Microinjection Of Kynurenic Acid Research Articles

Activation of nociception-sensitive ionotropic glutamate receptor-expressing rostroventrolateral medulla neurons by stimulation of cardiac afferents in rats.

Myocardial ischemia causes the release of bradykinin, which activates afferent nerve endings in the ventricular epicardium. This elicits a sympathetically mediated increase in arterial pressure and heart rate, referred to as the cardiogenic sympathetic afferent reflex. The rostroventrolateral medulla (RVLM) is a key sympathetic brain stem site for regulating cardiovascular activity. This study aimed to determine the importance of non-barosensitive nociception sympathetic activity and the role of glutamate receptor activation of RVLM neurons in the cardiogenic sympathetic afferent reflex. We tested the hypothesis that inhibition of barosensitive sympathetic activity attenuates but does not abolish the reflex response to cardiac visceral afferents. Renal sympathetic nerve activity (RSNA), arterial pressure, and heart rate responses to epicardial bradykinin application were recorded in anesthetized rats before and after bilateral RVLM microinjection of either GABAA agonist muscimol, ionotropic glutamate receptor antagonist kynurenic acid, N-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-5- phosphonopentanoic acid (AP5), or non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Baroreceptor loading-induced inhibition of barosensitive activity attenuated the bradykinin-induced RSNA response (93 ± 14% increase) and tachycardia (18 ± 3 bpm). While RVLM muscimol microinjection abolished the RSNA response (1.6 ± 4.2% from baseline, 0.49 ± 0.38 μV*s), surprisingly, it did not abolish the tachycardia (27 ± 4 bpm). Kynurenic acid microinjection blocked the arterial pressure and RSNA responses, while AP5 or CNQX only attenuated the responses. These data suggest that nociception-sensitive sympathetic activity that does not appear to be barosensitive is also involved in the cardiogenic sympathetic afferent reflex. Importantly, while muscimol and kynurenic acid abolished the arterial pressure and RSNA response, neither affected the tachycardia, suggesting an alternate cardiac pathway independent of RVLM.

Influence of Microinjection of Kynurenic acid into the Pre-Botzinger Complex on Swallowing in the Cat

Recent evidence supports the involvement of the pre-BÖtzinger complex (pre-BOT) in spatiotemporal features of swallowing and swallowing-breathing coordination in mice. We hypothesized that pharmacological disruption of glutamate neurotransmission in this brainstem region would perturb the swallow motor pattern in anesthetized cats. Electromyograms (EMGs) of upper airway muscles were recorded in anesthetized, spontaneously breathing cats (n=5). Swallowing was induced by injection of 3 cc of water into the oropharynx. Multi-barrel micropipettes were employed to inject artificial cerebrospinal fluid or kynurenic acid (KYN; 50 mM, 44±3 nL per injection) bilaterally into the region of the pre-BOT. Microinjection sites were confirmed histologically by detection of fluorescent beads that were suspended in the microinjectate. Blocking glutamate-related neuronal excitation by bilateral microinjections of the nonspecific glutamate receptor antagonist kynurenic acid had no effect on the number of swallows induced by water injection (control 2.3±0.3; KYN 3.0±0.7; P<0.32). Swallow durations were significantly increased following microinjections of KYN from 691±54 ms to 812±81 ms (p<0.04). KYN significantly reduced the magnitudes of geniohyoid (control 97±3 % of median; Kyn 47±12% of control median, p<0.02), thyrohyoid (control 100±3% of median; KYN 69±8% of control median, p<0.01), and posterior cricoarytenoid (control 103±2% of median; KYN 74±8% of control median, p<0.04) muscle EMGs during swallowing. There was no significant effect of microinjection of KYN on the magnitudes of thyroartytenoid, mylohyoid, or thyropharyngeus electromyograms during swallowing. The results suggest that disruption of glutamate neurotransmission in the preBOT has preferential effects consistent with actions on motor drive to hypoglossal and ventrolateral motoneuron pools that participate in swallowing. Lengthening of swallow duration induced by KYN supports a role for the preBOT in modulating the excitability of the swallow oscillatory circuit. NIH HL163008; NIH HL 131716; HL 155721. 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.

Stimulation of Auricular Vagal Nerves Attenuates Pressor Cardiovascular Responses through Influence on Medullary nuclei

Stimulation of the auricular branch of the vagus nerve (ABVN) is used to manage cardiovascular disorders, including elevated blood pressure. However, precise mechanisms underlying its effects remain unclear. One previous study showed that ABVN stimulation with acupuncture at the center of the inferior concha (the “heart acupoint,” CO15) activates neurons in the nucleus tractus solitarii (NTS) through vagal inputs. Still, its actions on NTS processing and downstream influence on the nucleus regulating cardiovascular function have not been studied. Thus, the present study evaluated the effect of ABVN stimulation on pressor cardiovascular responses and the underlying neural mechanisms associated with medullary nuclei. We hypothesized that ABVN stimulation with acupuncture activates the NTS and ultimately inhibits rostral ventrolateral medulla (rVLM), a critical region controlling cardiovascular function, through opioid and GABA mechanisms. In Sprague‐Dawley rats, repeated pressor cardiovascular reflexes were induced by repetitive gastric distension (GD) under anesthesia. ABVN stimulation with manual acupuncture was performed at the CO15 acupoint with gentle rotation of the acupuncture needle at a frequency of □2 Hz for four min every 10 min during a 30 min period, 2 min in each side of the ear. During sham‐ABVN stimulation, the acupuncture needle was inserted into the same site without subsequent rotation of the needle. Unilateral microinjection of the chemical (50 nl) was conducted in the NTS and rVLM. We found that GD‐induced pressor cardiovascular reflexes significantly attenuated by ABVN stimulation (n=8; P<0.05), but neither by sham‐ABVN stimulation nor mechanical stimulation with the acupuncture needle applied at the HX9 acupoint, which is located at the ear tip overlying lesser occipital nerve, the auricular somatic nerve. The modulation of pressor reflexes by ABVN stimulation was significantly (P<0.05) reversed by microinjection of kynurenic acid (25 mM; n=7) into the NTS as well as naloxone (1 mM; a non‐selective opioid receptor antagonist; n=8) or gabazine (27 mM; a GABAA receptor antagonist; n=8) into the rVLM, but not by their vehicles, 0.9% normal saline. Furthermore, in rats subjected to no GD intervention, we noted that ABVN stimulation induced markedly increase in c‐Fos expression in both NTS and rVLM, compared to sham‐ABVN stimulation. Our data suggest that ABVN stimulation with manual acupuncture excites the NTS through a glutamatergic mechanism, which, in turn, attenuate excitatory cardiovascular reflexes evoked by visceral stimulation through GABA and opioids in the rVLM.Support or Funding InformationThe National Center for Complementary & Integrative Health, R01 grant AT009347 and diversity supplement award to this grant.

Maternal protein restriction affects cardiovascular, but not respiratory response to L-glutamate microinjection into the NTS of conscious rats

ABSTRACT Purpose: Dysregulation of glutamatergic neurotransmission (GN) is linked to sympathetic-respiratory overactivity and hypertension. We investigated whether maternal protein restriction is able to alter GN into the nucleus of the solitary tract (NTS) in adult offspring. Methods: Wistar rat dams were fed with control (NP; 17% protein) or low-protein (LP; 8% protein) diet during pregnancy and lactation, and their offspring were evaluated at 70-90d old. Direct measurements of mean arterial pressure (MAP), heart rate (HR), respiratory frequency (RF) and respiratory (RV) and cardiac (CV) variabilities were assessed in consciousness. The evaluation of GN into NTS over cardiovascular system were assessed by microinjections of unilateral glutamate (L-glu 0.5 nmol/100nL) and bilateral kynurenic acid (Kyn 2.5 nmol/50nL). The NP and LP groups were compared using unpaired Student's t-test where p < 0.05 was considered significant. Results: The LP exhibited higher MAP at rest (p = 0.03) and after L-glu microinjection (p = 0.04), as well as an increase over HR after Kyn microinjection when compared to the NP (p = 0.049). In the RV, the LP group showed an increase of the component-standard deviation 1 (p = 0.037) at rest. In the CV, the LP presented an increase of the low frequency (LF) component of the pulse interval (PI) (p = 0.034), a decrease of high frequency (HF) of the PI (p = 0.034), beyond an increased LF/HF ratio of the PI (p = 0.027) when compared to the NP. The kynurenic acid microinjection did not produce changes in RV or CV (p > 0.05). Conclusions: Altered GN into the NTS may contribute to augmented blood pressure in protein-restricted offspring.

High‐Fat Diet Induced Modulation of Glutamatergic Currents in Neurons of the Dorsal Motor Nucleus of the Vagus Increases Gastric Tone and Motility

Understanding how vagally‐mediated neurocircuits are affected by alterations in diet is crucial to addressing dysregulated feeding behaviors and energy balance, as well as their role in the development of obesity. We have demonstrated previously that acute high fat diet (HFD) exposure modulates glutamatergic neurotransmission to vagal efferent motoneurons. The purpose of this study was to test the hypothesis that acute HFD increases activation of synaptic NMDA receptors (NMDA‐R) on gastric‐projecting dorsal motor nucleus of the vagus (DMV) neurons, which increases vagal efferent drive to the stomach.Sprague Dawley rats (4–8 weeks of age) of both sexes were fed a control diet (14% kcal from fat) throughout the study, or a HFD (60% kcal from fat) for 3–5 days prior to experimentation. Daily food intake was measured from singly‐housed rats for 4 days prior and throughout exposure to HFD. Whole cell patch clamp recordings of gastric‐projecting DMV neurons were made from thin (300μm) brainstem slices. The effects of the NMDA‐R antagonist, AP5 (25μM) on amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) as well as action potential firing (AP) firing rate were examined. In vivo recordings of gastric motility and tone were made in response to microinjection of the non‐selective ionotropic glutamate antagonist, kynurenic acid (100pmol/60nL) and AP5 (500pmol/60nL).HFD exposure had no significant effect on caloric intake (control 55±4kcal/24hr vs acute HFD 59±9kcal/24hr; p&gt;0.05) during the acute (3–5 day) period of study. After acute exposure to HFD, however, 9/15 DMV neurons responded to AP5 with a significant decrease in mEPSC amplitude and frequency (24.6±2.9pA vs 17.5±1.7pA; 1.4±0.3 pulses per second (p.p.s) vs 0.5±0.1p.p.s, respectively P&lt;0.05 for each) as compared to only 1/18 control neurons. (18.7±1.3pA vs 18.0±1.3pA, P&gt;0.05). In control conditions, AP5 had no effect on AP firing rate on 7/10 neurons (40.0±4.8 events/30sec vs 47.9±6.5 events/30sec, P&gt;0.05), however, firing rate was decreased significantly in 5/5 neurons following acute HFD exposure (27.4±4.87 events/30sec vs 21.1±13.6 events/30sec, P&lt;0.05). Brainstem microinjection of kynurenic acid had no effect on antral motility in control rats (102.5±17.6% of baseline, P&gt;0.05), but significantly reduced motility following acute HFD exposure (59.9±1.10% of baseline, P&lt;0.05).These data suggest that acute HFD exposure increases NMDA‐R activation, increases DMV neuronal excitability and, subsequently, vagal efferent control of gastric motility. Since this NMDA‐R activation is observed only during periods of caloric balance, these results suggest that plasticity within vagal neurocircuit glutamatergic signaling may be a critical homeostatic response to altered diet that helps maintain appropriate feeding behavior and caloric intake to defend against the development of obesity.Support or Funding InformationNSF IOS1148079 to Kirsteen BrowningThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glutamate receptors of the A5 region modulate cardiovascular responses evoked from the dorsomedial hypothalamic nucleus and perifornical area.

To assess the possible function of glutamate in the interaction between the dorsomedial hypothalamic nucleus-perifornical area (DMH-PeF) and the A5 pontine region (A5), cardiovascular and respiratory changes were studied in response to electrical stimulation of the DMH-PeF (1ms pulses, 30-50μA given at 100Hz for 5s) before and after the microinjection of kynurenic acid (non-specific glutamate receptor antagonist; 50nl, 5nmol), MK-801 (NMDA receptor antagonist; 50nl, 50nmol), CNQX (non-NMDA receptor antagonist; 50nl, 50nmol) or MCPG (metabotropic glutamate receptor antagonist; 50nl, 5nmol) within the A5 region. DMH-PeF electrical stimulation elicited a pressor (p< 0.001) and tachycardic response (p< 0.001) which was accompanied by an inspiratory facilitation characterised by an increase in respiratory rate (p< 0.001) due to a decrease in expiratory time (p< 0.01). Kynurenic acid within the A5 region decreased the tachycardia (p< 0.001) and the intensity of the blood pressure response (p< 0.001) to DMH-PeF stimulation. After the microinjection of MK-801 and CNQX into the A5 region, the magnitude of the tachycardia and the pressor response were decreased (p< 0.05 and p< 0.01; p< 0.001 and p< 0.05, respectively). After MCPG microinjection into the A5 region, a decrease in the tachycardia (p< 0.001) with no changes in the pressor response was observed during DMH-PeF stimulation. The respiratory response elicited by DMH-PeF stimulation was not changed after the microinjection of kynurenic acid, MK-801, CNQX or MCPG within the A5 region. These results suggest that A5 region glutamate receptors play a role in the cardiovascular response elicited from the DMH-PeF. The possible mechanisms involved in these interactions are discussed.

Microinjection of kynurenic acid in the rostral nucleus of the tractus solitarius disrupts spatiotemporal aspects of mechanically induced tracheobronchial cough.

The importance of neurons in the nucleus of the solitary tract (NTS) in the production of coughing was tested by microinjections of the nonspecific glutamate receptor antagonist kynurenic acid (kyn; 100 mM in artificial cerebrospinal fluid) in 15 adult spontaneously breathing anesthetized cats. Repetitive coughing was elicited by mechanical stimulation of the intrathoracic airway. Electromyograms (EMG) were recorded from inspiratory parasternal and expiratory transversus abdominis (ABD) muscles. Bilateral microinjections of kyn into the NTS rostral to obex [55 ± 4 nl total in 2 locations (n = 6) or 110 ± 4 nl total in 4 locations (n = 5)], primarily the ventrolateral subnucleus, reduced cough number and expiratory cough efforts (amplitudes of ABD EMG and maxima of esophageal pressure) compared with control. These microinjections also markedly prolonged the inspiratory phase, all cough-related EMG activation, and the total cough cycle duration as well as some other cough-related time intervals. In response to microinjections of kyn into the NTS rostral to the obex respiratory rate decreased, and there were increases in the durations of the inspiratory and postinspiratory phases and mean blood pressure. However, bilateral microinjections of kyn into the NTS caudal to obex as well as control vehicle microinjections in the NTS location rostral to obex had no effect on coughing or cardiorespiratory variables. These results are consistent with the existence of a critical component of the cough rhythmogenic circuit located in the rostral ventral and lateral NTS. Neuronal structures of the rostral NTS are significantly involved specifically in the regulation of cough magnitude and phase timing.NEW & NOTEWORTHY The nucleus of the solitary tract contains significant neuronal structures responsible for control of 1) cough excitability, 2) motor drive during cough, 3) cough phase timing, and 4) cough rhythmicity. Significant elimination of neurons in the solitary tract nucleus results in cough apraxia (incomplete and/or disordered cough pattern). The mechanism of the cough impairment is different from that for the concomitant changes in breathing.

Alterations in the rostral ventromedial medulla after the selective ablation of μ-opioid receptor expressing neurons

The rostral ventromedial medulla (RVM) exerts both inhibitory and excitatory controls over nociceptive neurons in the spinal cord and medullary dorsal horn. Selective ablation of mu-opioid receptor (MOR)-expressing neurons in the RVM using saporin conjugated to the MOR agonist dermorphin-saporin (derm-sap) attenuates stress and injury-induced behavioral hypersensitivity, yet the effect of RVM derm-sap on the functional integrity of the descending inhibitory system and the properties of RVM neurons remain unknown. Three classes of RVM neurons (on-cells, off-cells, and neutral cells) have been described with distinct responses to noxious stimuli and MOR agonists. Using single unit recording in lightly anesthetized rats, RVM neurons were characterized after microinjections of derm-sap or saporin. Derm-sap treatment resulted in a reduction in on-cells and off-cells when compared to saporin controls (P < 0.05). The number of neutral cells remained unchanged. After derm-sap treatment, RVM microinjections of the glutamate receptor agonist homocysteic acid increased tail-flick latencies, whereas the MOR agonist DAMGO had no effect. Furthermore, electrical stimulation of the periaqueductal gray produced analgesia in both derm-sap and saporin controls with similar thresholds. Microinjection of kynurenic acid, a glutamate receptor antagonist, into the RVM disrupted periaqueductal gray stimulation-produced analgesia in both saporin-treated and derm-sap-treated rats. These results indicate that MOR-expressing neurons in the RVM are not required for analgesia produced by either direct or indirect activation of neurons in the RVM.

Excitatory amino acid receptors in the dorsomedial hypothalamic area contribute to the chemoreflex tachypneic response

This study evaluated the effect of blockade of the excitatory amino acid (EAA) receptors in the dorsomedial hypothalamic (DMH) area on the ventilatory and cardiovascular responses of the chemoreflex activation in conscious rats. Bilateral microinjection of kynurenic acid (2.7nmol, n=6) into the DMH area reduced the tachypneic (+264±13 versus +204±14 cpm, P<0.05) and pressor (+52±5 versus +31±6mmHg, P<0.05) components of chemoreflex but had no effect on the bradycardic component (−214±7 versus −244±17bpm) of the chemoreflex. The magnitudes of the reduction in pressor and tachypneic chemoreflex responses were not significantly correlated (r=0.308, P=0.330). These data indicate that neurons located in the DMH area are activated by chemoreflex; that this activation is mediated via EAA receptors; and that it is essential for the full expression of the respiratory component of the chemoreflex.

Functional neural-bone marrow pathways: implications in hypertension and cardiovascular disease.

Treatment-resistant hypertension (TRHT) is characterized by persistently high arterial blood pressure (BP), partly as a result of a dysfunctional autonomic nervous system (ANS), wherein sympathetic drive/norepinephrine spillover is increased and parasympathetic drive is decreased.1–3 The difficulty in treatment of TRHT arises precisely from this partly neurogenic component because the available drug therapies do not target the central nervous system (CNS) directly. Because of this and despite recent advances in techniques such as renal denervation and carotid baroreceptor activation,4,5 successful treatment and long-term control of TRHT remain challenging.6 In an attempt to develop more effective treatments, many groups are investigating specific causes of TRHT. A large body of experimental evidence implicates both genetic and environmental influences, such as salt sensitivity and elevated systemic renin–angiotensin system (RAS) activity7–14 in the pathophysiology of this disease. Furthermore, a majority of studies point to dysregulations in the activity within the cardiorespiratory brain regions as a reason for increased sympathetic and decreased parasympathetic drive to the peripheral organs,14–21 resulting in end-organ damage,21–25 vascular/endothelial dysfunction,26,27 and hormonal imbalance,28 which perpetuate the pathophysiology and complicate treatment strategies. Despite our increasing understanding of TRHT, the origins of this brain dysregulation remain largely unknown. Recently, the activity of the immune system29–31 and neuroimmune pathways in patients with hypertension and animal models of hypertension has been highlighted.32–36 Studies suggest that both the sympathetic and the parasympathetic arms of the ANS can exert their influence on the activity of the immune organs, tissues, and cells, and that it is the dysfunctional ANS-immune communication that may lead to hypertension and CVD.35–40 The aim of this review is to summarize the latest advances in this …

Acute pancreatitis decreases the sensitivity of pancreas‐projecting dorsal motor nucleus of the vagus neurones to group II metabotropic glutamate receptor agonists in rats

Recent studies have shown that pancreatic exocrine secretions (PES) are modulated by dorsal motor nucleus of the vagus (DMV) neurones, whose activity is finely tuned by GABAergic and glutamatergic synaptic inputs. Group II metabotropic glutamate receptors (mGluR) decrease synaptic transmission to pancreas-projecting DMV neurones and increase PES. In the present study, we used a combination of in vivo and in vitro approaches aimed at characterising the effects of caerulein-induced acute pancreatitis (AP) on the vagal neurocircuitry modulating pancreatic functions. In control rats, microinjection of bicuculline into the DMV increased PES, whereas microinjections of kynurenic acid had no effect. Conversely, in AP rats, microinjection of bicuculline had no effect, whereas kynurenic acid decreased PES. DMV microinjections of the group II mGluR agonist APDC and whole cell recordings of excitatory currents in identified pancreas-projecting DMV neurones showed a reduced functional response in AP rats compared to controls. Moreover, these changes persisted up to 3 weeks following the induction of AP. These data demonstrate that AP increases the excitatory input to pancreas-projecting DMV neurones by decreasing the response of excitatory synaptic terminals to group II mGluR agonist.

Brainstem mechanisms controlling cardiovascular reflexes in channel catfish

Microinjections of kynurenic acid and kainic acid into the general visceral nucleus (nGV), homologous to the mammalian nucleus tractus solitarius of the medulla, in anesthestized, spontaneously breathing catfish were used to identify central areas and mechanisms controlling resting normoxic heart rate and blood pressure and the cardiovascular responses to hypoxia. Kynurenic acid, an antagonist of ionotropic glutamate receptors, significantly reduced resting normoxic heart rate but did not block the bradycardia associated with aquatic hypoxia. Kainic acid (an excitotoxic glutamatergic receptor agonist) also significantly reduced normoxic heart rate, but blocked the hypoxia-induced bradycardia. Neither kynurenic acid nor kainic acid microinjections affected blood pressure in normoxia or hypoxia. The results of this study indicate that glutamatergic receptors in the nGV are involved in the maintenance of resting heart rate and the destruction of these neurons with kainic acid abolishes the bradycardia associated with aquatic hypoxia.

Participation of 5-HT and AT1Receptors within the Rostral Ventrolateral Medulla in the Maintenance of Hypertension in the Goldblatt 1 Kidney-1 Clip Model

The hypothesis that changes in neurotransmission within the rostral ventrolateral medulla (RVLM) are important to maintain the high blood pressure (BP) was tested in Goldblatt one kidney-one clip hypertension model (1K-1C). Male Wistar rats were anesthetized (urethane 1.2 g/kg, i.v.), and the effects of bilateral microinjections into the RVLM of the following drugs were measured in 1K-1C or control groups: glutamate (0.1 mol/L, 100 nL) and its antagonist kynurenic acid (0.02 mol/L, 100 nL), the angiotensin AT1 receptor antagonist candesartan (0.01 mol/L, 100 nL), and the nonselective 5-HT receptor antagonist methiothepin (0.06 mol/L, 100 nL). Experiments in 1K-1C rats were performed 6 weeks after surgery. In anesthetized rats glutamate response was larger in hypertensive than in normotensive rats (H: Δ67 ± 6.5; N: Δ43 ± 3.54 mmHg). In contrast, kynurenic acid microinjection into the RVLM did not cause any change in BP in either group. The blockade of either AT1 or 5-HT receptors within the RVLM decreased BP only in 1K-1C rats. A largest depressor response was caused by 5-HT receptor blockade. The data suggest that 5-HT and AT1 receptors act tonically to drive RVLM in 1K-1C rats, and these actions within RVLM contribute to the pathogenesis of this model of hypertension.

Ionotropic but not metabotropic glutamatergic receptors in the Locus Coeruleus modulate the hypercapnic ventilatory response in unanesthetized rats

Central chemoreception is an important mechanism by which changes in tissue CO2/H+ regulate breathing, and the Locus coeruleus (LC) is one of many putative central chemoreceptor sites. Here, we studied the contribution of LC glutamatergic receptors on ventilatory, cardiovascular and thermal responses to hypercapnia. To this end, we measured pulmonary ventilation (VE), body temperatures (Tb), mean arterial pressure (MAP) and heart rate (fH) of male Wistar rats before and after microinjection of Kynurenic Acid (KY, an ionotropic glutamate receptors antagonist, 10 mM) or α‐methyl‐4‐carboxyphenylglycine (MCPG, a metabotropic glutamate receptor antagonist, 10 ηM) unilateraly into the LC, followed by 60 minutes of air or hypercapnia exposure (7% CO2). Ventilatory response to hypercapnia was higher in animals treated with KY intra‐LC (1899 ± 83 mL kg−1 min−1) compared to the control group (1141 ± 64 mL kg−1 min−1; P &lt; 0.01). However, the MCPG treatment within the LC had no effect on the hypercapnia‐induced hyperpnea. The cardiovascular and thermal control were not affected by hypercapnia or by the injection of KY and MCPG in the LC. These data suggest that the glutamate acting on ionotropic but not metabotropic receptors in the LC exerts an inibitory modulation of hypercapnia‐induced hyperpnea.Financial support: FAPESP, CNPq, PROPe.

Ionotropic but not metabotropic glutamatergic receptors in the locus coeruleus modulate the hypercapnic ventilatory response in unanaesthetized rats

Central chemoreceptors are important to detect changes of CO2/H(+), and the Locus coeruleus (LC) is one of the many putative central chemoreceptor sites. Here, we studied the contribution of LC glutamatergic receptors on ventilatory, cardiovascular and thermal responses to hypercapnia. To this end, we determined pulmonary ventilation (V(E)), body temperatures (T(b)), mean arterial pressure (MAP) and heart rate (HR) of male Wistar rats before and after unilateral microinjection of kynurenic acid (KY, an ionotropic glutamate receptor antagonist, 10 nmol/0.1 μL) or α-methyl-4-carboxyphenylglycine (MCPG, a metabotropic glutamate receptor antagonist, 10 nmol/0.1 μL) into the LC, followed by 60 min of air breathing or hypercapnia exposure (7% CO2). Ventilatory response to hypercapnia was higher in animals treated with KY intra-LC (1918.7 ± 275.4) compared with the control group (1057.8 ± 213.9, P < 0.01). However, the MCPG treatment within the LC had no effect on the hypercapnia-induced hyperpnea. The cardiovascular and thermal controls were not affected by hypercapnia or by the injection of KY and MCPG in the LC. These data suggest that glutamate acting on ionotropic, but not metabotropic, receptors in the LC exerts an inhibitory modulation of hypercapnia-induced hyperpnea.

Changes in GABAergic inputs in the paraventricular nucleus maintain sympathetic vasomotor tone in chronic heart failure

The paraventricular nucleus (PVN) of the hypothalamus is an important region of the brain involved in the regulation of sympathetic vasomotor tone. Accumulating evidence supports the idea that a change in hypothalamic γ-aminobutyric acid (GABA)-ergic inhibitory and glutamatergic excitatory inputs contribute to the exacerbated sympathetic drive in chronic heart failure (HF). The purpose of this study was to determine whether a possible imbalance between glutamatergic and GABAergic inputs to the PVN contributes to increased sympathetic outflow in HF in two different sympathetic territories. Renal (RSNA) and splanchnic sympathetic nerve activity (SSNA), mean arterial blood pressure (MAP) and heart rate were recorded from urethane-anesthetized HF or sham rats. The NMDA-glutamate and GABA-A receptor densities within the PVN were quantified in HF and sham rats by autoradiography. Bilateral microinjection of kynurenic acid (4nmol) into the PVN decreased MAP and RSNA and SSNA in HF but not in sham rats. Furthermore, in response to GABA-A blockade in the PVN by bicuculline (400 pmol), hypertension and SSNA were reduced in HF compared to sham. The quantification of ionotropic NMDA receptors and GABA-A receptors in the PVN showed a significant reduction of GABA-A in HF rats; however, the NMDA density in the PVN did not differ between groups. Thus, this study provides evidence that the sympathoexcitation is maintained by an imbalance between GABAergic and glutamatergic inputs in the PVN in HF. The reduced GABAergic input results in relatively augmented glutamatergic actions in the PVN of HF rats.

Hypoxia-induced hypothermia mediated by the glutamatergic transmission in the lateral preoptic area

Hypoxia evokes a regulated decrease in the body core temperature, which response is mediated, at least in part, by noradrenaline (NA) and nitric oxide (NO) in the rostromedial preoptic area (POA) of the hypothalamus. In the accompanying paper, it was shown that glutamatergic activation of the lateral POA also evokes hypothermic responses. Here, I tested the hypothesis that the glutamatergic transmission in the lateral POA is critically involved in the neural mechanism of hypoxia-induced hypothermia. Hypoxic ventilation (10% O2–90% N2, 5min) as well as a single microinjection of NA (50pmol) or the NO donor sodium nitroprusside (8.4nmol) into the rostromedial POA evoked an increase in the tail skin temperature and a decrease in the colonic temperature in urethane–chloralose-anesthetized, neuromuscularly blocked, artificially ventilated rats. All of these responses were greatly attenuated by pretreatment with multiple microinjections of kynurenic acid (10nmol, four locations), a nonselective glutamate receptor antagonist, but not by those with saline solution, in the bilateral rostral and central parts of the lateral POA. These results suggest that the NA- and NO-sensitive structure in the rostromedial POA activated the glutamatergic transmission in the lateral POA to mediate hypoxia-induced hypothermia.

Glutamatergic neurotransmission in the hypothalamus PVN on heart rate variability in exercise trained rats

The paraventricular nucleus of hypothalamus (PVN) is a well known site of integration for autonomic and cardiovascular responses, and the glutamate neurotransmitter plays an important role. The aim of our study was to evaluate the cardiovascular parameters and autonomic modulation by means of spectral analysis after ionotropic glutamate receptor inhibition in the PVN in conscious sedentary (S) or swimming trained (ST) rats. After exercise training protocol, adult male Wistar rats, instrumented with guide cannulae to PVN and artery and vein catheters were submitted to mean arterial pressure (MAP) and heart rate (HR) recording. At baseline, physical training induced a resting bradycardia (S: 379 ± 3, ST: 349 ± 2 bpm, Pb<0.05) and promoted adaptations in HRV characterized by an increase of HF in normalized values and a decrease of LF in absolute and normalized units compared with the sedentary group. Microinjection of kynurenic acid (KYNA) in the PVN of sedentary and trained rats promoted decreases in MAP and HR, but the decrease in HR was smaller in the trained animals (ΔHRS: -48 ± 7, ST: -28 ± 4 bpm, Pb<0.05). Furthermore, the differences in baseline parameters of pulse interval, found between sedentary and trained animals, disappeared after KYNA microinjection in the PVN. Our data suggest that the cardiovascular and autonomic adaptations to the heart induced by exercise training may involve glutamatergic mechanisms in the PVN.

Parabrachial complex glutamate receptors modulate the cardiorespiratory response evoked from hypothalamic defense area

To characterize the possible role of glutamate in the interaction between Hypothalamic Defense Area (HDA) and Parabrachial complex (PBc) nuclei, cardiorespiratory changes were analyzed in response to electrical stimulation of the HDA (1 ms pulses, 30-50 μA given at 100 Hz for 5s) before and after the microinjection of the nonspecific glutamate receptor antagonist kynurenic acid (50 nl, 5 nmol), NMDA receptor antagonist MK-801 (50 nl, 50 nmol), non-NMDA receptor antagonist CNQX (50 nl, 50 nmol) or metabotropic glutamate receptor antagonist MCPG (50 nl, 5 nmol) within the PBc. HDA stimulation evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (p<0.001) due to a decrease in expiratory time (p<0.01). The respiratory response was accompanied by a pressor (p<0.001) and a tachycardic response (p<0.001). Kynurenic acid within the lateral parabrachial region (lPB) abolished the tachycardia (p<0.001) and decreased the magnitude of blood pressure response (p<0.001) to HDA stimulation. Similarly, the magnitude of the tachycardia and the pressor response was decreased after the microinjection of MK-801 (p<0.01 and p<0.001, respectively) and CNQX (p<0.05 in both cases) into the lPB. Kynurenic acid microinjection in this region produced an inhibition of the tachypnea (p<0.001) to HDA stimulation but the respiratory response persisted unchanged after MK-801 or CNQX microinjection into the lPB. Kynurenic acid within the medial parabrachial region (mPB) abolished the tachycardia (p<0.01) and decreased the magnitude of the pressor response (p<0.001) to HDA stimulation. MK-801 and CNQX microinjection in this region decreased the magnitude of the tachycardia (p<0.05, in both cases) and pressor response (p<0.05, in both cases). The respiratory response evoked by HDA stimulation was not changed after the microinjection of kynurenic acid, MK-801 or CNQX within the mPB. No changes were observed in the cardiorespiratory response evoked to HDA stimulation after MCPG microinjection within lPB and mPB. These results indicate that glutamate PBc receptors are involved in the cardiorespiratory response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.

Sympathoexcitation during chemoreflex active expiration is mediated byl-glutamate in the RVLM/Bötzinger complex of rats

The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla/Bötzinger/pre-Bötzinger complexes (RVLM/BötC/pre-BötC) on the respiratory modulation of sympathoexcitatory response to peripheral chemoreflex activation (chemoreflex) was evaluated in the working heart-brain stem preparation of juvenile rats. We identified different types of baro- and chemosensitive presympathetic and respiratory neurons intermingled within the RVLM/BötC/pre-BötC. Bilateral microinjections of kynurenic acid (KYN) into the rostral aspect of RVLM (RVLM/BötC) produced an additional increase in frequency of the phrenic nerve (PN: 0.38 ± 0.02 vs. 1 ± 0.08 Hz; P < 0.05; n = 18) and hypoglossal (HN) inspiratory response (41 ± 2 vs. 82 ± 2%; P < 0.05; n = 8), but decreased postinspiratory (35 ± 3 vs. 12 ± 2%; P < 0.05) and late-expiratory (24 ± 4 vs. 2 ±1%; P < 0.05; n = 5) abdominal (AbN) responses to chemoreflex. Likewise, expiratory vagal (cVN; 67 ± 6 vs. 40 ± 2%; P < 0.05; n = 5) and expiratory component of sympathoexcitatory (77 ± 8 vs. 26 ± 5%; P < 0.05; n = 18) responses to chemoreflex were reduced after KYN microinjections into RVLM/BötC. KYN microinjected into the caudal aspect of the RVLM (RVLM/pre-BötC; n = 16) abolished inspiratory responses [PN (n = 16) and HN (n = 6)], and no changes in magnitude of sympathoexcitatory (n = 16) and expiratory (AbN and cVN; n = 10) responses to chemoreflex, producing similar and phase-locked vagal, abdominal, and sympathetic responses. We conclude that in relation to chemoreflex activation 1) ionotropic glutamate receptors in RVLM/BötC and RVLM/pre-BötC are pivotal to expiratory and inspiratory responses, respectively; and 2) activation of ionotropic glutamate receptors in RVLM/BötC is essential to the coupling of active expiration and sympathoexcitatory response.