Brainstem Dbh+ neurons control allergen-induced airway hyperreactivity.

Exaggerated airway constriction triggered by repeated exposure to allergen, also called hyperreactivity, is a hallmark of asthma. Whereas vagal sensory neurons are known to function in allergen-induced hyperreactivity1-3, the identity of downstream nodes remains poorly understood. Here we mapped a full allergen circuit from the lung to the brainstem and back to the lung. Repeated exposure of mice to inhaled allergen activated the nuclei of solitary tract (nTS) neurons in a mast cell-, interleukin-4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA sequencing, followed by RNAscope assay at baseline and allergen challenges, showed that a Dbh+ nTS population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted hyperreactivity whereas chemogenetic activation promoted it. Viral tracing indicated that Dbh+ nTS neurons project to the nucleus ambiguus (NA) and that NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that directly drive airway constriction. Delivery of noradrenaline antagonists to the NA blunted hyperreactivity, suggesting noradrenaline as the transmitter between Dbh+ nTS and NA. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. This knowledge informs how neural modulation could be used to control allergen-induced airway hyperreactivity.

Dissociable hindbrain GLP1R circuits for satiety and aversion.

The most successful obesity therapeutics, glucagon-like peptide-1 receptor (GLP1R) agonists, cause aversive responses such as nausea and vomiting1,2, effects that may contribute to their efficacy. Here, we investigated the brain circuits that link satiety to aversion, and unexpectedly discovered that the neural circuits mediating these effects are functionally separable. Systematic investigation across drug-accessible GLP1R populations revealed that only hindbrain neurons are required for the efficacy of GLP1-based obesity drugs. In vivo two-photon imaging of hindbrain GLP1R neurons demonstrated that most neurons are tuned to either nutritive or aversive stimuli, but not both. Furthermore, simultaneous imaging of hindbrain subregions indicated that area postrema (AP) GLP1R neurons are broadly responsive, whereas nucleus of the solitary tract (NTS) GLP1R neurons are biased towards nutritive stimuli. Strikingly, separate manipulation of these populations demonstrated that activation of NTSGLP1R neurons triggers satiety in the absence of aversion, whereas activation of APGLP1R neurons triggers strong aversion with food intake reduction. Anatomical and behavioural analyses revealed that NTSGLP1R and APGLP1R neurons send projections to different downstream brain regions to drive satiety and aversion, respectively. Importantly, GLP1R agonists reduce food intake even when the aversion pathway is inhibited. Overall, these findings highlight NTSGLP1R neurons as a population that could be selectively targeted to promote weight loss while avoiding the adverse side effects that limit treatment adherence.

Brainstem neuronal responses to transcutaneous auricular and cervical vagus nerve stimulation in rats.

Transcutaneous auricular vagus nerve stimulation (taVNS) targets subcutaneous axons in the auricular branch of the vagus nerve at the outer ear. Its non-invasive nature makes it a potential treatment for various disorders. taVNS induces neuromodulatory effects within the nucleus of the solitary tract (NTS), and due to its widespread connectivity, the NTS acts as a gateway to elicit neuromodulation in both higher-order brain regions and other brainstem nuclei (e.g. spinal trigeminal nucleus; Sp5). Our objective was to examine stimulation parameters on single-neuron electrophysiological responses in α-chloralose-anaesthetized Sprague-Dawley rats within NTS and Sp5. taVNS was also compared to traditional cervical VNS (cVNS) on single neuronal activation. Specifically, electrophysiological extracellular recordings were evaluated for a range of frequency and intensity parameters (20-250 Hz, 0.5-1.0 mA). Neurons were classified as positive, negative or non-responders based on increased activity, decreased activity or no response during stimulation, respectively. Frequency-dependent analysis showed that 20 and 100 Hz generated the highest proportion of positive responders in NTS and Sp5 with 1.0 mA intensities eliciting the greatest magnitude of response. Comparisons between taVNS and cVNS revealed similar parameter-specific activation for caudal NTS neuronal populations; however, individual neurons showed different activation profiles. The latter suggests that cVNS and taVNS send afferent input to NTS via different neuronal pathways. This study demonstrates differential parameter-specific taVNS responses and begins an investigation of the mechanisms responsible for taVNS modulation. Understanding the neuronal pathways responsible for eliciting neuromodulatory effects will enable more tailored taVNS treatments in various clinical disorders. KEY POINTS: Transcutaneous auricular vagus nerve stimulation (taVNS) offers a non-invasive alternative to invasive cervical vagus nerve stimulation (cVNS) by activating vagal afferents in the ear to induce neuromodulation. Our study evaluated taVNS effects on neuronal firing patterns in the nucleus of the solitary tract (NTS) and spinal trigeminal nucleus (Sp5) and found that 20 and 100 Hz notably increased neuronal activity during stimulation in both nuclei. Increasing taVNS intensity not only increased the number of neurons responding in Sp5 but also increased the magnitude of response, suggesting a heightened sensitivity to taVNS compared to NTS. Comparisons between cVNS and taVNS revealed similar overall activation but different responses on individual neurons, indicating distinct neural pathways. These results show parameter-specific and nuclei-specific responses to taVNS and confirm that taVNS can elicit responses comparable to cVNS at the neuronal level, but it does so through different neuronal pathways.

Optical perturbation of Agtr1a-containing neurons and afferents within the caudal nucleus of the solitary tract modulates sodium intake.

Angiotensin-II (Ang-II) production is driven by deviations in blood volume and osmolality, and serves the role of regulating blood pressure and fluid intake to maintain cardiovascular and hydromineral homeostasis. These actions are mediated by Ang-II acting on its type 1a receptor (AT1aR) within the central nervous system and periphery. Of relevance, AT1aR are expressed on sensory afferents responsible for conveying cardiovascular information to the nucleus of the solitary tract (NTS). We have previously determined that optical excitation of neurons and vagal afferents within the NTS that express AT1aR (referred to as NTSAT1aR) mimics the perception of increased vascular stretch and induces compensatory responses to restore blood pressure. Here, we test whether NTSAT1aR are also involved in the modulation of water and sodium intake. We directed the light-sensitive excitatory channelrhodopsin-2 (ChR2) or inhibitory halorhodopsin (Halo) to Agtr1a-containing neurons and measured water and sodium chloride (NaCl) intake in the presence and absence of optical stimulation within the NTS during various challenges to fluid homeostasis. Optical perturbation of NTSAT1aR modulates NaCl intake, such that excitation attenuates, whereas inhibition increases intake. This effect is only observed in the water-deprived condition, suggesting that NTSAT1aR are involved in the regulation of sodium intake during an imbalance in both the intracellular and extracellular fluid compartments. Furthermore, optical excitation of NTSAT1aR increases c-Fos expression within oxytocinergic neurons of the paraventricular nucleus of the hypothalamus (PVN), indicating that the regulation of sodium intake by NTSAT1aR may be mediated by oxytocin. Collectively, these results reveal that NTSAT1aR are sufficient and necessary to modulate sodium intake relative to perceived changes in vascular stretch.

Mapping of afferent and efferent connections of phenylethanolamine N-methyltransferase-expressing neurons in the nucleus tractus solitarii.

OBJECTIVE: Phenylethanolamine N-methyltransferase (PNMT)-expressing neurons in the nucleus tractus solitarii (NTS) contribute to the regulation of autonomic functions. However, the neural circuits linking these neurons to other brain regions remain unclear. This study aims to investigate the connectivity mechanisms of the PNMT-expressing neurons in the NTS (NTSPNMT neurons). METHODS: The methodologies employed in this study included a modified rabies virus-based retrograde neural tracing technique, conventional viral anterograde tracing, and immunohistochemical staining procedures. RESULTS: A total of 43 upstream nuclei projecting to NTSPNMT neurons were identified, spanning several key brain regions including the medulla oblongata, pons, midbrain, cerebellum, diencephalon, and telencephalon. Notably, dense projections to the NTSPNMT neurons were observed from the central amygdaloid nucleus, paraventricular nucleus of the hypothalamus, area postrema, and the gigantocellular reticular nucleus. In contrast, the ventrolateral medulla, lateral parabrachial nucleus, and lateral hypothalamic area were identified as the primary destinations for axon terminals originating from NTSPNMT neurons. Additionally, reciprocal projections were evident among 21 nuclei, primarily situated within the medulla oblongata. CONCLUSION: Our research findings demonstrate that NTSPNMT neurons form extensive connections with numerous nuclei, emphasizing their essential role in the homeostatic regulation of vital autonomic functions.

Acid-sensing ion channel 1 in nucleus tractus solitarii neurons contributes to the enhanced CO2-stimulated cardiorespiratory effect in spontaneously hypertensive rats.

AIMS: Activation of central respiratory chemoreceptors provides excitatory drive to both respiratory and sympathetic outputs. The enhanced respiratory-sympathetic coupling contributes to the onset and development of hypertension. However, the specific central targets and molecular mechanisms involved in this process remain elusive. This study aimed to investigate the role of acid-sensing ion channel 1 (ASIC1) in nucleus tractus solitarii (NTS) neurons in CO2-stimulated cardiorespiratory effects in spontaneously hypertensive rats (SHRs). MAIN METHODS: Respiration and blood pressure of conscious rats were recorded by whole-body plethysmography and telemetry, respectively. Western blot was used to detect the expression difference of ASIC1 protein in NTS region between Wistar-Kyoto (WKY) rats and SHRs. Excitability of NTS neurons were assessed by extracellular recordings. KEY FINDINGS: Compared to WKY rats, the enhanced CO2-stimulated cardiopulmonary effect and up-regulation of ASIC1 in the NTS were already observed in 4-week-old prehypertensive SHRs. Furthermore, specific blockade of ASIC1 effectively attenuated the CO2-stimulated increase in firing rate of NTS neurons in anesthetized adult SHRs. Intracerebroventricular injections of the ASIC1a blocker PcTx1 or knockdown Asic1 in NTS neurons significantly reduced the heightened CO2-stimulated ventilatory response, and diminished the CO2-stimulated increase in arterial pressure and heart rate in adult SHRs. SIGNIFICANCE: These findings showed that dysregulated ASIC1 signaling in the NTS contribute to the exaggerated CO2-stimulated cardiorespiratory effects observed in SHRs.

Orexin Facilitates the Peripheral Chemoreflex via Corticotropin-Releasing Hormone Neurons Projecting to the Nucleus of the Solitary Tract.

We previously showed that orexin neurons are activated by hypoxia and facilitate the peripheral chemoreflex (PCR)-mediated hypoxic ventilatory response (HVR), mostly by promoting the respiratory frequency response. Orexin neurons project to the nucleus of the solitary tract (nTS) and the paraventricular nucleus of the hypothalamus (PVN). The PVN contributes significantly to the PCR and contains nTS-projecting corticotropin-releasing hormone (CRH) neurons. We hypothesized that in male rats, orexin neurons contribute to the PCR by activating nTS-projecting CRH neurons. We used neuronal tract tracing and immunohistochemistry (IHC) to quantify the degree that hypoxia activates PVN-projecting orexin neurons. We coupled this with orexin receptor (OxR) blockade with suvorexant (Suvo, 20 mg/kg, i.p.) to assess the degree that orexin facilitates the hypoxia-induced activation of CRH neurons in the PVN, including those projecting to the nTS. In separate groups of rats, we measured the PCR following systemic orexin 1 receptor (Ox1R) blockade (SB-334867; 1 mg/kg) and specific Ox1R knockdown in PVN. OxR blockade with Suvo reduced the number of nTS and PVN neurons activated by hypoxia, including those CRH neurons projecting to nTS. Hypoxia increased the number of activated PVN-projecting orexin neurons but had no effect on the number of activated nTS-projecting orexin neurons. Global Ox1R blockade and partial Ox1R knockdown in the PVN significantly reduced the PCR. Ox1R knockdown also reduced the number of activated PVN neurons and the number of activated tyrosine hydroxylase neurons in the nTS. Our findings suggest orexin facilitates the PCR via nTS-projecting CRH neurons expressing Ox1R.

Populations of Hindbrain Glucagon-Like Peptide 1 (GLP1) Neurons That Innervate the Hypothalamic PVH, Thalamic PVT, or Limbic Forebrain BST Have Axon Collaterals That Reach All Central Regions Innervated by GLP1 Neurons.

Neurons in the caudal nucleus of the solitary tract (cNTS) and intermediate reticular nucleus (IRt) that express the glucagon gene (Gcg) give rise to glucagon-like peptide 1 (GLP1)-immunopositive axons in the spinal cord and many subcortical brain regions. Central GLP1 receptor signaling contributes to motivated behavior and stress responses in rats and mice, in which hindbrain GLP1 neurons are activated to express c-Fos in a metabolic state-dependent manner. The present study examined whether GLP1 inputs to distinct brain regions arise from distinct subsets of Gcg-expressing neurons, and mapped the distribution of axon collaterals arising from projection-defined GLP1 neural populations. Using our Gcg-Cre knock-in rat model, Cre-dependent adeno-associated virus (AAV) tracing was conducted in adult male and female rats to compare axonal projections of IRt versus cNTS GLP1 neurons. Overlapping projections were observed in all brain regions that receive GLP1 input, with the caveat that cNTS injections produced Cre-dependent labeling of some IRt neurons, and vice versa. In additional experiments, specific diencephalic or limbic forebrain nuclei were microinjected with Cre-dependent retrograde AAVs (AAVrg) that expressed reporters to fully label the axon collaterals of transduced GLP1 neurons. AAVrg injected into each forebrain site labeled Gcg-expressing neurons in both the cNTS and IRt. The collective axon collaterals of labeled neurons entered the spinal cord and every brain region previously reported to contain GLP1-positive axons. These results indicate that the axons of GLP1 neural populations that innervate the thalamic paraventricular nucleus, paraventricular nucleus of the hypothalamus, and/or bed nucleus of the stria terminalis collectively innervate all central regions that receive GLP1 axonal input.

Oxytocin and corticotropin-releasing hormone exaggerate nucleus tractus solitarii neuronal and synaptic activity following chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) in rodents mimics the hypoxia-induced elevation of blood pressure seen in individuals experiencing episodic breathing. The brainstem nucleus tractus solitarii (nTS) is the first site of visceral sensory afferent integration, and thus is critical for cardiorespiratory homeostasis and its adaptation during a variety of stressors. In addition, the paraventricular nucleus of the hypothalamus (PVN), in part through its nTS projections that contain oxytocin (OT) and/or corticotropin-releasing hormone (CRH), contributes to cardiorespiratory regulation. Within the nTS, these PVN-derived neuropeptides alter nTS activity and the cardiorespiratory response to hypoxia. Nevertheless, their contribution to nTS activity after CIH is not fully understood. We hypothesized that OT and CRH would increase nTS activity to a greater extent following CIH, and co-activation of OT+CRH receptors would further magnify nTS activity. Our data show that compared to their normoxic controls, 10 days' CIH exaggerated nTS discharge, excitatory synaptic currents and Ca2+ influx in response to CRH, which were further enhanced by the addition of OT. CIH increased the tonic functional contribution of CRH receptors, which occurred with elevation of mRNA and protein. Together, our data demonstrate that intermittent hypoxia exaggerates the expression and function of neuropeptides on nTS activity. KEY POINTS: Episodic breathing and chronic intermittent hypoxia (CIH) are associated with autonomic dysregulation, including elevated sympathetic nervous system activity. Altered nucleus tractus solitarii (nTS) activity contributes to this response. Neurons originating in the paraventricular nucleus (PVN), including those containing oxytocin (OT) and corticotropin-releasing hormone (CRH), project to the nTS, and modulate the cardiorespiratory system. Their role in CIH is unknown. In this study, we focused on OT and CRH individually and together on nTS activity from rats exposed to either CIH or normoxia control. We show that after CIH, CRH alone and with OT increased to a greater extent overall nTS discharge, neuronal calcium influx, synaptic transmission to second-order nTS neurons, and OT and CRH receptor expression. These results provide insights into the underlying circuits and mechanisms contributing to autonomic dysfunction during periods of episodic breathing.

Swallowing-like activity elicited in neonatal rat medullary slice preparation.

Swallowing is induced by a central pattern generator in the nucleus tractus solitarius (NTS). We aimed to create a medullary slice preparation to elucidate the neural architecture of the central pattern generator of swallowing (Sw-CPG) and record its neural activities. Experiments were conducted on 2-day-old Sprague-Dawley rats (n = 46). The brainstem-spinal cord was transected at the pontomedullary and cervicothoracic junctions; the medulla was sliced transversely at thicknesses of 600, 700, or 800 µm. The rostral end of the slice was 100 µm rostral to the vagus nerve. We recorded hypoglossal nerve activity and electrically stimulated the vagus nerve or microinjected bicuculline methiodide (BIC) into the NTS. The 800-µm slices generated both rhythmic respiratory activity and electrically elicited neural activity. The 700-µm slices generated only respiratory activity, while the 600-µm slices did not generate any neural activity. BIC microinjection into the NTS in 800-µm slices resulted in the typical activity that closely resembled the swallowing activity reported in other experiments. This swallowing-like activity consistently lengthened the respiratory interval. Despite complete inhibition of respiratory activity, weak swallowing-like activity was observed under bath application of a non-NMDA receptor antagonist. Contrastingly, bath application of NMDA receptor antagonists resulted in a complete loss of swallowing-like activity and no change in respiratory activity. These results suggest that the 800-µm medullary slice preparation contains both afferent and efferent neural circuits and pattern generators of swallowing activity. Additionally, NMDA receptors may be necessary for generating swallowing activity. This medullary slice preparation can therefore elucidate Sw-CPG neural networks.

SubSol-HIe is an AMPK-dependent hypoxia-responsive subnucleus of the nucleus tractus solitarius that coordinates the hypoxic ventilatory response and protects against apnoea in mice.

Functional magnetic resonance imaging (fMRI) suggests that the hypoxic ventilatory response is facilitated by the AMP-activated protein kinase (AMPK), not at the carotid bodies, but within a subnucleus (Bregma -7.5 to -7.1 mm) of the nucleus tractus solitarius that exhibits right-sided bilateral asymmetry. Here, we map this subnucleus using cFos expression as a surrogate for neuronal activation and mice in which the genes encoding the AMPK-α1 (Prkaa1) and AMPK-α2 (Prkaa2) catalytic subunits were deleted in catecholaminergic cells by Cre expression via the tyrosine hydroxylase promoter. Comparative analysis of brainstem sections, relative to controls, revealed that AMPK-α1/α2 deletion inhibited, with right-sided bilateral asymmetry, cFos expression in and thus activation of a neuronal cluster that partially spanned three interconnected anatomical nuclei adjacent to the area postrema: SolDL (Bregma -7.44 mm to -7.48 mm), SolDM (Bregma -7.44 mm to -7.48 mm) and SubP (Bregma -7.48 mm to -7.56 mm). This approximates the volume identified by fMRI. Moreover, these nuclei are known to be in receipt of carotid body afferent inputs, and catecholaminergic neurons of SubP and SolDL innervate aspects of the ventrolateral medulla responsible for respiratory rhythmogenesis. Accordingly, AMPK-α1/α2 deletion attenuated hypoxia-evoked increases in minute ventilation (normalised to metabolism), reductions in expiration time, and increases sigh frequency, but increased apnoea frequency during hypoxia. The metabolic response to hypoxia in AMPK-α1/α2 knockout mice and the brainstem and spinal cord catecholamine levels were equivalent to controls. We conclude that within the brainstem an AMPK-dependent, hypoxia-responsive subnucleus partially spans SubP, SolDM and SolDL, namely SubSol-HIe, and is critical to coordination of active expiration, the hypoxic ventilatory response and defence against apnoea.

Baroreflex afferent function is a part of insights of Leptin-mediated blood pressure reduction and Leptin-resistance hypertension.

The aim of this study is to verify the impact of Leptin in blood pressure (BP) regulation and Leptin-resistance in metabolic/neurogenic hypertension through baroreflex afferents and dysregulation. Artery BP/heart rate (HR) were measured while nodose (NG) microinjection of Leptin, membrane depolarization/inward current were obtained by whole-cell patch from NG neurons isolated from adult female rats. Baroreflex sensitivity (BRS) tested with PE/SNP, distribution/expression of Leptin/receptors in the NG/nucleus tractus solitary (NTS) examined using immumostaining and qRT-PCR, and serum concentrations of Leptin/NE measured by ELISA were observed in control and high fructose-drinking induced hypertension (HTN-HFD) rats. The results showed that BP was significantly/dose-dependently reduced by Leptin NG microinjection likely through direct excitation of female-specific subpopulation of Ah-type neurons showing a potent membrane depolarization/inward currents. Sex-specific distribution/expression of OB-Ra/OB-Rb in the NG were detected with estrogen-dependent manner, similar observations were also confirmed in the NTS. As expected, BRS was dramatically decreased in the presence of PE/SNP in both male and female rats except for the female with PE at given concentrations. Additionally, serum concentration of Leptin was elevated in HFD-HTN model rats of either sex with more obvious in females. Under hypertensive condition, the mean fluorescent density of OB-R and mRNA expression for OB-Ra/OB-Rb in the NG/NTS were significantly down-regulated. These results have demonstrated that Leptin play a role in dominant parasympathetic drive via baroreflex afferent activation to buffer Leptin-mediated sympathetic activation systemically and Leptin-resistance is an innegligible mechanism for metabolic/neurogenic hypertension through baroreflex afferent dysregulation.

A single-nuclei paired multiomic analysis of the human midbrain reveals age- and Parkinson's disease-associated glial changes.

Age is the primary risk factor for Parkinson's disease (PD), but how aging changes the expression and regulatory landscape of the brain remains unclear. Here we present a single-nuclei multiomic study profiling shared gene expression and chromatin accessibility of young, aged and PD postmortem midbrain samples. Combined multiomic analysis along a pseudopathogenesis trajectory reveals that all glial cell types are affected by age, but microglia and oligodendrocytes are further altered in PD. We present evidence for a disease-associated oligodendrocyte subtype and identify genes lost over the aging and disease process, including CARNS1, that may predispose healthy cells to develop a disease-associated phenotype. Surprisingly, we found that chromatin accessibility changed little over aging or PD within the same cell types. Peak-gene association patterns, however, are substantially altered during aging and PD, identifying cell-type-specific chromosomal loci that contain PD-associated single-nucleotide polymorphisms. Our study suggests a previously undescribed role for oligodendrocytes in aging and PD.

Control of breathing by orexinergic signaling in the nucleus tractus solitarii.

Orexin signaling plays a facilitatory role in respiration. Abnormalities in orexin levels correlate with disordered breathing patterns and impaired central respiratory chemoreception. Nucleus tractus solitarii (NTS) neurons expressing the transcription factor Phox2b contribute to the chemoreceptive regulation of respiration. However, the extent to which orexinergic signaling modulates respiratory activity in these Phox2b-expressing NTS neurons remains unclear. In the present study, the injection of orexin A into the NTS significantly increased the firing rate of the phrenic nerve. Further analysis using fluorescence in situ hybridization and immunohistochemistry revealed that orexin 1 receptors (OX1Rs) were primarily located in the ventrolateral subdivision of the NTS and expressed in 25% of Phox2b-expressing neurons. Additionally, electrophysiological recordings showed that exposure to orexin A increased the spontaneous firing rate of Phox2b-expressing neurons. Immunostaining experiments with cFos revealed that the OX1R-residing Phox2b-expressing neurons were activated by an 8% CO2 stimulus. Crucially, OX1R knockdown in these NTS neurons notably blunted the ventilatory response to 8% CO2, alongside an increase in sigh-related apneas. In conclusion, orexinergic signaling in the NTS facilitates breathing through the activation of OX1Rs, which induces the depolarization of Phox2b-expressing neurons. OX1Rs are essential for the involvement of Phox2b-expressing NTS neurons in the hypercapnic ventilatory response.

Single-nucleus RNA velocity reveals critical synaptic and cell-cycle dysregulations in neuropathologically confirmed Alzheimer's disease.

Typical differential single-nucleus gene expression (snRNA-seq) analyses in Alzheimer's disease (AD) provide fixed snapshots of cellular alterations, making the accurate detection of temporal cell changes challenging. To characterize the dynamic cellular and transcriptomic differences in AD neuropathology, we apply the novel concept of RNA velocity to the study of single-nucleus RNA from the cortex of 60 subjects with varied levels of AD pathology. RNA velocity captures the rate of change of gene expression by comparing intronic and exonic sequence counts. We performed differential analyses to find the significant genes driving both cell type-specific RNA velocity and expression differences in AD, extensively compared these two transcriptomic metrics, and clarified their associations with multiple neuropathologic traits. The results were cross-validated in an independent dataset. Comparison of AD pathology-associated RNA velocity with parallel gene expression differences reveals sets of genes and molecular pathways that underlie the dynamic and static regimes of cell type-specific dysregulations underlying the disease. Differential RNA velocity and its linked progressive neuropathology point to significant dysregulations in synaptic organization and cell development across cell types. Notably, most of the genes underlying this synaptic dysregulation showed increased RNA velocity in AD subjects compared to controls. Accelerated cell changes were also observed in the AD subjects, suggesting that the precocious depletion of precursor cell pools might be associated with neurodegeneration. Overall, this study uncovers active molecular drivers of the spatiotemporal alterations in AD and offers novel insights towards gene- and cell-centric therapeutic strategies accounting for dynamic cell perturbations and synaptic disruptions.

GFRAL Is Widely Distributed in the Brain and Peripheral Tissues of Mice.

In 2017, four independent publications described the glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as receptor for the growth differentiation factor 15 (GDF15, also MIC-1, NAG-1) with an expression exclusively in the mice brainstem area postrema (AP) and nucleus tractus solitarii (NTS) where it mediates effects of GDF15 on reduction of food intake and body weight. GDF15 is a cell stress cytokine with a widespread expression and pleiotropic effects, which both seem to be in contrast to the reported highly specialized localization of its receptor. This discrepancy prompts us to re-evaluate the expression pattern of GFRAL in the brain and peripheral tissues of mice. In this detailed immunohistochemical study, we provide evidence for a more widespread distribution of this receptor. Apart from the AP/NTS region, GFRAL-immunoreactivity was found in the prefrontal cortex, hippocampus, nucleus arcuatus and peripheral tissues including liver, small intestine, fat, kidney and muscle tissues. This widespread receptor expression, not taken into consideration so far, may explain the multiple effects of GDF-15 that are not yet assigned to GFRAL. Furthermore, our results could be relevant for the development of novel pharmacological therapies for physical and mental disorders related to body image and food intake, such as eating disorders, cachexia and obesity.

Stress integration by an ascending adrenergic-melanocortin circuit.

Stress is thought to be an important contributing factor for eating disorders; however, neural substrates underlying the complex relationship between stress and appetite are not fully understood. Using in vivo recordings from awake behaving mice, we show that various acute stressors activate catecholaminergic nucleus tractus solitarius (NTSTH) projections in the paraventricular hypothalamus (PVH). Remarkably, the resulting adrenergic tone inhibits MC4R-expressing neurons (PVHMC4R), which are known for their role in feeding suppression. We found that PVHMC4R silencing encodes negative valence in sated mice and is required for avoidance induced by visceral malaise. Collectively, these findings establish PVHMC4R neurons as an effector of stress-activated brainstem adrenergic input in addition to the well-established hypothalamic-pituitary-adrenal axis. Convergent modulation of stress and feeding by PVHMC4R neurons implicates NTSTH → PVHMC4R input in stress-associated appetite disorders.

Vagotomy blunts cardiorespiratory responses to vagal afferent stimulation via pre- and postsynaptic effects in the nucleus tractus solitarii.

Viscerosensory information travels to the brain via vagal afferents, where it is first integrated within the brainstem nucleus tractus solitarii (nTS), a critical contributor to cardiorespiratory function and site of neuroplasticity. We have shown that decreasing input to the nTS via unilateral vagus nerve transection (vagotomy) induces morphological changes in nTS glia and reduces sighs during hypoxia. The mechanisms behind post-vagotomy changes are not well understood. We hypothesized that chronic vagotomy alters cardiorespiratory responses to vagal afferent stimulation via blunted nTS neuronal activity. Male Sprague-Dawley rats (6 weeks old) underwent right cervical vagotomy caudal to the nodose ganglion, or sham surgery. After 1 week, rats were anaesthetized, ventilated and instrumented to measure mean arterial pressure (MAP), heart rate (HR), and splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA, respectively). Vagal afferent stimulation (2-50 Hz) decreased cardiorespiratory parameters and increased neuronal Ca2+ measured by in vivo photometry and in vitro slice imaging of nTS GCaMP8m. Vagotomy attenuated both these reflex and neuronal Ca2+ responses compared to shams. Vagotomy also reduced presynaptic Ca2+ responses to stimulation (Cal-520 imaging) in the nTS slice. The decrease in HR, SSNA and PhrNA due to nTS nanoinjection of exogenous glutamate also was tempered following vagotomy. This effect was not restored by blocking excitatory amino acid transporters. However, the blunted responses were mimicked by NMDA, not AMPA, nanoinjection and were associated with reduced NR1 subunits in the nTS. Altogether, these results demonstrate that vagotomy induces multiple changes within the nTS tripartite synapse that influence cardiorespiratory reflex responses to afferent stimulation. KEY POINTS: Multiple mechanisms within the nucleus tractus solitarii (nTS) contribute to functional changes following vagal nerve transection. Vagotomy results in reduced cardiorespiratory reflex responses to vagal afferent stimulation and nTS glutamate nanoinjection. Blunted responses occur via reduced presynaptic Ca2+ activation and attenuated NMDA receptor expression and function, leading to a reduction in nTS neuronal activation. These results provide insight into the control of autonomic and respiratory function, as well as the plasticity that can occur in response to nerve damage and cardiorespiratory disease.

GABAergic and glutamatergic inputs to the medulla oblongata and locus coeruleus noradrenergic pathways are critical for seizures and postictal antinociception neuromodulation.

We investigated the participation of the nucleus of the tractus solitarius (NTS) in tonic‒clonic seizures and postictal antinociception control mediated by NMDA receptors, the role of NTS GABAergic interneurons and noradrenergic pathways from the locus coeruleus (LC) in these phenomena. The NTS-lateral nucleus reticularis paragigantocellularis (lPGi)-LC pathway was studied by evaluating neural tract tracer deposits in the lPGi. NMDA and GABAergic receptors agonists and antagonists were microinjected into the NTS, followed by pharmacologically induced seizures. The effects of LC neurotoxic lesions caused by DSP-4, followed by NTS-NMDA receptor activation, on both tonic‒clonic seizures and postictal antinociception were also investigated. The NTS is connected to lPGi neurons that send outputs to the LC. Glutamatergic vesicles were found on dendrites and perikarya of GABAergic interneurons in the NTS. Both tonic‒clonic seizures and postictal antinociception are partially dependent on glutamatergic-mediated neurotransmission in the NTS of seizing rats in addition to the integrity of the noradrenergic system since NMDA receptor blockade in the NTS and intrathecal administration of DSP-4 decrease the postictal antinociception. The GABAA receptor activation in the NTS decreases both seizure severity and postictal antinociception. These findings suggest that glutamatergic inputs to NTS-GABAergic interneurons, in addition to ascending and descending noradrenergic pathways from the LC, are critical for the control of both seizures and postictal antinociception.

Vagus nerve stimulation inhibits cortical spreading depression via glutamate-dependent TrkB activation mechanism in the nucleus tractus solitarius.

BACKGROUND: Vagus nerve stimulation (VNS) was recently found to inhibit cortical spreading depression (CSD), the underlying mechanism of migraine aura, through activation of the nucleus tractus solitarius (NTS), locus coeruleus (LC) and dorsal raphe nucleus (DRN). The molecular mechanisms underlying the effect of VNS on CSD in these nuclei remain to be explored. We hypothesized that VNS may activate glutamate receptor-mediated tropomyosin kinase B (TrkB) signaling in the NTS, thereby facilitating the noradrenergic and serotonergic neurotransmission to inhibit CSD. METHODS: To investigate the role of TrkB and glutamate receptors in non-invasive VNS efficacy on CSD, a validated KCl-evoked CSD rat model coupled with intra-NTS microinjection of selective antagonists, immunoblot and immunohistochemistry was employed. RESULTS: VNS increased TrkB phosphorylation in the NTS. Inhibition of intra-NTS TrkB abrogated the suppressive effect of VNS on CSD and CSD-induced cortical neuroinflammation. TrkB was found colocalized with glutamate receptors in NTS neurons. Inhibition of glutamate receptors in the NTS abrogated VNS-induced TrkB activation. Moreover, the blockade of TrkB in the NTS attenuated VNS-induced activation of the LC and DRN. CONCLUSIONS: VNS induces the activation of glutamate receptor-mediated TrkB signaling in the NTS, which might modulate serotonergic and norepinephrinergic innervation to the cerebral cortex to inhibit CSD and cortical inflammation.