NTS Neurons Research Articles

Extensive Inhibitory Gating of Viscerosensory Signals by a Sparse Network of Somatostatin Neurons.

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.

Acute lung injury in neonatal rats causes postsynaptic depression in nucleus tractus solitarii second-order neurons

Acute Lung Injury (ALI) alters pulmonary reflex responses, in part due to changes in modulation within the lung and airway neuronal control networks. We hypothesized that synaptic efficacy of nucleus tractus solitarii (nTS) neurons, receiving input from lung, airway, and other viscerosensory afferent fibers, would decrease following ALI. Sprague Dawley neonatal rats (postnatal days 9–11) were given intratracheal installations of saline or bleomycin (a well-characterized model that reproduces the pattern of ALI) and then, one week later, in vitro slices were prepared for whole-cell and perforated whole-cell patch-clamp experiments (postnatal days 16–21). In preparations from ALI rats, 2nd-order nTS neurons had significantly decreased amplitudes of both spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs), compared to saline controls. Rise and decay times of sEPSCs were slower in whole-cell recordings from ALI animals. Similarly, the amplitude of tractus solitarii evoked EPSCs (TS-eEPSCs) were significantly lower in 2nd-order nTS neurons from ALI rats. Overall these results suggest the presence of postsynaptic depression at TS-nTS synapses receiving lung, airway, and other viscerosensory afferent tractus solitarii input after bleomycin-induced ALI.

Mapping the populations of neurotensin neurons in the male mouse brain

Neurotensin (Nts) is a neuropeptide implicated in the regulation of many facets of physiology, including cardiovascular tone, pain processing, ingestive behaviors, locomotor drive, sleep, addiction and social behaviors. Yet, there is incomplete understanding about how the various populations of Nts neurons distributed throughout the brain mediate such physiology. This knowledge gap largely stemmed from the inability to simultaneously identify Nts cell bodies and manipulate them in vivo. One means of overcoming this obstacle is to study NtsCre mice crossed onto a Cre-inducible green fluorescent reporter line (NtsCre;GFP mice), as these mice permit both visualization and in vivo modulation of specific populations of Nts neurons (using Cre-inducible viral and genetic tools) to reveal their function. Here we provide a comprehensive characterization of the distribution and relative densities of the Nts-GFP populations observed throughout the male NtsCre;GFP mouse brain, which will pave the way for future work to define their physiologic roles. We also compared the distribution of Nts-GFP neurons with Nts-In situ Hybridization (Nts-ISH) data from the adult mouse brain. By comparing these data sets we can distinguish Nts-GFP populations that may only transiently express Nts during development but not in the mature brain, and hence which populations may not be amenable to Cre-mediated manipulation in adult NtsCre;GFP mice. This atlas of Nts-GFP neurons will facilitate future studies using the NtsCre;GFP line to describe the physiological functions of individual Nts populations and how modulating them may be useful to treat disease.

Neurotensin Enhances Locomotor Activity and Arousal and Inhibits Melanin-Concentrating Hormone Signaling

Background: Hypothalamic neurotensin (Nts)-secreting neurons regulate fundamental physiological processes including metabolism and feeding. However, the role of Nts in modulation of locomotor activity, sleep, and arousal is unclear. We previously identified and characterized Nts neurons in the zebrafish hypothalamus. Materials and Methods: In order to study the role of Nts, nts mutant (nts–/–), and overexpressing zebrafish were generated. Results: The expression of both nts mRNA and Nts protein was reduced during the night in wild-type zebrafish. Behavioral assays revealed that locomotor activity was decreased during both day and night, while sleep was increased exclusively during the nighttime in nts–/– larvae. Likewise, inducible overexpression of Nts increased arousal in hsp70:Gal4/uas:Nts larvae. Furthermore, the behavioral response to light-to-dark transitions was reduced in nts–/– larvae. In order to elucidate potential contenders that may mediate Nts action on these behaviors, we profiled the transcriptome of 6 dpf nts–/– larvae. Among other genes, the expression levels of melanin-concentrating hormone receptor 1b were increased in nts–/– larvae. Furthermore, a portion of promelanin-concentrating hormone 1 (pmch1) and pmch2 neurons expressed the nts receptor. In addition, expression of the the two zebrafish melanin-concentrating hormone (Mch) orthologs, Mch1 and Mch2, was increased in nts–/– larvae. Conclusion: These results show that the Nts and Mch systems interact and modulate locomotor activity and arousal.

Expression and Function of Transient Receptor Potential Ankyrin 1 Ion Channels in the Caudal Nucleus of the Solitary Tract.

The nucleus of the solitary tract (NTS) receives visceral information via the solitary tract (ST) that comprises the sensory components of the cranial nerves VII, IX and X. The Transient Receptor Potential Ankyrin 1 (TRPA1) ion channels are non-selective cation channels that are expressed primarily in pain-related sensory neurons and nerve fibers. Thus, TRPA1 expressed in the primary sensory afferents may modulate the function of second order NTS neurons. This hypothesis was tested and confirmed in the present study using acute brainstem slices and caudal NTS neurons by RT-PCR, immunostaining and patch-clamp electrophysiology. The expression of TRPA1 was detected in presynaptic locations, but not the somata of caudal NTS neurons that did not express TRPA1 mRNA or proteins. Moreover, caudal NTS neurons did not show somatodendritic responsiveness to TRPA1 agonists, while TRPA1 immunostaining was detected only in the afferent fibers. Electrophysiological recordings detected activation of presynaptic TRPA1 in glutamatergic terminals synapsing on caudal NTS neurons evidenced by the enhanced glutamatergic synaptic neurotransmission in the presence of TRPA1 agonists. The requirement of TRPA1 for modulation of spontaneous synaptic activity was confirmed using TRPA1 knockout mice where TRPA1 agonists failed to alter synaptic efficacy. Thus, this study provides the first evidence of the TRPA1-dependent modulation of the primary afferent inputs to the caudal NTS. These results suggest that the second order caudal NTS neurons act as a TRPA1-dependent interface for visceral noxious-innocuous integration at the level of the caudal brainstem.

0073 Activation of Leptin Receptor Positive Neurons in the Nucleus of The Solitary Tract (NTS) Alleviates Sleep Disordered Breathing in Obese Mice

Obstructive Sleep Apnea (OSA) is a major cause of morbidity and mortality with high prevalence. The loss of motor input related to deeper sleep stages lead to recurrent pharyngeal collapse (apneas and hypopneas), leading to oxyhemoglobin desaturations and arousals. There is still no effective pharmacotherapy for OSA. Leptin is a powerful respiratory stimulant and long isoform of leptin receptor, LepRb in the brain is an important therapeutic target in sleep disordered breathing. Male LepRb-Cre mice with diet induced obesity (DIO) underwent bilateral stereotactic administration to NTS of an adeno-associated virus harboring the excitatory Cre-dependent Designer Receptor Exclusively Activated by Designer Drug (DREADD) rAAV5.2-hSyn-DIO-hM3(Gq)-mCherry (Virovek, 1 x 1013 vg/ml, 250 nl) (n = 4) and headmount with EEG and nuchal EMG leads under 1-2% isoflurane. Four-six weeks later, sleep studies were performed after DREADD ligand (JH37160) or saline i.p. injection in a randomized blinded manner. Treatment groups were crossed-over and sleep studies were repeated a week later. We successfully targeted NTS with DREADDs. JH37160 dramatically increased non-flow limited minute ventilation (VE) from 0.92±1.32 mL/min/g to 1.34±0.39 mL/min/g in NREM sleep and from 0.89±0.43 mL/min/g to 1.24±0.53 mL/min/g in REM sleep without change in body temperature suggesting up-regulation of ventilatory control rather than a metabolic response. There was no decrease in the frequency of obstructed breaths. However, upper airway obstruction significantly improved which was manifested by an increase in maximal inspiratory flow (from 2.1±7.3 mL/s to 3.8±0.9 mL/s in NREM and from 2.5±0.8 mL/s to 3.6±0.8 mL/s in REM) and minute ventilation (from 0.73±0.3 mL/min/g to 1.29±0.4 mL/min/g in NREM and from 0.96±0.46 mL/min/g to 1.19±0.49 mL/min/g in REM) during obstructed breathing. This improvement resulted in striking decreases in the oxyhemoglobin desaturation index, which decreased from 37.1 to 4.4 events/hr in REM sleep. Activation of LepRb+ neurons in NTS alleviates sleep disordered breathing in DIO mice affecting both ventilatory and upper airway control. NIH R01 HL128970, NIH R01 138932, FAPESP: 2018/08758-3, CNPq: 142073/2017-2

ACUTE EFFECT OF ALDOSTERONE ON THE MEMBRANE POTENTIAL IN NEURONS OF THE NUCLEUS OF THE SOLITARY TRACT

IntroductionA population of neurons in the nucleus of the solitary tract (NTS) expresses both the mineralocorticoid receptor (MR) and the enzyme 11β‐hydroxysteroid dehydrogenase type 2 (HSD2), making these neurons responsive to aldosterone, not to glucocorticoids. HSD2 neurons in the NTS contribute to the development of sodium appetite which is stimulated by a combination of body sodium deficiency and low blood volume, a condition that activates the renin‐angiotensin‐aldosterone system. The effect of aldosterone in the NTS to induce sodium appetite may involve non‐genomic mechanisms, independent of nuclear MR, gene transcription and protein synthesis.ObjectiveIn the present study, we investigated the acute effect of aldosterone on the membrane potential of NTS neurons in brainstem slices of rats.MethodologyMale Holtzman rats (150 to 250 g) were decapitated and the brainstem rapidly removed and submerged in ice‐cold artificial cerebrospinal fluid (aCSF) with osmolality of 300–310 mosmol/KgH2O and pH 7.4. Brainstem coronal slices were obtained and then placed in the recording chamber, which was continuously perfused with oxygenated aCSF with or without aldosterone at room temperature. Whole‐cell patch‐clamp recordings of NTS neurons were done in current‐clamp mode to measure the membrane potential. Data are shown as mean ± SEM, and statistical significance was determined by unpaired Student's t‐test using the GraphPad Prism software. Experimental protocols were approved by the Ethics Committee for Animal Care and Use (CEUA 13/2018).ResultsAldosterone (1 □M) caused cell depolarization and increased firing frequency of action potentials (0.8 ± 0.2 Hz, *** p < 0.001), in silent NTS neurons. Only 30% of the recorded neurons (6 of 20 neurons) showed a reversible depolarization in response to aldosterone and repolarization upon washout.ConclusionThe present results suggest that aldosterone, acting via non‐genomic mechanisms, can depolarize and increase the firing frequency of action potentials in NTS neurons in vitro. These cells (possibly HSD2 neurons) might be those involved in the development of sodium appetite induced by aldosterone.Support or Funding InformationFunding: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The amplitudes of miniature excitatory postsynaptic currents in NTS neurons from female rats vary as a function of the estrus cycle

Very little information exists regarding how glutamatergic transmission within the nucleus of the solitary tract (NTS) of females might vary as a function of the estrus cycle. Spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded in caudal NTS slices (250μM thick) superfused with TTX (1 um) and gabazine (25 um) under voltage clamp. Female rats were studied at different stages of the estrus cycle determined by vaginal smear and the number of neurons studied at each stage of the estrus cycle is as follows: estrus n=16; metestrus n=14; diestrus n=22. Male rats (n=33) were also studied for comparison. All mEPSCs were blocked by superfusion of the slice with CNQX (10 um). The amplitude of mEPSCs was significantly lower in diestrus (16.53 ± 1.01 pA) compared to estrus (23.30 ± 2.12 pA), metestrus (19.95 ± 1.13 pA), and males (20.49 ± 1.29 pA) (all p<0.035). The frequency of mEPSCs did not vary across the estrus cycle in females however mEPSC frequency was significantly lower in males (2.74 ± 0.28 Hz) compared to estrus (4.23 ± 0.63 Hz, p=.010) and metestrus (4.34 ± 0.61 Hz, p=.005). The frequency of mEPSCs did not differ between males and females in diestrus (3.68 ± 0.54 Hz, p=.085). mEPSC rise time did not differ between the 3 female groups and males (all p>.111). mEPSC decay time was significantly greater in estrus (9.00 ± 0.75 ms) compared to males (7.50 ± 0.39 ms, p=.038) but decay time during estrus did not differ from that in metestrus (7.58 ± 0.61 ms) and diestrus (7.59 ± 0.41 ms) (both p>.088). These results suggest that glutamatergic transmission in NTS neurons in female rats can vary as a function of the estrus cycle. During diestrus plasma estrogen levels are rising and during diestrus we observed mEPSCs with amplitudes lower than during other phases of the estrus cycle or in males. This suggests that estrogen may have a post‐synaptic effect that reduces NTS neuronal responses to glutamatergic inputs. Preliminary whole cell recordings from 3 NTS neurons in a brain slice from estrogen receptor alpha‐td tomato reporter mice lend support to this interpretation as superfusion of the slice with 100 nm 17β‐estradiol reversibly reduced mEPSC amplitude from 19–29%.Support or Funding InformationThis work was supported by HL088052This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Responses of Caudal NTS Neurons to Acute Tissue Hypoxia

The neurons of caudal nucleus tractus solitarii (nTS) initiate an integrated response to hypoxia by virtue of its afferent inputs and efferent projections. Furthermore, these neurons are also subjected to tissue hypoxia. In this study we investigated the effects of acute tissue hypoxia on intracellular Ca2+ ([Ca2+]i) levels in neurons from rat caudal nTS. Adult male Sprague‐Dawley rats were anesthetized with isoflurane and the hindbrain removed and cut into 250 μm thick sections. The NTS slices were then incubated for 45 min with 10 μM Fura‐2AM and 30 μL of F127 at room temperature and then washed for 20 min in artificial cerebrospinal fluid (aCSF) bubbled with 95% O2/5%CO2. A single slice was transferred to the recording chamber on an upright epifluorescent microscope and superfused with normal aCSF at a rate of 2.5 ml/min. Acute hypoxia was established by exposing hindbrain slices containing nTS to aCSF bubbled with 95% N2/5% CO2. The slice was superfused with either 20 μM nifedipine, 10 μM ruthenium red, or 10 μM ryanodine dissolved in aCSF. Cell viability was confirmed by 63 mM KCl. Fluorescence of Fura‐2AM was excited by epi‐illumination with light provided by a 75 W Xenon lamp band‐pass filtered alternatively at 340 or 380 nm. Emission light pass through a barrier filter (510 nm). Pairs of 340 and 380 nm images were acquired at intervals of 5 s and analyzed off‐line with NIS‐Elements AR 3.2 software. All images were captured with a charge‐coupled device (CCD) camera. A total of 20 sections were examined from 9 rats and an average of 4 cells/slice were analyzed in each section. Five minutes of hypoxic aCSF triggered a 41.37±20.95 % reversible increase in 340/380 ratio normalized to KCl relative to baseline with a latency of 56.9 s ± 25.1 s. This increase was due to calcium influx from extracellular compartment as removing calcium from extracellular bathing solution abolished the response. Nifedipine reduced the increase in [Ca2+]i during hypoxia by 14.62±11.88% (n=25 cells, p<0.05). Neither ryanodine nor ruthenium red altered the [Ca2+]i response to hypoxia. These findings suggest that the elevated [Ca2+]i as a response to acute tissue hypoxia in caudal NTS is dependent, at least in part, upon influx through L‐type voltage gated calcium channel. Other types of Ca channels, ROS generation, and mitochondrial release could also contribute to the response and are being investigated. Hypoxia‐induced elevations in intracellular Ca are likely to alter caudal NTS neuronal function under hypoxic conditions.Support or Funding InformationSupported by HL088052This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression and Function of Oxytocin Signaling on Nodose Sensory Neuron Potassium Currents after Chronic Intermittent Hypoxia (CIH)

Hypothalamic‐derived oxytocin (OT) increases systemically and in the dorsal vagal complex in response to a variety of stimuli and stressors, such as hypoxia. Within the nucleus tractus solitarii (nTS), OT modifies baroreflex sensitivity suggestive of an influence of OT on presynaptic afferents originating in the nodose ganglia or postsynaptic nTS neurons. nTS slice studies demonstrated an excitatory influence of OT on afferent terminals, and nodose neurons express the OT receptor (OTR) whose activation elevates somal calcium. The hypoxic stressor obstructive sleep apnea, and its CIH animal model, elevates blood pressure and alters heart rate variability. During CIH, however, enhancing OT in the dorsal vagal complex reduces blood pressure. To determine the mechanism(s) by which OT and OTR modulates sensory afferent‐nTS activity and mediated reflexes normally and after CIH we used immunohistochemistry, immunoblot, and electrophysiology on nodose ganglia tissue. Nodose ganglia of 5–6 week old male and female Sprague‐Dawley rats were studied after 10 days of Normoxia (Norm, 21% O2) or CIH (alternating 21% & 6% O2, 8 hr/day). Immunohistochemistry of cryostat‐sectioned (10 μm) nodose ganglia demonstrated OTRs in a subset of Norm somas and fibers. Immunoblot demonstrated a 0.52 fold reduction in OTR protein after CIH vs. Norm (n=4 each). OT plasma levels were reduced in CIH (n=12) when compared to Norm (n=10). To determine the functional effects of these changes, nodose neurons were dissociated, and 2–5 hours after isolation outward potassium currents (IK) were evoked via depolarizing voltages (10 mV steps, −70 to +60 mV, 140 ms) from a holding potential of −60 mV. The IK‐voltage relationship was measured in the presence of vehicle or 600 nM OT with or without the application of the OTR antagonist L‐368,899 (1 μM). OT‐sensitive IK was also characterized by the application of the K+ channel blockers Tetraethylammonium (TEA, 10 mM) or 4 ‐Aminopyridine (4‐AP, 5 mM), prior to and during the application of vehicle or 600 nM OT. In male nodose neurons, vehicle application after Norm and CIH did not alter IK, confirming no time dependent changes in IK. By contrast, OT decreased IK after Norm (n=7) and CIH (n=9), with the attenuation more prominent in CIH. The application of the OTR antagonist or TEA prior to and during 600 nM OT blocked the reduction of IK in CIH (n=5–7) and Norm (n=4–5). Conversely, 4‐AP did not block the OT‐reduction in IK after CIH (n=4) and Norm (n=6). To determine if there was a sex‐dependent influence of OT on IK, female nodose neurons were examined. OT induced a similar reduction of IK as males (n=5). Taken together, these data demonstrate OTR in nodose ganglia neurons and its activation via OT induces decreases IK that are consistent with increasing excitability in CIH. These data may also shed light on the influence of OT blood pressure control.Support or Funding InformationRO1 HL128454 & HL098602This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ionotropic and Metabotropic Glutamate Receptors Contribute to Cardiorespiratory Responses to Inhibition of Astrocytic Excitatory Amino Acid Transporters

The nucleus tractus solitarii (nTS) is the initial site of termination of cardiorespiratory afferents, which transmit signals through release of glutamate (Glu). Afferent information is integrated extensively in the nTS, thus shaping its output and cardiorespiratory responses. We have shown that inhibition of astrocytic excitatory amino acid transporter 2 (EAAT2) in the nTS increased ambient [Glu] and altered basal and reflex cardiorespiratory function. These effects were partially blocked by ionotropic glutamate receptor (iGluR) antagonism. We hypothesized that the responses remaining after iGluR blockade were due to activation of metabotropic (m)GluRs. To test this, Inactin‐anesthetized rats were instrumented to record mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic and phrenic nerve activity (SSNA; PhNA), and for microinjections into the nTS. The general EAAT inhibitor TFB‐TBOA (10 μM, 90 nl) was microinjected unilaterally into the nTS. GluR antagonists or vehicle (aCSF) were then microinjected, followed by two additional injections of TBOA. Unilateral nTS microinjection of TBOA decreased MAP, HR, PhNA and SSNA, consistent with effects of increased [Glu] in the nTS. Following aCSF, additional injections of TBOA produced augmented as well as prolonged cardiorespiratory responses. Prior blockade of iGluRs with a cocktail (90 nl) of NBQX (2 mM) and AP5 (10 mM) reduced the response to EAAT inhibition. However, it did not prevent the augmented effects of repetitive TBOA, indicating a role for other receptors. The effects of combined blockade of iGluRs and Group II/III mGluRs (MSOP, 100 mM + eGlu, 10 mM; 90 nl total) were similar to those of iGluR antagonism alone. In contrast, combined blockade of iGluRs and Group I mGluRs (LY367385, 10 mM; 90 nl total) blunted the cardiorespiratory effects of nTS EAAT inhibition, and markedly attenuated enhanced responses to subsequent injection of TBOA. These data are consistent with the concept that nTS inhibition of astrocytic EAATs increases [Glu], producing cardiorespiratory responses similar to excitation of nTS neurons involved in baroreflex function. These effects of EAAT inhibition are mediated primarily by iGluRs and Group I mGluRs. It is possible that Group II/III mGluRs also contribute via other mechanisms.Support or Funding InformationSupported by HL132836 and HL128454This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiovascular Deconditioning Induces Changes in the Excitatory Neurotransmission and Active Properties of Nucleus Tractus Solitarii Neurons

Hindlimb‐unloading (HU) in rats induces cardiovascular deconditioning analogous to that observed in individuals exposed to microgravity or bed rest. Among other physiological changes, HU rats exhibit autonomic imbalance, increased baseline heart rate and altered baroreflex and chemoreflex. Considering that the nucleus tractus solitariis (nTS) is an important central region for autonomic control and integration of cardiovascular reflex function, in this study we aimed to investigate the effects of HU on glutamatergic synaptic transmission and intrinsic properties of nTS neurons. For this purpose, stainless steel wire rings were positioned in the tail of male Sprague‐Dawley rats (3 weeks) and after one week of recovery the rings was connected to a suspension apparatus. HU rats were suspended at an angle of 30–35° for two weeks with free access to food and water, while control animals were maintained under normal postural conditions. After this period, coronal brainstem slices were obtained and the whole‐cell patch clamp technique was performed to evaluate the electrophysiological properties of nTS neurons. The effectiveness of the HU protocol was confirmed by significant hindlimb muscle atrophy. Adrenal weight was comparable between HU and control rats. Resting membrane potential of nTS neurons from HU rats was comparable to neurons from controls. However, HU reduced the percentage of neurons that exhibited spontaneous firing activity (Control: 46% vs HU: 11%). In addition, HU induced a significant increase in the action potential rheobase and A‐type potassium current of nTS neurons, which may contribute to altered basal firing rate. Furthermore, the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSC) was significantly higher in the neurons of HU rats in comparison to the control. Taken together the data indicated that HU increases glutamatergic neurotransmission and reduces the excitability of NTS neurons. Therefore, the results suggest that changes in neuronal activity and synaptic transmission in the nTS may contribute to the cardiovascular reflex alterations observed in HU rats.Support or Funding InformationSupport: HL132836This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pre- and post-inspiratory neurons change their firing properties in female rats exposed to chronic intermittent hypoxia

Obstructive sleep apnea patients face episodes of chronic intermittent hypoxia (CIH), which has been suggested as a causative factor for increased sympathetic activity (SNA) and hypertension. Female rats exposed to CIH develop hypertension and exhibit changes in respiratory–sympathetic coupling, marked by an increase in the inspiratory modulation of SNA. We tested the hypothesis that enhanced inspiratory-modulation of SNA is dependent on carotid bodies (CBs) and are associated with changes in respiratory network activity. For this, in CIH-female rats we evaluated the effect of CBs ablation on respiratory–sympathetic coupling, recorded from respiratory neurons in the working heart–brainstem preparation and from NTS neurons in brainstem slices. CIH-female rats had an increase in peripheral chemoreflex response and in spontaneous excitatory neurotransmission in NTS. CBs ablation prevents the increase in inspiratory modulation of SNA in CIH-female rats. Pre-inspiratory/inspiratory (Pre-I/I) neurons of CIH-female rats have a reduced firing frequency. Post-inspiratory neurons are active for a longer period during expiration in CIH-female rats. Further, using the computational model of a brainstem respiratory–sympathetic network, we demonstrate that a reduction in Pre-I/I neuron firing frequency simulates the enhanced inspiratory SNA modulation in CIH-female rats. We conclude that changes in respiratory–sympathetic coupling in CIH-female rats is dependent on CBs and it is associated with changes in firing properties of specific respiratory neurons types.

Distinct Subsets of Lateral Hypothalamic Neurotensin Neurons are Activated by Leptin or Dehydration

The lateral hypothalamic area (LHA) is essential for ingestive behavior but it remains unclear how LHA neurons coordinate feeding vs. drinking. Most LHA populations promote food and water consumption but LHA neurotensin (Nts) neurons preferentially induce water intake while suppressing feeding. We identified two molecularly and projection-specified subpopulations of LHA Nts neurons that are positioned to coordinate either feeding or drinking. One subpopulation co-expresses the long form of the leptin receptor (LepRb) and is activated by the anorectic hormone leptin (NtsLepRb neurons). A separate subpopulation lacks LepRb and is activated by dehydration (NtsDehy neurons). These molecularly distinct LHA Nts subpopulations also differ in connectivity: NtsLepRb neurons project to the ventral tegmental area and substantia nigra compacta but NtsDehy neurons do not. Intriguingly, the LHA Nts subpopulations cannot be discriminated via their classical neurotransmitter content, as we found that all LHA Nts neurons are GABAergic. Collectively, our data identify two molecularly- and projection-specified subpopulations of LHA Nts neurons that intercept either leptin or dehydration cues, and which conceivably could regulate feeding vs. drinking behavior. Selective regulation of these LHA Nts subpopulations might be useful to specialize treatment for ingestive disorders such as polydipsia or obesity.

Hyperglycemia Induced by Chronic Restraint Stress in Mice Is Associated With Nucleus Tractus Solitarius Injury and Not Just the Direct Effect of Glucocorticoids.

Chronic restraint stress (CRS) can affect hypothalamic-pituitary-adrenal (HPA) axis activity and increase glucocorticoid levels. Glucocorticoids are stress hormones that regulate multiple aspects of energy homeostasis. Stress also impairs glucose tolerance. The aim of this study was to investigate the cause of insulin-resistant hyperglycemia during CRS. We produced the CRS models (a 7-day restraint followed by a 3-day free moving procedure, total of 4 cycles for 40 days) in mice, detected the parameters related to glucose metabolism, and compared them to those of the dexamethasone (DEX) injection (0.2 mg/kg i.p., also a 4 cycle procedure as the CRS). The results showed that the CRS induced a moderate (not higher than 11 mmol/L) and irreversible insulin-resistant hyperglycemia in about 1/3 of the individuals, and all the restrained mice had adrenal hypertrophy. CRS induced the apoptosis of neurons in the anterior part of commissural subnucleus of nucleus tractus solitarius (acNTS) in the hyperglycemic mice, and acNTS mechanical damage also led to insulin-resistant hyperglycemia. In contrast, in the DEX-treated mice, adrenal gland atrophy was evident. The glucose and insulin tolerance varied with the delay of determination. DEX exposure in vivo does not induce the apoptosis of neurons in NTS. This study indicates that restraint stress and DEX induce metabolic disorders through different mechanisms. During CRS, injury (apoptosis) of glucose-sensitive acNTS neurons cause dysregulation of blood glucose. This study also suggests the mouse restraint stress model has value as a potential application in the study of stress-induced hyperglycemia.

Corticotropin-releasing hormone projections from the paraventricular nucleus of the hypothalamus to the nucleus of the solitary tract increase blood pressure.

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.

Neuronal activity of the medulla oblongata revealed by manganese-enhanced magnetic resonance imaging in a rat model of gastroesophageal reflux-related cough.

We investigated neuronal activity of the medulla oblongata during gastroesophageal reflux-related cough (GERC). A rat model of GERC was generated by perfusing HCl into lower esophagus and inducing cough with citric acid. The HCl group rat was received HCl perfusion without citric acid-induced cough. The saline control rat was perfused with saline instead and cough was induced. Citric acid-induced cough rat was only induced by citric acid. Blank group rats were fed normally. Fos expressions were observed in medulla oblongata nuclei using immunohistochemistry. Manganese-enhanced magnetic resonance imaging (MEMRI) was performed to detect the Mn(2+) signal following intraperitoneal injection of MnCl(2). HCl perfusion and citric acid-induced cough caused Fos expressions in the nucleus of solitary tract (nTS), dorsal motor nucleus of the vagus (DMV), paratrigeminal nucleus (Pa5), and intermediate reticular nucleus (IRt), which was higher than HCl group, saline control group, citric acid-induced cough group, and blank group. A high Mn(2+) signal was also observed in most of these nuclei in model rats, compared with blank group animals. The Mn(2+) signal was also higher in the HCl, saline and citric acid-induced cough group animals, compared with blank group animals. The study showed medulla oblongata neurons were excited in a HCl perfusion and citric acid-induced cough rat model, and nTS, DMV, Pa5 and IRt neurons maybe involved in the cough process and signal integrate.

Activation of lateral hypothalamic area neurotensin-expressing neurons promotes drinking

Animals must ingest water via drinking to maintain fluid homeostasis, yet the neurons that specifically promote drinking behavior are incompletely characterized. The lateral hypothalamic area (LHA) as a whole is essential for drinking behavior but most LHA neurons indiscriminately promote drinking and feeding. By contrast, activating neurotensin (Nts)-expressing LHA neurons (termed LHA Nts neurons) causes mice to immediately drink water with a delayed suppression of feeding. We therefore hypothesized that LHA Nts neurons are sufficient to induce drinking behavior and that these neurons specifically bias for fluid intake over food intake. To test this hypothesis we used designer receptors exclusively activated by designer drugs (DREADDs) to selectively activate LHA Nts neurons and studied the impact on fluid intake, fluid preference and feeding. Activation of LHA Nts neurons stimulated drinking in water-replete and dehydrated mice, indicating that these neurons are sufficient to promote water intake regardless of homeostatic need. Interestingly, mice with activated LHA Nts neurons drank any fluid that was provided regardless of its palatability, but if given a choice they preferred water or palatable solutions over unpalatable (quinine) or dehydrating (hypertonic saline) solutions. Notably, acute activation of LHA Nts neurons robustly promoted fluid but not food intake. Overall, our study confirms that activation of LHA Nts neurons is sufficient to induce drinking behavior and biases for fluid intake. Hence, LHA Nts neurons may be important targets for orchestrating the appropriate ingestive behavior necessary to maintain fluid homeostasis.This article is part of the Special Issue entitled ‘Hypothalamic Control of Homeostasis’.

Hypoxia activates a neuropeptidergic pathway from the paraventricular nucleus of the hypothalamus to the nucleus tractus solitarii.

The paraventricular nucleus of the hypothalamus (PVN) contributes to both autonomic and neuroendocrine function. PVN lesion or inhibition blunts cardiorespiratory responses to peripheral chemoreflex activation, suggesting that the PVN is required for full expression of these effects. However, the role of efferent projections to cardiorespiratory nuclei and the neurotransmitters/neuromodulators that are involved is unclear. The PVN sends dense projections to the nucleus tractus solitarii (nTS), a region that displays neuronal activation following hypoxia. We hypothesized that acute hypoxia activates nTS-projecting PVN neurons. Using a combination of retrograde tracing and immunohistochemistry, we determined whether hypoxia activates PVN neurons that project to the nTS and examined the phenotype of these neurons. Conscious rats underwent 2 h normoxia (21% O2, n = 5) or hypoxia (10% O2, n = 6). Hypoxia significantly increased Fos immunoreactivity in nTS-projecting neurons, primarily in the caudal PVN. The majority of activated nTS-projecting neurons contained corticotropin-releasing hormone (CRH). In the nTS, fibers expressing the CRH receptor corticotropin-releasing factor receptor 2 (CRFR2) were colocalized with oxytocin (OT) fibers and were closely associated with hypoxia-activated nTS neurons. A separate group of animals that received a microinjection of adeno-associated virus type 2-hSyn-green fluorescent protein (GFP) into the PVN exhibited GFP-expressing fibers in the nTS; a proportion of these fibers displayed OT immunoreactivity. Thus, nTS CRFR2s appear to be located on the fibers of PVN OT neurons that project to the nTS. Taken together, our findings suggest that PVN CRH projections to the nTS may modulate nTS neuronal activation, possibly via OTergic mechanisms, and thus contribute to chemoreflex cardiorespiratory responses.

Spontaneous activities in baroreflex afferent pathway contribute dominant role in parasympathetic neurocontrol of blood pressure regulation.

To study the dominant role of parasympathetic inputs at cellular level of baroreflex afferent pathway and underlying mechanism in neurocontrol of blood pressure regulation. Whole-cell patch-clamp and animal study were conducted. For the first time, we demonstrated the spontaneous activities from resting membrane potential in myelinated A- and Ah-type baroreceptor neurons (BRNs, the 1st-order), but not in unmyelinated C-types, using vagus-nodose slice of adult female rats. These data were further supported by the notion that the spontaneous synaptic currents could only be seen in the pharmacologically and electrophysiologically defined myelinated A- and Ah-type baroreceptive neurons (the 2nd-order) of NTS using brainstem slice of adult female rats. The greater frequency and the larger amplitude of the spontaneous excitatory postsynaptic currents (EPSCs) compared with the inhibitory postsynaptic currents (IPSCs) were only observed in Ah-types. The ratio of EPSCs:IPSCs was estimated at 3:1 and higher. These results confirmed that the afferent-specific spontaneous activities were generated from baroreflex afferent pathway in female-specific subpopulation of myelinated Ah-type BRNs in nodose and baroreceptive neurons in NTS, which provided a novel insight into the dominant role of sex-specific baroreflex-evoked parasympathetic drives in retaining a stable and lower blood pressure status in healthy subjects, particularly in females. The data from current investigations establish a new concept for the role of Ah-type baroreceptor/baroreceptive neurons in controlling blood pressure stability and provide a new pathway for pharmacological intervention for hypertension and cardiovascular diseases.