Microinjection Of N-methyl-D-aspartic Acid Research Articles

Dorsal raphe nucleus 5-Hydroxytryptamine 2A receptors are critical for the organisation of panic attack-like defensive behaviour and unconditioned fear-induced antinociception elicited by the chemical stimulation of superior colliculus neurons

Microinjections of N-methyl-d-aspartic acid (NMDA) in the midbrain tectum structures produce panic attack-like defensive behaviours, followed by an antinociceptive response. It has been suggested that fear-related defensive responses organised by brainstem neurons can be modulated by 5-hydroxytryptamine (5-HT). However, there is a shortage of studies showing the role of dorsal raphe nucleus (DRN) 5-HT2A receptors in the modulation of panic-like behaviour and fear-induced antinociception organised by the superior colliculus (SC). The purpose of this study was to investigate the participation of DRN 5-HT2A receptors in the modulation of panic attack-like behaviour and antinociception evoked by intra-SC injections of NMDA. In experiment I, the animals received microinjections of physiological saline or NMDA (6, 9 and 12 nmol) in the deep layers of the SC (dlSC). In experiment II, the most effective dose of NMDA (12 nmol) or vehicle was preceded by microinjections of vehicle or the 5-HT2A receptor selective antagonist R-96544 at different concentrations (0.5, 5 and 10 nM) in the DRN. Both proaversive and antinociceptive effects elicited by intra-dlSC injections of NMDA were attenuated by DRN pretreatment with R-96544. In addition, a morphological analysis showed that 5-HT2A receptors are present in GABAergic interneurons in the DRN. Taken together, these findings suggest that DRN 5-HT2A receptors are critical for the modulation of both panic attack-like defensive behaviour organised by SC neurons and unconditioned fear-induced antinociception. A possible interaction between serotonergic inputs, GABAergic interneurons and serotonergic outputs from the DRN was also considered.

Centrally mediated ejaculatory response via sympathetic outflow in rats: role of N-methyl-D-aspartic acid receptors in paraventricular nucleus.

Ejaculation is mediated by a spinal generator, which integrates inputs related to the sexual activity and coordinates sympathetic, parasympathetic, and motor outflow. Previous clinical studies indicate that primary premature ejaculation is related to the hyperactivity of the sympathetic nervous system. In this study, we explored the roles of N-methyl-D-aspartic acid (NMDA) receptors in paraventricular nucleus of the hypothalamus (PVN) on ejaculatory responses and its potential mechanism in the rats. We found that microinjection of 0.20nmol NMDA into the PVN reduced the latency of intromission and facilitated ejaculation during copulation. Moreover, delayed ejaculation and intromission were observed after the rats were microinjected with NMDA receptor antagonist D (-)-2-Amino-5-phosphonopentanoic acid (AP-5). However, we discovered that microinjection of NMDA into PVN significantly increased baseline lumbar splanchnic nerve activity (LSNA), and the NMDA dose was positively correlated with the increased LSNA (r=0.875, p=0.04). Meanwhile, the plasma norepinephrine level in rats injected with NMDA was much higher than that in rats injected with saline (1453.4±136.4pg/mL vs. 492.3±36.8pg/mL, p<0.01). Additionally, AP-5 reduced the baseline LSNA and abrogated the enhancing activity of NMDA in LSNA. Thus, we propose that NMDA receptors in PVN may facilitate ejaculation through enhancing the activity of sympathetic system.

GABA and glycine receptors in the nucleus ambiguus mediate tachycardia elicited by chemical stimulation of the hypothalamic arcuate nucleus.

We have previously reported that stimulation of the hypothalamic arcuate nucleus (ARCN) by microinjections of N-methyl-d-aspartic acid (NMDA) elicits tachycardia, which is partially mediated via inhibition of vagal inputs to the heart. The neuronal pools and neurotransmitters in them mediating tachycardia elicited from the ARCN have not been identified. We tested the hypothesis that the tachycardia elicited from the ARCN may be mediated by inhibitory neurotransmitters in the nucleus ambiguus (nAmb). Experiments were done in urethane-anesthetized, artificially ventilated, male Wistar rats. In separate groups of rats, unilateral and bilateral microinjections of muscimol (1 mM), gabazine (0.01 mM), and strychnine (0.5 mM) into the nAmb significantly attenuated tachycardia elicited by unilateral microinjections of NMDA (10 mM) into the ARCN. Histological examination of the brains showed that the microinjections sites were within the targeted nuclei. Retrograde anatomic tracing from the nAmb revealed direct bilateral projections from the ARCN and hypothalamic paraventricular nucleus to the nAmb. The results of the present study suggest that tachycardia elicited by stimulation of the ARCN by microinjections of NMDA is mediated via GABAA and glycine receptors located in the nAmb.

Cardiovascular responses to microinjections of endomorphin-2 into the nucleus of the solitary tract are attenuated in the spontaneously hypertensive rat

Stimulation of µ1-opioid receptors (M1ORs) in the medial nucleus solitarius (mNTS) by endomorphin-2 (EM2) elicits decreases in mean arterial pressure (MAP), heart rate (HR) and greater splanchnic nerve activity (GSNA) in Wistar rats. We tested the hypothesis that EM2-induced responses in the mNTS may be attenuated in the spontaneously hypertensive rat (SHR). Experiments were carried out in urethane-anesthetized, artificially ventilated, adult male SHR and Wistar-Kyoto rats (WKY). Alterations in responses to chemical stimulation of the hypothalamic arcuate nucleus (ARCN) after bilateral blockade of M1ORs in the mNTS were also studied. In SHR, microinjections of EM2 into the mNTS elicited smaller decreases in MAP, HR and GSNA compared to those elicited in WKY; smaller cardiovascular responses in SHR can be explained by lower expression of M1OR mRNA in the NTS of SHR compared to WKY. Decreases in MAP and GSNA and increases in HR were elicited by microinjections of N-methyl-d-aspartic acid (NMDA) into the ARCN of WKY. Bilateral blockade of M1ORs in the mNTS attenuated the decreases in MAP and GSNA and exaggerated the increases in HR elicited by the ARCN stimulation in WKY but not in SHR. Tonic inhibitory activity of neuropeptide Y/gamma-aminobutyric acid (NPY/GABA) neurons in the ARCN is attenuated in SHR; this observation may explain increases in MAP, GSNA and HR elicited by microinjections of NMDA into the ARCN of SHR. These results demonstrate that attenuation of EM2-induced responses in the mNTS of SHR may contribute to the excitatory responses elicited by ARCN stimulation in SHR.

Tonic γ-Aminobutyric Acid–ergic Activity in the Hypothalamic Arcuate Nucleus Is Attenuated in the Spontaneously Hypertensive Rat

We tested the hypothesis that tonic γ-aminobutyric acid–ergic activity in the hypothalamic arcuate nucleus (ARCN) modulates blood pressure control and attenuation of this inhibitory activity contributes to hypertension in the spontaneously hypertensive rats (SHR). Mean arterial pressure (MAP), heart rate (HR), and greater splanchnic nerve activity (GSNA) were recorded in urethane-anesthetized, artificially ventilated, adult male SHR and Wistar-Kyoto rats (WKY). Microinjections of gabazine into the ARCN elicited significantly smaller increases in MAP, HR, and GSNA in baroreceptor-intact SHR compared with baroreceptor-intact WKY. Attenuation of the responses to gabazine in SHR persisted, despite lowering of their baseline MAP to levels of WKY or barodenervation. Microinjections of N-methyl- d -aspartic acid (NMDA) into the ARCN elicited decreases in MAP and GSNA and increases in HR in baroreceptor-intact WKY. However, after microinjections of gabazine into the ARCN, microinjections of NMDA into the same nucleus elicited pressor responses in baroreceptor-intact WKY. In barodenervated WKY, increases in MAP and GSNA were elicited by ARCN stimulation by NMDA and the increases in HR were exaggerated. In baroreceptor-intact SHR, ARCN stimulation by NMDA elicited increases in MAP, GSNA, and HR which persisted, despite lowering of baseline MAP or barodenervation. Increases in MAP and GSNA elicited by ARCN stimulation by NMDA in barodenervated SHR were significantly greater than corresponding increases in barodenervated WKY. These results indicated that attenuated γ-aminobutyric acid–ergic activity in the ARCN and impaired baroreflex function may contribute to increases in blood pressure and sympathetic nerve activity after ARCN stimulation by NMDA and elevation of baseline blood pressure in SHR.

Estrogen attenuates antinociception produced by stimulation of Kölliker–Fuse nucleus in the rat

This is the first demonstration of sex-related differences in the alpha2-adrenoceptor-mediated antinociceptive effects produced by stimulation of an endogenous noradrenergic pathway. Electrical or chemical (substance P) stimulation of Kölliker-Fuse nucleus (KF, A7) is known to produce antinociception mediated by alpha2-adrenoceptors in the spinal cord. KF stimulation has also been shown to inhibit the responses of nociceptive neurons in the dorsal horn of the medulla and the spinal cord. We investigated whether KF stimulation produces sex-specific modulation of trigeminal nociception. The N-methyl-D-aspartic acid (NMDA)-induced nociceptive behavior was employed as an index of nociception. Microinjection of NMDA (2 nmol/10 microL) in the trigeminal region produced nociceptive scratching behavior that was confined to the orofacial region. Male and ovariectomized (OVX) Sprague-Dawley rats were implanted with a guide cannula dorsal to the KF nucleus and a PE-10 cannula in the trigeminal region dorsal to obex. Nociceptive testing was conducted after 5-7 days of recovery. A group of ovariectomized rats (OVX+E) was treated with estradiol benzoate 48 h prior to nociceptive testing. There were no significant differences in the number of NMDA-induced scratches or duration between the male, OVX and OVX+E groups. Microinjection of substance P (3.7 pmol/0.5 microL) in the KF significantly reduced the number of NMDA-induced scratches and their duration in male and OVX groups; these were restored to control levels by yohimbine (30 microg/15 microL), an alpha2-adrenoceptor antagonist. However, KF stimulation failed to inhibit the NMDA-induced scratching behavior in the OVX+E group. We conclude that stimulation of KF produces estrogen-dependent modulation of nociception.

Microinjection of glutamate into dorsal motor nucleus of the vagus excites gallbladder motility through NMDA receptor - nitric oxide - cGMP pathway.

We have reported that both glutamate and nitric oxide (NO) participated in the regulation of gallbladder motility in dorsal motor nucleus of the vagus (DMV). The aim of this study is to investigate the type of receptor in DMV that mediates the excitatory effect of glutamate on gallbladder motility and the correlation between the glutamate and NO. A frog bladder connected with a force transducer was inserted into the gallbladder to record the change of gallbladder pressure. Glutamate (65 mmol L(-1), 100 nL) microinjected into DMV significantly increased the strength of gallbladder phasic contraction. This effect was abolished by ketamine (180 mmol L(-1), 100 nL), the specific N-methyl-d-aspartic acid (NMDA) receptor antagonist, but was not influenced by 6-cyaon-7-nitroquinoxaline-2,3-(1H,4H)-dione (CNQX) (180 mmol L(-1), 100 nL), the non-NMDA ionotropic receptor antagonist. N(G)-nitro-l-arginine-emthyl (l-NAME) (1 mol L(-1), 100 nL), the nitric oxide synthase (NOS) inhibitor, reversed the excitatory effect of glutamate on gallbladder motility. Microinjection of sodium nitroprusside (SNP), the NO donor, into DMV enhanced the gallbladder motility, and this effect was not modulated by ketamine. Microinjection of NMDA (5 mmol L(-1), 100 nL) increased the strength of gallbladder phasic contraction, and this effect was attenuated by methylene blue (100 mmol L(-1), 100 nL), the soluble guanylate cyclase inhibitor. These results suggest that glutamate regulate the gallbladder motility through the NMDA receptor - NO - cGMP pathway in DMV.

Role of paraventricular nucleus in regulation of sympathetic nerve frequency components.

Autospectral and coherence analyses were used to determine the role of and interactions between paraventricular nucleus (PVN) nitric oxide, gamma-aminobutyric acid (GABA), and the N-methyl-D-aspartic acid (NMDA)-glutamate receptor in regulation of sympathetic nerve discharge (SND) frequency components in anesthetized rats. Four observations were made. First, PVN microinjection of bicuculline (BIC) (GABA(A) receptor antagonist), but not single PVN injections of NMDA (excitatory amino acid) or N(G)-monomethyl-L-arginine (L-NMMA; a nitric oxide synthase inhibitor), altered SND frequency components. Second, combined PVN microinjections of L-NMMA and NMDA changed the SND bursting pattern; however, the observed pattern change was different from that produced by PVN BIC and not observed after sinoaortic denervation. Third, PVN microinjection of kynurenic acid prevented and reversed BIC-induced changes in the SND bursting pattern. Finally, vascular resistance (renal and splenic) was significantly increased after PVN BIC microinjection despite the lack of change in the level of renal and splenic SND. These data demonstrate that the PVN contains the neural substrate for altering SND frequency components and suggest complex interactions between specific PVN neurotransmitters and between PVN neurotransmitters and the arterial baroreceptor reflex in SND regulation.

Activation of different vestibular subnuclei evokes differential respiratory and pressor responses in the rat.

Activation of the vestibular system can either increase or decrease ventilation. The objectives of the present study were to clarify whether these different responses are the result of activating different vestibular subnuclei, by addressing three questions. Do neurones within the medial, lateral and spinal vestibular nuclei (VN(M), VN(L) and VN(S), respectively) function differently in respiratory modulation? Is the ventral medullary nucleus gigantocellularis (NGC) required to fully express the VN-mediated respiratory responses? Is glutamate, by acting on N-methyl-D-aspartic acid (NMDA) receptors in the vestibular subnuclei, capable of modulating respiration? In anaesthetized, tracheotomized and spontaneously breathing rats, electrical stimuli (< 10 s) applied in the VN(L) and VN(S) significantly elevated ventilation by 35 % and 30 % (P < 0.05), respectively. However, VN(M) stimulation produced statistically significant (P < 0.05) changes that differed depending upon the stimulation site: either ventilatory inhibition (by 40 % in 57 % of the trials) or excitation (by 55 % in 43 % of trials), and which were often accompanied by a pressor response. These electrical-stimulation-evoked cardiorespiratory responses were almost eliminated following microinjection of ibotenic acid into the stimulation sites (P < 0.05) or bilaterally into the NGC (P < 0.05). As compared to vehicle, microinjection of NMDA into the unilateral VN(M), VN(L) and VN(S) significantly increased ventilation to 74 %, 58 % and 60 % (P < 0.05), respectively, with no effect on arterial blood pressure. These data suggest that neurones within the vestibular subnuclei play different roles in cardiorespiratory modulation, and that the integrity of the NGC is essential for the full expression of these VN-mediated responses. The evoked respiratory excitatory responses are probably mediated by glutamate acting on NMDA receptors, whereas the neurotransmitters involved in VN(M)-mediated respiratory inhibition and hypertension remain unknown.

NMDA-mediated increase in renal sympathetic nerve discharge within the PVN: role of nitric oxide.

The paraventricular nucleus (PVN) of the hypothalamus is an important site of integration in the central nervous system for sympathetic outflow. Both glutamate and nitric oxide (NO) play an important role in the regulation of sympathetic nerve activity. The purpose of the present study was to examine the interaction of NO and glutamate within the PVN in the regulation of renal sympathetic nerve activity in rats. Renal sympathetic nerve discharge (RSND), arterial blood pressure (BP), and heart rate (HR) were measured in response to administration of N-methyl-D-aspartic acid (NMDA) and N(G)-monomethyl-L-arginine (L-NMMA) into the PVN. We found that microinjection of NMDA (25, 50, and 100 pmol) into the PVN increased RSND, BP, and HR in a dose-dependent manner, reaching 53 +/- 9%, 19 +/- 3 mmHg, and 32 +/- 12 beats/min, respectively, at the highest dose. These responses were significantly enhanced by prior microinjection of L-NMMA. On the other hand, inhibition of NO within the PVN by microinjection of L-NMMA also induced increases in RSND, BP, and HR in a dose-dependent manner, reaching 48 +/- 6.5%, 11 +/- 4 mmHg, and 55 +/- 16 beats/min, respectively, at the highest dose. This sympathoexcitatory response was eliminated by prior microinjection of DL-2-amino-5-phosphonovaleric acid, an antagonist of the NMDA receptor. Furthermore, with the use of the push-pull technique, perfusion of glutamate (0.5 micromol) or NMDA (0.1 nmol) into the PVN induced an increase in NO release. In conclusion, our data indicate that NMDA receptors within the PVN mediate an excitatory effect on renal sympathetic nerve activity, arterial BP, and HR. NO in the PVN, which is released by activation of the NMDA receptor, also inhibits NMDA-mediated increases in sympathetic nerve activity. This negative feedback of NO on the glutamate system within the PVN may play an important role in maintaining the overall balance and tone of sympathetic outflow in normal and pathophysiological conditions known to have increased sympathetic tone.

Effects of microinjection of [formula omitted] acid into the nucleus tractus solitarii on cerebral blood flow in anesthetized rats

N- Methyl- d-aspartic acid (NMDA) (10 pmol in 100 nl of 0.9% sodium chloride solution) was microinjected into the nucleus tractus solitarii (NTS) of urethane-anesthetized, paralyzed and artificially ventilated rats, and cerebral blood flow (CBF) was determined using a combination of labeled microspheres. Moderate hypertension within the upper limit of cerebral autoregulation was induced by blood transfusion in order to measure CBF at normotension. Arterial blood pressure (ABP) was decreased by unilateral microinjection into the NTS in these rats but remained within normotensive range. The CBF in the cerebral cortex ipsilateral to the stimulated NTS significantly ( P < 0.01) decreased from 38 ± 4 (mean ± S.E.M) to 27 ± 4 ml · min −1 · (100 g) −1( n = 9). The cerebrovascular resistance (CVR) in the cerebral cortex ipsilateral to the stimulated NTS significantly ( P < 0.01) increased from 2.6 ± 0.3 to 4.1 ± 0.7 mmHg per [ml · min −1 · (100 g) −1]. Blockade of NMDA receptors in the NTS with d,l-2-amino-5-phosphonovalerate (AP5, 500 pmol) abolished the CBF decrease and CVR increase responses elicited by microinjection of NMDA into the NTS ( n = 9). Blockade of non-NMDA receptors in the NTS with 6,7-dinitro-quinoxaline-2,3-dione (DNQX, 100 pmol) had little effect on the CBF decrease and CVR increase responses elicited by microinjection of NMDA into the NTS ( n = 10). Microinjection of the vehicle solution into the NTS had no effects on cerebral circulation ( n = 7). Cerebral autoregulation was well maintained at moderate hypertension induced by blood transfusion and at normotension returned from moderate hypertension following controlled hemorrhage ( n = 8). These results suggest that the NMDA receptors in the NTS may be involved in the control of cerebral circulation.

Identification of cardiovascular neurons in the rostral ventromedial medulla in anesthetized rats.

Recent studies have identified a region in the rostral ventromedial medulla (RVMM) of rats that appears to be involved in cardiovascular function. Since these studies used either microinjection of lidocaine or electrical stimulation, the exact contribution of intrinsic neurons as opposed to fibers of passage could not be determined. The present study was performed to map the location of neurons in RVMM from which changes in mean arterial pressure could be elicited by the microinjection of the excitatory amino acid analogue N-methyl-D-aspartic acid (NMDA) (20 ng/50 nl), which selectively activates cell bodies in barbiturate-anesthetized rats. Microinjection of NMDA into RVMM most often (53%) elicited pressor responses (31 +/- 7 mm Hg). On the basis of these responses, RVMM was determined to encompass a large portion of the nucleus gigantocellularis 0.5-1.5 mm lateral to the midline, 0.5-3.5 mm above the ventral surface, and extending from the rostral to the caudal pole of the facial nucleus. Depressor responses (-21 +/- 3 mm Hg) were found at all levels of RVMM but were most concentrated and of the largest magnitude in the rostral and caudal poles of RVMM. Microinjection of the inhibitory neurotransmitter glycine (500 mM) was used to determine whether neurons in RVMM were contributing to the maintenance of arterial pressure. Microinjection of glycine decreased arterial pressure (-15 +/- 2 mm Hg) throughout most of RVMM. Unexpectedly, increases in mean arterial pressure (24 +/- 3 mm Hg) were elicited by microinjection of glycine into the same region in RVMM in which NMDA most frequently elicited pressor responses.(ABSTRACT TRUNCATED AT 250 WORDS)