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PubMed2000

Developmental sex differences in calbindin-D(28K) and calretinin immunoreactivity in the neonatal rat hypothalamus.

The proteins calbindin-D(28K) and calretinin buffer intracellular calcium and are speculated to be involved in the integration of neuronal signaling. Using Western blot analysis, we compared the levels of calbindin-D(28K) and calretinin in the developing male and female rat hypothalamus on postnatal days (PN) 0, PN2, PN4, PN6, PN8, and PN10. Analysis of variance (ANOVA) of mean calbindin levels indicated a significant effect of sex (p </=.001) and age (p </=. 0001) and a significant interaction (p </=.02). Post-hoc Neuman-Keuls analysis revealed that PN0 and PN2 males had significantly elevated calbindin levels over PN0 and PN2 females (p </=.05). ANOVA of mean calretinin levels from the same animals also indicated a significant effect of sex (p </=.002) and a significant interaction between sex and age (p </=.001). Post-hoc analysis indicated males had significantly elevated calretinin levels over PN0, PN4 (p </=.05) and PN6 (p </=.01) females. Immunocytochemical analyses indicated calbindin-immunopositive staining for cell bodies in the central subdivision of the medial preoptic nucleus, paraventricular nucleus, arcuate nucleus, and dorsomedial nucleus, and an area immediately surrounding the ventromedial nucleus (VMN). Calbindin immunoreactivity was absent from the ventrolateral VMN, but lightly stained cell bodies were observed in the dorsomedial VMN. The sex differences observed in calcium binding proteins parallel our previously observed sex differences in excitatory gamma-aminobutyric acid and glutamate early in development and may be related to mechanisms of sexual differentiation of the brain.

Aging↗
PubMed2000

Expression of ciliary neurotrophic factor activated by retinal Müller cells in eyes with NMDA- and kainic acid-induced neuronal death.

PURPOSE: To elucidate the role of retinal Muller cells in N-methyl-D-aspartate (NMDA)- or kainic acid (KA)induced retinal damage. METHODS: In experimental eyes, NMDA or KA was injected into the vitreous of rat eyes. Immunohistochemistry and western blot analysis were conducted to elucidate expression and localization of glial fibrillary acidic protein (GFAP) and ciliary neurotrophic factor (CNTF). In addition, the neuroprotective effects of CNTF were calculated by counting cells in the ganglion cell layer (GCL) and by measuring the thickness of the various retinal layers. RESULTS: Morphometric analysis of retinal damage in NMDA- and KA-injected eyes showed significant cell loss in the GCL and thinning of the inner plexiform layer (IPL) of the retina, but not of other retinal layers. Immunohistochemistry demonstrated disappearance and/or decrease in immunoreactivities of calbindin- and calretinin- positive cells and their neurites and upregulated expression of both GFAP and CNTF in experimental eyes. Western blot analysis showed an increase in protein expression for CNTF in retinas of experimental eyes. Confocal images and sequential localization demonstrated colocalization of CNTF and GFAP in the inner retinal layer and possibly in Muller cells. In addition, pretreatment with CNTF (1 microg) before the intravitreal injection of NMDA (or KA) demonstrated that CNTF has neuroprotective effects against NMDA- or KA-induced neuronal death in the retina. CONCLUSIONS: These studies revealed the upregulated expression of CNTF and GFAP in Muller cells in response to NMDA- and KA-induced neuronal death, suggesting that production of CNTF in Muller cells may be a part of the endogenous neuroprotective system in the retina.

Animals↗
PubMed2000

Patterns of calretinin, calbindin, and tyrosine-hydroxylase expression are consistent with the prosomeric map of the frog diencephalon.

This paper re-examines a previously published segmental map of the frog diencephalon (Puelles et al. [1996] Brain Behav.Evol. 47:279-310) by means of immunocytochemical mapping of calretinin, calbindin, and tyrosine hydroxylase. The distribution of neuronal populations, axon tracts, and neuropils immunoreactive for these markers was studied in adult specimens of Rana perezi and Xenopus laevis sectioned sagittally or horizontally. Emphasis was placed on study of the relationship of observed chemoarchitectural boundaries with the postulated overall prosomeric organization and the schema of nuclear subdivisions we reported previously, based on acetylcholinesterase histochemistry and Nissl pattern in Rana. The data reveal a large-scale correspondence with the segmental map in both species, although some differences were noted between Rana and Xenopus. Notably, retinorecipient neuropils were generally immunoreactive for calretinin only in Rana. Importantly, calretinin immunostaining underlines particularly well the transverse prosomeric boundaries of the dorsal thalamus. A number of nuclear subdivisions noted before with AChE were corroborated, and some novel subdivisions became apparent, particularly in the anterior nucleus of the dorsal thalamus and in the habenular complex. The mapping of tyrosine hydroxylase clarified the segmental distribution of the catecholaminergic cell groups in the frog forebrain, which is comparable to that observed in other vertebrates.

Animals↗
PubMed2000

Neurones in the adult rat anterior medullary velum.

The presence of neurones in the rat anterior medullary velum (AMV) has been investigated by using antibodies to the calcium-binding proteins, parvalbumin (PV), calretinin (CR), and calbindin-D28k (CB). Disparate populations of mainly GABAergic neurones were located in the rostral and caudal regions of the AMV. The rostral region of the AMV was characterised by GABAergic CR-labelled or PV-labelled neurones. CR-labelled neurones were bipolar or multipolar with round to ovoid somata (diameters between 8 and 12 microm), and rostrocaudally running dendrites forming a network. PV-labelled neurones had round somata (diameters between 6 and 10 microm) and were bi-tufted, with beaded dendrites. Both CR-labelled and PV-labelled dendrites formed punctate pericellular associations with unlabelled somatic profiles. In the caudal region of the AMV, PV-labelled neurones were GABAergic, multipolar cells, having round somata (diameters between 9 and 12 microm), with either beaded or nonbeaded dendrites forming a network of interconnecting dendrites. PV-labelled pericellular associations were made around both PV-labelled and unlabelled somatic profiles. CR labelled unipolar brush cells (UBCs) were not GABAergic. UBCs were characterised by a round to oval somata (10-15 microm in diameter) from which a single primary dendrite emerged to form a distal expansion having small terminal dendrites. From the distal expansion, there also appeared to be CR-labelled processes emanating and extending for up to 250 microm. CB occasionally labelled "Purkinje-like cells" (PLCs). The rat AMV is a more complex structure than first envisaged with the presence of predominantly inhibitory neurones expressing different calcium-binding proteins. Functional and anatomic aspects of this circuitry are further discussed.

Animals↗
PubMed2000

Cholinergic endings on various neurones containing calcium binding proteins and glutamic acid decarboxylase in the hippocampus of the rat.

Immunohistochemical study of the cholinergic innervation of the hippocampal cells containing glutamic acid decarboxylase (GAD) and calcium binding proteins: parvalbumin (PV), calbindin D28k (CB) and calretinin (CR) was conducted on 5 adult rat brains. Analysis of sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and respectively either GAD or PV, CB, CR, using confocal laser-scanning microscope shows that the intensive cholinergic innervations receive GAD, PV and CB-positive hippocampal cells. Cholinergic afferentiations of the CR-positive neurones are considerably fewer.

Acetylcholine↗
PubMed2002

Chemical coding and electrophysiology of enteric neurons expressing neurofilament 145 in guinea pig gastrointestinal tract.

Electrophysiologic recording and indirect immunofluorescence were combined to study localization of the medium-sized neurofilament 145 (NF145) component of the cytoskeleton in morphologically identified neurons in the myenteric and submucosal plexuses of the guinea pig enteric nervous system. Neuronal localization of chemical markers, including calbindin DK28, calretinin, nitric oxide synthase, choline-acetyltransferase, neuropeptide Y, serotonin, neurokinin 1 receptor protein, and somatostatin, was integrated with electrophysiologic and morphologic results for a more complete assessment. NF145 immunoreactivity (-IR) was present in ganglion cells with Dogiel type I morphology in the myenteric plexus of the stomach and small and large intestine. NF145-IR was not found in myenteric ganglion cells with Dogiel type II morphology. NF145-IR was not present in any of the ganglion cells in the submucosal plexus. NF145 was expressed in nerve fibers in both myenteric and submucosal plexuses. The majority of these fibers were identified as sympathetic postganglionic axons based on their disappearance in organotypic culture and on their expression of tyrosine hydroxylase. The myenteric ganglion cells with NF145-IR had electrophysiologic properties of S-type enteric neurons. NF145-IR was found in neurons with vasoactive intestinal peptide, serotonin, nitric oxide synthase, somatostatin, and neurokinin 1 receptor but not with neuropeptide Y or calbindin. The results in general suggest that NF145 is localized to distinct subsets of myenteric motor neurons and interneurons. Absence of NF145 from ganglion cells in the submucosal plexus is an example of differences between myenteric and submucosal components of the enteric nervous system.

Action Potentials↗
PubMed2002

I(h) channels contribute to the different functional properties of identified dopaminergic subpopulations in the midbrain.

Dopaminergic (DA) midbrain neurons in the substantia nigra (SN) and ventral tegmental area (VTA) are involved in various brain functions such as voluntary movement and reward and are targets in disorders such as Parkinson's disease and schizophrenia. To study the functional properties of identified DA neurons in mouse midbrain slices, we combined patch-clamp recordings with either neurobiotin cell-filling and triple labeling confocal immunohistochemistry, or single-cell RT-PCR. We discriminated four DA subpopulations based on anatomical and neurochemical differences: two calbindin D28-k (CB)-expressing DA populations in the substantia nigra (SN/CB+) or ventral tegmental area (VTA/CB+), and respectively, two calbindin D28-k negative DA populations (SN/CB-, VTA/CB-). VTA/CB+ DA neurons displayed significantly faster pacemaker frequencies with smaller afterhyperpolarizations compared with other DA neurons. In contrast, all four DA populations possessed significant differences in I(h) channel densities and I(h) channel-mediated functional properties like sag amplitudes and rebound delays in the following order: SN/CB- --> VTA/CB- --> SN/CB+ --> VTA/CB+. Single-cell RT-multiplex PCR experiments demonstrated that differential calbindin but not calretinin expression is associated with differential I(h) channel densities. Only in SN/CB- DA neurons, however, I(h) channels were actively involved in pacemaker frequency control. In conclusion, diversity within the DA system is not restricted to distinct axonal projections and differences in synaptic connectivity, but also involves differences in postsynaptic conductances between neurochemically and topographically distinct DA neurons.

Action Potentials↗
PubMed2002

The distribution and morphology of calbindin D28K- and calretinin-immunoreactive neurons in the visual cortex of mouse.

We studied the distribution and morphology of calbindin D28K- and calretinin-immunoreactive (IR) neurons in the mouse visual cortex with immunocytochemistry. Most of the calbindin D28K-IR neurons were located in layers II/III and V, while calretinin-IR neurons were predominantly located in layers II/III. The labeled neurons showed variations in morphology. The majority of the calbindin D28K-IR neurons were stellate and round or oval cells with multipolar dendrites. The majority of calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicular to the pial surface. In the mouse visual cortex, 20.2% of calbindin D28K-IR neurons contained calretinin and 27.2% of calretinin-IR neurons contained calbindin D28K. These results indicate that the calcium-binding proteins, calbindin D28K and calretinin are distributed in specific layers and in selective cell types of the mouse visual cortex.

Animals↗
PubMed2000

Developmental study of rat vestibular neuronal circuits during a spaceflight of 17 days.

The aim of this study was to investigate the potential plasticity of the vestibular system, in structural and biochemical terms, at the level of the gravity receptors (the sensory hair cells), the primary neurons relaying the sensory signals (the vestibular ganglion neurons) and their projections into the vestibular nuclei. We studied the biochemical differentiation of the sensory cells and of the vestibular ganglion by investigating which calcium-binding proteins were present. We studied the development of peripheral synaptic connections of the efferent system by investigating the distribution of CGRP (calcitonin-gene related-peptide) and we also studied the cerebellar synaptic connections in the vestibular nuclei, as identified by the presence of calbindin. Putative changes were studied after a 17-day episode of microgravity (Neurolab STS-90), in developing rats between postnatal days 8 and 25. The extent to which these changes could be caused by alterations in gravity was determined by examining sensory and nervous structures not involved in gravity detection, the cochlea and the cochlear nuclei.

Acoustic Maculae↗
PubMed2003

Co-localisation of NOS with calcium-binding proteins during the postnatal development of the rat claustrum.

An immunocytochemical double-staining method was applied in order to study the co-localisation of nitric oxide synthase (NOS) with three calcium-binding proteins, calbindin D28k (CB), calretinin (CR) and parvalbumin (PV) in the claustrum of the rat during the first 4 months of life (postnatal days: PO-P120). The co-localisation of NOS/PV and NOS/CB is reported. These neurons fall into the category of non-pyramidal cells. Double-labelled NOS/CB neurons are observed in the claustrum starting from P4, whereas double-labelled NOS/PV neurons are observed from P14 onwards. The percentages of double-labelled neurons increase in relation to the age. Double-labelled NOS/CB and NOS/PV neurons, although they do not constitute a numerous population, play an important role in the process of maturation of the claustrum. This is confirmed by the occurrence of these types of neurons at definite stages of maturation and by the increase in their number.

Aging↗
PubMed2003

Resonant synchronization in heterogeneous networks of inhibitory neurons.

Brain rhythms arise through the synchronization of neurons and their entrainment in a regular firing pattern. In this process, networks of reciprocally connected inhibitory neurons are often involved, but what mechanism determines the oscillation frequency is not completely understood. Analytical studies predict that the emerging frequency band is primarily constrained by the decay rate of the unitary IPSC. We observed a new phenomenon of resonant synchronization in computer-simulated networks of inhibitory neurons in which the synaptic current has a delayed onset, reflecting finite spike propagation and synaptic transmission times. At the resonant level of network excitation, all neurons fire synchronously and rhythmically with a period approximately four times the mean delay of the onset of the inhibitory synaptic current. The amplitude and decay time constant of the synaptic current have relatively minor effects on the emerging frequency band. By varying the axonal delay of the inhibitory connections, networks with a realistic synaptic kinetics can be tuned to frequencies from 40 to >200 Hz. This resonance phenomenon arises in heterogeneous networks with, on average, as few as five connections per neuron. We conclude that the delay of the synaptic current is the primary parameter controlling the oscillation frequency of inhibitory networks and propose that delay-induced synchronization is a mechanism for fast brain rhythms that depend on intact inhibitory synaptic transmission.

Action Potentials↗
PubMed2003

Immunocytochemical localization of neurons containing the AMPA GluR2/3 subunit in the hamster visual cortex.

AMPA glutamate receptors play a crucial role in brain functions such as synaptic plasticity and development. We have studied the chemo-architecture of the AMPA glutamate receptor subtype GluR2/3 in the hamster visual cortex by immunocytochemistry and compared it with the distribution of the calcium-binding proteins, calbindin D28K and calretinin. Anti-GluR2/3-immunoreactive (IR) neurons were predominantly located in layers II/III, V, and VI, and the majority of the labeled neurons were round or oval. However, many pyramidal cells in layer V were also labeled. Two-color immunofluorescence revealed that none of the GluR2/3-IR neurons contained calbindin D28 K or calretinin. Thus specific layers of neurons express the GluR2/3 subunit and these do not correlate with expression of calbindin D28K and calretinin.

Animals↗
PubMed2004

Co-existence of calcium-binding proteins and gamma-aminobutyric acid or glycine in neurons of the rat medullary dorsal horn.

BACKGROUND: We investigated the co-expression of calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV, a combination of the three is referred to as CaBPs) with gamma-aminobutyric acid (GABA) or glycine in neurons of the rat medullary dorsal horn (MDH). METHODS: Immunofluorescence histochemical double-staining for CaBPs and GABA or glycine was performed on the sections from rat MDH. RESULTS: CB-, CR-, PV-, GABA- and glycine-like immunoreactive (LI) neurons were differentially observed in all layers of the MDH, but particularly in lamina II. Neurons that exhibited immunoreactivity for both CaBPs and GABA or glycine were also observed mainly in lamina II. A few of them were found in laminae I and III. The percentages of neurons which co-expressed CB/GABA or CB/glycine out of the total numbers of CB- and GABA-LI neurons or CB- and glycine-LI neurons were 5.3% and 12.1% or 4.1% and 10.0%, respectively. The ratios of CR/GABA or CR/glycine co-existing neurons out of the total numbers of CR- and GABA-LI neurons or CR- and glycine-LI neurons were 5.8% and 7.6% or 4.4% and 7.1%, respectively. The rates of PV/GABA or PV/glycine co-localized neurons out of the total numbers of PV- and GABA-LI neurons or PV- and glycine-LI neurons were 11.1% and 5.1% or 9.9% and 5.1%, respectively. CONCLUSION: The results indicate that some neurons in the MDH contain both CaBPs and GABA or glycine.

Animals↗
PubMed2004

[Effect of rabies virus infection on the expression of parvalbumin, calbindin and calretinin in mouse cerebral cortex].

Some clinical features of rabies and experimental evidence from cell culture and laboratory animals suggest impairment of gabaergic neurotransmission. Several types of gabaergic neurons occur in the cerebral cortex. They can be identified by three neuronal markers: the calcium binding proteins (CaBPs) parvalbumin (PV), calbindin (CB) and calretinin (CR). Rabies virus spreads throughout the cerebral cortex; however, rabies cytopathic effects on gabaergic neurons are unknown. The expression of calcium-binding proteins (CaBPs) parvalbumin (PV), calbindin (CB) and calretinin (CR) was studied in the frontal cortex of mice. The effect of gabaergic neurons was evaluated immunohistochemically. The distribution patterns of CaBPs in normal mice and in mice infected with 'fixed' or 'street' rabies virus were compared. PV was found in multipolar neurons located in all cortical layers except layer I, and in pericellular clusters of terminal knobs surrounding the soma of pyramidal neurons. CB-immunoreactivity was distributed in two cortical bands. One was composed of round neurons enclosed by a heavily labeled neuropil; this band corresponds to supragranular layers II and III. The other was a weakly stained band of neuropil which contained scattered multipolar CB-ir neurons; this corresponds to infragranular layers V and VI. The CR-ir neurons were bipolar fusiform cells located in all layers of cortex, but concentrated in layers II and III. A feature common to samples infected with both types of viruses was a more intense immunoreactivity to PV in contrast to normal samples. The infection with 'street' virus did not cause additional changes in the expression of CaBPs. However, the infection with 'fixed' virus produced a remarkable reduction of CB-immunoreactivity demonstrated by the loss of CB-ir neurons and low neuropil stain in the frontal cortex. In addition, the size of CR-ir neurons in the cingulate cortex was decreased.

Animals↗
PubMed2004

Nitric oxide synthase and calcium-binding protein-containing neurons in the hamster visual cortex.

The distribution and morphology of neurons containing neuronal nitric oxide synthase (NOS), and calcium-binding proteins calbindin D28K and calretinin in the hamster visual cortex were compared by immunocytochemistry. Staining for NOS, calbindin D28K and calretinin was seen both in the specific layers and in the selective cell types. The densest concentration of anti-NOS-immunoreactive (IR) neurons was found in layer VI. Most of the calbindin D28K-IR neurons were located in layers II/III and V while the calretinin-IR neurons were predominantly located in layers II/III. The labeled neurons varied in morphology. The large majority of NOS-IR neurons were round or oval cells with many dendrites coursing in all directions. The majority of the calbindin D28K-IR neurons were stellate and round or oval cells with multipolar dendrites. The majority of the calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicular to the pial surface. Our study showed that 14.7% and 27.5% of the NOS-IR cells in the hamster visual cortex contained calbindin D28K or calretinin, respectively. These results indicate that NOS, calbindin and calretinin are located in specific layers and specific cell types and the vast majority of NOS-containing neurons are limited to neurons that do not express calbindin D28K or calretinin.

Animals↗
PubMed2005

Open field stress and neurons containing calcium-binding proteins in the piriform cortex of the rat.

In the present study we wanted to check whether the expression of the c-Fos protein (the marker of cellular activity) appears in cells containing calcium-binding proteins (CaBPs) in animals exposed to the open field test. Eight adult Wistar rats were examined. In the first step the open field test was applied throughout 10 minutes. After perfusional fixation brains were frozen and cut on the cryostat in the coronal plane and stained with the standard immunohistochemical method. Sections were double stained for c-Fos and CaBPs: parvalbumin (PV), calbindin (CB), calretinin (CR). c-Fos positive cells were localized predominantly in layers II and III of the piriform cortex (PC). The double labeling study showed that neurons containing CaBPs are rarely c-Fos-immunoreactive. Often PV-positive and CB-positive fibers surround c-Fos-positive neurons in layers II and III in a form of a basket. It seems that cells containing CaBPs are not directly involved in the response to aversive stimuli but cells containing those calcium-binding proteins might influence directly c-Fos positive neurons of PC.

Animals↗