Probes for INS

RESULTS Microscopic analysis revealed that the full-length _HTT_ mRNA (_FL-HTT_) was retained in RNA nuclear clusters together with the incompletely spliced _HTT1a_ transcript. These clusters were not observed in zQ175 HD mouse model where, instead, _FL-Htt_ and _Htt1a_ mRNAs were detected as mostly cytoplasmic molecules. Immunohistochemistry showed a progressive appearance of aggregated HTT in nuclei in the cortex, striatum, hippocampus and cerebellum. HTRF indicated that the level of exon 1 HTT was highest in the cerebellum. Soluble mutant exon 1 HTT decreased with age, with concomitant increase in aggregated HTT. In YAC128 MEFs, _HTT1a_ was detected and ASOs targeting _HTT_ were efficient in lowering _HTT_ levels in this model system.

Understanding the processes of inflammation and tissue regeneration after injury is of great importance. For a long time, macrophages have been known to play a central role during different stages of inflammation and tissue regeneration. However, the molecular and cellular mechanisms by which they exert their effects are as yet mostly unknown. While in vitro macrophages have been characterized, recent progress in macrophage biology studies revealed that macrophages in vivo exhibited distinctive features. Actually, the precise characterization of the macrophages in vivo is essential to develop new healing treatments and can be approached via in situ analyses. Nowadays, the characterization of macrophages in situ has improved significantly using antigen surface markers and cytokine secretion identification resulting in specific patterns. This review aims for a comprehensive overview of different tools used for in situ macrophage identification, reporter genes, immunolabeling and in situ hybridization, discussing their advantages and limitations.

Autism spectrum disorders (ASDs) are a group of highly inheritable neurodevelopmental disorders. Functional mutations in TRIO, especially in the GEF1 domain, are strongly implicated in ASDs, whereas the underlying neurobiological pathogenesis and molecular mechanisms remain to be clarified. Here we characterize the abnormal morphology and behavior of embryonic migratory interneurons (INs) upon Trio deficiency or GEF1 mutation in mice, which are mediated by the Trio GEF1-Rac1 activation and involved in SDF1α/CXCR4 signaling. In addition, the migration deficits are specifically associated with altered neural microcircuit, decreased inhibitory neurotransmission, and autism-like behaviors, which are reminiscent of some features observed in patients with ASDs. Furthermore, restoring the excitatory/inhibitory (E/I) imbalance via activation of GABA signaling rescues autism-like deficits. Our findings demonstrate a critical role of Trio GEF1 mediated signaling in IN migration and E/I balance, which are related to autism-related behavioral phenotypes.

Vesicular transporter proteins are an essential component of the presynaptic machinery that regulates neurotransmitter storage and release. They also provide a key point of control for homeostatic signaling pathways that maintain balanced excitation and inhibition following changes in activity levels, including the onset of sensory experience. To advance understanding of their roles in the developing auditory forebrain, we tracked the expression of the vesicular transporters of glutamate (VGluT1, VGluT2) and GABA (VGAT) in primary auditory cortex (A1) and medial geniculate body (MGB) of developing mice (P7, P11, P14, P21, adult) before and after ear canal opening (~P11-P13). RNA sequencing, in situ hybridization, and immunohistochemistry were combined to track changes in transporter expression and document regional patterns of transcript and protein localization. Overall, vesicular transporter expression changed the most between P7 and P21. The expression patterns and maturational trajectories of each marker varied by brain region, cortical layer, and MGB subdivision. VGluT1 expression was highest in A1, moderate in MGB, and increased with age in both regions. VGluT2 mRNA levels were low in A1 at all ages, but high in MGB, where adult levels were reached by P14. VGluT2 immunoreactivity was prominent in both regions. VGluT1 + and VGluT2 + transcripts were co-expressed in MGB and A1 somata, but co-localization of immunoreactive puncta was not detected. In A1, VGAT mRNA levels were relatively stable from P7 to adult, while immunoreactivity increased steadily. VGAT + transcripts were rare in MGB neurons, whereas VGAT immunoreactivity was robust at all ages. Morphological changes in immunoreactive puncta were found in two regions after ear canal opening. In the ventral MGB, a decrease in VGluT2 puncta density was accompanied by an increase in puncta size. In A1, perisomatic VGAT and VGluT1 terminals became prominent around the neuronal somata. Overall, the observed changes in gene and protein expression, regional architecture, and morphology relate to-and to some extent may enable-the emergence of mature sound-evoked activity patterns. In that regard, the findings of this study expand our understanding of the presynaptic mechanisms that regulate critical period formation associated with experience-dependent refinement of sound processing in auditory forebrain circuits.

Reliable biomarkers are needed to avoid diagnostic delay and its devastating effects in patients with primary central nervous system (CNS) lymphoma (PCNSL). We analysed the discriminating sensitivity and specificity of myeloid differentiation primary response (88) (MYD88) L265P mutation (mut-MYD88) and interleukin-10 (IL-10) in cerebrospinal fluid (CSF) of both patients with newly diagnosed (n = 36) and relapsed (n = 27) PCNSL and 162 controls (118 CNS disorders and 44 extra-CNS lymphomas). The concordance of MYD88 mutational status between tumour tissue and CSF sample and the source of ILs in PCNSL tissues were also investigated. Mut-MYD88 was assessed by TaqMan-based polymerase chain reaction. IL-6 and IL-10 messenger RNA (mRNA) was assessed on PCNSL biopsies using RNAscope technology. IL levels in CSF were assessed by enzyme-linked immunosorbent assay. Mut-MYD88 was detected in 15/17 (88%) PCNSL biopsies, with an 82% concordance in paired tissue-CSF samples. IL-10 mRNA was detected in lymphomatous B cells in most PCNSL; expression of IL-6 transcripts was negligible. In CSF samples, mut-MYD88 and high IL-10 levels were detected, respectively, in 72% and 88% of patients with newly diagnosed PCNSL and in 1% of controls; conversely, IL-6 showed a low discriminating sensitivity and specificity. Combined analysis of MYD88 and IL-10 exhibits a sensitivity and specificity to distinguish PCNSL of 94% and 98% respectively. Similar figures were recorded in patients with relapsed PCNSL. In conclusion, high detection rates of mut-MYD88 and IL-10 in CSF reflect, respectively, the MYD88 mutational status and synthesis of this IL in PCNSL tissue. These biomarkers exhibit a very high sensitivity and specificity in detecting PCNSL both at initial diagnosis and relapse. Implications of these findings in patients with lesions unsuitable for biopsy deserve to be investigated.

Although the immunogenicity of formalin-fixed paraffin-embedded tissue sections can decrease during storage and transport, the exact mechanism of antigenic loss and how to prevent it are not clear. Herein, we investigated changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), E-cadherin, and Ki-67 in human breast tissue microarray (TMA) tissue sections stored for up to 3 months in dry and wet conditions. The positive rates of ER and PR expression were minimally changed after 3 months of storage, but the Allred scores of ER and PR stored in humid conditions decreased remarkably in comparison to fresh-cut tissue. The HER-2 antigenicity and RNA integrity of breast TMA sections stored in dry conditions diminished gradually with storage time, whereas the immunoreactivity and RNA quality of HER-2 in humid conditions decreased sharply as storage length increased. The area and intensity of E-cadherin staining in tissue sections stored in dry conditions did not change significantly and were minimally changed after 3 months, respectively. In contrast, the area and intensity of E-cadherin staining in tissue sections stored in humid conditions decreased significantly as storage length increased. Finally, the Ki-67 labeling index of tissue sections stored for 3 months in dry (9% decrease) and wet (31.9% decrease) conditions was decreased in comparison to fresh sections. In conclusion, these results indicate that water is a crucial factor for protein and RNA degradation in stored tissue sections, and detailed guidelines are required in the clinic.

Dopamine neurons in the substantia nigra (SN) and ventral tegmental area (VTA) play a central role in the reinforcing properties of abused drugs including methamphetamine and cocaine. Chronic effects of psychostimulants in the SN/VTA also involve non-dopaminergic transmitters, including glutamate and the stress-related peptide corticotropin-releasing factor (CRF). In the SN/VTA, astrocytes express a variety of membrane-bound neurotransmitter receptors and transporters that influence neurotransmission. CRF receptor type 2 (CRF2) activity in the VTA is important for stress-induced relapse and drug-seeking behaviour, but the localization of its effects is incompletely understood. Here, we first identified CRF2 transcript in astrocytes of the SN/VTA using RNA-Seq in Aldh1l1;NuTRAP mice and confirmed it using in situ hybridization (RNAscope) in wild-type mice. We then used immunofluorescence to quantify the astrocytic marker protein S100β, glial-specific glutamate/aspartate transporter GLAST, and CRF2 in the SN/VTA following 12 days of treatment (i.p.) with methamphetamine (3 mg/kg), cocaine (10 mg/kg), or saline. We observed a significant decrease in GLAST immunofluorescence in brains of psychostimulant treated mice compared with saline controls. In addition, we observed increased labelling of CRF2 in drug treated groups, a decrease in the number of S100β positive cells, and an increase of co-staining of CRF2 with both S100β and tyrosine hydroxylase (dopamine neurons). Our results suggest a significant interaction between CRF2, GLAST, and astrocytes in the midbrain that emerges with repeated exposure to psychostimulants. These findings provide rationale for future investigation of astrocyte-based strategies for altering cellular and circuit function in response to stress and drug exposure.

Over the past two decades, opioid abuse has risen especially among women. In both sexes hippocampal neural circuits involved in associative memory formation and encoding of motivational incentives are critically important in the transition from initial drug use to drug abuse/dependence. The opioid circuit particularly the mossy fiber pathway, are crucial for associative memory processes important for addiction. Our anatomical studies, especially those utilizing electron microscopic immunocytochemistry, have provided unique insight into sex differences in the distribution of opioid peptides and receptors in specific hippocampal circuits and how these distributions are altered following stress and oxycodone-associative learning processes. Here we review the hippocampal opioid system in rodents with respect to ovarian hormones effects and baseline sex differences then sex differences following acute and chronic stress. Next, we review sex differences in the hippocampal opioid system in unstressed and chronically stressed rats following oxycodone conditioned place preference. We show that opioid peptides and receptors are distributed within hippocampal circuits in females with elevated estrogen states in a manner that would enhance sensitivity to endogenous and exogenous opioids. Moreover, chronic stress primes the opioid system in females in a manner that would promote opioid-associative learning processes. In contrast, chronic stress has limited effects on the opioid system in males and reduces its capacity to support opioid-mediated learning processes. Interestingly, acute stress appears to prime males for opioid associative learning. On a broader scale the findings highlighted in this review have important implications in understanding sex differences in opioid drug use and abuse.

Sustained virological response (SVR) to the treatment of recurrent HCV in liver transplant recipients has excellent clinical outcomes; however, little is known about the effects on allograft histology. The study aimed to assess the histology of the allograft liver. In this single-center, retrospective cohort study, patients with recurrent hepatitis C (HCV) in allograft liver who were cured with antiviral therapy between 2010 and 2016 were identified. Biopsies were reviewed by two liver pathologists blinded to the treatment and SVR status. Paired analysis was performed to compare pre- and post-treatment histological features. Of the 62 patients analyzed, 22 patients received PEGylated interferon/ribavirin (IFN) therapy, while 40 patients received direct-acting antiviral agents (DAA). The mean age was 57 years, 24% were female, and 79% were Caucasian. RNA in situ hybridization testing for HCV and HEV was negative in all the tested patients. Significant reduction in the inflammatory grade of post-treatment biopsy specimens was noted in all subjects (n = 57; p < 0.001) and in the IFN group (n = 21; p = 0.001) but not in the DAA group (p = 0.093). Of all subjects, 21% had worsening stage, 31% had improvement, and 48% had no change in stage. Of the treatment groups, 27% in the IFN and 17% in the DAA groups had worsening stage; however, the results were not statistically significant in all subjects or by treatment modality. Persistent inflammatory infiltrates and fibrosis was noted in allograft tissue of patients cured with DAA. Significant improvement in grade was noted in the IFN group, without a significant change in stage.

Nociceptive markers in mice have been identified in two distinct peptidergic and nonpeptidergic neurons in the dorsal root ganglion (DRG) and distributed in different laminae of the dorsal horn of the spinal cord. Recently, however, a study in humans showed a significant overlapping in these two populations. In this study, we investigated the distribution of various nociceptive markers in the lumbar DRG and spinal cord of the dromedary camel. Immunohistochemical data showed a remarkable percentage of total neurons in the DRG expressed IB4 binding (54.5%), calcitonin gene-related peptide (CGRP; 49.5%), transient receptor potential vanilloid 1 (TRPV1; 48.2%), and nitric oxide synthase (NOS; 30.6%). The co-localization data showed that 89.6% and 74.0% of CGRP- and TRPV1-labeled neurons, respectively, were IB4 positive. In addition, 61.6% and 84.2% of TRPV1- and NOS-immunoreactive neurons, respectively, were also co-localized with CGRP. The distribution of IB4, CGRP, TRPV1, substance P, and NOS immunoreactivities in the spinal cord were observed in lamina I and outer lamina II (IIo). Quantitative data showed that 82.4% of IB4-positive nerve terminals in laminae I and IIo were co-localized with CGRP, and 86.0% of CGRP-labeled terminals were co-localized with IB4. Similarly, 85.1% of NOS-labeled nerve terminals were co-localized with CGRP. No neuropeptide Y (NPY) or cholecystokinin (CCK) immunoreactivities were detected in the DRG, and no co-localization between IB4, NPY, and CCK were observed in the spinal cord. Our results demonstrate marked convergence of nociceptive markers in the primary afferent neurons in camels, which is similar to humans rather than the mouse. The data also emphasizes the importance of interspecies differences when selecting ideal animal models for studying nociception and treating chronic pain.

Chronic pain is under-managed in individuals over 65 years of age due to a dearth of knowledge regarding the impact of age on opioid efficacy in the elderly. We have previously shown that advanced age and sex alter morphine modulation of persistent inflammatory pain (induced by intraplantar administration of Complete Freund's adjuvant (CFA)), such that morphine potency is highest in adult male rats (2mos), with EC50 values 2-fold higher in aged males (18mos) and females regardless of age. Age-induced reductions in morphine potency were accompanied by reduced mu opioid receptor (MOR) expression in the ventrolateral periaqueductal gray (vlPAG), a CNS region critical in pain modulation. The present studies further explore the impact of age on opioid signaling within the PAG. MOR affinity, availability, and G-protein activation were assessed using radioligand binding assays and GTPγS assays in vlPAG tissue from adult and aged, male and female rats collected 72h following CFA administration. Regulation of opioid induced G-protein signaling was assessed using RNAscope to analyze mRNA expression of Regulator of G-Protein Signaling (RGS) proteins RGS4 and RGS9-2. We find that aged males and females (adult and aged) exhibit reduced vlPAG MOR binding potential and reduced G-protein activation efficiency compared to adult males, suggesting age- and sex- differences in MOR machinery drive reduced opioid potency. RNAscope revealed increased expression of RGS4 and RGS9-2 in the vlPAG of aged animals compared to adults, indicating that MOR signaling is subject to greater negative regulation in the aged vlPAG. The observed age-related reductions in vlPAG MOR agonist binding and opioid induced G-protein activation, along with the observed increase in vlPAG RGS expression have significant implications in pain management in the aged population. Our novel findings elucidate several mechanisms mediating reduced morphine potency in aged animals, and identify potential targets to improve pain management in the elderly. R01DA041529-04.

Background A nonsynonymous single nucleotide polymorphism in the neuropeptide S receptor 1 (NPSR1) gene (rs324981) results in isoleucine to asparagine substitution at amino acid 107. In humans, the ancestral variant (NPSR1 I107) is associated with increased anxiety sensitivity and risk of panic disorder, while the human-specific variant (NPSR1 N107) is considered protective against excessive anxiety. In rodents, neurobiological constituents of the NPS system have been analyzed in detail and praised for their anxiolytic-like effects. However, implication for the human situation remains unclear as rodents carry only the ancestral NPSR1 I107 variant. Methods We hypothesized that phenotypic correlates of NPSR1 variants manifest in fear-related circuits in the amygdala. We used CRISPR/Cas9-mediated gene editing to generate a “humanized” mouse strain, where individuals express either NPSR1 I107 or N107. Results Stimulation of NPSR1 evoked excitatory responses in principal neurons of the anterior basal amygdala (aBA) with significant difference in magnitude between genotypes, resulting in synaptic disinhibition of putative extinction neurons in posterior BA in mice expressing the human-specific hypofunctional N107 but not the ancestral I107 variant. N107 mice displayed improved extinction of conditioned fear, which was phenocopied after pharmacological antagonism of NPSR1 in aBA of I107 mice. Differences in fear extinction between male and female mice related to an interaction of Npsr1 genotype and salience of fear training. Conclusions In conclusion, the NPS system regulates extinction circuits in the amygdala depending on Npsr1 genotype, contributing to sex-specific differences in fear extinction and high anxiety sensitivity of individuals bearing the ancestral NPSR1 I107 variant.