Single Cell Scale Neuronal and Glial Gene Expression and Putative Cell Phenotypes and Networks in the Nucleus Tractus Solitarius in an Alcohol Withdrawal Time Series

Alcohol withdrawal syndrome (AWS) is characterized by neuronal hyperexcitability, autonomic dysregulation, and severe negative emotion. The nucleus tractus solitarius (NTS) likely plays a prominent role in the neurological processes underlying these symptoms as it is the main viscerosensory nucleus in the brain. The NTS receives visceral interoceptive inputs, influences autonomic outputs, and has strong connections to the limbic system and hypothalamic-pituitary-adrenal axis to maintain homeostasis. Our prior analysis of single neuronal gene expression data from the NTS shows that neurons exist in heterogeneous transcriptional states that form distinct functional subphenotypes. Our working model conjectures that the allostasis secondary to alcohol dependence causes peripheral and central biological network decompensation in acute abstinence resulting in neurovisceral feedback to the NTS that substantially contributes to the observed AWS. We collected single noradrenergic and glucagon-like peptide-1 (GLP-1) neurons and microglia from rat NTS and measured a subset of their transcriptome as pooled samples in an alcohol withdrawal time series. Inflammatory subphenotypes predominate at certain time points, and GLP-1 subphenotypes demonstrated hyperexcitability post-withdrawal. We hypothesize such inflammatory and anxiogenic signaling contributes to alcohol dependence via negative reinforcement. Targets to mitigate such dysregulation and treat dependence can be identified from this dataset.

Prostaglandin E2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 channels

Background Cough hypersensitivity is a major characteristic feature associated with several types of cough, including chronic cough, but its underlying mechanisms remain to be fully understood. Inflammatory mediators, such as prostaglandin E2 (PGE2), have been implicated in both peripheral induction and sensitization of the cough reflex. In this study, using a conscious guinea pig model of cough, we investigated whether PGE2 can sensitize the cough reflex via central actions and, if so, via which mechanisms. Methods All drugs were administered by intracerebroventricular (i.c.v.) route and whole-body plethysmograph set-up was used for both induction, using aerosolized citric acid (0.2 M), and recording of cough. Immunohistochemistry was performed to confirm the expression of NaV 1.8 channels in the nucleus tractus solitarius (nTS). Results We show that both PGE2 and the non-selective EP1/EP3 agonist, sulprostone, dose-dependently enhanced the citric acid-induced cough (P ≤ 0.001, P ≤ 0.01, respectively). Pretreatment with the EP1 antagonist, ONO-8130, did not affect the sulprostone-induced cough sensitization, whilst the EP3 antagonist, L-798,106, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, treatment with either the EP2 agonist, butaprost or the EP4 agonist, L-902,688, had no effect on cough sensitization. Additionally, pretreatment with either the TRPV1 antagonist, JNJ-17203212 or the TRPA1 antagonist, HC-030031, alone or in combination, nor with the NaV 1.1, 1.2, 1.3, 1.4, 1.6 and 1.7 channel blocker, tetrodotoxin, had any effect on the cough. In contrast, pretreatment with the NaV 1.8 antagonist, A-803467, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, NaV 1.8 channels were shown to be expressed in the nTS. Conclusion Collectively, our findings show that PGE2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 but independently of TRPV1,TRPA1 and TTX-sensitive sodium channel activation. These results indicate that PGE2 plays an important role in central sensitization of the cough reflex and suggest that central EP3 receptors and/or NaVv 1.8 channels may represent novel antitussive molecular targets. Graphical Abstract

Fibroblast Growth Factor 19 Increases the Excitability of Pre-Motor Glutamatergic Dorsal Vagal Complex Neurons From Hyperglycemic Mice

Intracerebroventricular administration of the protein hormone fibroblast growth factor 19 (FGF19) to the hindbrain produces potent antidiabetic effects in hyperglycemic mice that are likely mediated through a vagal parasympathetic mechanism. FGF19 increases the synaptic excitability of parasympathetic motor neurons in the dorsal motor nucleus of the vagus (DMV) from hyperglycemic, but not normoglycemic, mice but the source of this synaptic input is unknown. Neurons in the area postrema (AP) and nucleus tractus solitarius (NTS) express high levels of FGF receptors and exert glutamatergic control over the DMV. This study tested the hypothesis that FGF19 increases glutamate release in the DMV by increasing the activity of glutamatergic AP and NTS neurons in hyperglycemic mice. Glutamate photoactivation experiments confirmed that FGF19 increases synaptic glutamate release from AP and NTS neurons that connect to the DMV in hyperglycemic, but not normoglycemic mice. Contrary to expectations, FGF19 produced a mixed effect on intrinsic membrane properties in the NTS with a trend towards inhibition, suggesting that another mechanism was responsible for the observed effects on glutamate release in the DMV. Consistent with the hypothesis, FGF19 increased action potential-dependent glutamate release in the NTS in hyperglycemic mice only. Finally, glutamate photoactivation experiments confirmed that FGF19 increases the activity of glutamatergic AP neurons that project to the NTS in hyperglycemic mice. Together, these results support the hypothesis that FGF19 increases glutamate release from AP and NTS neurons that project to the DMV in hyperglycemic mice. FGF19 therefore modifies the local vago-vagal reflex circuitry at several points. Additionally, since the AP and NTS communicate with several other metabolic regulatory nuclei in the brain, FGF19 in the hindbrain may alter neuroendocrine and behavioral aspects of metabolism, in addition to changes in parasympathetic output.

Hindbrain Administration of Oxytocin Reduces Food Intake, Weight Gain and Activates Catecholamine Neurons in the Hindbrain Nucleus of the Solitary Tract in Rats

Existing studies show that CNS oxytocin (OT) signaling is important in the control of energy balance, but it is unclear which neurons may contribute to these effects. Our goals were to examine (1) the dose-response effects of acute OT administration into the third (3V; forebrain) and fourth (4V; hindbrain) ventricles to assess sensitivity to OT in forebrain and hindbrain sites, (2) the extent to which chronic 4V administration of OT reduces weight gain associated with the progression of diet-induced obesity, and (3) whether nucleus tractus solitarius (NTS) catecholamine neurons are downstream targets of 4V OT. Initially, we examined the dose-response effects of 3V and 4V OT (0.04, 0.2, 1, or 5 μg). 3V and 4V OT (5 μg) suppressed 0.5-h food intake by 71.7 ± 6.0% and 60 ± 12.9%, respectively. 4V OT (0.04, 0.2, 1 μg) reduced food intake by 30.9 ± 12.9, 42.1 ± 9.4, and 56.4 ± 9.0%, respectively, whereas 3V administration of OT (1 μg) was only effective at reducing 0.5-h food intake by 38.3 ± 10.9%. We subsequently found that chronic 4V OT infusion, as with chronic 3V infusion, reduced body weight gain (specific to fat mass) and tended to reduce plasma leptin in high-fat diet (HFD)-fed rats, in part, through a reduction in energy intake. Lastly, we determined that 4V OT increased the number of hindbrain caudal NTS Fos (+) neurons (156 ± 25) relative to vehicle (12 ± 3). The 4V OT also induced Fos in tyrosine hydroxylase (TH; marker of catecholamine neurons) (+) neurons (25 ± 7%) relative to vehicle (0.8 ± 0.3%). Collectively, these findings support the hypothesis that OT within the hindbrain is effective at reducing food intake, weight gain, and adiposity and that NTS catecholamine neurons in addition to non-catecholaminergic neurons are downstream targets of CNS OT.

The Carotid Body Detects Circulating Tumor Necrosis Factor-Alpha to Activate a Sympathetic Anti-Inflammatory Reflex

Recent evidence has suggested that the carotid bodies might act as immunological sensors, detecting pro-inflammatory mediators and signalling to the central nervous system, which, in turn, orchestrates autonomic responses. Here, we demonstrated that the TNF-α receptor type I is expressed in the carotid bodies of rats. The systemic administration of TNF-α increased carotid body afferent discharge and activated glutamatergic neurons in the nucleus tractus solitarius (NTS) that project to the rostral ventrolateral medulla (RVLM), where the majority of pre-sympathetic neurons reside. The activation of these neurons was accompanied by generalized activation of the sympathetic nervous system. Carotid body ablation blunted the TNF-α-induced activation of RVLM-projecting NTS neurons and the increase in splanchnic sympathetic nerve activity. Finally, plasma and spleen levels of cytokines after TNF-α administration were higher in rats subjected to either carotid body ablation or splanchnic sympathetic denervation. Collectively, our findings indicate that the carotid body detects circulating TNF-α to activate a counteracting sympathetic anti-inflammatory mechanism.

MRI Patterns Distinguish AQP4 Antibody Positive Neuromyelitis Optica Spectrum Disorder From Multiple Sclerosis

Neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) are inflammatory diseases of the CNS. Overlap in the clinical and MRI features of NMOSD and MS means that distinguishing these conditions can be difficult. With the aim of evaluating the diagnostic utility of MRI features in distinguishing NMOSD from MS, we have conducted a cross-sectional analysis of imaging data and developed predictive models to distinguish the two conditions. NMOSD and MS MRI lesions were identified and defined through a literature search. Aquaporin-4 (AQP4) antibody positive NMOSD cases and age- and sex-matched MS cases were collected. MRI of orbits, brain and spine were reported by at least two blinded reviewers. MRI brain or spine was available for 166/168 (99%) of cases. Longitudinally extensive (OR = 203), “bright spotty” (OR = 93.8), whole (axial; OR = 57.8) or gadolinium (Gd) enhancing (OR = 28.6) spinal cord lesions, bilateral (OR = 31.3) or Gd-enhancing (OR = 15.4) optic nerve lesions, and nucleus tractus solitarius (OR = 19.2), periaqueductal (OR = 16.8) or hypothalamic (OR = 7.2) brain lesions were associated with NMOSD. Ovoid (OR = 0.029), Dawson's fingers (OR = 0.031), pyramidal corpus callosum (OR = 0.058), periventricular (OR = 0.136), temporal lobe (OR = 0.137) and T1 black holes (OR = 0.154) brain lesions were associated with MS. A score-based algorithm and a decision tree determined by machine learning accurately predicted more than 85% of both diagnoses using first available imaging alone. We have confirmed NMOSD and MS specific MRI features and combined these in predictive models that can accurately identify more than 85% of cases as either AQP4 seropositive NMOSD or MS.

NTS Prlh overcomes orexigenic stimuli and ameliorates dietary and genetic forms of obesity

Calcitonin receptor (Calcr)-expressing neurons of the nucleus tractus solitarius (NTS; CalcrNTS cells) contribute to the long-term control of food intake and body weight. Here, we show that Prlh-expressing NTS (PrlhNTS) neurons represent a subset of CalcrNTS cells and that Prlh expression in these cells restrains body weight gain in the face of high fat diet challenge in mice. To understand the relationship of PrlhNTS cells to hypothalamic feeding circuits, we determined the ability of PrlhNTS-mediated signals to overcome enforced activation of AgRP neurons. We found that PrlhNTS neuron activation and Prlh overexpression in PrlhNTS cells abrogates AgRP neuron-driven hyperphagia and ameliorates the obesity of mice deficient in melanocortin signaling or leptin. Thus, enhancing Prlh-mediated neurotransmission from the NTS dampens hypothalamically-driven hyperphagia and obesity, demonstrating that NTS-mediated signals can override the effects of orexigenic hypothalamic signals on long-term energy balance.