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.     

Calbindin D-28k immunoreactivity in the rat accessory olfactory bulb

The distribution pattern and the morphology of calbindin D-28k-immunoreactive neurons were studied in the accessory olfactory bulb of the rat using a monoclonal antibody and the avidin-biotin-immunoperoxidase method. Positive neurons were observed in all layers but the vomeronasal nerve layer. Scarce monodendritic periglomerular neurons were calbindin D-28k-immunoreactive. Different morphological types of short-axon cells were calbindin D-28k-immunostained, with different degrees of intensity, in the boundary between the internal and external plexiform layer. In addition, deep short-axon cells located in the granule cell layer were calbindin D-28k-immunopositive. By contrast, previous studies described all cells in the rat accessory olfactory bulb as calbindin D-28k-immunonegative. The staining pattern in the rat accessory olfactory bulb showed both similarities and differences with the distribution pattern of the same calcium-binding protein in the main olfactory bulb.     

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.     

Complementary Distribution of Calbindin D-28k and Parvalbumin in the Basal Forebrain and Midbrain of the Squirrel Monkey

The distribution of cell bodies expressing either calbindin D-28k or parvalbumin immunoreactivity in the basal forebrain and midbrain of squirrel monkeys (Saimiri sciureus) was studied on contiguous sections incubated with monoclonal antibodies raised against calbindin or parvalbumin. In the nucleus accumbens, medium-sized calbindin-positive neurons formed two cell bridges joining the ventral part of the striatum to the olfactory tubercle, whereas medium-sized parvalbumin-positive cells in the same area were much less numerous and more uniformly distributed. The medial and dorsal septal nuclei contained a small number of elongated calbindin-positive neurons and only a few parvalbumin-immunoreactive cells. In the nucleus of the diagonal band of Broca, calbindin and parvalbumin were found to label two distinct but closely intermingled neuronal populations. In the striatum, medium-sized calbindin-immunoreactive cells occurred in very large numbers and appeared to be confined to the extrastriosomal matrix. Medium-sized, parvalbumin-immunoreactive neurons were also present in the striatum but they were less numerous than the calbindin-positive cells. The calbindin-positive neurons in the dorsal portion of the striatum were less intensely stained than those in the ventral portion, whereas this pattern did not occur for neurons expressing parvalbumin immunoreactivity. At the pallidal level, neurons in both segments were devoid of calbindin but displayed a very strong parvalbumin immunoreactivity. Most of the large neurons of the nucleus basalis of Meynert were strongly calbindin-immunoreactive and many of them invaded dorsally the medullary laminae of the pallidal complex. The neurons of the subthalamic nucleus were markedly enriched with parvalbumin but displayed only light calbindin staining. In the substantia nigra/ventral tegmental area complex, calbindin-immunoreactive cells abounded in the ventral tegmental area and in the dorsal tier of the pars compacta of the substantia nigra, but were absent in the ventral tier of the pars compacta and in the entire pars reticulata of the substantia nigra. In contrast, numerous parvalbumin-immunoreactive neurons occurred in the pars reticulata and pars lateralis, but none were found in the pars compacta and ventral tegmental area. These findings reveal that the patterns of calbindin and parvalbumin distribution in primate basal forebrain and midbrain are strikingly complementary, suggesting a synergistic role for these calcium-binding proteins in basal forebrain and midbrain function.     

Calbindin D-28k as a marker for the associative cortical territory of the striatum in macaque

An immunohistochemical study was made to investigate the topographic distribution of calbindin D-28k in relation to the associative and sensorimotor cortical territories in the macaque. An intense calbindin-staining was found in the caudate nucleus and ventromedial putamen, i.e., in the associative striatum. In contrast, only a weak immunoreaction was found in the dorsolateral, sensorimotor, putamen. Calbindin immunoreactivity thus appears as a specific marker for the associative striatum.     

Calbindin D-28k- and substance P-immunoreactive primary sensory neurons: Peripheral projections in chick hindlimbs

The peripheral projections of two distinct subpopulations of primary sensory neurons, expressing either calbindin D-28k or substance P, were studied in chick hindlimbs by immunodetecting calbindin D-28k with a rabbit antiserum and substance P with a mouse monoclonal antibody. Calbindin D-28k-immunoreactive axons provided an innervation restricted to specific mechanoreceptors such as muscle spindles, Herbst and Merkel corpuscles, or collars of feather follicles but were absent from Golgi tendon organs. In contrast, substance P-positive axons spread out diffusely in muscles and skin, formed loose plexuses, and extended free branches to the endomysium, arteries, superficial dermis, or dermal pulp of feather follicles. The present results show that calbindin D-28k- and substance P-immunoreactive primary sensory neurons provide distinct modes of innervation to selective targets in peripheral tissues. The results suggest a possible correlation between CaBP-expressing nerve endings and rapidly adapting mechanoreceptors. © 1993 Wiley-Liss, Inc.     

Alteration in Levels of Expression of Brain Calbindin D-28k and Calretinin mRNA in Genetically Epilepsy-Prone Rats

Summary: Variations in the concentration of free calcium in neurons is believed to play a major role in regulating neuronal excitability. Because calcium-binding proteins such as calbindin D-28k and calretinin help to regulate intracellular calcium, we investigated the possibility that the expression of these proteins may be affected in genetically epilepsy-prone rats (GEPRs). The mRNA levels of both proteins were compared across several brain regions using in situ hybridization histochemistry and Northern blot analysis with semiquantitation by optical density measures on autoradiograms from two GEPR strains that differ in the severity of audiogenic seizures (GEPR9 and GEPR3) and from Sprague-Dawley rats. Results revealed a lower level of expression in calbindin D-28k mRNA in the caudate putamen-accumbens nuclei in GEPR3 (-30%) and GEPR9 (-60%) relative to controls. The calbindin D-28k mRNA level was also lower in the reuniens nucleus of the thalamus (-41% in GEPR3; -34% in GEPR9). The calretinin mRNA level was lower in the substantia nigra compacta of both GEPR rat strains (-31% in GEPR3 and -34% in GEPR9 relative to controls). No changes in mRNA were detected in other brain regions expressing calbindin D-28k or calretinin mRNA. These results indicate that the expression of these related calcium-binding proteins is altered in the GEPRs before the induction of seizures. This initial defect could alter either the calcium-buffering capacity or regulation of calcium-mediated processes by these proteins and thus play a role in the molecular cascade of events inducing the genetic susceptibility to, and the generalization of, seizures in these rat strains.     

Calbindin D-28K in the dopaminergic mesocortical projection of a monkey (Aotus trivirgatus)

Injections of fluorescent dyes were made in the prefrontal and motor cortex of owl monkeys and retrogradely labeled neurons in the mesencephalon were analyzed for tyrosine hydroxylase and calbindin-D28K immunostaining. Numbers of mesocortical dopaminergic neurons in the dorsal substantia nigra compacta and in the ventral tegmental area also contain calbindin-D28K. This cortically projecting calbindin-D28K containing subpopulation of the dopaminergic mesencephalic cells may be characterized by different electrophysiological properties and a lesser vulnerability to cell death.     

Calbindin D-28k immunoreactivity increases in the hippocampus after long-term treatment of soy isoflavones in middle-aged ovariectomized and male rats

This article investigates the long-term effects of soybean isoflavones (ISO) on the changes of calbindin D-28k (CB) immunoreactivity in the hippocampus in middle-aged ovariectomized female rats as well as middle-aged control female and male rats to identify any correlation between calcium and phytoestrogens. In the CA1 region, CB immunoreactivity in the ovariectomized females was similar to that of the control females, whereas CB immunoreactivity in the males was significantly lower than that of the control females. In the dentate gyrus, CB immunoreactivity in the ovariectomized females and males was significantly lower than that of the control females. CB immunoreactivity in all groups was increased dose-dependently after ISO treatment in the CA1 region and dentate gyrus. This result suggests that ISO treatment enhances the expression of CB immunoreactivity in the hippocampus in the middle-aged rats.     

Quantitative Distribution of Parvalbumin, Calretinin, and Calbindin D-28k Immunoreactive Neurons in the Visual Cortex of Normal and Alzheimer Cases

The distribution of parvalbumin (PV), calretinin (CR), and calbindin (CB) immunoreactive neurons was studied with the help of an image analysis system (Vidas/Zeiss) in the primary visual area 17 and associative area 18 (Brodmann) of Alzheimer and control brains. In neither of these areas was there a significant difference between Alzheimer and control groups in the mean number of PV, CR, or CB immunoreactive neuronal profiles, counted in a cortical column going from pia to white matter. Significant differences in the mean densities (numbers per square millimeter of cortex) of PV, CR, and CB immunoreactive neuronal profiles were not observed either between groups or areas, but only between superficial, middle, and deep layers within areas 17 and 18. The optical density of the immunoreactive neuropil was also similar in Alzheimer and controls, correlating with the numerical density of immunoreactive profiles in superficial, middle, and deep layers. The frequency distribution of neuronal areas indicated significant differences between PV, CR, and CB immunoreactive neuronal profiles in both areas 17 and 18, with more large PV than CR and CB positive profiles. There were also significantly more small and less large PV and CR immunoreactive neuronal profiles in Alzheimer than in controls. Our data show that, although the brain pathology is moderate to severe, there is no prominent decrease of PV, CR and CB positive neurons in the visual cortex of Alzheimer brains, but only selective changes in neuronal perikarya.     

Parvalbumin- and calbindin D28k-immunoreactive neurons in the hippocampal formation of the macaque monkey.

Calcium-binding proteins calbindin D28k (CaBP) and parvalbumin (PV) were localized in neurons of the monkey hippocampal formation. CaBP immunoreactivity is present in all granule cells and in a large proportion of CA1 and CA2 pyramidal neurons, as well as in a distinct population of local circuit neurons. In the dentate gyrus, CaBP-immunoreactive nongranule cells are present in the molecular layer and in the hilar region, but they do not include the pyramidal basket cells at the hilar border. In the Ammon's horn, CaBP-positive, nonpyramidal neurons are more frequent in the CA3 area than in any other parts of the hippocampal formation. They are concentrated in the strata oriens and pyramidale of areas CA1-3, whereas only a few small neurons were found in the strata lucidum and radiatum of CA3 and in the stratum moleculare of the CA1 area. PV is exclusively present in local circuit neurons both in the dentate gyrus and in Ammon's horn. In the dentate gyrus the presumed basket cells at the hilar border exhibit PV immunoreactivity. In the hilar region and molecular layer only a relatively small number of cells are immunoreactive for PV. Most of these PV-positive cell bodies are located in the inner half of the molecular layer, with occasional horizontal cells at the hippocampal fissure. In Ammon's horn, strata oriens and pyramidale of areas CA1-3 contain a large number of PV-positive cells. There are no PV-immunoreactive cells in the strata lucidum, radiatum, or lacunosum moleculare. The CaBP- and PV-containing neurons form different subpopulations of cells in the monkey hippocampal formation. With the exception of a basket cell type in the monkey dentate gyrus, the CaBP- and PV-positive cell types were found to be remarkably similar in rodents and primates.     

Thalamic and Basal Forebrain Afferents Modulate the Development of Parvalbumin and Calbindin D28k Immunoreactivity in the Barrel Cortex of the Rat

In the adult barrel cortex of the rat the calcium-binding proteins calbindin D28k (CALB) and parvalbumin (PARV) are found in separate populations of GABAergic nonpyramidal neurons. In layers II to IV of the barrel cortex most PARV-immunoreactive neurons are likely to derive from a subpopulation of CALB-immunoreactive neurons whose CALB immunoreactivity ceases when they begin to express PARV between the second and third postnatal weeks. The aim of this study was to investigate the influence of subcortical afferents on the neurochemical differentiation of cortical PARV- and CALB-immunoreactive nonpyramidal neurons during development of the barrel cortex. We produced unilateral excitotoxic lesions with a single injection of ibotenic acid (0.5 μ, 0.05 M) in different subcortical nuclei in 7-to 8-day-old rats. Lesions involving the ventroposterior thalamic nuclei resulted in delayed development of PARV and CALB immunoreactivity in the barrel cortex. One week after ibotenic acid injections a transient decrease in the number of PARV-immunoreactive neurons in layer IV was observed, together with increased numbers of CALB-immunoreactive neurons in all cortical layers. The number of nonpyramidal neurons displaying coexistence of PARV and CALB in the lesioned hemisphere also increased compared with the numbers in the control hemisphere or control littermates. In contrast, lesions affecting the globus pallidus, zona incerta and reticular thalamic nucleus transiently increased the number of PARV-immunoreactive neurons in layers II and III, but had no effect on the number of CALB-positive cells. From 3 weeks onwards no differences were found between control and iesioned hemispheres after injections into either the ventroposterior thalamic nuclei or the magnocellular basal forebrain. These results suggest that CALB and PARV expression in nonpyramidal cortical neurons can be reversibly modulated in opposite directions by different cortical afferents during postnatal development.     

Calbindin D-28k and choline acetyltransferase are expressed by different neuronal populations in pedunculopontine nucleus but not in nucleus basalis in squirrel monkeys

Single- and double-immunostaining procedures were used to study the distribution of the acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) and the calcium binding protein calbindin D-28k in the nucleus basalis of Meynert (nbM) and in the pedunculopontine nucleus (PPN) of the squirrel monkey (Saimiri sciureus). As expected from previous studies in other primates, including humans, the nbM in the squirrel monkey is enriched with large ChAT-immunoreactive neurons that form clusters in the substantia innominata. Some ChAT-positive neurons are also scattered more dorsally within the internal and external medullary laminae of the pallidal complex. A smaller number of calbindin-immunoreactive cells occur in the same locations and their mean cross-sectional somatic area (424 microns 2) is not significantly different from that of the ChAT-immunoreactive cells (450 microns 2). Furthermore, 60% of the ChAT-immunopositive cells in the nbM display calbindin immunoreactivity. Most of these double-immunoreactive neurons occur in the typical clusters of the nbM, whereas the large neurons scattered in between the clusters display ChAT immunoreactivity only. In the PPN, ChAT-positive neurons are scattered around and partly within the superior cerebellar peduncle and also form a dense cluster in the lateral portion of the mesopontine tegmentum. Calbindin-immunoreactive cells also abound around the superior cerebellar peduncle, but they are more sparsely distributed and cover a larger sector of the tegmentum than the ChAT-positive neurons. These calbindin-immunoreactive cells are significantly smaller (200 microns 2) than the ChAT-immunoreactive cells (471 microns 2) and no double-immunostained neurons are present in the PPN.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Calbindin D-28k is expressed in the microvascular basal lamina in the ventral horn at early time after transient spinal cord ischemia in the rabbit

Much evidence has been accumulated that the increased expression of calbindin D-28k (CB) is involved in the blockade of calcium-evoked excitotoxicity in cerebral ischemia. We investigated the expression of CB in the basal lamina of microvessels in the ventral horn of the rabbit spinal cord after transient spinal cord ischemia. Spinal cord sections at the level of L7 were immunostained using monoclonal antibody raised against CB at light and electron microscopic levels. CB immunoreactivity was detected in the basal lamina of microvessels at 30 min after ischemic insult. By 3 h after ischemia, CB immunoreactivity was increased in the basal lamina of the microvessels. CB immunoreactivity began to decrease at 6 h after ischemia and nearly disappeared at 48 h after ischemic insult. For calcium detection in the blood vessels of spinal cord, we conducted an alizarin red staining. Alizarin red reactivity was detected in some microvessels at 3 h after ischemic insult. Our results suggest that the ectopic expression of CB in the microvascular basal laminae may be associated with the buffering of calcium in the endothelial cells of microvessels after ischemic damage.     

Calbindin D28k-containing splanchnic and cutaneous dorsal root ganglion neurons of the rat

Calbindin D28k (CaBP)-containing splanchnic and cutaneous sensory neurons in the rat dorsal root ganglia (DRGs) were investigated immunocytochemically in combination with a fluorescent dye tracer (Fluoro gold). About 15% of the DRG neurons at Th9-10 levels showed CaBP-like immunoreactivity. Eighty-four % of the splanchnic sensory neurons were immunoreactive to CaBP, while only 3% of the cutaneous sensory neurons were. The diameters of the splanchnic and cutaneous sensory neurons containing CaBP were 23.4 +/- 6.3 microns and 38.4 +/- 8.8 microns, respectively. Splanchnic sensory neurons containing CaBP were sensitive to capsaicin while cutaneous ones were not. These findings suggest that CaBP-containing splanchnic and cutaneous sensory neurons constitute different subgroups among the DRG neurons at the lower thoracic level.     

Parvalbumin- and calbindin D28k-immunoreactive neurons in the superficial layers of the spinal cord dorsal horn of rat

Immunohistochemical techniques were utilized to investigate the distribution and morphology of neurons containing the calcium binding proteins parvalbumin (PV) and calbindin D28k (CaBP) in the superficial layers of rat spinal cord. Most PV-immunoreactive (PV-IR) neurons were restricted to a 25 to 60 microns thick band straddling the border between lamina II and III. Positive somata had long rostrocaudally oriented dendrites confined to narrow sagittally arranged sheets within this band and axons that entered lamina II or the superficial portions of lamina III. Long varicose axons, presumed to originate from these cells, were moderately distributed in Lissauer's tract and lamina II. CaBP-immunoreactive (CaBP-IR) neurons were found within lamina I and throughout lamina II. Large calibre PV-IR and CaBP-IR axons were seen in the dorsal column and the lateral funiculus. Dorsal rhizotomy or neonatal capsaicin treatment appeared to have no effect on PV-IR and CaBP-IR elements in the superficial lumbar dorsal horn. However, dorsal rhizotomy reduced the number of positive axons in the dorsal column and in deeper lamina of the dorsal horn. These results add to the known lamination patterns of the superficial dorsal horn and point to the existence of a lamina defined by PV-positive neurons at the lamina II/III border. These neurons may have electrophysiological characteristics attributed to PV- or CaBP-containing neurons elsewhere in the CNS.     

Calbindin D28K-immunoreactivity identifies distinct subpopulations of sympathetic pre- and postganglionic neurons in the rat

Neurons performing the same function can be identified immunohistochemically because they often share the same neurochemistry. The distribution of calcium-binding proteins, like calbindin, has been used previously to identify functional subpopulations of neurons in many parts of the nervous system. In this study we have investigated the distribution of calbindin D28K-immunoreactivity in subpopulations of sympathetic preganglionic neurons in the intermediolateral nucleus of the rat spinal cord. The majority of calbindin D28K-immunoreactive preganglionic neurons also had co-localised nitric oxide synthase, although a population of preganglionic neurons in the mid- to low thoracic intermediolateral nucleus expressed only calbindin D28K-immunoreactivity. Retrograde-tracing studies showed that calbindin D28K-immunoreactive neurons projected to the superior cervical and stellate ganglia, with smaller numbers of cells projecting to the lumbar sympathetic chain and superior mesenteric ganglia. Very few calbindin D28K-immunoreactive neurons projected to the inferior mesenteric ganglion, and none projected to the adrenal medulla. The distribution of calbindin D28K-immunoreactive terminals and postganglionic neurons in the superior cervical and stellate ganglia was also investigated. Many postganglionic neurons were calbindin D28K-immunoreactive, and most of these lacked neuropeptide Y-immunoreactivity. Calbindin D28K-immunoreactive nerve terminals were common and formed dense pericellular baskets around many postganglionic neurons, including some of those that were calbindin D28K-immunoreactive, but only rarely formed pericellular baskets around neuropeptide Y-immunoreactive neurons. The function of some of the classes of postganglionic neurons that were the target of calbindin D28K-immunoreactive preganglionic terminals was determined by combining immunohistochemistry with retrograde-tracer injections into a range of peripheral tissues. Calbindin D28K-immunoreactive nerve terminals, with co-localised nitric oxide synthase-immunoreactivity, surrounded secretomotor neurons projecting to the submandibular salivary gland and pilomotor neurons projecting to skin, but did not surround neurons projecting to brown fat or vasomotor neurons projecting to the skin, muscle, or salivary glands. J. Comp. Neurol. 386:245–259, 1997. © 1997 Wiley-Liss, Inc.     

Parvalbumin-, calretinin- and calbindin-D28k-immunoreactivity and GABA in a forebrain region involved in auditory filial imprinting

The distribution and morphology of neurons containing the Ca-binding proteins parvalbumin (PV), calbindin-D28k (CaBP) and calretinin (CaR) are described in a rostral forebrain region (MNH) of the chick, known to be involved in auditory filial imprinting. PV immunoreactivity is chiefly a marker for numerous large to medium-sized neurons in the neostriatal part of MNH. They show patchy staining of their dendrites, but PV-positive spines are not visible. CaBP is represented in a different neuron population with on the average slightly smaller-sized somata, which carry long, spiny, CaBP-positive dendrites. In contrast to PV and CaBP, CaR immunoreactivity is a marker chiefly for neuropil in MNH but only for few stained neurons. They may be spiny and show the largest size variations. The density of CaR-immunoreactive neuropil is highest in the hyperstriatal part of MNH. Double immunostaining for PV and CaBP reveals that these proteins are expressed mostly in different neuron populations, with only few neurons containing both proteins. These neuron populations appear to form an interconnected network within MNH. A possible relationship between the expression of either Ca-binding protein and the presence of the inhibitory transmitter GABA is also examined. The GABA-antibody labels scattered, very small to medium-sized neurons and dense punctate neuropil. The comparison of the area histograms of somata reveals an overlap with all 3 Ca-binding protein containing cell populations, except for a large proportion of small GABA-positive neurons. The characteristics of immunostained neuron populations are compared to the previously described 3 Golgi-types of neurons in MNH, and possibilities of a functional implication of the proteins in MNH plasticity are examined.