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The thalamic reticular nucleus receives axons from the thalamic sensory nuclei and the cerebral cortex. The visual part of this nucleus in carnivores is the perigeniculate nucleus located dorsal to the lateral geniculate nucleus. The perigeniculate nucleus participates in the modulation of visual processing and in the transition of synchronized slow rhythmicity during sleep into desynchronized high-frequency activity during arousal and consists of inhibitory neurons. The main neurochemical markers for perigeniculate neurons are glutamic acid decarboxylase and Casup2+/sup-binding protein parvalbumin. Previous studies of postnatal development focused on the morphological features of the perigeniculate nucleus; however, its neurochemistry remains poorly understood. In this study, we focused on the postnatal development of perigeniculate neurons using immunohistochemical labeling of parvalbumin, two related Casup2+/sup-binding proteins (calretinin and calbindin), glutamic acid decarboxylase, and a common neuronal protein, NeuN, in kittens that were 0-123 days old and in adult cats. In parallel with the well-known dominant neuronal populations expressing parvalbumin and GAD67 and persisting until adulthood, transient populations expressing calretinin and calbindin were observed. The calbindin-positive neurons were similar to the main perigeniculate population and showed close morphological features and parvalbumin coexpression. In contrast, the calretinin-positive neurons differed in their morphological characteristics and did not express GAD67, thus distinguishing them from the majority of perigeniculate neurons. A possible link between these populations was revealed, and the development of thalamocortical processing is discussed.

Retrosplenial cortex (RSC) is a brain region involved in critical cognitive functions including memory, planning, and spatial navigation and is commonly affected in neurodegenerative diseases. Subregions of RSC are typically described as Brodmann areas 29 and 30, which are defined by cytoarchitectural features. Using immunofluorescence, we studied the distributions of neurons immunoreactive for NeuN, latexin, and calcium binding proteins (calbindin, calretinin, and parvalbumin) in RSC of Carollia perspicillata, Seba's short-tailed fruit bat. We observed that latexin was specifically present in areas 29a and 29b but not 29c and 30. We further identified distribution patterns of calcium binding proteins that group areas 29a and 29b separately from areas 29c and 30. We conclude first that latexin is a useful marker to classify subregions of RSC and second that these subregions contain distinct patterns of neuronal immunoreactivity for calcium binding proteins. Given the long lifespan of Carollia, bat RSC may be a useful model in studying age-related neurodegeneration.

The superior colliculus (SC) is a layered midbrain structure with functions that include polysensory and sensorimotor integration. Here, we describe the distribution of different immunohistochemically identified classes of neurons in the SC of adult marmoset monkeys (Callithrix jacchus). Neuronal nuclei (NeuN) staining was used to determine the overall neuronal density in the different SC layers. In addition, we studied the distribution of neurons expressing different calcium-binding proteins (calbindin [CB], parvalbumin [PV] and calretinin [CR]). Our results indicate that neuronal density in the SC decreases from superficial to deep layers. Although the neuronal density within the same layer varies little across the mediolateral axis, it tends to be lower at rostral levels, compared to caudal levels. Cells expressing different calcium-binding proteins display differential gradients of density according to depth. Both CB- and CR-expressing neurons show markedly higher densities in the stratum griseum superficiale (SGS), compared to the stratum opticum and intermediate and deep layers. However, CR-expressing neurons are twice as common as CB-expressing neurons outside the SGS. The distribution of PV-expressing cells follows a shallow density gradient from superficial to deep layers. When normalized relative to total neuronal density, the proportion of CR-expressing neurons increases between the superficial and intermediate layers, whereas that of CB-expressing neurons declines toward the deep layers. The proportion of PV-expressing neurons remains constant across layers. Our data provide layer-specific and accurate estimates of neuronal density, which may be important for the generation of biophysical models of how the primate SC transforms sensory inputs into motor signals.

In the current study, we examined the number, distribution, and aspects of the neurochemical identities of infracortical white matter neurons, also termed white matter interstitial cells (WMICs), in the brains of a southern lesser galago (Galago moholi), a black-capped squirrel monkey (Saimiri boliviensis boliviensis), and a crested macaque (Macaca nigra). Staining for neuronal nuclear marker (NeuN) revealed WMICs throughout the infracortical white matter, these cells being most dense close to inner cortical border, decreasing in density with depth in the white matter. Stereological analysis of NeuN-immunopositive cells revealed estimates of approximately 1.1, 10.8, and 37.7 million WMICs within the infracortical white matter of the galago, squirrel monkey, and crested macaque, respectively. The total numbers of WMICs form a distinct negative allometric relationship with brain mass and white matter volume when examined in a larger sample of primates where similar measures have been obtained. In all three primates studied, the highest densities of WMICs were in the white matter of the frontal lobe, with the occipital lobe having the lowest. Immunostaining revealed significant subpopulations of WMICs containing neuronal nitric oxide synthase (nNOS) and calretinin, with very few WMICs containing parvalbumin, and none containing calbindin. The nNOS and calretinin immunopositive WMICs represent approximately 21% of the total WMIC population; however, variances in the proportions of these neurochemical phenotypes were noted. Our results indicate that both the squirrel monkey and crested macaque might be informative animal models for the study of WMICs in neurodegenerative and psychiatric disorders in humans.

BACKGROUND The cytologic evaluation of serous effusions to distinguish malignant cells from reactive mesothelial cells (RMCs)was an enormous challenge. The purpose of this study was to investigate the diagnostic value of glucose transporter 1 (GLUT1) and calretinin (CR) in serous effusions of patients with malignant and in order to significantly ameliorate the diagnostic accuracy. METHODS The expressions of GLUT1 and CR were measured by streptavidin-peroxidase (S-P) immunocytochemical technique in serous effusions of 183 patients with malignant and in 95 patients with benign diseases. RESULTS The positive ratio of GLUT1 was 91.8% (168/183) in serous effusions from patients with malignant and 5.3% (5/95) in benign diseases, they had a significant difference (P

Past studies find that chronic stress alters inhibitory, GABAergic circuitry of neurons in distinct hippocampal subregions. Less clear is whether these effects persist weeks after chronic stress ends, and whether these effects involve changes in the total number of hippocampal GABAergic neurons or modulates the function of specific GABAergic subtypes. A transgenic mouse line (VGAT:Cre Ai9) containing an indelible marker for GABAergic neurons (tdTomato) throughout the brain was used to determine whether chronic stress alters total GABAergic neuronal number or the expression of two key GABAergic cell subtypes, calretinin expressing (CR+) and somatostatin expressing (SOM+) neurons, and whether these changes endure weeks later. Male and female mice were chronically stressed in wire mesh restrainers for 6h/d/21d (Str) or not (Con), and then allowed a 3 week rest period (Str-Rest) and compared to those without a rest period (Str-NoRest). Epifluorescent microscope images of immunohistochemistry-processed brains were quantified to estimate the total number of fluorescently-labeled hippocampal GABAergic neurons and the proportion that were CR+ or SOM+. Neither chronic stress nor sex altered the total number of GABAergic cells. In contrast, chronic stress reduced the expression of CR+ in the CA3 region of the hippocampus in both males and females, with robust reductions in the DG region of males, but not females, and these changes reversed following a rest period. Chronic stress also reduced the proportion of hippocampal SOM+ neurons and this reduction persisted even with a rest period. These results show chronic stress dynamically reduced CR expression without changing total inhibitory neuronal number and point to CR as a potential new lead to understand mechanisms by which chronic stress alters hippocampal function.

The visual system of cetaceans is at best poorly understood. With a handful of electrophysiological studies and a limited number of histological preparations from well-preserved specimen, the investigation of the principles underlying the cortical organization in cetaceans remains a challenge. In the course of our current investigation, we identified the transition from V2 to V1 in the long-finned pilot whale Globicephala melas, only recognizable through immunocytochemistry, and a similar if not homologue transition in the sheep Ovis aries. Our results emphasize the importance of differential pattern recognition in which the application of different markers uncovers a diversity in a delphinid's cortex, formerly widely considered as uniform and archetypal. In fact, the evidence that we present suggests the existence of relatively unacknowledged areas beyond the well-known sensory territories in cetaceans.

The midline thalamus bi-directionally connects the medial prefrontal cortex (mPFC) and hippocampus (HC) creating a unique cortico-thalamo-cortico circuit fundamental to memory and executive function. While the anatomical connectivity of midline thalamus has been thoroughly investigated, little is known about its cellular organization within each nucleus. Here we used immunohistological techniques to examine cellular distributions in the midline thalamus based on the calcium binding proteins parvalbumin (PV), calretinin (CR), and calbindin (CB). We also examined these calcium binding proteins in a population of reuniens cells known to project to both mPFC and HC using a dual fluorescence retrograde adenoassociated virus (AAV) based tracing approach. These dual reuniens mPFC-HC projecting cells, in particular, are thought to be important for synchronizing mPFC and HC activity. First, we confirmed the absence of PV+ neurons in the midline thalamus. Second, we found a common pattern of CR+ and CB+ cells throughout midline thalamus with CR+ cells running along the nearby third ventricle (3V) and penetrating the midline. CB+ cells were consistently more lateral and toward the middle of the dorsal-ventral extent of the midline thalamus. Notably, single-labeled CR+ and CB+ zones were partially overlapping and included dual-labeled CR+/CB+ cells. Within RE, we also observed a CR and CB subzone specific diversity. Interestingly, dual mPFC-HC projecting neurons in RE expressed none of the calcium binding proteins examined, but were contained in nests of CR+ and CB+ cells. Overall, the midline thalamus was well organized into CR+ and CB+ rich zones distributed throughout the region, with dual mPFC-HC projecting cells in reuniens representing a unique cell population. These results provide a cytoarchitectural organization in the midline thalamus based on calcium binding protein expression, and sets the stage for future cell-type specific interrogations of the functional role of these different cell populations in mPFC-HC interactions.

In this study we performed a neurochemical characterization of the hypothalamus in the developing alpaca (Vicugna pacos) with the aim of revealing the distributions of immunoreactive (-ir) cells containing parvalbumin (PV), calbindin (CB), calretinin (CR), the somatostatin (SOM), the enzyme aromatase P450 (P450Arom), the estrogen receptor α (ER-α), and estrogen receptor β (ER-β) in embryonal stages, early fetal age, and in the newborn. This analysis has been carried out on embryos at 20, 30, 45 days, fetuses at 90 days, and newborn alpaca. Our immunohistochemical results revealed no cells-ir throughout the embryonic hypothalami of 20, 30, and 45 days. On the fetal stage of 90 days, SOM-ir cells were observed in the lateral hypothalamus and the ventromedial nuclei of the tuberal region. We checked for the presence of P450Arom-ir cells in the periventricular area and dorsomedial hypothalamic nucleus of the tuberal region. In these fetal stages, no PV-ir, CB-ir, CR-ir or ERs-ir cells were identified. In the newborn, the PV-ir, CB-ir, CR-ir, and SOM-ir cells were detected in both the anterior and tuberal hypothalamic area. The P450Arom-ir cells the ER-α-ir and ER-β-ir cells were found in the anterior hypothalamus. Our results offer a contribution in the future purpose to obtain a time-expression pattern of the considered markers in alpaca during gestation and represents a foundation for future investigations on the alpaca brain to define the cross talk between PV, CB, CR, P450Arom, SOM, and ERs in the hypothalamus, the strategic region for the control of the reproductive behavior.

Background Cytopathological examination of pleural effusions is a fast and minimally invasive method for verification of the presence of neoplastic cells. We report our 2-year experience using a categorised diagnostic system and reporting risks of malignancy (ROMs) for each defined category. Methods Cytological reports of patients between November 2016 and October 2018 were collected, with results primarily classified into a five-tiered classification scheme. Immunohistochemistry markers used in cytology and their results were also recorded. Final agreement to histology and overall test performance was calculated for cases with available concomitant (up to 3 months) pleural biopsies. Results A total of 519 samples from 385 patients were collected, being 29 (5.6%) classified as non-diagnostic, 291 (56%) as negative, 28 (5.4%) as atypical, 30 (5.8%) as suspicious and 141 (27.2%) as positive. Most requested markers were calretinin, TTF1, Ber-EP4 and Gata-3, being conclusive in 45 (76.3%) cases. Total cyto-histological agreement was achieved in 49 (80.3%) specimens, with an overall sensitivity and specificity of 69.4% and 93.3%, respectively. Positive predictive value was 96.2% and negative predictive value was of 56%. ROM for each diagnostic category was 50% for non-diagnostic, 44% for negative, 50% for atypical, 83.3% for suspicious and 96.2% for positive. Conclusions Our 2-year retrospective study has shown a high specificity and positive predictive value for pleural cytology. The use of a five-tiered system has also shown to be highly effective, with a concordantly progressive higher ROM for the assigned diagnostic categories.

Lungfishes are the closest living relatives of land vertebrates, and their neuroanatomical organization is particularly relevant for deducing the neural traits that have been conserved, modified, or lost with the transition from fishes to land vertebrates. The immunohistochemical localization of calbindin (CB) and calretinin (CR) provides a powerful method for discerning segregated neuronal populations, fiber tracts, and neuropils and is here applied to the brains of Neoceratodus and Protopterus, representing the two extant orders of lungfishes. The results showed abundant cells containing these proteins in pallial and subpallial telencephalic regions, with particular distinct distribution in the basal ganglia, amygdaloid complex, and septum. Similarly, the distribution of CB and CR containing cells supports the division of the hypothalamus of lungfishes into neuromeric regions, as in tetrapods. The dense concentrations of CB and CR positive cells and fibers highlight the extent of the thalamus. As in other vertebrates, the optic tectum is characterized by numerous CB positive cells and fibers and smaller numbers of CR cells. The so-called cerebellar nucleus contains abundant CB and CR cells with long ascending axons, which raises the possibility that it could be homologized to the secondary gustatory nucleus of other vertebrates. The corpus of the cerebellum is devoid of CB and CR and cells positive for both proteins are found in the cerebellar auricles and the octavolateralis nuclei. Comparison with other vertebrates reveals that lungfishes share most of their features of calcium binding protein distribution with amphibians, particularly with salamanders.

Introduction Precise cytological diagnosis of pelvic high-grade serous carcinoma (HGSC) in ascites is important for tumour staging, therapeutic decision-making and prognostic evaluation. However, it can often be difficult to distinguish metastatic HGSC cells from reactive mesothelial cells based on morphology alone. Immunocytochemical analysis of ascites cell blocks has been used to obtain accurate diagnosis and provide a reliable basis for treatment decisions in the clinic. This study was performed to determine whether a panel of antibodies is necessary to achieve high specificity and sensitivity for the identification of HGSC cells. Methods Ascites samples from 70 cases (70/253, 27.7%) of histologically confirmed HGSC were postoperatively collected from 2012 to 2015 and were immunocytochemically analysed. Results The sensitivity and specificity of Ber-EP4 (a marker of HGSC) for detecting HGSC was 85.7% and 82.1%, respectively, whereas the sensitivity and specificity of HBME-1 for identifying mesothelial cells was 100% and 68.3%, respectively. To improve the rate of detection further of HGSC, 29 cases of ascites were also stained for E-cadherin (a marker of HGSC) and calretinin (a marker of mesothelial cells). The combination of Ber-EP4 and E-cadherin as markers of adenocarcinoma cells increased the sensitivity and specificity for HGSC detection to 100% and 88.9%, respectively. Meanwhile, the sensitivity and specificity for mesothelial cell identification increased to 100% and 90%, respectively, when HBME-1 and calretinin were combined. Conclusion This panel of complementary biomarkers is valuable and ideal for the differential diagnosis of HGSC based on ascites cytology.

In human neuropsychiatric disorders, there are functional and anatomical abnormalities of GABAergic interneurons in each temporal cortex subregion. Furthermore, accumulation of amyloid-β is observed in the temporal cortex in the early stages of Alzheimer's disease. Each subregion of the temporal cortex has an important role in coordinating the input and output of the hippocampus. When subregions of the temporal cortex are impaired, memory and learning ability decrease. GABAergic interneurons control excitatory neurons, forming the cortico-cortical and cortico-hippocampal networks. However, in temporal cortex subregions, details of the distribution and developmental processes of GABAergic interneurons and perineuronal nets (PNNs) have not been elucidated. Here we examined the development of GABAergic interneurons and PNNs in mouse temporal cortex subregions. Results indicate that temporal cortex GABAergic interneurons have developmental stages different to those of the primary sensory cortex. In addition, the density of PNNs in the temporal cortex is lower than that in the sensory cortex. Furthermore, we found that the Wisteria floribunda agglutinin-reactive extracellular matrix molecule is present in the upper level of layer 1 of the temporal cortex. These results support the idea that mouse temporal cortex subregions develop differently from other cortical regions and have region-specific characteristics after maturation. The present study results suggested that the structure of the temporal cortex is significantly different from the sensory cortex and that temporal cortex may be highly vulnerable to neuropsychiatric and neurodegenerative disorders.

The complex neuroanatomical connections of the inferior colliculus (IC) and its major subdivisions offer a juxtaposition of segregated processing streams with distinct organizational features. While the tonotopically layered central nucleus (CNIC) is well-documented, less is known about functional compartments in the neighboring lateral cortex (LCIC). In addition to a laminar framework, LCIC afferent-efferent patterns suggest a multimodal mosaic, consisting of a patchy modular network with surrounding extramodular domains. The present study utilizes several neurochemical markers that reveal an emerging LCIC modular-extramodular microarchitecture. In newborn and post-hearing C57BL/6J and CBA/CaJ mice, histochemical and immunocytochemical stains were performed for acetylcholinesterase (AChE), nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), glutamic acid decarboxylase (GAD), cytochrome oxidase (CO), and calretinin (CR). Discontinuous layer 2 modules are positive for AChE, NADPH-d, GAD, and CO throughout the rostrocaudal LCIC. While not readily apparent at birth, discrete cell clusters emerge over the first postnatal week, yielding an identifiable modular network prior to hearing onset. Modular boundaries continue to become increasingly distinct with age, as surrounding extramodular fields remain largely negative for each marker. Alignment of modular markers in serial sections suggests each highlight the same periodic patchy network throughout the nascent LCIC. In contrast, CR patterns appear complementary, preferentially staining extramodular LCIC zones. Double-labeling experiments confirm that NADPH-d, the most consistent developmental modular marker, and CR label separate, non-overlapping LCIC compartments. Determining how this emerging modularity may align with similar LCIC patch-matrix-like Eph/ephrin guidance patterns, and how each interface with, and potentially influence developing multimodal LCIC projection configurations is discussed. This article is protected by copyright. All rights reserved.

The SLC8A1 (solute carrier family 8, member 1) gene, encoding Na+/Ca2+ exchanger, is essential in regulating calcium reabsorption and homeostasis. Calcium homeostasis plays a key role in cell proliferation and apoptosis. We hypothesized that polymorphisms in five calcium-regulating genes (SLC8A1, ATP2B1, CALB1, CALB2, and CABP1) interact with calcium intake in relation to the risk of colorectal neoplasia. A two-phase (discovery and replication) study was conducted within the Tennessee Colorectal Polyp Study, including a total of 1275 cases and 2811 controls. In Phase I, we identified six out of 135 SNPs that significantly interacted with calcium intake in relation to adenoma risk. In Phase II, the calcium intake by rs4952490 (SLC8A1) interaction was replicated (P interaction = 0.048). We found an inverse association between calcium intake (1000-2000 mg/day) and colorectal adenomas, particularly for multiple/advanced adenomas, among the G-allele carriers but not among homozygous carriers of the common variant (A) in rs4952490. In the joint analysis of SLC8A1, KCNJ1 and SLC12A1 SNPs, carriers of variant alleles in at least two genes and with calcium intake above the DRI (1000 mg/day) were approximately 30-57% less likely to have adenomas than those whose calcium intake was below the DRI. The association was stronger for multiple/advanced adenomas. No association was found among those who did not carry any variant alleles in these genes when calcium intake was below 2500 mg per day. These findings, if confirmed, may provide a new avenue for the personalized prevention of colorectal adenoma and cancer. This article is protected by copyright. All rights reserved

Objective Focal cortical dysplasia (FCD) is a major cause of pharmacoresistant focal epilepsy. Little is known about the pathomechanisms underlying the characteristic cytoarchitectural abnormalities associated with FCD. In the present study, a broad panel of markers identifying layer-specific neuron subpopulations was applied to characterize dyslamination and structural alterations in FCD with balloon cells (FCD 2b). Methods Pan-neuronal neuronal nuclei (NeuN) and layer-specific protein expression (Reelin, Calbindin, Calretinin, SMI32 (nonphosphorylated neurofilament H), Parvalbumin, transducin-like enhancer protein 4 (TLE4), and Vimentin) was studied by immunohistochemistry on paraffin sections of FCD2b cases (n = 22) and was compared to two control groups with (n = 7) or without epilepsy (n = 4 postmortem cases). Total and layer-specific neuron densities were systematically quantified by cell counting considering age at surgery and brain region. Results We show that in FCD2b total neuron densities across all six cortical layers were not significantly different from controls. In addition, we present evidence that a basic laminar arrangement of layer-specific neuron subtypes was preserved despite the severe disturbance of cortical structure. SMI32-positive pyramidal neurons showed no significant difference in total numbers, but a reduction in layers III and V. The densities of supragranular Calbindin- and Calretinin-positive interneurons in layers II and III were not different from controls, whereas Parvalbumin-expressing interneurons, primarily located in layer IV, were significantly reduced in numbers when compared to control cases without epilepsy. In layer VI, the density of TLE4-positive projection neurons was significantly increased. Altogether, these data show that changes in cellular composition mainly affect deep cortical layers in FCD2b. Significance The application of a broad panel of markers defining layer-specific neuronal subpopulations revealed that in FCD2b neuronal diversity and a basic laminar arrangement are maintained despite the severe disturbance of cytoarchitecture. Moreover, it showed that Parvalbumin-positive, inhibitory interneurons are highly vulnerable in contrast to other interneuron subtypes, possibly related to the epileptic condition.

Objective: Febrile seizures (FS) are fever-associated convulsions, being the most common seizure disorder in early childhood. A subgroup of these children later develops epilepsy characterized by a hyperexcitable neuronal network in the hippocampus. Hippocampal excitability is regulated by the hippocampal dentate gyrus (DG) where postnatal neurogenesis occurs. Experimental FS increase the survival of newborn hippocampal dentate granule cells (DGCs), yet the significance of this neuronal subpopulation to the hippocampal network remains unclear. In the current study, we characterized the temporal maturation and structural integration of these post-FS born DGCs in the DG. Methods: Experimental FS were induced in 10-day-old rat pups. The next day, retroviral particles coding for enhanced green fluorescent protein (eGFP) were stereotactically injected in the DG to label newborn cells. Histochemical analyses of eGFP expressing DGCs were performed one, 4, and 8 weeks later and consisted of the following: (1) colocalization with neurodevelopmental markers doublecortin, calretinin, and the mature neuronal marker NeuN; (2) quantification of dendritic complexity; and (3) quantification of spine density and morphology. Results: At neither time point were neurodevelopmental markers differently expressed between FS animals and normothermia (NT) controls. One week after treatment, DGCs from FS animals showed dendrites that were 66% longer than those from NT controls. At 4 and 8 weeks, Sholl analysis of the outer 83% of the molecular layer showed 20-25% more intersections in FS animals than in NT controls (p < 0.01). Although overall spine density was not affected, an increase in mushroom-type spines was observed after 8 weeks. Significance: Experimental FS increase dendritic complexity and the number of mushroom- type spines in post-FS born DGCs, demonstrating a more mature phenotype and suggesting increased incoming excitatory information. The consequences of this hyperconnectivity to signal processing in the DG and the output of the hippocampus remain to be studied. We greatly appreciate the kindness of H. van Praag for providing plasmids used for virus production. Furthermore, we would like to thank Rik Paesen for his help with the confocal microscope and Petra Bex and Rosette Beenaerts for their assistance with the virus production and immunohisto-chemistry. This research was financially supported by a 'Bijzonder Onderzoeksfonds' grant from Hasselt University. Nick Smisdom was supported by a post-doctoral scholarship of the Research Foundation - Flanders (FWO-Vlaanderen).

For patients with incurable neurodegenerative disorders such as Huntington's (HD) and Parkinson's disease, cell transplantation has been explored as a potential treatment option. Here, we present the first clinicopathological study of a patient with HD in receipt of cell-suspension striatal allografts who took part in the NEST-UK multicenter clinical transplantation trial. Using various immunohistochemical techniques, we found a discrepancy in the survival of grafted projection neurons with respect to grafted interneurons as well as major ongoing inflammatory and immune responses to the grafted tissue with evidence of mutant huntingtin aggregates within the transplant area. Our results indicate that grafts can survive more than a decade post-transplantation, but show compromised survival with inflammation and mutant protein being observed within the transplant site. Ann Neurol 2018;84:950-956.

Malignant mesothelioma is an aggressive and therapy-resistant neoplasm arising from mesothelial cells. Evidence suggests that the major pathology associated with asbestos-induced mesothelioma is local iron overload. In the present study, we induced iron-induced mesothelioma in rats based on previous reports. Ten Wistar rats were given ferric saccharate and nitrilotriacetate i.p. for 5 days a week. Five of the ten rats exhibited widespread mesotheliomas in the peritoneum and tunica vaginalis. The tumor cells showed positive immunostaining for calretinin, wilms tumor-1, podoplanin and the oxidative DNA marker 8-hydroxy-2′-deoxyguanosine. In three of the five rats with mesothelioma, array-based comparative genomic hybridization analysis identified a common chromosomal deletion mapped to the chromosomal 4q31 locus, which encompasses the TBXAS1 gene. Downregulation of the TBXAS1 gene was confirmed using quantitative PCR. TBXAS1 gene expression was also reduced in three of four human malignant pleural mesothelioma cell lines compared with normal bronchial epithelial cells. Immunohistochemistry revealed that TBXAS1 expression was weakly positive and positive in five and three out of eight human malignant mesothelioma samples, respectively. In conclusion, TBXAS1 gene expression was downregulated in rats with iron-induced mesothelioma. The relationship between iron overload and TBXAS1 downregulation should be pursued further.

Bowen's disease (BD)/carcinoma is a type of squamous cell carcinoma of the skin, however, the possibility of adnexal differentiation (the development of sebaceous carcinoma or porocarcinoma) occurring in BD/carcinoma has been suggested. We herein describe a case of BD with superficial invasive carcinoma, which showed a clear cell focus, demonstrating tricholemmal differentiation. This clear cell focus showed the following findings: primarily composed of clear cells, somewhat columnar clear cells aligned in a palisade along a discernible basement membrane, tricholemmal keratinization and glycogen contained within the cells. In addition, the immunohistochemical profile in this clear cell focus, namely, negative staining for cytokeratin (CK)1 and positive staining for CK17 and calretinin in the inner cells of the neoplastic lobule, corresponded to that of the outer root sheath cells. This case suggested that adnexal differentiation can rarely occur within true BD/carcinoma, although adnexal carcinomas are commonly associated with a simple bowenoid change.

Human epidemiological data suggest a link between anesthesia exposure in early postnatal life and subsequent lasting neurocognitive alterations. Understanding the underlying mechanisms of this potential association is of paramount importance in an attempt to develop protective strategies. While general anesthetics are powerful modulators of GABAergic neurotransmission, little is known about the impact of these drugs on developing GABAergic networks. Here we addressed this issue by evaluating the impact of a 6-h-long midazolam exposure on the development of calbindin-, calretinin- and parvalbumin-expressing GABAergic interneurons.Physiological expression patterns of calbindin-, calretinin-, and parvalbumin-positive neurons as well as the impact of a 6-h-long midazolam exposure on these cell populations were evaluated in the medial prefrontal cortex of Wistar rats at defined stages of the brain growth spurt using stereological analysis. Activated caspase-3 immunohistochemistry was used to quantify apoptotic death in controls and midazolam-treated subjects.In control animals, the number of parvalbumin expressing cells significantly (p0.01) increased while those of calbindin positive populations significantly (p0.01) decreased between postnatal day 10 and 20. Expression of calretinin remained constant during this period. Immediately following exposure, midazolam induced neuroapoptosis at both early (postnatal day 5, p=0.016) and later (postnatal day 15, p=0.025) stages of brain development. While this did not diminish overall neuronal density in the medial prefrontal cortex, exposure at P5 led to a subsequent increase in the number of parvalbumin positive neurons in lower cortical layers, and midazolam administration at P15 increased the number of both parvalbumin and calretinin expressing neurons 5 days following exposure.These observations demonstrate that midazolam exposure can impair the physiological differentiation patterns of GABAergic interneurons during the brain growth spurt. Considering the important role of GABAergic networks in neuronal physiology, these data provide us with one potential mechanism that could account for the lasting neurobehavioral and cognitive deficits observed in the context of anesthesia exposure in the early postnatal period.

Aims The significance and pathogenesis of irregular or asynchronous maturation within endometrial glands remains uncertain. The aim of this study was to investigate differences in epithelial hormone receptor immunoreactivity and stromal cell calretinin, CD34 and p16 expression in morphologically normal secretory endometrium and in asynchronous (non-secretory) endometrial glands (AEGs). Methods and results Nineteen consecutive endometrial specimens showing AEGs were examined. The mean age of the patients was 42.8 years, and the most common presenting symptom was menorrhagia. Immunohistological expression of oestrogen receptor (ER) α, of ERβ and of progesterone receptor (PR) were compared in normal secretory glands and in AEGs. Stromal cell expression of calretinin, CD34 and p16 was also investigated. In contrast to normal secretory glands, the epithelial cells lining AEGs were usually ERα/PR-positive and showed significantly increased ERβ expression. Altered calretinin and CD34 expression within functional layer stromal cells was seen in five and two cases, respectively, but there were no differences in stromal cell immunoreactivity around AEGs. Conclusions The most common clinical symptom associated with AEGs in this study was menorrhagia. Aberrant hormone receptor expression in AEGs suggests a localized, possibly clonal, defect in epithelial maturation. There were no immunophenotypic changes to suggest that AEGs are related to a primary endometrial stromal deficiency.

Objective This study was designed to evaluate whether exogenous CRT was beneficial for alleviating MR-induced injury by suppressing ER stress in rat MMECs. Methods MMECs were pretreated with CRT (25 pg/mL) for 12 hours, followed by the exposure to 2.856 GHz radiation at a mean power density of 30 mW/cm2 for six minutes. MR-induced injury in MMECs was evaluated by LDH leakage, apoptosis, and cell viability analysis. The expression of GRP78, CRT, CHOP, Bcl-2, and Bax were examined by Western blot analysis to reflect ER stress response and ER stress-related apoptosis. Results MR induced marked MMECs injury, as shown by increased LDH leakage and apoptosis rate and decreased cell viability. MR also induced excessive ER stress, characterized by increased expression of GRP78 and CRT, and ER stress-related apoptotic signaling as well, as shown by the upregulation of CHOP and Bax and the downregulation of Bcl-2. Exogenous CRT pretreatment remarkably attenuated MR-induced cell apoptosis and LDH leakage, ER stress, and activation of the ER stress-related apoptotic signaling. Conclusions Exogenous CRT attenuates MR-induced ER stress-related apoptosis by suppressing CHOP-mediated apoptotic signaling pathways in MMECs.

GABAergic inhibitory interneurons control fundamental aspects of neuronal network function. Their functional roles are assumed to be defined by the identity of their input synapses, the architecture of their dendritic tree, the passive and active membrane properties and finally the nature of their postsynaptic targets. Indeed, interneurons display a high degree of morphological and physiological heterogeneity. However, whether their morphological and physiological characteristics are correlated and whether interneuron diversity can be described by a continuum of GABAergic cell types or by distinct classes has remained unclear. Here we perform a detailed morphological and physiological characterization of GABAergic cells in the dentate gyrus, the input region of the hippocampus. To achieve an unbiased and efficient sampling and classification we used knock-in mice expressing the enhanced green fluorescent protein (eGFP) in glutamate decarboxylase 67 (GAD67)-positive neurons and performed cluster analysis. We identified five interneuron classes, each of them characterized by a distinct set of anatomical and physiological parameters. Cross-correlation analysis further revealed a direct relation between morphological and physiological properties indicating that dentate gyrus interneurons fall into functionally distinct classes which may differentially control neuronal network activity. © 2013 The Authors. Hippocampus Published by Wiley Periodicals, Inc.

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expansion of CAG trinucleotide repeats encoding for polyglutamine (polyQ) in the huntingtin (Htt) gene. Despite considerable effort, the mechanisms underlying the toxicity of the mutated Htt protein remains largely uncertain. To identify novel therapeutic targets, we recently employed the approach of tandem affinity purification and discovered that calretinin (Cr), a member of the EF-hand family of calcium-binding proteins, is preferentially associated with mHtt, although it also interacts with wild-type Htt. These observations were supported by coimmunoprecipitation and by colocalization of Cr with mHtt in neuronal cultures. Over- expression of Cr reduced mHtt-caused cytotoxicity in both non-neuronal and neuronal cell models of HD, whereas knockdown of Cr expression in the cells enhanced mHtt-caused neuronal cell death. In addition, over-expression of Cr was also associated with reduction of intracellular free calcium and activation of Akt. These results suggest that Cr may be a potential therapeutic target for treatment of HD.

The advantages of the embryonic chick as a model for studying neural development range from the relatively low cost of fertilized eggs to the rapid rate of development. We investigated in ovo cerebellar development in the chick, which has a nearly identical embryonic period as the mouse (19–22 days). We focused on three antigens: Calbindin (CB), Zebrin II (ZII), and Calretinin (CR), and our results demonstrate asynchronous expression patterns during cerebellar development. Presumptive CB+ Purkinje cells are first observed at embryonic day (E)10 in clusters in posterior cerebellum. At E12, corresponding with global expression of CB across the cerebellum, Purkinje cells began to express ZII. By E14–E16, Purkinje cells disperse into a monolayer and develop a pattern of alternating immunopositive and immunonegative ZII stripes. CR is initially expressed by clusters of presumptive Purkinje cells in the nodular zone at E8. However, this expression is transient and at later stages, CR is largely confined to the granule and molecular layers. Before hatch (E18–E20), Purkinje cells adopt a morphologically mature phenotype with complex dendritic arborizations. Comparing this data to that seen in mice, we found that the sequence of Purkinje cell formation, protein expression, and development is similar in both species, but these events consistently begin ∼5–7 days earlier in the precocial chick cerebellum, suggesting an important role for heterochrony in neurodevelopment. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Chondrosteans represent an ancient lineage in ray-finned bony fishes and hence in jawed vertebrates. This immunohistochemical study in the brain of the Siberian sturgeon reports the neuronal distribution of three cytosolic calcium-binding proteins: calbindin-D28k (CB), calretinin (CR), and parvalbumin (PV). CB and CR are widely expressed in different neuron subsets distributed throughout the sturgeon brain. Studies using double immunofluorescence reveal a wide co-distribution of CB and CR in the brain nuclei but scarce co-localization at cellular level. In the forebrain, CR- and CB-immunoreactive (ir) populations were observed in the olfactory bulbs, in pallial and subpallial telencephalic areas, and in some diencephalic nuclei. CR-ir cells were also observed in the posterior tubercle and CB-ir cells in the preglomerular complex. At midbrain and hindbrain basal levels, CB-ir and CR-ir cell bodies were mainly distributed in periventricular areas. In the cerebellum, CB and CR cells were co-localized in some granular cell subsets in laterodorsal and dorsolateral regions, and in some Purkinje-like cells. CB-ir and CR-ir fibers were mainly observed in the olfactory bulbs, hypothalamus, and habenula, and in fiber tracts that coursed in the optic tectum and through the mesencephalic and rhombencephalic basal areas. With regard to PV, the sturgeon brain showed a rather limited distribution of PV-ir perikarya and fibers. Thus, CR, CB, and PV allowed the identification of subpopulations of neurons not distinguished on the basis of cytoarchitecture alone, which provided a better understanding of the anatomical organization of the sturgeon brain. These results reveal numerous shared features with teleosts, but also important differences. J. Comp. Neurol. 520:2086–2122, 2012. © 2011 Wiley Periodicals, Inc.

The anchovy retina is unique among vertebrates in being designed for polarization-contrast vision and has a marked cone area in the ventrotemporal quadrant for acute vision in the frontal field of view. Whereas cone topography and fine structure have been well examined in the European anchovy, the morphological organization of its inner retina is poorly understood so far. Using fluorescence staining of cell nuclei in combination with antibody stainings, the 3D pattern of cell nuclei was recorded coevally in all three retinal cell layers by confocal laser scanning microscopy, and the topographies of several neuron types were mapped separately across the retina. Cell classification and quantification succeeded for rods, cones, three types of horizontal cells (H1-3), rod and cone bipolars, amacrines, ganglion cells, and displaced amacrines as well as Müller cells; moreover, the morphology of H1 horizontal cells (calretinin-positive), rod bipolars (protein kinase C-positive), and a subtype of cone bipolars (parvalbumin-positive) is shown. Most cell types follow the cone topography, with a cell density maximum in the ventrotemporal quadrant (e.g., 730 cones, 2,800 cone bipolars, 230 GCL cells per 10(4) μm(2) ), showing their affiliation to the photopic system, except rods and rod bipolars (showing a cell density minimum in the cone area). The correlation of density maps of different cell types provides insight into convergence and divergence patterns in cone and rod pathways.

Immunohistochemical methods were used to characterize the expression of two calcium-binding proteins, calretinin (CR) and S100, in the olfactory rosette of the adult zebrafish. These proteins are expressed in different sets of sensory neurons, and together represent a large proportion of these cells. Double immunofluorescence for CR and Gα(olf) protein, and CR immunoelectron microscopy, indicated that most CR-immunoreactive (ir) cells were ciliary neurons. Differential S100- and CR-ir projections to glomerular fields of the olfactory bulb were also observed, although these projections overlap in some glomeruli. Application of the carbocyanine dye DiI to either S100-ir or CR-ir glomerular regions led to labeling of cells mostly similar to S100-ir and CR-ir neurons, respectively. Instead, these bulbar regions project to similar telencephalic targets. On the other hand, antibodies against keyhole limpet hemocyanin (KLH)-stained numerous sensory cells in the olfactory rosette, including cells that were CR- and S100-negative. This antiserum also stained most primary bulbar projections and revealed extrabulbar olfactory primary projections coursing to the ventral area of the telencephalon through the medial olfactory tract. This extrabulbar projection was confirmed by tract-tracing with DiI. A loose association of this extrabulbar primary olfactory projection and the catecholaminergic populations of the ventral area was also observed with double tyrosine hydroxylase/KLH-like immunohistochemistry. Comparison between KLH-like-ir pathways and the structures revealed by FMRFamide immunohistochemistry (a marker of terminal ganglion cells and fibers) indicated that the KLH-like-ir extrabulbar projection was different from the terminal nerve system. The significance of the extrabulbar olfactory projection of zebrafish is discussed.

In this study we analyzed the effects in the inferior colliculus of a unilateral ablation of the auditory cortex in rats. Variations in both calretinin immunoreactivity and protein levels determined by Western blot suggest that such lesions induce changes in the regulation of this calcium-binding protein. Stereological counts of calretinin-immunoreactive neurons in the inferior colliculus 15, 90, and 180 days after the lesion showed a progressive increase in the number of immunoreactive neurons, with a parallel increase in the intensity of staining. Two hundred forty days after the cortical lesion, both the number of immunoreactive neurons and the staining intensity had returned to control values. The effects of the cortical lesion on calretinin regulation are more intense in those inferior colliculus subdivisions more densely innervated by the corticocollicular projection. This finding, along with the time course of calretinin regulation suggests that degeneration of the descending projection is linked to calretinin regulation in the inferior colliculus. We hypothesize, based on the role of calretinin, that the observed increase in immunoreactivity levels seen in the inferior colliculus after lesioning of the auditory cortex may be related to altered excitability in deafferented neurons. Our finding, may reflect adaptive mechanisms to changes in calcium influx and excitability in inferior colliculus neurons induced by lesions of the descending projection from the cortex to the inferior colliculus. J. Comp. Neurol. 518:4261–4276, 2010. © 2010 Wiley-Liss, Inc.

Malignant deciduoid mesothelioma (MDM) is a rare variant of epithelioid mesothelioma. This type of tumor might be associated with the asbestos exposure and carries a poor prognosis in general. MDM was first described by Nascimento et al. in 1994 in a peritoneal lesion of a young woman. And its diagnosis is frequently mistaken with florid mesothelial hyperplasia and peritoneal deciduosis. There are 44 MDM cases reported in the literature up today. A 59-year-old woman patient referred to our clinic was identified with an abdominal mass. Computed tomography of whole abdomen of the patient showed a mass with the widest transverse dimension of 65 × 60 mm at abdominal bifurcation in the mesenteric region. The patient was diagnosed with MDM after the cytopathological examination of the fine needle aspiration biopsy performed from the mass. Consequently, she received a total mass excision and right hemicolectomy under general anesthesia. The cytomorphological appearance of the ascitic fluid is detailed for the first time by Gillespie et al. and is described only in two manuscripts. In the present study, we aimed to report a case of a 59-year-old woman since she was diagnosed with MDM and because her cytological findings were further supported by histomorphological and immunohistochemical evaluations of the operation material obtained from the patient. Diagn. Cytopathol. 2011;39:402–408. © 2010 Wiley-Liss, Inc.

Previous studies have suggested that the patterns of innervation and high interconnectivity of the piriform cortex (PC) provide for strong olfactory hippocampal memory; however, these same attributes may create high seizurogenic tendencies. Thus, understanding this wiring is important from a physiological and pathophysiological perspective. Distinct interneurons expressing differing calcium binding proteins (CBPs), parvalbumin (PV), calbindin (CB), and calretinin (CR), have been shown to exist in PC. However, a comprehensive examination of the distribution and innervation patterns of these neurons has not been done. Thus the purpose of this study was to combine the analysis of the CBP cell localization with analysis of their innervation patterns. Each type was differentially localized in the three layers of the PC. Only CR-positive neurons were found in layer 1. PV and CB are coexpressed in layers 2-3, most expressing both PV and CB. A morphological estimate of the dendritic extent for each subtype showed that PV and PV/CB cells demonstrated equally wide, horizontal and vertical arborizations, whereas CB cells had wide horizontal and restricted vertical arborizations. CR cells had restricted horizontal and very long vertical arborizations. Postsynaptic morphological targeting was also found to be specific, namely, PV(+) and PV/CB(+) nerve terminals (NTs) innervate perisomatic regions of principal cells. CR(+) NTs innervate only dendrites of principal cells, and CB(+) NTs innervate both somata and dendrites of principal cells. These data show highly complex innervation patterns for all of the CBP interneurons of the PC and form a basis for further studies in the plasticity of this region.

Information about the position and movement of the head in space is coded by vestibular receptors and relayed to four nuclei that comprise the vestibular nuclear complex (VNC). Many additional brainstem nuclei are involved in the processing of vestibular information, receiving signals either directly from the eighth nerve or indirectly via projections from the VNC. In cats, squirrel monkeys, and macaque monkeys, we found neurochemically defined subdivisions within the medial vestibular nucleus (MVe) and within the functionally related nucleus prepositus hypoglossi (PrH). In humans, different studies disagree about the borders, sizes, and possible subdivisions of the vestibular brainstem. In an attempt to clarify this organization, we have begun an analysis of the neurochemical characteristics of the human using brains from the Witelson Normal Brain Collection and standard techniques for antigen retrieval and immunohistochemistry. Using antibodies to calbindin, calretinin, parvalbumin, and nitric oxide synthase, we find neurochemically defined subdivisions within the MVe similar to the subdivisions described in cats and monkeys. The neurochemical organization of PrH is different. We also find unique neurochemical profiles for several structures that suggest reclassification of nuclei. These data suggest both quantitative and qualitative differences among cats, monkeys, and humans in the organization of the vestibular brainstem. These results have important implications for the analysis of changes in that organization subsequent to aging, disease, or loss of input. J. Comp. Neurol. 518:872–895, 2010. © 2009 Wiley-Liss, Inc.

Mechanical stimuli affect differentiation of specific cell types in several organs of mouse fetuses that develop without any skeletal musculature. To that end, we employed Myf5(-/-):MyoD(-/-) mouse embryos that completely lack skeletal musculature, and analyzed the development of sensory fields in the inner ear. Amyogenic fetuses lack skeletal muscles that move the chain of three middle ear ossicles which normally transfers sound vibrations. They also cannot tilt their head, which prevents the perception of angular acceleration. While our findings in the spiral organ of Corti are surprisingly normal, our results show that the development of cristae ampullaris, vestibular sensory fields sensitive to the angular acceleration, was the most affected. In cristae, hair cells and supporting cells were significantly smaller in the mutant embryos, but hair cells completely lacked tenascin, while supporting cells were more numerous. In maculae, supporting cells were significantly smaller but more numerous in the mutants. Here, we propose that our finding of a specific type I hair cell absence in the mutant's crista may now be employed in the identification of a profile of genes specific for the lacking cell type.

Cerebral selenium (Se) deficiency is associated with neurological phenotypes including seizures and ataxia. We wanted to define whether neurons require selenoprotein expression and which selenoproteins are most important, and explore the possible pathomechanism. Therefore, we abrogated the expression of all selenoproteins in neurons by genetic inactivation of the tRNA[Ser](Sec) gene. Cerebral expression of selenoproteins was significantly diminished in the mutants, and histological analysis revealed progressive neurodegeneration. Developing interneurons failed to specifically express parvalbumin (PV) in the mutants. Electrophysiological recordings, before overt cell death, showed normal excitatory transmission, but revealed spontaneous epileptiform activity consistent with seizures in the mutants. In developing cortical neuron cultures, the number of PV(+) neurons was reduced on combined Se and vitamin E deprivation, while other markers, such as calretinin (CR) and GAD67, remained unaffected. Because of the synergism between Se and vitamin E, we analyzed mice lacking neuronal expression of the Se-dependent enzyme glutathione peroxidase 4 (GPx4). Although the number of CR(+) interneurons remained normal in Gpx4-mutant mice, the number of PV(+) interneurons was reduced. Since these mice similarly exhibit seizures and ataxia, we conclude that GPx4 is a selenoenzyme modulating interneuron function and PV expression. Cerebral SE deficiency may thus act via reduced GPx4 expression.-Wirth, E. K., Conrad, M., Winterer, J., Wozny, C., Carlson, B. A., Roth, S., Schmitz, D., Bornkamm, G. W., Coppola, V., Tessarollo, L., Schomburg, L., Köhrle, J., Hatfield, D. L., Schweizer, U. Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration.

Retinitis pigmentosa reflects a family of diseases that result in retinal photoreceptor death and functional blindness. The natural course of retinal changes secondary to photoreceptor degeneration involves anatomical remodeling (cell process alterations and soma displacement) and neurochemical remodeling. Anatomical remodeling predominantly occurs late in the disease process and cannot explain the significant visual deficits that occur very early in the disease process. Neurochemical remodeling includes modified glutamate receptor disposition and altered responses secondary to functional activation of glutamate receptors. We investigated the neurochemical remodeling of retinal neurons in the rd/rd (rd1) mouse retina by tracking the functional activation of glutamate receptors with a cation probe, agmatine. We provide evidence that bipolar cells and amacrine cells undergo selective remodeling of glutamate receptors during the early phases of retinal degeneration. These early neurochemical changes in the rd/rd mouse retina include the expression of aberrant functional ionotropic glutamate receptors on the cone ON bipolar cells from postnatal day 15 (P15), poor functional activation of metabotropic glutamate receptors on both rod and cone ON bipolar cells throughout development/degeneration, and poor functional activation of N-methyl-D-aspartate receptors on amacrine cells from P15. Our results suggest that major neurochemical remodeling occurs prior to anatomical remodeling, and likely accounts for the early visual deficits in the rd/rd mouse retina.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been implicated in the inflammation-dependent sensitization of nociceptors. Because the periodontal ligament (PDL) contains numerous nociceptors and mechanoceptors, phosphorylation of ERK1/2 was investigated in nerve fibers of the PDL to elucidate the role of constitutive local activation of ERK1/2 in peripheral sensitization.Decalcified free-floating sections of rat molars with PDL were incubated using total (t)-ERK1/2 and phosphorylated (p)-ERK1/2 antibodies. For identification of nerve fibers in the PDL, double staining was performed using protein gene product 9.5 (PGP 9.5) with p-ERK1/2. To test whether p-ERK1/2 activated in sensory and mechanoreceptive terminals, double incubations were performed using p-ERK1/2 with calcitonin gene-related peptide (CGRP) and with calretinin. Labeled nerve fibers were quantified by the point-counting method.In cervical, midroot, and apical zones of the PDL, t-ERK1/2- and p-ERK1/2-labeled nerve fibers were found in close association with blood vessels. The p-ERK1/2-labeled free nerve fibers were often detected in cervical and apical areas of the PDL. In nerve fibers of the PDL, p-ERK1/2 was colocalized with PGP 9.5, CGRP, and calretinin.The perivascular distribution of t-ERK1/2 and p-ERK1/2 in nerve fibers in the PDL is compatible with a role for the constitutive activation of ERK1/2 in the neural regulation of blood vessels in the PDL. The colocalizations of p-ERK1/2 with CGRP and calretinin indicate that ERK1/2 is constitutively activated in a subpopulation of sensory and mechanoreceptive nerve terminals in the PDL.

The patterns of hippocampal neuronal loss and rewiring of the dentate gyrus (DG) were studied in the mouse model of temporal lobe epilepsy at 2 months after pilocarpine-induced status epilepticus (PISE). NeuN immunocytochemistry showed two patterns of neuronal damage, i.e., type 1 with partial loss of pyramidal neurons in CA3 area and type 2 with almost compete loss of CA3 pyramidal neurons. Anterograde tracing with Phaseolus vulgaris leucoagglutinin (PHA-L) demonstrated that, at different rostrocaudal segments of the hippocampus, associational and commissural connections in the DG changed differently between mice with type 1 vs. type 2 neuronal loss. Calretinin (CR)-immunopositive mossy cells in ventral hilus and its fibers in inner molecular layer of bilateral DG remained in mice with type 1 but almost disappeared in mice with type 2 neuronal loss, which was further supported by retrograde labeling with cholera toxin subunit B (CTB) showing colocalization of CTB with CR in the ventral hilus of bilateral DG in mice with type 1 neuronal loss, which was lost in those with type 2 neuronal loss. Furthermore, the sprouted PHA-L-immunopositive en passant and terminal boutons from the DG were found in CA1 area to contact with surviving calbindin-, CR-, and parvalbumin-immunopositive neurons. The present study therefore suggests that different patterns of neuronal loss in CA3 area may be linked to different axon reorganizations in the DG.

We studied how the noncompetitive NMDA receptor antagonist MK801 affected different stages of adult hippocampal neurogenesis in mice, and investigated how the activation of benzodiazepine receptors with diazepam interacted with the effects of MK801 on the precursor cells in the adult dentate gyrus. Our findings were: (i) one single MK801 application increased precursor cell proliferation and adult neurogenesis but not gliogenesis 4 weeks later; (ii) the number of label-retaining precursor cells decreased after MK801 (with P = 0.06); (iii) the pro-neurogenic effect included increased cell cycle entry of precursor cells as well as completed cell divisions, except in type-2a cells; (iv) NMDA receptor blockade also increased the number of nestin-GFP-expressing cells expressing calretinin; (v) diazepam alone had a very similar effect on overall precursor cell proliferation to that of MK801 alone; and (vi) diazepam, when co-applied with MK801, abolished the suppression of divisions of type-2a cells induced by MK801 alone, suppressed the MK801-induced effect on proliferation of type-2b cells and had no influence on the effects of MK801 on type-3 cells, but did suppress the increased number of nestin-GFP-positive cells expressing calretinin. From these results we hypothesize that, depending on the precursor cell stage, NMDA-dependent neurotransmission has distinct effects that are partly antagonized and partly enhanced by GABAergic input. We propose that NMDA receptor-dependent signalling maintains the precursor cells in the dentate gyrus while blocking initial stages of development and promoting more advanced stages.

A general pattern of organization of the forebrain shared by amphibians, mainly anurans, and amniotes has been proposed considering the relative topography of the territories, their connectivity, and their neurochemistry. These criteria were needed because the amphibians possess limited cell migration from the ventricle that precludes a parcellation into circumscribed nuclei. In the present study we have tested the identity of most newly described forebrain territories in anurans and urodeles with regard to their content in calbindin-D28k (CB) and calretinin (CR). By means of immunohistochemistry, these proteins were demonstrated to be particularly abundant and specifically distributed in the amphibian forebrain and were extremely useful markers for delineating nuclear boundaries otherwise indistinguishable. In the telencephalon, labeled cells in the pallium allowed the identification of particular regions with marked differences between anurans and urodeles, whereas the subpallium showed more conservative patterns of distribution. In particular, the components of the amygdaloid complex and the basal ganglia were distinctly labeled. The distribution in the nonevaginated secondary prosencephalon and diencephalon showed abundant common features between anurans and urodeles, highlighted using the prosomeric model for the comparison. In the pretectum, thalamus, and prethalamus of urodeles, the CB and CR staining was particularly suitable for the identification of diverse structures within the simple periventricular gray layer. However, the analysis across species also revealed a considerable degree of heterogeneity, even within comparatively well-defined neuronal populations. Therefore, the content of a particular calcium binding protein in a neuronal group is not a fully reliable criterion for considering homologies. J. Comp. Neurol. 511:187–220, 2008. © 2008 Wiley-Liss, Inc.

Adenomatoid tumors are benign mesothelial neoplasms most commonly found in the male and female genital tracts. Extragenital adenomatoid tumors are rare, most of them being solitary tumors. To our knowledge, only one case of multiple extragenital adenomatoid tumors, involving the liver and peritoneum, has been reported to date. Here we report another case of multiple extragenital adenomatoid tumors involving the mesocolon and omentum. A 47-year-old woman presented with a delayed menstrual period. Ultrasonography revealed a left adnexal mass, and surgical resection was attempted at a local hospital. The patient was transferred to our hospital without resection due to the intraoperative finding of multiple peritoneal tumors. At our hospital, an 8.0x7.5x6.0 cm tumor at the mesocolon of the sigmoid colon and three omental nodules measuring up to 2.5x2.0x1.7 cm were resected. Grossly, they were well circumscribed, gray-tan and elastic with small cystic spaces. Microscopically, they were composed of tubules and anastomosing channels lined by flattened or cuboidal cells with bland nuclei. Immunohistochemically, the tumor cells were positive for pan-cytokeratin AE1/AE3, vimentin, cytokeratin 5/6 and calretinin. The postoperative course was uneventful, and the patient was well 10 months after the operation. Despite their rarity, adenomatoid tumors should be included in the differential diagnosis of multiple intra-abdominal tumors.

We have shown previously that N-(4-hydroxyphenyl)retinamide (4HPR, fenretinide), a retinoic acid derivative, induces neuronal differentiation in cultured human retinal pigment epithelial (RPE) cells [Chen et al., J. Neurochem., 84 (2003), 972]. We asked the question whether the mitogen-activated protein kinase (MAPK) pathway is involved in the regulation of the 4HPR-induced neuronal differentiation of RPE (ARPE-19) cells. When we treated ARPE-19 cells with 4HPR, c-Raf and MEK1/2 kinase were activated resulting in activation of the downstream effector ERK1/2 and of SAPK/JNK. By blocking the upstream kinase MEK1/2 with specific inhibitor U0126 we abrogated the 4HPR-induced phosphorylation of ERK1/2 and SAPK/JNK, indicating that the neuronal differentiation occurs through a positive cross-talk between the ERK and the SAPK/JNK pathways. Both U0126 and the suppression of ERK1/2 expression with small interfering RNA effectively blocked the 4HPR-induced neuronal differentiation of RPE cells and the expression of calretinin. The activated ERK1/2 then induced a sequential activation of p90RSK, and increase in phosphorylation of transcription factors c-fos and c-jun leading to transcriptional activation of AP-1. Taken together, our results clearly demonstrate that c-Raf/MEK1/2 signaling cascade involving ERK1/2 plays a central role in mediating the 4HPR-induced neuronal differentiation and calretinin expression in the human ARPE-19 retinal pigment epithelial cell line.

A substantial problem in research concerned with axonal repair is the use of a wide variety of lesion models and the complexity associated with the respective in vivo lesion paradigms. Organotypic slice cultures are a potential in vitro alternative because the cytoarchitectonic tissue organization is well preserved and the slices can be maintained in culture for several weeks. Until now no spinal cord slice culture model for the study of axonal growth has been available. Here we present a spinal cord slice culture model that is well suited for the study of axonal growth. The spinal cord slices were cut not in the transverse but in the sagittal longitudinal plane such that several spinal cord segments were included in the slice culture. In these cultures the typical ventro-dorsal polarity of the spinal cord was maintained and intrinsic spinal cord axons formed a strong fibre tract extending along the longitudinal axis of the slice. The axons became myelinated during the culture period and synaptic contacts were present in these cultures. After mechanical lesions the intrinsic spinal cord axons had a substantial potential for axonal growth and regeneration. The number of regenerating axons crossing the lesion site decreased with increasing maturation of the culture, but even in mature cultures a small number of crossing fibres were present. This slice culture model could provide an important tool for several aspects of spinal cord research in the fields of axonal growth and regeneration, synapse formation, formation of intrinsic spinal cord circuits and myelination.

Different subclasses of γ-aminobutyric acid (GABA) cortical neurons can be distinguished by their content of neuropeptides such as somatostatin (SST), or calcium-binding proteins such as calretinin (CR). SST, but not CR, neurons have been reported to be altered in the prefrontal cortex (PFC) of subjects with schizophrenia. Understanding the functional significance of the SST neuron disturbances in schizophrenia requires knowledge of the specialized synaptic circuitry of these neurons relative to that of CR neurons. Consequently, we used immuno-electron microscopy to examine the synaptic type and postsynaptic targets of SST-immunoreactive (IR) axon terminals in monkey PFC and compared these findings with similar data for CR-IR axon terminals. SST-IR axon terminals formed exclusively symmetric synapses and contacted only dendritic shafts (86%) and dendritic spines (14%), whereas CR-IR terminals also formed synapses with cell bodies. The postsynaptic targets of SST-IR axon terminals also differed across layers with synapses onto dendritic spines more frequent in the superficial (20%) than in the deep (8%) layers. Dual-label immunoelectron microscopy revealed that CR-IR axon terminals targeted GABA-IR dendritic shafts with a greater frequency (60%) than did SST-IR axon terminals (21.5%). Conversely, SST-IR axon terminals contacted unlabeled dendritic shafts, presumably belonging to pyramidal neurons, more frequently than did CR-IR axon terminals (57% vs. 19%, respectively). This specialized synaptic circuitry of SST neurons in the primate PFC suggests that the alterations of these neurons in schizophrenia is likely to have distinct functional consequences. Synapse 62:456–465, 2008. © 2008 Wiley-Liss, Inc.

BACKGROUND. Immunohistochemistry is helpful in distinguishing metastatic carcinoma from atypical mesothelial cells; however, it is not useful in differentiating atypical mesothelial cells from malignant mesothelial cells. K homolog domain containing protein overexpressed in cancer (KOC), a member of the insulin-like growth factor mRNA-binding protein (IMP) family, also known as L523S and IMP3, is expressed during embryogenesis and in various malignancies. Using a mouse monoclonal antibody (L523S) against KOC, KOC expression was investigated in malignant tumors and reactive mesothelial cells in serous effusions. METHODS. Seventy-six cases with paraffin-embedded pleural, pericardial, and peritoneal serous effusion cell blocks including 60 malignant serous effusions (11 malignant pleural mesotheliomas and 49 metastatic carcinomas) and benign pleural effusions (14 cases with reactive mesothelial cells and 2 cases with atypical cells with uncertain significance) were selected for immunohistochemical analysis with L523S, calretinin, and CK5/6. RESULTS. Immunohistochemical studies showed that positive staining for KOC of variable degrees of intensity was observed in 47 of 60 cases in malignant serous effusions including 10 of 11 mesotheliomas and 36 of 49 metastatic carcinomas. The associated reactive mesothelial cells were negative for KOC but positive for calretinin and CK5/6. All 11 malignant mesotheliomas exhibited positivity for calretinin, and 9 of 11 cases had CK5/6 staining. In addition, 16 cases that were originally diagnosed either as pleural effusions with reactive mesothelial cells (14) or atypical cells with uncertain significance (2) were also tested for KOC expression. Interestingly, 3 of 16 cases exhibited various degrees of positivity for KOC, 2 of which were diagnosed as lung adenocarcinoma with a recurrence after tumor resection and 1 as malignant pleural mesothelioma. CONCLUSIONS. Anti-L523S antibody is a useful marker for the detection of malignant cells in serous effusions and it can have significant utility in differentiating reactive mesothelial cells from malignant mesothelioma and metastatic carcinoma in combination with calretinin and CK5/6 staining. Cancer (Cancer Cytopathol) 2008. © 2007 American Cancer Society.

Bipolar neurons (BNs) in the vestibular ganglion (VG) connect vestibular hair cells with the central nervous system (CNS). Disturbed function and cell loss in central vestibular target areas or in the vestibular periphery involve BNs either retro- or anterogradely. However, the impact of central vestibular disturbances or hair cell defects on the maintenance of BNs is poorly understood. In the present study the volume of the VG, the size and total number of BNs, and the number of BNs expressing the calcium-binding protein calretinin (Calr) were quantified stereologically in the cerebellar mutants purkinje cell degeneration (pcd/pcd), weaver (wv/wv), and Lurcher (Lc/+), and in the vestibular mutants jerker (je/je), shaker-1 (sh/sh), and Varitint-waddler (Va/+). In all the different mutant mice investigated the total number of BNs did not differ from that of wildtypes. In contrast, the number of Calr-positive BNs was significantly reduced in je/je (23%) and sh/sh (33%) mutants. Reduced cell size was apparent in sh/sh mutants and the volume of the VG significantly decreased in je/je mice. Calr was virtually absent from calyx endings in the vestibular periphery of je/je, sh/sh, and Va/+ mutants, whereas in wildtypes and cerebellar mutants many calyces displayed intense Calr labeling. These results imply that the survival of BNs is apparently unaffected by the peripheral and central target defects found in the mutants investigated. Whether the decrease in Calr expression may reflect biochemical adaptations in response to input disturbances or a specific loss of large BNs is discussed.

The entorhino-hippocampal pathway is the major excitatory input from neurons of the entorhinal cortex on both ipsilateral and contralateral hippocampus/dentate gyrus. This fiber tract consists of the alvear path, the perforant path and a crossed commissural projection. In this study, the histogenesis and development of the various subsets of the entorhino-hippocampal projection have been investigated. DiI, DiO, Fast Blue tracing and calretinin immunocytochemistry as well as were carried out with pre and postnatal rats at different developmental stages. The alvear path and the commissural pathway start to develop as early as embryonic day E16, while the first perforant afferents reach the stratum lacunosum-moleculare of the hippocampus at E17 and at outer molecular layer of the denate gyrus at postnatal day 2. Retrograde tracing with DiI identifies entorhinal neurons in layer II–IV as the developmental origin of the entorhino-hippocampal pathway. Furthermore, calretinin immunocytochemistry revealed transitory Cajal-Retzius cells in the stratum lacunosum-moleculare of the hippocampus from E16. DiI labeling of entorhinal cortex fibers and combined calretinin-immunocytochemistry reveal a close relationship between Cajal-Retzius cells and entorhinal afferents. This temporal and spatial relationship suggests that Cajal-Retzius cell serves as a guiding cue for entorhinal afferents at early cortical development.

Olfactory sensory information is processed and integrated by circuits within the olfactory bulb. Golgi morphology suggests the olfactory bulb contains several major neuronal classes. However, an increasingly diverse collection of neurochemical markers have been localized in subpopulations of olfactory bulb neurons. While the mouse is becoming the animal model of choice for olfactory research, little is known about the proportions of neurons expressing and coexpressing different neurochemical markers in this species. Here we characterize neuronal populations in the mouse main olfactory bulb, focusing on glomerular populations. Immunofluorescent labeling for: 1) calretinin, 2) calbindin D-28K (CB), 3) parvalbumin, 4) neurocalcin, 5) tyrosine hydroxylase (TH), 6) the 67-kDa isoform of GAD (GAD67), and 7) the neuronal marker NeuN was performed in mice expressing green fluorescent protein under the control of the glutamic acid decarboxylase 65kDa (GAD65) promoter. Using unbiased stereological cell counts we estimated the total numbers of cells and neurons in the bulb and the number and percentage of neurons expressing and coexpressing different neurochemical populations in each layer of the olfactory bulb. Use of a genetic label for GAD65 and immunohistochemistry for GAD67 identified a much larger percentage of GABAergic neurons in the glomerular layer (55% of all neurons) than previously recognized. Additionally, while many glomerular neurons expressing TH or CB coexpress GAD, the majority of these neurons preferentially express the GAD67 isoform. These data suggest that the chemospecific populations of neurons in glomeruli form distinct subpopulations and that GAD isoforms are preferentially regulated in different neurochemical cell types.

The mammillary bodies (MB) are important relay nuclei within limbic and extralimbic connections. They are known to play important roles in memory formation and are affected in alcoholism and vitamin B1 deficiency. Their strategic position linking temporo-limbic to cortico-thalamic brain structures make the MB a candidate brain structure for alterations in schizophrenia. We studied 15 postmortem brains of schizophrenics and 15 matched control brains. Brain sections were stained either with Heidenhain-Woelcke, glutamic acid decarboxylase (GAD), calretinin, or parvalbumin. We determined the volumes of the MB and performed cell countings using stereological principles and a computerized image analysis system. The volumes of MB do not differ between schizophrenics and controls. However, in schizophrenia the number of neurons as well as the resulting neuronal densities was significantly reduced on both sides (on left side by 38.9%, on right side by 22%). No changes were seen in the number of GAD-expressing or calretinin-containing neurons, whereas the number of parvalbumin-immunoreactive MB neurons was reduced by more than 50% in schizophrenia. This cell loss (as a result of developmental malformation and/or neurodegeneration) points to a prominent involvement of the MB in the pathomorphology of schizophrenia. Parvalbumin-immunoreactive GABAergic interneurons have been reported to be diminished in schizophrenia. However, in the MB parvalbumin labels a subpopulation of glutamate/aspartate-containing neurons projecting mainly to the anterior thalamus. Thus, our data provide new evidence for impaired limbic circuits in schizophrenia.

Pioneering work indicates that the final position of neurons in specific layers of the mammalian cerebral cortex is determined primarily by birthdate. Glutamatergic projection neurons are born in the cortical proliferative zones of the dorsal telencephalon, and follow an “inside-out” neurogenesis gradient: later-born cohorts migrate radially past earlier-born neurons to populate more superficial layers. GABAergic interneurons, the major source of cortical inhibition, comprise a heterogeneous population and are produced in proliferative zones of the ventral telencephalon. Mechanisms by which interneuron subclasses find appropriate layer-specific cortical addresses remain largely unexplored. Major cortical interneuron subclasses can be identified based on expression of distinct calcium-binding proteins including parvalbumin, calretinin, or calbindin. We determined whether cortical layer-patterning of interneurons is dependent on phenotype. Parvalbumin-positive interneurons populate cortical layers with an inside-out gradient, and birthdate is isochronous to projection neurons in the same layers. In contrast, another major GABAergic subtype, labeled using calretinin, populates the cerebral cortex using an opposite “outside-in” gradient, heterochronous to neighboring neurons. In addition to birthdate, phenotype is also a determinant of cortical patterning. Discovery of a cortical subpopulation that does not follow the well-established inside-out gradient has important implications for mechanisms of layer formation in the cerebral cortex. J. Comp. Neurol. 501:369–380, 2007. © 2007 Wiley-Liss, Inc.