Effect of light on the calretinin and calbindin expression in skin club cells of adult zebrafish.

In the last decade, zebrafish has been used as a model for the study of several human skin diseases. The epidermis of Danio rerio is composed of keratinocytes and two types of secretory cells: mucous cells and club cells. Club cells have multiple biological functions and among them may be important in the protection against ultraviolet damage through the proliferative response or through the increased production of protective substances. Calcium-binding proteins such as calbindin D28K and calretinin are used as markers of nervous and enteric nervous systems, but they are present in numerous other cells. These proteins are involved in a wide variety of cell activities, such as cytoskeletal organization, cell motility and differentiation, cell cycle regulation and neuroprotective function. In this study we demonstrated, for the first time, the presence of calretinin and calbindin D28K in skin club cells of Danio rerio exposed to different wavelengths by immunohistochemistry analysis. Exposure to white-blue light and blue light causes the expression and colocalization of calbindin-D28K and calretinin. These proteins were moderately expressed and no colocalization was observed in the club cells of the control fish. In zebrafish exposed to continuous darkness for 10 days, in the club cells the two antibodies did not detect any proteins specifically. These results demonstrate that calbindin and calretinin could be involved in the pathophysiology of skin injury due to exposure to short-wavelength visible light spectrums.

GABAergic Medial Septal Neurons with Low-Rhythmic Firing Innervating the Dentate Gyrus and Hippocampal Area CA3.

The medial septum implements cortical theta oscillations, a 5-12 Hz rhythm associated with locomotion and paradoxical sleep reflecting synchronization of neuronal assemblies such as place cell sequence coding. Highly rhythmic burst-firing parvalbumin-positive GABAergic medial septal neurons are strongly coupled to theta oscillations and target cortical GABAergic interneurons, contributing to coordination within one or several cortical regions. However, a large population of medial septal neurons of unidentified neurotransmitter phenotype and with unknown axonal target areas fire with a low degree of rhythmicity. We investigated whether low-rhythmic-firing neurons (LRNs) innervated similar or different cortical regions to high-rhythmic-firing neurons (HRNs) and assessed their temporal dynamics in awake male mice. The majority of LRNs were GABAergic and parvalbumin-immunonegative, some expressing calbindin; they innervated interneurons mostly in the dentate gyrus (DG) and CA3. Individual LRNs showed several distinct firing patterns during immobility and locomotion, forming a parallel inhibitory stream for the modulation of cortical interneurons. Despite their fluctuating firing rates, the preferred firing phase of LRNs during theta oscillations matched the highest firing probability phase of principal cells in the DG and CA3. In addition, as a population, LRNs were markedly suppressed during hippocampal sharp-wave ripples, had a low burst incidence, and several of them did not fire on all theta cycles. Therefore, CA3 receives GABAergic input from both HRNs and LRNs, but the DG receives mainly LRN input. We propose that distinct GABAergic LRNs contribute to changing the excitability of the DG and CA3 during memory discrimination via transient disinhibition of principal cells.SIGNIFICANCE STATEMENT For the encoding and recall of episodic memories, nerve cells in the cerebral cortex are activated in precisely timed sequences. Rhythmicity facilitates the coordination of neuronal activity and these rhythms are detected as oscillations of different frequencies such as 5-12 Hz theta oscillations. Degradation of these rhythms, such as through neurodegeneration, causes memory deficits. The medial septum, a part of the basal forebrain that innervates the hippocampal formation, contains high- and low-rhythmic-firing neurons (HRNs and LRNs, respectively), which may contribute differentially to cortical neuronal coordination. We discovered that GABAergic LRNs preferentially innervate the dentate gyrus and the CA3 area of the hippocampus, regions important for episodic memory. These neurons act in parallel with the HRNs mostly via transient inhibition of inhibitory neurons.

Three Types of A11 Neurons Project to the Rat Spinal Cord.

The A11 dopaminergic cell group is the only group among the A8-A16 dopaminergic cell groups that includes neurons innervating the spinal cord, and a decrease in dopaminergic transmission at the spinal cord is thought to contribute to the pathogenesis of restless legs syndrome. However, the mechanisms regulating the neuronal activity of A11 dopaminergic neurons remain to be elucidated. Unraveling the neuronal composition, distribution and connectivity of A11 neurons would provide insights into the mechanisms regulating the spinal dopaminergic system. To address this, we performed immunohistochemistry for calcium-binding proteins such as calbindin (Calb) and parvalbumin (PV), in combination with the retrograde tracer Fluorogold (FG) injected into the spinal cord. Immunohistochemistry for Calb, PV, or tyrosine hydroxylase (TH), a marker for dopaminergic neurons, revealed that there were at least three types of neurons in the A11 region: neurons expressing Calb, TH, or both TH and Calb, whereas there were no PV-immunoreactive (IR) cell bodies. Both Calb- and PV-IR processes were found throughout the entire A11 region, extending in varied directions depending on the level relative to bregma. We found retrogradely labeled FG-positive neurons expressing TH, Calb, or both TH and Calb, as well as FG-positive neurons lacking both TH and Calb. These findings indicate that the A11 region is composed of a variety of neurons that are distinct in their neurochemical properties, and suggest that the diencephalospinal dopamine system may be regulated at the A11region by both Calb-IR and PV-IR processes, and at the terminal region of the spinal cord by Calb-IR processes derived from the A11 region.

Development of Calbindin- and Calretinin-Immunopositive Neurons in the Enteric Ganglia of Rats.

Calbindin D28 K (CB) and calretinin (CR) are the members of the EF-hand family of calcium-binding proteins that are expressed in neurons and nerve fibers of the enteric nervous system. CB and CR are expressed differentially in neuronal subpopulations throughout the central and peripheral nervous systems and their expression has been used to selectively target specific cell types and isolate neuronal networks. The present study presents an immunohistochemical analysis of CB and CR in the enteric ganglia of small intestine in rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 60-day-old, 1-year-old, and 2-year-old). The data obtained suggest a number of age-dependent changes in CB and CR expression in the myenteric and submucous plexuses. In the myenteric plexus, the lowest percentage of CB-immunoreactive (IR) and CR-IR neurons was observed at birth, after which the number of IR cells increased in the first 10 days of life. In the submucous plexus, CB-IR and CR-IR neurons were observed from 10-day-old onwards. The percentage of CR-IR and CB-IR neurons increased in the first 2 months and in the first 20 days, respectively. In all animals, the majority of the IR neurons colocalized CR and CB. From the moment of birth, the mean of the cross-sectional area of the CB-IR and CR-IR neuronal profiles was larger than that of CB- and CR-negative cells.

The ontogenetic development of neurons containing calcium-binding proteins in the septum of the guinea pig: Late onset of parvalbumin immunoreactivity versus calbindin and calretinin.

The study describes the immunoreactivity of calbindin (CB), calretinin (CR) and parvalbumin (PV), their distribution pattern and the co-distribution of CB and CR as well as CB and PV in the septum of the guinea pig during development. Immunohistochemistry was conducted on embryonic (E40, E50, E60), newborn (P0) and postnatal (P5, P10, P20, P40, P100) guinea pig brains. The presence of both CB and CR was detected at E40, while PV began to be observed at E60. Immunoreactivity for CB was constant throughout ontogeny. In contrast to CR immunoreactivity, PV immunoreactivity was higher in the postnatal stages than in the prenatal and newborn stages. Double immunostaining showed that CB co-localized with CR from E40 onwards, while with PV from P5 onwards, suggesting that CB co-operates with these proteins in the guinea pig septum during different periods of ontogeny. Our results also indicate that among the studied CaBPs, CB exhibited the highest immunoreactivity during both embryonic and postnatal development.

Structural development and dorsoventral maturation of the medial entorhinal cortex.

We investigated the structural development of superficial-layers of medial entorhinal cortex and parasubiculum in rats. The grid-layout and cholinergic-innervation of calbindin-positive pyramidal-cells in layer-2 emerged around birth while reelin-positive stellate-cells were scattered throughout development. Layer-3 and parasubiculum neurons had a transient calbindin-expression, which declined with age. Early postnatally, layer-2 pyramidal but not stellate-cells co-localized with doublecortin - a marker of immature neurons - suggesting delayed functional-maturation of pyramidal-cells. Three observations indicated a dorsal-to-ventral maturation of entorhinal cortex and parasubiculum: (i) calbindin-expression in layer-3 neurons decreased progressively from dorsal-to-ventral, (ii) doublecortin in layer-2 calbindin-positive-patches disappeared dorsally before ventrally, and (iii) wolframin-expression emerged earlier in dorsal than ventral parasubiculum. The early appearance of calbindin-pyramidal-grid-organization in layer-2 suggests that this pattern is instructed by genetic information rather than experience. Superficial-layer-microcircuits mature earlier in dorsal entorhinal cortex, where small spatial-scales are represented. Maturation of ventral-entorhinal-microcircuits - representing larger spatial-scales - follows later around the onset of exploratory behavior.

Enteric plexuses of two choline-acetyltransferase transgenic mouse lines: chemical neuroanatomy of the fluorescent protein-expressing nerve cells.

We studied cholinergic circuit elements in the enteric nervous system (ENS) of two distinct transgenic mouse lines in which fluorescent protein expression was driven by the choline-acetyltransferase (ChAT) promoter. In the first mouse line, green fluorescent protein was fused to the tau gene. This construct allowed the visualization of the fiber tracts and ganglia, however the nerve cells were poorly resolved. In the second mouse line (ChATcre-YFP), CRE/loxP recombination yielded cytosolic expression of yellow fluorescent protein (YFP). In these preparations the morphology of enteric neurons could be well studied. We also determined the neurochemical identity of ENS neurons in muscular and submucous layers using antibodies against YFP, calretinin (CALR), calbindin (CALB), and vasoactive intestinal peptide (VIP). Confocal microscopic imaging was used to visualize fluorescently-conjugated secondary antibodies. In ChATcre-YFP preparations, YFP was readily apparent in somatodendritic regions of ENS neurons. In the myenteric plexus, YFP/CALR/VIP staining revealed that 34% of cholinergic cells co-labeled with CALR. Few single-stained CR-positive cells were observed. Neither YFP nor CALR co-localized with VIP. In GFP/CALB/CALR staining, all co-localization combinations were represented. In the submucosal plexus, YFP/CALR/VIP staining revealed discrete neuronal populations. However, in separate preparations, double labeling was observed for YFP/CALR and CALR/VIP. In YFP/CALR/CALB staining, all combinations of double staining and triple labeling were verified. In conclusion, the neurochemical coding of ENS neurons in these mouse lines is consistent with many observations in non-transgenic animals. Thus, they provide useful tools for physiological and pharmacological studies on distinct neurochemical subtypes of ENS neurons.

The cocaine- and amphetamine-regulated transcript, calbindin, calretinin and parvalbumin immunoreactivity in the medial geniculate body of the guinea pig.

The purpose of this study was to describe the distribution and colocalization of cocaine- and amphetamine-regulated transcript (CART) and three calcium-binding proteins (calbindin, calretinin and parvalbumin) in each main division of the medial geniculate body (MGB) in the guinea pig. From low to moderate CART immunoreactivity was observed in all divisions of the MGB, although in most of its length only fibers and neuropil were labeled. A small number of CART immunoreactive somata were observed in the caudal segment of the MGB. The central parts of all divisions contained a distinctly smaller number of CART immunoreactive fibers relative to their outer borders, where CART fibers formed patchy clusters. As a whole, the intense CART immunoreactive borders formed a shell around the weakly CART labeled core. Double-labeling immunofluorescence showed that CART did not colocalize with either calbindin, calretinin or parvalbumin, whose immunoreactivity was predominantly restricted to perikarya. The distribution pattern of calretinin was more similar to that of calbindin than to that of parvalbumin. Calretinin and calbindin exhibited higher immunoreactivity in the medial and dorsal divisions of the MGB, where parvalbumin staining was low. In general, although parvalbumin exhibited the weakest immunoreactivity of all studied Ca(2+) binding proteins, it was most highly expressed in the ventral division of the MGB. Our results indicate that CART could be involved in hearing, although its immunoreactivity in the medial geniculate complex was not as intense as in other sensory brain regions. In the guinea pig the heterogeneous and complementary pattern of calbindin, calretinin and parvalbumin is evident, however, the overlap in staining appears to be more extensive than that seen in other rodents.

N-methyl-N-nitrosourea during late gestation results in concomitant but reversible progenitor cell reduction and delayed neurogenesis in the hippocampus of rats.

N-Methyl-N-nitrosourea (MNU) is an alkylating agent having genotoxic potential to cause gene mutations and antiproliferative cytotoxic activity on developing brains to cause microcephaly by mid-gestational exposure in rodents. This study investigated the transient genotoxic and cytocidal effect of MNU at the beginning of the subgranular zone (SGZ) formation in the hippocampal dentate gyrus on neurogenesis in later life using rats. Pregnant rats were injected with MNU at 0 (vehicle controls), 1 or 3mg/kg body weight intraperitoneally from gestational day (GD) 18 to GD 20 once a day. In offspring, effects were observed at 3mg/kg. Fetal brains on GD 21 after the last MNU injection increased TUNEL(+) apoptotic cells in the tertiary germinal matrices. At postnatal day (PND) 21 on weaning, offspring displayed decrease of doublecortin (Dcx)(+) cells and cell proliferation in the SGZ and increase of calbindin (Calb1)(+) interneurons in the dentate hilus. Postnatal single bromodeoxyuridine (BrdU) injection on PND 3 resulted in an increase of BrdU(+)/Dcx(+) cells. On PND 77, Dcx(+) cells recovered in number, but cell proliferation increased in the SGZ. Thus, late-gestational maternal MNU exposure may induce reversible reductions of type-3 progenitor and/or immature granule cells and cell proliferation on weaning in response to progenitor cell apoptosis, as well as delayed neurogenesis due to cell cycle arrest. Increases of Calb1(+) interneurons on weaning and SGZ cell proliferation later on may reflect compensatory mechanism for MNU-induced aberrant neurogenesis. Considering the lack of effects on PND 77, MNU may mainly target transient populations of highly proliferative progenitor cells without affecting their stem cells to undergo progenitor production. Protective and plasticity mechanism may be operated against genotoxic agents on hippocampal neurogenesis.

Grid-layout and theta-modulation of layer 2 pyramidal neurons in medial entorhinal cortex.

Little is known about how microcircuits are organized in layer 2 of the medial entorhinal cortex. We visualized principal cell microcircuits and determined cellular theta-rhythmicity in freely moving rats. Non-dentate-projecting, calbindin-positive pyramidal cells bundled dendrites together and formed patches arranged in a hexagonal grid aligned to layer 1 axons, parasubiculum, and cholinergic inputs. Calbindin-negative, dentate-gyrus-projecting stellate cells were distributed across layer 2 but avoided centers of calbindin-positive patches. Cholinergic drive sustained theta-rhythmicity, which was twofold stronger in pyramidal than in stellate neurons. Theta-rhythmicity was cell-type-specific but not distributed as expected from cell-intrinsic properties. Layer 2 divides into a weakly theta-locked stellate cell lattice and spatiotemporally highly organized pyramidal grid. It needs to be assessed how these two distinct principal cell networks contribute to grid cell activity.

Colocalization pattern of calbindin and cocaine- and amphetamine-regulated transcript in the mammillary body-anterior thalamic nuclei axis of the guinea pig.

The study describes for the first time the colocalization pattern of calbindin (CB) and cocaine- and amphetamine-regulated transcript (CART) in the mammillary body (MB) and anterior thalamic nuclei (ATN) - structures connected in a topographically organized manner by the mammillothalamic tract (mtt). Immunohistochemical study was performed on fetal (E40, E50, E60), newborn (P0) and postnatal (P20, P80) brains of the guinea pig, but the coexistence pattern of the substances was invariable throughout the examined developmental stages. CB and CART colocalized in the perikarya of the lateral part of the medial mammillary nucleus (MMl), whereas in its medial part (MMm) only CB was detected. In the mtt, which originates from the MB, both the substances were present and colocalized in single fibers. Next, fibers from the mtt spread toward the ATN in a particular way: fibers containing CB ran to both the anteromedial thalamic nucleus (AM) and anteroventral thalamic nucleus (AV), while fibers containing CART ran mostly to the latter one. In the ventral part of AV, CB and CART colocalized vastly in the neuropil. The lateral mammillary nucleus and anterodorsal thalamic nucleus were virtually devoid of CB- and CART-positive structures. Based on the known connections between the MB and ATN, we conclude that the studied substances may cooperate in the MMl-AV part of the axis and CB plays a significant role in the MMm-AM part.

Cortical inputs innervate calbindin-immunoreactive interneurons of the rat basolateral amygdaloid complex.

The present study was undertaken to shed light on the synaptic organization of the rat basolateral amygdala (BLA). The BLA contains multiple types of GABAergic interneurons that are differentially connected with extrinsic afferents and other BLA cells. Previously, it was reported that parvalbumin immunoreactive (PV(+) ) interneurons receive strong excitatory inputs from principal BLA cells but very few cortical inputs, implying a prevalent role in feedback inhibition. However, because prior physiological studies indicate that cortical afferents do trigger feedforward inhibition in principal cells, the present study aimed to determine whether a numerically important subtype of interneurons, expressing calbindin (CB(+) ), receives cortical inputs. Rats received injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHAL) in the perirhinal cortex or adjacent temporal neocortex. Light and electron microscopic observations of the relations between cortical inputs and BLA neurons were performed in the lateral (LA) and basolateral (BL) nuclei. Irrespective of the injection site (perirhinal or temporal neocortex) and target nucleus (LA or BL), ~90% of cortical axon terminals formed asymmetric synapses with dendritic spines of principal BLA neurons, while 10% contacted the dendritic shafts of presumed interneurons, half of which were CB(+) . Given the previously reported pattern of CB coexpression among GABAergic interneurons of the BLA, these results suggest that a subset of PV-immunonegative cells that express CB, most likely the somatostatin-positive interneurons, are important mediators of cortically evoked feedforward inhibition in the BLA.

Distribución de calbindina y parvoalbúmina y efecto del virus de la rabia sobre su expresión en la médula espinal de ratones / Calbindin and parvalbumin distribution in spinal cord of normal and rabies-infected mice

Introducción. Aunque se trata de una enfermedad infecciosa del sistema nervioso, poco se conoce sobre los mecanismos patogénicos de la infección con el virus de la rabia. En particular, son escasos los estudios sobre su histopatología en la médula espinal. Objetivo. Estudiar la distribución de las proteínas calbindina y parvoalbúmina, en la médula espinal de ratones y evaluar el efecto de la infección con el virus de la rabia sobre su expresión. Materiales y métodos. Se inocularon ratones con virus de la rabia, por vía intracerebral o intramuscular, y se extrajo la médula espinal para hacer cortes transversales, los cuales se sometieron a tratamiento inmunohistoquímico con anticuerpos monoclonales para revelar la presencia de las dos proteínas en ratones normales y en animales infectados. Se llevó a cabo el análisis cualitativo y cuantitativo de la inmunorreacción de las dos proteínas. Resultados. Las proteínas calbindina y parvoalbúmina se distribuyeron de manera diferencial en las láminas de Rexed. La infección con el virus de la rabia produjo una disminución en la expresión de calbindina. Por el contrario, la infección provocó un incremento en la expresión de parvoalbúmina. El efecto de la rabia sobre las dos proteínas fue similar al comparar las dos vías de inoculación. Conclusión. El efecto diferencial de la infección con el virus de la rabia sobre calbindina y parvoalbúmina en la médula espinal de ratones, es similar al reportado anteriormente para áreas encefálicas. Esto sugiere uniformidad en su respuesta a la infección en todo el sistema nervioso central y es un aporte importante para el conocimiento de la patogénesis de la rabia.

Introduction: Rabies is a fatal infectious disease of the nervous system; however, the knowledge about the pathogenic neural mechanisms in rabies is scarce. In addition, there are few studies of rabies pathology of the spinal cord. Objective: To study the distribution of calcium binding proteins calbindin and parvalbumin and assessing the effect of rabies virus infection on their expression in the spinal cord of mice. Materiales y methods: Mice were inoculated with rabies virus, by intracerebral or intramuscular route. The spinal cord was extracted to perform some crosscuts which were treated by immunohistochemistry with monoclonal antibodies to reveal the presence of the two proteins in normal and rabies infected mice. We did qualitative and quantitative analyses of the immunoreactivity of the two proteins. Results: Calbindin and parvalbumin showed differential distribution in Rexed laminae. Rabies infection produced a decrease in the expression of calbindin. On the contrary, the infection caused an increased expression of parvalbumin. The effect of rabies infection on the two proteins expression was similar when comparing both routes of inoculation. Conclusion: The differential effect of rabies virus infection on the expression of calbindin and parvalbumin in the spinal cord of mice was similar to that previously reported for brain areas. This result suggests uniformity in the response to rabies infection throughout the central nervous system. This is an important contribution to the understanding of the pathogenesis of rabies.

Distribution of parvalbumin, calbindin and calretinin containing neurons and terminal networks in relation to sleep associated nuclei in the brain of the giant Zambian mole-rat (Fukomys mechowii).

To broaden the understanding of the neural control and evolution of the sleep-wake cycle in mammals, the distribution and interrelations of sleep associated nuclei with neurons and terminal networks expressing the calcium-binding proteins parvalbumin, calbindin and calretinin were explored in a rodent that lacks a significant visual system. The sleep-associated nuclei explored include the cholinergic basal forebrain and pontine nuclei, the catecholaminergic locus coeruleus complex, the serotonergic dorsal raphe nuclear complex, the hypothalamic orexinergic nuclei, and the thalamic reticular nucleus. Zambian mole-rat brains were sectioned and stained in a one in nine series for Nissl, myelin, choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), serotonin (5HT), orexin (OrxA), calbindin (CB), calretinin (CR) and parvalbumin (PV). We observed that while the density of immunopositive calbindin (CB+) neurons and terminal networks varied in the different sleep related nuclei, they were found in all nuclei apart from the compact and diffuse subdivisions of the subcoeruleus, which lacked CB+ neurons but evinced a CB+ terminal network. The density of calretinin immunopositive (CR+) neurons and terminal networks varied between the sleep related nuclei, but was present in all nuclei examined. Neurons and terminal networks associated with PV immunoreactivity were the most sparsely distributed in these nuclei, but were present in the majority of nuclei. The thalamic reticular nucleus had the highest density of PV+ neurons and terminal networks, while PV+ neurons were absent in the cholinergic pontine nuclei, and PV+ neurons and terminal networks were absent in the orexinergic nuclei. The increased presence of neurons and terminal networks expressing the calcium binding proteins in comparison to that seen in the laboratory rat, specifically in the brainstem, may account for the prominent muscle twitches during REM sleep previously observed in this subterranean African rodent.

[Calbindin and parvalbumin distribution in spinal cord of normal and rabies-infected mice]. / Distribución de calbindina y parvoalbúmina y efecto del virus de la rabia sobre su expresión en la médula espinal de ratones.

INTRODUCTION: Rabies is a fatal infectious disease of the nervous system; however, the knowledge about the pathogenic neural mechanisms in rabies is scarce. In addition, there are few studies of rabies pathology of the spinal cord. OBJECTIVE: To study the distribution of calcium binding proteins calbindin and parvalbumin and assessing the effect of rabies virus infection on their expression in the spinal cord of mice. MATERIALES Y METHODS: Mice were inoculated with rabies virus, by intracerebral or intramuscular route. The spinal cord was extracted to perform some crosscuts which were treated by immunohistochemistry with monoclonal antibodies to reveal the presence of the two proteins in normal and rabies infected mice. We did qualitative and quantitative analyses of the immunoreactivity of the two proteins. RESULTS: Calbindin and parvalbumin showed differential distribution in Rexed laminae. Rabies infection produced a decrease in the expression of calbindin. On the contrary, the infection caused an increased expression of parvalbumin. The effect of rabies infection on the two proteins expression was similar when comparing both routes of inoculation. CONCLUSION: The differential effect of rabies virus infection on the expression of calbindin and parvalbumin in the spinal cord of mice was similar to that previously reported for brain areas. This result suggests uniformity in the response to rabies infection throughout the central nervous system. This is an important contribution to the understanding of the pathogenesis of rabies.