Calcium-binding Protein calbindin-D28k Research Articles

Upregulated expression of oncomodulin, the beta isoform of parvalbumin, in perikarya and axons in the diencephalon of parvalbumin knockout mice

The calcium-binding proteins parvalbumin, calbindin D-28k, calretinin and calcineurin are present in subsets of GABAergic gigantic calyciform presynaptic terminals of the reticular thalamic nucleus (RTN). Previously it was hypothesized that GABA and calcium-binding proteins including parvalbumin are not only colocalized in the same neuron subpopulation, but that GABA synthesis and parvalbumin expression could be also genetically regulated by a common mechanism. Moreover, parvalbumin expression levels could influence GABA synthesis. For this, we analyzed GABA immunoreactivity in RTN gigantic calyciform presynaptic terminals of parvalbumin–deficient (PV−/−) mice. With respect to GABA immunoreactivity we found no differences compared to wild–type animals. However, using a polyclonal parvalbumin antibody raised against full-length rat muscle parvalbumin on brain sections of PV−/− mice, we observed paradoxical parvalbumin immunoreactivity in partly varicose axons in the diencephalon, mainly in the lamina medullaris externa surrounding the thalamus. A detailed immunohistochemical, biochemical and molecular biological analysis revealed this immunoreactivity to be the result of an upregulation of oncomodulin (OM), the mammalian beta isoform of parvalbumin in PV−/− mice. In addition, OM was present in a sparse subpopulation of neurons in the thalamus and in the dentate gyrus. OM expression has not been observed before in neurons of the mammalian brain; its expression was restricted to outer hair cells in the organ of Corti. Our results indicate that the absence of parvalbumin has no major effect on the GABA-synthesizing system in RTN presynaptic terminals excluding a direct effect of parvalbumin on this regulation. However, a likely homeostatic mechanism is induced resulting in the upregulation of OM in selected axons and neuronal perikarya. Our results warrant further detailed investigations on the putative role of OM in the brain.

Quantitative analysis of neurons with Kv3 potassium channel subunits, Kv3.1b and Kv3.2, in macaque primary visual cortex

Voltage-gated potassium channels that are composed of Kv3 subunits exhibit distinct electrophysiological properties: activation at more depolarized potentials than other voltage-gated K+ channels and fast kinetics. These channels have been shown to contribute to the high-frequency firing of fast-spiking (FS) GABAergic interneurons in the rat and mouse brain. In the rodent neocortex there are distinct patterns of expression for the Kv3.1b and Kv3.2 channel subunits and of coexpression of these subunits with neurochemical markers, such as the calcium-binding proteins parvalbumin (PV) and calbindin D-28K (CB). The distribution of Kv3 channels and interrelationship with calcium-binding protein expression has not been investigated in primate cortex. We used immunoperoxidase and immunofluorescent labeling and stereological counting techniques to characterize the laminar and cell-type distributions of Kv3-immunoreactive (ir) neurons in macaque V1. We found that across the cortical layers approximately 25% of both Kv3.1b- and Kv3.2-ir neurons are non-GABAergic. In contrast, all Kv3-ir neurons in rodent cortex are GABAergic (Chow et al. [1999] J Neurosci. 19:9332-9345). The putatively excitatory Kv3-ir neurons were mostly located in layers 2, 3, and 4b. Further, the proportion of Kv3-ir neurons that express PV or CB also differs between macaque V1 and rodent cortex. These data indicate that, within the population of cortical neurons, a broader population of neurons, encompassing cells of a wider range of morphological classes may be capable of sustaining high-frequency firing in macaque V1.

Bergmann Glial S100B Activates Myo-inositol Monophosphatase 1 and Co-localizes to Purkinje Cell Vacuoles in SCA1 Transgenic Mice

Spinocerebellar ataxia-1 (SCA1) is a late onset neurodegenerative disease caused by the expansion of a polyglutamine repeat within ataxin-1 protein. The toxic effects triggered by mutant ataxin-1 result in degeneration of the neurons in cerebellum, brain stem and spinocerebellar tracts. The targeted overexpression of mutant ataxin-1 in cerebellar Purkinje cells (PCs) of the SCA1 transgenic mice results in the formation of cytoplasmic vacuoles in PCs. These vacuoles appear early on before the onset of behavioral abnormalities. Interestingly, we found that vacoules contain S100B and vimentin proteins, which normally localize to neighboring Bergmann glia (BG). Further, immunohistochemical and specialized silver stain analysis revealed that vacuolar formation is associated with alterations in the morphology of dendritic spines of PCs. To gain insights into the mechanisms of vacuolar formation, we investigated if vacuoles in SCA1 PCs have an autophagic origin or are a consequence of some other event. We examined the expression levels (by Western blotting) of microtubule-associated protein light chain 3 (LC3)-I and LC3-II, and the degradation levels of p62 (a LC3 partner) in the cerebellar fractions prepared from pre-symptomatic SCA1 and age-matched wild-type mice. No p62 degradation was observed; however, LC3-II/(LC3-I + LC3-II) ratios were significantly altered in SCA1 mice indicating changes in the autophagic flux. In addition, LC3 localized to PC vacuoles. Further, we observed a co-localization of myo-inositol monophosphatase 1 (IMPA1) with S100B in PC vacuoles. IMPA1 is present in PC spines and has been implicated in autophagy. In vitro studies using purified IMPA1 and S100B demonstrated that S100B interacted with and activated IMPA1. Both apo and Ca(2+)-bound S100B were found to activate IMPA1, depending on substrate concentration. IMPA1 is regulated by another calcium-binding protein calbindin-D28k (CaB), since we reported earlier that the CaB levels are reduced in SCA1 PCs, the activation of IMPA1 by S100B may modulate CaB-dependent inositol signaling. This may cause BG-PC interface to degenerate resulting in vacuolar formation. In sum, these data indicate that vacuoles appearing early in SCA1 PCs could be developing through some unknown autophagic mechanism.

Neuronal subtype identity in the rat auditory brainstem as defined by molecular profile and axonal projection

The nuclei of the auditory brainstem harbor a diversity of neuronal cell types and are interconnected by excitatory as well as inhibitory ascending, descending, and commissural pathways. Classically, neurons have been characterized by size and shape of their cell body and by the geometry of their dendrites. Our study is based on the use of axonal tracers in combination with immunocytochemistry to identify and distinguish neuronal subtypes by their molecular signature in dorsal and ventral cochlear nucleus, lateral superior olive, medial superior olive, medial nucleus of the trapezoid body, and inferior colliculus of the adult rat. The presumed neurotransmitters glutamate, glycine, and GABA were used alongside the calcium-binding proteins parvalbumin, calretinin, and calbindin-D28k as molecular markers. Our data provide distinct extensions to previous characterizations of neuronal subtypes and reveal regularities and differences across auditory brainstem nuclei that are discussed for their functional implications.

Reduction of Calbindin D-28k-Immunoreactive Neurons in the Dog Dentate Gyrus

The calcium binding protein calbindin D-28k (CB) plays an important role in modulating the activity of neurons in the dentate gyrus. We observed CB immunoreactivity in the dentate gyrus of dogs of various ages (German shepherds). In the 1-year-old group, CB immunoreactivity was detected in almost all of granule cells with poor processes. In the 8-year-old group, the number of CB-immunoreactive ((+)) neurons in the granule cell layer was significantly reduced (73.2% vs. 1-year-old group), while CB(+) cell bodies and fibers were well developed. In the 10-year-old group, the number of CB(+) neurons was further reduced by 31.3% when compared to that in the 1-year-old group. This finding demonstrates that the number of CB(+) neurons decreases in the aged dog brain and this may be associated with reduction of function in the dentate gyrus.

Glutamate Receptors GluR1 and GluR4 in the Hamster Superior Colliculus: Distribution and Co-localization with Calcium-Binding Proteins and GABA

We investigated the distributions of AMPA glutamate receptor subtypes GluR1 and GluR4 in the hamster superior colliculus (SC) with antibody immunocytochemistry and the effect of enucleation on these distributions. We compared these labelings to those of GluR2/3 in our previous report (Park et al., 2004, Neurosci Res., 49:139–155) and calcium-binding proteins calbindin D28K, calretinin, parvalbumin, and GABA. Anti-GluR1-immunoreactive (IR) cells were scattered throughout the SC. By contrast, anti-GluR4-IR cells formed distinct clusters within the lower lateral stratum griseum intermediale (SGI) and lateral stratum album intermediale (SAI). The GluR1- and GluR4-IR neurons varied in size and morphology. The average diameter of the GluR1-IR cells was 13.00 µm, while the GluR4-IR cells was 20.00 µm. The large majority of IR neurons were round or oval cells, but they also included stellate, vertical fusiform and horizontal cells. Monocular enucleation appeared to have no effect on the GluR1 and GluR4 immunoreactivity. Some GluR1-IR cells expressed calbindin D28K (9.50%), calretinin (6.59%), parvalbumin (2.53%), and GABA (20.54%). By contrast, no GluR4-IR cells expressed calcium-binding proteins or GABA. Although the function of the AMPA receptor subunits in SC is not yet clear, the distinct segregation of the GluR subunits, its differential colocalization with calcium-binding proteins and GABA, and differential responses to enucleation suggest the functional diversity of the receptor subunits in visuo-motor integration in the SC.

Oestrogen Receptor α and β Immunoreactive Cells in the Suprachiasmatic Nucleus of Mice: Distribution, Sex Differences and Regulation by Gonadal Hormones

Oestrogen regulates various aspects of circadian rhythm physiology. The presence of oestrogen receptors within the suprachiasmatic nucleus (SCN), the principal circadian oscillator, indicates that some actions of oestrogen on circadian functions may be exerted at that site. The present study analysed sex differences, topographic distribution, and neurochemical phenotype of neurones expressing the alpha and beta subtypes of oestrogen receptors (ERalpha and ERbeta) in the mouse SCN. We found that relatively few neurones in the SCN are immunoreactive (IR) for ERalpha (approximately 4.5% in females and 3% in males), but five- to six-fold more SCN neurones express ERbeta. ER-IR neurones are primarily in the shell subdivision of the nucleus and show differences between the sexes, significantly greater numbers being found in females. Treatment of male or female gonadectomised mice with oestradiol benzoate for 24 h substantially reduced the number of ERbeta-IR neurones, but not ERalpha-IR neurones. Double-labelling immunocytochemical experiments to characterise the phenotype of the oestrogen-receptive neurones showed the presence of the calcium-binding proteins calretinin or calbindin D28K in approximately 12% and 10%, respectively, of ERalpha-IR neurones. A higher proportion (approximately 38%) of ERbeta-IR neurones contains calbindin D28K; a few (approximately 2%) express calretinin or vasopressin. These double-labelled cells appear primarily in the shell subdivision of the SCN. Neither vasoactive intestinal polypeptide- nor gastrin releasing peptide-immunoreactivity was observed in ER-IR neurones. These data indicate that the primary target cells for oestrogen are in the shell subdivision of the nucleus. The sexually differentiated expression and distribution of ERalpha and ERbeta in various cell populations of the SCN suggest multiple modes of oestrogen signalling within this nucleus, which may modulate circadian functions.

P3-402: Age-related loss of calbindin identifies basal forebrain cholinergic neurons destined to degenerate in Alzheimer's disease

The reasons for the vulnerability of select neuronal populations in Alzheimer's Disease (AD) remain unknown. We have used the basal forebrain cholinergic neurons (BFCN), which are vulnerable to degeneration early in the course of AD, to determine of the causes of selective neuronal vulnerability. We have observed a selective loss of the calcium binding protein calbindin-D28K from the human BFCN in the course of normal aging. The calcium dysregulation caused by the loss of calbindin may contribute to the loss of BFCN in AD. In the present set of experiments, we investigated whether the BFCN which degenerate in AD are those which lose calbindin in the course of aging. Immunohistochemical methods were used in postmortem brains from young (<65 years), old and AD cases. Cell counting techniques were used to determine the numbers of immunoreactive neurons. The number of choline acetyltransferase (ChAT) and low affinity neurotrophin receptor (p75NTR) immunoreactive BFCN remained constant in old individuals when compared with the young and displayed a significant decrease in AD. In contrast, the number of calbindin-positive BFCN showed a significant decrease in normal aged individuals when compared with the young and remained constant in AD. The percentage of calbindin-positive BFCN in the aged was significantly lower when compared with the young. Of great interest, the percentage of the remaining BFCN in AD which contained calbindin immunoreactivity displayed a significant increase when compared with the normal aged, indicating that the majority of surviving BFCN in AD are calbindin-positive. Western blot analysis showed no increase in the total levels of calbinidin protein in AD basal forebrain, suggesting that the maintained calbindin immunoreactivity in AD BFCN is not due to reactive upregulation. These observations indicate that the loss of cholinergic neurons in AD occurs preferentially in the BFCN which lose their calbindin in the course of normal aging and that the BFCN which contain calbindin survive the neurodegenerative process. It is likely that loss of calbindin during aging deprives the BFCN from the calcium buffering capacity of this protein, leaving them vulnerable to degeneration due to calcium toxicity in AD.

Calcium-binding proteins in the circadian centers of the common marmoset ( Callithrix jacchus) and the rock cavy ( Kerodon rupestris) brains

The hypothalamic suprachiasmatic nucleus (SCN) and the thalamic intergeniculate leaflet (IGL) are considered to be the main centers of the mammalian circadian timing system. In primates, the IGL is included as part of the pregeniculate nucleus (PGN), a cell group located mediodorsally to the dorsal lateral geniculate nucleus. This work was carried out to comparatively evaluate the immunohistochemical expression of the calcium-binding proteins calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR) into the circadian brain districts of the common marmoset and the rock cavy. In both species, although no fibers, terminals or perikarya showed PV-immunoreaction (IR) into the SCN, CB-IR perikarya labeling was detected throughout the SCN rostrocaudal extent, seeming to delimit its cytoarchitectonic borders. CR-IR perikarya and neuropil were noticed into the ventral and dorsal portions of the SCN, lacking immunoreactivity in the central core of the marmoset and filling the entire nucleus in the rock cavy. The PGN of the marmoset presented a significant number of CB-, PV-, and CR-IR perikarya throughout the nucleus. The IGL of the rocky cavy exhibited a prominent CB- and CR-IR neuropil, showing similarity to the pattern found in other rodents. By comparing with literature data from other mammals, the results of the present study suggest that CB, PV, and CR are differentially distributed into the SCN and IGL among species. They may act either in concert or in a complementary manner in the SCN and IGL, so as to participate in specific aspects of the circadian regulation.

Distinct immunohistochemically defined areas in the medial amygdala in the developing and adult mouse

The medial amygdala has been considered a subpallial structure, but various studies have shown that is a somewhat complex structure expressing both pallial and subpallial gene markers. In this regard, we analyzed the immunohistochemical expression of the neurotransmitter GABA, the vesicular glutamate transporter type 2 (VGLUT2), the neuronal nitric oxide synthase (nNOS), and the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the developing and adult mouse medial amygdala. From intermediate embryonic stages on, neurochemical data show a distinctive superficial region forming a band all along the medial amygdalar surface. This superficial band displays a strong VGLUT2-immunoreactive neuropil and numerous CR-immunoreactive fibers, as well as some nNOS-, CR- or CB-positive cells. In contrast, the superficial region of the posterior medial amygdala appears to be non-GABA immunoreactive. This band in the posterior medial amygdala matches a Tbr1-expressing territory. Our results also show differences between dorsal and ventral parts of the postnatal and adult posterior medial amygdala. Especially, a compact cell aggregate of nNOS immunoreactive cells was found in the ventral portion of the medial amygdala, whereas the dorsal part is occupied by scattered weakly stained cells. Comparison of our results with gene expression patterns and fiber-tracing studies, let us to propose that the superficial band is a pallial derivative, whereas the dorsal and ventral nuclei of the posterior medial amygdala receive each neurons from different subpallial compartments, and the latter one a subset of excitatory, pallial projection neurons.

Calbindin-D28k and calretinin as markers of retinal neurons in the anuran amphibian Rana perezi

In the present study we have analyzed the distribution of the calcium binding proteins calbindin-D28k (CB) and calretinin (CR) immunoreactive cells in the retina of the anuran Rana perezi using poly- and mono-clonal antibodies that were proven to be specific in the amphibian brain, without cross-reactivity. Double immunohistofluorescence techniques were used to demonstrate colocalization of both proteins in the same retinal cells. In addition, retrograde tracing experiments from the optic nerve were conducted to labeled ganglion cells and these were observed in combination with CB and/or CR immunohistochemistry. Cells containing CB were identified as all cones, scattered bipolar and amacrine cells together with cells in the ganglion cell layer. The pattern of CR immunoreactivity was strikingly different. Abundant cells contained CR in the inner retinal layers including horizontal, bipolar and amacrine cells, and cells in the ganglion cell layer. By means of double immunohistochemistry it was found that only subpopulations of amacrine cells and cells in the ganglion cell layer contained both CB and CR. Tracing from the optic nerve revealed retrogradely labeled ganglion cells with different morphologies and most of them contained CB and/or CR. All these data taken together suggest that in amphibians CB and CR are distinctly and precisely distributed in retinal neurons showing, however, peculiar features not observed previously in other vertebrates.

Characterization of cochlear nucleus principal cells of Meriones unguiculatus and Monodelphis domestica by use of calcium-binding protein immunolabeling

Antibodies directed against calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k and calretinin were used as neuronal markers to identify and characterize different principal cell types in the mammalian cochlear nucleus. For this purpose, double immunofluorescence labeling and the combination of CaBP-labeling with pan-neuronal markers were applied to analyze the CaBPs distribution in neurons of the cochlear nucleus (CN) of the Mongolian gerbil ( Meriones unguiculatus) and the gray short-tailed opossum ( Monodelphis domestica). Despite of the fact, that these two mammalian species are not closely related, principal cell types in the CN of the two species showed many corresponding morphological features and similarities in immunolabeling of the CaBPs. Parvalbumin seems not to be suited as a differential neuronal marker in the CN since it is expressed by almost all neurons. In contrast, calbindin and calretinin were more restricted to specific cell types and showed a mostly complementary labeling pattern. As one of the most interesting findings, calbindin and calretinin were predominantly found in subpopulations of globular bushy cells and octopus cells in the ventral CN. Such a neuron-specific CaBP-expression in subpopulations of morphologically defined cell types argues for a more refined classification of CN cell types in Meriones and Monodelphis. Additionally, other cell types (cartwheel cells, unipolar brush cells, fusiform cells) were marked with calbindin or calretinin as well. Calretinin staining was predominantly observed in auditory nerve fibers and their endings including endbulbs of Held in Meriones. Spherical bushy cells showed a different calretinin-immunolabeling in Meriones and Monodelphis. This species-specific difference may be related to adaptive differences in auditory function.

Increased calbindin-D28K immunoreactivity in rat cerebellar Purkinje cell with excitatory amino acids agonists is not dependent on protein synthesis.

The calcium binding protein Calbindin-D28K (CaBP) is abundantly expressed in cerebellar Purkinje cells and show increased immunoreactivity (CaBP-IR) when challenged with glutamate or an analog agonist for the ionotropic glutamate receptor (iGluR). Here we report that t-ACPD, a metabotropic glutamate receptor (mGluR) agonist, produced small increases in CaBP-IR which was potentiated by a mGluR antagonist The increase in CaBPIR was not due to de novo protein synthesis because the translational inhibitors (cycloheximide and emetine) or transciptional inhibitors (actinomycine-D and a-amanitine), did not prevent the EAA enhanced CaBP-IR. The CaBP-IR in the PC appears to be coupled to the ionotropic rather than the metabotropic glutamate receptors, but the latter become effective in the presence of their blocker, L-AP3. The results suggest that CaBP may increase its IR through a conformational change of the protein itself.

Role of tissue transglutaminase type 2 in calbindin–D28k interaction with ataxin-1

Spinocerebellar ataxia-1 (SCA1) is caused by the expansion of a polyglutamine repeats within the disease protein, ataxin-1. The mutant ataxin-1 precipitates as large intranuclear aggregates in the affected neurons. These aggregates may protect neurons from mutant protein and/or trigger neuronal degeneration by encouraging recruitment of other essential proteins. Our previous studies have shown that calcium binding protein calbindin-D28k (CaB) associated with SCAl pathogenesis is recruited to ataxin-l aggregates in Purkinje cells of SCAl mice. Since our recent findings suggest that tissue transglutaminase 2 (TG2) may be involved in crosslinking and aggregation of ataxin-l, the present study was initiated to determine if TG2 has any role in CaB-ataxin-l interaction. The guinea pig TG2 covalently crosslinked purified rat brain CaB. Time dependent progressive increase in aggregation produced large multimers, which stayed on top of the gel. CaB interaction with ataxin-l was studied using HeLa cell lysates expressing GFP and GFP tagged ataxin-l with normal and expanded polyglutamine repeats (Q2, Q30 and Q82). The reaction products were analyzed by Western blots using anti-polyglutamine, CaB or GFP antibodies. CaB interacted with ataxin-1 independent of TG2 as the protein-protein crosslinker DSS stabilized CaB-ataxin-l complex. TG2 crosslinked CaB preferentially with Q82 ataxin-1. The crosslinking was inhibited with EGTA or TG2 inhibitor cystamine. The present data indicate that CaB may be a TG2 substrate. In addition, aggregates of mutant ataxin-l may recruit CaB via TG2 mediated covalent crosslinking, further supporting the argument that ataxin-l aggregates may be toxic to neurons.

Chemical parcellation of the anterior thalamic nuclei in the human brain

The anterior thalamic nuclei (ATN) encompass a large region of the anteromedial aspect of the human thalamus. Three ATN have been classically described: anteroventral (AV), anteromedial (AM) and anterodorsal (AD). The present study has carried out histochemical and immunohistochemical procedures in the ATN of normal individuals to analyze whether these nuclei are chemically distinct. The markers used in this study were acetylcholinesterase (AChE), limbic system-associated membrane protein (LAMP), the calcium binding proteins calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR), and the neuropeptides substance P (SP) and enkephalin (ENK). Other cytoarchitectural and myeloarchitectural techniques, specifically Nissl and Gallyas stainings, were used to delineate the boundaries of the ATN. The main findings of this study are: 1) AChE was very abundant in the AD and was irregular or heterogeneously distributed in the AV and AM; 2) LAMP immunoreactive (ir) neuropil was present throughout the ATN and its distribution was heterogeneous in the AV and AM; 3) the ATN harbored CB-, PV- and CR-ir neurons and neuropil; and, 4) the neuropeptide analysis revealed numerous SP positive varicose fibers scattered throughout the ATN in contrast to very few ENK-ir varicose fibers. These morphological findings describe a heterogeneous chemical anatomy in the human ATN which may reflect regional differences in the functional organization of the ATN with respect to the other thalamic nuclei and the cerebral cortex.

Differences in locomotor behavior revealed in mice deficient for the calcium-binding proteins parvalbumin, calbindin D-28k or both

We investigated the role of the two calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CB) in the locomotor activity and motor coordination using null-mutant mice for PV (PV−/−), CB (CB−/−) or both proteins (PV−/−CB−/−). These proteins are expressed in distinct, mainly non-overlapping populations of neurons of the central and peripheral nervous system and PV additionally in fast-twitch muscles. In a test measuring repeated locomotor activity during 18–20 days, the analysis revealed a slightly increased activity in mice lacking either protein, while the lack of both decreased the number of beams crossed during active periods. An increase in the characteristic speed during the first 8 days could be attributed to PV-deficiency, while the elimination of CB in CB−/− and double-KO mice decreased the percentage of fast movements at all time points. In the latter, additionally a reduction of the fastest speed was observed. The alterations in locomotor activity (fast movements, fastest speed) strongly correlate with the impairment in locomotor coordination in mice deficient for CB evidenced in the runway assay and the rotarod assay. The graded locomotor phenotype (CB > PV) is qualitatively correlated with alterations in Purkinje cell firing reported previously in these mice. The presence or absence of either protein did not affect the spontaneous locomotor activity when animals were placed in a novel environment and tested only once for 30 min. In summary, the lack of these calcium-binding proteins yields characteristic, yet distinct phenotypes with respect to locomotor activity and coordination.

Immunohistochemical and hodological characterization of calbindin‐D28k‐containing neurons in the spinal cord of the turtle, Pseudemys scripta elegans

Neurons and fibers containing the calcium-binding protein calbindin-D28k (CB) were studied by immunohistochemical techniques in the spinal cord of adult and juvenile turtles, Pseudemys scripta elegans. Abundant cell bodies and fibers immunoreactive for CB were widely and distinctly distributed throughout the spinal cord. Most neurons and fibers were labeled in the superficial dorsal horn, but numerous cells were also located in the intermediate gray and ventral horn. In the dorsal horn, most CB-containing cells were located in close relation to the synaptic fields formed by primary afferents, which were not labeled for CB. Double immunohistofluorescence demonstrated distinct cell populations in the dorsal horn labeled only for CB or nitric oxide synthase, whereas in the dorsal part of the ventral horn colocalization of nitric oxide synthase was found in about 6% of the CB-immunoreactive cells in this region. Choline acetyltransferase immunohistochemistry revealed that only about 2% of the neurons in the dorsal part of the ventral horn colocalized CB, whereas motoneurons were not CB-immunoreactive. The involvement of CB-containing neurons in ascending spinal projections to the thalamus, tegmentum, and reticular formation was demonstrated combining the retrograde transport of dextran amines and immunohistochemistry. Similar experiments demonstrated supraspinal projections from CB-containing cells mainly located in the reticular formation but also in the thalamus and the vestibular nucleus. The revealed organization of the neurons and fibers containing CB in the spinal cord of the turtle shares distribution and developmental features, colocalization with other neuronal markers, and connectivity with other tetrapods and, in particular with mammals.

EARLY SOCIAL ISOLATION DECREASES THE EXPRESSION OF CALBINDIN D-28K AND DENDRITIC BRANCHING IN THE MEDIAL PREFRONTAL CORTEX OF THE RAT

Social isolation in rodents is the most well characterized animal model for early stressful experiences and their neurobehavioral consequences. The present study analyzed the effects of early social isolation on the expression of the calcium binding protein calbindin-D28k (CAD) and dendritic arborization in the medial prefrontal cortex (mPFC) of the rat. Sprague-Dawley male rats were reared either under isolation or social conditions from 21 to 51 postnatal days. At the end of this period the animals were behaviorally evaluated in the open-field test, sacrificed, and mPFC serial sections were processed either for immunocytochemical labeling against CAD or Golgi-Cox-Sholl staining. Isolated-reared rats exhibited a dramatic decrease in the number of CAD immunoreactive neurons and a significant dendritic atrophy of layer II/III pyramidal cells in association with a reduced exploratory behavior.

Chemoarchitecture of the middle temporal visual area in the marmoset monkey (Callithrix jacchus): Laminar distribution of calcium‐binding proteins (calbindin, parvalbumin) and nonphosphorylated neurofilament

We studied the distributions of interneurons containing the calcium-binding proteins parvalbumin and calbindin D-28k, as well as that of pyramidal neurons containing nonphosphorylated neurofilament (NNF), in the middle temporal visual area (MT) of marmoset monkeys. The distributions of these classes of cells in MT are distinct from those found in adjacent areas. Similar to the primary visual area (V1), in MT, calbindin-immunopositive neurons can be objectively classified into "dark" and "light" subtypes based on optical density of stained cell bodies. Calbindin-positive dark neurons are particularly concentrated in layers 2 and 3, whereas light neurons have a more widespread distribution. In addition, a subcategory of calbindin-positive dark neuron, characterized by a "halo" of stained processes surrounding the cell body, is found within and around layer 4 of MT and V1. These cells are rare in most other visual areas. In comparison, parvalbumin-immunopositive cells in area MT have a relatively homogeneous distribution, although with a trend toward higher spatial density in lower layer 3, and are relatively uniform in terms of density of staining. Finally, MT shows a characteristic trilaminar distribution of NNF-immunopositive pyramidal cells, with stained cell bodies evident in layers 3, 5, and 6. Although the laminar distribution of cells stained for the three markers overlap to some extent, these subcategories can be readily distinguished in terms of morphology, including cell body size. Chemoarchitectural parallels observed between MT and V1 suggest comparable physiological requirements and neuronal circuitry.

Anomalous Inhibitory Circuits in Cortical Tubers of Human Tuberous Sclerosis Complex Associated With Refractory Epilepsy: Aberrant Expression of Parvalbumin and Calbindin-D28k in Dysplastic Cortex

Damage or loss of inhibitory cortical gamma-aminobutyric acid (GABA)ergic interneurons is associated with impaired inhibitory control of neocortical pyramidal cells, leading to hyperexcitability and epileptogenesis. The calcium binding proteins parvalbumin and calbindin-D(28k) are expressed in subpopulations of GABAergic local circuit neurons in the neocortex and can serve as neuronotypic markers. Parvalbumin and calbindin-D(28k) facilitate the neuron's ability to sustain firing and provide neuroprotection. The goal of this study was to assess the hitherto unknown status of inhibitory interneurons in cortical tubers of human tuberous sclerosis complex. Surgically excised cortical tubers from three patients with tuberous sclerosis complex were evaluated immunohistochemically with antibodies to parvalbumin and calbindin-D(28k). Cortical specimens from young patients with intractable seizures, including microdysgenesis (n = 3), postischemic cortical scarring (n = 1), porencephaly (n = 1), postictal gliosis (n = 3), and low-grade neuronal or glial tumors (n = 5), were also examined for comparison. In cortical tubers, calcium binding protein immunoreactivities (calbindin-D(28k) > parvalbumin) were present in medium- or large-size dysplastic neurons, whereas giant or ballooned cells were parvalbumin or calbindin-D(28k) negative. In microdysgenesis, a nearly normal number of parvalbumin-positive neurons and a decreased number of calbindin-D(28k)-positive neurons were present. In peritumoral but more so in gliotic cortex, a coordinate decrease of parvalbumin and calbindin-D(28k) immunoreactivities was present. Our findings indicate that the expression of parvalbumin or calbindin-D(28k) by subpopulations of dysplastic neurons in cortical tubers is aberrant and denotes dysfunctional inhibitory circuits inept for excitoprotection.