Morphometric multivariate analysis of GABAergic neurons containing calretinin and neuronal nitric oxide synthase in the mouse hippocampus

Several studies reported the morphology of calretinin-positive (CR+) neurons and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) labeled or neuronal nitric oxide synthase-positive (nNOS+) neurons in the rodent hippocampus, where these neurons showed similar morphological features. In addition, a previous study reported the frequent colocalization of CR and NADPH-d in the rat hippocampus. In this study, we aimed to examine whether CR+ neurons and nNOS+ neurons belong to a same morphological subpopulation of GABAergic neurons in the mouse hippocampus. Neurons were immunocytochemically classified into three groups, i.e., CR+/nNOS−, CR−/nNOS+ and CR+/nNOS+ groups. The present morphometric analysis was performed in the mouse Ammon’s horn, because CR+/nNOS+ neurons were rarely found in the mouse dentate gyrus. We selected three morphometric parameters, i.e., soma area, soma form factor (FF) and number of primary dendrites. Dunnett’s post-hoc analysis revealed that soma area, soma FF and number of primary dendrites were significantly larger in CR−/nNOS+ group than in CR+/nNOS− and CR+/nNOS+ groups. The morphometric data of CR+/nNOS+ group were quite similar to those of CR+/nNOS− group. The morphometric multivariate logistic regression analysis revealed that these three morphometric parameters were independent significant variables to discriminate between CR+/nNOS− and CR−/nNOS+ groups, and the majority of CR+/nNOS− and CR−/nNOS+ groups were correctly classified from the morphometric features. The present results clearly indicate that CR+/nNOS− neurons and CR−/nNOS+ neurons belong to different morphological subpopulations, and lead us to speculate that they might play different functional roles in the hippocampal circuit. The further application of morphometric multivariate analysis would be valuable to understand the functional roles of chemically defined neurons in the various brain regions.     

Interneuron deficits in patients with the Miller-Dieker syndrome

Lissencephaly is characterized by a thickened cortex and loss of gyri, resulting in the brain having a smooth surface. Patients with lissencephaly frequently exhibit epilepsy and mental retardation, conditions often associated with a defect in inhibitory neurons. While lissencephaly has traditionally been considered a disorder of radial migration, recent data indicate interneurons migrate non-radially, while projection neurons migrate radially. To determine if an interneuron defect, and therefore a non-radial migration defect, exists in patients with lissencephaly, we studied the calretinin-expressing interneuron subpopulation in the brains from two fetuses and two children with lissencephaly and a deletion involving 17p13 deletion (Miller-Dieker syndrome) along with age-matched controls. Our data indicate fetuses with the Miller-Dieker syndrome have a significant (tenfold) reduction in the number of calretinin-expressing interneurons present, whereas minimal reductions in the number of calretinin-expressing interneurons are present in children with this disorder. These data parallel those seen in the Lis1^+/– mouse model of human lissencephaly, and are consistent with a non-radial cell migration defect in humans. Thus, when considering the pathogenesis of human lissencephaly and the clinical manifestations in these patients, defects in both non-radial cell migration (inhibitory interneurons) and radial migration (excitatory projection neurons) must be considered.     

Calretinin is not a marker for subdivisions within the suprachiasmatic nucleus

In this study, we report the immunocytochemical localization of the calcium-binding protein calretinin (CAL) in the suprachiasmatic nuclei (SCN) of male and female rodents including rats, mice, golden hamsters, and Arvicanthis niloticus. The results revealed that CAL is present in different subdivisions of the SCN in the different species studied and CAL can, therefore, not be considered a marker for particular subdivisions within the SCN. No differences were found between males and females.     

Relative sparing of calretinin containing neurons in the substantia nigra of 6-OHDA treated rat parkinsonian model

A certain calcium binding protein (CaBP) has been known to exert a neuroprotective effect in various neurodegenerative diseases. Using the 6-OHDA induced rat Parkinsonian model, we examined if calretinin (CR), one of CaBP family, could play the similar role in the Parkinson's disease because CR is profusely localized in dopaminergic neurons of the substantia nigra pars compacta (SNPC) of the rat. Employing immunohistochemical analyses, we found that the survival rate of CR neurons was significantly higher than that of tyrosine hydroxylase (TH) neurons in the SNPC of the Parkinsonian rat. Furthermore double-labeled fluorescent microscopy revealed that almost all surviving TH neurons were also positive to CR. Our data suggest that CR-positive neurons are less vulnerable to 6-OHDA and CR in the dopaminergic neurons may have a protective function for survival of these neurons in the experimentally induced Parkinsonian rat.     

Differential expression of calcium-binding proteins in the red nucleus of the developing and adult human brain

The adult human red nucleus consists of two parts: (1) the parvocellular part, which is clearly separated from (2) the magnocellular part. The latter and its rubrospinal projection is known to be rudimentary in the adult human brain. Information concerning the fetal or neonatal features of the red nucleus is sparse. This study is aimed at providing a detailed account of the distribution of three calcium-binding proteins: calretinin (CR), calbindin (CB), and parvalbumin (PV), which are known to be expressed in distinct neuronal populations. Special attention has been paid to transient phenomena. CB was the most abundant protein in the magnocellular part in fetal and perinatal brains; immunoreactive (ir) neurons appeared numerous and densely packed. In the adult only few and widely spaced ir nerve cells were present. CR-expression largely corresponds to that of CB, except that fewer neurons were immunolabelled. In double- labellings the majority of neurons expressed both CB and CR; a moderate number of nerve cells solely expressing CR was present in the magnocellular part. PV-ir fibers and a moderate number of small cells were observed in the fetal, perinatal as well as the adult parvocellular part. A few PV-ir neurons were seen in the magnocellular part of the fetal and perinatal brains. Our results indicated that: (1) the magnocellular and parvocellular parts of the red nucleus were well-demarcated portions from fetal life onwards, thus a dominance of the parvocellular part over the magnocellular occurred during development; (2) the magnocellular part was more prominent in the fetal period than in adulthood; (3) neurons in the red nucleus were heterogeneous with respect to the immunoreactivities towards the three calcium-binding proteins examined; (4) the transient prominence of the magnocellular part might be a substrate for a specific transitory pattern of motor behaviour. Accepted: 7 September 2000     

A comparison of the distribution of GABA-ergic neurons in cortices representing different sensory modalities

It is well known that sensory receptive field properties are shaped by inhibitory processes. Given the physiological and perceptual distinctions among the different sensory modalities, it might be expected that the contribution of GABA-ergic inhibition to the process would vary from area to area, depending on the sensory modality represented. Furthermore, as receptive field properties become progressively more complex at higher cortical levels, differences in the inhibitory contributions to these computations would be reflected in differences in GABA-ergic neuronal distribution. These possibilities were examined in the cortices surrounding the cat Anterior Ectosylvian Sulcus (AES) which contains higher order visual (AEV), somatosensory (SIV) and auditory (Field AES) representations, and is located between the lower-level primary (AI) and secondary auditory (AII) and somatosensory (SII) areas. Using standard immunocytochemical and light-microscopic techniques, the distribution of GABA-ergic neurons (and their co-localized calcium-binding proteins: calbindin (CB), calretinin (CR) and parvalbumin (PV)) was determined for each area. When normalized for differences in cortical thickness, the depth distribution of each of the immunopositive types was plotted. These data confirmed that there were striking differences in the distribution of GABA-, CB-, CR- and PV-positive neurons. However, the laminar organization for a given marker was remarkably similar for the different subregions, irrespective of modality or hierarchical level. These data indicate that, instead of underlying processing differences among different sensory and hierarchical representations, the distribution of GABA-ergic inhibitory neurons reveals common organizational features across sensory cortex.     

Associational sprouting in the mouse fascia dentata after entorhinal lesion in vitro

Collateral sprouting is a form of neuronal plasticity observed in brain following injury. In order to establish an in vitro model of collateral sprouting, entorhino-hippocampal slice cultures were prepared from brain of C57BL/6 mouse pups (P1-4) and incubated for 14-16 days in vitro. Thereafter, entorhino-hippocampal fibers were cut and the outer molecular layer of the fascia dentata was denervated. At this age, entorhino-hippocampal fibers do not regenerate, as could be shown using anterograde tracing with Miniruby. Sprouting of associational mossy cell axons was monitored using calretinin-immunocytochemistry. Control and lesioned entorhino-hippocampal slices were studied at 1, 5, and 10 days postlesion. Whereas only the inner portion of the molecular layer was occupied by calretinin-positive mossy cell axons in controls and after 1 and 5 days postlesion, the entire width of the molecular layer was occupied by associational fibers by 10 days postlesion. Thus, robust sprouting of associational mossy cell axons occurs in response to entorhinal denervation in vitro. Using organotypic entorhino-hippocampal slices of genetically engineered mice, this sprouting model can be used to identify molecules involved in the regulation of sprouting following brain injury.     

Age-related changes in calbindin-D28k,calretinin, and parvalbumin-immunoreactive neurons in the human cerebral cortex

Calbindin-D(28k) (CB), calretinin (CRT), and parvalbumin (PV) are high-affinity cytosolic calcium (Ca(2+)) binding proteins (CBP) that have been found to regulate intracellular calcium concentrations in neurons through their buffering capacity and to protect neurons from insults that induce elevations of intracellular Ca(2+). In earlier studies we observed a substantial and neurochemically specific loss of CB from the human basal forebrain cholinergic neurons (BFCN) in the course of normal aging. In the present experiments we expanded our investigation of age-related changes in calcium binding proteins in the human brain by investigating the status of CB-, CRT-, and PV-positive neurons in 17 cortical areas. There was a trend toward a decrease in the number of CB-immunoreactive neurons in all areas studied. However, this trend reached significance in only 4 areas in which the loss of CB-positive neurons ranged between 20 and 46%. Immunoreactivity for CRT was also decreased in many areas and this difference reached significance in three regions (26-37%). Cortical neurons displaying PV immunoreactivity did not show an age-related change. Comparison with other neurochemically specific cortical neurons indicated a similar age-related loss of nonphosphorylated neurofilament and NADPH-d activity in only a few cortical areas. In contrast, neuronal acetylcholinesterase activity was increased in a few cortical areas. These observations indicate that loss of CBP-positive neurons occurs in restricted cortical regions and is not a specific change as other neurochemically specific neurons also display restricted age-related changes. Furthermore, the age-related changes in cortical CBP-positive neurons appear to be considerably smaller than similar changes in the BFCN. The age-related depletion of CBPs is likely to deprive neurons from the capacity to buffer intracellular calcium and thus to leave them vulnerable to pathological processes that can cause increased intracellular calcium and lead to their degeneration.     

Distribution of calcium-binding protein immunoreactivities in the guinea pig auditory brainstem

This study was intended to provide an overview of the distribution of calcium-binding proteins in the rodent auditory brainstem. We based our observations on immunohistochemical material obtained in the guinea pig, a species widely used in auditory research in which a mapping of calcium-binding proteins in the auditory brainstem is still missing. Differences in the amounts of these proteins throughout the auditory brainstem were further analyzed semiquantitatively. Parvalbumin was present in most neurons and their axon terminals throughout the ascending auditory brainstem. Nuclei that surround the main relay nuclei of the ascending auditory pathway lacked labeling. Calretinin staining was prominent in spherical and globular cells of the cochlear nucleus, in their axon terminals in the superior olivary complex, and in principal cells of the medial superior olive. Measures of optical densities showed that auditory neurons involved in sound localization had the highest calretinin labeling levels. Calbindin D-28k was present in cartwheel cells of the dorsal cochlear nucleus, in almost all neurons of the medial nucleus of the trapezoid body, and in globular cells in the ventral nucleus of the lateral lemniscus. The labeling patterns for calretinin and calbindin D-28k were non-overlapping throughout the auditory brainstem. This was also evident in the ventral nucleus of the lateral lemniscus where calbindin D-28k-immunoreactive terminals were found in the medial portion, while the calretinin-immunoreactive terminals were observed in the lateral portion. This study presents the first direct and comprehensive comparison of these three calcium-binding proteins in the auditory brainstem of a rodent. Each antibody yields a unique staining pattern that provides a basis for further defining neuronal populations. In addition, since their axons are also selectively stained, auditory nuclei can further be compartmentalized based on different terminal fields. These immunoreactivities have provided clues to the complex structure of the auditory brainstem.     

Parvalbumin, calretinin and carbonic anhydrase in the trigeminal and spinal primary neurons of the rat

The cell-body size of parvalbumin-immunoreactive (-ir) primary neurons was measured in the trigeminal (TG) and lumber dorsal root ganglia (DRG). In the DRG, parvalbumin-ir was mostly detected in large cells (94% in the range of 600–2800 μm²). Parvalbumin-ir TG cells were smaller than similar DRG cells and yet parvalbumin-ir TG cells of < 400 μm² (2.86%) were rare. Trichrome stains for parvalbumin, calretinin (CR) and carbonic anhydrase (CA), and for parvalbumin, calcitonin gene-related peptide (CGRP) and CA were performed to estimate possible overlap of these substances. Virtually all parvalbumin-ir DRG cells contained CA activity while a small subpopulation (28.5%) of CR-ir DRG cells lacked CA activity. All the CR-ir DRG cells that exhibited CA were also ir for parvalbumin. 31.1% of parvalbumin-ir DRG cells exhibited CR-ir while 71.5% of CR-ir DRG cells showed parvalbumin-ir. All the CR-ir DRG cells of < 400 μm² lacked CA activity and parvalbumin-ir while all those of > 800 μm² exhibited both activities. 30% of CR-ir DRG cells in the size range of 400–800 μm² co-expressed CA. DRG cel co-expressing parvalbumin and CGRP were rare (1%). As was the case for the DRG, most of parvalbumin-ir TG cells exhibited CA activity (89.24%) and lacked CGRP-ir (96.6%). CR-ir TG cells were also subdivided into two groups; one with and the other without co-expression of CA. Unlike in the DRG, however, co-expression of parvalbumin and CR could never be detected in the TG.