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.

Variation in the gene Tas1r3 reveals complex temporal properties of mouse brainstem taste responses to sweeteners.

The gene Tas1r3 codes for the protein T1R3, which dimerizes with T1R2 to form a sweetener-binding receptor in taste cells. Tas1r3 influences sweetener preferences in mice, as shown by work with a 129.B6-Tas1r3 segregating congenic strain on a 129P3/J (129) genetic background; members of this strain vary in whether they do or do not have one copy of a donor fragment with the C57BL/6ByJ (B6) allele for Tas1r3 (B6/129 and 129/129 mice, respectively). Taste-evoked neural responses were measured in the nucleus of the solitary tract (NST), the first central gustatory relay, in B6/129 and 129/129 littermates, to examine how the activity dependent on the T1R2/T1R3 receptor is distributed across neurons and over time. Responses to sucrose were larger in B6/129 than in 129/129 mice, but only during a later, tonic response portion (>600 ms) sent to different cells than the earlier, phasic response. Similar results were found for artificial sweeteners, whose responses were best considered as complex spatiotemporal patterns. There were also group differences in burst firing of NST cells, with a significant positive correlation between bursting prevalence and sucrose response size in only the 129/129 group. The results indicate that sweetener transduction initially occurs through T1R3-independent mechanisms, after which the T1R2/T1R3 receptor initiates a separate, spatially distinct response, with the later period dominating sweet taste perceptions and driving sugar preferences. Furthermore, the current data suggest that burst firing is distributed across NST neurons nonrandomly and in a manner that may amplify weak incoming gustatory signals.

The baroreflex afferent pathway plays a critical role in H2S-mediated autonomic control of blood pressure regulation under physiological and hypertensive conditions.

Hydrogen sulfide (H2S), which is closely related to various cardiovascular disorders, lowers blood pressure (BP), but whether this action is mediated via the modification of baroreflex afferent function has not been elucidated. Therefore, the current study aimed to investigate the role of the baroreflex afferent pathway in H2S-mediated autonomic control of BP regulation. The results showed that baroreflex sensitivity (BRS) was increased by acute intravenous NaHS (a H2S donor) administration to renovascular hypertensive (RVH) and control rats. Molecular expression data also showed that the expression levels of critical enzymes related to H2S were aberrantly downregulated in the nodose ganglion (NG) and nucleus tractus solitarius (NTS) in RVH rats. A clear reduction in BP by the microinjection of NaHS or L-cysteine into the NG was confirmed in both RVH and control rats, and a less dramatic effect was observed in model rats. Furthermore, the beneficial effects of NaHS administered by chronic intraperitoneal infusion on dysregulated systolic blood pressure (SBP), cardiac parameters, and BRS were verified in RVH rats. Moreover, the increase in BRS was attributed to activation and upregulation of the ATP-sensitive potassium (KATP) channels Kir6.2 and SUR1, which are functionally expressed in the NG and NTS. In summary, H2S plays a crucial role in the autonomic control of BP regulation by improving baroreflex afferent function due at least in part to increased KATP channel expression in the baroreflex afferent pathway under physiological and hypertensive conditions.

Acute Visceral Pain in Rats: Vagal Nerve Block Compared to Bupivacaine Administered Intramuscularly.

BACKGROUND: Visceral and parietal peritoneum layers have different sensory innervations. Most visceral peritoneum sensory information is conveyed via the vagus nerve to the nucleus of the solitary tract (NTS). We already showed in animal models that intramuscular (i.m.) injection of local anesthetics decreases acute somatic and visceral pain and general inflammation induced by aseptic peritonitis. The goal of the study was to compare the effects of parietal block, i.m. bupivacaine, and vagotomy on spinal cord and NTS stimulation induced by a chemical peritonitis. METHODS: We induced peritonitis in rats using carrageenan and measured cellular activation in spinal cord and NTS under the following conditions, that is, a parietal nerve block with bupivacaine, a chemical right vagotomy, and i.m. microspheres loaded with bupivacaine. Proto-oncogene c-Fos (c-Fos), cluster of differentiation protein 11b (CD11b), and tumor necrosis factor alpha (TNF-α) expression in cord and NTS were studied. RESULTS: c-Fos activation in the cord was inhibited by nerve block 2 hours after peritoneal insult. Vagotomy and i.m. bupivacaine similarly inhibited c-Fos activation in NTS. Forty-eight hours after peritoneal insult, the number of cells expressing CD11b significantly increased in the cord (P = .010). The median difference in the effect of peritonitis compared to control was 30 cells (CI95, 13.5-55). TNF-α colocalized with CD11b. Vagotomy inhibited this microglial activation in the NTS, but not in the cord. This activation was inhibited by i.m. bupivacaine both in cord and in NTS. The median difference in the effect of i.m. bupivacaine added to peritonitis was 29 cells (80% increase) in the cord and 18 cells (75% increase) in the NTS. Our study underlines the role of the vagus nerve in the transmission of an acute visceral pain message and confirmed that systemic bupivacaine prevents noxious stimuli by inhibiting c-Fos and microglia activation. CONCLUSIONS: In rats receiving intraperitoneal carrageenan, i.m. bupivacaine similarly inhibited c-Fos and microglial activation both in cord and in the NTS. Vagal block inhibited activation only in the NTS. Our study underlines the role of the vagus nerve in the transmission of an acute visceral pain message and confirmed that systemic bupivacaine prevents noxious stimuli. This emphasizes the effects of systemic local anesthetics on inflammation and visceral pain.

Sexual dimorphism in rats exposed to maternal high fat diet: alterations in medullary sympathetic network.

Exposure to high fat diet during perinatal period (PHFD) leads to neuroplastic changes in autonomic circuits, however, the role of gender has been incompletely understood. This study aims to investigate (i) short, and (ii) long-term effects of PHFD on autonomic outflow, and (iii) sexual dimorphic variations emerge at adulthood. Male and female rats were fed a control diet (13.5 % kcal from fat) or PHFD (60 % kcal from fat) from embryonic day-14 to postnatal day-21. To assess changes in autonomic outflow, heart rate variability (HRV) was analyzed at 10- and 20-week-old ages. Expressions of tyrosine hydroxylase (TH), metabotropic glutamate2/3 receptor (mGlu2/3R), N-methyl-D-aspartate1 receptor (NMDA1R), and gamma aminobutyric acidA receptor (GABAAR) were evaluated by immunohistochemistry. PHFD did not affect the body weight of 4-, 10-or 20-week-old male or female offsprings. PHFD significantly increased the sympathetic marker low frequency (LF) component, and sympatho-vagal balance (LF:HF) only in 10-week-old PHFD males. Compared with control, the propranolol-induced (4 mg·kg- 1, ip) decline in LF was observed more prominently in PHFD rats, however, these changes were found to be restored at the age of 20 weeks. In caudal ventrolateral medulla and nucleus tractus solitarius, expression of mGlu2/3R was downregulated in PHFD males, whereas no change was detected in NMDA1R. The number of GABAAR-expressing TH-immunoreactive cells was decreased in rostral ventrolateral medulla of PHFD males. The findings of this study suggest that exposure to maternal high-fat diet could lead to autonomic imbalance with increased sympathetic tone in the early adulthood of male offspring rats without developing obesity.

Thrombin action on astrocytes in the hindbrain of the rat disrupts glycemic and respiratory control.

Severe trauma can produce a postinjury "metabolic self-destruction" characterized by catabolic metabolism and hyperglycemia. The severity of the hyperglycemia is highly correlated with posttrauma morbidity and mortality. Although no mechanism has been posited to connect severe trauma with a loss of autonomic control over metabolism, traumatic injury causes other failures of autonomic function, notably, gastric stasis and ulceration ("Cushing's ulcer"), which has been connected with the generation of thrombin. Our previous studies established that proteinase-activated receptors (PAR1; "thrombin receptors") located on astrocytes in the autonomically critical nucleus of the solitary tract (NST) can modulate gastric control circuit neurons to cause gastric stasis. Hindbrain astrocytes have also been implicated as important detectors of low glucose or glucose utilization. When activated, these astrocytes communicate with hindbrain catecholamine neurons that, in turn, trigger counterregulatory responses (CRR). There may be a convergence between the effects of thrombin to derange hindbrain gastrointestinal control and the hindbrain circuitry that initiates CRR to increase glycemia in reaction to critical hypoglycemia. Our results suggest that thrombin acts within the NST to increase glycemia through an astrocyte-dependent mechanism. Blockade of purinergic gliotransmission pathways interrupted the effect of thrombin to increase glycemia. Our studies also revealed that thrombin, acting in the NST, produced a rapid, dramatic, and potentially lethal suppression of respiratory rhythm that was also a function of purinergic gliotransmission. These results suggest that the critical connection between traumatic injury and a general collapse of autonomic regulation involves thrombin action on astrocytes.

NTS and VTA oxytocin reduces food motivation and food seeking.

Oxytocin (OT) is a neuropeptide whose central receptor-mediated actions include reducing food intake. One mechanism of its behavioral action is the amplification of the feeding inhibitory effects of gastrointestinal (GI) satiation signals processed by hindbrain neurons. OT treatment also reduces carbohydrate intake in humans and rodents, and correspondingly, deficits in central OT receptor (OT-R) signaling increase sucrose self-administration. This suggests that additional processes contribute to central OT effects on feeding. This study investigated the hypothesis that central OT reduces food intake by decreasing food seeking and food motivation. As central OT-Rs are expressed widely, a related focus was to assess the role of one or more OT-R-expressing nuclei in food motivation and food-seeking behavior. OT was delivered to the lateral ventricle (LV), nucleus tractus solitarius (NTS), or ventral tegmental area (VTA), and a progressive ratio (PR) schedule of operant reinforcement and an operant reinstatement paradigm were used to measure motivated feeding behavior and food-seeking behavior, respectively. OT delivered to the LV, NTS, or VTA reduced 1) motivation to work for food and 2) reinstatement of food-seeking behavior. Results provide a novel and additional interpretation for central OT-driven food intake inhibition to include the reduction of food motivation and food seeking.

Estradiol treatment attenuates high fat diet-induced microgliosis in ovariectomized rats.

Consumption of a high fat diet (HFD) increases circulating free fatty acids, which can enter the brain and promote a state of microgliosis, as defined by a change in microglia number and/or morphology. Most studies investigating diet-induced microgliosis have been conducted in male rodents despite well-documented sex differences in the neural control of food intake and neuroimmune signaling. This highlights the need to investigate how sex hormones may modulate the behavioral and cellular response to HFD consumption. Estradiol is of particular interest since it exerts a potent anorexigenic effect and has both anti-inflammatory and neuroprotective effects in the brain. As such, the aim of the current study was to investigate whether estradiol attenuates the development of HFD-induced microgliosis in female rats. Estradiol- and vehicle-treated ovariectomized rats were fed either a low-fat chow diet or a 60% HFD for 4 days, after which they were perfused and brain sections were processed via immunohistochemistry for microglia-specific Iba1 protein. Four days of HFD consumption promoted microgliosis, as measured via an increase in the number of microglia in the arcuate nucleus (ARC) of the hypothalamus and nucleus of the solitary tract (NTS), and a decrease in microglial branching in the ARC, NTS, lateral hypothalamus (LH), and ventromedial hypothalamus. Estradiol replacement attenuated the HFD-induced changes in microglia accumulation and morphology in the ARC, LH, and NTS. We conclude that estradiol has protective effects against HFD-induced microgliosis in a region-specific manner in hypothalamic and hindbrain areas implicated in the neural control of food intake.

Enhancement of excitatory transmission in NTS neurons projecting to ventral medulla of rats exposed to sustained hypoxia is blunted by minocycline.

KEY POINTS: Rats subjected to sustained hypoxia (SH) present increases in arterial pressure (AP) and in glutamatergic transmission in the nucleus tractus solitarius (NTS) neurons sending projections to ventrolateral medulla (VLM). Treatment with minocycline, a microglial inhibitor, attenuated the increase in AP in response to SH. The increase in the amplitude of glutamatergic postsynaptic currents in the NTS-VLM neurons, induced by postsynaptic mechanisms, was blunted by minocycline treatment. The number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS of minocycline-treated rats. The data show that microglial recruitment/proliferation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the observed increase in AP. ABSTRACT: Short-term sustained hypoxia (SH) produces significant autonomic and respiratory adjustments and triggers activation of microglia, the resident immune cells in the brain. SH also enhances glutamatergic neurotransmission in the NTS. Here we evaluated the role of microglial activation induced by SH on the cardiovascular changes and mainly on glutamatergic neurotransmission in NTS neurons sending projections to the ventrolateral medulla (NTS-VLM), using a microglia inhibitor (minocycline). Direct measurement of arterial pressure (AP) in freely moving rats showed that SH (24 h, fraction of inspired oxygen ( FI,O2 ) 0.1) in vehicle and minocycline (30 mg/kg i.p. for 3 days)-treated groups produced a significant increase in AP in relation to control groups under normoxic conditions, but this increase was significantly lower in minocycline-treated rats. Whole-cell patch-clamp recordings revealed that the active properties of the membrane were comparable among the groups. Nevertheless, the amplitudes of glutamatergic postsynaptic currents, evoked by tractus solitarius stimulation, were increased in NTS-VLM neurons of SH rats. Changes in asynchronous glutamatergic currents indicated that the observed increase in amplitude was due to postsynaptic mechanisms. These changes were blunted in the SH group previously treated with minocycline. Using immunofluorescence, we found that the number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS neurons of minocycline-treated rats. Our data support the concept that microglial activation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the increase in AP observed in this experimental model.

Altered A-type potassium channel function in the nucleus tractus solitarii in acquired temporal lobe epilepsy.

Sudden unexpected death in epilepsy (SUDEP) is among the leading causes of death in people with epilepsy. Individuals with temporal lobe epilepsy (TLE) have a high risk for SUDEP because the seizures are often medically intractable. Neurons in the nucleus tractus solitarii (NTS) have been implicated in mouse models of SUDEP and play a critical role in modulating cardiorespiratory and autonomic output. Increased neuronal excitability of inhibitory, GABAergic neurons in the NTS develops during epileptogenesis, and NTS dysfunction has been implicated in mouse models of SUDEP. In this study we used the pilocarpine-induced status epilepticus model of TLE (i.e., pilo-SE mice) to investigate the A-type voltage-gated K+ channel as a potential contributor to increased excitability in GABAergic NTS neurons during epileptogenesis. Compared with age-matched control mice, pilo-SE mice displayed an increase in spontaneous action potential frequency and half-width 9-12 wk after treatment. Activity of GABAergic NTS neurons from pilo-SE mice showed less sensitivity to 4-aminopyridine. Correspondingly, reduced A-type K+ current amplitude was detected in these neurons, with no change in activation or inactivation kinetics. No changes were observed in Kv4.1, Kv4.2, Kv4.3, KChIP1, KChIP3, or KChIP4 mRNA expression. These changes contribute to the increased excitability in GABAergic NTS neurons that develops in TLE and may provide insight into potential mechanisms contributing to the increased risk for cardiorespiratory collapse and SUDEP in this model. NEW & NOTEWORTHY Sudden unexpected death in epilepsy (SUDEP) is a leading cause of death in epilepsy, and dysfunction in central autonomic neurons may play a role. In a mouse model of acquired epilepsy, GABAergic neurons in the nucleus tractus solitarii developed a reduced amplitude of the A-type current, which contributes to the increased excitability seen in these neurons during epileptogenesis. Neuronal excitability changes in inhibitory central vagal circuitry may increase the risk for cardiorespiratory collapse and SUDEP.

Neuroinflammation in the NTS is associated with changes in cardiovascular reflexes during systemic inflammation.

BACKGROUND: Lipopolysaccharide (LPS)-induced systemic inflammation (SI) is associated with neuroinflammation in the brain, hypotension, tachycardia, and multiple organs dysfunctions. Considering that during SI these important cardiovascular and inflammatory changes take place, we measured the sensitivity of the cardiovascular reflexes baroreflex, chemoreflex, and Bezold-Jarisch that are key regulators of hemodynamic function. We also evaluated neuroinflammation in the nucleus tractus solitarius (NTS), the first synaptic station that integrates peripheral signals arising from the cardiovascular and inflammatory status. METHODS: We combined cardiovascular recordings, immunofluorescence, and assays of inflammatory markers in male Wistar rats that receive iv administration of LPS (1.5 or 2.5 mg kg-1) to investigate putative interactions of the neuroinflammation in the NTS and in the anteroventral preoptic region of the hypothalamus (AVPO) with the short-term regulation of blood pressure and heart rate. RESULTS: LPS induced hypotension, tachycardia, autonomic disbalance, hypothermia followed by fever, and reduction in spontaneous baroreflex gain. On the other hand, during SI, the bradycardic component of Bezold-Jarisch and chemoreflex activation was increased. These changes were associated with a higher number of activated microglia and interleukin (IL)-1ß levels in the NTS. CONCLUSIONS: The present data are consistent with the notion that during SI and neuroinflammation in the NTS, rats have a reduced baroreflex gain, combined with an enhancement of the bradycardic component of Bezold-Jarisch and chemoreflex despite the important cardiovascular impairments (hypotension and tachycardia). These changes in the cardiac component of Bezold-Jarisch and chemoreflex may be beneficial during SI and indicate that the improvement of theses reflexes responsiveness though specific nerve stimulations may be useful in the management of sepsis.

Effects of acute and chronic nicotine on catecholamine neurons of the nucleus of the solitary tract.

Nicotine is an addictive drug that has broad effects throughout the brain. One site of action is the nucleus of the solitary tract (NTS), where nicotine initiates a stress response and modulates cardiovascular and gastric function through nicotinic acetylcholine receptors (nAChRs). Catecholamine (CA) neurons in the NTS influence stress and gastric and cardiovascular reflexes, making them potential mediators of nicotine's effects; however nicotine's effect on these neurons is unknown. Here, we determined nicotine's actions on NTS-CA neurons by use of patch-clamp techniques in brain slices from transgenic mice expressing enhanced green fluorescent protein driven by the tyrosine hydroxylase promoter (TH-EGFP). Picospritzing nicotine both induced a direct inward current and increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in NTS-CA neurons, effects blocked by nonselective nAChR antagonists TMPH and MLA. The increase in sEPSC frequency was mimicked by nAChRα7 agonist AR-R17779 and blocked by nAChRα7 antagonist MG624. AR-R17779 also increased the firing of TH-EGFP neurons, an effect dependent on glutamate inputs, as it was blocked by the glutamate antagonist NBQX. In contrast, the nicotine-induced current was mimicked by nAChRα4ß2 agonist RJR2403 and blocked by nAChRα4ß2 antagonist DHßE. RJR2403 also increased the firing rate of TH-EGFP neurons independently of glutamate. Finally, both somatodendritic and sEPSC nicotine responses from NTS-CA neurons were larger in nicotine-dependent mice that had under gone spontaneous nicotine withdrawal. These results demonstrate that 1) nicotine activates NTS-CA neurons both directly, by inducing a direct current, and indirectly, by increasing glutamate inputs, and 2) NTS-CA nicotine responsiveness is altered during nicotine withdrawal.

Angiotensin II inhibits DDAH1-nNOS signaling via AT1R and µOR dimerization to modulate blood pressure control in the central nervous system.

G protein-coupled receptors (GPCRs) are important drug targets. Blocking angiotensin II (Ang II) type 1 receptor signaling alleviates hypertension and improves outcomes in patients with heart failure. Changes in structure and trafficking of GPCR, and desensitization of GPCR signaling induce pathophysiological processes. We investigated whether Ang II, via induction of AT1R and µ-opioid receptor (µOR) dimerization in the nucleus tractus solitarius (NTS), leads to progressive hypertension. Ang II signaling increased µOR and adrenergic receptor α2A (α2A-AR) heterodimer levels and decreased expression of extracellular signal-regulated kinases 1/2T202/Y204, ribosomal protein S6 kinaseT359/S363, and nNOSS1416 phosphorylation. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression was abolished in the NTS of adult spontaneously hypertensive rats (SHRs). Endomorphin-2 was overexpressed in NTS of adult SHRs compared with that in 6-week-old Wistar-Kyoto rats (WKY). Administration of µOR agonist into the NTS of WKY increased blood pressure (BP), decreased nitric oxide (NO) production, and decreased DDAH1 activity. µOR agonist significantly reduced the activity of DDAH1 and decreased neuronal NO synthase (nNOS) phosphorylation. The AT1R II inhibitor, losartan, significantly decreased BP and abolished AT1R-induced formation of AT1R and µOR, and α2A-AR and µOR, heterodimers. Losartan also significantly increased the levels of nNOSS1416 phosphorylation and DDAH1 expression. These results show that Ang II may induce expression of endomorphin-2 and abolished DDAH1 activity by enhancing the formation of AT1R and µOR heterodimers in the NTS, leading to progressive hypertension.

Effects of progesterone on hypoxia-induced inhibition of excitatory synaptic transmission in the rat nucleus tractus solitarius.

The present study evaluated the ability of progesterone to alleviate the synaptic transmission disturbed by hypoxia in the nucleus tractus solitarius (NTS). Hypoxia with N2 inhibited spontaneous and tractus solitarius-evoked excitatory postsynaptic currents (sEPSCs and eEPSCs) in NTS neurons of the rat brainstem slice. An additional application of progesterone counteracted the hypoxia-induced inhibition of sEPSCs and eEPSCs without affecting the baseline currents. This effect of progesterone occurred rapidly and reversibly. Progesterone had neither effect on sEPSCs nor eEPSCs in normoxia. These results suggest that progesterone restores hypoxia-induced disturbance of the NTS glutamatergic transmission, presumably by a presynaptic, non-genomic mechanism.

Involvement of the cAMP-Dependent Pathway in Dextromethorphan-Induced Inhibition of Spontaneous Glutamate Transmission in the Nucleus Tractus Solitarius Neurons of Guinea Pigs.

Dextromethorphan (DEX) presynaptically decreases glutamatergic transmission in second-order neurons of the nucleus tractus solitarius (TS). To clarify the inhibitory mechanism of DEX, the present study examined the interaction of DEX with cAMP. The effects of DEX on miniature and TS-evoked excitatory postsynaptic currents (mEPSCs and eEPSCs) were recorded under activation of the cAMP-dependent pathway using the brainstem slices. An increase in cAMP by forskolin counteracted the inhibitory effect of DEX on mEPSCs. Eight-Bromo-cAMP and N-ethylmaleimide also attenuated the DEX effect. However, forskolin had negligible effects on the DEX-induced inhibition of eEPSCs. This suggests that DEX decreases spontaneous glutamate release by inhibiting the cAMP-dependent pathway and synchronous release by another unknown mechanism.

Glutamatergic activation of A1 and A2 noradrenergic neurons in the rat brain stem.

AIM: To analyze the effects of glutamatergic agonists and antagonists on the activation of the A1 and A2 noradrenergic neurons localized in caudal ventrolateral medulla and nucleus tractus solitarii, respectively. METHODS: Rats were injected with glutamatergic agonists - kainic acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or N-methyl-D-aspartic acid (NMDA), and the brain sections were prepared for immunohistochemistry. Before agonist injections, antagonists - 6-cyano-7-nitroquinoxaline-2,3-dione or dizocilpine were administered. The expression of c-Fos, as the neuronal activation marker, and tyrosine hydroxylase (TH), as the marker of noradrenergic neurons was assessed with dual immunohistochemistry. The percentage of c-Fos-positive noradrenergic neurons relative to all TH-positive neurons in the respective areas of the brain stem was calculated. RESULTS: All three glutamatergic agonists significantly increased the number of the c-Fos-positive noradrenergic neurons in both the A1 and A2 area when compared with control animals. Kainic acid injection activated about 57% of TH-positive neurons in A1 and 40% in A2, AMPA activated 26% in A1 and 38% in A2, and NMDA 77% in A1 and 22% in A2. The injections of appropriate glutamatergic antagonists greatly decreased the number of activated noradrenergic neurons. CONCLUSION: Our results suggest that noradrenergic neurons are regulated and/or activated by glutamatergic system and that these neurons express functional glutamate receptors.

Selective Deletion of Sodium Salt Taste during Development Leads to Expanded Terminal Fields of Gustatory Nerves in the Adult Mouse Nucleus of the Solitary Tract.

Neuronal activity plays a key role in the development of sensory circuits in the mammalian brain. In the gustatory system, experimental manipulations now exist, through genetic manipulations of specific taste transduction processes, to examine how specific taste qualities (i.e., basic tastes) impact the functional and structural development of gustatory circuits. Here, we used a mouse knock-out model in which the transduction component used to discriminate sodium salts from other taste stimuli was deleted in taste bud cells throughout development. We used this model to test the hypothesis that the lack of activity elicited by sodium salt taste impacts the terminal field organization of nerves that carry taste information from taste buds to the nucleus of the solitary tract (NST) in the medulla. The glossopharyngeal, chorda tympani, and greater superficial petrosal nerves were labeled to examine their terminal fields in adult control mice and in adult mice in which the α-subunit of the epithelial sodium channel was conditionally deleted in taste buds (αENaC knockout). The terminal fields of all three nerves in the NST were up to 2.7 times greater in αENaC knock-out mice compared with the respective field volumes in control mice. The shapes of the fields were similar between the two groups; however, the density and spread of labels were greater in αENaC knock-out mice. Overall, our results show that disruption of the afferent taste signal to sodium salts disrupts the normal age-dependent "pruning" of all terminal fields, which could lead to alterations in sensory coding and taste-related behaviors. SIGNIFICANCE STATEMENT: Neural activity plays a major role in the development of sensory circuits in the mammalian brain. To date, there has been no direct test of whether taste-elicited neural activity has a role in shaping central gustatory circuits. However, recently developed genetic tools now allow an assessment of how specific taste stimuli, in this case sodium salt taste, play a role in the maturation of the terminal fields in the mouse brainstem. We found that the specific deletion of sodium salt taste during development produced terminal fields in adults that were dramatically larger than in control mice, demonstrating for the first time that sodium salt taste-elicited activity is necessary for the normal maturation of gustatory inputs into the brain.

Excitatory Hindbrain-Forebrain Communication Is Required for Cisplatin-Induced Anorexia and Weight Loss.

Cisplatin chemotherapy is commonly used to treat cancer despite severe energy balance side effects. In rats, cisplatin activates nucleus tractus solitarius (NTS) projections to the lateral parabrachial nucleus (lPBN) and calcitonin-gene related peptide (CGRP) projections from the lPBN to the central nucleus of the amygdala (CeA). We demonstrated previously that CeA glutamate receptor signaling mediates cisplatin-induced anorexia and body weight loss. Here, we used neuroanatomical tracing, immunofluorescence, and confocal imaging to demonstrate that virtually all NTS→lPBN and lPBN→CeA CGRP projections coexpress vesicular glutamate transporter 2 (VGLUT2), providing evidence that excitatory projections mediate cisplatin-induced energy balance dysregulation. To test whether lPBN→CeA projection neurons are required for cisplatin-induced anorexia and weight loss, we inhibited these neurons chemogenetically using a retrograde Cre-recombinase-expressing canine adenovirus-2 in combination with Cre-dependent inhibitory Designer Receptors Exclusive Activated by Designer Drugs (DREADDs) before cisplatin treatment. Inhibition of lPBN→CeA neurons attenuated cisplatin-induced anorexia and body weight loss significantly. Using a similar approach, we additionally demonstrated that inhibition of NTS→lPBN neurons attenuated cisplatin-induced anorexia and body weight loss significantly. Together, our data support the view that excitatory hindbrain-forebrain projections are necessary for cisplatin's untoward effects on energy intake, elucidating a key neuroanatomical circuit driving pathological anorexia and weight loss that accompanies chemotherapy treatment. SIGNIFICANCE STATEMENT: Chemotherapy treatments are commonly used to treat cancers despite accompanying anorexia and weight loss that may limit treatment adherence and reduce patient quality of life. Strikingly, we lack a neural understanding of, and effective treatments for, chemotherapy-induced anorexia and weight loss. The current data characterize the excitatory nature of neural projections activated by cisplatin in rats and reveal the necessity of specific hindbrain-forebrain projections for cisplatin-induced anorexia and weight loss. Together, these findings help to characterize the neural mechanisms mediating cisplatin-induced anorexia, advancing opportunities to develop better-tolerated chemotherapies and adjuvant therapies to prevent anorexia and concurrent nutritional deficiencies during cancer treatment.

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.

Improved glucose homeostasis in male obese Zucker rats coincides with enhanced baroreflexes and activation of the nucleus tractus solitarius.

Young adult male obese Zucker rats (OZR) develop insulin resistance and hypertension with impaired baroreflex-mediated bradycardia and activation of nucleus tractus solitarius (NTS). Because type 1 diabetic rats also develop impaired baroreflex-mediated NTS activation, we hypothesized that improving glycemic control in OZR would enhance compromised baroreflexes and NTS activation. Fasting blood glucose measured by telemetry was comparable in OZR and lean Zucker rats (LZR) at 12-17 wk. However, with access to food, OZR were chronically hyperglycemic throughout this age range. By 15-17 wk of age, tail samples yielded higher glucose values than those measured by telemetry in OZR but not LZR, consistent with reports of exaggerated stress responses in OZR. Injection of glucose (1g/kg ip) produced larger rises in glucose and areas under the curve in OZR than LZR. Treatment with metformin (300 mg·kg-1·day-1) or pioglitazone (5 mg·kg-1·day-1) in drinking water for 2-3 wk normalized fed glucose levels in OZR with no effect in LZR. After metformin treatment, area under the curve for blood glucose after glucose injection was reduced in OZR and comparable to LZR. Hyperinsulinemia was slightly reduced by each treatment in OZR, but insulin was still greatly elevated compared with LZR. Neither treatment reduced hypertension in OZR, but both treatments significantly improved the blunted phenylephrine-induced bradycardia and NTS c-Fos expression in OZR with no effect in LZR. These data suggest that restoring glycemic control in OZR enhances baroreflex control of heart rate by improving the response of the NTS to raising arterial pressure, even in the presence of hyperinsulinemia and hypertension.