Zhi-Ling Guo

Long-loop pathways in cardiovascular electroacupuncture responses

We have shown that electroacupuncture (EA) at P 5-6 (overlying median nerves) activates arcuate (ARC) neurons, which excite the ventrolateral periaqueductal gray (vlPAG) and inhibit cardiovascular sympathoexcitatory neurons in the rostral ventrolateral medulla (rVLM). To investigate whether the ARC inhibits rVLM activity directly or indirectly, we stimulated the splanchnic nerve to activate rVLM neurons. Micropipettes were inserted in the rVLM, vlPAG, and ARC for neural recording or injection. Microinjection of kainic acid (KA; 1 mM, 50 nl) in the ARC blocked EA inhibition of the splanchnic nerve stimulation-induced reflex increases in rVLM neuronal activity. Microinjection of d,l-homocysteic acid (4 nM, 50 nl) in the ARC, like EA, inhibited reflex increases in the rVLM neuronal discharge. The vlPAG neurons receive convergent input from the ARC, splanchnic nerve, P 5-6, and other acupoints. Microinjection of KA bilaterally into the rostral vlPAG partially reversed rVLM neuronal responses and cardiovascular inhibition during d,l-homocysteic acid stimulation of the ARC. On the other hand, injection of KA into the caudal vlPAG completely reversed these responses. We also observed that ARC neurons could be antidromically activated by stimulating the rVLM, and that ARC perikarya was labeled with retrograde tracer that had been microinjected into the rVLM. These neurons frequently contained beta-endorphin and c-Fos, activated by EA stimulation. Therefore, the vlPAG, particularly, the caudal vlPAG, is required for ARC inhibition of rVLM neuronal activation and subsequent EA-related cardiovascular activation. Direct projections from the ARC to the rVLM, which serve as an important source of beta-endorphin, appear also to exist.

Electroacupuncture induces c-Fos expression in the rostral ventrolateral medulla and periaqueductal gray in cats: relation to opioid containing neurons

Our previous studies have shown that electroacupuncture (EA) at the Neiguan-Jianshi (P5-P6) acupoints inhibits sympathetic outflow and attenuates excitatory visceral cardiovascular reflexes through enkephalin- or beta-endorphin-related opioid receptors in the rostral ventrolateral medulla (rVLM). It is not known whether EA at these acupoints activates neurons containing enkephalin or beta-endorphin in the rVLM as well as in the periaqueductal gray (PAG) that are involved in EA-mediated central neural regulation of sympathetic activity. The present study evaluated activated neurons in the rVLM and PAG by detecting c-Fos immunoreactivity, and identified the relationship between c-Fos nuclei and neuronal structures containing enkephalin or beta-endorphin in these regions. To enhance the detection of cell bodies containing enkephalin or beta-endorphin, colchicine (90-100 microg/kg) was injected into the subarachnoid space in anesthetized cats 28-30 h prior to EA or the sham-operated control for EA. Following bilateral barodenervation and cervical vagotomy, EA (1-4 mA, 2 Hz, 0.5 ms) was performed at the P5-P6 acupoints (overlying median nerve; n=7) for 30 min. Identical procedures, with the exception of electrical stimulation, were carried out in five control animals. EA decreased blood pressure (BP) in four of seven cats (5-15 mm Hg) while the sham procedure for EA produced no responses. Perikarya containing enkephalin were found in the rVLM and rarely in the PAG, while no cell bodies labeled with beta-endorphin were identified in either region. Compared to animals in the control group, more c-Fos immunoreactivity, located principally in close proximity to fibers containing enkephalin or beta-endorphin, was observed in the rVLM and ventrolateral PAG (vlPAG) in EA-treated cats. Moreover, neurons double-labeled with c-Fos and enkephalin in the rVLM were significantly increased in cats following EA stimulation (P<0.05). These data indicate that EA at the P5-P6 acupoints activates neurons in the rVLM and vlPAG. These activated neurons contain enkephalin in the rVLM, and most likely interact with nerve fibers containing enkephalin or beta-endorphin in both the rVLM and vlPAG. The results from this study provide the first anatomical evidence showing that EA at the P5-P6 acupoints has the potential to influence neuronal structures (perikarya, axons and/or dendrites) containing enkephalin or beta-endorphin in specific regions of the brain stem. These neurons likely form the substrate for EAs influence on sympathoexcitatory cardiovascular reflexes.

Serotonergic Projection from Nucleus Raphe Pallidus to Rostral Ventrolateral Medulla Modulates Cardiovascular Reflex Responses during Acupuncture

We have demonstrated that stimulation of somatic afferents during electroacupuncture (EA) inhibits sympathoexcitatory cardiovascular rostral ventrolateral medulla (rVLM) neurons and reflex responses. Furthermore, EA at P5-P6 acupoints over the median nerve on the forelimb activate serotonin (5-HT)-containing neurons in the nucleus raphe pallidus (NRP). The present study, therefore, examined the role of the NRP and its synaptic input to neurons in the rVLM during the modulatory influence of EA. Since serotonergic neurons in the NRP project to the rVLM, we hypothesized that the NRP facilitates EA inhibition of the cardiovascular sympathoexcitatory reflex response through activation of 5-HT1A receptors in the rVLM. Animals were anesthetized and ventilated, and heart rate and blood pressure were monitored. We then inserted microinjection and recording electrodes in the rVLM and NRP. Application of bradykinin (10 microg/ml) on the gallbladder every 10 min induced consistent excitatory cardiovascular reflex responses. Stimulation with EA at P5-P6 acupoints reduced the increase in blood pressure from 41+/-4 to 22+/-4 mmHg for more than 70 min. Inactivation of NRP with 50 nl of kainic acid (1 mM) reversed the EA-related inhibition of the cardiovascular reflex response. Similarly, blockade of 5-HT1A receptors with the antagonist WAY-100635 (1 mM, 75 nl) microinjected into the rVLM reversed the EA-evoked inhibition. In the absence of EA, NRP microinjection of dl-homocysteic acid (4 nM, 50 nl), to mimic EA, reduced the cardiovascular and rVLM neuronal excitatory reflex response during stimulation of the gallbladder and splanchnic nerve, respectively. Blockade of 5-HT1A receptors in the rVLM reversed the NRP dl-homocysteic acid inhibition of the cardiovascular and neuronal reflex responses. Thus activation of the NRP, through a mechanism involving serotonergic neurons and 5-HT1A receptors in the rVLM during somatic stimulation with EA, attenuates sympathoexcitatory cardiovascular reflexes.

Expression of c-Fos in Arcuate Nucleus Induced by Electroacupuncture: relations to neurons containing opioids and glutamate

Electroacupuncture (EA) at the Neiguan-Jianshi acupoints (P5-P6, overlying the median nerve) attenuates sympathoexcitatory reflexes probably through the opioid system. The arcuate nucleus (ARC) within hypothalamus is an important brain area that produces opioid peptides. Physiological studies have demonstrated that the predominant response to EA is excitation in the ARC and that excitatory projections from the ARC to the ventrolateral periaqueductal gray during EA at P5-P6 contribute to inhibition of sympathoexcitatory cardiovascular reflexes. These data imply that ARC neurons activated by EA also may contain excitatory neurotransmitters. Thus, the present study evaluated activation of the ARC induced by EA at P5-P6, in relation to the opioid system and glutamate, by detecting c-Fos, an immediate early gene, opioid peptides and vesicular glutamate transporter 3 (VGLUT3). To enhance detection of perikarya containing the opioid peptides, colchicine (90-100 microg/kg) was administered in cats 28-30 h before EA or the sham-operated control. EA was performed at P5-P6 for 30 min. Compared to controls (n=5), c-Fos-positive cells and neurons double-labeled with c-Fos and beta-endorphin, enkephalin or VGLUT3 in the ARC were significantly increased in EA-treated cats (n=6; all P<0.05). Moreover, neurons triple-labeled with c-Fos, beta-endorphin and VGLUT3 were noted in this region following EA stimulation, but not in controls. Thus, EA at P5-P6 activates neurons in the ARC, some of which contain opioids as well as glutamate or both. The results imply that EA at P5-P6 has the potential to influence ARC neurons containing multiple neuronal substances that subsequently modulate cardiovascular function.

Responses of opioid and serotonin containing medullary raphe neurons to electroacupuncture

The midline medulla oblongata, which includes the nucleus raphe obscurus, raphe magnus and raphe pallidus (NRP), is involved in regulation of cardiovascular responses. Opioids and serotonin (5-HT) are thought to function as important neurotransmitters in this region. We previously have demonstrated that electroacupuncture (EA) at the Neiguan-Jianshi acupoints (P5-P6, overlying the median nerves) attenuates sympathoexcitatory blood pressure reflexes through its influence on several brain regions. However, the role of these three raphe nuclei in the acupuncture responses is unknown. In baroreceptor denervated and vagotomized cats, the present study evaluated c-Fos activation in the raphe nuclei induced by EA and examined its relationship to enkephalin and 5-HT. To enhance detection of perikarya containing enkephalin, colchicine (90-100 microg/kg) was administered into the subarachnoid space in anesthetized cats 28-30 h before the placement of acupuncture needles at P5-P6 acupoints with or without electrical stimulation for 30 min. Perikarya containing the opioid and 5-HT were found in the raphe nuclei of all animals following application of colchicine. Compared to controls without electrical stimulation (n=5), c-Fos immunoreactivity and neurons double-labeled with c-Fos and either enkephalin or 5-HT were found more frequently in all three midline medullary nuclei, especially in NRP (n=6, all P<0.05) of EA-treated cats. Moreover, neurons triple-labeled with c-Fos, enkephalin and 5-HT were noted frequently in the NRP following EA stimulation. These results suggest that the medullary raphe nuclei, particularly the NRP, process somatic signals during EA and participate in EA-related modulation of cardiovascular function through an opioid or serotonergic mechanism.

Medullary pathways involved in cardiac sympathoexcitatory reflexes in the cat

Stimulation of cardiac sympathetic afferents evokes excitatory cardiovascular reflexes. However, the exact regions in the brain that integrate these reflexes have not been identified. Expression of c-Fos in the neurons provides a useful marker of the activated neurons. In the present study, we examined the response of c-Fos within the medulla of the cat to chemical stimulation of cardiac sympathetic afferents. After bilateral sinoaortic denervation and cervical vagotomy, we applied bradykinin (BK, 1-10 microg, n=7) six times to the anterior ventricular surface every 20 min. We observed consistent increases in blood pressure and heart rate while the vehicle for BK (0.9% saline, n=6) produced no responses. Ninety minutes after the end of the sixth treatment, transcardial perfusion was performed with 4% paraformaldehyde and the brainstem was harvested for immunohistochemical staining. Compared to the control animals, we noted Fos immunoreactive neurons in the nucleus of the solitary tract, lateral tegmental field, caudal and rostral ventrolateral medulla (VLM), and vestibular nucleus in the BK-treated cats (all P<0.05). Fos immunoreactivity was found in catecholaminergic neurons of the VLM. These findings indicate that the activated neurons in the medulla, especially in the VLM, are involved in integration of cardiac-cardiovascular sympathoexcitatory reflexes.

Nitric oxide in rostral ventrolateral medulla regulates cardiac-sympathetic reflexes: role of synthase isoforms

Our previous studies have shown that nitric oxide (NO) synthase (NOS)-containing neurons in the rostral ventrolateral medulla (rVLM) are activated during cardiac sympathoexcitatory reflexes (Refs. 12 and 13). However, the precise function of NO in the rVLM in regulation of these reflexes has not been defined. Three isoforms of NOS, including neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), are located in the rVLM. We explored the role of NO, derived from different NOS isoforms in the rVLM, in processing cardiac-sympathetic reflexes using whole animal reflex and electrophysiological approaches. We found that, in anesthetized cats, increased mean arterial blood pressure and renal sympathetic nerve activity elicited by epicardial application of bradykinin (BK; 1-10 microg/ml, 50 microl) were significantly attenuated following unilateral rVLM microinjection of the nonselective NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (50 nmol/50 nl), or a specific nNOS inhibitor, 7-nitroindazole (7-NI; 5-10 pmol/50 nl; both P < 0.05). In contrast, the responses of mean arterial blood pressure and renal sympathetic nerve activity to cardiac BK stimulation were unchanged by unilateral rVLM microinjection of N(omega)-nitro-D-arginine methyl ester (inactive isomer of N(omega)-nitro-L-arginine methyl ester, 50 nmol/50 nl), 3-6% methanol (7-NI vehicle), N(6)-(1-iminoethyl)-L-lysine (250 pmol/50 nl; iNOS inhibitor), or N(5)-(1-iminoethyl)-L-ornithine (250 nmol/50 nl; eNOS inhibitor). Furthermore, in separate cats, we noted that iontophoresis of 7-NI (0.1 mM) reduced the increased discharge of cardiovascular sympathoexcitatory rVLM neurons in response to cardiac stimulation with BK (P < 0.05). These neurons were characterized by their responses to inputs from baroreceptors, and their cardiac rhythmicity was determined through frequency and time domain analyses, correlating their discharge to arterial blood pressure and cardiac sympathetic efferent nerve activity. These data suggest that NO, specifically nNOS, mediates sympathetic cardiac-cardiovascular responses through its action in the rVLM.

Activation of nitric oxide-producing neurons in the brain stem during cardiac sympathoexcitatory reflexes in the cat.

Our previous studies have shown that selective inhibition of nitric oxide in the brain reduces pressor responses to activation of cardiac sympathetic afferents, thus suggesting that nitric oxide is involved in central regulation of cardiac-cardiovascular sympathoexcitatory reflexes. Central neural regions in which nitric oxide-producing neurons are activated during these reflexes have not been well characterized. In the present study, we located nitric oxide-producing neurons in the brain stem activated by the input from cardiac sympathetic afferents by detecting colocalization of c-Fos immunoreactivity with nitric oxide synthesizing neurons. Expression of c-Fos has been used as a marker of activated neurons. Nitric oxide-producing neurons were identified by histochemical labeling of nicotine adenine dinucleotide phosphate-diaphorase (NADPH-d). In anesthetized cats with bilateral barodenervation and cervical vagotomy, bradykinin (1-10 microg in 0.1 ml; n=6) was applied to the anterior surface of the left ventricle six times every 20 minutes. Repetitive application of bradykinin consistently increased blood pressure, while the vehicle for bradykinin (0.9% saline, n=5) produced no responses. A substantial fraction (6-27%) of NADPH-d positive neurons displayed Fos immunoreactivity in the nucleus of the solitary tract, caudal and rostral ventral lateral medulla, lateral tegmental field, locus coeruleus and parabrachial nucleus in the bradykinin-treated cats. However, either no or rare (1-4%) double-labeled cells were found in these regions in control animals. Thus, nitric oxide-producing neurons are activated in several regions in the brain stem during stimulation of cardiac sympathetic afferents by bradykinin. Our data suggest that nitric oxide functions as a neurotransmitter/modulator in these areas to regulate the cardiac sympathoexcitatory reflexes.

Central pathways in the pons and midbrain involved in cardiac sympathoexcitatory reflexes in cats.

Activation of cardiac sympathetic afferents elicits pain and excitatory cardiovascular reflexes including acute hypertension and tachyarrhythmias. Our previous studies have shown that specific regions in the medulla, such as the nucleus of solitary tract and ventrolateral medulla, are involved in central regulation of cardiac sympathoexcitatory reflexes. However, the contributions of supramedullary nuclei to these reflexes have not been characterized. In the present study, we located activated neurons in the pons and midbrain induced by inputs from cardiac sympathetic afferents by detecting their c-Fos immunoreactivity. In anesthetized cats with bilateral carotid denervation and cervical vagotomy, epicardial application of bradykinin (1-10 microg, in 0.1 ml; n=7) was performed on the anterior surface of the left ventricle six times, every 20 min. Repetitive application of bradykinin caused consistent excitatory cardiovascular reflexes characterized by increases in blood pressure and heart rate. No responses were evoked by the vehicle for bradykinin (0.9% saline, n=7). Compared to control cats, c-Fos immunoreactive cells were significantly increased (P<0.05) in the rostral pons, caudal and intermediate midbrain in the bradykinin-treated cats. The specific areas activated include the parabrachial nucleus, Kölliker-Fuse nucleus, locus coeruleus, dorsal nucleus of raphe, and dorsal, lateral and ventrolateral periaqueductal gray. From these results we suggest that cardiovascular-related regions in the pons and midbrain form part of a long loop in central integration of cardiac sympathoexcitatory reflexes.

Involvement of nuclei in the hypothalamus in cardiac sympathoexcitatory reflexes in cats.

The hypothalamus is considered to be an important area in the central regulation of cardiovascular function. However, its role in processing excitatory cardiovascular reflexes induced by stimulation of cardiac afferents has not been established. In the present study, using c-Fos immunoreactivity, we located neurons in the hypothalamus activated by inputs from cardiac sympathetic afferents. Following bilateral barodenervation and cervical vagotomy in anesthetized cats, bradykinin (BK, 1-10 microg, in 0.1 ml; n=7) was applied repetitively (6x, every 20 min) to the anterior epicardial surface of the left ventricle. This chemical stimulation caused consistent excitatory cardiovascular reflexes characterized by increases in blood pressure (BP) and heart rate (HR), while the vehicle for BK (0.9% saline, n=6) produced no such responses. Compared to control cats, c-Fos immunoreactive cells were significantly increased (P<0.05) in the arcuate nucleus (ARC), dorsal hypothalamic area (HDA), dorsomedial nucleus, paraventricular hypothalamic nucleus (PVN) and periventricular nucleus in the BK-treated animals. More neurons double-labeled with c-Fos and nitric oxide synthase (NOS) were observed in the PVN following epicardial application of BK (P<0.05). There was no significant increase in co-localization of these two labelings in the other nuclei. These results suggest that several nuclei in the hypothalamus respond to activation of cardiac sympathetic afferents, leading to sympathoexcitatory reflexes. Nitric oxide (NO) may function as a neurotransmitter or as a neuromodulator in the PVN during these cardiac-cardiovascular responses.