Probes for INS

The lateral habenula (LHb) has a key role in integrating a variety of neural circuits associated with reward and aversive behaviors. There is limited information about how the different cell types and neuronal circuits within the LHb coordinate physiological and motivational states. Here, we report a cell type in the medial division of the LHb (LHbM) in male rats that is distinguished by: (1) a molecular signature for GABAergic neurotransmission (Slc32a1/VGAT) and estrogen receptor (Esr1/ERα) expression, at both mRNA and protein levels, as well as the mRNA for vesicular glutamate transporter Slc17a6/VGLUT2, which we term the GABAergic estrogen-receptive neuron (GERN); (2) its axonal projection patterns, identified by in vivo juxtacellular labeling, to both local LHb and to midbrain modulatory systems; and (3) its somatic expression of receptors for vasopressin, serotonin and dopamine, and mRNA for orexin receptor 2. This cell type is anatomically located to receive afferents from midbrain reward (dopamine and serotonin) and hypothalamic water and energy homeostasis (vasopressin and orexin) circuits. These afferents shared the expression of estrogen synthase (aromatase) and VGLUT2, both in their somata and axon terminals. We demonstrate dynamic changes in LHbM VGAT+ cell density, dependent upon gonadal functional status, that closely correlate with motivational behavior in response to predator and forced swim stressors. The findings suggest that the homeostasis and reward-related glutamatergic convergent projecting pathways to LHbMC employ a localized neurosteroid signaling mechanism via axonal expression of aromatase, to act as a switch for GERN excitation/inhibition output prevalence, influencing depressive or motivated behavior.

The Neuregulin (NRG) family of ErbB ligands is comprised of numerous variants originating from the use of different genes, alternative promoters and splice variants. NRGs have generally been thought to be transported to axons and presynaptic terminals where they signal via ErbB3/4 receptors in paracrine or juxtacrine mode. However, we recently demonstrated that unprocessed pro-NRG2 accumulates on cell bodies and proximal dendrites, and that NMDAR activity is required for shedding of its ectodomain by metalloproteinases. Here we systematically investigated the subcellular distribution and processing of major NRG isoforms in rat hippocampal neurons. We show that NRG1 isotypes I and II, which like NRG2 are single-pass transmembrane proteins with an Ig-like domain, share the same subcellular distribution and ectodomain shedding properties. We furthermore show that NRG3, like CRD-NRG1, is a dual-pass transmembrane protein that harbors a second transmembrane domain near its amino-terminus. Both NRG3 and CRD-NRG1 cluster on axons through juxtacrine interactions with ErbB4 present on GABAergic interneurons. Interestingly, while single-pass NRGs accumulate as unprocessed pro-forms, axonal puncta of CRD-NRG1 and NRG3 are comprised of processed protein. Mutations of CRD-NRG1 and NRG3 that render them resistant to BACE cleavage, as well as BACE inhibition, result in the loss of axonal puncta and in the accumulation of unprocessed proforms in neuronal soma. Together, these results define two groups of NRGs with distinct membrane topologies and fundamentally different targeting and processing properties in central neurons. The implications of this functional diversity for the regulation of neuronal processes by the NRG/ErbB pathway are discussed.SIGNIFICANCE STATEMENTNumerous Neuregulins are generated through the use of different genes, promoters and alternative splicing, but the functional significance of this evolutionary conserved diversity remains poorly understood. Here we show that NRGs can be categorized by their membrane topologies. Single-pass Neuregulins such as NRG1 types I/II and NRG2 accumulate as unprocessed pro-forms on cell bodies, and their ectodomains are shed by metalloproteinases in response to NMDA receptor activation. By contrast, dual-pass CRD-NRG1 and NRG3 are constitutively processed by BACE and accumulate on axons where they interact with ErbB4 in juxtacrine mode. These findings reveal a previously unknown functional relationship between membrane topology, protein processing and subcellular distribution, and suggest that single- and dual-pass NRGs regulate neuronal functions in fundamentally different ways.

Four immunohistochemistry (IHC) diagnostic assays have been approved for tumour PD-L1 protein assessment in the clinic. However, mRNA detection by in situ hybridisation (ISH) could be utilised as an alternative to protein detection. Detecting spatial changes in gene expression provides vital prognostic and diagnostic information, particularly in immune oncology where the phenotype, cellular infiltration and immune activity status may be associated with patient survival. Translation of mRNA expression to a clinically relevant cut off or threshold is challenging due to variability between assays and the detection of different analytes. These studies aim to confirm the suitability of formalin fixed paraffin embedded (FFPE) tissue sections for use with RNA ISH. A comparison of mRNA expression and protein expression may inform the suitability of mRNA as a patient selection biomarker in a similar manner to IHC and provide evidence of a suitable scoring algorithm. Ninety patient samples, thirty for each indication of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma (UC), previously assessed using the VENTANA PD-L1 (SP263) Assay were chosen to represent a wide dynamic range of percentage tumour cell staining (TCIHC). Expression of mRNA was assessed by ISH using the RNAScope 2.5 assay and probe CD274/PD-L1 (Advanced Cell Diagnostics) including kit provided positive and negative control probes. Brightfield whole slide images of tissues were captured. The percentage of tumour cells with PD-L1 mRNA expression (%TCmRNA) and mean punctate dots/tumour cell were determined using image analysis. Differences in RNA expression between the IHC derived TCIHC≥25% and <25% groups were assessed using t-tests. For each indication, a receiver-operating characteristic (ROC) analysis identified thresholds for patient classification using %TCmRNA and dots/tumour cell, with reference to TCIHC≥25%. Eighty-six samples were successfully tested; 3 failed due to insufficient control probe staining, 1 due to lack of tumour. Percent TCmRNA staining using RNAScope demonstrated statistical significance (at α = 0.05) in the PD-L1 high (TCIHC ≥25%) vs the PD-L1 low (TCIHC <25%) groups for NSCLC, HNSCC, and UC. The number of punctate dots/tumour cell was significantly higher in the PD-L1 high vs the PD-L1 low groups for NSCLC and HNSCC but not UC. For %TCmRNA; ROC analysis identified thresholds of: NSCLC 18.0%, HNSCC 31.8%, UC 25.8%. For dots/tumour cell, thresholds were: NSCLC 0.26, HNSCC 0.53, UC 0.45. Routine tissue fixation and processing is suitable for RNA detection using RNAScope. PD-L1 mRNA extent and level is associated with PD-L1 status determined by IHC. Threshold optimisation for %TCmRNA and mean dots/tumour cell results in high specificity to IHC PD-L1 classification, but only moderate sensitivity.

Shank2 is an abundant postsynaptic scaffolding protein implicated in neurodevelopmental and psychiatric disorders, including autism spectrum disorders (ASD). Deletion of Shank2 in mice has been shown to induce social deficits, repetitive behaviors, and hyperactivity, but the identity of the cell types that contribute to these phenotypes has remained unclear. Here, we report a conditional mouse line with a Shank2 deletion restricted to parvalbumin (PV)-positive neurons (Pv-Cre;Shank2fl/fl mice). These mice display moderate hyperactivity in both novel and familiar environments and enhanced self-grooming in novel, but not familiar, environments. In contrast, they showed normal levels of social interaction, anxiety-like behavior, and learning and memory. Basal brain rhythms in Pv-Cre;Shank2fl/fl mice, measured by electroencephalography, were normal, but susceptibility to pentylenetetrazole (PTZ)-induced seizures was decreased. These results suggest that Shank2 deletion in PV-positive neurons leads to hyperactivity, enhanced self-grooming and suppressed brain excitation.

Gene expression analysis is essential for understanding the rich repertoire of cellular functions. With the development of sensitive molecular tools such as single-cell RNA sequencing, extensive gene expression data can be obtained and analyzed from various tissues. Single-molecule fluorescence in situ hybridization (smFISH) has emerged as a powerful complementary tool for single-cell genomics studies because of its ability to map and quantify the spatial distributions of single mRNAs at the subcellular level in their native tissue. Here, we present a detailed method to study the copy numbers and spatial localizations of single mRNAs in the cochlea and inferior colliculus. First, we demonstrate that smFISH can be performed successfully in adult cochlear tissue after decalcification. Second, we show that the smFISH signals can be detected with high specificity. Third, we adapt an automated transcript analysis pipeline to quantify and identify single mRNAs in a cell-specific manner. Lastly, we show that our method can be used to study possible correlations between transcriptional and translational activities of single genes. Thus, we have developed a detailed smFISH protocol that can be used to study the expression of single mRNAs in specific cell types of the peripheral and central auditory systems.

Osteoblast-lineage cells in bone human were recently shown to colonize eroded bone surfaces and to closely interact with osteoclasts. They proved identical with reversal cells and are believed to differentiate into bone forming osteoblasts thereby coupling resorption and formation. However, they also exert catabolic activity that contributes to osteoclastic bone resorption, but this has not received much attention. Herein, we used co-cultures of primary human osteoblast-lineage cells and human osteoclasts derived from peripheral blood monocytes to investigate whether a catabolic activity of osteoblast-lineage cells may impact on osteoclastic bone resorption. Through a combination of immunofluorescence, in-situ hybridization, and time-lapse we show that MMP-13 expressing osteoblast-lineage cells are attracted to and closely interact with bone resorbing osteoclasts. This close interaction results in a strong and significant increase in the bone resorptive activity of osteoclasts - especially those making trenches. Importantly, we show that osteoclastic bone resorption becomes sensitive to inhibition of matrix metalloproteinases in the presence, but not in the absence, of osteoblast-lineage cells. We propose that this may be due to the direct action of osteoblast-lineage-derived MMP-13 on bone resorption.

Sensory experience influences the establishment of neural connectivity through molecular mechanisms that remain unclear. Here, we employ single-nucleus RNA sequencing to investigate the contribution of sensory-driven gene expression to synaptic refinement in the dorsal lateral geniculate nucleus of the thalamus, a region of the brain that processes visual information. We find that visual experience induces the expression of the cytokine receptor Fn14 in excitatory thalamocortical neurons. By combining electrophysiological and structural techniques, we show that Fn14 is dispensable for early phases of refinement mediated by spontaneous activity but that Fn14 is essential for refinement during a later, experience-dependent period of development. Refinement deficits in mice lacking Fn14 are associated with functionally weaker and structurally smaller retinogeniculate inputs, indicating that Fn14 mediates both functional and anatomical rearrangements in response to sensory experience. These findings identify Fn14 as a molecular link between sensory-driven gene expression and vision-sensitive refinement in the brain.

Despite generally being a reinforcing drug of abuse, amphetamine (amph) also produces effects such as hypophagia and conditioned taste avoidance (CTA), which may indicate that amph acts as an aversive homeostatic stressor. Stress-responsive prolactin-releasing peptide (PrRP)-positive noradrenergic and glucagon-like peptide-1 (GLP-1)-positive neurons in the caudal nucleus of the solitary tract (cNTS) are modulated by metabolic state, and are prime candidates for mediating amph-induced hypophagia and CTA. The present study used dual immunolabeling and fluorescent in situ hybridization (RNAscope) to examine acute amph-induced activation of cFos expression in phenotypically-identified cNTS neurons in ad lib-fed vs. overnight-fasted male Sprague Dawley rats. We also examined the impact of food deprivation on amph-induced CTA. Compared to control saline treatment, amph activated significantly more cNTS neurons, including PrRP-negative noradrenergic (NA) neurons, GABAergic neurons, and glutamatergic neurons, but not PrRP or GLP-1 neurons. Amph also increased neural activation within a subset of central cNTS projection targets, including the lateral parabrachial nucleus and central amygdala, but not the paraventricular hypothalamus. Food deprivation did not alter amph-induced neural activation or impact the ability of amph to support CTA. These findings indicate that PrRP-negative NA and other cNTS neurons are recruited by acute amph treatment regardless of metabolic state, and may participate in amph-induced hypophagia and CTA.