Probes for INS

Current understanding of the contribution of C1 neurons to blood pressure (BP) regulation derives predominantly from experiments carried out in anesthetized animals or reduced ex vivo preparations. Here we use ArchaerhodopsinT3.0 (ArchT) loss-of-function optogenetics to explore BP regulation by C1 neurons in intact unanesthetized rats. Using a lentivirus that expresses ArchT under the Phox2b-activated promoter PRSx8 (PRSx8-ArchT), ∼65% of transduced neurons were C1 (balance retrotrapezoid nucleus, RTN). Other rats received CaMKII-ArchT3.0 AAV2 (CaMKII-ArchT) which transduced C1 neurons and larger numbers of unidentified glutamatergic and GABAergic cells.Under anesthesia, ArchT-photoactivation reduced sympathetic nerve activity and BP and silenced/strongly inhibited most (7/12) putative C1 neurons. In unanesthetized PRSx8-ArchT-treated rats breathing room air, bilateral ArchT-photoactivation caused a very small BP reduction that was only slightly larger under hypercapnia (6% FiCO2) but was greatly enhanced during hypoxia (10 and 12% FiO2), after sino-aortic denervation, or during isoflurane anesthesia. Degree of hypotension correlated with percentage of ArchT-transduced C1 cells. ArchT-photoactivation produced similar BP changes in CaMKII-ArchT-treated rats. Photoactivation in PRSX8-ArchT rats reduced breathing frequency (FR); in CamKII-ArchT rats FR increased.We conclude that the BP drop elicited by ArchT activation resulted from C1 inhibition and was unrelated to breathing changes. C1 neurons have low activity under normoxia but their activation is important to BP stability during hypoxia or anesthesia and contributes greatly to the hypertension caused by baroreceptor deafferentation. Finally, C1 neurons are marginally activated by hypercapnia and the large breathing stimulation caused by this stimulus has very little impact on resting BP.SIGNIFICANCE STATEMENTC1 neurons (C1) are glutamatergic/peptidergic/catecholaminergic neurons located in the medulla oblongata, which may operate as a switchboard for differential, behavior-appropriate, activation of selected sympathetic efferents. Based largely on experimentation in anesthetized or reduced preparations, a rostrally-located subset of C1 may contribute to both BP stabilization and dysregulation (hypertension). Here we used Archaerhodopsin-based loss-of-function optogenetics to explore the contribution of these neurons to BP in conscious rats. The results suggest that C1 contributes little to resting BP under normoxia or hypercapnia, C1 discharge is continuously restrained by arterial baroreceptors and C1 activation is critical to stabilize BP under hypoxia or anesthesia. This optogenetic approach could also be useful to explore the role of C1 during specific behaviors or in hypertensive models.

Pain is a multidimensional experience and negative affect, or how much the pain is "bothersome", significantly impacts the sufferers' quality of life. It is well established that the kappa opioid system contributes to depressive and dysphoric states, but whether this system contributes to the negative affect precipitated by the occurrence of chronic pain remains tenuous. Using a model of persistent pain, we show by quantitative RT-PCR, florescence in situ hybridization, western blotting and GTPgS autoradiography an upregulation of expression and the function of kappa opioid receptors (KORs) and its endogenous ligand dynorphin in the mesolimbic circuitry in animals with chronic pain compared to surgical controls. Using in vivo microdialysis and microinjection of drugs into the mesolimbic dopamine system, we demonstrate that inhibiting KORs reinstates evoked dopamine release and reward related behaviors in chronic pain animals. Chronic pain enhanced KOR agonist-induced place aversion in a sex-dependent manner. Using various place preference paradigms, we show that activation of KORs drives pain aversive states in male but not female mice. However, KOR antagonist treatment was effective in alleviating anxiogenic and depressive affective-like behaviors in both sexes. Finally, ablation of KORs from dopamine neurons using AAV-TH-cre in KORloxP mice prevented pain-induced aversive states as measured by place aversion assays. Our results strongly support the use of KOR antagonists as therapeutic adjuvants to alleviate the emotional, tonic-aversive component of chronic pain, which is argued to be the most significant component of the pain experience that impacts patients' quality of life.Significance StatementWe show that KORs are sufficient to drive the tonic-aversive component of chronic pain - the emotional component of pain that is argued to significantly impact a patient's quality of life. The impact of our study is broadly relevant to affective disorders associated with disruption of reward circuitry and thus likely contributes to many of the devastating sequelae of chronic pain, including the poor response to treatment of many patients, debilitating affective disorders (other disorders including anxiety and depression that demonstrate high co-morbidity with chronic pain) and substance abuse. Indeed, co-existing psychopathology increases pain intensity, pain-related disability and effectiveness of treatments (Jamison and Edwards, 2013).

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.