Microinjection Of Kynurenic Acid Research Articles

The cardiovascular responses to chemoreflex activation are not changed by isolated blockade of NMDA or non‐NMDA receptors in the dorsomedial hypothalamus

Studies from our laboratory have shown that microinjection of kynurenic acid, a non‐selective antagonist at NMDA and non‐NMDA receptors, into the dorsomedial hypothalamus (DMH) reduced the pressor response and abolished the behavioral response of chemoreflex. This study evaluated the effect of selective blockade of NMDA or non‐NMDA receptors on the cardiovascular responses of the chemoreflex. Bilateral microinjection of AP‐5 into the DMH produced no significant changes on the pressor response (+47 ± 2 mmHg vs 44 ± 3 mmHg) and bradycardia (−245 ± 24 vs −244 ± 27 bpm) of the chemoreflex; however, it was able to reduce the cardiovascular responses induced by NMDA (+21 ± 1 vs 8 ± 2 mmHg; +135 ± 13 vs 69 ± 18 bpm). Bilateral microinjection of CNQX into the DMH also produced no changes on the pressor response (+44 ± 9 vs +36 ± 8 mmHg) and bradycardia (−228 ± 34 vs −219 ± 32 bpm) of the chemoreflex, but significantly reduced the cardiovascular responses induced by AMPA (+21 ± 2 vs +5 ± 2 mmHg; +111 ± 5 vs +40 ± 17 bpm). These results indicate that isolated blockade of NMDA receptors or non‐NMDA receptors of the DMH does not promote changes in cardiovascular responses to chemoreflex. Support: CNPq/FAPEMIG.

Microinjection of kynurenic acid (KA) into the medial reticular formation elicits behavior‐dependent effects on cough and swallow motor patterns in the anesthetized cat

Kynurenic acid (KA) affects multiple receptors and is elevated in the brainstem during neurological diseases, such as Alzheimer's and Parkinson's. Aspiration pneumonia associated with dysphagia and dystussia is common in these disorders. The medial reticular formation (MRF) caudal to obex is important for cough coordination. We speculated this region is also important for swallow. Electromyograms of geniohyoid (GH), thyroarytenoid (ThAr), upper esophageal sphincter (UES), thyrohyoid (TH), parasternal (PS) and transversus abdominis (TA) were recorded in anesthetized cats. Cough and swallow were elicited by tracheobronchial stimulation and intraoral water injection respectively. KA (50nL; 50mM) was microinjected bilaterally at 6 locations within the MRF. During swallow, KA induced differential effects on cranial motor activity (decreased: TH, ThAr, and GeHy and increased: UES), but the spinal motor component (PS) did not change. During cough, cranial motor drive did not change, but there was a decrease in motor drive to spinal motoneuron pools (PS, TA). The results support multiple effects of KA including direct actions at/near the injections and secondary synaptic effects on anatomically distant neural assemblies. The data support the existence of common neuronal assemblies in the MRF for swallow and cough. Support: HL 89104; HL 103415.

Ionotropic glutamate receptors in the external lateral parabrachial nucleus participate in processing cardiac sympathoexcitatory reflexes

Stimulation of cardiac sympathetic afferents during myocardial ischemia with metabolites such as bradykinin (BK) evokes sympathoexcitatory reflex responses and activates neurons in the external lateral parabrachial nucleus (elPBN). The present study tested the hypothesis that this region in the pons processes sympathoexcitatory cardiac reflexes through an ionotropic glutamate receptor mechanism. The ischemic metabolite BK (0.1-1 μg) was injected into the pericardial space of anesthetized and bilaterally vagotomized or intact cats. Hemodynamic and renal sympathetic nerve activity (RSNA) responses to repeated administration of BK before and after unilateral 50-nl microinjections of kynurenic acid (Kyn; 25 mM), 2-amino-5-phosphonopentanoic acid (AP5; 25 mM), and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzol(F)quinoxaline (NBQX; 10 mM) into the elPBN were recorded. Intrapericardial BK evoked significant increases in mean arterial pressure (MAP) and RSNA in seven vagotomized cats. After blockade of glutamate receptors with the nonselective glutamate receptor antagonist Kyn, the BK-evoked reflex increases in MAP (50 ± 6 vs. 29 ± 2 mmHg) and RSNA (59 ± 8.6 vs. 29 ± 4.7%, before vs. after) were significantly attenuated. The BK-evoked responses returned to pre-Kyn levels 85 min after the application of Kyn. Similarly, BK-evoked reflex responses were reversibly attenuated by blockade of glutamate N-methyl-d-aspartate (NMDA) receptors with AP5 (n = 5) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors with NBQX (n = 5). In contrast, we observed that the repetitive administration of BK evoked consistent reflex responses including MAP and RSNA before and after microinjection of 50 nl of the artificial cerebrospinal fluid vehicle into the elPBN in five animals. Microinjection of glutamate receptor antagonists into regions outside the elPBN did not alter BK-induced reflex responses. Microinjection of Kyn into the elPBN reversibly attenuated BK-induced reflex responses in four vagus intact animals. These data are the first to show that NMDA and AMPA ionotropic glutamate receptors in the elPBN play an important role in processing cardiac excitatory reflex responses.

Microinjection of kynurenic acid (KA) into the medial reticular formation elicits dysphagic swallow motor patterns in the anesthetized cat

Kynurenic acid (KA), an endogenous compound with activities at multiple neurotransmitter receptors, is found in higher concentrations in the central nervous system of patients with Alzheimer's and Parkinson's disease. A leading cause of death in these disorders is aspiration pneumonia, caused by dysphagia and dystussia. The medial reticular formation (MRF) caudal to obex is important for cough coordination. We speculate this region is also important for swallow. Electromyograms (EMG) of geniohyoid (GH), thyroarytenoid (ThAr), upper esophageal sphincter (UES), and thyrohyoid (TH) were recorded in anesthetized cats. Swallowing was elicited by injection of water into the pharynx. KA (50nL; 50mM) was microinjected bilaterally at 4 locations within the MRF. There was significant suppression of EMG magnitudes for TH, ThAr, and GH during swallow 20 min after microinjections. At 2 hours post-injections, EMG magnitudes began returning to control values. However, disruption of temporal coordination of the EMGs persisted. Complete resolution of the suppressive effect and temporal discoordination during swallow was observed at 4 hours. We conclude that neurons within the MRF are important for control of laryngeal and pharyngeal motoneuron activity during swallow. These results support the hypothesis that the swallow control network includes MRF neurons caudal to obex. Supported by NIH HL 89104; HL 103415.

Role of glutamate receptors in the lateral parabrachial nucleus in regulation of cardiac sympathoexcitatory reflexes

Stimulation of cardiac sympathetic afferents by ischemic metabolites like bradykinin (BK) evokes sympathoexcitatory reflex responses and activates neurons in the external lateral parabrachial nucleus (elPBN). The present study tested the hypothesis that the elPBN processes these reflexes through a glutamatergic mechanism. BK (0.1–1 μg) was injected into pericardial sack of anesthetized and bilateral vagotomized cats. Hemodynamic and renal sympathetic nerve activity (RSNA) responses to repeated intrapericardial BK before and after microinjection of kynurenic acid (Kyn, 25 mM, 50 nl) into the elPBN were recorded. Intrapericardial BK application evoked significant increases in mean blood pressure (MAP, 50 ± 6 mmHg) and RSNA (59 ± 8.6 %) in six cats. Kyn in the elPBN reduced BK-evoked reflex responses including MAP (50 ± 6 vs. 29 ± 2 mmHg, before vs. after) and RSNA (59 ± 8.6 vs. 29 ± 4.7 %, before vs. after). In contrast, inadvertent microinjection into areas surrounding the elPBN did not alter the responses to intrapericardial BK (MAP, 49 ± 3 vs. 51 ± 4 mmHg; RSNA, 64 ± 4 vs. 62 ± 2%, before vs. after). These data suggest that glutamate receptors in the elPBN play an important role in processing cardiac sympathoexcitatory reflex responses (supported by NIH HL-66217).

Glutamate receptors in ventral medulla are essential for expiratory and inspiratory responses to chemoreflex activation in unanesthetized rats

The role of glutamate receptors in Bötzinger (BotC) and pre-Bötzinger complex (pre-BotC) on chemoreflex-evoked sympathetic/pressor and respiratory responses (KCN) was evaluated in awake rats and in situ working heart-brainstem preparation (WHBP). In awake rats (n=7) bilateral microinjections of Kynurenic acid (KYN, 160 mM) into BotC enhanced tachypneic (426±11 vs 204±11 cpm; p<0,05) and reduced pressor (13±1 vs 55±3 mmHg; p<0,05) responses to chemoreflex. In pre-BotC of awake rats KYN produced respiratory arrest. In the WHBP the expiration was evaluated by recording the abdominal nerve (ABD) and the inspiration by recording hypoglossal (HN) and phrenic nerves (PN). KYN (200 mM) microinjected into BotC in the WHBP (n=8) enhanced the frequency of PN (0.89±0.03 vs 0.42±0.02 Hz; p<0.05) and pre-inspiratory HN responses (83±9 vs 45±5 %; P<0.05) but decreased the sympathoexcitatory (22±2 vs 95±9 %; p<0.05) and ABD (35±2 vs 76±5%; P<0.05) responses to chemoreflex activation. KYN microinjected into the pre-BotC in the WHBP (n=7) abolished the inspiratory responses (PN and HN activities), but produced no changes in the sympathoexcitatory and expiratory responses to chemoreflex activation. We conclude that in unanesthetized models glutamate receptors in BotC and pre-BotC are essential for the processing of expiratory and inspiratory responses to chemoreflex activation. Supported by CAPES, CNPq and FAPESP.

Asymmetrical changes in lumbar sympathetic nerve activity following stimulation of the sciatic nerve in rat

Noxious stimulation of the leg increases hind limb blood flow (HBF) to the ipsilateral side and decreases to the contralateral in rat. Whether or not this asymmetrical response is due to direct control by sympathetic terminals or mediated by other factors such as local metabolism and hormones remains unclear. The aim of this study was to compare responses in lumbar sympathetic nerve activity, evoked by stimulation of the ipsilateral and contralateral sciatic nerve (SN). We also sought to determine the supraspinal mechanisms involved in the observed responses. In anesthetized and paralyzed rats, intermittent electrical stimulation (1 mA, 0.5 Hz) of the contralateral SN evoked a biphasic sympathoexcitation. Following ipsilateral SN stimulation, the response is preceded by an inhibitory potential with a latency of 50 ms ( N = 26). Both excitatory and inhibitory potentials are abolished following cervical C1 spinal transection ( N = 6) or bilateral microinjections of muscimol ( N = 6) in the rostral ventrolateral medulla (RVLM). This evidence is suggestive that both sympathetic potentials are supraspinally mediated in this nucleus. Blockade of RVLM glutamate receptors by microinjection of kynurenic acid ( N = 4) selectively abolished the excitatory potential elicited by ipsilateral SN stimulation. This study supports the physiological model that activation of hind limb nociceptors evokes a generalized sympathoexcitation, with the exception of the ipsilateral side where there is a withdrawal of sympathetic tone resulting in an increase in HBF.

Excitatory amino acid receptors in the dorsomedial hypothalamus are involved in the cardiovascular and behavioural chemoreflex responses

The present study investigated the role of the dorsomedial hypothalamus (DMH) on cardiovascular and behavioural responses of chemoreflex activation in conscious rats. The arterial chemoreflex was activated by potassium cyanide (KCN, 40 μg, i.v.) before and after bilateral microinjection of lidocaine (2%) or kynurenic acid (2.7 nmol) into the DMH. Locomotor activity was measured to assess the chemoreflex behavioural response. Bilateral microinjection of lidocaine into the DMH produced a significant reduction in the pressor response induced by chemoreflex activation (+51 ± 4 versus +34 ± 5 mmHg, n = 5, P < 0.05). A similar reduction in the pressor chemoreflex response was also observed after microinjection of kynurenic acid into the DMH (+50 ± 3 versus +22 ± 5 mmHg, n = 6, P < 0.05). Strikingly, the behaviour/locomotor activity induced by chemoreflex activation was virtually abolished after blockade of excitatory amino acid receptors in the DMH with kynurenic acid (44 ± 6 versus 5 ± 4 cm, n = 6, P < 0.05). There was no correlation between the reduction in pressor and behavioural chemoreflex responses (r = -0.186, P > 0.05). The bradycardic response of the chemoreflex was not altered by lidocaine or kynurenic acid microinjected into the DMH. These results strongly suggest that the excitatory amino acid receptors in the DMH are essential for full expression of the behavioural response of the chemoreflex and participate, at least in part, in the integration of the pressor response of the chemoreflex in conscious rats.

Nucleus raphé pallidus participates in midbrain-medullary cardiovascular sympathoinhibition during electroacupuncture

We have shown that electroacupuncture (EA) inhibits sympathoexcitatory rostral ventrolateral medulla (rVLM) neurons and reflex responses following activation of a long-loop pathway in the arcuate nucleus and ventrolateral periaqueductal gray (vlPAG). Additionally, EA at P 5-6 acupoints (overlying the median nerve) activates serotonin-containing neurons in the nucleus raphé pallidus (NRP), which, in turn, inhibit rVLM neurons. Although direct projections from the vlPAG to the rVLM exist, it is uncertain whether an indirect pathway through the NRP serves an important role in vlPAG-rVLM cardiovascular modulation. Therefore, the splanchnic nerve (SN) was stimulated to induce cardiovascular sympathoexcitatory reflexes, and EA was applied at P 5-6 acupoints in α-chloralose-anesthetized cats. A single-barreled recording electrode was inserted into the NRP or rVLM. Microinjection of DL-homocysteic acid (DLH) into the vlPAG increased the NRP neuronal response to SN stimulation (5 ± 1 to 12 ± 2 spikes/30 stim). Likewise, EA at P 5-6 for 30 min increased the NRP response to SN stimulation (3 ± 1 to 10 ± 2 spikes/30 stim), an effect that could be blocked by microinjection of kynurenic acid (KYN) into the caudal vlPAG. Furthermore, the reflex increase in blood pressure induced by application of bradykinin to the gallbladder and the rVLM cardiovascular presympathetic neuronal response to SN stimulation was inhibited by injection of DLH into the vlPAG, a response that was reversed by injection of KYN into the NRP. These results indicate that EA activates the vlPAG, which excites the NRP to, in turn, inhibit rVLM presympathetic neurons and reflex cardiovascular sympathoexcitatory responses.

Nucleus Raphe Pallidus in EA inhibition of rostral ventrolateral medulla and cardiovascular excitatory responses

EA at P5‐6 acupoints (median nerve) excites the arcuate nucleus and ventrolateral periaqueductal gray (vlPAG), and inhibits sympathetic cardiovascular rostral ventrolateral medulla (rVLM) neurons and associated reflex responses. The present study investigated the role of the NRP in EA inhibition of rVLM neurons and whether EA and activity from the vlPAG to the rVLM passes through the NRP. In [alpha]‐chloralose anesthetized cats the splanchnic nerve (SN) was stimulated to induce cardiovascular sympathoexcitatory reflexes and EA was applied at P 5‐6 acupoints. A recording electrode was inserted into the NRP or rVLM. Microinjection of dl‐homocysteic acid (DLH), but not normal saline (NS), into vlPAG increased the NRP neuronal response to SN stimulation (5±1 to 12±1 spikes/30 stim). EA at P 5‐6 for 30 min increased the NRP response to SN stimulation (5±1 to 14±3 spikes/30 stim). EA excitation of the NRP response was blocked by microinjection of kynurenic acid (KYN) into the caudal vlPAG. Furthermore, the reflex increase in BP induced by application of bradykinin on gallbladder and the rVLM neuronal response to SN stimulation was inhibited (~58%) by injection of DLH into the vlPAG, a response that could be blocked by injection of KYN into the NRP. Thus, EA activates the arcuate and vlPAG, which excites the NRP and, in turn, inhibits the rVLM activity and sympathoexcitatory cardiovascular reflex responses. (HL‐63313)

Glutamatergic transmission is altered in the caudal NTS of rats submitted to chronic intermittent hypoxia

We hypothesize that chronic intermittent hypoxia (CIH) alters the glutamatergic mechanisms in neurons of the caudal aspect of nucleus of tractus solitarius (cNTS) of rats. Using in situ working heart‐brainstem preparations of rats submitted to CIH (6% O2, for 40 s every 9 min, 8 h/day; n=9) or normoxia (control, n=7) for 10 days, basal and chemoreflex‐induced changes in phrenic and sympathetic nerve activities were evaluated after microinjections of kynurenic acid (KYN, 250 mM) in the cNTS. KYN in the cNTS produced large increases in phrenic burst frequency (0.3±0.1 vs 0.8±0.1 Hz) and duration (0.6±0.1 vs 0.9±0.1 s) in control rats, while in CIH rats the increase in the frequency was smaller (0.3±0.1 vs 0.6±0.1 Hz) and no changes were observed in the duration (0.6±0.1 vs 0.7±0.1 sec). Besides, the sympathoexcitatory response to chemoreflex activation (KCN, 0.05%) was reduced in control rats (90±11 vs 56±6 %), but not in CIH rats (112±9 vs 119±10 %) after KYN in the cNTS, whilst no changes were observed in the tachypneic response in both groups. Accordingly, the expression of NMDAR1 in the cNTS, examined by immunoblotting, was higher in CIH (n=3) than in control rats (n=3)(p=0.02). Altogether, the data suggest that glutamatergic neurotransmission in the cNTS is increased after CIH exposure, which may contribute to the respiratory and sympathetic dysfunctions observed in this experimental model. Supported by FAPESP and CNPq.

Glutamatergic Antagonism in the NTS Decreases Post-Inspiratory Drive and Changes Phrenic and Sympathetic Coupling During Chemoreflex Activation

For a better understanding of the processing at the nucleus tractus solitarius (NTS) level of the autonomic and respiratory responses to peripheral chemoreceptor activation, herein we evaluated the role of glutamatergic neurotransmission in the intermediate (iNTS) and caudal NTS (cNTS) on baseline respiratory parameters and on chemoreflex-evoked responses using the in situ working heart-brain stem preparation (WHBP). The activities of phrenic (PND), cervical vagus (cVNA), and thoracic sympathetic (tSNA) nerves were recorded before and after bilateral microinjections of kynurenic acid (Kyn, 5 nmol/20 nl) into iNTS, cNTS, or both simultaneously. In WHBP, baseline sympathetic discharge markedly correlated with phrenic bursts (inspiration). However, most of sympathoexcitation elicited by chemoreflex activation occurred during expiration. Kyn microinjected into iNTS or into cNTS decreased the postinspiratory component of cVNA and increased the duration and frequency of PND. Kyn into iNTS produced no changes in sympathoexcitatory and tachypneic responses to peripheral chemoreflex activation, whereas into cNTS, a reduction of the sympathoexcitation, but not of the tachypnea, was observed. The pattern of phrenic and sympathetic coupling during the chemoreflex activation was an inspiratory-related rather than an expiratory-related sympathoexcitation. Kyn simultaneously into iNTS and cNTS produced a greater decrease in postinspiratory component of cVNA and increase in frequency and duration of PND and abolished the respiratory and autonomic responses to chemoreflex activation. The data show that glutamatergic neurotransmission in the iNTS and cNTS plays a tonic role on the baseline respiratory rhythm, contributes to the postinspiratory activity, and is essential to expiratory-related sympathoexcitation observed during chemoreflex activation.

Microinjection of kynurenic acid induces airway smooth muscle relaxation in ferrets

Beside extensive study central mechanisms and neuronal networks that control airway constriction and relaxation remain unclear and poorly understood. In the present study realized in anesthetized, paralyzed and artificially ventilated ferrets we sought to determine whether nicotinic acetylcholine receptors (nAChRs) dominate respiratory drive under control conditions due to suppression of N‐methyl‐D‐aspartate (NMDA) receptors. We observed that blockade of cholinesterase activity by administration of eserine is followed by increased cholinergic outflow and airway smooth muscle tone (ASMT). Microinjection of kynurenic acid (KYNA) as NMDA receptor blockers, into the region of the ventrolateral medulla where airway related vagal preganglionic neurons (AVPNs) are located, significantly decreased the reflex changes in tracheal tone. KYNA, a product of tryptophan metabolism has neuroprotective and neuroinhibitory properties that have been attributed to its action as competitive antagonist at the glycine site on NMDA receptors. Furthermore, KYNA non‐competitively inhibits alpha7 nicotinic acetylcholine presynaptic receptors (nAChRs), inhibiting glutamate release, and regulates the expression of alpha4beta2 nAChR. In our physiological experiments performed under in vivo conditions we conclude that stimulation or inhibition of AVPNs by eserine or KYNA was manifested with increase or decrease, respectively, of pressure in tracheal segment (Ptseg), lung resistance (RL), or dynamic compliance (Cdyn). Our results indicate that nAChRs might be targets for KYNA and suggest a functionally significant cross reaction between the nicotinic cholinergic system and kynurenine pathway in AVPNs and respiratory drive.

Glutamatergic mechanisms on the interaction of sympathetic and respiratory responses to chemoreflex activation in the NTS

We evaluated the effect of the ionotropic glutamatergic receptor antagonism in different areas of the NTS on baseline respiratory parameters and on chemoreflex responses using an in situ working heart‐brainstem preparation, in which were recorded the activity of the phrenic (PNA) and thoracic sympathetic (tSNA) nerves. Bilateral microinjections of kynurenic acid (KYN 250 mM) were performed into intermediate (iNTS, 0.2‐0.3 mm rostral from calamus scriptorius, CS) and caudal NTS (cNTS, 0.1‐0.3 mm caudal from CS). KYN into cNTS evoked a significant increase in duration and frequency of phrenic burst discharge associated with a slight attenuation of the sympathoexcitation (ΔtSNA: 114±12 vs 67±17 %), but not the tachypnea, evoked by peripheral chemoreflex activation (KCN 0.05%, 50 μL). Besides, KYN microinjected simultaneously into iNTS and cNTS produced higher increases in frequency and duration of phrenic bursts with a significant attenuation of both sympathoexcitatory (ΔtSNA: 103±11 vs 23±5 %) and tachypneic (ΔPNA: 0.4±0.1 vs 0.02±0.06 Hz) responses to chemoreflex activation. The data show that L‐glutamate in iNTS and cNTS plays a tonic inhibitory role on the respiratory activity and the changes in the respiratory pattern due to the glutamate receptor antagonism in the NTS may affect the processing of the ventilatory and sympathetic responses to chemoreflex activation. Supported by FAPESP and CNPq.

Glutamatergic Inputs in the Hypothalamic Paraventricular Nucleus Maintain Sympathetic Vasomotor Tone in Hypertension

The paraventricular nucleus (PVN) of the hypothalamus is critical to the regulation of sympathetic output. The PVN hyperactivity is known to cause increased sympathetic nerve activity in spontaneously hypertensive rats (SHRs). The purpose of this study was to determine whether glutamatergic input to the PVN contributes to heightened sympathetic outflow in hypertension. Lumbar sympathetic nerve activity, mean arterial blood pressure, and heart rate were recorded from anesthetized SHRs and Wistar-Kyoto (WKY) rats. Bilateral microinjection of an N-methyl-D-aspartate receptor antagonist, 2-amino-5-phosphonopentanoic acid, or a non-N-methyl-D-aspartate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, into the PVN dose-dependently decreased lumbar sympathetic nerve activity, mean arterial blood pressure, and heart rate in SHRs but not in WKY rats. Bilateral microinjection of kynurenic acid into the PVN also significantly decreased lumbar sympathetic nerve activity, mean arterial blood pressure, and heart rate in SHRs but not in WKY rats. Furthermore, microinjection of gabazine, a specific GABA(A) receptor antagonist, into the PVN increased lumbar sympathetic nerve activity, mean arterial blood pressure, and heart rate in both SHRs and WKY rats. Notably, this response was significantly attenuated in SHRs compared with that in WKY rats. In addition, kynurenic acid abolished the sympathoexcitatory and pressor responses to microinjection of gabazine into the PVN in both SHRs and WKY rats. Thus, this study provides new functional evidence that resting sympathetic vasomotor tone is maintained by tonic glutamatergic input in the PVN in SHRs. Removal of GABAergic inhibition results in augmented glutamatergic input in the PVN, which probably constitutes an important source of excitatory drive to the brain stem vasomotor neurons in hypertension.

Chemoreflex sympathoexcitation was not altered by the antagonism of glutamate receptors in the commissural nucleus tractus solitarii in the working heart–brainstem preparation of rats

The changes in thoracic sympathetic nerve activity, heart rate and frequency of phrenic nerve discharge in response to chemoreflex activation before and after bilateral microinjections of glutamate receptor antagonists into the comissural nucleus tractus solitarii (cNTS) were evaluated in the working heart-brainstem preparation of rats. Microinjections of kynurenic acid (KYN, 250 mM), (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG, 100 mM), or KYN plus MCPG into the cNTS were performed in three different groups. These microinjections into the cNTS did not affect the increase in the thoracic sympathetic nerve activity elicited by chemoreflex activation (KYN, 54 +/- 3 versus 51 +/- 2%, n = 11; MCPG, 48 +/- 5 versus 54 +/- 5%, n = 7; and KYN plus MCPG, 57 +/- 6 versus 55 +/- 3%, n = 5). The increase in the frequency of the phrenic nerve discharge in response to chemoreflex activation was also not affected by KYN (0.28 +/- 0.02 versus 0.30 +/- 0.04 Hz), MCPG (0.27 +/- 0.03 versus 0.27 +/- 0.04 Hz), or KYN plus MCPG (0.30 +/- 0.04 versus 0.20 +/- 0.03 Hz). The bradycardic response to chemoreflex activation was significantly reduced after microinjection of KYN at 2 (-220 +/- 16 versus -50 +/- 6 beats min(-1)) and 10 min (-220 +/- 16 versus -65 +/- 9 beats min(-1)) and after microinjection of KYN plus MCPG into the NTS it was abolished at 2 (-192 +/- 14 versus -2 +/- 1 beats min(-1)) and 10 min (-192 +/- 14 versus -4 +/- 2 beats min(-1)). These data support the hypothesis that the neurotransmission of the sympathoexcitatory and respiratory components of the chemoreflex in the cNTS involves neurotransmitters other than L-glutamate and also the concept that the parasympathetic component of this reflex is mediated by L-glutamate.

Chemoreflex sympatho‐excitation in the working heart‐brainstem preparation (WHBP) of rat was not affected by the antagonism of glutamate receptors in the commissural nucleus tractus solitarius (NTS).

The changes in thoracic sympathetic nerve activity (tSNA), heart rate (HR) and frequency of phrenic nerve discharge (PND) in response to chemoreflex activation before and after bilateral microinjections of glutamate receptor antagonists into the NTS were evaluated in the WHBP. Microinjections of kynurenic acid (KYN, 250 mM), MCPG (100 mM) or KYN plus MCPG into the NTS were performed in 3 different groups. Microinjections of KYN (54±3 vs 51±2 %, n=11), MCPG (48±5 vs 54±5 %, n=7) or KYN plus MCPG (57±6 vs 55±3 %, n=5) into the NTS did not affect the increase in the tSNA elicited by chemoreflex activation. The increase in the frequency of the PND in response to chemoreflex activation was also not affected by microinjections of glutamate receptor antagonists: KYN (0.68±0.03 vs 0.76±0.05 Hz), MCPG (0.60±0.03 vs 0.61±0.03 Hz) and KYN plus MCPG (0.62±0.03 vs 0.62±0.02 Hz). The bradycardic response to chemoreflex activation was significantly reduced at 2 (−220±16 vs −50±6 bpm) and 10 min (−220±16 vs −65±9 bpm) after microinjection of KYN and was abolished at 2 (−192±14 vs −2±1 bpm) and 10 min (−192±14 vs −4±2 bpm) after microinjections of KYN plus MCPG into the NTS. These data support the hypothesis that the neurotransmission of the sympatho-excitatory and respiratory components of the chemoreflex in the NTS involves neurotransmitters other than L-glutamate and also the concept that the parasympathetic component of this reflex is mediated by L-glutamate. Supported by CNPq and FAPESP.

Central nervous mechanisms in the generation of the pattern of breathing.

The role of the Bötzinger complex (BötC) and the pre-Bötzinger complex (pre-BötC) in the genesis of the breathing pattern was investigated in anesthetized, vagotomized, paralysed and artificially ventilated rabbits making use of bilateral microinjections of kainic acid (KA) and excitatory amino acid (EAA) receptor antagonists. KA microinjections into either the BötC or the pre-BötC transiently eliminated respiratory rhythmicity in the presence of tonic phrenic activity (tonic apnea). Rhythmic activity resumed as low-amplitude, high-frequency irregular oscillations, superimposed on tonic inspiratory activity and displayed a progressive, although incomplete recovery. Microinjections of kynurenic acid (KYN) and D(-)-2-amino-5-phosphonopentanoic acid (D-AP5) into the BötC caused a pattern of breathing characterized by low-amplitude, high-frequency irregular oscillations and subsequently tonic apnea. Responses to KYN and D-AP5 in the pre-BötC were similar, although less pronounced than those elicited by these drugs in the BötC and never characterized by tonic apnea. Microinjections of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) into the BötC and the pre-BötC induced much less intense responses mainly consisting of increases in respiratory frequency. The results show that the investigated medullary regions play a prominent role in the genesis of the normal pattern of breathing through the endogenous activation of EAA receptors.

Glutamatergic receptors of the rostral ventrolateral medulla are involved in the ventilatory response to hypoxia

Rostral ventrolateral medulla (RVLM) is a region in the brainstem that is involved in the physiologic responses to hypoxia (i.e. hyperventilation and regulated hypothermia) and contains l-glutamate receptors. Therefore, we examined the effects of blocked of glutamatergic receptors in the RVLM on hypoxic hyperventilation and regulated hypothermia. Ventilation (V(E)) and body temperature (T(b)) were measured before and after bilaterally microinjection of kynurenic acid (KYN, 5 nmol/100 nl, an ionotropic glutamatergic receptors antagonist) and alpha-methyl-4-carboxyphenylglycine (MCPG, 10 nmol/100 nl, a metabotropic glutamatergic receptors antagonist) into the RVLM, followed by a 60-min period of hypoxia exposure. Control rats received microinjection of saline (vehicle). KYN or MCPG into the RVLM did not change V(E) and T(b) under normoxia, but reduced the hypoxic hyperventilation due to a lower tidal volume, although regulated hypothermia persisted. These data suggest that glutamatergic receptors in the RVLM are involved in the ventilatory response to hypoxia, exercising an excitatory modulation of the RVLM neurons, but play no role in hypoxia-induced hypothermia.

Antagonism of glutamate receptors in the intermediate and caudal NTS of awake rats produced no changes in the hypertensive response to chemoreflex activation

The role of excitatory amino acid (EAA) receptors in the neurotransmission of the sympathoexcitatory component of the chemoreflex (pressor response) in the intermediate and caudal aspects of the commissural nucleus tractus solitarii (NTS) of awake rats was evaluated. Microinjection of kynurenic acid, a non-selective antagonist of EAA receptors, into the intermediate and caudal commissural NTS produced a large increase in the baseline mean arterial pressure (MAP), which may reduce the magnitude of the pressor response to chemoreflex activation. To avoid this problem sodium nitroprusside (SNP) was infused (i.v.) after microinjections of kynurenic acid (2 nmol/50 nl) into the NTS, in order to normalize the MAP and then the chemoreflex was activated and the magnitude of the pressor response evaluated. Microinjection of kynurenic acid into the intermediate (bilaterally) and caudal (midline) commissural NTS ( n=6) produced a significant increase in baseline MAP (103±5 vs. 137±6 mm Hg) normalized by SNP infusion (107±4 mm Hg) and under this experimental condition the pressor response to chemoreflex activation was not statistically different in relation to the control (37±7 vs. 44±6 mm Hg). Bilateral microinjections of kynurenic acid into the caudal NTS ( n=8) also produced a significant increase in baseline MAP (109±4 vs. 145±6 mm Hg) normalized by SNP infusion (109±6 mm Hg). After normalization of MAP, the pressor response to chemoreflex activation at 3 (34±6 mm Hg) and 10 min (37±6 mm Hg) was also not different in relation to the control (46±5 mm Hg). These data indicate that the antagonism of EAA receptors simultaneously in the intermediate (bilateral) and caudal (midline) commissural NTS or only in the caudal commissural NTS (bilateral) of awake rats had no effect on the hypertensive response to chemoreflex activation. We suggest that neurotransmitter other than l-glutamate may take part in the neurotransmission of the sympathoexcitatory component of the chemoreflex at the NTS level.