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.     

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.     

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.     

Development of Calbindin-D28k Immunoreactive Neurons in the Embryonic Chick Lumbosacral Spinal Cord

The development of immunoreactivity for the calcium-binding protein calbindin-D28k (CaB) was investigated in the embryonic and hatched chick lumbosacral spinal cord. CaB-immunoreactive neurons were revealed in the dorsal and ventral horns as well as in the intermediate grey matter from early stages of neuronal development. CaB immunoreactivity was first detected in large neurons in the presumptive dorsal horn at embyronic day 5, while small neurons in the lateral dorsal horn were the last to appear, at embryonic day 10. We have identified and traced the morphological maturation of six CaB-immunoreactive cell groups, three in the dorsal horn and three in the ventral horn. In the dorsal horn these groups were (1) large neurons in the lateral dorsal horn (laminae I and IV), (2) small neurons in the lateral dorsal horn (lamina II), and (3) small neurons in the medial dorsal horn (lamina III). All three groups were present throughout the entire length of the lumbosacral spinal cord and showed persistent CaB immunoreactivity. In the ventral horn, CaB-immunoreactive neurons were classified into the following three categories: (1) Neurons dorsal to the lateral motor column (lamina VII). These neurons were present exclusively in the upper lumbosacral segments (LS1 – 3), and they showed steady CaB immunoreactivity during their maturation. (2) Neurons at the dorsomedial aspect of the lateral motor column (at the border of laminae VII and IX). This population of neurons was characteristic of the lower segments of the lumbosacral cord (LS5 – 7) and presented transient CaB expression. (3) Neurons within the lateral motor column (lamina IX). These neurons were dispersed throughout the length of the lumbosacral spinal cord. They were three to four times more numerous in the upper than in the lower lumbosacral segments, and their numbers declined throughout LS1 – 7 as the animal matured. The characteristic features of the development of neurons immunoreactive for CaB are discussed and correlated with previous neuroanatomical and physiological studies concerning sensory and motor functions of the developing chick spinal cord.     

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.     

Postnatal development of parvalbumin immunoreactivity in the cerebral cortex of the cat

Parvalbumin immunoreactivity in the developing neocortex of the cat progresses following specific laminar, areal, and, in a particular area, roughly anteroposterior gradients. Parvalbumin immunoreactivity first occurs in basket cells and later in chandelier neurons. Pyramid-like immunoreactive neurons are also transitorily observed from the second to the third week in layer V of the auditory association-related areas. Parvalbumin-immunoreactive neurons first appear in the primary somatosensory cortex and primary auditory and visual areas, followed by the primary motor and polysensory association areas and, finally, the auditory association areas and cortical areas related to the limbic system. In addition to cortical neurons, three fiber systems are immunolabeled with antiparvalbumin antibodies: thalamocortical, callosal, and ipsilateral corticocortical. Parvalbumin-immunoreactive thalamocortical fibers appear during the first month of postnatal life. Parvalbumin-immunoreactive callosal and ipsilateral corticocortical fibers are seen from the fourth postnatal week onward. Because all parvalbumin-immunoreactive cortical neurons in adulthood are nonpyramidal inhibitory cells, the present findings suggest that a number of ipsilateral corticocortical and callosal connections may be inhibitory. © 1994 Wiley-Liss, Inc.     

Parvalbumin,calbindin D-28k,and calretinin immunoreactivity in the ascending auditory pathway of horseshoe bats

In the subcortical auditory system of Rhinolophus rouxi, antibodies directed against the calcium-binding proteins parvalbumin, calbindin D-28k, and calretinin yield partly overlapping and partly complementary labeling patterns which are described in detail for each nucleus. The most general features of the labeling patterns are that: (1) Parvalbumin is a potent marker for large and heterogenous populations of cells and puncta (presumed axon terminals) throughout the auditory pathway. (2) Immunostaining with the monoclonal calbindin-antiserum was typically absent or sparse in most auditory brainstem centers, but prominent in auditory nerve fibers and in cells of the medial geniculate body (MGB). (3) Calretinin label is abundant but more restricted to subsets of auditory nuclei or subpopulations of cells than parvalbumin. (4) Calcium-binding proteins are useful markers to define particular subregions or cell types in auditory nuclei: for example, (i) different labeling patterns are obtained within the nuclei of the lateral lemniscus and adjacent tegmental zones; (ii) in the inferior colliculus both calbindin- and calretinin-antisera yield similar regional specific staining patterns, but label different cell types; (iii) subregions of the medial geniculate body have characteristic profiles of calcium-binding proteins; and (iv) analyses of different nuclei showed that there is no simple common denominator for cells characterized by the expression of particular calcium-binding proteins, nor does labeling correspond in a straightforward way with specific functional systems. (5) there are profound differences between the calbindin labeling patterns seen in Rhinolophus and those in other mammals.     

Vulnerability of Midbrain Dopaminergic Neurons in Calbindin-D28k-deficient Mice: Lack of Evidence for a Neuroprotective Role of Endogenous Calbindin in MPTPtreated and Weaver Mice

Calbindin-D_28k (calbindin) is an intracellular calcium binding protein of unknown in vivo function. It is abundantly expressed in many populations of neurons, and it can, presumably by buffering calcium overload, protect cells against excitotoxic damage. In the midbrain, calbindin is preferentially expressed in those dopamine neurons which are spared from degeneration in Parkinson's disease and its animal models. Whether calbindin itself determines neuronal vulnerability is questioned in other lesion models where calbindin expression is not positively correlated with neuronal resistance. To study the possible neuroprotective role of calbindin in vivo, we generated calbindin-deficient mice by gene targeting and assessed the viability of midbrain dopamine neurons in both a chemical and a genetic lesion paradigm. Tyrosine hydroxylase-immunoreactive neurons were counted in calbindin null-mutant mice treated with the neurotoxin 1 -methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and in a calbindin-deficient weaver strain (homozygous for weaver and the calbindin null mutation). The extent and pattern of neuron loss observed in MPTP-treated wild-type and homozygous weaver mice were as previously described. Surprisingly, no significant differences were observed between MPTP-treated calbindin null mutants and their wild-type littermates, or between calbindin-weaver double mutant mice and weaver mice. Thus, in all four groups the same subpopulation of tyrosine hydroxylase-positive midbrain neurons (i.e. those normally containing calbindin) were preferentially spared. Calretinin, a closely related calcium-binding protein, which is also expressed in some midbrain dopamine neurons, was not up-regulated in these surviving neurons. These findings indicate that the resistance of calbindin-containing neurons in the MPTP and weaver models is not causally related to the expression of calbindin, and that endogenous calbindin is not required for protection of these neurons.     

Distribution of GABAergic interneurons immunoreactive for calretinin, calbindin D28K, and parvalbumin in the cerebral cortex of the lizard Podarcis hispanica.

The types and distribution of cells containing three calcium-binding proteins, calretinin, calbindin D28K, and parvalbumin, have been studied by immunocytochemistry in different areas of the cerebral cortex of lizards. Cross-reactivity of the antisera has been excluded by demonstrating the existence of several cell groups immunoreactive for one but not the other two calcium-binding proteins. In the dorsal and dorsomedial cortices all three proteins coexist in a single subpopulation of gamma-aminobutyric acid (GABA)ergic neurons, the terminals of which form pericellular baskets around cell bodies of bipyramidal neurons. The somata of these neurons are largely restricted to the cellular and inner plexiform layers, but the dendrites usually penetrate all layers, allowing the neurons to sample input from all possible sources. A small number of parvalbumin-containing neurons in the outer plexiform layer do not contain the other two proteins. The medial cortex, which is likely to be homologous to the mammalian dentate gyrus, only contains parvalbumin-immunoreactive neurons. The dendritic trees of these cells appear to avoid the Timm-positive fields receiving input from zinc-rich fiber collaterals, originating from principal cells. The lateral cortex contains calbindin D28K-immunoreactive GABAergic neurons, which lack the other two calcium-binding proteins. These neurons have horizontally running dendrites in the outer plexiform layer, but their axon terminals could not be visualized. The present study uncovered important similarities and differences between the lizard and the mammalian archicortex in the types of neurons containing calcium-binding proteins. As in mammals, different cell types evolved in the lizard to inhibit the perisomatic versus the distal dendritic region of principal cells, the calcium-binding protein-containing neurons being responsible for the former, and neuropeptide-containing neurons for the latter. The results also suggest that further neurochemical diversion of GABAergic interneurons coupled to a functional specialization took place during phylogenetic development from reptiles to mammals.     

Postnatal Development of GABA Neurons in the Rat Somatosensory Barrel Cortex: A Quantitative Study

As an estimate of the numerical importance of GABA-containing neurons during development, their quantitative distribution was analysed in the primary somatosensory cortex of rats between postnatal days (P) 5 and 60, using the disector method and GABA postembedding immunocytochemistry. In relation to the overall number of neurons in the barrel field cortex, the proportion of GABA neurons showed an early significant decrease between P5 and P10 from 14 to 11%, most likely due to termination of transient expression of GABA by some cells. It then remained stable until P20, after which it started slowly but steadily to increase, reaching 14% of the total at P60. The absolute number of GABA neurons also increased by nearly 50% during that period, whereas the number of all neurons remained constant. These changes are seemingly due to a subpopulation of neurons, shown to be of small size, which express GABA late in development. Thus, anatomical adjustments of the cortical GABA system may be observed at least until the end of the second postnatal month, reflecting both delayed maturation and adaptation of this inhibitory circuitry. We suggest the existence of three subpopulations of cortical GABA neurons depending on the time of onset and the regulation of their GABA expression: (i) neurons which express GABA before completion of migration and thus provide for its neurotrophic influence. (ii) neurons which express GABA immediately after completion of migration and build up the cortical inhibitory circuitry, and (iii) neurons which express GABA later in development and represent a substrate of experience dependent plasticity.     

Distribution of calbindin-D28k immunoreactivity in the monkey temporal lobe: The amygdaloid complex

Calbindin-D_28k is a calcium-binding protein located in a variety of neuronal cell types in many regions of the central nervous system. In the present study, we describe the distribution of calbindin-D_28k-immunoreactive cells, fibers, and terminals in the monkey amygdaloid complex. Calbindin-D_28k-immunoreactive neurons could be divided into four major cell types. Neurons of the first three cell types demonstrated clearly stained dendrites that were either aspiny or had a few spines on their distal portions. Type 1 cells were small, stellate, or multipolar and found throughout the amygdala. Type 2 cells were large, multipolar and were most commonly found in the deep nuclei, particularly in the lateral nucleus, intermediate division of the basal nucleus, accessory basal nucleus and in the periamygdaloid cortex. Type 3 cells were fusiform, of various sizes, and were found throughout the amygdala. Type 4 cells were quite large and lightly stained; the dendrites of these cells were usually unstained. The size, shape, and location of Type 4 labeled cell bodies suggested that they might be the large, modified pyramidal cells that constitute the projection neurons of the amygdala. Type 4 cells were observed primarily in the lateral, basal, and accessory basal nuclei and in the periamygdaloid cortex. Calbindin-D_28k-immunoreactive fibers and terminals were difficult to observe in the amygdala partly because of a diffuse, finely granular neuropil labeling that was particularly dense in the anterior cortical and medial nuclei, in the central nucleus, and in the periamygdaloid cortex. The neuropil labeling was substantially lighter in the lateral, basal, and accessory basal nuclei. Conspicuous linear profiles resembling the “calbindin bundles” of the neocortex were evident in large numbers in the accessory basal nucleus, the medial portion of the parvicellular division of the basal nucleus, in the amygdalohippocampal area, and in the periamygdaloid cortex. There were calbindin-D_28k-positive fibers in the stria terminalis and in the ventral amygdalofugal pathway. When the distributions of calbindin-D_28k and parvalbumin immunoreactivity in the monkey amygdaloid complex were compared, it appeared that the overall distribution of these two calcium-binding proteins was generally complementary rather than overlapping. © 1993 Wiley-Liss, Inc.     

Pyramidal cell dendrites are the primary targets of calbindin D28k-immunoreactive interneurons in the hippocampus

The axonal arborization and postsynaptic targets of calbindin D28k (CB)-immunoreactive nonprincipal neurons have been studied in the rat dorsal hippocampus. Two types of neurons were distinguished on the basis of soma location, the characteristics of the dendritic tree, and the axon arborisation pattern. Type I cells were located in stratum radiatum of the CA1 and CA3 regions and occasionally in strata pyramidale and oriens. These cells had multipolar or bitufted dendritic trees primarily located in stratum radiatum. Their axons could be followed for a considerable distance, arborised within stratum radiatum, and were covered with regularly spaced small boutons. As demonstrated with postembedding immunogold staining, their axon terminals were γ-aminobutyric acid (GABA) immunoreactive, and formed symmetrical synapses pre-dominantly on proximal and distal dendrites of pyramidal cells (28% and 58%, respectively), and occasionally on spines (9%) or on GABA-positive dendrites (5%). Type II cells were found exclusively in stratum oriens of the CA1 and CA3 regions and possessed large, fusiform cell bodies and long, horizontally oriented dendrites. Their axon initial segments turned towards the alveus and disappeared in a myelin sheet, which was often possible to follow into the white matter. We conclude that type I CB-immunoreactive cells are likely to represent a major source of inhibitory synapses in the dendritic region of pyramidal cells, which are responsible for the control of dendritic electrogenesis. The distribution of local collaterals of type II cells—if they have any—remains unknown, but their main axon is likely to project to the medial septum. © 1996 Wiley-Liss, Inc.     

Parvalbumin and calbindin D-28k in the entopeduncular nucleus,subthalamic nucleus,and substantia nigra of the rat as revealed by double-immunohistochemical methods

The cellular localization of calbindin D-28k (CB) and parvalbumin (PV) was analyzed by means of double-immunohistochemical techniques applied to single sections in the entopeduncular nucleus (EP), the subthalamic nucleus (STh), and the substantia nigra (SN) of the rat. In EP, PV-positive cells abounded centrally, where CB immunostaining was minimal. The medial and ventral sectors of EP were markedly enriched with CB neurophil but devoid of PV-positive cells. CB-positive neurons abounded particularly in the rostral pole of EP. In STh, PV-positive neurons and neuropil were concentrated in the lateral two thirds of this nucleus. Only a few PV-positive cells were detected in sectors of STh devoid of PV-positive neuropil. The STh was completely devoid of CB immunostaining. In the rostral two thirds of SN, PV-positive neurons were largely confined to the lateral half of the pars reticulata (SNR), and occurred more ventrally and medially in the caudal third. Intense CB-immunoreactive neuropil was found in medial and dorsal parts of rostral SNR, and CB-positive cells were observed in the SN pars compacta and the ventral tegmental area. PV and CB cells were also observed in the pars lateralis of SN. The markedly heterogeneous pattern of distribution of PV and CB in EP, STh, and SN suggests that these two calcium-binding proteins may label distinct functional domains in each of these three components of the rat basal ganglia. Synapse 25:359–367, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of calcium-binding proteins expressed in cultured hNT neurons and hNT neurons transplanted into the rat striatum

An alternative source of cells for neural transplantation and brain repair that has many characteristics of immature neurons is the hNT neuron, derived from an embryonal human teratocarcinoma (NTera2) cell line that is terminally differentiated in vitro with retinoic acid. The majority of hNT neurons are GABAergic in cell culture. We have determined the calcium-binding protein (CBP) phenotypes of hNT neurons for three CBPs, calretinin (CR), calbindin D-28K (CB), and parvalbumin (PV), in cell culture and after transplantation into the rat striatum. In cell culture, 95% of all cell profiles were human nuclear matrix antigen (NuMA) positive. PV-positive hNT neurons constituted 50% of all neuron-like profiles, with CB+ and CR+ constituting 14 and 6% of cells, respectively. In contrast, when the striatal grafts were examined after 30 days survival using confocal microscopy, only 10% of hNT neurons immunopositive for NuMA were PV+; 19% were CB+/NuMA+, approximately the same percentage as was seen in vitro, and 82% of grafted hNT neurons were CR+. These results suggest that hNT neurons can be subdivided into at least three subpopulations based on the CBP phenotype that they express and that there is a CBP phenotypic shift following transplantation. Three related hypotheses are proposed to account for this phenotypic shift of hNT neurons after transplantation: (a) selective survival of the CR+ subpopulation of hNT neurons, (b) selective transitory quiescence of the transplanted PV+ cells due to transplantation stress, or (c) dedifferentiation of the hNT neurons following transplantation, which may allow them to respond to local environmental cues during the engraftment process.     

Local circuit neurons immunoreactive for calretinin,calbindin D-28k or parvalbumin in monkey prefronatal cortex: Distribution and morphology

In the cerebral cortex, local circuit neurons provide critical inhibitory control over the activity of pyramidal neurons, the major class of excitatory efferent cortical cells. The calciumbinding proteins, calretinin, calbindin, and parvalbumin, are expressed in a variety of cortical local circuit neurons. However, in the primate prefrontal cortex, relatively little is known, especially with regard to calretinin, about the specific classes or distribution of local circuit neurons that contain these calcium-binding proteins. In this study, we used immunohistochemical techniques to characterize and compare the morphological features and distribution in macaque monkey prefrontal cortex of local circuit neurons that contain each of these calcium-binding proteins. On the basis of the axonal features of the labeled neurons, and correlations with previous Golgi studies, calretinin appeared to be present in double-bouquet neurons, calbindin in neurogliaform neurons and Martinotti cells, and parvalbumin in chandelier and wide arbor (basket) neurons. Calretinin was also found in other cell populations, such as a distinctive group of large neurons in the infragranular layers, but it was not possible to assign these neurons to a known cell class. In addition, although the animals studied were adults, immunoreactivity for both calretinin and calbindin was found in Cajal-Retzius neurons of layer I. Dual labeling studies confirmed that with the exception of the Cajal-Retzius neurons, each calcium-binding protein was expressed in separate populations of prefrontal cortical neurons. Comparisons of the laminar distributions of the labeled neurons also indicated that these calcium-binding proteins were segregated into discrete neuronal populations. Calretinin-positive neurons were present in greatest density in deep layer I and layer II, calbindin-immuno-reactive cells were most dense in layers II-superficial III, and parvalbumin-containing neurons were present in greatest density in the middle cortical layers. In addition, the relative density of calretinin-labeled neurons was approximately twice that of the calbindin- and parvalbumin-positive neurons. However, within each group of labeled neurons, their laminar distribution and relative density did not differ substantially across regions of the prefrontal cortex. These findings demonstrate that calretinin, calbindin, and parvalbumin are markers of separate populations of local circuit neurons in monkey prefrontal cortex, and that they may be useful tools in unraveling the intrinsic inhibitory circuitry of the primate prefrontal cortex in both normal and disease states.     

Differential Distribution of Tau Proteins in Developing Cat Cerebral Cortex and Corpus Callosum

During the postnatal development of cat visual cortex and corpus callosum the molecular composition of tau proteins varied with age. In both structures, they changed between postnatal days 19 and 39 from a set of two juvenile forms to a set of at least two adult variants with higher molecular weights. During the first postnatal week, tau proteins were detectable with TAU-1 antibody in axons of corpus callosum and visual cortex, and in some perikarya and dendrites in the visual cortex. At later ages, tau proteins were located exclusively within axons in all cortical layers and in the corpus callosum. Dephosphorylation of postnatal day 11 cortical tissue by alkaline phosphatase strongly increased tau protein immunoreactivity on Western blots and in numerous perikarya and dendrites in all cortical layers, in sections, suggesting that some tau forms had been unmasked. During postnatal development the intensity of this phosphate-dependent somatodendritic staining decreased, but remained in a few neurons in cortical layers II and III. On blots, the immunoreactivity of adult tau to TAU-1 was only marginally increased by dephosphorylation. Other tau antibodies (TAU-2, B19 and BR133) recognized two juvenile and two adult cat tau proteins on blots, and localized tau in axons or perikarya and dendrites in tissue untreated with alkaline phosphatase. Tau proteins in mature tissue were soluble and not associated with detergent-resistant structures. Furthermore, dephosphorylation by alkaline phosphatase resulted in the appearance of more tau proteins in soluble fractions. Therefore tau proteins seem to alter their degree of phosphorylation during development. This could affect microtubule stability as well as influence axonal and dendritic differentiation.     

Localization of parvalbumin,calretinin, and calbindin D-28k in identified extraocular motoneurons and internuclear neurons of the cat

Calcium-binding proteins have been shown to be excellent markers of specific neuronal populations. We aimed to characterize the expression of calcium-binding proteins in identified populations of the cat extraocular motor nuclei by means of immunohistochemistry against parvalbumin, calretinin, and calbindin D-28k. Abducens, medial rectus, and trochlear motoneurons were retrogradely labeled with horseradish peroxidase from their corresponding muscles. Oculomotor and abducens internuclear neurons were retrogradely labeled after horseradish peroxidase injection into either the abducens or the oculomotor nucleus, respectively. Parvalbumin staining produced the highest density of immunoreactive terminals in all extraocular motor nuclei and was distributed uniformly. Around 15–20% of the motoneurons were moderately stained with antibody against parvalbumin, but their axons were heavily stained, indicating an intracellular segregation of parvalbumin. Colchicine administration increased the number of parvalbumin-immunoreactive motoneurons to approximately 85%. Except for a few calbindin-immunoreactive trochlear motoneurons (1%), parvalbumin was the only marker of extraocular motoneurons. Oculomotor internuclear neurons identified from the abducens nucleus constituted a nonuniform population, because low percentages of the three types of immunostaining were observed, calbindin being the most abundant (28.5%). Other interneurons located within the boundaries of the oculomotor nucleus were mainly calbindin-immunoreactive. The medial longitudinal fascicle contained numerous parvalbumin- and calretinin-immunoreactive but few calbindin-immunoreactive axons. The majority of abducens internuclear neurons projecting to the oculomotor nucleus (80.7%) contained calretinin. Moreover, the distribution of calretinin-immunoreactive terminals in the oculomotor nucleus overlapped that of the medial rectus motoneurons and matched the anterogradely labeled terminal field of the abducens internuclear neurons. Parvalbumin immunostained 42% of the abducens internuclear neurons. Colocalization of parvalbumin and calretinin was demonstrated in adjacent semithin sections, although single-labeled neurons were also observed. Therefore, calretinin is proven to be a good marker of abducens internuclear neurons. From all of these data, it is concluded that parvalbumin, calretinin, and calbindin D-28k selectively delineate certain neuronal populations in the oculomotor system and constitute valuable tools for further analysis of oculomotor function under normal and experimental conditions. J. Comp. Neurol. 390:377–391, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of Nerve Growth Factor (NGF) Release from Hippocampal Neurons: Evidence for a Constitutive and an Unconventional Sodium-dependent Regulated Pathway

We investigated the mechanism of neuronal nerve growth factor (NGF) release with regard to the potential function of NGF as a mediator of neuronal plasticity in the CNS. The analysis was performed in hippocampal slices and in primary cultures of hippocampal neurons, transiently transfected with an NGF cDNA construct to increase the level of NGF expression. In both systems there was activity-dependent NGF release initiated by high potassium (KCl), veratridine, glutamate or carbachol. Replacement of 90% of sodium in the medium with N -methyl-glucamine strongly reduced this release. The KCl- and veratridine-initiated NGF release was suppressed by tetrodotoxin; release by glutamate was less sensitive to tetrodotoxin but was sodium-dependent. The glutamate effect could be inhibited by GYK152644, an antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, but not by MK-801, an antagonist of NMDA receptors. The activity-dependent release of NGF did not depend on extracellular Ca²⁺, but was sensitive to the intracellular Ca²⁺ chelator bis-( o -aminophenoxy)-ethane- N,N,N',N' -tetraacetic acid tetra(acetoxymethyl)-ester, and to depletion of intracellular calcium stores. Conversely, mobilization of Ca²⁺ from intracellular stores with caffeine and thapsigargin mimicked the effect of depolarization. Basal NGF release could be reduced by either temperature block (15°C) or tetrodotoxin to ˜50%. The combination of both treatments reduced NGF release to below the detection limit, suggesting that basal release has constitutive and regulated components, the latter presumably resulting from spontaneous activity of interconnected neurons.