Probes for INS

Seasonal changes in day length (photoperiod) affect numerous physiological functions. The suprachiasmatic nucleus (SCN)-paraventricular nucleus (PVN) axis plays a key role in processing photoperiod-related information. Seasonal variations in SCN and PVN neurotransmitter expression have been observed in humans and animal models. However, the molecular mechanisms by which the SCN-PVN network responds to altered photoperiod is unknown. Here, we show in mice that neuromedin S (NMS) and vasoactive intestinal polypeptide (VIP) neurons in the SCN display photoperiod-induced neurotransmitter plasticity. In vivo recording of calcium dynamics revealed that NMS neurons alter PVN network activity in response to winter-like photoperiod. Chronic manipulation of NMS neurons is sufficient to induce neurotransmitter switching in PVN neurons and affects locomotor activity. Our findings reveal previously unidentified molecular adaptations of the SCN-PVN network in response to seasonality and the role for NMS neurons in adjusting hypothalamic function to day length via a coordinated multisynaptic neurotransmitter switching affecting behavior.

Evidence is emerging that immune responses not only play a part in the central nervous system (CNS) in diseases but may also be relevant for healthy conditions. We discovered a major role for the interleukin-4 (IL-4)/IL-4 receptor alpha (IL-4Rα) signaling pathway in synaptic processes, as indicated by transcriptome analysis in IL-4Rα-deficient mice and human neurons with/without IL-4 treatment. Moreover, IL-4Rα is expressed presynaptically, and locally available IL-4 regulates synaptic transmission. We found reduced synaptic vesicle pools, altered postsynaptic currents, and a higher excitatory drive in cortical networks of IL-4Rα-deficient neurons. Acute effects of IL-4 treatment on postsynaptic currents in wild-type neurons were mediated via PKCγ signaling release and led to increased inhibitory activity supporting the findings in IL-4Rα-deficient neurons. In fact, the deficiency of IL-4Rα resulted in increased network activity in vivo, accompanied by altered exploration and anxiety-related learning behavior; general learning and memory was unchanged. In conclusion, neuronal IL-4Rα and its presynaptic prevalence appear relevant for maintaining homeostasis of CNS synaptic function.

Select neuronal populations display steady rhythmic neuronal firing that provides tonic excitation to drive downstream networks and behaviors. In noradrenergic neurons of the locus coeruleus (LC), circadian neurons of the suprachiasmatic nucleus (SCN), and CO2/H+-activated neurons of the brainstem retrotrapezoid nucleus (RTN), large subthreshold membrane potential oscillations contribute to the pacemaker-like action potential discharge. The oscillations and firing in LC and SCN involve contributions from leak sodium (NALCN) and L-type calcium channels while recent work from RTN suggested an additional pivotal role for a secondary calcium-activated and voltage-gated cationic current sensitive to TRPM4 channel blockers. Here, we tested whether TRPM4 contributes to subthreshold oscillations in mouse LC and SCN. By RNAscope in situ hybridization, Trpm4 transcripts were detected in both cell groups. In whole-cell recordings from acute slice preparations, prominent voltage-dependent membrane potential oscillations were revealed in LC and SCN after blocking action potentials. These oscillations were inhibited by two chemically-distinct blockers of TRPM4, 9-phenanthrol (9-pt) and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). Under whole-cell voltage clamp, inward currents evoked by oscillation voltage waveforms were inhibited in LC by blocking L-type calcium channels and TRPM4. These data implicate TRPM4 in the large subthreshold membrane potential oscillations that underlie tonic action potential discharge in LC and SCN, providing a voltage-dependent and calcium-dependent cationic current to augment the depolarizing inward Na+ and Ca2+ currents previously associated with this distinctive electroresponsive property.

Dynamic regulation of G-protein-coupled receptor (GPCR) kinase 2 (GRK2) expression restores cellular function by protecting from overstimulation via GPCR and non-GPCR signaling. In the primary afferent neurons, GRK2 negatively regulates nociceptive tone. The present study tested the hypothesis that induction of GRK2 in the primary afferent neurons contributes to the resolution of acute pain after tissue injury. GRK2 expression in the dorsal root ganglion (DRG) was analyzed at 1 and 7 days after the incision. Intraperitoneal administration of a GRK2 inhibitor was performed 7 days post-incision in male Sprague-Dawley rats who underwent plantar incisions to analyze the pain-related behavioral effect of the GRK2 inhibitor. Separately, GRK2 expression was analyzed after injecting insulin-like growth factor 1 (IGF1) into the rat hind paw. In addition, an IGF1 receptor (IGF1R) inhibitor was administered in the plantar incision rats to determine its effect on the incision-induced hyperalgesia and GRK2 expression. Plantar incision induced an increase in GRK2 in the DRG at 7 days, but not at 1 day post-incision. Acute hyperalgesia after the plantar incision disappeared by 7 days post-incision. Intraperitoneal injection of the GRK2 inhibitor at this time reinstated mechanical hyperalgesia, although the GRK2 inhibitor did not produce hyperalgesia in naive rats. After the incision, IGF1 expression increased in the paw, but not in the DRG. Intraplantar injection of IGF1 increased GRK2 expression in the ipsilateral DRG. IGF1R inhibitor administration prevented both the induction of GRK2 and resolution of hyperalgesia after the plantar incision. These findings demonstrate that induction of GRK2 expression driven by tissue IGF1 has potent analgesic effects and produces resolution of hyperalgesia after tissue injury. Dysregulation of IGF1-GRK2 signaling could potentially lead to failure of the spontaneous resolution of acute pain and, hence, development of chronic pain after surgery.

Forming long-term memories is crucial for adaptive behavior and survival in changing environments. The molecular consolidation processes which underlie the formation of these long-term memories are dependent on protein synthesis in excitatory and SST-expressing neurons. A centrally important, parallel process to this involves the removal of the memory constraint quinone reductase 2 (QR2), which has been recently shown to enhance memory consolidation for novel experiences in the cortex and hippocampus, via redox modulation. However, it is unknown within which cell type in the cortex removal of QR2 occurs, nor how this affects neuronal function. Here, we use novel taste learning in the mouse anterior insular cortex (aIC) to show that similarly to mRNA translation, QR2 removal occurs in excitatory and SST-expressing neurons. Interestingly, both novel taste and QR2 inhibition reduce excitability specifically within SST, but not excitatory neurons. Furthermore, reducing QR2 expression in SST, but not in PV or excitatory neurons, is sufficient to enhance taste memory. Thus, QR2 mediated intrinsic property changes of SST interneurons in the aIC is a central removable factor to allow novel taste memory formation. This previously unknown involvement of QR2 and SST interneurons in resetting aIC activity hours following learning, describes a molecular mechanism to define cell circuits for novel information. Therefore, the QR2 pathway in SST interneurons provides a fresh new avenue by which to tackle age-related cognitive deficits, while shedding new light onto the functional machinations of long-term memory formation for novel information.

Many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), lead to the selective degeneration of discrete cell types in the CNS despite the ubiquitous expression of many genes linked to disease. Therapeutic advancement depends on understanding the unique cellular adaptations that underlie pathology of vulnerable cells in the context of disease-causing mutations. Here, we employ bacTRAP molecular profiling to elucidate cell type-specific molecular responses of cortical upper motor neurons in a preclinical ALS model. Using two bacTRAP mouse lines that label distinct vulnerable or resilient projection neuron populations in motor cortex, we show that the regulation of oxidative phosphorylation (Oxphos) pathways is a common response in both cell types. However, differences in the baseline expression of genes involved in Stem and the handling of reactive oxygen species likely lead to the selective degeneration of the vulnerable cells. These results provide a framework to identify cell-type-specific processes in neurodegenerative disease.

Introduction Information processing in cortical cell microcircuits involves regulation of excitatory pyramidal (PYR) cells by inhibitory Somatostatin- (SST), Parvalbumin- (PV), and Vasoactive intestinal peptide- (VIP) expressing interneurons. Human post-mortem and rodent studies show impaired PYR-cell dendritic morphology and decreased SST-cell markers in MDD or after chronic stress. However, knowledge of coordinated changes across microcircuit cell-types is virtually absent. Methods We investigated the transcriptomic effects of unpredictable chronic mild stress (UCMS) on distinct microcircuit cell-types in the medial prefrontal cortex (Cingulate regions 24a/b and 32) in mice. C57Bl/6 mice, exposed to UCMS or control housing for five weeks, were assessed for anxiety- and depressive-like behaviors. Microcircuit cell-types were laser-microdissected and processed for RNA-sequencing. Results UCMS induced predicted elevations in behavioral emotionality in mice. DESeq2 analysis revealed unique differentially-expressed genes in each cell-type after UCMS. Pre-synaptic functions, oxidative stress response, metabolism, and translational regulation were differentially dysregulated across cell-types, whereas nearly all cell-types showed downregulated post-synaptic gene signatures. Across the cortical microcircuit, we observed a shift from a distributed transcriptomic coordination across cell-types in controls towards UCMS-induced increased coordination between PYR-, SST- and PV-cells, and hub-like role for PYR-cells. Lastly, we identified a microcircuit-wide coexpression network enriched in synaptic, bioenergetic, and oxidative stress response genes that correlated with UCMS-induced behaviors. Conclusions These findings suggest cell-specific deficits, microcircuit-wide synaptic reorganization, and a shift in cells regulating the cortical excitation-inhibition balance, suggesting increased coordinated regulation of PYR-cells by SST- and PV-cells.

Experience-dependent plasticity of synapses modulates information processing in neural circuits and is essential for cognitive functions. The genome, via non-coding enhancers, was proposed to control information processing and circuit plasticity by regulating experience-induced transcription of genes that modulate specific sets of synapses. To test this idea, we analyze here the cellular and circuit functions of the genomic mechanisms that control the experience-induced transcription of Igf1 (insulin-like growth factor 1) in vasoactive intestinal peptide (VIP) interneurons (INs) in the visual cortex of adult mice. We find that two sensory-induced enhancers selectively and cooperatively drive the activity-induced transcription of Igf1 to thereby promote GABAergic inputs onto VIP INs and to homeostatically control the ratio between excitation and inhibition (E/I ratio)-in turn, this restricts neural activity in VIP INs and principal excitatory neurons and maintains spatial frequency tuning. Thus, enhancer-mediated activity-induced transcription maintains sensory processing in the adult cortex via homeostatic modulation of E/I ratio.

Schizophrenia is one of the most widespread and complex mental disorders. To characterize the impact of schizophrenia, we performed single-nucleus RNA sequencing (snRNA-seq) of >220,000 neurons from the dorsolateral prefrontal cortex of patients with schizophrenia and matched controls. In addition, >115,000 neurons were analyzed topographically by immunohistochemistry. Compositional analysis of snRNA-seq data revealed a reduction in abundance of GABAergic neurons and a concomitant increase in principal neurons, most pronounced for upper cortical layer subtypes, which was substantiated by histological analysis. Many neuronal subtypes showed extensive transcriptomic changes, the most marked in upper-layer GABAergic neurons, including down-regulation in energy metabolism and up-regulation in neurotransmission. Transcription factor network analysis demonstrated a developmental origin of transcriptomic changes. Last, Visium spatial transcriptomics further corroborated upper-layer neuron vulnerability in schizophrenia. Overall, our results point toward general network impairment within upper cortical layers as a core substrate associated with schizophrenia symptomatology.

Neuropathic pain affects up to 10% of the total population and no specific target is ideal for therapeutic need. The sodium leak channel (NALCN), a non-selective cation channel, mediates the background Na+ leak conductance and controls neuronal excitability and rhythmic behaviors. Here, we show that increases of NALCN expression and function in dorsal root ganglion (DRG) and dorsal spinal cord contribute to chronic constriction injury (CCI)-induced neuropathic pain in rodents. NALCN current and neuronal excitability in acutely isolated DRG neurons and spinal cord slices of rats were increased after CCI which were decreased to normal levels by NALCN-siRNA. Accordingly, pain-related symptoms were significantly alleviated by NALCN-siRNA-mediated NALCN knockdown and completely prevented by NALCN-shRNA-mediated NALCN knockdown in rats or by conditional NALCN knockout in mice. Our results indicate that increases in NALCN expression and function contribute to CCI-induced neuronal sensitization; therefore, NALCN may be a novel molecular target for control of neuropathic pain.

In mammals, the hypothalamic suprachiasmatic nucleus (SCN) functions as the central circadian pacemaker, orchestrating behavioral and physiological rhythms in alignment to the environmental light/dark cycle. The neurons that comprise the SCN are anatomically and functionally heterogeneous, but despite their physiological importance, little is known about the pathways that guide their specification and differentiation. Here, we report that the stem/progenitor cell transcription factor, Sex determining region Y-box 2 (Sox2), is required in the embryonic SCN to control the expression of SCN-enriched neuropeptides and transcription factors. Ablation of Sox2 in the developing SCN leads to downregulation of circadian neuropeptides as early as embryonic day (E) 15.5, followed by a decrease in the expression of two transcription factors involved in SCN development, Lhx1 and Six6, in neonates. Thymidine analog-retention assays revealed that Sox2 deficiency contributed to reduced survival of SCN neurons during the postnatal period of cell clearance, but did not affect progenitor cell proliferation or SCN specification. Our results identify SOX2 as an essential transcription factor for the proper differentiation and survival of neurons within the developing SCN.