Probes for INS

Dravet syndrome (DS) is a form of epilepsy with a high incidence of sudden unexpected death in epilepsy (SUDEP). Respiratory failure is a leading cause of SUDEP, and DS patients' frequently exhibit disordered breathing. Despite this, mechanisms underlying respiratory dysfunction in DS are unknown. We found that mice expressing a DS-associated Scn1a missense mutation (A1783V) conditionally in inhibitory neurons (Slc32a1cre/+::Scn1aA1783V fl/+; defined as Scn1aΔE26) exhibit spontaneous seizures, die prematurely and present a respiratory phenotype including hypoventilation, apnea, and a diminished ventilatory response to CO2. At the cellular level in the retrotrapezoid nucleus (RTN), we found inhibitory neurons expressing the Scn1a A1783V variant are less excitable, whereas glutamatergic chemosensitive RTN neurons, which are a key source of the CO2/H+-dependent drive to breathe, are hyper-excitable in slices from Scn1aΔE26 mice. These results show loss of Scn1a function can disrupt respiratory control at the cellular and whole animal levels.

Voluntary urination ensures that waste is eliminated when safe and socially appropriate, even without a pressing urge. Uncontrolled urination, or incontinence, is a common problem with few treatment options. Normal urine release requires a small region in the brainstem known as Barrington's nucleus (Bar), but specific neurons that relax the urethral sphincter and enable urine flow are unknown. Here we identify a small subset of Bar neurons that control the urethral sphincter in mice. These excitatory neurons express estrogen receptor 1 (BarESR1), project to sphincter-relaxing interneurons in the spinal cord and are active during natural urination. Optogenetic stimulation of BarESR1 neurons rapidly initiates sphincter bursting and efficient voiding in anesthetized and behaving animals. Conversely, optogenetic and chemogenetic inhibition reveals their necessity in motivated urination behavior. The identification of these cells provides an expanded model for the control of urination and its dysfunction.

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.

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope™ revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting Ih and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.

Ventral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2+ neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2+ neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure.