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 EA's influence on sympathoexcitatory cardiovascular reflexes.     

Medullary and spinal cord projections from cardiovascular responsive sites in the rostral ventromedial medulla

The rostral ventromedial medulla (RVMM) is a sympathoexcitatory area. However, little is known about its efferent projections. In this study, biotinylated dextran amine (BDA) or Phaseolus vulgaris leucoagglutinin (PHA-L) were used to investigate the medullary and spinal cord projections from pressor sites in RVMM. Initially, RVMM was systematically explored in urethane-anesthetized rats using microinjection of L-glutamate for sites that elicited increases in arterial pressure. A pressor area was identified that included the rostral magnocellular reticular and rostral lateral paragigantocellular reticular nuclei. In the second series of experiments, BDA or PHA-L was iontophoretically injected into RVMM pressor sites. Anterograde labeling was observed throughout the brainstem and spinal cord, bilaterally, but with an ipsilateral predominance. Dense labeling was observed within the nucleus of the solitary tract (NTS); the greatest density of labeling was observed in the caudal dorsolateral, medial, and ventrolateral subnuclei. Additionally, light to moderately dense labeling was found within the nucleus substantia gelatinosus and commissural nucleus. In the nucleus ambiguus/ventrolateral medullary (Amb/VLM) region, the density of labeling was greatest in caudal regions. Within Amb, most of the labeling was localized to its external formation. Anterograde labeling was also found throughout the spinal cord. In the thoracolumbar segments, dense axonal labeling was observed within the dorsolateral funiculus. These labeled axons innervated the intermediolateral nucleus and the central autonomic area. Taken together, these data suggest that RVMM neurons elicit increases in sympathetic activity by likely providing a direct excitatory input to spinal sympathetic preganglionic neurons, and by a direct inhibitory input to medullary cardioinhibitory and depressor areas.     

Prefrontal,accumbal [shell] and ventral striatal mechanisms in jaw movements and non-cyclase-coupled dopamine D1-like receptors

The effect on jaw movements of intracerebral injections of the dopamine D1-like receptor agents SK&F 83959 (3-methyl-6-chloro-7,8-dihydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), SK&F 38393 ([R]-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) and SCH 23390 ([R]-3-methyl-7-chloro-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) and of injections of the dopamine D2-like receptor agonist quinpirole into the ventrolateral striatum, accumbens shell or prefrontal cortex were studied. SK&F 38393 and SK&F 83959 injected into the ventrolateral striatum synergised with i.v. quinpirole; in the shell of accumbens, SK&F 38393 evidenced weaker synergism with quinpirole, while SK&F 83959 did not synergise with it; neither agent synergised with quinpirole in the prefrontal cortex. Co-injection of SCH 23390 or SK&F 83959 into the prefrontal cortex antagonised jaw movements induced by injection of SK&F 83959 into the ventrolateral striatum in combination with i.v. quinpirole. Injection of SK&F 83959 + quinpirole into the ventrolateral striatum, but not into the accumbens shell, resulted in synergism. These findings indicate a primary, but not exclusive, role for ventral striatal, non-cyclase-coupled dopamine D1-like receptors in the induction of jaw movements. These processes appear to require tonic activity of prefrontal cyclase-linked dopamine D1A [and/or D1B] receptors.     

Motor cortical control of cardiovascular bulbar neurones projecting to spinal autonomic areas

There is evidence that the motor cortex is involved in cardiovascular adjustments associated with somatic motor activity, as it has functional connections with the ventrolateral medulla, a brainstem region critically involved in the control of blood pressure and the regulation of plasma catecholamine levels. The ventrolateral medulla sends projections to the spinal intermediolateral nucleus, where preganglionic neurones controlling heart and blood vessels (T2 segment) and adrenal medulla (T8 segment) are found. The aim of the present study was to determine whether electrical stimulation of the rat motor cortex induces cardiovascular responses and Fos expression in ventrolateral medulla neurones projecting to the T2 and T8 segments. After a set of experiments designed to record cardiovascular parameters (blood pressure and plasma catecholamine levels), injections of retrograde tracer (Fluorogold) were performed in the intermediolateral nucleus of two groups of rats, at the T2 or at the T8 segmental levels. Five days later, the motor cortex was stimulated in order to induce Fos expression in the ventrolateral medulla. Stimulation of the motor cortex induced: (1). hypotension and a significant decrease in plasma noradrenaline levels, and (2). a significant increase in the number of the double-labelled neurones in the rostral ventrolateral medulla projecting to T2. These data demonstrate that cardiovascular adjustments, preparatory to, or concomitant with, motor activity may be initiated in the motor cortex and transmitted to cardiac and vasomotor spinal preganglionic neurones, via the ventrolateral medulla.     

Differential regulation of brown adipose and splanchnic sympathetic outflows in rat: roles of raphe and rostral ventrolateral medulla neurons

1. The medullary premotor neurons determining the sympathetic outflow regulating cardiac function and vasoconstriction are located in the rostral ventrolateral medulla (RVLM). The present study sought evidence for an alternative location for the sympathetic premotor neurons determining the sympathetic nerve activity (SNA) controlling brown adipose tissue (BAT) metabolism and thermogenesis. 2. The tonic discharge on sympathetic nerves is determined by the inputs to functionally specific sympathetic preganglionic neurons from supraspinal populations of premotor neurons. Under normothermic conditions, BAT SNA was nearly silent, while splanchnic (SPL) SNA, controlling mesenteric vasoconstriction, exhibited sustained large-amplitude bursts. 3. The rostral raphe pallidus (RPa) contains potential sympathetic premotor neurons that project to the region of sympathetic preganglionic neurons in the thoracic spinal cord. Disinhibition of neurons in RPa elicited a dramatic increase in BAT SNA, with only a small rise in SPL SNA. 4. Splanchnic SNA was strongly influenced by the baroreceptor reflex, as indicated by a high coherence with the arterial pressure wave, a significant amplitude modulation over the time-course of the cardiac cycle and a marked inhibition of SPL SNA during a sustained increase in arterial pressure. When activated, the bursts in BAT SNA exhibited no correlation with arterial pressure and were not affected by increases in arterial pressure. 5. Because these characteristics and reflex responses in sympathetic outflow have been shown to arise from the on-going or altered discharge of sympathetic premotor neurons, the marked differences between SPL and BAT SNA provide strong evidence supporting the hypothesis that vasoconstriction and thermogenesis (metabolism) are controlled by distinct populations of sympathetic premotor neurons, the former in the RVLM and the latter, potentially, in the RPa.     

家兔大鼠延髓呼吸中枢压力感受区的细胞构筑

24只家兔和10只大鼠的延髓呼吸中枢压力感受区(BSA),经HE、Cajal等染色及免疫组化技术作细胞构筑学研究。BSA在软脑膜2～82μm之下,范围为390μm×490μm×1000μm,由中、小型多极和梭形神经元构成,属B1/35-羟色胺能神经元。其表层的胶质纤维及神经纤维与脑表面平行呈板层状排列。BSA富于毛细血管。     

Analysis of the receptor involved in the central hypotensive effect of rilmenidine and moxonidine

The aim of this study was to determine whether α₂-adrenoceptors or imidazoline I₁-receptors are responsible for the central sympathoinhibition produced by rilmenidine and moxonidine, two clonidine-like antihypertensive drugs. Rilmenidine and moxonidine were compared with the indirectly acting α₂-adrenoceptor agonist α-methyldopa. Three antagonists were used. Yohimbine and SK & F86466 were used as selective α₂-adrenoceptor antagonists. They were compared with efaroxan which is also an α₂-adrenoceptor antagonist, but, in addition, possesses affinity for imidazoline I₁-receptors. According to some but not all studies, the affinity of efaroxan for I₁-receptors is much higher than its affinity for α₂-adrenoceptors. Drugs were administered into the cisterna cerebellomedullaris of conscious rabbits by a catheter implanted previously under halothane anaesthesia. Rilmenidine (10 μg kg^–1), moxonidine (0.3 μg kg^–1) and α-methyldopa (0.4 mg kg^–1) lowered blood pressure and the plasma noradrenaline concentration; the degree of sympathoinhibition produced by the three agonists was very similar. When injected after the agonists, efaroxan (0.1–14 μg kg^–1; cumulative doses), yohimbine (0.4–14 μg kg^–1) and SK & F86466 (0.4–44 μg kg^–1) counteracted the effects of the agonists on blood pressure and the plasma noradrenaline concentration. Efaroxan was about tenfold more potent than yohimbine and SK & F86466 at antagonizing the hypotensive effects of α-methyldopa. Similarly, efaroxan was two- to tenfold more potent than yohimbine and SK & F86466 against rilmenidine and moxonidine. Finally, efaroxan was about as potent against α-methyldopa as against rilmenidine and moxonidine. The results confirm previous observations that selective α₂-adrenoceptor antagonists are capable of completely antagonizing effects of rilmenidine and moxonidine. The effects of the α₂-adrenoceptor antagonist with an additional high affinity for imidazoline I₁-receptors, efaroxan, can also be explained by blockade of α₂-adrenoceptors. Efaroxan was more potent against rilmenidine and moxonidine than the selective α₂-adrenoceptor antagonists. This was probably due to the fact that the affinity of efaroxan for α₂-adrenoceptors is higher than the affinity of yohimbine and SK & F86466, since efaroxan was also the most potent of the three antagonists against the indirectly acting α₂-adrenoceptor agonist α-methyldopa. The observation that efaroxan was equally potent against rilmenidine and moxonidine and against α-methyldopa suggests that the same receptors were involved in the effects of the three agonists, α₂-adrenoceptors; this observation is not compatible with the high I₁/α₂ selectivity of efaroxan and the hypothesis that rilmenidine and moxonidine activate I₁-receptors, whereas α-methyldopa activates α₂-adrenoceptors. Thus, the data do not indicate involvement of I₁ imidazoline receptors in the central sympathoinhibition elicited by ril-menidine and moxonidine in rabbits. It is likely that ril-menidine and moxonidine produce sympathoinhibition by activating the same receptors which are activated by the indirectly acting catecholamine α-methyldopa, namely α₂-adrenoceptors. Received: 7 December 1998 / Accepted: 2 February 1999     

Rapid effects of corticosterone on cardiovascular neurons in the rostral ventrolateral medulla of rats

The present study has explored possible fast actions of corticosteroid hormones on activity of cardiovascular neurons of the rostral ventrolateral medulla. Experiments were conducted in 60 urethane-anesthetized, artificially ventilated adult rats. Extracellular recordings of unitary firings were made from the RVLM with multi- or single-barreled microelectrodes. Barosensitive cardiovascular neurons were identified through activation of the baroreceptor reflex by electrical stimulation of the aortic nerve and by intravenous injection of phenylephrine. In 52 barosensitive cardiovascular neurons, iontophoretically applied corticosterone sulfate increased the ongoing activity of 30 (57.7%) neurons, the other 22 (42.3%) neurons being unaffected. In 16 bulbospinal pre-sympathetic neurons, iontophorized corticosterone increased the firing rate of 12 neurons. Intravenously applied corticosterone (0.2 mg) increased the firing rates of 5 out of 12 bulbospinal pre-sympathetic neurons. The average baseline activity of cardiovascular neurons sensitive to corticosterone was found to be significantly less than that of the cardiovascular neurons insensitive to corticosterone. In 64 non-cardiovascular neurons, the firing rate of 13 (20.3%) neurons increased, 23 (36.0%) decreased and 28 (43.7%) remained unchanged following local application of corticosterone. The changes in firing rates of RVLM neurons following application of corticosterone occurred rapidly and were dependent on the doses of the agent. RU-38486 was able to reduce or block the rapid effects of corticosterone on cardiovascular and non-cardiovascular neurons. The results demonstrated that corticosterone may fast, non-genomically, modulate the activity of central regulators of the cardiovascular system and suggested that fast non-genomic actions of corticosteroid hormones may be an important mechanism in the integration of the autonomic nervous and the cardiovascular systems during some conditions such as stress.     

µ1‐Opioid receptors in the dorsomedial and ventrolateral columns of the periaqueductal grey matter are critical for the enhancement of post‐ictal antinociception

Generalised tonic and tonic–clonic seizures are followed by significant increase in nociceptive thresholds in both laboratory animals and humans. The endogenous opioid peptides play a role in antinociceptive signalling, and the periaqueductal grey matter (PAG) is recruited to induce analgesia. Thus, the aim of this investigation was to evaluate the role of µ₁‐opioid receptors in the dorsomedial (dm) and ventrolateral (vl) columns of PAG in post‐ictal antinociception. Pentylenetetrazole (PTZ; 64 mg/kg), which is an ionotropic GABA‐mediated Cl^? influx antagonist, was intraperitoneally (IP) administered to induce tonic–clonic seizures in Wistar rats. The tail‐flick test was used to measure the nociceptive threshold. Microinjections of naltrexone (5.0 µg/0.2 µL), which is a non‐selective opioid receptor antagonist, in both dmPAG and vlPAG decreased the tonic–clonic seizure‐induced antinociception in seizing animals from 10 to 120 min after seizures. Furthermore, microinjections of the µ₁‐opioid receptor‐selective antagonist naloxonazine (5.0 µg/0.2 µL) into the dmPAG decreased post‐ictal antinociception immediately after convulsive reactions and from 10 to 90 min after seizures. However, vlPAG‐pretreatment with naloxonazine at the same concentration decreased the post‐ictal antinociception 30 min after the onset of tonic–clonic seizures and the nociceptive threshold returned to basal values 120 min after seizures. These findings indicate that µ₁‐opioid receptor‐signalling mechanisms in both dmPAG and vlPAG play a relevant role in the organisation of post‐ictal antinociception. In addition, µ₁‐opioid receptors in the dmPAG rather than in vlPAG seem to be more critically recruited during the antinociception induced by generalised tonic–clonic seizures.     

Participation of the Prolactin-Releasing Peptide-Containing Neurones in Caudal Medulla in Conveying Haemorrhagic Stress-Induced Signals to the Paraventricular Nucleus of the Hypothalamus

The prolactin-releasing peptide (PrRP) has been proposed to be a co-transmitter or modulator of noradrenaline (NA) because it colocalises with NA in the A1 (in the ventrolateral reticular formation) and A2 (in the nucleus of the solitary tract; NTS) cell groups in the caudal medulla. The baroreceptor signals, originating from the great vessels, are transmitted primarily to the NTS, and then part of the signals is conveyed to the hypothalamic neuroendocrine neurones via the ascending NA neurones. The hypotensive haemorrhagic paradigm was employed to examine whether the PrRP-containing neurones in the caudal medulla participate in conveying signals to the hypothalamic neuroendocrine neurones. Among the caudal medullary A1 or A2 neurones, the majority of the PrRP-immunoreactive (-ir) neurones became c-Fos-ir at 2 h after hypotensive haemorrhage. Hypothalamic corticotrophin-releasing hormone-ir neurones and vasopressin-ir neurones became c-Fos positive in parallel with the activation of medullary PrRP-ir neurones. After delivery of retrograde tracer fluorogold (FG) to the paraventricular nucleus of the hypothalamus (PVN), part of the PrRP/FG double-labelled neurones in the A1 and A2 became c-Fos-ir after haemorrhage, demonstrating that PrRP-ir neurones participate in conveying the haemorrhagic stress-induced signals from the medulla to the PVN. PrRP and/or NA were microinjected directly to the PVN of conscious rats, and they presented a synergistic action on arginine vasopressin release, whereas an additive action was observed for adrenocorticotrophin release. These results suggest that the PrRP-containing NA neurones in the caudal medulla may relay the haemorrhagic stress-induced medullary inputs to the hypothalamic neuroendocrine neurones.