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PubMed2004

Immunocytochemically defined interneuron populations in the hippocampus of mouse strains used in transgenic technology.

Transgenic mice are overtaking the role of model animals in neuroscience. They are used in developmental, anatomical, and physiological as well as experimental neurology. However, most results on the organization of the nervous system derive from the rat. The rat hippocampus and its neuronal elements have been thoroughly investigated, revealing remarkable functional and morphological diversity and specificity among hippocampal interneurons. Our aim was to examine the properties of distinct hippocampal interneuron populations, i.e., those immunoreactive for calcium-binding proteins (parvalbumin, calbindin, and calretinin), neuropeptides (cholecystokinin, neuropeptide Y, somatostatin, vasoactive intestinal polypeptide), and certain receptors (metabotropic glutamate receptor 1alpha, cannabinoid receptor type 1) in four strains of mice widely used in transgenic technology, and to compare their properties to those in the rat. Our data indicate that the distribution as well as the dendritic and axonal arborization of mouse interneurons immunoreactive for the different markers was identical in the examined mouse strains, and in most respects are similar to the features found in the rat. The postsynaptic targets of neurons terminating in the perisomatic (parvalbumin), proximal (calbindin), and distal (somatostatin) dendritic region, as well as on other interneurons (calretinin), also matched those found in the rat. However, a few significant differences could also be observed between the two species in addition to the already described immunoreactivity of mossy cells for calretinin: the absence of spiny calretinin-immunoreactive interneurons in the CA3 region, sparse contacts between calretinin-immunoreactive interneurons, and the axon staining for somatostatin and neuropil labeling for cholecystokinin. We can conclude that the morphofunctional classification of interneurons established in the rat is largely valid for mouse strains used in transgenic procedures.

Animals↗
PubMed2001

Nitric oxide synthase-positive neurons in the rat superior colliculus: colocalization of NOS with NMDAR1 glutamate receptor, GABA, and parvalbumin.

We analyzed the potential input and output components of nitric oxide synthase (NOS)-containing neurons in the rat superior colliculus (SC). To identify whether NOS-positive neurons receive glutamatergic input we investigated the colocalization of NOS with NMDA receptor subunit R1 (NMDAR1). In addition, to examine whether putative nitric oxide synthesizing neurons represent a neurochemically specific or distinct subpopulation of cells in the SC we studied the colocalization of NOS with the neurotransmitter GABA, the calcium-binding proteins parvalbumin, calbindin and calretinin and with neuropeptides such as somatostatin, substance P and neuropeptide Y. We found that 90% of NOS-positive neurons in the superficial layers of the rat SC express NMDAR1. Nearly 20% of the population of nitridergic neurons also expresses GABA and 15% of them express parvalbumin. NOS-positive neurons in the superior colliculus did not contain calretinin, calbindin or either of the neuropeptides tested. The results of this study show that the capacity for synthesizing NO in the SC is largely restricted to neurons that receive glutamatergic inputs and that some of these neurons express GABA or parvalbumin.

Animals↗
PubMed2006

Glial cell line-derived neurotrophic factor promotes the arborization of cultured striatal neurons through the p42/p44 mitogen-activated protein kinase pathway.

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival or differentiation of several types of neurons. This study examines GDNF-induced signal transduction and biological effects in cultured striatal neurons. Results show that GDNF addition to striatal cultures transiently increased the protein levels of phosphorylated p42/p44, but did not change the levels of phosphorylated Akt. GDNF effects on phosphorylated p42/p44 levels were blocked by the mitogen-activated protein kinase (MAPK) pathway specific inhibitors (PD98059 and U0126). Activation of the p42/p44 MAPK pathway by GDNF led to an increase in the degree of dendritic arborization and axon length of both GABA- and calbindin-positive neurons but had no effect on their survival and maturation. These GDNF-mediated effects were suppressed in the presence of the inhibitor of the MAPK pathway (PD98059). Furthermore, the addition of the phosphatidylinositol 3-kinase pathway specific inhibitor (LY294002) blocked GDNF-mediated striatal cell differentiation suggesting that the basal activity of this pathway is needed for the effects of GDNF. Our results indicate that treatment of cultured striatal cells with GDNF specifically activates the p42/p44 MAPK pathway, leading to an increase in the arborization of GABA- and calbindin-positive neurons.

Animals↗
PubMed2006

Developmental changes in Ca2+-regulated functions of early postnatal Purkinje neurons.

Ca(2+) influx through L-type Ca(2+) channels regulates several different cellular processes in developing Purkinje neurons, including activation of transcription factors and expression of cellular proteins. In the current studies, we examined the age dependence of these actions of Ca(2+) during the early developmental period. Purkinje neurons acutely isolated from postnatal day 4-8 rat pups were studied. We also examined the sensitivity of the Ca(2+)-regulated processes to a toxic environmental factor (ethanol) known to show age-dependent actions on developing Purkinje neurons. Results show that Ca(2+) activation of the transcription factor cAMP-responsive element binding protein (CREB) and Ca(2+)-induced alterations in the level of the apoptotic enzyme caspase 3 show both dose and age dependence in the early-developing Purkinje neurons. Interestingly, the age dependence was opposite for the two proteins. Ca(2+) regulation of calbindin, a Ca(2+) binding protein, was dose dependent but showed little age dependence. Exposure to ethanol altered Ca(2+) activation of pCREB in an age-dependent manner but did not alter Ca(2+) regulation of caspase 3 or calbindin levels. Taken together, these results show that the downstream effects of Ca(2+) signaling have age-dependent components during early Purkinje neuron development. This age dependence may play an important role in the normal developmental program and could contribute to the critical window of sensitivity observed for certain toxic agents during early development.

Age Factors↗
PubMed1998

Electrostatic coupling to pH-titrating sites as a source of cooperativity in protein-ligand binding.

This paper describes an alternative mechanism for the cooperative binding of charged ligands to proteins. The ligand-binding sites are electrostatically coupled to protein side chains that can undergo protonation and deprotonation. The binding of one ligand alters the protein's protonation equilibrium in a manner that makes the the binding of the second ligand more favorable. This mechanism requires no conformational change to produce a cooperative effect, although it is not exclusive of conformational change. We present a theoretical description of the mechanism, and calculations on three kinds of systems: A model system containing one protonation site and two ligand-binding sites; a model system containing two protonation sites and two ligand-binding sites; and calbindin D9k, which contains two Ca2+-binding sites and 30 protonation sites. For the one-protonation-site model, it is shown that the influence of the protonation site can only be cooperative. The competition of this effect with the anticooperative effect of ligand-ligand repulsion is studied in detail. For the two-protonation site model, the effect can be either cooperative or, in special cases, anticooperative. For calbindin D9k, the calculations predict that six protonation sites in or near the ligand-binding sites make a cooperative contribution that approximately cancels the anticooperative effect of Ca2+-Ca2+ repulsion, accounting for more than half of the total cooperative effect that is needed to overcome repulsion and produce the net cooperativity observed experimentally. We argue that cooperative mechanisms of the kind described here are likely when there is more than one ligand-binding site in a protein domain.

Binding Sites↗
PubMed1996

Analysis of binding of the 1,25-dihydroxyvitamin D3 receptor to positive and negative vitamin D response elements.

The binding of the 1,25-dihydroxyvitamin D3 receptor to the vitamin D response elements (VDREs) in the rat osteocalcin (OSC-DRE), mouse osteopontin (MOP-DRE), rat calbindin D-9k (CaBP-DRE), and human parathyroid hormone genes (PTH-DRE) was studied. Binding of VDR to the three positive VDREs is cooperative. The degree of cooperativity is highest with the calbindin VDRE compared with either the OSC-DRE or the MOP-DRE. This cooperativity is largely absent in the case of the negative element, the PTH-DRE. The VDR binds in order of decreasing affinity to PTH-DRE > OSC-DRE = MOP-DRE > CaBP-DRE. Thus, the greatest affinity is associated with the lowest degree of cooperativity. Further study has revealed that the PTH-VDRE actually consists of two repeat elements like all other VDREs and is not a single six-base sequence. A nuclear factor has also been found that binds downstream from the GGTTCA element in the PTH promoter. The binding site of this factor overlaps the PTH-DRE nucleotide sequence.

Animals↗
PubMed1989

Two classes of cortical GABA neurons defined by differential calcium binding protein immunoreactivities.

Calcium ions play a key role in many aspects of neuronal behavior and certain calcium binding proteins that may influence this behavior are differentially distributed in the central nervous system. In this study it is shown that immunoreactivity for calbindin-28 and for parvalbumin is localized in separate populations of inhibitory GABA interneurons in all areas of the neocortex of Old World monkeys. Virtually all GABA neurons show immunoreactivity for one or other calcium binding protein but, except for a few cells in layer IV, GABA cells do not show immunoreactivity for both proteins. Among the two cell populations, parvalbumin immunoreactivity characterizes basket neurons while calbindin immunoreactivity characterizes double bouquet neurons. These findings suggest that the two GABA cell types differ in their regulation of calcium homeostasis and may yield clues to their different roles in intracortical circuitry.

Animals↗
PubMed1992

Abnormal local-circuit neurons in epilepsia partialis continua associated with focal cortical dysplasia.

A limited cortical resection including the rolandic fissure and the pre- and postcentral cortical regions was carried out in a patient suffering from epilepsia partialis continua resistant to antiepileptic drugs. The histological examination revealed several foci of very large neurons distributed with no laminar organization in the depth of the rolandic fissure and in the crown of the primary motor and primary somatosensory areas; these lesions were consistent with focal cortical dysplasia. In addition, decreased numbers of neurons, astrocytosis and proliferation of capillaries, compatible with chronic tissue necrosis, were found in the inferior regions of the banks of the rolandic fissure. Subpopulations of local-circuit neurons were examined with parvalbumin, calbindin D-28k and somatostatin immunocytochemistry. Focal areas of cortical dysplasia contained abnormal immunoreactive neurons. Huge parvalbumin-immunoreactive cells were distributed at random and resembled axo-axonic (chandelier) and basket neurons. Abnormal calbindin D-28k-immunoreactive cells were reminiscent of double-bouquet neurons and multipolar cells. Very large somatostatin-immunoreactive cells were seldom observed in the dysplastic foci. On the other hand, areas of tissue necrosis displayed massive reduction of immunoreactive cells and fibers. Abnormalities in the morphology and distribution of local-circuit (inhibitory) neurons observed here for the first time in focal cortical dysplasia may have a pivotal role in the appearance and prolongation of electrical discharges and continuous motor signs in human focal epilepsy.

Adolescent↗
PubMed1995

Digoxigenylated primary antibodies for sensitive dual-peroxidase labelling of neural markers.

This study extends the application of the digoxigenin-anti-digoxigenin (DIG) technique to immunocytochemistry by using digoxigenin-tagged primary antibodies. Certain features of this technique when applied to non-radioactive in situ hybridization, such as the absence of endogeneous digoxigenin immunoreactivity in animal tissues, seem to be advantageous also for its application to immunocytochemistry. Thus, the present work is focused on dual-peroxidase staining experiments based on digoxigenylated antibodies directed against glial fibrillary acidic protein, parvalbumin, and calbindin, in a straightforward combination with conventional cytochemical methods. The protocols include the concomitant detection of two antigens, for which only primary antibodies from one animal species are available, with differently haptenized antibodies (e.g., biotinylated anti-calbindin and digoxigenylated anti-parvalbumin). The versatility of the DIG technique is exemplified by the combination of lectin and immunocytochemical procedures for the detection of astrocytes and microglia, and the simultaneous visualization of perineuronal nets and parvalbumin-containing neurons in the rat brain.

Animals↗
PubMed2001

Enhanced neurogenesis after transient global ischemia in the dentate gyrus of the rat.

The dentate gyrus is one of the few areas of the mammalian brain where new neurons are continuously produced in adulthood. Certain insults such as epileptic seizures and ischemia are known to enhance the rate of neuronal production. We analyzed this phenomenon using the temporary occlusion of the two carotid arteries combined with arterial hypotension as a method to induce ischemia in rats. We measured the rate of cell production and their state of differentiation with a mitotic indicator, bromodeoxyuridine (BrdU), in combination with the immunohistochemical detection of neuronal markers. One week after the ischemic episode, the cell production in dentate gyrus was increased two- to threefold more than the basal level seen in control animals. Two weeks after ischemia, over 60% of these cells became young neurons as determined by colabeling with BrdU and a cytoplasmic protein (CRMP-4) involved in axonal guidance during development. Five weeks after the ischemia, over 60% of new neurons expressed calbindin, a calcium-binding protein normally expressed in mature granule neurons. In addition to more cells being generated, a greater proportion of all new cells remained in the differentiated but not fully mature state during the 2- to 5-week period after ischemia. The maturation rate of neurons as determined by the calbindin labeling and by the rate of migration from a proliferative zone into the granule cell layer was not changed when examined 5 weeks after ischemia. The results support the hypothesis that survival of dentate gyrus after ischemia is linked with enhanced neurogenesis. Additional physiological stimulation after ischemia may be exploited to stimulate maturation of new neurons and to offer new therapeutic strategies for promoting recovery of neuronal circuitry in the injured brain.

Animals↗
PubMed1998

Efferent synaptic connections of dopaminergic neurons grafted into the caudate nucleus of experimentally induced parkinsonian monkeys are different from those of control animals.

This study investigated the question of whether grafted dopamine cells in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys form synapses and, if they do, whether their postsynaptic targets were the same as those in control monkeys or in previous studies in rats. Electron-microscopic single immunostaining was performed for tyrosine hydroxylase on vibratome sections prepared from the head of the caudate nucleus of controls and MPTP-treated African green monkeys (Cercopithecus aethiops sabaeus) that received a graft. Furthermore, correlated light- and electron-microscopic double immunostaining was carried out for tyrosine hydroxylase and calbindin in the same brain area of MPTP-treated plus grafted animals. In control monkeys, the majority (97%) of dopamine boutons terminate on spines that were also synaptic targets of immunonegative boutons forming asymmetric synaptic contacts: synaptic triads. In MPTP-treated, grafted animals, the majority of transplanted dopamine cells terminate on dendritic shafts (67%) and somata (32%), and only a few (1.33%) form axospine synapses. The results of the double immunostaining experiments indicated that these newly formed axosomatic and axodendritic synapses are associated with calbindin-immunoreactive, medium-sized, spiny striatonigral projection neurons. These observations indicate that: (1) dopamine from transplanted embryonic tissue acts via synaptic contacts on host neurons; (2) the primary synaptic targets of transplanted dopamine cells are not spines but dendrites and somata of host neurons; (3) these target neurons are the same as in control animals; and (4) comparing these observations with results of control and grafted rats, there are major species differences between rats and monkeys in the dopamine innervation of both control and transplanted animals.

Animals↗
PubMed2000

Temporal lobe epilepsy with and without psychosis: exploration of hippocampal pathology including that in subpopulations of neurons defined by their content of immunoreactive calcium-binding proteins.

We have investigated relationships between hippocampal/temporal lobe neuropathology and psychosis in subjects with temporal lobe epilepsy, paying particular attention to possible differences in density of hippocampal neurons immunoreactive for calcium-binding proteins. There was a trend for a greater prevalence of left handedness in the psychotic (n = 6) than the non-psychotic (n = 26) cases (P = 0.0504). Psychotic cases also differed from non-psychotic ones in having: (1) more focal lesions outside the hippocampus (P = 0.006); (2) less severe CAI neuron loss (P = 0.015); and (3) a trend, after Bonferroni correction, for a higher density of calbindin-immunoreactive neurons in the CA4 (P = 0.022). An additional finding was that dentate granule cell dispersion was significantly associated with the presence of a reduced density of calretinin-immunoreactive neurons in CA4 (P = 0.002) and with a more severe loss of CA4 neurons visible with Nissl stain (P = 0.003). Thus, cases of temporal lobe epilepsy with psychosis were distinguishable on the basis of a higher density of calbindin-reactive neurons in CA4 as well as on more general aspects of their pathology.

Adolescent↗
PubMed2003

Villin is a possible marker of receptor cells in frog taste organs.

We investigated lingual taste organs of four frog species mainly by means of fluorescence immunohistochemistry for villin, calbindin, and serotonin. Cells immunoreactive for villin appeared in the taste organs of all the species used. These villin-immunostained cells were basoapically elongated in shape and extended up to the apical surface. They were also immunoreactive for calbindin. On the other hand, serotonin-immunoreactive cells, identified as Merkel-like basal cells, were immunonegative for villin. Considering the present results combined with those of studies by other research groups, the villin-immunostained cells were postulated to function as taste receptors.

Animals↗
PubMed2006

Nucleus accumbens subregions: hodological and immunohistochemical study in the domestic chick (Gallus domesticus).

The nucleus accumbens was identified in avian species some time ago. However, the precise localization and extent of this nucleus is still a matter of controversy. We have used immunolabeling against calbindin, neuropeptide Y, and DARPP-32 (dopamine- and adenosine-related phosphoprotein, 32 kDa) for the selective marking of putative accumbens subdivisions and have followed the anterograde transport of biotinylated dextran amine injected to the nucleus tractus solitarii region of 7-day-old domestic chicks. The nucleus accumbens extending between rostrocaudal atlas coordinates A 10.6 and A 8.8 can be subdivided into the core and shell, the core corresponding to the ventromedial and juxtaventricular medial striatum laterodorsal to the bed nucleus of stria terminalis, and the shell representing an arched region situated ventrally and ventrolaterally to the core. Immunoreactivity to both calbindin and neuropeptide Y is more intense in the shell than in the core division. DARPP-32 immunolabeling does not differ in the two divisions but is markedly weaker in the bed nucleus of stria terminalis, enabling the separation of this nucleus from the surrounding accumbens subdivisions. Fibers from the nucleus solitarius predominantly terminate in the shell division, similar to the situation described in mammals. Whereas the suggested core lies entirely within the boundary of the medial striatum, the shell seems partially to overlap the ventral pallidum. We have been unable to subdivide the remaining part of accumbens lying rostral to A 10.6 into a putative shell and core by the methods employed in the present study. This region probably corresponds to the rostral pole of the nucleus accumbens.

Animals↗
PubMed1998

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine.

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.

Animals↗
PubMed1998

Quantitative analysis of inputs to somatostatin-immunoreactive descending interneurons in the myenteric plexus of the guinea-pig small intestine.

Somatostatin immunoreactivity occurs in a specific subgroup of cholinergic descending interneurons in the myenteric plexus of the guinea-pig small intestine. In the present work, we made light- and electron-microscopic investigations of chemically defined inputs to these neurons, in order that the origins of the connections of other neurons with them could be deduced. Somatostatin-immunoreactive synapses and close contacts were found on the cell bodies and filamentous processes of somatostatin neurons; these were 84% of all inputs. It is thus confirmed that this class of interneuron forms chains that project anally. Descending interneurons with immunoreactivity for nitric oxide synthase provided 14% of inputs to somatostatin-immunoreactive descending interneurons. An antiserum against a calcium-binding protein, calbindin, was used as marker for the majority of intrinsic primary afferent neurons, AH/Dogiel type II neurons; this class of neurons provided only 2.5% of the inputs to somatostatin-immunoreactive descending interneurons. We conclude that somatostatin-immunoreactive descending interneurons are involved in the conduction of impulses distally along the full length of the small intestine, but receive only a minor input from calbindin-immunoreactive primary afferent neurons.

Animals↗
PubMed1993

Heterogeneous distribution of D1, D2 and D5 receptor mRNAs in monkey striatum.

The primate striatum has a compartmental organization reflected both in the topography of its afferent projections and in the segregation of its morphologically similar but neurochemically distinct efferent neurons. Discretely projecting mesostriatal neurons release dopamine (DA) which modulates the responses of striatal neurons to other afferent inputs. Multiple DA receptor (DAR) subtypes have been cloned and characterized and mapping their cellular expression is crucial for understanding the influence of DA on striatal function. We report the distribution of mRNAs for D1, D2 and D5 DAR subtypes (D2R, D2R and D5R) in the striatum of cynomolgus monkeys (Macaca fascicularis) studied by in situ hybridization histochemistry (ISH) using monkey-specific cRNA probes. Adjacent sections were stained for calbindin immunoreactivity to distinguish striosomal and matrix compartments for comparison with the patterns obtained with ISH. In the caudate nucleus, D1R mRNA was concentrated in calbindin-poor striosomes where dense grain clusters were seen overlying the majority of medium-sized neurons (diameter approximately 15 microns). D1R mRNA localization was relatively homogeneous in the putamen. By contrast, the distributions of D2R and D5R mRNAs showed no clear preference for the striosomal or matrix compartments of either caudate nucleus or putamen. In the ventral striatum (nucleus accumbens, olfactory tubercle and ventral portions of caudate nucleus and putamen), expression of D1R and D2R mRNA was sparse relative to dorsal striatum, while D5R mRNA expression was roughly equal in ventral and dorsal striatum. Circumscribed zones of hybridization associated with islands of tightly packed small cells occurred with all three DAR mRNA subtypes in the ventral striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Calretinin-immunoreactive neurons in the normal human temporal cortex and in Alzheimer's disease.

Calretinin-containing neurons (CR) were visualized by immunocytochemistry in the human temporal cortex. The morphology of calretinin-positive neurons ranged from bipolar, bitufted, fusiform to double bouquet cells, whose long axis was parallel to the radial axis of the cortex. Calretinin-immunoreactive cells were more abundant in layers II, III and less frequent in layer VI and white matter. In layer I, large horizontal neurons resembling Cajal-Retzius cells were observed. Layers IV and V contained few labeled cells. The CR-immunoreactive neuropil was abundant, especially in supragranular layers. However, the most prominent feature of the pattern of calretinin staining was the presence of long, vertically oriented bundles of calretinin-immunoreactive processes. These bundles formed a widespread, regular columnar system descending throughout layers II to VI. Despite the virtually identical morphological features of CR-immunoreactive neurons and certain calbindin-immunoreactive neurons, colocalization studies for both antibodies against calretinin and calbindin, revealed little coexistence (in supragranular layers) or none (in infragranular layers). Thus, double bouquet cells could be considered as forming a chemically heterogeneous neuronal population. In addition, four brains from patients with Alzheimer's disease were immunostained for calretinin. No major differences from normal brains were found; the distribution, morphology and the characteristic, vertically oriented bundles resembled those described in normal brains. These data suggest that these calcium-binding protein-containing interneurons are present in normal human brain and that they are resistant to degeneration in Alzheimer's disease.

Aged↗