic stage, both parvalbumin and calbindin D-28k immunoreactivity markedly diminished or disappeared in the anterior horn. Immunoblotting analysis revealed a significant reduction of immunoreactivity to parvalbumin antibody in transgenic mice compared with the controls. In the brain stem, parvalbumin-positive oculomotor and abducens neurons and the calbindin D-28k-positive sixth nucleus were well-preserved in transgenic mice as well as in the controls. Thus, the diffuse and severe loss of parvalbumin immunoreactivity of large motor neurons even at early stages in SOD1-transgenic mice and the absence of calbindin D-28k immunoreactivity of normal large motor neurons suggest that these calcium-binding proteins may contribute to selective vulnerability and an early loss of function of large motor neurons in this SOD1-transgenic mouse model.     

Calbindin D-28k Protein and mRNA Localization in the Rat Brain

After the discovery of calretinin, a protein with high sequence homology to calbindin D-28k, the validity of immunohistochemical results obtained using polyclonal antibodies for this protein, was in question. In order to validate the previous results on the localization of calbindin D-28k in the brain, we localized the protein by highly specific monoclonal antibodies and revealed its mRNA histochemically by in situ hybridization. In general there was good agreement between the results obtained using these two different techniques and those reported in previous publications. The concordance was particularly impressive for the cerebral cortex, basal ganglia, basal nucleus of Meynert, hippocampus, thalamus, cerebellum and superior colliculus. In the amygdala and hypothalamus the low spatial resolution of in situ hybridization did not allow precise definition of some nuclei displaying a positive reaction for the protein. In the rhombencephalon, cells of the parabrachial nuclei and the dorsal raphe nucleus expressed calbindin D-28k. Neurons in the dorsal horn of the spinal cord and some horizontal cells of the retina were tagged with both methods. The only discrepancy was the presence of immunoreactive ependymal cells, whereas mRNA never occurred in cells lining the ventricles. Thus, the combined approach has established the widespread distribution of cells expressing calbindin D-28k in the rat brain.     

Calretinin in neurochemically well-defined cell populations of rabbit retina

In the rabbit retina, parvalbumin has been localized selectively to AII amacrine cells, while 28 kDa calbindin could be detected in horizontal cells, in one type of depolarizing cone bipolar cell and a population of wide-field amacrine cells. The distribution of the third neuronal calcium binding protein, calretinin, however, has not been studied to date in detail in the rabbit retina. Therefore in this study we aimed to describe the overall distribution of calretinin in the different retinal layers and the possible colocalization pattern with other neurochemical marker molecules. A few cone photoreceptor cells were found to be labeled, whereas the outer plexiform layer was free from immunoreactive elements. In the most proximal row of the inner nuclear layer amacrine cells were labeled, while more distally a few cells emitted beaded axon-like processes, toward the outer retina. There were large (18–28 μm in diameter) cells labeled in the ganglion cell layer, of which many apparently had their axon stained. Some of the calretinin immunoreactive amacrine cells (the AII neurons) also contained parvalbumin. Colocalization of calretinin and 28 kDa calbindin could not be ascertained in the same amacrine cell populations, nor was tyrosine hydroxylase present in calretinin-containing cells. There was partial colocalization of calretinin in the γ-aminobutyric acid-positive amacrine cell population. Parvalbumin containing ganglion cells were also positive for calretinin; however, the calretinin-positive ganglion cells were more numerous. γ-Aminobutyric acid could be colocalized in some calretinin-positive neurons of the ganglion cell layer.