Caudal NTS Research Articles

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.

Glutamatergic and purinergic transmitters and astrocyte modulation in the synaptic transmission in the NTS of rats exposed to short-term sustained hypoxia.

There is evidence that astrocytes modulate synaptic transmission in the nucleus tractus solitarius (NTS) interacting with glutamatergic and purinergic mechanisms. Here, using in situ working heart-brainstem preparations, we evaluated the involvement of astrocyte and glutamatergic/purinergic neurotransmission in the processing of autonomic and respiratory pathways in the NTS of control and rats exposed to sustained hypoxia (SH). Baseline autonomic and respiratory activities and the responses to chemoreflex activation (KCN) were evaluated before and after microinjections of fluorocitrate (FCt, an astrocyte metabolic inhibitor), kynurenic acid, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (PPADS) (nonselective antagonists of glutamatergic and purinergic receptors) into the rostral aspect of the caudal commissural NTS. FCt had no effects on the baseline parameters evaluated but reduced the bradycardic response to chemoreflex activation in SH rats. FCt combined with kynurenic acid and PPADS in control rats reduced the baseline duration of expiration, which was attenuated after SH. FCt produced a large increase in PN frequency discharge in control rats, which was reduced after SH, indicating a reduction in the astrocyte modulation after SH. The data show that 1) the bradycardic component of the peripheral chemoreflex is reduced in SH rats after astrocytes inhibition, 2) the inhibition of astrocytes in the presence of double antagonists in the NTS affects the modulation of baseline duration of expiration in control but not in SH rats, and 3) the autonomic and respiratory responses to chemoreflex activation are mediated by glutamatergic and purinergic receptors in the rostral aspect of the caudal commissural NTS.NEW & NOTEWORTHY Our findings indicate that the neurotransmission of autonomic and respiratory components of the peripheral chemoreflex in the nucleus tractus solitarius (NTS) is mediated by glutamatergic and purinergic mechanisms and reveal a selective involvement of NTS astrocytes in controlling the chemoreflex parasympathetic response in rats exposed to sustained hypoxia (SH) and the baseline duration of expiration mainly in control rats, indicating a selective role for astrocytes modulation in the NTS of control and SH rats.

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

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

Electrophysiological responses to appetitive and consummatory behavior in the rostral nucleus tractus solitarius in awake, unrestrained rats.

As the intermediate nucleus in the brainstem receiving information from the tongue and transmitting information upstream, the rostral portion of the nucleus tractus solitarius (rNTS) is most often described as a "taste relay". Although recent evidence implicates the caudal NTS in a broad neural circuit involved in regulating ingestion, there is little information about how cells in the rNTS respond when an animal is eating solid food. Single cells in the rNTS were recorded in awake, unrestrained rats as they explored and ate solid foods (Eating paradigm) chosen to correspond to the basic taste qualities: milk chocolate for sweet, salted peanuts for salty, Granny Smith apples for sour and broccoli for bitter. A subset of cells was also recorded as the animal licked exemplars of the five basic taste qualities: sucrose, NaCl, citric acid, quinine and MSG (Lick paradigm). Most cells were excited by exploration of a food-filled well, sometimes responding prior to contact with the food. In contrast, cells that were excited by food well exploration became significantly less active while the animal was eating the food. Most cells were broadly tuned across foods, and those cells that were recorded in both the Lick and Eating paradigms showed little correspondence in their tuning across paradigms. The preponderance of robust responses to the appetitive versus the consummatory phase of ingestion suggests that multimodal convergence onto cells in the rNTS may be used in decision making about ingestion.

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.

Alleviating Hypertension by Selectively Targeting Angiotensin Receptor-Expressing Vagal Sensory Neurons.

Cardiovascular homeostasis is maintained, in part, by neural signals arising from arterial baroreceptors that apprise the brain of blood volume and pressure. Here, we test whether neurons within the nodose ganglia that express angiotensin type-1a receptors (referred to as NGAT1aR) serve as baroreceptors that differentially influence blood pressure (BP) in male and female mice. Using Agtr1a-Cre mice and Cre-dependent AAVs to direct tdTomato to NGAT1aR, neuroanatomical studies revealed that NGAT1aR receive input from the aortic arch, project to the caudal nucleus of the solitary tract (NTS), and synthesize mechanosensitive ion channels, Piezo1/2 To evaluate the functionality of NGAT1aR, we directed the fluorescent calcium indicator (GCaMP6s) or the light-sensitive channelrhodopsin-2 (ChR2) to Agtr1a-containing neurons. Two-photon intravital imaging in Agtr1a-GCaMP6s mice revealed that NGAT1aR couple their firing to elevated BP, induced by phenylephrine (i.v.). Furthermore, optical excitation of NGAT1aR at their soma or axon terminals within the caudal NTS of Agtr1a-ChR2 mice elicited robust frequency-dependent decreases in BP and heart rate, indicating that NGAT1aR are sufficient to elicit appropriate compensatory responses to vascular mechanosensation. Optical excitation also elicited hypotensive and bradycardic responses in ChR2-expressing mice that were subjected to deoxycorticosterone acetate (DOCA)-salt hypertension; however, the duration of these effects was altered, suggestive of hypertension-induced impairment of the baroreflex. Similarly, increased GCaMP6s fluorescence observed after administration of phenylephrine was delayed in mice subjected to DOCA-salt or chronic delivery of angiotensin II. Collectively, these results reveal the structure and function of NGAT1aR and suggest that such neurons may be exploited to discern and relieve hypertension.

Synaptic loss and gliosis in the nucleus tractus solitarii with streptozotocin-induced Alzheimer’s disease

Obstructive sleep apnea is highly prevalent in Alzheimer’s disease (AD). However, brainstem centers controlling respiration have received little attention in AD research, and mechanisms behind respiratory dysfunction in AD are not understood. The nucleus tractus solitarii (nTS) is an important brainstem center for respiratory control and chemoreflex function. Alterations of nTS integrity, like those shown in AD patients, likely affect neuronal processing and adequate control of breathing. We used the streptozotocin-induced rat model of AD (STZ-AD) to analyze cellular changes in the nTS that corroborate previously documented respiratory dysfunction. We used 2 common dosages of STZ (2 and 3 mg/kg STZ) for model induction and evaluated the early impact on cell populations in the nTS. The hippocampus served as control region to identify site-specific effects of STZ. There was significant atrophy in the caudal nTS of the 3 mg/kg STZ-AD group only, an area known to integrate chemoafferent information. Also, the hippocampus had significant atrophy with the highest STZ dosage tested. Both STZ-AD groups showed respiratory dysfunction along with multiple indices for astroglial and microglial activation. These changes were primarily located in the caudal and intermediate nTS. While there was no change of astrocytes in the hippocampus, microglial activation was accompanied by a reduction in synaptic density. Together, our data demonstrate that STZ-AD induces site-specific effects on all major cell types, primarily in the caudal/intermediate nTS. Both STZ dosages used in this study produced a similar outcome and can be used for future studies examining the initial symptoms of STZ-AD.

Nucleus Tractus Solitarius Neurons Activated by Hypercapnia and Hypoxia Lack Mu Opioid Receptor Expression.

Impaired chemoreflex responses are a central feature of opioid-induced respiratory depression, however, the mechanism through which mu opioid receptor agonists lead to diminished chemoreflexes is not fully understood. One brainstem structure involved in opioid-induced impairment of chemoreflexes is the nucleus of the solitary tract (NTS), which contains a population of neurons that express mu opioid receptors. Here, we tested whether caudal NTS neurons activated during the chemoreflex challenge express mu opioid receptors and overlap with neurons activated by opioids. Using genetic labeling of mu opioid receptor-expressing neurons and cFos immunohistochemistry as a proxy for neuronal activation, we examined the distribution of activated NTS neurons following hypercapnia, hypoxia, and morphine administration. The main finding was that hypoxia and hypercapnia primarily activated NTS neurons that did not express mu opioid receptors. Furthermore, concurrent administration of morphine with hypercapnia induced cFos expression in non-overlapping populations of neurons. Together these results suggest an indirect effect of opioids within the NTS, which could be mediated through mu opioid receptors on afferents and/or inhibitory interneurons.

NTS Glutamate Signaling in the Chemoreflex Axis of an Alzheimer's Disease Rat Model

Respiratory dysfunction is commonly observed in Alzheimer’s disease (AD) and likely due to neuronal alterations of the respiratory network in the brain. The streptozotocin (STZ) ‐induced AD model mimics respiratory problems seen in humans and exhibits neuronal hyperactivity in the nucleus tractus solitarii (nTS), the central integration site for respiratory afferents in the brainstem. This ongoing study looks at the changes of glutamate signaling within the chemoreflex axis as a possible mechanism for nTS hyperactivity and respiratory dysfunction in STZ‐AD.AD was induced by intracerebroventricular injection of 2 mg/kg STZ in 6‐week old male Sprague Dawley rats. After 14 days, caudal nTS neurons within close proximity of DiI‐labeled chemoafferent terminals were recorded using the patch clamp technique. Electrical stimulation of the tractus solitarii (TS) generated glutamatergic excitatory postsynaptic currents (EPSCs) that were compared between experimental groups. Consistent with enhanced glutamatergic signaling, TS‐EPSC amplitude was increased in STZ‐AD. High TS stimulation frequency induced a time and frequency dependent depression that was similar between groups, indicating no change in presynaptic vesicle turnover or postsynaptic receptor desensitization. On the other hand, asynchronous EPSCs following high frequency stimulation seemed enhanced in STZ‐AD, supporting an increased excitatory tone in the nTS. Immunohistochemical analysis of the caudal nTS revealed no change in neuron number but showed a reduced synaptic density in the AD model. Astrocyte number and morphology were unchanged between groups. However, microglia, which are able to influence glutamate‐handling of astrocytes, showed an activated phenotype.Overall, our data show enhanced glutamatergic signaling in the chemoreflex axis contributing to nTS hyperactivity of the STZ‐AD model. nTS hyperactivity may lead to the observed respiratory dysfunction in this model and potentially constitutes a mechanism for sleep disordered breathing in AD patients.

A Novel Organ-Specific Approach to Selectively Target Sensory Afferents Innervating the Aortic Arch.

The brain maintains cardiovascular homeostasis, in part, via the arterial baroreflex which senses changes in blood pressure (BP) at the level of the aortic arch. Sensory afferents innervating the aortic arch employ baroreceptors to convert stretch exerted on the arterial wall into action potentials carried by the vagus nerve to second order neurons residing within the nucleus of the solitary tract (NTS). Although the baroreflex was described more than 80 years ago, the specific molecular, structural, and functional phenotype of the baroreceptors remain uncharacterized. This is due to the lack of tools that provide the genetic and target organ specificity that is required to selectively characterize baroreceptor afferents. Here, we use a novel approach to selectively target baroreceptors. Male mice on a C57BL/6J background were anesthetized with isoflurane, intubated, and artificially ventilated. Following sternotomy, the aortic arch was exposed, and a retrograde adeno-associated virus was applied to the aortic arch to direct the expression of channelrhoropsin-2 (ChR2) and/or tdTomato (tdTom) to sensory afferents presumably functioning as baroreceptors. Consistent with the structural characteristics of arterial baroreceptors, robust tdTom expression was observed in nerve endings surrounding the aortic arch, within the fibers of the aortic depressor and vagus nerves, cell bodies of the nodose ganglia (NDG), and neural projections to the caudal NTS (cNTS). Additionally, the tdTom labeled cell bodies within the NDG also expressed mRNAs coding for the mechanically gated ion channels, PIEZO-1 and PIEZO-2. In vitro electrophysiology revealed that pulses of blue light evoked excitatory post-synaptic currents in a subset of neurons within the cNTS, suggesting a functional connection between the labeled aortic arch sensory afferents and second order neurons. Finally, the in vivo optogenetic stimulation of the cell bodies of the baroreceptor expressing afferents in the NDG produced robust depressor responses. Together, these results establish a novel approach for selectively targeting sensory neurons innervating the aortic arch. This approach may be used to investigate arterial baroreceptors structurally and functionally, and to assess their role in the etiology or reversal of cardiovascular disease.

Cholecystokinin system is involved in the anorexigenic effect of peripherally applied palmitoylated prolactin-releasing peptide in fasted mice.

Prolactin-releasing peptide (PrRP) has been proposed to mediate the central satiating effects of cholecystokinin (CCK) through the vagal CCK1 receptor. PrRP acts as an endogenous ligand of G protein-coupled receptor 10 (GPR10), which is expressed at the highest levels in brain areas related to food intake regulation, e.g., the paraventricular hypothalamic nucleus (PVN) and nucleus of the solitary tract (NTS). The NTS and PVN are also significantly activated after peripheral CCK administration. The aim of this study was to determine whether the endogenous PrRP neuronal system in the brain is involved in the central anorexigenic effect of the peripherally administered CCK agonist JMV236 or the CCK1 antagonist devazepide and whether the CCK system is involved in the central anorexigenic effect of the peripherally applied lipidized PrRP analog palm-PrRP31 in fasted lean mice. The effect of devazepide and JMV236 on the anorexigenic effects of palm-PrRP31 as well as devazepide combined with JMV236 and palm-PrRP31 on food intake and Fos cell activation in the PVN and caudal NTS was examined. Our results suggest that the anorexigenic effect of JMV236 is accompanied by activation of PrRP neurons of the NTS in a CCK1 receptor-dependent manner. Moreover, while the anorexigenic effect of palm-PrRP31 was not affected by JMV236, it was partially attenuated by devazepide in fasted mice. The present findings indicate that the exogenously influenced CCK system may be involved in the central anorexigenic effect of peripherally applied palm-PrRP31, which possibly indicates some interaction between the CCK and PrRP neuronal systems.

SPARC: AAV‐Mediated Reporter Expression in Vagal Afferent Subsets Using Cell‐type Specific Cre Mice

Vagal afferent nerves carry information from peripheral organs to the brainstem, where they synapse with central networks that govern physiological function through reflexes. Vagal afferent nerves are known to control respiratory functions. There are various subtypes of afferent nerves depending on its their developmental origin, expression of certain ion channels/receptors, stimuli and conduction velocities. Here, we used cell type specific Cre knock‐in strains in combination with AAV carrying Cre‐sensitive reporter allele to label specific subsets of vagal afferent nerves and determine their brainstem projection.In this study, Trpv1 and Tac1 Cre strains were crossed with Floxed Rosa‐Ai9 tdTomato to generate mouse models expressing tdTomato in TRPV1 or Tac1 expressing cells/nerves in the presence of Cre. First, unilateral vagal injection of AAV9‐Flex‐EGFP (Cre sensitive) was carried out in TRPV1‐ and Tac1‐CreRosa strains. Next, unilateral vagal injection of AAV9‐hSyn‐EGFP (constitutively active) and AAV9‐Flex‐tdTomao (Cre‐dependent tdTomato expression) was carried out into the TRPV1‐Cre strain. Mice (6 to 8‐week‐old) received vagal injection of AAV virus and housed for four weeks. Vagal ganglia and brainstem were collected and cryosectioned sequentially. Immunohistochemistry was performed using anti‐Trpv1, anti‐DsRed and anti‐GFP, and images were taken using confocal microscope.We observed tdTomato and EGFP labeled‐neurons in vagal ganglia of TRPV1‐ and Tac1‐CreRosa strains that received AAV‐Flex‐EGFP injection. 62% of tdTomato expressing cells also expressed EGFP, and less than 1% of EGFP(+) cells were negative for tdTomato in the TRPV1‐CreRosa strain. In the Tac1‐CreRosa strain, the majority of tdTomato and EGFP expression was seen in jugular ganglion. 60% of tdTomato expressing neurons co‐expressed EGFP. In medulla of both TRPV1‐Cre and Tac1‐CreRosa strains, the majority of EGFP expressing nerves were seen in the nucleus of solitary tract (nTS) and in the area postrema (AP) and paratrigeminal nucleus (Pa5). In TRPV1‐CreRosa strain, EGFP+ nerves mainly innervated SolG, SolDL and SolC in the nTS. In Tac1‐CreRosa strain, some EGFP+ nerves were observed in SolG, SolDL and SolC in caudal nTS, while others were seen in SolM, SolV and SolVL subnuclei in the rostral nTS. Next, unilateral intraganglionic co‐injection of AAV9‐hSyn‐EGFP and AAV9‐Flex‐tdTomato, caused tdTomato in 70% of EGFP(+) vagal neurons, while only 2% expressed tdTomato without EGFP. In the brainstem, most of the nociceptive vagal afferents (tdTomato+ population) innervated SolG, SolDL and SolC. Non‐nociceptive population (only expressing EGFP expression) was observed in SolM, SolV and SolVL subnuclei.Taken together, our approach with the unilateral intraganglionic injections of AAV, into the vagal ganglia, carrying Cre‐dependent reporter allele allows the visualization of specific subsets of neurons/nerves in the vagal ganglia and the brainstem.Support or Funding InformationThis study is funded by National Institutes of Health Common Fund SPARC OT2 (2016–2019): “Functional mapping of peripheral and central circuits for airway protection and breathing” (grant no.: OT2OD023854).

Chronic Intermittent Hypoxia Enhances GABAergic and Glycinergic Signaling in NTS Neurons

In our past studies, spontaneous miniature IPSPs (mIPSCs) recorded from caudal NTS neurons surrounding the tractus solitarius appeared to be mainly mediated by GABA in normal rats. However, the averaged rise time and decay time are significant shorter in NTS neurons from rats after 7 days Chronic Intermittent Hypoxia (CIH) exposure as compared to normoxic, control rats (p<0.001). This study test whether CIH alters evoked inhibition in the NTS by recruiting a glycine mediated mechanism. The inhibitory postsynaptic currents(eIPSCs) evoked by electrical stimulation of the tractus solitarius were examined with whole cell patch‐clamp recordings on NTS second‐order neurons identified by DiA labeling of carotid bodies in a horizontal brainstem slice preparation from CIH exposed and control rats. All experiments were performed in the lateral NTS at the level of the area postrema. The results indicate that NTS neurons from the CIH rats had variable peak amplitudes from 67.6 to 617.28 pA compared with neurons from controls (47.15 to 284.07 pA). CIH significantly increased eIPSC amplitudes (33 neurons in CIH: −189.84 ± 22.76 pA vs 34 neurons in control: −124.3 ± 11.63 pA, p<0.05). This was accompanied by faster eIPSC kinetics (max rise slope of CIH: −150.54 ± 13.47 pA/ms vs control: −106.04 ± 8.82 pA/ms, p<0.01: and max decay slope of CIH: 53.24 ± 1.67 pA/ms vs control: 47.58 ± 1.41 pA/ms, p=0.01) that were significantly increased in neurons from CIH rats as compared to controls. To discriminate among glycine, GABA, or dual eIPSCs, we applied 3 μm strychnine (glyR antagonist) or 25 μm gabazine (GABAAR antagonist) in the presence of 10 μm CNQX (a competitive AMPA/kainate receptor antagonist). Baseline eIPSCs were recorded and either gabazine or strychnine was added to the perfusion solution to differentiate GABA‐mediated current and glycine‐mediated current. Of the 12 neurons from control rats, 7 neurons were gabazine‐sensitive, 1 neuron was strychnine‐sensitive, and 4 neurons had both GABA‐mediated and glycine‐mediated currents. In the 13 neurons from CIH rats, there were 4 gabazine‐sensitive neurons, 3 strychnine sensitive neurons and 6 neurons sensitive to both gabazine and strychnine. The glyR‐mediated eIPSCs expressed faster rise and decay kinetics. In NTS neurons from normoxic control rats, most eIPSCs appear to be GABA mediated while neurons from CIH rats demonstrated glycine and mixed eIPSCs. Our findings suggest that evoked glycinergic signaling in the NTS may be enhanced by seven days of CIH exposure.Support or Funding InformationThis work was supported by P01 HL088052

Effect of Streptozotocin (STZ) Concentration on Chemoreflex Function and Morphology of the Nucleus Tractus Solitarii in STZ‐Induced Alzheimer’s Disease

Alzheimer’s disease (AD) is associated with deterioration of memory and cognition due to changes in brain areas important for these functions. The majority of AD patients also suffer multiple episodes of breathing cessations (apneas) during sleep. AD‐related sleep apnea may be associated with the decline of brainstem centers that control breathing and chemoreflex function. The nucleus tractus solitarii (nTS) is a key region for respiratory control and any AD‐related deterioration of this nucleus critically impacts its function.In this study, we used the streptozotocin (STZ) ‐induced rat model for sporadic AD (6‐week old rats, N = 6–8 rats/group) and compared chemoreflex dysfunction to indices of morphological alterations in the nTS. Particularly, we compared data obtained two weeks following injections of either 2 mg/kg or 2× 1.5 mg/kg STZ. Peripheral chemoreflex function was tested with 10% O2 in a plethysmography chamber. While increased respiratory rate thirty minutes into hypoxia was significantly blunted with both STZ dosages, animals receiving 2 mg/kg STZ partially compensated with larger tidal volumes. Hence, minute ventilation (rate x volume) was only significantly decreased in animals that received 2× 1.5 mg/kg STZ. Consistent with previously published data analyzing memory dysfunction in this model, there was a trend for reduced overall hippocampal size for both STZ concentrations. Immunohistochemical analysis of neuronal density (Anti‐NeuN antibody) in the CA1 region showed no difference between both STZ concentrations and control. However, synaptic density (Anti‐Synaptophysin antibody) was significantly reduced in both STZ concentrations. The overall size of the caudal nTS (4 sections each 180 μm apart) in the brainstem exhibited a greater reduction in 2× 1.5 mg/kg STZ. NeuN density was not altered between STZ concentrations and control. Interestingly, despite the stronger decline of chemoreflex function and nTS size with 2× 1.5 mg/kg STZ, synaptic density in the nTS (calamus scriptorius area) yielded a significant reduction for 2 mg/kg STZ only.Our study shows that reduced nTS size and synaptic density may be underlying the early changes of chemoreflex dysfunction in this model of sporadic AD. 2× 1.5 mg/kg STZ seemed to have a greater impact on respiratory function and nTS size. However, the varying change in synaptic density of each STZ concentration may be due to regional differences, since nTS size at calamus scriptorius did not differ between groups. Future studies will incorporate analysis of more caudal and rostral sections.Support or Funding InformationTruScholar and MoLSAMP grant from Truman State University (TSU) to CMH, GIASR from TSU to MW, seed money from A.T. Still University to TDO

Neurochemical Characterization of Brainstem Pro-Opiomelanocortin Cells.

Genetic research has revealed pro-opiomelanocortin (POMC) to be a fundamental regulator of energy balance and body weight in mammals. Within the brain, POMC is primarily expressed in the arcuate nucleus of the hypothalamus (ARC), while a smaller population exists in the brainstem nucleus of the solitary tract (POMCNTS). We performed a neurochemical characterization of this understudied population of POMC cells using transgenic mice expressing green fluorescent protein (eGFP) under the control of a POMC promoter/enhancer (PomceGFP). Expression of endogenous Pomc mRNA in the nucleus of the solitary tract (NTS) PomceGFP cells was confirmed using fluorescence-activating cell sorting (FACS) followed by quantitative PCR. In situ hybridization histochemistry of endogenous Pomc mRNA and immunohistochemical analysis of eGFP revealed that POMC is primarily localized within the caudal NTS. Neurochemical analysis indicated that POMCNTS is not co-expressed with tyrosine hydroxylase (TH), glucagon-like peptide 1 (GLP-1), cholecystokinin (CCK), brain-derived neurotrophic factor (BDNF), nesfatin, nitric oxide synthase 1 (nNOS), seipin, or choline acetyltransferase (ChAT) cells, whereas 100% of POMCNTS is co-expressed with transcription factor paired-like homeobox2b (Phox2b). We observed that 20% of POMCNTS cells express receptors for adipocyte hormone leptin (LepRbs) using a PomceGFP:LepRbCre:tdTOM double-reporter line. Elevations in endogenous or exogenous leptin levels increased the in vivo activity (c-FOS) of a small subset of POMCNTS cells. Using ex vivo slice electrophysiology, we observed that this effect of leptin on POMCNTS cell activity is postsynaptic. These findings reveal that a subset of POMCNTS cells are responsive to both changes in energy status and the adipocyte hormone leptin, findings of relevance to the neurobiology of obesity.

Mapping of Sensory Nerve Subsets within the Vagal Ganglia and the Brainstem Using Reporter Mice for Pirt, TRPV1, 5-HT3, and Tac1 Expression.

Vagal afferent sensory nerves, originating in jugular and nodose ganglia, are composed of functionally distinct subsets whose activation evokes distinct thoracic and abdominal reflex responses. We used Cre-expressing mouse strains to identify specific vagal afferent populations and map their central projections within the brainstem. We show that Pirt is expressed in virtually all vagal afferents; whereas, 5-HT3 is expressed only in nodose neurons, with little expression in jugular neurons. Transient receptor potential vanilloid 1 (TRPV1), the capsaicin receptor, is expressed in a subset of small nodose and jugular neurons. Tac1, the gene for tachykinins, is expressed predominantly in jugular neurons, some of which also express TRPV1. Vagal fibers project centrally to the nucleus tractus solitarius (nTS), paratrigeminal complex, area postrema, and to a limited extent the dorsal motor nucleus of the vagus. nTS subnuclei preferentially receive projections by specific afferent subsets, with TRPV1+ fibers terminating in medial and dorsal regions predominantly caudal of obex, whereas TRPV1− fibers terminate in ventral and lateral regions throughout the rostral–caudal aspect of the medulla. Many vagal Tac1+ afferents (mostly derived from the jugular ganglion) terminate in the nTS. The paratrigeminal complex was the target of multiple vagal afferent subsets. Importantly, lung-specific TRPV1+ and Tac1+ afferent terminations were restricted to the caudal medial nTS, with no innervation of other medulla regions. In summary, this study identifies the specific medulla regions innervated by vagal afferent subsets. The distinct terminations provide a neuroanatomic substrate for the diverse range of reflexes initiated by vagal afferent activation.

Phenotyping neurons activated in the mouse brain during restoration of salt debt

Salt overconsumption contributes to hypertension, which is a major risk factor for stroke, heart and kidney disease. Characterising neuronal pathways that may control salt consumption is therefore important for developing novel approaches for reducing salt overconsumption. Here, we identify neurons within the mouse central amygdala (CeA), lateral parabrachial nucleus (LPBN), intermediate nucleus of the solitary tract (iNTS), and caudal NTS (cNTS) that are activated and display Fos immunoreactivity in mice that have consumed salt in order to restore a salt debt, relative to salt replete and salt depleted controls. Double-label immunohistochemical studies revealed that salt restoring mice had significantly greater densities of activated enkephalin neurons within the CeA and iNTS, while statistically significant changes within the LPBN and cNTS were not observed. Furthermore, within the CeA, restoration of salt debt conferred a significant increase in the density of activated calretinin neurons, while there was no change relative to control groups in the density of activated neurons that co-expressed protein kinase C delta (PKC-δ). Taken together, these studies highlight the importance of opioid systems within the CeA and iNTS in neuronal processes associated with salt restoration, and may aid the development of future pharmacological and other strategies for reducing salt overconsumption.

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.

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.

Colocalization of A2a but not A1 adenosine receptors with GABA-ergic neurons in cardiopulmonary chemoreflex network in the caudal nucleus of the solitary tract.

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 A1 and A2a pre‐ and postsynaptic receptors, respectively. However, adenosine A2a 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 A1 (inhibitor) and A2a (activator) receptors powerfully inhibits the cardiopulmonary chemoreflex (CCR) at the level of the caudal NTS. A1 receptors most likely inhibit glutamate release in the CCR network, whereas A2a receptors facilitate NTS GABA‐ergic mechanisms which in turn inhibit CCR glutamatergic transmission. Therefore, we hypothesized that A2a receptors are located on NTS GABA‐ergic neurons/terminals whereas A1 receptors may be located on NTS glutamatergic neurons/terminals. We investigated this hypothesis using double immunofluorescent staining for A2a or A1 adenosine receptors and GABA synthesizing enzyme, GAD67, in 30 μm thick, floating, medullary rat sections. We found that A2a adenosine receptors are localized within the GABA‐ergic cells in the caudal NTS, whereas A1 adenosine receptors are absent from these neurons. Instead, A1 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 A2a, but not A1 receptors are located on GABA‐ergic neurons.