Probes for INS

Acetylcholine is a potent neuromodulator capable of modifying patterns of acoustic information flow. In auditory cortex, cholinergic systems have been shown to increase salience/gain while suppressing extraneous information. However, the mechanism by which cholinergic circuits shape signal processing in the auditory thalamus (medial geniculate body, MGB) is poorly understood. The present study, in male Fischer Brown Norway rats, seeks to determine the location and function of presynaptic neuronal nicotinic acetylcholine receptors (nAChRs) at the major inputs to MGB and characterize how nAChRs change during aging. In vitro electrophysiological/optogenetic methods were used to examine responses of MGB neurons following activation of nAChRs during a paired-pulse paradigm. Presynaptic nAChR activation increased responses evoked by stimulation of excitatory corticothalamic and inhibitory tectothalamic terminals. Conversely, nAChR activation appeared to have little effect on evoked responses from inhibitory thalamic reticular nucleus and excitatory tectothalamic terminals. In situ hybridization data showed nAChR subunit transcripts in GABAergic inferior colliculus neurons and glutamatergic auditory cortical neurons supporting the present slice findings. Responses to nAChR activation at excitatory corticothalamic and inhibitory tectothalamic inputs were diminished by aging. These findings suggest that cholinergic input to the MGB increases the strength of tectothalamic inhibitory projections, potentially improving signal-to-noise ratio and signal detection while increasing corticothalamic gain, which may facilitate top-down identification of stimulus identity. These mechanisms appear negatively affected by aging, potentially diminishing speech perception in noisy environments. Cholinergic inputs to the MGB appear to maximize sensory processing by adjusting both top-down and bottom-up mechanisms in conditions of attention and arousal.Significance StatementThe pedunculopontine tegmental nucleus (PPTg) is the source of cholinergic innervation for sensory thalamus and is a critical part of an ascending arousal system which controls the firing mode of thalamic cells based on attentional demand. The present study describes the location and impact of aging on presynaptic neuronal nicotinic receptors (nAChRs) within the circuitry of the auditory thalamus (medial geniculate body; MGB). We show that nAChRs are located on ascending inhibitory and descending excitatory presynaptic inputs onto MGB neurons, likely selectively increasing gain and improving temporal clarity. In addition, we show that aging has a deleterious effect on nAChR efficacy. Cholinergic dysfunction at the level of MGB may negatively impact speech understanding in the elderly population.

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.

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.

Central leptin action is sufficient to restore euglycemia in insulinopenic type 1 diabetes (T1D); however, the underlying mechanism remains poorly understood. To examine the role of intracellular STAT3 pathways, we used LepRs/s mice with disrupted leptin-pSTAT3 signaling to test the effect of central leptin on euglycemia restoration. These mice developed STZ-induced T1D, which was surprisingly not associated with hyperglucagonemia, a typical manifestation in T1D. Further, leptin action on euglycemia restoration was abrogated in these mice, which was associated with refractory hypercorticosteronemia. To examine the role of fast-acting neurotransmitters glutamate and γ-aminobutyric acid (GABA), two major neurotransmitters in the brain, from LepR neurons, we used mice with disrupted release of glutamate, GABA or both from LepR neurons. Surprisingly, all mice responded normally to leptin-mediated euglycemia restoration, which was associated with expected correction from hyperglucagonemia and hyperphagia. In contrast, mice with loss of glutamate and GABA appeared to develop an additive obesity effect over those with loss of single neurotransmitter release. Thus, our study reveals that STAT3 signaling, but not fast-acting neurotransmitter release, is required for leptin action on euglycemia restoration, and that hyperglucagonemia is not required for T1D.