Probes for INS

Versican expression promotes tumor growth by destabilizing focal cell contacts, thus impeding cell adhesion and facilitating cell migration. It not only presents or recruits molecules to the cell surface, but also modulates gene expression levels and coordinates complex signal pathways. Previously, we suggested that the interaction between versican and human epidermal growth factor receptors may be directly associated with tumor aggressiveness. Thus, the expression of EGFR and HER-2 in these neoplasms may contribute to a better understanding of the progression mechanisms in malignant mammary tumors. The purpose of this study was to correlate the gene and protein expressions of EGFR and HER2 by RNA In Situ Hybridization (ISH) and immunohistochemistry (IHC), respectively, and their relationship with the versican expression in carcinomas in mixed tumors and carcinosarcomas of the canine mammary gland. The results revealed that EGFR mRNA expression showed a significant difference between in situ and invasive carcinomatous areas in low and high versican expression groups. Identical results were observed in HER-2 mRNA expression. In immunohistochemistry analysis, neoplasms with low versican expression showed greater EGFR immunostaining in the in situ areas than in invasive areas, even as the group presenting high versican expression displayed greater EGFR and HER-2 staining in in situ areas. Significant EGFR and HER-2 mRNA and protein expressions in in situ carcinomatous sites relative to invasive areas suggest that these molecules play a role during the early stages of tumor progression.

By virtue of their extensive axonal arborisation and perisomatic synaptic targeting, cortical inhibitory Parvalbumin (PV) cells strongly regulate principal cell output and plasticity and modulate experience-dependent refinement of cortical circuits during development. An interesting aspect of PV cell connectivity is its prolonged maturation time course, which is completed only by end of adolescence. The p75 neurotrophin receptor (p75NTR) regulates numerous cellular functions, however its role on cortical circuit development and plasticity remains elusive, mainly because localizing p75NTR expression with cellular and temporal resolution has been challenging.By using RNAscope and a modified version of the Proximity Ligation Assay, we found that p75NTR expression in PV cells decreases between the second and fourth postnatal week, at a time when PV cell synapse numbers increase dramatically. Conditional knockout of p75NTR in single PV neurons in vitro and in PV cell networks in vivo causes precocious formation of PV cell perisomatic innervation and perineural nets around PV cell somata, therefore suggesting that p75NTR expression modulates the timing of maturation of PV cell connectivity in the adolescent cortex.Remarkably, we found that PV cells still express p75NTR in adult mouse cortex of both sexes and that its activation is sufficient to destabilize PV cell connectivity and to restore cortical plasticity following monocular deprivation in vivo. Altogether, our results show that p75NTR activation dynamically regulates PV cell connectivity, and represents a novel tool to foster brain plasticity in adults.SIGNIFICANCE STATEMENTIn the cortex, inhibitory, GABA-releasing neurons control the output and plasticity of excitatory neurons. Within this diverse group, parvalbumin-expressing (PV) cells form the larger inhibitory system. PV cell connectivity develops slowly, reaching maturity only at the end of adolescence, however the mechanisms controlling the timing of its maturation are not well understood. We discovered that the expression of the neurotrophin receptor p75NTR in PV cells inhibits the maturation of their connectivity in a cell autonomous fashion, both in vitro and in vivo and that p75NTR activation in adult PV cells promotes their remodelling and restores cortical plasticity. These results reveal a new p75NTR function in the regulation of the time course of PV cell maturation and in limiting cortical plasticity.

Although Netos are considered auxiliary subunits critical for kainate receptor (KAR) function, direct evidence for their regulation of native KARs is limited. Because Neto KAR regulation is GluK subunit/Neto isoform specific, such regulation must be determined in cell-type-specific contexts. We demonstrate Neto1/2 expression in somatostatin (SOM)-, cholecystokinin/cannabinoid receptor 1 (CCK/CB1)-, and parvalbumin (PV)-containing interneurons. KAR-mediated excitation of these interneurons is contingent upon Neto1 because kainate yields comparable effects in Neto2 knockouts and wild-types but fails to excite interneurons or recruit inhibition in Neto1 knockouts. In contrast, presynaptic KARs in CCK/CB1 interneurons are dually regulated by both Neto1 and Neto2. Neto association promotes tonic presynaptic KAR activation, dampening CCK/CB1 interneuron output, and loss of this brake in Neto mutants profoundly increases CCK/CB1 interneuron-mediatedinhibition. Our results confirm that Neto1 regulates endogenous somatodendritic KARs in diverse interneurons and demonstrate Neto regulation of presynaptic KARs in mature inhibitory presynaptic terminals.

Objective:
To investigate in situ mRNA expression of HER 2 oncogene in breast cancers with equivocal immunohistochemical results , and to explore the potential feasibility of RNAscope technique in evaluating HER2 status in breast cancers .Methods Sixty-nine FFPE samples of invasive ductal breast cancer with equivocal HER 2 immunohistochemistry results ( IHC 2+) were collected from surgical excisions from Peking Union Medical College Hospital between June 2010 and June 2013.HER2 status and in situ mRNA expression were tested by fluorescence in situ hybridization ( FISH) and RNAscope respectively using tissue microarray constructed from tumor paraffin blocks .The results of HER2 mRNA expression were scored 0 to 4 ( from low to high levels ) according to mRNA expression in 100 cancer cells .HER2 mRNA expression was evaluated in two groups of patients , with positive and negative FISH results .Results Twenty-three of the 69 samples were FISH positive, including 16 samples that were scored 4 by RNAscope (70%,16/23), 6 samples were scored 3 ( 26%,6/23 ) and one sample was scored 2 ( 4%,1/23 ) .High in situ mRNA expression (score 4 or 3) were observed in 96%of HER2 FISH positive samples.All of samples that were scored 4 by RNAscope were FISH positive .Forty-six samples were FISH negative , including 17 samples that were scored 3 by RNAscope (37%,17/46), 25 samples were scored 2 (54%,25/46), and 4 samples were scored 1 (9%,4/46).Conclusions Breast cancer with HER2 IHC 2 +could be further classified according to in situ mRNA expression status .Among them, RNAscope score of 4 could be one of the interpretation criteria for re-testing IHC 2+samples.In situ detection of HER2 mRNA may be an additional candidate method of confirmation for HER 2 gene amplification or protein overexpression , and has potential clinical utility.