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A sexually dimorphic network of brain regions controls learning and production of song in zebra finches. How this specialized song system evolved is unknown. To start addressing this question, we focused on developmental differences between the sexes, using the expression of the calcium-binding protein calbindin (CB) during embryonic to adult stages to map out the early development of Area X, a male-specific striatal structure. We related this pattern to the expression of three transcription factors, Pax6 and Islet1 to delineate the striatal radial domains, and Nkx2.1 as a marker for cells of pallidal origin. An incipient Area X-CB+ domain became discernable at embryonic day 13 in the Islet1-ventral striatal field. This region contained many Nkx2.1-expressing cells with a morphology characteristic of migrating cells. Eight days after hatching (PHD) CB staining clearly delineated Area X. Another CB+ structure formed around PHD5 at the subpallial/pallial boundary. We call it the CB+striatal capsule (CB-StC) and discuss its relation with the previously described striatal capsule in vertebrates. The CB cell population in both Area X and CB-StC, but not in the surrounding striatum, colocalized with the striatal medium spiny neurons (MSNs) marker, D1-receptor associated signaling protein dopamine-and-cAMP-regulated phosphoprotein of 32 kDa, DARPP32. In females, CB-positive cells were also present in the rostral striatum but did not coalesce into an Area X-like structure. We discuss possible reasons for CB expression in MSNs in songbirds and mammals, but not described in chicken striatum., (Copyright © 2012 Wiley Periodicals, Inc.)

Amygdalar intrinsic inhibitory networks comprise several subpopulations of gamma-aminobutyric acidergic neurons, each characterized by distinct morphological features and clusters of functionally relevant neurochemical markers. In rodents, the calcium-binding proteins parvalbumin (PVB) and calbindin D28k (CB) are coexpressed in large subpopulations of amygdalar interneurons. PVB-immunoreactive (-IR) neurons have also been shown to be ensheathed by perineuronal nets (PNN), extracellular matrix envelopes believed to affect ionic homeostasis and synaptic plasticity. We tested the hypothesis that differential expression of these three markers may define distinct neuronal subpopulations within the human amygdala. Toward this end, triple-fluorescent labeling using antisera raised against PVB and CB as well as biotinylated Wisteria floribunda lectin for detection of PNN was combined with confocal microscopy. Among the 1,779 PVB-IR neurons counted, 18% also expressed CB, 31% were ensheathed in PNN, and 7% expressed both CB and PNN. Forty-four percent of PVB-IR neurons did not colocalize with either CB or PNN. The distribution of each of these neuronal subgroups showed substantial rostrocaudal gradients. Furthermore, distinct morphological features were found to characterize each neuronal subgroup. In particular, significant differences relative to the distribution and morphology were detected between PVB-IR neurons expressing CB and PVB-IR neurons wrapped in PNNs. These results indicate that amygdalar PVB-IR neurons can be subdivided into at least four different subgroups, each characterized by a specific neurochemical profile, morphological characteristics, and three-dimensional distribution. Such properties suggest that each of these neuronal subpopulations may play a specific role within the intrinsic circuitry of the amygdala., (Copyright 2006 Wiley-Liss, Inc.)

Background and Objectives: The incidence of malignant peritoneal mesothelioma (MPM) is rising. Our aim was to present our experience with this entity in order to increase the awareness about this disease to avoid misdiagnosis., Methods: Records of seven patients with histologically confirmed MPM were retrospectively reviewed. Demographic and clinicopathological findings were studied in detail., Results: There were two females and 5 males; mean age was 50.3 years (range 16-73). Asbestos exposure was recorded in two patients, familial Mediterranean fever in one and previous radiation in one. Main presentations were abdominal pain and distension. None of the patients was diagnosed preoperatively. The average delay in diagnosis was 10 months. Calretinin expression was identified in all tumors. Three patients were treated with cytoreductive surgery combined with systemic chemotherapy. Two patients who remain alive were young female patients who were diagnosed by laparoscopic incidental findings and were treated with cytoreductive surgery combined with hyperthermic intraoperative intraperitoneal chemotherapy (HIIC). Median survival was 19.7 months. The average survival time of the five patients who died of their diseases was 10.2 months., Conclusions: An awareness of MPM is important to prevent misdiagnosis. Immunohistochemistry has an important role in confirming the diagnosis. MPM remains a difficult therapeutic challenge. Thorough cytoreductive surgery is the cornerstone of current treatment while HIIC is a promising strategy in suitable patients.

The number of cerebellar Purkinje cells is increased by over 40% in young transgenic mice that overexpress a human Bcl-2 transgene (Hu-Bcl-2). To determine whether the Bcl-2-mediated rescue of Purkinje cells persists through life, the numbers of Purkinje cells were estimated in 6-, 12-, 18-, and 24-month-old Hu-Bcl-2 transgenic mice and age-matched controls. In addition, the expression of four markers for Purkinje cell differentiation, calbindin (CaBP), the 67-kDa isoform of glutamic acid decarboxylase (GAD67), gamma-aminobutyric acid transaminase (GABA-T), and the NMDA-R1 receptor subtype (NMDA-NR1) was analyzed in 6-month-old Hu-Bcl-2 transgenics and controls to determine whether overexpression of Bcl-2 and rescue from naturally occurring cell death affects the normal differentiation of Purkinje cells. The estimates of Purkinje cell numbers showed that the number of Purkinje cells in the Hu-Bcl-2 transgenics declines after 6 months to approach wild-type values by 18 months. Although the exogenous human BCL-2 is still expressed in Purkinje cells at 24 months, the expression levels of human BCL-2 appear to decline significantly after 6 months, suggesting that survival of the supernumary Purkinje cells depends on the sustained overexpression of Bcl-2. All the Purkinje cells in the Hu-Bcl-2 transgenic mice appeared to express normal levels of the differentiation markers analyzed so there was no evidence for a class of Purkinje cells that do not differentiate normally when rescued from naturally occurring cell death., (Copyright 2004 Wiley-Liss, Inc.)

Calbindin-D9k (CaBP-9k) is a member of intracellular calcium binding proteins, which have a high affinity to calcium. CaBP-9k is mainly expressed in the mammalian intestine, uterus and placenta, and is regulated in tissue- and species-specific manners. Previous studies have shown that CaBP-9k expression is mainly controlled by steroid hormones and their receptors. Thus, we further investigated the expression and regulation of CaBP-9k during an estrus cycle in the pig uterus by Northern blot and immunoblot analysis in this study. In addition, serum levels of estrogen (E2) and progesterone (P4) were measured using ELISA. The CaBP-9k mRNA is highly expressed in the porcine uterus during a luteal phase compared to a follicular phase, and its mRNA level in a luteal phase is increased up to 10-fold compared to a follicular phase. In parallel to the level of CaBP-9k mRNA, the CaBP-9k protein is also dominantly expressed in the porcine uterus, and strongly expressed in the epithelium and glands of the porcine uterus during a luteal phase. Although, the localization of the CaBP-9k protein is scarcely detected at follicular phase, it is dominantly expressed in the porcine uterus during a luteal phase. In addition, the serum P4 level was significantly increased during a luteal phase compared to a follicular phase, whereas no difference was observed in E2 levels between follicular and luteal phases, indicating that the ratio of P4/E2 is remarkably increased in porcine uterus during a luteal phase compared to a follicular phase. In conclusion, these results suggest that P4 may play an important role in the up-regulation of CaBP-9k gene in the porcine uterus in a luteal phase, which is unlike the condition in the rat uterus. In addition, the porcine CaBP-9k may be dominantly expressed in the epithelium and glandular structure of pig uterus during a luteal phase. It may also be differentially regulated during this cycle presumably by steroid hormones, especially up-regulated P4 levels in this tissue., (Copyright 2004 Wiley-Liss, Inc.)

The monkey cingulo-parahippocampal isthmus was identified recently in the depths and lateral bank of the anterior calcarine fissure but was not characterized fully. Cytoarchitectonic and immunohistochemical results presented here reveal that the isthmus is composed of four cortical areas. These include the presubiculum of the isthmus (PrSi), parasubiculum of the isthmus (PaSi), area 29 of the isthmus (area 29i) and area prostriata (Pro), which has anterior (Pro-a) and posterior (Pro-p) divisions. The PrSi, characterized by dense calbindin+ (CB+) neuropil in layer III, merges with area 29i at approximately the middle portion of the isthmus; the latter lacking the CB+ neuropil. The PaSi, characterized by a cell-free lamina dissecans and light parvalbumin+ labeling, is observed in the ventral isthmus. The Pro, located posterior to area 29i and PaSi, and anterior to area 17, has an incipient layer IV, but the density of granule cells gradually increase toward area 17. Pro-a has an incipient layer IV, contains few SMI-32+ neurons, and adjoins area 30 dorsally. The latter also has an incipient layer IV but contains, in contrast, more SMI-32+ neurons. Pro-p has a clear but thin layer IV, contains a small number of SMI-32+ neurons, and adjoins both area 23 and area 18 dorsally and area 18 ventrally. Compared with Pro-p, area 23 contains many more SMI-32+ neurons, whereas area 18 contains far more SMI-32+ neurons. These findings reveal that the isthmus is a key cortical zone connecting both the cingulate and parahippocampal gyri, but also the limbic and visual cortices. Emphasizing the former only, which has been the tendency historically, underestimates the anatomic complexity of the isthmus, and likely, its functional correlates., (Copyright 2002 Wiley-Liss, Inc.)

We used transgenic reporter mice to determine whether brain regions that respond to retinoic acid (RA) during development do so in maturity. We focused on two prominent sites of embryonic RA signaling: the dorsal spinal cord and the olfactory bulb. In the mature dorsal spinal cord, expression of a direct repeat 5 RA response element (DR5-RARE) transgene is seen in interneurons in laminae I and II, as well as in ependymal cells around the central canal. In the olfactory bulb, DR5-RARE transgene-expressing neurons are seen in the mature granule cell and periglomerular layers, as well as in cells in the subventricular zone of the forebrain-the established source for newly generated granule and periglomerular neurons. In addition, there are transgene-labeled neurons in a small number of other brain regions. These include the spinal trigeminal nucleus, area postrema, habenula, amygdala, and the cerebral cortex. Thus, a distinct type of RA-mediated gene expression, detected with the DR5-RARE reporter transgene, defines neurons, subependymal, or ependymal cells in discrete locations throughout the neuraxis. Some of these cells--particularly those in the spinal cord and olfactory bulb--are found in central nervous system regions that receive local RA signals early in development, and retain a significant amount of functional or structural plasticity in the adult., (Copyright 2002 Wiley-Liss, Inc.)

The unipolar brush cell (UBC) is a type of glutamatergic interneuron in the granular layer of the cerebellum. The UBC brush and a single mossy fiber (MF) terminal contact each other within a cerebellar glomerulus, forming a giant synapse. Many UBCs receive input from extrinsic MFs, whereas others are innervated by intrinsic mossy terminals formed by the axons of other UBCs. In all mammalian species so far examined, the vestibulocerebellum is enriched of UBCs that are strongly immunoreactive for the calcium binding protein calretinin (CR) in both the somatodendritic and axonal compartment. UBCs have postsynaptic ionotropic glutamate receptors and extrasynaptic metabotropic glutamate receptors that immunocytochemically highlight their somatodendritic compartment and brush, respectively. In this study on the mouse cerebellum, we present evidence that immunoreactivities to CR and mGluR1alpha define two distinct UBC subsets with partly overlapping distributions in lobule X (the nodulus). In sections double-labeled for CR and mGluR1alpha, the patterns of distributions of CR(+)/mGluR1alpha(-) UBCs and CR(-)/mGluR1alpha(+) UBCs differed along the mediolateral and dorsoventral axes of the folium. Moreover, mGluR1alpha(+) UBCs outnumbered CR(+) UBCs. Both UBC subsets were mGluR2/3, GluR2/3, and NMDAR1 immunoreactive. The different distribution patterns of the two UBC subsets within lobule X suggest that expression of CR or mGluR1alpha by UBCs may be afferent-specific and related to the terminal fields of different vestibular MF afferents., (Copyright 2002 Wiley-Liss, Inc.)

We examined the expression of calcium binding proteins parvalbumin (PV), calretinin (CR), and calbindin D28K (CB), and neuronal nitric oxide synthase (nNOS) in gamma-aminobutyric acid (GABA)ergic neurons of the mouse hippocampus, with particular reference to areal and dorsoventral differences. First, we estimated the colocalization of the calcium binding proteins and nNOS. GABAergic neurons containing both PV and nNOS, i.e., PV-immunoreactive (-IR)/nNOS-IR neurons, were rare in Ammon's horn but frequent in the dentate gyrus (DG). CR-IR/nNOS-IR neurons and CB-IR/nNOS-IR neurons were frequent in Ammon's horn but rare in the DG. In the entire hippocampus, the percentage of CR-IR neurons containing nNOS was significantly higher at the ventral level (44.3%) than at the dorsal level (17.0%). The percentage of CB-IR neurons containing nNOS was also significantly higher at the ventral level (42.3%) than at the dorsal level (29.3%). Next, we estimated the numerical densities (NDs) of calcium binding protein-containing GABAergic neurons. The ND of PV-IR neurons was comparable at the dorsal (1.16 x 10(3)/mm(3)) and ventral levels (1.23 x 10(3)/mm(3)), respectively. The ND of CR-IR neurons was less at the dorsal level (0.52 x 10(3)/mm(3)) than at the ventral level (0.64 x 10(3)/mm(3)). The ND of CB-IR neurons was also less at the dorsal level (0.91 x 10(3)/mm(3)) than at the ventral level (1.57 x 10(3)/mm(3)). Overall, approximately half of the GABAergic neurons contained one of the three calcium binding proteins (45% at the dorsal level and 47% at the ventral level). These data establish a baseline for examining potential roles of GABAergic neurons in hippocampal network activity in mice., (Copyright 2002 Wiley-Liss, Inc.)

The mouse cerebellar cortex is subdivided by an elaborate array of parasagittal and transverse boundaries. The relationship between these two orthogonal patterns of compartmentation is understood poorly. We have combined the use of adult and perinatal molecular markers of compartmentation-zebrin II, calbindin, and an L7/pcp-2-lacZ transgene-to resolve some of these issues. Our results indicate that the adult cerebellar vermis is divided along the rostrocaudal axis by three transverse boundaries: through the rostral face of lobule VI, in the caudal half of lobule VII, and across the posterolateral fissure between lobules IX and X. These three boundaries subdivide the vermis into four transverse zones: the anterior zone (lobules I-V), the central zone (lobules VI-VII), the posterior zone (lobules VIII-IX), and the nodular zone (lobule X). The same zones and boundaries also can be identified in the newborn cerebellum. The parasagittal organization is different in each zone: a unique combination of Purkinje cell phenotypes is found in each transverse zone both in the neonate and the adult, and different zones have distinct developmental time tables. Furthermore, the parasagittal bands of Purkinje cells revealed in the adult cerebellar cortex by using antizebrin II immunocytochemistry are discontinuous across the transverse boundaries. These data suggest that the transverse zones of the vermis form first during development and that parasagittal compartmentation develops independently in each transverse zone., (Copyright 1999 Wiley-Liss, Inc.)

The expression of the calcium-binding protein calretinin (CR) in the chick brainstem auditory nuclei angularis (NA), laminaris (NL), and magnocelularis (NM) was studied during normal development and after deafening by surgical removal of the otocyst (embryonic precursor of the inner ear) or columella (middle ear ossicle). CR mRNA was localized by in situ hybridization by using a radiolabeled oligonucleotide chick CR probe. CR immunoreactivity (CR-IR) was localized on adjacent tissue sections. CR mRNA signal in the auditory nuclei was expressed at comparable levels at embryonic day (E)9 and E11 and increased thereafter to reach the highest levels in posthatch chicks. CR-IR neurons were apparent in NM and NA at E11 and in NL by E13, and CR-IR increased in all three auditory nuclei thereafter. Neither unilateral nor bilateral otocyst removal caused detectable changes in the intensity of CR mRNA expression or CR-IR in the auditory nuclei at any of the several ages examined. Similarly, columella removal at posthatching day 2 or 3 failed to significantly affect CR mRNA or CR-IR levels at 3 hours, 1 day, or 3-4 days survival times. We conclude that cochlear nerve input is not necessary for expression of either calretinin mRNA or protein and that the profound decrease in sound-evoked activity caused by columella removal does not affect the maintenance of CR expression after hatching.

Using an antibody against calbindin-28kD, we have studied the spatial pattern of expression of this protein in the superior colliculi (SC) of four strains of mature laboratory rats. In all four strains, calbindin-expressing cells (CECs) formed horizontally oriented tiers in the retinorecipient and intermediate gray layers but were diffusely distributed throughout the deep layers. Ontogenetically, calbindin-28kD was expressed for the first time in the retinorecipient layers at postconceptional day 20 (PCD 20), by cells located in the rostrolateral region where the first born retinal ganglion cells (RGCs) are represented. Although on the day of birth (PCD 22/23), the CECs were distributed more widely, they were still absent in the most medial part of the SC, that is, the region where the latest born RGCs are represented. The spatial distribution of CECs became adultlike only by PCD 29, that is, at the end of the period of the naturally occurring death of the RGCs. Monocular eye enucleations on PCD 23 prevented the expression of calbindin in the medial fifth of the retinorecipient layers of the contralateral SC, while the unilateral removal of the visual cortices had no discernable effect on the numbers and distribution of the CECs in either SC. Thus, the spatiotemporal pattern of ontogenetic expression of calbindin-28kD in the retinorecipient layers of SC reflects the spatiotemporal pattern of generation of the RGCs, and the retinal input appears to induce neuronal expression of calbindin-28kD in these layers.

Calbindin and non-phosphorylated neurofilament proteins were assessed in hippocampus following a unilateral intracerebroventricular kainic acid injection at 4, 26, and 60 days post-lesion, using immunocytochemical expression. The density of calbindin-positive non-pyramidal neurons throughout the hippocampus showed no significant alteration at 4 days post-lesion, a significant decrease at 26 days post-lesion, and a partial recovery at 60 days post-lesion. In addition, calbindin immunoreactivity was dramatically reduced at 26 days post-lesion in the CA1 pyramidal and dentate granule cell layers and the mossy fibers, bilaterally. Although not significant statistically, most of these reductions showed signs of reversal at 60 days post-lesion except the CA1 pyramidal cell layer where the dramatic reductions persisted. Neurofilaments were also altered throughout the post-lesion period, particularly in abnormal expression of non-phosphorylated neurofilament proteins in mossy fibers. The apparent return of calbindin immunoreactivity in non-pyramidal neurons by 60 days post-lesion suggests that recovery from the lesion may involve remaining neuronal elements which either become reactivated with time or have the capability to express normal levels of calbindin with re-innervation. On the other hand, prolonged calbindin reductions in superficial CA1 pyramidal cells suggest sustained down-regulation of calbindin expression owing to persistent reductions in the activity of these neurons. The temporal correlation of the expression of non-phosphorylated neurofilaments in mossy fibers with their sprouting response following target loss suggests a potential role for non-phosphorylated neurofilaments in neuronal plasticity involving axonal sprouting. Alternatively, it may also suggest that injury-induced neurofilament modifications are either conducive or permissive for axonal sprouting.

In the present study, we attempted to trace the development of the striatal matrix by analyzing the ontogenetic expression of calbindin-D28K (calbindin), a calcium binding protein selectivity expressed in medium-sized neurons of the matrix compartment of the mature rat's caudoputamen. The localization of calbindin was documented in a series of developing rat brains, as was the compartmental location of these cells relative to tyrosine hydroxylase (TH)-immunostained dopamine islands, sites of future striosomes. Medium-sized striatal neurons appeared in the striatum at embryonic day (E) 20, and from their first appearance, the calbindin-positive neurons had highly heterogeneous distributions. They first formed a latticework of patches and bands in a ventral region of the caudoputamen. By postnatal day (P) 7, this early calbindin-positive lattice had evolved into a mosaic in which circumscript pockets of low calbindin-like immunoreactivity appeared in more extensive calbindin-rich surrounds. With further development, the mosaic gradually encroached on all but the dorsolateral caudoputamen, a district that is calbindin-poor at adulthood. A special lateral branch of the striatal calbindin system was also identified, distinct from the rest of the calbindin-positive mosaic in several developmental characteristics. In the parts of the caudoputamen where the developing calbindin system and dopamine island system were both present, the dopamine islands invariably lay in calbindin-poor zones. Most dopamine islands, however, only filled parts of the corresponding calbindin-poor zones. Moreover, there were some calbindin-poor zones for which TH-positive dopamine islands could not be detected. Thus during development, calbindin was expressed in the extrastriosomal matrix of the striatum, but the matrix could be divided into calbindin-rich and calbindin-poor zones. In the calbindin-rich regions, there were patches of especially intense calbindin expression and zones of weaker expression. These results suggest that there is neurochemical heterogeneity in the striatal matrix during the prolonged developmental period in which the early calbindin-positive lattice expands to form the calbindin-positive matrix of the mature striatum. Surprisingly, calbindin expression in the matrix, although eventually distributed in strictly complementary fashion to striosomes, does not originate as a system complementary to dopamine islands. The prolonged disparity between the borders of dopamine islands and calbindin-poor zones, and the different spatiotemporal schedules of development of the islands and the calbindin gaps suggest instead that the final match between the borders of striosomes and surrounding matrix results from dynamic processes occurring early in postnatal development. Candidate mechanisms for the gradual adjustment of these borders are proposed.