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Introduction: Vagus nerve stimulation (VNS) is clinically useful for treating epilepsy, depression, and chronic pain. Currently, cervical VNS (cVNS) treatment is well-established, while auricular VNS (aVNS) is under development. Vagal stimulation regulates functions in diverse brain regions; therefore, it is critical to better understand how electrically-evoked vagal inputs following cVNS and aVNS engage with different brain regions., Objective: As vagus inputs are predominantly transmitted to the nucleus of tractus solitarius (NTS), we directly compared the activation of NTS neurons by cVNS or aVNS and the brain regions directly projected by the activated NTS neurons in mice., Methods: We adopted the targeted recombination in active populations method, which allows for the activity-dependent, tamoxifen-inducible expression of mCherry-a reporter protein-in neurons specifically associated with cVNS or aVNS., Results: cVNS and aVNS induced comparable bilateral mCherry expressions in neurons within the NTS, especially in its caudal section (cNTS). However, the numbers of mCherry-expressing neurons within different subdivisions of cNTS was distinctive. In both cVNS and aVNS, anterogradely labeled mCherry-expressing axonal terminals were similarly observed across different areas of the forebrain, midbrain, and hindbrain. These terminals were enriched in the rostral ventromedial medulla, parabrachial nucleus, periaqueductal gray, thalamic nuclei, central amygdala, and the hypothalamus. Sex difference of cVNS- and aVNS-induced labeling of NTS neurons was modest., Conclusion: The central projections of mCherry-expressing cNTS terminals are comparable between aVNS and cVNS, suggesting that cVNS and aVNS activate distinct but largely overlapping projections into the brain through the cNTS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The most successful obesity therapeutics, glucagon-like peptide-1 receptor (GLP1R) agonists, cause aversive responses such as nausea and vomiting 1,2 , 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 NTS GLP1R neurons triggers satiety in the absence of aversion, whereas activation of AP GLP1R neurons triggers strong aversion with food intake reduction. Anatomical and behavioural analyses revealed that NTS GLP1R and AP GLP1R 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 NTS GLP1R neurons as a population that could be selectively targeted to promote weight loss while avoiding the adverse side effects that limit treatment adherence., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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 hyperreactivity 1-3 , 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., (© 2024. The Author(s).)

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 (NTS PNMT 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 NTS PNMT neurons were identified, spanning several key brain regions including the medulla oblongata, pons, midbrain, cerebellum, diencephalon, and telencephalon. Notably, dense projections to the NTS PNMT 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 NTS PNMT neurons. Additionally, reciprocal projections were evident among 21 nuclei, primarily situated within the medulla oblongata., Conclusion: Our research findings demonstrate that NTS PNMT neurons form extensive connections with numerous nuclei, emphasizing their essential role in the homeostatic regulation of vital autonomic functions., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

The termination of a meal is controlled by dedicated neural circuits in the caudal brainstem. A key challenge is to understand how these circuits transform the sensory signals generated during feeding into dynamic control of behaviour. The caudal nucleus of the solitary tract (cNTS) is the first site in the brain where many meal-related signals are sensed and integrated 1-4 , but how the cNTS processes ingestive feedback during behaviour is unknown. Here we describe how prolactin-releasing hormone (PRLH) and GCG neurons, two principal cNTS cell types that promote non-aversive satiety, are regulated during ingestion. PRLH neurons showed sustained activation by visceral feedback when nutrients were infused into the stomach, but these sustained responses were substantially reduced during oral consumption. Instead, PRLH neurons shifted to a phasic activity pattern that was time-locked to ingestion and linked to the taste of food. Optogenetic manipulations revealed that PRLH neurons control the duration of seconds-timescale feeding bursts, revealing a mechanism by which orosensory signals feed back to restrain the pace of ingestion. By contrast, GCG neurons were activated by mechanical feedback from the gut, tracked the amount of food consumed and promoted satiety that lasted for tens of minutes. These findings reveal that sequential negative feedback signals from the mouth and gut engage distinct circuits in the caudal brainstem, which in turn control elements of feeding behaviour operating on short and long timescales., (© 2023. The Author(s).)

Circadian regulation of autonomic reflex pathways pairs physiological function with the daily light cycle. The brainstem nucleus of the solitary tract (NTS) is a key candidate for rhythmic control of the autonomic nervous system. Here we investigated circadian regulation of NTS neurotransmission and synaptic throughput using patch-clamp electrophysiology in brainstem slices from mice. We found that spontaneous quantal glutamate release onto NTS neurons showed strong circadian rhythmicity, with the highest rate of release during the light phase and the lowest in the dark, that were sufficient to drive day/night differences in constitutive postsynaptic action potential firing. In contrast, afferent evoked action potential throughput was enhanced during the dark and diminished in the light. Afferent-driven synchronous release pathways showed a similar decrease in release probability that did not explain the enhanced synaptic throughput during the night. However, analysis of postsynaptic membrane properties revealed diurnal changes in conductance, which, when coupled with the circadian changes in glutamate release pathways, tuned synaptic throughput between the light and dark phases. These coordinated pre-/postsynaptic changes encode nuanced control over synaptic performance and pair NTS action potential firing and vagal throughput with time of day. KEY POINTS: Vagal afferent neurons relay information from peripheral organs to the brainstem nucleus of the solitary tract (NTS) to initiate autonomic reflex pathways as well as providing important controls of food intake, digestive function and energy balance. Vagally mediated reflexes and behaviours are under strong circadian regulation. Diurnal fluctuations in presynaptic vesicle release pathways and postsynaptic membrane conductances provide nuanced control over NTS action potential firing and vagal synaptic throughput. Coordinated pre-/postsynaptic changes represent a fundamental mechanism mediating daily changes in vagal afferent signalling and autonomic function., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The nervous system uses various coding strategies to process sensory inputs. For example, the olfactory system uses large receptor repertoires and is wired to recognize diverse odours, whereas the visual system provides high acuity of object position, form and movement 1-5 . Compared to external sensory systems, principles that underlie sensory processing by the interoceptive nervous system remain poorly defined. Here we developed a two-photon calcium imaging preparation to understand internal organ representations in the nucleus of the solitary tract (NTS), a sensory gateway in the brainstem that receives vagal and other inputs from the body. Focusing on gut and upper airway stimuli, we observed that individual NTS neurons are tuned to detect signals from particular organs and are topographically organized on the basis of body position. Moreover, some mechanosensory and chemosensory inputs from the same organ converge centrally. Sensory inputs engage specific NTS domains with defined locations, each containing heterogeneous cell types. Spatial representations of different organs are further sharpened in the NTS beyond what is achieved by vagal axon sorting alone, as blockade of brainstem inhibition broadens neural tuning and disorganizes visceral representations. These findings reveal basic organizational features used by the brain to process interoceptive inputs., (© 2022. The Author(s).)

Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours 1 . Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated 2-4 . Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS 2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS-AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1 + neurons in the NTS-AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection., (© 2022. The Author(s).)

Obstructive sleep apnea (OSA) is recurrent obstruction of the upper airway due to the loss of upper airway muscle tone during sleep. OSA is highly prevalent, especially in obesity. There is no pharmacotherapy for OSA. Previous studies have demonstrated the role of leptin, an adipose-tissue-produced hormone, as a potent respiratory stimulant. Leptin signaling via a long functional isoform of leptin receptor, LEPR b , in the nucleus of the solitary tract (NTS), has been implicated in control of breathing. We hypothesized that leptin acts on LEPR b positive neurons in the NTS to increase ventilation and maintain upper airway patency during sleep in obese mice. We expressed designer receptors exclusively activated by designer drugs (DREADD) selectively in the LEPR b positive neurons of the NTS of Lepr b -Cre-GFP mice with diet-induced obesity (DIO) and examined the effect of DREADD ligand, J60, on tongue muscle activity and breathing during sleep. J60 was a potent activator of LEPR b positive NTS neurons, but did not stimulate breathing or upper airway muscles during NREM and REM sleep. We conclude that, in DIO mice, the stimulating effects of leptin on breathing during sleep are independent of LEPR b signaling in the NTS.

Neuronatin (Nnat) is involved in the regulation of cellular molecular signaling and appears to be also linked to metabolic processes. The gastrointestinal peptides cholecystokinin (CCK) and bombesin (BN) have an effect on the short-term inhibition of food intake and induce neuronal activation in different brain nuclei, prominently in the nucleus of the solitary tract (NTS) involved in the modulation of food intake. The aim of the study was to examine if Nnat immunoreactivity is detectable in the NTS, and whether peripheral CCK-8S or BN cause c-Fos activation of Nnat neurons. Non-fasted male Sprague-Dawley rats received an intraperitoneal (i.p.) injection of 5.2 or 8.7 nmol CCK-8S/kg or 26 or 32 nmol BN/kg (n = 4 all groups) or vehicle solution (0.15 M NaCl; n = 7). The number of c-Fos neurons was determined 90 min post injection in the NTS and dorsal motor nucleus of the vagus (DMV). We observed Nnat immunoreactive neurons in the NTS and DMV. CCK-8S (25-fold and 51-fold, p = 0.025 and p = 0.001) and BN (31-fold and 59-fold, p = 0.007 and p = 0.001) at both doses increased the number of c-Fos positive neurons in the NTS. CCK and BN did not show a significant effect in the DMV. Both doses of CCK-8S (24-fold and 48-fold p = 0.011 and p = 0.001) and bombesin (31-fold and 56-fold, p = 0.002 and p = 0.001) increased the number of activated Nnat neurons in the NTS (p = 0.001) compared to the vehicle group, while in the DMV no significant increase of c-Fos activation was detected. In conclusion, i.p. injected CCK-8S or BN induce an increased neuronal activity in NTS Nnat neurons, giving rise that Nnat may play a role in the regulation of food intake mediated by peripheral CCK-8S or BN., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Leptin signaling within the nucleus of the solitary tract (NTS) contributes to the control of food intake, and injections of leptin into the NTS reduce meal size and increase the efficacy of vagus-mediated satiation signals. Leptin receptors (LepRs) are expressed by vagal afferents as well as by a population of NTS neurons. However, the electrophysiological properties of LepR-expressing NTS neurons have not been well characterized, and it is unclear how leptin might act on these neurons to reduce food intake. To address this question, we recorded from LepR-expressing neurons in horizontal brain slices containing the NTS from male and female LepR-Cre X Rosa-tdTomato mice. We found that the vast majority of NTS LepR neurons received monosynaptic innervation from vagal afferent fibers and LepR neurons exhibited large synaptic NMDA receptor (NMDAR)-mediated currents compared with non-LepR neurons. During high-frequency stimulation of vagal afferents, leptin increased the size of NMDAR-mediated currents, but not AMPAR-mediated currents. Leptin also increased the size of evoked EPSPs and the ability of low-intensity solitary tract stimulation to evoke action potentials in LepR neurons. These effects of leptin were blocked by bath applying a competitive NMDAR antagonist (DCPP-ene) or by an NMDAR channel blocker applied through the recording pipette (MK-801). Last, feeding studies using male rats demonstrate that intra-NTS injections of DCPP-ene attenuate reduction of overnight food intake following intra-NTS leptin injection. Our results suggest that leptin acts in the NTS to reduce food intake by increasing NMDAR-mediated currents, thus enhancing NTS sensitivity to vagal inputs. SIGNIFICANCE STATEMENT Leptin is a hormone that critically impacts food intake and energy homeostasis. The nucleus of the solitary tract (NTS) is activated by vagal afferents from the gastrointestinal tract, which promotes termination of a meal. Injection of leptin into the NTS inhibits food intake, while knockdown of leptin receptors (LepRs) in NTS neurons increases food intake. However, little was known about how leptin acts in the NTS neurons to inhibit food intake. We found that leptin increases the sensitivity of LepR-expressing neurons to vagal inputs by increasing NMDA receptor-mediated synaptic currents and that NTS NMDAR activation contributes to leptin-induced reduction of food intake. These findings suggest a novel mechanism by which leptin, acting in the NTS, could potentiate gastrointestinal satiation signals., (Copyright © 2020 the authors.)

Objective: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are used as anti-diabetic drugs and are approved for obesity treatment. However, GLP-1RAs also affect heart rate (HR) and arterial blood pressure (ABP) in rodents and humans. Although the activation of GLP-1 receptors (GLP-1R) is known to increase HR, the circuits recruited are unclear, and in particular, it is unknown whether GLP-1RAs activate preproglucagon (PPG) neurons, the brain source of GLP-1, to elicit these effects., Methods: We investigated the effect of GLP-1RAs on heart rate in anaesthetized adult mice. In a separate study, we manipulated the activity of nucleus tractus solitarius (NTS) PPG neurons (PPG NTS ) in awake, freely behaving transgenic Glu-Cre mice implanted with biotelemetry probes and injected with AAV-DIO-hM3Dq:mCherry or AAV-mCherry-FLEX-DTA., Results: Systemic administration of the GLP-1RA Ex-4 increased resting HR in anaesthetized or conscious mice, but had no effect on ABP in conscious mice. This effect was abolished by β-adrenoceptor blockade with atenolol, but unaffected by the muscarinic antagonist atropine. Furthermore, Ex-4-induced tachycardia persisted when PPG NTS neurons were ablated, and Ex-4 did not induce expression of the neuronal activity marker cFos in PPG NTS neurons. PPG NTS ablation or acute chemogenetic inhibition of these neurons via hM4Di receptors had no effect on resting HR. In contrast, chemogenetic activation of PPG NTS neurons increased resting HR. Furthermore, the application of GLP-1 within the subarachnoid space of the middle thoracic spinal cord, a major projection target of PPG neurons, increased HR., Conclusions: These results demonstrate that both systemic application of Ex-4 or GLP-1 and chemogenetic activation of PPG NTS neurons increases HR. Ex-4 increases the activity of cardiac sympathetic preganglionic neurons of the spinal cord without recruitment of PPG NTS neurons, and thus likely recapitulates the physiological effects of PPG neuron activation. These neurons therefore do not play a significant role in controlling resting HR and ABP but are capable of inducing tachycardia and so are likely involved in cardiovascular responses to acute stress., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Taste receptor cells use multiple signaling pathways to detect chemicals in potential food items. These cells are functionally grouped into different types: Type I cells act as support cells and have glial-like properties; Type II cells detect bitter, sweet, and umami taste stimuli; and Type III cells detect sour and salty stimuli. We have identified a new population of taste cells that are broadly tuned to multiple taste stimuli including bitter, sweet, sour, and umami. The goal of this study was to characterize these broadly responsive (BR) taste cells. We used an IP3R3-KO mouse (does not release calcium (Ca2+) from internal stores in Type II cells when stimulated with bitter, sweet, or umami stimuli) to characterize the BR cells without any potentially confounding input from Type II cells. Using live cell Ca2+ imaging in isolated taste cells from the IP3R3-KO mouse, we found that BR cells are a subset of Type III cells that respond to sour stimuli but also use a PLCβ signaling pathway to respond to bitter, sweet, and umami stimuli. Unlike Type II cells, individual BR cells are broadly tuned and respond to multiple stimuli across different taste modalities. Live cell imaging in a PLCβ3-KO mouse confirmed that BR cells use this signaling pathway to respond to bitter, sweet, and umami stimuli. Short term behavioral assays revealed that BR cells make significant contributions to taste driven behaviors and found that loss of either PLCβ3 in BR cells or IP3R3 in Type II cells caused similar behavioral deficits to bitter, sweet, and umami stimuli. Analysis of c-Fos activity in the nucleus of the solitary tract (NTS) also demonstrated that functional Type II and BR cells are required for normal stimulus induced expression., Competing Interests: The authors have declared that no competing interests exist.

Phoenixin (PNX) is a neuropeptide shown to play roles in the control of reproduction. The nucleus of the solitary tract (NTS), a critical autonomic integrating centre in the hindbrain, is one of many areas with dense expression of PNX. Using coronal NTS slices obtained from male Sprague-Dawley rats, the present study characterised the effects of PNX on both spike frequency and membrane potential of NTS neurones. Extracellular recordings demonstrated that bath-applied 10 nmol L -1 PNX increased the firing frequency in 32% of NTS neurones, effects which were confirmed with patch-clamp recordings showing that 50% of NTS neurones tested depolarised in response to application of the peptide. Surprisingly, the responsiveness to PNX in NTS neurones then declined suddenly to 9% (P < 0.001). This effect was subsequently attributed to stress associated with construction in our animal care facility because PNX responsiveness was again observed in slices from rats delivered and maintained in a construction-free facility. We then examined whether this loss of PNX responsiveness could be replicated in rats placed on a chronic stress regimen involving ongoing corticosterone (CORT) treatment in the construction-free facility. Slices from animals treated in this way showed a similar lack of neuronal responsiveness to PNX (9.1 ± 3.9%) within 2 weeks of CORT treatment. These effects were specific to PNX responsiveness because CORT treatment had no effect on the responsiveness of NTS neurones to angiotensin II. These results are the first to implicate PNX with respect to directly controlling the excitability of NTS neurones and also provide intriguing data showing the plasticity of these effects associated with environmental and glucocorticoid stress levels of the animal., (© 2020 British Society for Neuroendocrinology.)

A role for glial cells in brain circuits controlling feeding has begun to be identified with hypothalamic astrocyte signaling implicated in regulating energy homeostasis. The nucleus of the solitary tract (NTS), within the brainstem dorsal vagal complex (DVC), integrates vagal afferent information from the viscera and plays a role in regulating food intake. We hypothesized that astrocytes in this nucleus respond to, and influence, food intake. Mice fed high-fat chow for 12 hr during the dark phase showed NTS astrocyte activation, reflected in an increase in the number (65%) and morphological complexity of glial-fibrillary acidic protein (GFAP)-immunoreactive cells adjacent to the area postrema (AP), compared to control chow fed mice. To measure the impact of astrocyte activation on food intake, we delivered designer receptors exclusively activated by designer drugs (DREADDs) to DVC astrocytes (encompassing NTS, AP, and dorsal motor nucleus of the vagus) using an adeno-associated viral (AAV) vector (AAV-GFAP-hM3Dq&#95;mCherry). Chemogenetic activation with clozapine-N-oxide (0.3 mg/kg) produced in greater morphological complexity in astrocytes and reduced dark-phase feeding by 84% at 4 hr postinjection compared with vehicle treatment. hM3Dq-activation of DVC astrocytes also reduced refeeding after an overnight fast (71% lower, 4 hr postinjection) when compared to AAV-GFAP-mCherry expressing control mice. DREADD-mediated astrocyte activation did not impact locomotion. hM3Dq activation of DVC astrocytes induced c-FOS in neighboring neuronal feeding circuits (including in the parabrachial nucleus). This indicates that NTS astrocytes respond to acute nutritional excess, are involved in the integration of peripheral satiety signals, and can reduce food intake when activated., (© 2019 The Authors. Glia published by Wiley Periodicals, Inc.)

To understand hindbrain pathways involved in the control of food intake, we examined roles for calcitonin receptor (CALCR)-containing neurons in the NTS. Ablation of NTS Calcr abrogated the long-term suppression of food intake, but not aversive responses, by CALCR agonists. Similarly, activating Calcr NTS neurons decreased food intake and body weight but (unlike neighboring Cck NTS cells) failed to promote aversion, revealing that Calcr NTS neurons mediate a non-aversive suppression of food intake. While both Calcr NTS and Cck NTS neurons decreased feeding via projections to the PBN, Cck NTS cells activated aversive CGRP PBN cells while Calcr NTS cells activated distinct non-CGRP PBN cells. Hence, Calcr NTS cells suppress feeding via non-aversive, non-CGRP PBN targets. Additionally, silencing Calcr NTS cells blunted food intake suppression by gut peptides and nutrients, increasing food intake and promoting obesity. Hence, Calcr NTS neurons define a hindbrain system that participates in physiological energy balance and suppresses food intake without activating aversive systems., Competing Interests: Declaration of Interests J.L.T. and C.J.R. are past or present employees of MedImmune, LLC and hold stock in AstraZeneca. The other authors declare that they have no conflicts of interest relevant to this manuscript., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Glucose is the essential energy source for the brain, whose deficit, triggered by energy deprivation or therapeutic agents, can be fatal. Increased appetite is the key behavioral defense against hypoglycemia; however, the central pathways involved are not well understood. Here, we describe a glucoprivic feeding pathway by tyrosine hydroxylase (TH)-expressing neurons from nucleus of solitary tract (NTS), which project densely to the hypothalamus and elicit feeding through bidirectional adrenergic modulation of agouti-related peptide (AgRP)- and proopiomelanocortin (POMC)-expressing neurons. Acute chemogenetic inhibition of arcuate nucleus (ARC)-projecting NTS TH neurons or their target, AgRP neurons, impaired glucoprivic feeding induced by 2-Deoxy-D-glucose (2DG) injection. Neuroanatomical tracing results suggested that ARC-projecting orexigenic NTS TH neurons are largely distinct from neighboring catecholamine neurons projecting to parabrachial nucleus (PBN) that promotes satiety. Collectively, we describe a circuit organization in which an ascending pathway from brainstem stimulates appetite through key hunger neurons in the hypothalamus in response to hypoglycemia., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Stress responses are coordinated by widespread neural circuits. Homeostatic and psychogenic stressors activate preproglucagon (PPG) neurons in the caudal nucleus of the solitary tract (cNTS) that produce glucagon-like peptide-1; published work in rodents indicates that these neurons play a crucial role in stress responses. While the axonal targets of PPG neurons are well established, their afferent inputs are unknown. Here we use retrograde tracing with cholera toxin subunit b to show that the cNTS in male and female mice receives axonal inputs similar to those reported in rats. Monosynaptic and polysynaptic inputs specific to cNTS PPG neurons were revealed using Cre-conditional pseudorabies and rabies viruses. The most prominent sources of PPG monosynaptic input include the lateral (LH) and paraventricular (PVN) nuclei of the hypothalamus, parasubthalamic nucleus, lateral division of the central amygdala, and Barrington's nucleus (Bar). Additionally, PPG neurons receive monosynaptic vagal sensory input from the nodose ganglia and spinal sensory input from the dorsal horn. Sources of polysynaptic input to cNTS PPG neurons include the hippocampal formation, paraventricular thalamus, and prefrontal cortex. Finally, cNTS-projecting neurons within PVN, LH, and Bar express the activation marker cFOS in mice after restraint stress, identifying them as potential sources of neurogenic stress-induced recruitment of PPG neurons. In summary, cNTS PPG neurons in mice receive widespread monosynaptic and polysynaptic input from brain regions implicated in coordinating behavioral and physiological stress responses, as well as from vagal and spinal sensory neurons. Thus, PPG neurons are optimally positioned to integrate signals of homeostatic and psychogenic stress. SIGNIFICANCE STATEMENT Recent research has indicated a crucial role for glucagon-like peptide-1-producing preproglucagon (PPG) neurons in regulating both appetite and behavioral and autonomic responses to acute stress. Intriguingly, the central glucagon-like peptide-1 system defined in rodents is conserved in humans, highlighting the translational importance of understanding its anatomical organization. Findings reported here indicate that PPG neurons receive significant monosynaptic and polysynaptic input from brain regions implicated in autonomic and behavioral responses to stress, as well as direct input from vagal and spinal sensory neurons. Improved understanding of the neural pathways underlying the recruitment of PPG neurons may facilitate the development of novel therapies for the treatment of stress-related disorders., (Copyright © 2019 Holt et al.)

Background: Elevated circulating levels of the divergent transforming growth factor-beta (TGFb) family cytokine, growth differentiation factor 15 (GDF15), acting through its CNS receptor, glial-derived neurotrophic factor receptor alpha-like (GFRAL), can cause anorexia and weight loss leading to anorexia/cachexia syndrome of cancer and other diseases. Preclinical studies suggest that administration of drugs based on recombinant GDF15 might be used to treat severe obesity. However, the role of the GDF15-GFRAL pathway in the physiological regulation of body weight and metabolism is unclear. The critical site of action of GFRAL in the CNS has also not been proven beyond doubt. To investigate these two aspects, we have inhibited the actions of GDF15 in mice started on high-fat diet (HFD)., Methods: The actions of GDF15 were inhibited using two methods: (1) Groups of 8 mice under HFD had their endogenous GDF15 neutralised by monoclonal antibody treatment, (2) Groups of 15 mice received AAV-shRNA to knockdown GFRAL at its hypothesised major sites of action, the hindbrain area postrema (AP) and the nucleus of the solitary tract (NTS). Metabolic measurements were determined during both experiments., Conclusions: Treating mice with monoclonal antibody to GDF15 shortly after commencing HFD results in more rapid gain of body weight, adiposity and hepatic lipid deposition than the control groups. This is accompanied by reduced glucose and insulin tolerance and greater expression of pro-inflammatory cytokines in adipose tissue. Localised AP and NTS shRNA-GFRAL knockdown in mice commencing HFD similarly caused an increase in body weight and adiposity. This effect was in proportion to the effectiveness of GFRAL knockdown, indicated by quantitative analysis of hindbrain GFRAL staining. We conclude that the GDF15-GFRAL axis plays an important role in resistance to obesity in HFD-fed mice and that the major site of action of GDF15 in the CNS is GFRAL-expressing neurons in the AP and NTS.

Background: Central sensitization plays a pivotal role in the maintenance of chronic pain induced by chronic pancreatitis (CP). We hypothesized that the nucleus tractus solitarius (NTS), a primary central site that integrates pancreatic afferents apart from the thoracic spinal dorsal horn, plays a key role in the pathogenesis of visceral hypersensitivity in a rat model of CP., Aim: To investigate the role of the NTS in the visceral hypersensitivity induced by chronic pancreatitis., Methods: CP was induced by the intraductal injection of trinitrobenzene sulfonic acid (TNBS) in rats. Pancreatic hyperalgesia was assessed by referred somatic pain via von Frey filament assay. Neural activation of the NTS was indicated by immunohistochemical staining for Fos. Basic synaptic transmission within the NTS was assessed by electrophysiological recordings. Expression of vesicular glutamate transporters (VGluTs), N-methyl-D-aspartate receptor subtype 2B (NR2B), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subtype 1 (GluR1) was analyzed by immunoblotting. Membrane insertion of NR2B and GluR1 was evaluated by electron microscopy. The regulatory role of the NTS in visceral hypersensitivity was detected via pharmacological approach and chemogenetics in CP rats., Results: TNBS treatment significantly increased the number of Fos-expressing neurons within the caudal NTS. The excitatory synaptic transmission was substantially potentiated within the caudal NTS in CP rats (frequency: 5.87 ± 1.12 Hz in CP rats vs 2.55 ± 0.44 Hz in sham rats, P < 0.01; amplitude: 19.60 ± 1.39 pA in CP rats vs 14.71 ± 1.07 pA in sham rats; P < 0.01). CP rats showed upregulated expression of VGluT2, and increased phosphorylation and postsynaptic trafficking of NR2B and GluR1 within the caudal NTS. Blocking excitatory synaptic transmission via the AMPAR antagonist CNQX and the NMDAR antagonist AP-5 microinjection reversed visceral hypersensitivity in CP rats (abdominal withdraw threshold: 7.00 ± 1.02 g in CNQX group, 8.00 ± 0.81 g in AP-5 group and 1.10 ± 0.27 g in saline group, P < 0.001). Inhibiting the excitability of NTS neurons via chemogenetics also significantly attenuated pancreatic hyperalgesia (abdominal withdraw threshold: 13.67 ± 2.55 g in Gi group, 2.00 ± 1.37 g in Gq group, and 2.36 ± 0.67 g in mCherry group, P < 0.01)., Conclusion: Our findings suggest that enhanced excitatory transmission within the caudal NTS contributes to pancreatic pain and emphasize the NTS as a pivotal hub for the processing of pancreatic afferents, which provide novel insights into the central sensitization of painful CP., Competing Interests: Conflict-of-interest statement: The authors declare that there are no competing financial interests., (©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.)

Integration and modulation of primary afferent sensory information begins at the first terminating sites within the CNS, where central inhibitory circuits play an integral role. Viscerosensory information is conveyed to the nucleus of the solitary tract (NTS) where it initiates neuroendocrine, behavioral, and autonomic reflex responses that ensure optimal internal organ function. This excitatory input is modulated by diverse, local inhibitory interneurons, whose functions are not clearly understood. Here we show that, in male rats, 65% of somatostatin-expressing (SST) NTS neurons also express GAD67, supporting their likely role as inhibitory interneurons. Using whole-cell recordings of NTS neurons, from horizontal brainstem slices of male and female SST-yellow fluorescent protein (YFP) and SST-channelrhodopsin 2 (ChR2)-YFP mice, we quantified the impact of SST-NTS neurons on viscerosensory processing. Light-evoked excitatory photocurrents were reliably obtained from SST-ChR2-YFP neurons ( n = 16) and the stimulation-response characteristics determined. Most SST neurons (57%) received direct input from solitary tract (ST) afferents, indicating that they form part of a feedforward circuit. All recorded SST-negative NTS neurons ( n = 72) received SST-ChR2 input. ChR2-evoked PSCs were largely inhibitory and, in contrast to previous reports, were mediated by both GABA and glycine. When timed to coincide, the ChR2-activated SST input suppressed ST-evoked action potentials at second-order NTS neurons, demonstrating strong modulation of primary viscerosensory input. These data indicate that the SST inhibitory network innervates broadly within the NTS, with the potential to gate viscerosensory input to powerfully alter autonomic reflex function and other behaviors. SIGNIFICANCE STATEMENT Sensory afferent input is modulated according to state. For example the baroreflex is altered during a stress response or exercise, but the basic mechanisms underpinning this sensory modulation are not fully understood in any sensory system. Here we demonstrate that the neuronal processing of viscerosensory information begins with synaptic gating at the first central synapse with second-order neurons in the NTS. These data reveal that the somatostatin subclass of inhibitory interneurons are driven by visceral sensory input to play a major role in gating viscerosensory signals, placing them within a feedforward circuit within the NTS., (Copyright © 2019 the authors.)

This study aimed to determine the relative efficacy of the macronutrients, protein, fat and carbohydrate to induce satiation and satiety in rats in relation to macronutrient activation of neurons in the nucleus of the solitary tract (NTS). Male Sprague Dawley rats were schedule-fed twice a day for 2 h, receiving 100% of daily ad-libitum energy intake. On test day 1, 30 min before the first scheduled meal of the day, rats were gavaged with an 8 kcal isocaloric, isovolumetric solution of a glucose, lipid or peptone macronutrient solution or a non-caloric saline solution. To assess satiation, thirty minutes later rats were given access to food for 2 h and food intake determined. A second 2 h food access period 3 h later was used for assessment of satiety. On the second test day, rats were gavaged as before and killed 90 min after food presentation. Blood was collected for measurement of circulating metabolic markers. Brains were removed for analysis of c-Fos expression by in situ hybridization in the NTS. Rats which received saline consumed a similar amount of food compared to pre-gavage intakes. However, rats gavaged with a caloric macronutrient solution all reduced food intake by 18-20 kcal. Interestingly, the reduction in caloric intake was greater than the caloric value of the macronutrient solution gavaged and was sustained following the second scheduled meal. Quantification by in situ hybridization of c-Fos mRNA expression in the NTS 90 min post-gavage, showed a significant increase with each macronutrient, but was 24-29% higher with a lipid or peptone gavage compared to a glucose gavage. In conclusion, when delivered directly to the stomach, all macronutrients can be equally effective in inducing satiation with significant neuronal activation in the NTS of the hindbrain., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Key Points: Neurons from the brainstem nucleus of the tractus solitarius (NTS) participate in the counter-regulatory mechanisms in response to hypoglycaemia. ATP-sensitive potassium (K ATP ) channels are expressed in NTS neurons, and are partially open at rest in normoglycaemic 5 mM glucose. In normoglycaemic conditions, most NTS neurons depolarize in response to low external glucose (0.5 mM), via a voltage-dependent mechanism. Conversely, most NTS neurons incubated in hyperglycaemic 10 mM glucose do not respond to low glucose due to a more positive resting membrane potential caused by the closure of K ATP channels following increased intracellular metabolic ATP. Our findings show that in hyperglycaemic conditions, NTS neurons failed to sense rapid changes in external glucose, which could be related to hypoglycaemia-associated autonomic failure., Abstract: The nucleus of the tractus solitarius (NTS) is an integrative centre for autonomic counter-regulatory responses to hypoglycaemia. K ATP channels link the metabolic status of the neuron to its excitability. Here we investigated the influence of K ATP channels on the membrane potential of NTS neurons in normo- and hyperglycaemic external glucose concentrations, and after switching to a hypoglycaemic concentration, using in vitro electrophysiological recordings in brainstem slices. We found that in normoglycaemic (5 mM) glucose, tolbutamide, a K ATP channel antagonist, depolarized the membrane of most neurons, and this effect was observed in more hyperpolarized neurons. All neurons hyperpolarized after pharmacological activation of K ATP channels. Most NTS neurons depolarized in the presence of low glucose (0.5 mM), and this effect was only seen in hyperpolarized neurons. The effect of glucose was caused by a cationic current with a reversal potential around -50 mV. In the presence of hyperglycaemic glucose (10 mM), neurons were more depolarized, and fewer neurons responded to K ATP blockage. Application of 0.5 mM glucose solution to these neurons depolarized the membrane only in more hyperpolarized neurons. We conclude that NTS neurons present with K ATP channels open at rest in normoglycaemic conditions, and their membrane potential is affected by extracellular glucose. Moreover, NTS neurons depolarize the membrane in response to the application of a low glucose solution, but this effect is occluded by membrane depolarization triggered by K ATP blockage. Our data suggest a homeostatic regulation of the membrane potential by external glucose, and a possible mechanism related to the hypoglycaemia-associated autonomic failure., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

The nucleus tractus solitarii (NTS) is implicated in the control of breathing, but the neuronal phenotype and circuit mechanism involved in such a physiological function remain incompletely understood. This study focused on the respiratory role of paired-like homeobox 2b gene (Phox2b)-expressing NTS neurons and sought to determine whether selective stimulation of this set of neurons activates breathing in male mice. A Cre-dependent vector encoding a Gq-coupled human M3 muscarinic receptor (hM3Dq) was microinjected into the NTS of Phox2b-Cre transgenic mice. The hM3Dq-transduced neurons were pharmacologically activated in conscious mice while respiratory effects were measured by plethysmography. We demonstrate that chemogenetic stimulation of Phox2b-expressing NTS neurons significantly increased baseline minute volume via an increase in respiratory frequency rather than tidal volume. Chemogenetic stimulation also synergized with moderate CO 2 stimulation to enhance pulmonary ventilatory response. Selective ablation of Phox2b-expressing NTS neurons notably attenuated a hypercapnic ventilatory response. Moreover, histological evidence revealed that stimulation of Phox2b-expressing NTS neurons increased neuronal activity of the preBötzinger complex. Finally, we presented the neuroanatomical evidence of direct projection of Phox2b-expressing NTS neurons to putative respiratory central pattern generator. Overall, these findings suggest that selective activation of Phox2b-expressing NTS neurons potentiates baseline pulmonary ventilation via an excitatory drive to respiratory central pattern generator and this group of neurons is also required for the hypercapnic ventilatory response. SIGNIFICANCE STATEMENT The nucleus tractus solitarii (NTS) has been implicated in the control of breathing. The paired-like homeobox 2b gene (Phox2b) is the disease-defining gene for congenital central hypoventilation syndrome and is restrictively present in brainstem nucleus, including the NTS. Using a chemogenetic approach, we demonstrate herein that selective stimulation of Phox2b-expressing NTS neurons vigorously potentiates baseline pulmonary ventilation via an excitatory drive to respiratory central pattern generator in rodents. Genetic ablation of these neurons attenuates the hypercapnic ventilatory response. We also suggest that a fraction of Phox2b-expressing neurons exhibit CO 2 sensitivity and presumably function as central respiratory chemoreceptors. The methodology is expected to provide a future applicability to the patients with sleep-related hypoventilation or apnea., (Copyright © 2019 the authors.)

Neurons in the brainstem dorsal vagal complex integrate neural and humoral signals to coordinate autonomic output to viscera that regulate a variety of physiological functions, but how this circuitry regulates metabolism is murky. We tested the hypothesis that premotor, GABAergic neurons in the nucleus tractus solitarius (NTS) form a hindbrain micro-circuit with preganglionic parasympathetic motorneurons of the dorsal motor nucleus of the vagus (DMV) that is capable of modulating systemic blood glucose concentration. In vitro, neuronal activation or inhibition using either excitatory or inhibitory designer receptor exclusively activated by designer drugs (DREADDs) constructs expressed in GABAergic NTS neurons increased or decreased, respectively, action potential firing of GABAergic NTS neurons and downstream synaptic inhibition of the DMV. In vivo, DREADD-mediated activation of GABAergic NTS neurons increased systemic blood glucose concentration, whereas DREADD-mediated silencing of these neurons was without effect. The DREADD-induced hyperglycemia was abolished by blocking peripheral muscarinic receptors, consistent with the hypothesis that altered parasympathetic drive mediated the response. This effect was paralleled by elevated serum glucagon and hepatic phosphoenolpyruvate carboxykinase 1 (PEPCK1) expression, without affecting insulin levels or muscle metabolism. Activity in a hindbrain inhibitory microcircuit is sufficient to modulate systemic glucose concentration, independent of insulin secretion or utilization.

Theories of neural coding in the taste system typically rely exclusively on data gleaned from taste-responsive cells. However, even in the nucleus tractus solitarius (NTS), the first stage of central processing, neurons with taste selectivity coexist with neurons whose activity is linked to motor behavior related to ingestion. We recorded from a large ( n = 324) sample of NTS neurons recorded in awake rats, examining both their taste selectivity and the association of their activity with licking. All subjects were implanted with a bundle of microelectrodes aimed at the NTS and allowed to recover. Following moderate water deprivation, rats were placed in an experimental chamber where tastants or artificial saliva (AS) were delivered from a lick spout. Electrophysiological responses were recorded, and waveforms from single cells were isolated offline. Results showed that only a minority of NTS cells responded to taste stimuli as determined by conventional firing-rate measures. In contrast, most cells, including taste-responsive cells, tracked the lick pattern, as evidenced by significant lick coherence in the 5- to 7-Hz range. Several quantitative measures of taste selectivity and lick relatedness showed that the population formed a continuum, ranging from cells dominated by taste responses to those dominated by lick relatedness. Moreover, even neurons whose responses were highly correlated with lick activity could convey substantial information about taste quality. In all, data point to a blurred boundary between taste-dominated and lick-related cells in NTS, suggesting that information from the taste of food and from the movements it evokes are seamlessly integrated. NEW & NOTEWORTHY Neurons in the rostral nucleus of the solitary tract (NTS) are known to encode information about taste. However, recordings from awake rats reveal that only a minority of NTS cells respond exclusively to taste stimuli. The majority of neurons track behaviors associated with food consumption, and even strongly lick-related neurons could convey information about taste quality. These findings suggest that the NTS integrates information from both taste and behavior to identify food.

Activation of corticotropin-releasing hormone (CRH) type 2 receptors (CRHR2) in the nucleus of the solitary tract (NTS) contributes to the development of hypertension, but the source of CRH inputs to the NTS that increases blood pressure remains unknown. This study tested the hypothesis that activation of CRH-containing projections from the paraventricular nucleus of the hypothalamus (PVN) to the NTS increase blood pressure. We expressed channelrhodopsin 2 (ChR2), a light-sensitive ion channel, into CRH-containing neurons in the PVN. This was achieved by injecting Cre-inducible virus expressing ChR2 into the PVN of CRH-Cre mice. CRH-Cre mice are genetically modified mice expressing Cre recombinase only in neurons producing CRH. We found that optogenetic stimulation of CRH-containing somas in the PVN or CRH-containing fibers in the NTS originating from the PVN significantly increased blood pressure and heart rate. Microinjection of K-41498 (CRHR2 antagonist) into the NTS attenuated the pressor and tachycardiac responses induced by optogenetic stimulation of CRH-containing somas in the PVN. In vitro loose-patch recordings revealed that optogenetic stimulation of CRH-containing fibers in the NTS originating from the PVN significantly increased the discharge frequency of NTS neurons. This effect was attenuated by pretreatment of K-41498 and was abolished by pretreatment of kynurenic acid (nonselective glutamate receptor antagonist). These results suggest that activation of PVN-NTS CRH-containing projections increases blood pressure and heart rate. The cardiovascular responses may be mediated at least in part by the corelease of CRH and glutamate from NTS CRH-containing axons originating from the PVN. NEW & NOTEWORTHY Optogenetic stimulation of paraventricular nucleus of the hypothalamus (PVN) corticotropin-releasing hormone (CRH)-containing somas or nucleus of the solitary tract (NTS) CRH-containing fibers originating from the PVN increased blood pressure and heart rate. Corelease of CRH and glutamate from NTS CRH-containing axons originating from the PVN may contribute to the pressor and tachycardiac responses elicited by optogenetic stimulation of PVN CRH-containing somas.

Sodium deficiency elevates aldosterone, which in addition to epithelial tissues acts on the brain to promote dysphoric symptoms and salt intake. Aldosterone boosts the activity of neurons that express 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a hallmark of aldosterone-sensitive cells. To better characterize these neurons, we combine immunolabeling and in situ hybridization with fate mapping and Cre-conditional axon tracing in mice. Many cells throughout the brain have a developmental history of Hsd11b2 expression, but in the adult brain one small brainstem region with a leaky blood-brain barrier contains HSD2 neurons. These neurons express Hsd11b2, Nr3c2 (mineralocorticoid receptor), Agtr1a (angiotensin receptor), Slc17a6 (vesicular glutamate transporter 2), Phox2b, and Nxph4; many also express Cartpt or Lmx1b. No HSD2 neurons express cholinergic, monoaminergic, or several other neuropeptidergic markers. Their axons project to the parabrachial complex (PB), where they intermingle with AgRP-immunoreactive axons to form dense terminal fields overlapping FoxP2 neurons in the central lateral subnucleus (PBcL) and pre-locus coeruleus (pLC). Their axons also extend to the forebrain, intermingling with AgRP- and CGRP-immunoreactive axons to form dense terminals surrounding GABAergic neurons in the ventrolateral bed nucleus of the stria terminalis (BSTvL). Sparse axons target the periaqueductal gray, ventral tegmental area, lateral hypothalamic area, paraventricular hypothalamic nucleus, and central nucleus of the amygdala. Dual retrograde tracing revealed that largely separate HSD2 neurons project to pLC/PB or BSTvL. This projection pattern raises the possibility that a subset of HSD2 neurons promotes the dysphoric, anorexic, and anhedonic symptoms of hyperaldosteronism via AgRP-inhibited relay neurons in PB.

Adenosine operating in the nucleus of the solitary tract (NTS) may inhibit or facilitate neurotransmitter release from nerve terminals and directly inhibit or facilitate central neurons via A 1 and A 2a pre- and postsynaptic receptors, respectively. However, adenosine A 2a receptors, may also activate GABA-ergic neurons/terminals which in turn inhibit glutamatergic transmission in the NTS network. Our previous studies showed that adenosine operating via both A 1 (inhibitor) and A 2a (activator) receptors powerfully inhibits the cardiopulmonary chemoreflex (CCR) at the level of the caudal NTS. A 1 receptors most likely inhibit glutamate release in the CCR network, whereas A 2a receptors facilitate NTS GABA-ergic mechanisms which in turn inhibit CCR glutamatergic transmission. Therefore, we hypothesized that A 2a receptors are located on NTS GABA-ergic neurons/terminals whereas A 1 receptors may be located on NTS glutamatergic neurons/terminals. We investigated this hypothesis using double immunofluorescent staining for A 2a or A 1 adenosine receptors and GABA synthesizing enzyme, GAD67, in 30 μm thick, floating, medullary rat sections. We found that A 2a adenosine receptors are localized within the GABA-ergic cells in the caudal NTS, whereas A 1 adenosine receptors are absent from these neurons. Instead, A 1 receptors were located on non-GABA-ergic (likely glutamatergic) neurons/terminals in the caudal NTS. These data support our functional findings and the hypothesis that adenosine A 2a, but not A 1 receptors are located on GABA-ergic neurons., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Sensory-motor control of the pharyngeal swallow requires sensory afferent inputs from the pharynx and larynx evoked by introducing bolus into the pharynx. Patients with reduced sensitivity of the pharynx and larynx are likely to have a swallowing impairment, such as pre-swallow aspiration due to delayed swallow triggering. Interferential current stimulation applied to the neck is thought to improve the swallowing function of dysphagic patients, although the mechanism underlying the facilitatory effect of such stimulation remains unknown. In the present study, we examined the changes in the elicitability of swallowing due to the stimulation and the responses of the swallowing-related neurons in the nucleus tractus solitarius and in the area adjacent to the stimulation in decerebrate and paralyzed guinea pigs. The swallowing delay time was shortened by the stimulation, whereas the facilitatory effect of eliciting swallowing was attenuated by kainic acid injection into the nucleus tractus solitarius. Approximately half of the swallowing-related neurons responded to the stimulation. These data suggest that the interferential current stimulation applied to the neck could enhance the sensory afferent pathway of the pharynx and larynx, subserving excitatory inputs to the neurons of the swallowing pattern generator, thereby facilitating the swallowing reflex.

The nucleus of the solitary tract is a potential site for taste-visceral interactions. Connections from the caudal, visceral area of the nucleus (cNST) to the rostral, gustatory zone (rNST) have been described, but the phenotype of cells giving rise to the projection(s) and their distribution among rNST subdivisions are unknown. To determine these characteristics of the intrasolitary pathway, we injected pan-neuronal and floxed AAV viruses into the cNST of mice expressing cre in glutamatergic, GABAergic, or catecholaminergic neurons. Particular attention was paid to the terminal field distribution in rNST subdivisions by simultaneously visualizing P2X2 localized to gustatory afferent terminals. All three phenotypically identified pathways terminated in rNST, with the density greatest for glutamatergic and sparsest for catecholaminergic projections, observations supported by retrograde tracing. Interestingly, cNST neurons had more prominent projections to rNST regions medial and ventral to P2X2 staining, i.e., the medial and ventral subdivisions. In addition, GABAergic neurons projected robustly to the lateral subdivision and adjacent parts of the reticular formation and spinal trigeminal nucleus. Although cNST neurons also projected to the P2X2-rich central subdivision, such projections were sparser. These findings suggest that cNST visceral signals exert stronger excitatory and inhibitory influences on local autonomic and reflex pathways associated with the medial and ventral subdivisions compared to weaker modulation of ascending pathways arising from the central subdivision and ultimately destined for the forebrain., (© 2018 Wiley Periodicals, Inc.)

Glutamatergic neurons that express pre-proglucagon (PPG) and are immunopositive (+) for glucagon-like peptide-1 (i.e., GLP-1+ neurons) are located within the caudal nucleus of the solitary tract (cNTS) and medullary reticular formation in rats and mice. GLP-1 neurons give rise to an extensive central network in which GLP-1 receptor (GLP-1R) signaling suppresses food intake, attenuates rewarding, increases avoidance, and stimulates stress responses, partly via GLP-1R signaling within the cNTS. In mice, noradrenergic (A2) cNTS neurons express GLP-1R, whereas PPG neurons do not. In this study, confocal microscopy in rats confirmed that prolactin-releasing peptide (PrRP)+ A2 neurons are closely apposed by GLP-1+ axonal varicosities. Surprisingly, GLP-1+ appositions were also observed on dendrites of PPG/GLP-1+ neurons in both species, and electron microscopy in rats revealed that GLP-1+ boutons form asymmetric synaptic contacts with GLP-1+ dendrites. However, RNAscope confirmed that rat GLP-1 neurons do not express GLP-1R mRNA. Similarly, Ca 2+ imaging of somatic and dendritic responses in mouse ex vivo slices confirmed that PPG neurons do not respond directly to GLP-1, and a mouse crossbreeding strategy revealed that <1% of PPG neurons co-express GLP-1R. Collectively, these data suggest that GLP-1R signaling pathways modulate the activity of PrRP+ A2 neurons, and also reveal a local "feed-forward" synaptic network among GLP-1 neurons that apparently does not use GLP-1R signaling. This local GLP-1 network may instead use glutamatergic signaling to facilitate dynamic and potentially selective recruitment of GLP-1 neural populations that shape behavioral and physiological responses to internal and external challenges., (© 2018 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

Products of the proopiomelanocortin (POMC) prohormone regulate aspects of analgesia, reward, and energy balance; thus, the neurons that produce POMC in the hypothalamus have received considerable attention. However, there are also cells in the nucleus of the solitary tract (NTS) that transcribe Pomc, although low levels of Pomc mRNA and relative lack of POMC peptide products in the adult mouse NTS have hindered the study of these cells. Therefore, studies of NTS POMC cells have largely relied on transgenic mouse lines. Here, we set out to determine the amino acid (AA) transmitter phenotype of NTS POMC neurons by using Pomc-Gfp transgenic mice to identify POMC cells. We found that cells expressing the green fluorescent protein (GFP) represent a mix of GABAergic and glutamatergic cells as indicated by Gad2 and vesicular Glut2 ( vGlut2) mRNA expression, respectively. We then examined the AA phenotype of POMC cells labeled by a Pomc-Cre transgene and found that these are also a mix of GABAergic and glutamatergic cells. However, the NTS cells labeled by the Gfp- and Cre-containing transgenes represented distinct populations of cells in three different Pomc-Cre mouse lines. Consistent with previous work, we were unable to reliably detect Pomc mRNA in the NTS despite clear expression in the hypothalamus. Thus, it was not possible to determine which transgenic tool most accurately identifies NTS cells that may express Pomc or release POMC peptides, although the results indicate the transgenic tools for study of these NTS neurons can label disparate populations of cells with varied AA phenotypes.

Expression of the transcription factor FOXP2 is linked to brain circuits that control motor function and speech. Investigation of FOXP2 protein expression in respiratory areas of the ponto-medullary brainstem of adult rat revealed distinct rostro-caudal expression gradients. A high density of FOXP2 immunoreactive nuclei was observed within the rostral pontine Kölliker-Fuse nucleus, compared to low densities in caudal pontine and rostral medullary respiratory nuclei, including the: (i) noradrenergic A5 and parafacial respiratory groups; (ii) Bötzinger and pre-Bötzinger complex and; (iii) rostral ventral respiratory group. Moderate densities of FOXP2 immunoreactive nuclei were observed in the caudal ventral respiratory group and the nucleus retroambiguus, with significant density levels found in the caudal half of the dorsal respiratory group and the hypoglossal pre-motor area lateral around calamus scriptorius. FOXP2 immunoreactivity was absent in all cranial nerve motor nuclei. We conclude that FOXP2 expression in respiratory brainstem areas selectively delineates laryngeal and hypoglossal pre-motor neuron populations essential for the generation of sound and voice., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The nucleus of the tractus solitarius (NTS) is a brain stem region critical to many physiologic processes and has been implicated in addiction to multiple classes of abused drugs, including alcohol (EtOH). That said, the mechanism by which EtOH modulates NTS neurocircuit activity is not well characterized and has yet to be examined utilizing electrophysiologic methods in mouse models of alcohol use disorders. To begin to address this gap in knowledge, we sought to use whole-cell and cell-attached recordings to determine the mechanism of acute EtOH action on GABAergic and glutamatergic neurotransmission, as well as on action potential firing in the NTS of adult male, EtOH naïve mice. Bath application of EtOH (50mM) significantly enhanced the frequency of spontaneous inhibitory postsynaptic current events, while increasing the amplitude of these events in half of the neurons tested. This finding suggests a presynaptic mechanism of EtOH action on GABAergic transmission in the NTS as well as a postsynaptic mechanism in subsets of NTS neurons. EtOH application was further associated with a significant decrease in action potential firing in most, but not all, NTS neurons tested. EtOH induced a small but significant decrease in spontaneous excitatory postsynaptic current frequency, indicating that EtOH may also inhibit NTS glutamatergic signaling to some degree. Intriguingly, in vivo EtOH exposure (4g/kg IP) enhanced c-FOS colocalization with tyrosine hydroxylase via immunohistochemical methods, indicating that NTS norepinephrine neurons may be activated by acute EtOH exposure. Although future work is needed, the current data indicate that acute EtOH may enhance GABAergic signaling in local NTS circuits resulting in disinhibition of NTS norepinephrine neurons. Such a finding has important implications in understanding the role of the NTS in the development of alcoholism., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The neurobiological substrates that mediate the anorectic effects of both endogenous glucagon-like peptide-1 (GLP-1) and exogenous GLP-1 receptor (GLP-1R) agonists are an active area of investigation. As the lateral dorsal tegmental nucleus (LDTg) expresses the GLP-1R and represents a potential neuroanatomical hub connecting the nucleus tractus solitarius (NTS), the major central source of GLP-1, with the other nuclei in the midbrain and forebrain, we tested the hypothesis that GLP-1R signaling in the LDTg controls food intake. Direct activation of LDTg GLP-1R suppresses food intake through a reduction in average meal size and independent of nausea/malaise. Immunohistochemical data show that GLP-1-producing neurons in the NTS project to the LDTg, providing anatomical evidence of endogenous central GLP-1 in the LDTg. Pharmacological blockade of LDTg GLP-1Rs with exendin-(9-39) dose-dependently increases food intake and attenuates the hypophagic effects of gastric distension. As GLP-1 mimetics are administered systemically in humans, we evaluated whether peripherally administered GLP-1R agonists access the LDTg to affect feeding. Immunohistochemical data show that a systemically administered fluorescent GLP-1R agonist accesses the LDTg and is juxtaposed with neurons. Additionally, blockade of LDTg GLP-1Rs attenuates the hypophagic effects of a systemic GLP-1R agonist. Together, these data indicate that LDTg GLP-1R signaling controls energy balance and underscores the role of the LDTg in integrating energy balance-relevant signals to modulate feeding.

Homeostatic regulation of visceral organ function requires integrated processing of neural and neurohormonal sensory signals. The nucleus of the solitary tract (NTS) is the primary sensory nucleus for cranial visceral sensory afferents. Angiotensin II (ANG II) is known to modulate peripheral visceral reflexes, in part, by activating ANG II type 1A receptors (AT 1A R) in the NTS. AT 1A R-expressing NTS neurons occur throughout the NTS with a defined subnuclear distribution, and most of these neurons are depolarized by ANG II. In this study we determined whether AT 1A R-expressing NTS neurons receive direct visceral sensory input, and whether this input is modulated by ANG II. Using AT 1A R-GFP mice to make targeted whole cell recordings from AT 1A R-expressing NTS neurons, we demonstrate that two-thirds (37 of 56) of AT 1A R-expressing neurons receive direct excitatory, visceral sensory input. In half of the neurons tested (4 of 8) the excitatory visceral sensory input was significantly reduced by application of the transient receptor potential vallinoid type 1 receptor agonist, capsaicin, indicating AT 1A R-expressing neurons can receive either C- or A-fiber-mediated input. Application of ANG II to a subset of second-order AT 1A R-expressing neurons did not affect spontaneous, evoked, or asynchronous glutamate release from visceral sensory afferents. Thus it is unlikely that AT 1A R-expressing viscerosensory neurons terminate on AT 1A R-expressing NTS neurons. Our data suggest that ANG II is likely to modulate multiple visceral sensory modalities by altering the excitability of second-order AT 1A R-expressing NTS neurons.

Key Points: In the rat nucleus of the solitary tract (NTS), activation of astrocytic proteinase-activated receptor 1 (PAR1) receptors leads to potentiation of neuronal synaptic activity by two mechanisms, one TRPV1-dependent and one TRPV1-independent. PAR1-dependent activation of presynaptic TRPV1 receptors facilitates glutamate release onto NTS neurons. The TRPV1-dependent mechanism appears to rely on astrocytic release of endovanilloid-like molecules. A subset of NTS neurons excited by PAR1 directly project to the rostral ventral respiratory group. The PAR1 initiated, TRPV1-dependent modulation of synaptic transmission in the NTS contributes to regulation of breathing., Abstract: Many of the cellular and molecular mechanisms underlying astrocytic modulation of synaptic function remain poorly understood. Recent studies show that G-protein coupled receptor-mediated astrocyte activation modulates synaptic transmission in the nucleus of the solitary tract (NTS), a brainstem nucleus that regulates crucial physiological processes including cardiorespiratory activity. By using calcium imaging and patch clamp recordings in acute brain slices of wild-type and TRPV1 -/- rats, we show that activation of proteinase-activated receptor 1 (PAR1) in NTS astrocytes potentiates presynaptic glutamate release on NTS neurons. This potentiation is mediated by both a TRPV1-dependent and a TRPV1-independent mechanism. The TRPV1-dependent mechanism appears to require release of endovanilloid-like molecules from astrocytes, which leads to subsequent potentiation of presynaptic glutamate release via activation of presynaptic TRPV1 channels. Activation of NTS astrocytic PAR1 receptors elicits cFOS expression in neurons that project to respiratory premotor neurons and inhibits respiratory activity in control, but not in TRPV1 -/- rats. Thus, activation of astrocytic PAR1 receptor in the NTS leads to a TRPV1-dependent excitation of NTS neurons causing a potent modulation of respiratory motor output., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

By determining its cellular localization in the nucleus tractus solitarii (NTS), we sought anatomical support for a putative physiological role for acid-sensing ion channel Type 1 (ASIC1) in chemosensitivity. Further, we sought to determine the effect of a lesion that produces gliosis in the area. In rats, we studied ASIC1 expression in control tissue with that in tissue with gliosis, which is associated with acidosis, after saporin lesions. We hypothesized that saporin would increase ASIC1 expression in areas of gliosis. Using fluorescent immunohistochemistry and confocal microscopy, we found that cells and processes containing ASIC1-immunoreactivity (IR) were present in the NTS, the dorsal motor nucleus of vagus, and the area postrema. In control tissue, ASIC1-IR predominantly colocalized with IR for the astrocyte marker, glial fibrillary acidic protein (GFAP), or the microglial marker, integrin αM (OX42). The subpostremal NTS was the only NTS region where neurons, identified by protein gene product 9.5 (PGP9.5), contained ASIC1-IR. ASIC1-IR increased significantly (157 ± 8.6% of control, p < 0.001) in the NTS seven days after microinjection of saporin. As we reported previously, GFAP-IR was decreased in the center of the saporin injection site, but GFAP-IR was increased in the surrounding areas where OX42-IR, indicative of activated microglia, was also increased. The over-expressed ASIC1-IR colocalized with GFAP-IR and OX42-IR in those reactive astrocytes and microglia. Our results support the hypothesis that ASIC1 would be increased in activated microglia and in reactive astrocytes after injection of saporin into the NTS.

Angiotensin II acts via two main receptors within the central nervous system, with the type 1A receptor (AT 1A R) most widely expressed in adult neurons. Activation of the AT 1 R in the nucleus of the solitary tract (NTS), the principal nucleus receiving central synapses of viscerosensory afferents, modulates cardiovascular reflexes. Expression of the AT 1 R occurs in high density within the NTS of most mammals, including humans, but the fundamental electrophysiological and neurochemical characteristics of the AT 1A R-expressing NTS neurons are not known. To address this, we have used a transgenic mouse, in which the AT 1A R promoter drives expression of green fluorescent protein (GFP). Approximately one-third of AT 1A R-expressing neurons express the catecholamine-synthetic enzyme tyrosine hydroxylase (TH), and a subpopulation of these stained for the transcription factor paired-like homeobox 2b (Phox2b). A third group, comprising approximately two-thirds of the AT 1A R-expressing NTS neurons, showed Phox2b immunoreactivity alone. A fourth group in the ventral subnucleus expressed neither TH nor Phox2b. In whole cell recordings from slices in vitro, AT 1A R-GFP neurons exhibited voltage-activated potassium currents, including the transient outward current and the M-type potassium current. In two different mouse strains, both AT 1A R-GFP neurons and TH-GFP neurons showed similar AT 1A R-mediated depolarizing responses to superfusion with angiotensin II. These data provide a comprehensive description of AT 1A R-expressing neurons in the NTS and increase our understanding of the complex actions of this neuropeptide in the modulation of viscerosensory processing., (Copyright © 2017 the American Physiological Society.)

Cholecystokinin (CCK)-expressing neurons within the nucleus of the solitary tract (CCK NTS ) of the mouse are responsive to satiety signals and their chemogenetic activation suppresses appetite. Optogenetic activation of CCK NTS axon terminals within either the parabrachial nucleus (PBN) or the paraventricular nucleus of the hypothalamus (PVH) is sufficient to suppress feeding. An interesting dichotomy has been revealed when assessing the motivational valence of these two circuits. Activating CCK NTS cell bodies is aversive as demonstrated by conditioned taste aversion and place-preference assays. Activation of the CCK NTS →PBN pathway is also aversive; however, stimulating the CCK NTS →PVH pathway is appetitive when assayed using a real-time, place-preference task. Thus, these two projections from CCK NTS neurons reduce food intake through opposite motivational states; one pathway signals positive valence (CCK NTS →PVH) and the other signals negative valence (CCK NTS →PBN)., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Glucose is a crucial substrate essential for cell survival and function. Changes in glucose levels impact neuronal activity and glucose deprivation increases feeding. Several brain regions have been shown to respond to glucoprivation, including the nucleus of the solitary tract (NTS) in the brain stem. The NTS is the primary site in the brain that receives visceral afferent information from the gastrointestinal tract. The catecholaminergic (CA) subpopulation within the NTS modulates many homeostatic functions including cardiovascular reflexes, respiration, food intake, arousal, and stress. However, it is not known if they respond to changes in glucose. Here we determined whether NTS-CA neurons respond to changes in glucose concentration and the mechanism involved. We found that decreasing glucose concentrations from 5 mM to 2 mM to 1 mM, significantly decreased action potential firing in a cell-attached preparation, whereas increasing it back to 5 mM increased the firing rate. This effect was dependent on glutamate release from afferent terminals and required presynaptic 5-HT 3 Rs. Decreasing the glucose concentration also decreased both basal and 5-HT 3 R agonist-induced increase in the frequency of spontaneous glutamate inputs onto NTS-CA neurons. Low glucose also blunted 5-HT-induced inward currents in nodose ganglia neurons, which are the cell bodies of vagal afferents. The effect of low glucose in both nodose ganglia cells and in NTS slices was mimicked by the glucokinase inhibitor glucosamine. This study suggests that NTS-CA neurons are glucosensing through a presynaptic mechanism that is dependent on vagal glutamate release, 5-HT 3 R activity, and glucokinase., (Copyright © 2017 the American Physiological Society.)

The application of sodium cyanide (NaCN) to the carotid body receptors (CBR) (CBR stimulation) induces rapid blood hyperglycemia and an increase in brain glucose retention. The commissural nucleus tractus solitarius (cNTS) is an essential relay nucleus in this hyperglycemic reflex; it receives glutamatergic afferents (that also release brain derived neurotrophic factor, BDNF) from the nodose-petrosal ganglia that relays CBR information. Previous work showed that AMPA in NTS blocks hyperglycemia and brain glucose retention after CBR stimulation. In contrast, BDNF, which attenuates glutamatergic AMPA currents in NTS, enhances these glycemic responses. Here we investigated the combined effects of BDNF and AMPA (and their antagonists) in NTS on the glycemic responses to CBR stimulation. Microinjections of BDNF plus AMPA into the cNTS before CBR stimulation in anesthetized rats, induced blood hyperglycemia and an increase in brain arteriovenous (a-v) of blood glucose concentration difference, which we infer is due to increased brain glucose retention. By contrast, the microinjection of the TrkB antagonist K252a plus AMPA abolished the glycemic responses to CBR stimulation similar to what is observed after AMPA pretreatments. In BDNF plus AMPA microinjections preceding CBR stimulation, the number of c-fos immunoreactive cNTS neurons increased. In contrast, in the rats microinjected with K252a plus AMPA in NTS, before CBR stimulation, c-fos expression in cNTS decreased. The expression of AMPA receptors GluR2/3 did not change in any of the studied groups. These results indicate that BDNF in cNTS plays a key role in the modulation of the hyperglycemic reflex initiated by CBR stimulation., (Copyright © 2017. Published by Elsevier B.V.)

Objective: Glucagon-like peptide-1 (GLP-1) and 5-HT are potent regulators of food intake within the brain. GLP-1 is expressed by preproglucagon (PPG) neurons in the nucleus tractus solitarius (NTS). We have previously shown that PPG neurons innervate 5-HT neurons in the ventral brainstem. Here, we investigate whether PPG neurons receive serotonergic input and respond to 5-HT., Methods: We employed immunohistochemistry to reveal serotonergic innervation of PPG neurons. We investigated the responsiveness of PPG neurons to 5-HT using in vitro Ca 2+ imaging in brainstem slices from transgenic mice expressing the Ca 2+ indicator, GCaMP3, in PPG neurons, and cell-attached patch-clamp recordings., Results: Close appositions from 5-HT-immunoreactive axons occurred on many PPG neurons. Application of 20 μM 5-HT produced robust Ca 2+ responses in NTS PPG dendrites but little change in somata. Dendritic Ca 2+ spikes were concentration-dependent (2, 20, and 200 μM) and unaffected by blockade of glutamatergic transmission, suggesting 5-HT receptors on PPG neurons. Neither activation nor blockade of 5-HT 3 receptors affected [Ca 2+ ] i . In contrast, inhibition of 5-HT 2 receptors attenuated increases in intracellular Ca 2+ and 5-HT 2C receptor activation produced Ca 2+ spikes. Patch-clamp recordings revealed that 44% of cells decreased their firing rate under 5-HT, an effect blocked by 5-HT 1A receptor antagonism., Conclusions: PPG neurons respond directly to 5-HT with a 5-HT 2C receptor-dependent increase in dendritic [Ca 2+ ] i . Electrical responses to 5-HT revealed additional inhibitory effects due to somatic 5-HT 1A receptors. Reciprocal innervation between 5-HT and PPG neurons suggests that the coordinated activity of these brainstem neurons may play a role in the regulation of food intake.

Reactive oxygen species (ROS) play a profound role in cardiorespiratory function under normal physiological conditions and disease states. ROS can influence neuronal activity by altering various ion channels and transporters. Within the nucleus tractus solitarii (nTS), a vital brainstem area for cardiorespiratory control, hydrogen peroxide (H 2 O 2 ) induces sustained hyperexcitability following an initial depression of neuronal activity. The mechanism(s) associated with the delayed hyperexcitability are unknown. Here we evaluate the effect(s) of H 2 O 2 on cytosolic Ca 2+ (via fura-2 imaging) and voltage-dependent calcium currents in dissociated rat nTS neurons. H 2 O 2 perfusion (200 µM; 1 min) induced a delayed, slow, and moderate increase (~27%) in intracellular Ca 2+ concentration ([Ca 2+ ] i ). The H 2 O 2 -mediated increase in [Ca 2+ ] i prevailed during thapsigargin, excluding the endoplasmic reticulum as a Ca 2+ source. The effect, however, was abolished by removal of extracellular Ca 2+ or the addition of cadmium to the bath solution, suggesting voltage-gated Ca 2+ channels (VGCCs) as targets for H 2 O 2 modulation. Recording of the total voltage-dependent Ca 2+ current confirmed H 2 O 2 enhanced Ca 2+ entry. Blocking VGCC L, N, and P/Q subtypes decreased the number of cells and their calcium currents that respond to H 2 O 2 The number of responder cells to H 2 O 2 also decreased in the presence of dithiothreitol, suggesting the actions of H 2 O 2 were dependent on sulfhydryl oxidation. In summary, here, we have shown that H 2 O 2 increases [Ca 2+ ] i and its Ca 2+ currents, which is dependent on multiple VGCCs likely by oxidation of sulfhydryl groups. These processes presumably contribute to the previously observed delayed hyperexcitability of nTS neurons in in vitro brainstem slices., (Copyright © 2017 the American Physiological Society.)

Objective: Fos protein expression in catecholamine-synthesizing neurons of the substantia nigra (SN) pars compacta (SNC, A8), pars reticulata (SNR, A9), and pars lateralis (SNL), the ventral tegmental area (VTA, A10), the locus coeruleus (LC, A6) and subcoeruleus (sLC), the ventrolateral pons (PON-A5), the nucleus of the solitary tract (NTS-A2), the area postrema (AP), and the ventrolateral medulla (VLM-A1) was quantitatively evaluated aft er a single administration of asenapine (ASE) (designated for schizophrenia treatment) in male Wistar rats preconditioned with a chronic unpredictable variable mild stress (CMS) for 21 days. Th e aim of the present study was to reveal whether a single ASE treatment may 1) activate Fos expression in the brain areas selected; 2) activate tyrosine hydroxylase (TH)-synthesizing cells displaying Fos presence; and 3) be modulated by CMS preconditioning., Methods: Control (CON), ASE, CMS, and CMS+ASE groups were used. CMS included restraint, social isolation, crowding, swimming, and cold. Th e ASE and CMS+ASE groups received a single dose of ASE (0.3 mg/kg, s.c.) and CON and CMS saline (300 μl/rat, s.c.). The animals were sacrificed 90 min aft er the treatments. Fos protein and TH-labeled immunoreactive perikarya were analyzed on double labeled histological sections and enumerated on captured pictures using combined light and fluorescence microscope illumination., Results: Saline or CMS alone did not promote Fos expression in any of the structures investigated. ASE alone or in combination with CMS elicited Fos expression in two parts of the SN (SNC, SNR) and the VTA. Aside from some cells in the central gray tegmental nuclei adjacent to LC, where a small number of Fos profiles occurred, none or negligible Fos occurrence was detected in the other structures investigated including the LC and sLC, PON-A5, NTS-A2, AP, and VLM-A1. CMS preconditioning did not infl uence the level of Fos induction in the SN and VTA elicited by ASE administration. Similarly, the ratio between the amount of free Fos and Fos colocalized with TH was not aff ected by stress preconditioning in the SNC, SNR, and the VTA., Conclusions: Th e present study provides an anatomical/functional knowledge about the nature of the acute ASE treatment on the catecholamine-synthesizing neurons activity in certain brain structures and their missing interplay with the CMS preconditioning.

Previously, many different types of NTS barosensitive neurons were identified. However, the time course of NTS barosensitive neuronal activity (NA) in response to arterial pressure (AP) changes, and the relationship of NA-AP changes, have not yet been fully quantified. In this study, we made extracellular recordings of single NTS neurons firing in response to AP elevation induced by occlusion of the descending aorta in anesthetized rats. Our findings were that: 1) Thirty-five neurons (from 46 neurons) increased firing, whereas others neurons either decreased firing upon AP elevation, or were biphasic: first decreased firing upon AP elevation and then increased firing during AP decrease. 2) Fourteen neurons with excitatory responses were activated and rapidly increased their firing during the early phase of AP increase (early neurons); whereas 21 neurons did not increase firing until the mean arterial pressure changes (ΔMAP) reached near/after the peak (late neurons). 3) The early neurons had a significantly higher firing rate than late neurons during AP elevation at a similar rate. 4) Early neuron NA-ΔMAP relationship could be well fitted and characterized by the sigmoid logistic function with the maximal gain of 29.3. 5) The increase of early NA correlated linearly with the initial heart rate (HR) reduction. 6) The late neurons did not contribute to the initial HR reduction. However, the late NA could be well correlated with HR reduction during the late phase. Altogether, our study demonstrated that the NTS excitatory neurons could be grouped into early and late neurons based on their firing patterns. The early neurons could be characterized by the sigmoid logistic function, and different neurons may differently contribute to HR regulation. Importantly, the grouping and quantitative methods used in this study may provide a useful tool for future assessment of functional changes of early and late neurons in disease models.

Ventilatory acclimatization to hypoxia (VAH) is the time-dependent increase in ventilation, which persists upon return to normoxia and involves plasticity in both central nervous system respiratory centers and peripheral chemoreceptors. We investigated the role of glial cells in VAH in male Sprague-Dawley rats using minocycline, an antibiotic that inhibits microglia activation and has anti-inflammatory properties, and barometric pressure plethysmography to measure ventilation. Rats received either minocycline (45mg/kg ip daily) or saline beginning 1 day before and during 7 days of chronic hypoxia (CH, Pi O 2  = 70 Torr). Minocycline had no effect on normoxic control rats or the hypercapnic ventilatory response in CH rats, but minocycline significantly ( P < 0.001) decreased ventilation during acute hypoxia in CH rats. However, minocycline administration during only the last 3 days of CH did not reverse VAH. Microglia and astrocyte activation in the nucleus tractus solitarius was quantified from 30 min to 7 days of CH. Microglia showed an active morphology (shorter and fewer branches) after 1 h of hypoxia and returned to the control state (longer filaments and extensive branching) after 4 h of CH. Astrocytes increased glial fibrillary acidic protein antibody immunofluorescent intensity, indicating activation, at both 4 and 24 h of CH. Minocycline had no effect on glia in normoxia but significantly decreased microglia activation at 1 h of CH and astrocyte activation at 24 h of CH. These results support a role for glial cells, providing an early signal for the induction but not maintenance of neural plasticity underlying ventilatory acclimatization to hypoxia. NEW & NOTEWORTHY The signals for neural plasticity in medullary respiratory centers underlying ventilatory acclimatization to chronic hypoxia are unknown. We show that chronic hypoxia activates microglia and subsequently astrocytes. Minocycline, an antibiotic that blocks microglial activation and has anti-inflammatory properties, also blocks astrocyte activation in respiratory centers during chronic hypoxia and ventilatory acclimatization. However, minocycline cannot reverse ventilatory acclimatization after it is established. Hence, glial cells may provide signals that initiate but do not sustain ventilatory acclimatization., (Copyright © 2017 the American Physiological Society.)

Background: The effect of electroacupuncture (EA) is affected by both the acupuncture point selection and the frequency of stimulation. However, little is known regarding acupuncture point and simulation frequency selection. Neuronal activation of the nucleus of the solitary tract (NTS) is one of the important targets of EA for modulating gastrointestinal function. This study investigated the effects of various combinations of EA frequencies and acupuncture points on NTS neurons., Methods: Rats were randomly divided into normal, 2 Hz EA, 100 Hz EA and the alternate 2/100 Hz EA groups. Then rats in each group were randomly divided into the following two subgroups according to the acupuncture point: ST 36 group and ST 25 group. All the rats underwent electrode implantation surgery. Rats in all EA groups received one treatment with EA (a constant square wave at, 2 Hz,100 Hz or 2/100 Hz frequencies with intensities ranging from 1 to 2 mA), and NTS neuronal activation was recorded before and after EA treatment. Finally, to confirm the effect of EA on the NTS, minimal acupuncture was administered and its effect on NTS was detected., Results: ST 36 stimulated with 2 Hz EA significantly increased the population of excited NTS neurons and spike frequency. However, ST 36 stimulated with 100 Hz or 2/100 Hz EA produced only a transient effect on the activity of NTS neurons and did not induce any effect on the spike frequency. Furthermore, the excitatory effect of 100 Hz or 2/100 Hz EA on NTS neurons in the ST 36 group was lower than 2 Hz EA at the same point. When applied to ST 25, 2 Hz EA had no significant excitatory effect on NTS neurons or spike frequency. However, 100 Hz EA or 2/100 Hz EA at ST 25 decreased both NTS neuronal excitability and spike frequency. By comparing the effects of different EA combinations, it was shown 2 Hz EA applied to ST 36 had the strongest excitatory effect on NTS neurons, while 100 Hz EA applied to ST 25 had the greatest inhibitory effect. Minimal acupuncture stimulation produced no effect on NTS neurons., Conclusion: EA's effects on NTS were mainly affected by the acupuncture point selection, but the frequency of EA also played a role. Different combinations of acupuncture points and frequency selection may lead to different EA effects on NTS neuronal excitability.

Sensory inputs from the oropharynx terminate in both the trigeminal brainstem complex and the rostral part of the nucleus of the solitary tract (nTS). Taste information is conveyed via the facial and glossopharyngeal nerves, while general mucosal innervation is carried by the trigeminal and glossopharyngeal nerves. In contrast, the caudal nTS receives general visceral information largely from the vagus nerve. Although the caudal nTS shows clear morphological and molecularly delimited subdivisions, the rostral part does not. Thus, linking taste-induced patterns of activity to morphological subdivisions in the nTS is challenging. To test whether molecularly defined features of the rostral nTS correlate with patterns of taste-induced activity, we combined immunohistochemistry for markers of various visceral afferent and efferent systems with c-Fos-based activity maps generated by stimulation with a sour tastant, 30 mM citric acid. We further dissociated taste-related activity from activity arising from acid-sensitive general mucosal innervation by comparing acid-evoked c-Fos in wild-type and "taste blind" P2X 2 /P2X 3 double knockout (P2X-dbl KO) mice. In wild-type mice, citric acid stimulation evoked significant c-Fos activation in the central part of the rostral nTS-activity that was largely absent in the P2X-dbl KO mice. P2X-dbl KO mice, like wild-type mice, did exhibit acid-induced c-Fos activity in the dorsomedial trigeminal brainstem nucleus situated laterally adjacent to the rostral nTS. This dorsomedial nucleus also showed substantial innervation by trigeminal nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP), a marker for polymodal nociceptors, suggesting that trigeminal general mucosal innervation carries information about acids in the oral cavity. J. Comp. Neurol. 525:271-290, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)