NTS Neurons Research Articles

CCK expressing neurons in the NTS are directly activated by CCK-sensitive C-type vagal afferents.

Vagal sensory afferents carrying information from the gastrointestinal tract (GI) terminate in the nucleus of the solitary tract (NTS). Different subpopulations of NTS neurons then relay this information throughout the brain. Cholecystokinin (CCK) is a satiety peptide that activates vagal afferents in the GI. However, CCK is also expressed by neurons in the NTS, and activation of these neurons decreases food intake. What is less clear is how these NTS CCK neurons are activated by vagal afferents and what type of information they integrate about meal size and content. To address this, we identified NTS-CCK neurons by crossing CCK-IRES-Cre mice with floxed-Rosa-tdtomato mice and made a horizontal brain slice containing vagal afferents in the solitary tract (ST). Voltage clamp recordings of NTS-CCK neurons show that activation of the ST evokes excitatory post-synaptic currents (EPSCs) mediated by both AMPA and NMDA receptors. Analysis of these EPSCs revealed that 80% of NTS-CCK neurons receive direct, monosynaptic inputs, with many also receiving indirect, or polysynaptic, inputs. NTS-CCK neurons are sensitive to the TRPV1 agonist capsaicin, suggesting they are downstream of C-fibers. In addition, both CCK and a 5-HT3R agonist increased sEPSC frequency in NTS-CCK neurons, with 69% of NTS-CCK neurons sensitive to CCK and 42% to 5-HT3 receptor agonists, as well as 45% sensitive to both and 10% to neither. Taken together with previous studies, this suggests that NTS-CCK neurons are driven primarily by vagal afferents that are sensitive to CCK and are only weakly driven by those sensitive to 5-HT.

Brainstem opioid peptidergic neurons regulate cough reflexes in mice

Cough is a vital defensive reflex for expelling harmful substances from the airway. The sensory afferents for the cough reflex have been intensively studied. However, the brain mechanisms underlying the cough reflex remain poorly understood. Here, we developed a paradigm to quantitatively measure cough-like reflexes in mice. Using this paradigm, we found that prodynorphin-expressing (Pdyn+) neurons in the nucleus of the solitary tract (NTS) are critical for capsaicin-induced cough-like reflexes. These neurons receive cough-related neural signals from Trpv1+ vagal sensory neurons. The activation of Pdyn+ NTS neurons triggered respiratory responses resembling cough-like reflexes. Among the divergent projections of Pdyn+ NTS neurons, a glutamatergic pathway projecting to the caudal ventral respiratory group (cVRG), the canonical cough center, was necessary and sufficient for capsaicin-induced cough-like reflexes. These results reveal that Pdyn+ NTS neurons, as a key neuronal population at the entry point of the vagus nerve to the brainstem, initiate cough-like reflexes in mice.

Abstract P169: SGLT2 Colocalizes on the Nucleus Tractus Solitarii Neurons in Normotensive and Hypertensive Rats in a Location-Dependent Manner

Introduction/Background: Hypertension affects ~45% adults in the US and is associated with a high sodium diet. The nucleus tractus solitarii (nTS) in the brainstem influences blood pressure through the interplay between glutamate and GABA. Sodium-glucose cotransporter 2 (SGLT2) is a protein recently found within the nTS, and its expression in the nTS may impact blood pressure. In this study, we investigated the distribution of SGLT2 in the nTS of normotensive and hypertensive rats, focusing on its presence in glutamatergic (Glu) and GABAergic neurons. Within the nTS, increases in glutamate lowers blood pressure, and increases of GABA raises blood pressure. The interaction between SGLT2 and glutamate/GABAergic neurons within the nTS is not well-understood. Hypothesis: We hypothesize that SGLT2 colocalizes on both Glu and GABAergic nTS neurons but increases due to a high salt diet. Methods: Male Dahl-Salt Sensitive Rats were fed a normal 0.4% salt diet (NSD) for 2 weeks and randomly placed to a NSD or a high 4% salt diet (HSD) for 7 weeks. After 9 weeks total, the brainstem section containing the nTS of each rat was isolated at 9AM. Goals/Aims: We used immunohistochemistry to identify either Glu (CAMKII) and GABAergic (GAD67) and localize SGLT2. Using a confocal microscope, a Z-stack image was compiled at 40x magnification and colocalization analysis was completed to determine if SGLT2 is found greater on Glu or GABAergic neurons. Results: We examined 3 areas within the nTS: caudal, rostral, and mid (area postrema, a region where cardiorespiratory afferent information is received). We first found that SGLT2 colocalizes to different extents on both Glu and GABAergic nTS neurons. We used Pearson’s R correlation to determine the strength and linear relationship between cell type and SGLT2. In GABAergic neurons, we found a positive correlation (r=0.319) in NSD, which decreased significantly in HSD (p=0.013, t-test) in the nTS at area postrema. Conversely, within the caudal nTS, SGLT2 colocalizes to a greater extent on Glu neurons in HSD (r=0.434) and lower in NSD (r=0.308), (p=0.04, t-test). Conclusions: In our model, we find that SGLT2 colocalization with Glu and GABAergic neurons is present within the nTS in a location-dependent manner, which may affect its function in the development of hypertension. Further experiments exploring the SGLT2 within normotensive and hypertensive rats may better reveal the function of the protein within the nTS.

Opposing actions of co-released GABA and neurotensin on the activity of preoptic neurons and on body temperature.

Neurotensin (Nts) is a neuropeptide acting as a neuromodulator in the brain. Pharmacological studies have identified Nts as a potent hypothermic agent. The medial preoptic area, a region that plays an important role in the control of thermoregulation, contains a high density of neurotensinergic neurons and Nts receptors. The conditions in which neurotensinergic neurons play a role in thermoregulation are not known. In this study, optogenetic stimulation of preoptic Nts neurons induced a small hyperthermia. In vitro, optogenetic stimulation of preoptic Nts neurons resulted in synaptic release of GABA and net inhibition of the preoptic pituitary adenylate cyclase-activating polypeptide (Adcyap1) neurons firing activity. GABA-A receptor antagonist or genetic deletion of Slc32a1 (VGAT) in Nts neurons unmasked also an excitatory effect that was blocked by a Nts receptor 1 antagonist. Stimulation of preoptic Nts neurons lacking Slc32a1 resulted in excitation of Adcyap1 neurons and hypothermia. Mice lacking Slc32a1 expression in Nts neurons presented changes in the fever response and in the responses to heat or cold exposure as well as an altered circadian rhythm of body temperature. Chemogenetic activation of all Nts neurons in the brain induced a 4-5°C hypothermia, which could be blocked by Nts receptor antagonists in the preoptic area. Chemogenetic activation of preoptic neurotensinergic projections resulted in robust excitation of preoptic Adcyap1 neurons. Taken together, our data demonstrate that endogenously released Nts can induce potent hypothermia and that excitation of preoptic Adcyap1 neurons is the cellular mechanism that triggers this response.

The nucleus of solitary tract (NTS) synchronizes sleep-wake-state-dependent cortical activity through the parabrachial nucleus (PB) in rat

BackgroundThe medullary nucleus of solitary tract (NTS) and its afferents of vagus nerve have long been investigated in regulation of cortical activity and sleep promotion. However, the underlying neural circuit by which the NTS regulates electroencephalogram (EEG) and sleep remain unclear. As the NTS has a strong projection to the pontine arousal site, the parabrachial nucleus (PB), we proposed the NTS via the pontine parabrachial nucleus (PB) regulates cortical activity and sleep. MethodsWe bilaterally and directly stimulated the NTS neurons by chemogenetic approach and NTS terminals in the PB by optogenetic approach and examined changes in EEG and sleep in rats. ResultsOpto- and chemo-stimulation of the NTS and NTS-PB pathway altered neither sleep amounts nor patterns; however, both stimulations consistently increased EEG delta (0.5–4.0 Hz) EEG power during non-rapid-eye-movement (NREM) sleep and alpha-beta (10–30 Hz) EEG power during wake and REM sleep. ConclusionOur results indicate that the NTS via its projections to the PB synchronizes low frequency EEG during NREM sleep and high frequency EEG during wake and REM sleep. This pathway may serve the neural foundation for the vagus nerve stimulation (VNS) treating cortical disorders.

Norepinephrine Neurons in the Nucleus of the Solitary Tract Suppress Luteinizing Hormone Secretion in Female Mice.

Stress impairs fertility, at least in part, via inhibition of gonadotropin secretion. Luteinizing hormone (LH) is an important gonadotropin that is released in a pulsatile pattern in males and in females throughout the majority of the ovarian cycle. Several models of stress, including acute metabolic stress, suppress LH pulses via inhibition of neurons in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy cells) which form the pulse generator. The mechanism for inhibition of KNDy neurons during stress, however, remains a significant outstanding question. Here, we investigated a population of catecholamine neurons in the nucleus of the solitary tract (NTS), marked by expression of the enzyme dopamine beta-hydroxylase (DBH), in female mice. First, we found that a subpopulation of DBH neurons in the NTS is activated (express c-Fos) during metabolic stress. Then, using chemogenetics, we determined that activation of these cells is sufficient to suppress LH pulses, augment corticosterone secretion, and induce sickness-like behavior. In subsequent studies, we identified evidence for suppression of KNDy cells (rather than downstream signaling pathways) and determined that the suppression of LH pulses was not dependent on the acute rise in glucocorticoids. Together these data support the hypothesis that DBH cells in the NTS are important for regulation of neuroendocrine and behavioral responses to stress.

Viscerosensory signalling to the nucleus accumbens via the solitary tract nucleus.

The nucleus of the solitary tract (NTS) receives direct viscerosensory vagal afferent input that drives autonomic reflexes, neuroendocrine function and modulates behaviour. A subpopulation of NTS neurons project to the nucleus accumbens (NAc); however, the function of this NTS-NAc pathway remains unknown. A combination of neuroanatomical tracing, slice electrophysiology and fibre photometry was used in mice and/or rats to determine how NTS-NAc neurons fit within the viscerosensory network. NTS-NAc projection neurons are predominantly located in the medial and caudal portions of the NTS with 54 ± 7% (mice) and 65 ± 3% (rat) being TH-positive, representing the A2 NTS cell group. In horizontal brainstem slices, solitary tract (ST) stimulation evoked excitatory post-synaptic currents (EPSCs) in NTS-NAc projection neurons. The majority (75%) received low-jitter, zero-failure EPSCs characteristic of monosynaptic ST afferent input that identifies them as second order to primary sensory neurons. We then examined whether NTS-NAc neurons respond to cholecystokinin (CCK, 20 μg/kg ip) invivo in both mice and rats. Surprisingly, there was no difference in the number of activated NTS-NAc cells between CCK and saline-treated mice. In rats, just 6% of NTS-NAc cells were recruited by CCK. As NTS TH neurons are the primary source for NAc noradrenaline, we measured noradrenaline release in the NAc and showed that NAc noradrenaline levels declined in response to cue-induced reward retrieval but not foot shock. Combined, these findings suggest that high-fidelity afferent information from viscerosensory afferents reaches the NAc. These signals are likely unrelated to CCK-sensitive vagal afferents but could interact with other sensory and higher order inputs to modulate learned appetitive behaviours.

Brainstem Dbh+ neurons control allergen-induced airway hyperreactivity

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

Involvement of D1 dopamine receptor in the nucleus of the solitary tract of rats in stress-induced hypertension and exercise.

Chronic stress can cause hypertension, whereas daily exercise promotes healthy well being through destressing. Although the nucleus of the solitary tract (NTS) is involved in the development of hypertension, the molecular and physiological mechanisms of stress and exercise remain unclear. In this study, we tested whether gene expression in the NTS is altered by stress and daily exercise and whether this is involved in cardiovascular regulation. We have performed RT 2 Profiler PCR arrays targeting a panel of neurotransmitter receptor genes in the NTS of Wistar rats subjected to chronic restraint stress (1 h a day over 3 weeks) with or without voluntary wheel exercise. We also performed immunohistochemistry to determine whether the identified molecules were expressed at the protein level. Additionally, microinjection studies in anesthetized rats were performed to examine whether validated molecules exhibit physiological roles in cardiovascular regulation of the NTS. We observed that blood pressure was significantly increased by stress and the increase was suppressed by exercise. Using PCR analysis, we determined that the expression levels of four genes in the NTS, including the dopamine receptor D1 gene ( Drd1 ), were significantly affected by stress and suppressed by exercise. We also examined dopamine D1 receptor (D1R) expression in NTS neurons and found significantly greater expression in the stressed than nonstressed animals. Furthermore, the microinjection of a D1R agonist into the NTS in anesthetized rats induced hypotensive effects. These results suggest that NTS D1R plays a role in the counteracting processes of stress-induced hypertension.

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

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

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

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

Orexin 1 Receptors in The Paraventricular Nucleus of The Hypothalamus (PVN) Facilitate The Peripheral Chemoreflex

Increased sensitivity of the peripheral chemoreflex (PCR) to hypoxia is associated with cardiovascular-related diseases such as hypertension and heart failure. We previously showed that systemic blockade of both orexin 1 (Ox1R) and orexin 2 receptors reduced the PCR-mediated hypoxic ventilatory response (HVR), and that hypoxia activates PVN-projecting orexin neurons. The effect of OxR blockade on the HVR was strongest in the active phase of the circadian cycle and was associated with decreased numbers of activated neurons in the nucleus of the solitary tract (nTS) and nTS-projecting corticotropin-releasing neurons in the PVN. While the projection from the PVN to the nTS contributes to the PCR, the neuronal mechanism(s) activating this projection are unclear. We hypothesized that orexin acting at Ox1R in the PVN facilitates the PCR. To test this hypothesis, we first performed IHC to confirm the expression of Ox1R in the PVN. We then assessed the degree to which specific Ox1R blockade reduced the HVR (n=6; tested first with vehicle (100% DMSO), then Ox1R antagonist SB-334867, 1 mg/kg, i.p). To assess the contribution of PVN Ox1Rs to the PCR as well as the associated activation of neurons in the PVN and nTS, we specifically knocked down Ox1R expression in the PVN via nanoinjection of an Ox1R shRNA-GFP expressing AAV vector. Rats were exposed to graded hypoxia during the active phase, with inspired O2 taken from 21% to 11% over a ~5 min period. Rats were kept near-isocapnic by slightly increasing the inspired CO2. Subsets of rats were exposed to hypoxia to assess the effects of Ox1R on neuronal activation using c-Fos expression as a surrogate marker. Systemic Ox1R blockade significantly reduced the HVR compared to the control response (by ~30%; p=0.0027). Knockdown of Ox1R in the PVN also reduced the HVR (by ~20%; p=0.049) and reduced the number of c-Fos expressing PVN neurons in response to hypoxia (p=0.0232). More specifically, hypoxia induced c-Fos expression in fewer PVN neurons transfected with Ox1R shRNA (confirmed by GFP expression) than neurons from control rats transfected with scrambled RNA (p=0.0083). Ox1R knockdown in the PVN also significantly reduced the hypoxia-induced increase in number of c-Fos expressing tyrosine-hydroxylase (TH) nTS neurons (p=0.0303), suggesting that PVN Ox1Rs mediate hypoxia-induced activation of TH neurons in the nTS. These findings suggest that in the PVN, orexin acting at Ox1Rs mediate the facilitation of the PCR and the associated activation of the PVN and nTS neurons. Ox1R in the PVN could be a novel target to treat diseases associated with increased PCR sensitivity. Grants: 5R01HL098602 (to DK, EH and KC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modulation by Astrocytes of AMPA Currents in the NTS Neurons of Mice Is Reduced by Short Term-Sustained Hypoxia

Hypoxia due to reduction in the partial pressure of oxygen in the arterial blood (PaO2) activates the afferents of peripheral chemoreflex to produce cardiovascular and respiratory adjustments to bring PaO2 back to the normal range. Herein we evaluated the possible changes of astrocytic modulation on glutamatergic transmission in NTS neurons of mice submitted to short term-sustained hypoxia (SH). To reach this goal, we used Male C57Bl/6J mice (18-20g), which were submitted to SH (FiO2 0.1) or normoxia for 24 hours. Brainstem slices and whole-cell patch clamp technique were used to evaluate the synaptic transmission in NTS neurons. The intracellular calcium concentration in NTS astrocytes was evaluated by multiphoton microscopy and transgenic Aldh-CRE-GCAMP6f mice (Institutional Ethical Committee Approvals: #016/2018 and #1109/2022R1). SH increased the amplitude of TS-evoked AMPA currents [control: -72.8 ± 13.3 pA ( n = 12) vs SH: -120 ± 14.4 pA ( n = 21)] in NTS neurons with no changes on the rise- or decay-time. SH also increased the amplitude [control: 35.88 ± 7.9 pA ( n = 12) vs SH: 72.74 ± 15.47 pA ( n = 10)] and decay time [control: 61.8 ± 15.2 ms ( n = 9) vs SH: 286 ± 86.7 ms ( n = 10)] of TS evoked-NMDA currents in neurons, while the amplitude of extrasynaptic NMDA currents [control: 28.7 ± 7.4 pA ( n = 10) vs SH: 24.7 ± 3.2 pA ( n = 7)] was not affected. Perfusion with fluoracetate (FAC), a glial metabolism inhibitor, decreased the amplitude of TS-evoked AMPA currents in control group [aCSF: -54.29 ± 16.1 pA vs aCSF + FAC: -24.43 ± 6.82 pA ( n =9)] without affecting AMPA currents in SH neurons [aCSF: -124.5 ± 35.27 pA vs aCSF + FAC: -130.5 ± 50.16 pA ( n =8)]. The passive properties of astrocytes did not change after SH [RMP: -83.73 ± 11.85 mV ( n =10) vs −83.63 ± 11.86 mV ( n =10); Rinput: 123 ± 127.5 MΩ ( n =9) vs 208 ± 212.7 MΩ ( n =10)]. The increase in intracellular calcium concentration induced by AMPA in the astrocytes of control and SH mice was similar [Control: 112.8 ± 15.41 ΔF/F0 (n=68 ROIs) vs SH: 56.38 ± 18.31 ΔF/F0 (n=13 ROIs)]. The data shows that SH increased the evoked glutamatergic currents in NTS neurons of mice, probably due to a reduction in the astrocytic modulation of excitatory transmission by mechanisms apparently independent of astrocytic intracellular calcium. FAPESP (2018/15957-2, 2018/02336-0 and 2022/05237-8), CNPq (309338/2020-4) and CAPES-PROEX (23038.006922/2021-31). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Functional Impact of nTS Glutamate Stress on Respiration in an Alzheimer’s Disease Model

Alzheimer’s disease (AD) is closely associated with obstructive sleep apnea (OSA). Such hypoxic insults trigger glutamate release of chemoafferents into the nucleus tractus solitarii (nTS), an important upstream center of the chemoreflex. Potential alterations of glutamate handling in the nTS may lead to the respiratory dysfunction seen in AD patients. Using the streptozotocin (STZ)-induced rat model — an effective proxy for human AD — we studied the functional consequences of nTS glutamate stress on respiration. STZ-AD was induced in 6-week-old male Sprague Dawley rats via intracerebroventricular injections of 2 - 2.25 mg/kg STZ. After two weeks, EMG recordings of diaphragm activity served as surrogate for respiratory responses to glutamate microinjections (20 nL of 40 mM) into the caudal nTS. In a subset of rats, chemoafferent terminals were labeled with the fluorescent tracer DiI to permit patch clamp recordings of identified 2nd order nTS neurons participating in the chemoreflex loop. Evoked glutamatergic excitatory postsynaptic currents (TS-EPSCs) were generated via tractus solitarii stimulation at frequencies (10 - 40 Hz) typical for afferent discharge during hypoxia. Acute glutamate injections into the caudal nTS increased respiratory frequency by 15 - 20 breaths per minute. The response magnitude was similar between CTL and STZ-AD animals. Excitatory stress with repeated glutamate injections (5 min. apart) evoked a reliable respiratory response in CTL that slowly declined to ~65% from its maximum over the course of 10 injections. In contrast, the decline of respiratory response in STZ-AD was more pronounced and occurred significantly earlier than in CTL, indicating altered glutamate handling in the caudal nTS of STZ-AD rats. Next, we used patch clamp electrophysiology to analyze increased glutamate release of chemoafferents onto 2nd order nTS neurons. High frequency stimulation of chemoafferents induced a successive depression of TS-EPSC amplitude in nTS neurons. TS-EPSC depression was significantly stronger in STZ-AD and may be due to altered glutamate handling at the synapse. Western Blot analysis of STZ-AD brainstem tissue including the nTS revealed a significant increase in glial fibrillary acidic protein (GFAP) expression and a decrease of excitatory amino acid transporters (EAATs) expression, suggesting astrocyte involvement in compromised nTS glutamate handling in STZ-AD. In summary, the STZ-AD model showed reduced respiratory responses to glutamate stress in the caudal nTS. The altered stress response may come from enhanced depression of TS-EPSC amplitude at nTS neurons in the chemoreflex. Altered astrocytic glutamate removal from the synaptic cleft may contribute to TS-EPSC depression. Together, altered glutamate handling in a respiratory brainstem center may contribute to OSA in AD patients. NIH R15AG065927 (TDO & DO), KCOM Biomedical Graduate Program (RKT &TDO), ATSU Research Support (SKRC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic Vagotomy Blunts Cardiorespiratory Reflex Responses Through Pre- and Postsynaptic Effects in the Brainstem Nucleus Tractus Solitarii (nTS)

Sensory information from the heart, lungs, and gastrointestinal tract travels to the brain via afferents of the paired vagus nerves, where it is first integrated in the brainstem nTS that critically contributes to cardiorespiratory function. We have shown that reducing input to the nTS by unilaterally transecting the vagus nerve (vagotomy) induces astrocyte reactivity and microglial activation, and reduces sighs in hypoxia. However, the cardiorespiratory effects of vagotomy and contributing mechanisms are not well understood. We hypothesized that chronic vagotomy blunts cardiorespiratory reflex responses to vagal afferent stimulation via reduced nTS neuronal activity. Male Sprague-Dawley rats (6-wk) underwent right cervical vagotomy (or sham surgery) caudal to the nodose ganglion. After 7 days rats were anesthetized, ventilated, and instrumented to record mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA, respectively), which decreased in response to vagal afferent stimulation in sham animals. Vagotomy blunted these decreases in MAP, SSNA, and PhrNA compared to shams. In rats expressing the calcium indicator GCaMP8m in nTS neurons, vagotomy also reduced neuronal Ca2+ responses to afferent stimulation both in vivo (photometry) and in vitro (live slice imaging). We then hypothesized that these changes were due to altered signaling at the level of the tripartite synapse, where astrocytes enwrap the sensory afferent-nTS soma synapse. We found that there is likely a vagal afferent contribution to the altered reflex responses, as vagotomy reduced presynaptic Ca2+ responses in vagal afferent terminals to solitary tract stimulation in the brainstem slice. Glutamate agonists, which induce similar cardiorespiratory responses as vagal stimulation, were nanoinjected into the nTS to determine if there was a postsynaptic contribution to the blunted responses as well. Responses to glutamate injection were also blunted following vagotomy, indicating that astrocytic glutamate uptake or nTS soma glutamate receptor function may be altered. Blocking astrocytic excitatory amino acid transporters, which remove extracellular glutamate in the nTS induced similar baseline effects and did not restore cardiorespiratory responses to glutamate in vagotomy. However, nanoinjection of NMDA into the nTS to activate ionotropic glutamate receptors mimicked the blunted responses to glutamate following vagotomy, with no difference in responses to AMPA nanoinjection. Likewise, immunohistochemistry and Western blot showed that vagotomy reduced NMDA receptor subunit expression in the nTS. Together these results indicate that chronic vagotomy blunts cardiorespiratory responses to vagal afferent stimulation, likely through reduced presynaptic activation and decreased NMDA receptor expression and function. These studies will help improve our understanding of how afferent input induces neuroplasticity within the nTS, and the resulting impacts on cardiorespiratory function in health and disease. NIH HL132836, HL128454, HL98602, T32OD011126. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic intermittent hypoxia (CIH) enhances nTS sensitivity to oxytocin (OT) and corticotropin-releasing hormone (CRH) via effects at the nTS soma and on presynaptic release

OT and CRH neurons from the hypothalamus project to nucleus tractus solitarii (nTS) to influence its activity and response to hypoxia. CIH increases sympathetic nervous system activity, blood pressure, and respiration, and the nTS contributes to these responses. Yet, the influence of these OT or CRH inputs on nTS activity during CIH is unclear. We hypothesized that OT or CRH individually increases nTS activity, and their combined influence would further enhance activity. We also expected these responses to be enhanced by CIH. Male Sprague-Dawley rats (~3 wks) were exposed to 10d normoxia (21% O2) or CIH (~30s of 6% O2, 10x/hr, 8 h/day). Recorded events were examined during exogenous OT and CRH or their receptor block (OTR-x, CRHR2-x). As a time control, aCSF only was examined, which did not alter any events over the 40-min recording period. In nTS slices, extracellular neuronal discharge from multielectrode array recording was increased by OT and its co-application with CRH throughout the nTS after normoxia. Following CIH, OT and CRH alone, and their co-application increased firing rate, and the response to OT+CRH after CIH was greater than in normoxia slices. Fura-2 calcium imaging in dissociated nTS neurons demonstrated enhanced responses to OT+ CRH following CIH vs normoxia. In second-order nTS neurons recorded via patch clamp, OT and CRH induced spontaneous action potential discharge after CIH in quiescent neurons during baseline periods. Excitatory postsynaptic currents (EPSCs) were recorded to examine synaptic transmission. Spontaneous (s)EPSCs were not altered by OT and CRH after normoxia or CIH. Yet, only after CIH OTR-x and/or CRHR2-x reduced sEPSC amplitude, indicating tonic activation of these receptors maintain current amplitude. Afferent (TS)-EPSCs evoked at 0.5 and 20 Hz were not altered by OT and CRH or their receptor blockers in normoxia. However, CIH elevated TS-EPSCs in response to CRH and CRH+OT. Block of OTR and CRHR2 after CIH did not alter TS-EPSC amplitude, indicating their minimal tonic influence on sensory integration. After a 20Hz stimulation, the peak of asynchronous EPSCs increased in presence of CRH and OT+CRH only after CIH. We examined presynaptic Ca2+ in pre-labeled GCaMP6m afferent terminals in response to a 20 Hz stimulation. In normoxia-exposed slices, OT and CRH increased Ca2+ fluorescence. However, after CIH, the application of CRH and the co-application of OT+CRH decreased the terminal Ca2+ response. The expression of OT but not CRH fibers within the nTS decreased (immunohistochemistry) while OTR and CRHR2 mRNA increased (RNA Scope). The CRH and OT neuron populations within PVN were not altered after CIH (immunohistochemistry). Altogether, our data show that following CIH, OT and CRH augment neuronal activity via postsynaptic mechanisms while CRH also increases synaptic transmission through enhancing presynaptic release. Those alterations may contribute to elevated sympathetic response and blood pressure observed after CIH. NIH R01 HL098602 & R01 HL128454 (DDK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CCK-sensitive C fibers activate NTS leptin receptor-expressing neurons via NMDA receptors.

The hormone leptin reduces food intake through actions in the peripheral and central nervous systems, including in the hindbrain nucleus of the solitary tract (NTS). The NTS receives viscerosensory information via vagal afferents, including information from the gastrointestinal tract, which is then relayed to other central nervous system (CNS) sites critical for control of food intake. Leptin receptors (lepRs) are expressed by a subpopulation of NTS neurons, and knockdown of these receptors increases both food intake and body weight. Recently, we demonstrated that leptin increases vagal activation of lepR-expressing neurons via increased NMDA receptor (NMDAR) currents, thereby potentiating vagally evoked firing. Furthermore, chemogenetic activation of these neurons was recently shown to inhibit food intake. However, the vagal inputs these neurons receive had not been characterized. Here we performed whole cell recordings in brain slices taken from lepRCre × floxedTdTomato mice and found that lepR neurons of the NTS are directly activated by monosynaptic inputs from C-type afferents sensitive to the transient receptor potential vanilloid type 1 (TRPV1) agonist capsaicin. CCK administered onto NTS slices stimulated spontaneous glutamate release onto lepR neurons and induced action potential firing, an effect mediated by CCKR1. Interestingly, NMDAR activation contributed to the current carried by spontaneous excitatory postsynaptic currents (EPSCs) and enhanced CCK-induced firing. Peripheral CCK also increased c-fos expression in these neurons, suggesting they are activated by CCK-sensitive vagal afferents in vivo. Our results indicate that the majority of NTS lepR neurons receive direct inputs from CCK-sensitive C vagal-type afferents, with both peripheral and central CCK capable of activating these neurons and NMDARs able to potentiate these effects.

Multiple NTS neuron populations cumulatively suppress food intake.

Several discrete groups of feeding-regulated neurons in the nucleus of the solitary tract (nucleus tractus solitarius; NTS) suppress food intake, including avoidance-promoting neurons that express Cck (NTSCck cells) and distinct Lepr- and Calcr-expressing neurons (NTSLepr and NTSCalcr cells, respectively) that suppress food intake without promoting avoidance. To test potential synergies among these cell groups, we manipulated multiple NTS cell populations simultaneously. We found that activating multiple sets of NTS neurons (e.g. NTSLepr plus NTSCalcr [NTSLC], or NTSLC plus NTSCck [NTSLCK]) suppressed feeding more robustly than activating single populations. While activating groups of cells that include NTSCck neurons promoted conditioned taste avoidance (CTA), NTSLC activation produced no CTA despite abrogating feeding. Thus, the ability to promote CTA formation represents a dominant effect but activating multiple non-aversive populations augments the suppression of food intake without provoking avoidance. Furthermore, silencing multiple NTS neuron groups augmented food intake and body weight to a greater extent than silencing single populations, consistent with the notion that each of these NTS neuron populations plays crucial and cumulative roles in the control of energy balance. We found that silencing NTSLCK neurons failed to blunt the weight-loss response to vertical sleeve gastrectomy (VSG) and that feeding activated many non-NTSLCK neurons, however, suggesting that as-yet undefined NTS cell types must make additional contributions to the restraint of feeding.

TRPV1 enhances cholecystokinin signaling in primary vagal afferent neurons and mediates the central effects on spontaneous glutamate release in the NTS.

The gut peptide cholecystokinin (CCK) is released during feeding and promotes satiation by increasing excitation of vagal afferent neurons that innervate the upper gastrointestinal tract. Vagal afferent neurons express CCK1 receptors (CCK1Rs) in the periphery and at central terminals in the nucleus of the solitary tract (NTS). While the effects of CCK have been studied for decades, CCK receptor signaling and coupling to membrane ion channels are not entirely understood. Previous findings have implicated L-type voltage-gated calcium channels as well as transient receptor potential (TRP) channels in mediating the effects of CCK, but the lack of selective pharmacology has made determining the contributions of these putative mediators difficult. The nonselective ion channel transient receptor potential vanilloid subtype 1 (TRPV1) is expressed throughout vagal afferent neurons and controls many forms of signaling, including spontaneous glutamate release onto NTS neurons. Here we tested the hypothesis that CCK1Rs couple directly to TRPV1 to mediate vagal signaling using fluorescent calcium imaging and brainstem electrophysiology. We found that CCK signaling at high concentrations (low-affinity binding) was potentiated in TRPV1-containing afferents and that TRPV1 itself mediated the enhanced CCK1R signaling. While competitive antagonism of TRPV1 failed to alter CCK1R signaling, TRPV1 pore blockade or genetic deletion (TRPV1 KO) significantly reduced the CCK response in cultured vagal afferents and eliminated its ability to increase spontaneous glutamate release in the NTS. Together, these results establish that TRPV1 mediates the low-affinity effects of CCK on vagal afferent activation and control of synaptic transmission in the brainstem.NEW & NOTEWORTHY Cholecystokinin (CCK) signaling via the vagus nerve reduces food intake and produces satiation, yet the signaling cascades mediating these effects remain unknown. Here we report that the capsaicin receptor transient receptor potential vanilloid subtype 1 (TRPV1) potentiates CCK signaling in the vagus and mediates the ability of CCK to control excitatory synaptic transmission in the nucleus of the solitary tract. These results may prove useful in the future development of CCK/TRPV1-based therapeutic interventions.

A brainstem circuit for phonation and volume control in mice

Mammalian vocalizations are critical for communication and are produced through the process of phonation, in which expiratory muscles force air through the tensed vocal folds of the larynx, which vibrate to produce sound. Despite the importance of phonation, the motor circuits in the brain that control it remain poorly understood. In this study, we identified a subpopulation of ~160 neuropeptide precursor Nts (neurotensin)-expressing neurons in the mouse brainstem nucleus retroambiguus (RAm) that are robustly activated during both neonatal isolation cries and adult social vocalizations. The activity of these neurons is necessary and sufficient for vocalization and bidirectionally controls sound volume. RAm Nts neurons project to all brainstem and spinal cord motor centers involved in phonation and activate laryngeal and expiratory muscles essential for phonation and volume control. Thus, RAm Nts neurons form the core of a brain circuit for making sound and controlling its volume, which are two foundations of vocal communication.