AMPA-type glutamate receptors (AMPAR) mediate excitatory cochlear transmission. However, the unique roles of AMPAR subunits are unresolved. Lack of subunit GluA3 (Gria3KO) in male mice reduced cochlear output by 8-weeks of age. Since Gria3 is X-linked and considering sex differences in hearing vulnerability, we hypothesized accelerated presbycusis in Gria3KO females. Here, auditory brainstem responses (ABR) were similar in 3-week-old female Gria3WT and Gria3KO mice. However, when raised in ambient sound, ABR thresholds were elevated and wave-1 amplitudes were diminished at 5-weeks and older in Gria3KO. In contrast, these metrics were similar between genotypes when raised in quiet. Paired synapses were similar in number, but lone ribbons and ribbonless synapses were increased in female Gria3KO mice in ambient sound compared to Gria3WT or to either genotype raised in quiet. Synaptic GluA4:GluA2 ratios increased relative to Gria3WT, particularly in ambient sound, suggesting an activity-dependent increase in calcium-permeable AMPARs in Gria3KO. Swollen afferent terminals were observed by 5-weeks only in Gria3KO females reared in ambient sound. We propose that lack of GluA3 induces sex-dependent vulnerability to AMPAR-mediated excitotoxicity.

Both male and female zebrafish have a population of germline stem cells that produce gametes throughout the life of the fish. These cells localize to specific regions in the gonads and can be identified because they uniquely express the nanos2 gene, which encodes a conserved regulator of translation. A method is presented here for identifying germline stem cells in the ovary and testis using a combined protocol for whole-mount fluorescent RNA in situ hybridization to detect nanos2 mRNA and immunofluorescence to detect the pan-germ cell marker Vasa.

The constant exposure of ocular surface to external environment and then to several microbial agents is often related to the pathogenesis of various inflammatory eye disorders. In the present study α-Smooth Muscle Actin (α-SMA) and Langerin CD/207 expression and function was investigated in a rabbit corneal keratitis. The inflammation was induced by the secreted form of glycoprotein B (gB1s) of HSV-1, in an ex vivo rabbit corneal model. α-SMA is often used as a marker for myofibroblasts. In this study, for the first time, we show α-SMA positive corneal epithelial cells, during HSV-1 cornea inflammation, demonstrating a crucial role in wound healing, especially during remodeling phase. Furthermore, we show the presence of Dendritic Cells Langerin CD/207 positive, located mainly in the basal epithelial layer and in corneal stroma during the inflammatory processes. Our result validating the ex vivo organotypic rabbit corneal model, for the study about pathogenesis of HSV-1 ocular infection.

The antifibrotic potential of platelet-rich plasma (PRP) is controversial. This study examined the effects of PRP on in vitro transforming growth factor (TGF)-β1-induced differentiation of fibroblasts into myofibroblasts, the main drivers of fibrosis, and the involvement of vascular endothelial growth factor (VEGF)-A in mediating PRP-induced responses. The impact of PRP alone on fibroblast differentiation was also assessed. Myofibroblastic phenotype was evaluated by confocal fluorescence microscopy and western blotting analyses of α-smooth muscle actin (sma) and type-1 collagen expression, vinculin-rich focal adhesion clustering, and stress fiber assembly. Notch-1, connexin 43, and VEGF-A expression were also analyzed by RT-PCR. PRP negatively regulated fibroblast-myofibroblast transition via VEGF-A/VEGF receptor (VEGFR)-1-mediated inhibition of TGF-β1/Smad3 signaling. Indeed TGF-β1/PRP co-treated fibroblasts showed a robust attenuation of the myofibroblastic phenotype concomitant with a decrease of Smad3 expression levels. The VEGFR-1 inhibition by KRN633 or blocking antibodies, or VEGF-A neutralization in these cells prevented the PRP-promoted effects. Moreover PRP abrogated the TGF-β1-induced reduction of VEGF-A and VEGFR-1 cell expression. The role of VEGF-A signaling in counteracting myofibroblast generation was confirmed by cell treatment with soluble VEGF-A. PRP as single treatment did not induce fibroblast myodifferentiation. This study provides new insights into cellular and molecular mechanisms underpinning PRP antifibrotic action.

Both male and female zebrafish have a population of germ-line stem cells that produce gametes throughout the life of the fish. These cells localize to specific regions in the gonads and can be identified because they uniquely express the nanos2 gene, which encodes a conserved regulator of translation. A method is presented here for identifying germ-line stem cells in the ovary and testis using a combined protocol for whole-mount fluorescent RNA in situ hybridization to detect nanos2 mRNA and immunofluorescence to detect the pan-germ cell marker Vasa.

Previous studies show that actin-binding Rho activating protein (Abra) is expressed in cardiomyocytes and vascular smooth muscle cells. In this study, we investigated the expression profile of Abra in the central nervous system of normal adult rats by confocal immunofluorescence. Results showed that Abra immunostaining was located in neuronal nuclei, cytoplasm and processes in the central nervous system, with the strongest staining in the nuclei; in the cerebral cortex, Abra positive neuronal bodies and processes were distributed in six cortical layers including molecular layer, external granular layer, external pyramidal layer, internal granular layer, internal pyramidal layer and polymorphic layer; in the hippocampus, the cell bodies of Abra positive neurons were distributed evenly in pyramidal layer and granular layer, with positive processes in molecular layer and orien layer; in the cerebellar cortex, Abra staining showed the positive neuronal cell bodies in Purkinje cell layer and granular layer and positive processes in molecular layer; in the spinal cord, Abra-immunopositive products covered the whole gray matter and white matter; co-localization studies showed that Abra was co-stained with F-actin in neuronal cytoplasm and processes, but weakly in the nuclei. In addition, in the hippocampus, Abra was co-stained with F-actin only in neuronal processes, but not in the cell body. This study for the first time presents a comprehensive overview of Abra expression in the central nervous system, providing insights for further investigating the role of Abra in the mature central nervous system.

BACKGROUND: Altered gamma-aminobutyric acid (GABA) signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in patients with schizophrenia and schizoaffective disorder. Levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67-kDa isoform (GAD67) in the PFC have been consistently reported to be lower in patients with these disorders, but the status of the second GABA-synthesizing enzyme, glutamic acid decarboxylase 65-kDa isoform (GAD65), remains unclear.
METHODS: GAD65 messenger RNA (mRNA) levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. In a subset of subject pairs, GAD65 relative protein levels were quantified by confocal immunofluorescence microscopy.
RESULTS: Mean GAD65 mRNA levels were 13.6% lower in subjects with schizoaffective disorder but did not differ in subjects with schizophrenia relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein levels within subjects with schizoaffective disorder were not attributable to factors commonly comorbid with the diagnosis.
CONCLUSIONS: In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in patients with schizoaffective disorder relative to patients with schizophrenia, these findings may support an interpretation that GAD65 downregulation provides a homeostatic response complementary to GAD67 downregulation that serves to reduce inhibition in the face of lower PFC network activity.

Confocal laser scanning microscopy is an advanced technique for imaging tissue samples in vitro and in vivo at high optical resolution. The development of new fluorochrome variants do not only make it possible to perform multicolor flow cytometry of single cells, but in combination with high resolution laser scanning systems also to investigate the distribution of cells in lymphoid tissues by confocal immunofluorescence analyses, thus allowing the distinction of various cell populations directly in the tissue. Here, we provide a protocol for the visualization of at least six differently fluorochrome-labeled antibodies at the same time using a conventional confocal laser scanning microscope with four laser lines (405 nm, 488 nm, 555 nm, and 639 nm laser wavelength) in both murine and human tissue samples. We further demonstrate that compensation correction algorithms are not necessary to reduce spillover of fluorochromes into other channels when the used fluorochromes are combined according to their specific emission bands and the varying Stokes shift for co-excited fluorochromes with the same laser line.

Activation of proliferation of Schwann cells is crucial for axonal guidance and successful nerve regeneration following peripheral nerve injury (PNI). Considering melatonin plays an important role in proliferative regulation of central glial cells, the present study determined whether melatonin can effectively promote Schwann cell proliferation and improve nerve regeneration after PNI. The spontaneous immortalized rat Schwann cell line (RSC 96 cells) was first analyzed by quantitative polymerase chain reaction (QPCR) to detect the potential existence of melatonin receptors. The melatonin receptor-mediated signaling responsible for proliferation was examined by measuring the phosphorylation of extracellular signal-regulated kinases (ERK1/2) pathway. The in vivo model of PNI was performed by the end-to-side neurorrhaphy. The quantity of Schwann cells as well as the number of re-innervated motor end plates (MEP) on target muscles was examined to represent the functional recovery of injured nerves. QPCR results indicated that MT1 is the dominant receptor in Schwann cells. Immunoblotting and proliferation assay revealed an enhanced phosphorylation of ERK1/2 and increased number of RSC 96 cells following melatonin administration. Nonselective melatonin receptor antagonist (luzindole) treatment significantly suppressed all the above findings, suggesting that the proliferative effects of melatonin were mediated by a receptor-dependent pathway. In vivo results corresponded well with in vitro findings in which melatonin effectively increased the amount of proliferated Schwann cells and re-innervated MEP on target muscles following PNI. As melatonin successfully improves nerve regeneration by promoting Schwann cell proliferation, therapeutic use of melatonin may thus serve as a promising strategy to counteract the PNI-induced neuronal disability.

The aim of the study was to evaluate sialic acids and hyaluronan expression, anionic components important for the structure and function of the renal tubulointerstitial compartment, in the early stages of sepsis. Two groups of rats were used: (1) sham-operated controls; (2) cecal ligation and puncture (CLP) (polymicrobial sepsis model). A search for microbial growth was made in the peritoneal fluid to document infection. Tubular function was evaluated by means of urinary protein loss, urinary Na(+) and urea excretion. Kidney samples were processed to analyze histology, sialic acids (lectin histochemistry) and hyaluronan (immunohistochemistry) expression. Results showed increased urinary protein loss and fractional excretion of Na(+) and urea reduction in the CLP group. Histological changes, particularly in the cortex and in proximal tubules of the CLP group, were observed. In septic rats, compared to controls, sialic acids decreased in amount and their acetylation increased in the tubules, although to a lesser extent in the proximal portion. Hyaluronan was expressed in the medullary interstitium and in a few areas of cortex in controls. In septic rats it increased in the cortical interstitium and appeared in proximal tubules. These results suggest correlation between expression changes of anionic components and tubulointerstitium morphofunctional alterations during sepsis. A role of these molecules in protection/defense and repair processes may be suggested.