Sympathoinhibition from ventrolateral periaqueductal gray mediated by the caudal midline medulla

Activation of neurons in the ventrolateral region of the periaqueductal gray (vlPAG) can elicit a decrease in renal sympathetic nerve activity and blood pressure. The present study investigated whether the vlPAG-evoked sympathoinhibitory response depends on neurons in the caudal midline medulla (CMM). In pentobarbital-anesthetized rats, activation of neurons in the vlPAG evoked a decrease in renal sympathetic nerve activity to 29.4 +/- 4.8% below baseline levels and arterial blood pressure fell 8.9 +/- 1.6 mmHg (n = 20). Microinjection of the GABA agonist muscimol into sympathoinhibitory regions of the CMM significantly attenuated the vlPAG-evoked sympathoinhibition to 17.9 +/- 4.1% below baseline and the depressor response to 4.3 +/- 1.2 mmHg. At 65% (13/20) of the sites examined, the vlPAG-evoked sympathoinhibition was responsive to CMM muscimol microinjection and attenuated from 34.2% to 11.5%, with the depressor response reduced from 14.8 to 3 mmHg. Microinjection of muscimol at the remaining 35% of the CMM sympathoinhibitory sites was ineffective on the vlPAG-evoked sympathoinhibition and depressor response. These data indicate that sympathoinhibitory and hypotensive responses elicited by activation of neurons in the vlPAG can be mediated by neurons in the sympathoinhibitory region of the CMM. The finding that the vlPAG-evoked response is not affected by muscimol at all CMM sympathoinhibitory sites also suggests that sympathoinhibitory sites in the CMM are not homogeneous and can mediate functionally different responses.     

Neuronal organization of the periaqueductal gray matter in the cat midbrain

It is shown by the use of Golgi's method in Antonova's modification that the neuronal structure of the periaqueductal gray matter (PGM) in the frontal plane is characterized by the presence of small and medium-sized cells of "reticular type," which can be subdivided into three types: fusiform, triangular, and multipolar. On the basis of the visual distribution of these types of neurons and also of statistical analysis of 800 identified neurons, two regions can be distinguished: medial, directly surrounding the aqueduct of Sylvius, containing small neurons, among which the fusiform kind predominate significantly (P<0.001), and a lateral region with larger neurons, with significantly (P<0.001) more triangular cells. Neurons in the medial region show a characteristic and strong (P<0.001) tendency for their dendrites to be oriented toward the lumen of the aqueduct, and through them the physiologically active substances of the CSF may influence the functional activity of neurons of PGM.Central Research Institute of Reflex Therapy, Moscow City Council Main Health Board, Moscow. Translated from Neirofiziologiya, Vol. 16, No. 6, pp. 773–777, November–December, 1984.     

Evidence that hemorrhagic hypotension is mediated by the ventrolateral periaqueductal gray region

Severe hemorrhage lowers arterial pressure by suppressing sympathetic activity. This study tested the hypothesis that the decompensatory phase of hemorrhage is mediated by the ventrolateral periaqueductal gray (vlPAG), a region importantly involved in the autonomic and behavioral responses to stress and trauma. Neuronal activity in the vlPAG was inhibited with either lidocaine or cobalt chloride 5 min before hemorrhage (2.5 ml/100 g body wt) was initiated in conscious, unrestrained rats. Bilateral injection of lidocaine (0.5 microl of a 2% or 1 microl of a 5% solution) into the caudal vlPAG delayed the onset and reduced the magnitude of the hypotension produced by hemorrhage significantly. In contrast, inactivation of the dorsolateral PAG with lidocaine was ineffective. Cobalt chloride (5 mM; 0.5 microl), which inhibits synaptic transmission but not axonal conductance, also attenuated hemorrhagic hypotension significantly. Microinjection of lidocaine or cobalt chloride into the vlPAG of normotensive, nonhemorrhaged rats did not influence cardiovascular function. These data indicate that the vlPAG plays an important role in the response to hemorrhage.     

Excitatory projections from arcuate nucleus to ventrolateral periaqueductal gray in electroacupuncture inhibition of cardiovascular reflexes

We have shown that the modulatory effect of electroacupuncture (EA) on the blood pressure (BP) response induced by visceral organ stimulation is related to inhibition of cardiovascular neurons in the rostral ventrolateral medulla (rVLM) through a mechanism that involves opioids. This effect is long lasting and may involve a long-loop neural supraspinal pathway, including the arcuate nucleus (ARC), which is an important site of opioid neurotransmitter synthesis. Therefore, we evaluated the role of the hypothalamic ARC and its interaction with the midbrain ventrolateral periaqueductal gray (vlPAG) in the EA-BP response. The gallbladder of alpha-chloralose-anesthetized cats was stimulated to test for the influence of EA on splanchnic afferent-induced cardiovascular reflexes. Electrodes were placed around the splanchnic nerve (SN), and acupuncture needles were applied at P5-6 acupoints overlying the median nerve (MN). Electrophysiological recordings showed that spontaneous activity of ARC and vlPAG neurons was low (1.3 +/- 0.5 and 2.0 +/- 0.5 spikes/s, respectively). We observed a gradation of responses of ARC neurons to the stimulation of different acupoints, ranging from uniform responses of all neurons during stimulation of the P5-6, LI4-11, H5-6, and St2-G2 located over deep nerves to fewer responses during stimulation of LI6-7 and G37-39 located over superficial nerves. Microinjection of the excitatory amino acid dl-homocysteic acid (DLH 4 nM, 50 nl) into the ARC augmented the responses of vlPAG neurons, whereas microinjection of kainic acid (KA 1 mM, 50 nl) to deactivate neurons in the ARC decreased vlPAG responses to SN stimulation. Thirty minutes of EA at P5-6 increased the SN-evoked discharge of vlPAG neurons (7.0 +/- 1.2 to 14.3 +/- 3.0 spikes/30 stimuli), a response that was blocked by microinjection of KA into the ARC. Microinjection of DLH into the ARC, like EA, inhibited (30 min) the reflex increase in BP induced by application of bradykinin (BK) to the gallbladder, whereas microinjection of KA into the ARC blocked the inhibitory influence of EA at P5-6 on the BK-induced BP response. These results suggest that excitatory projections from the ARC to the vlPAG are essential to the EA inhibition of the reflex increase in BP induced by SN or gallbladder visceral afferent stimulation.     

Sympathoinhibition from ventrolateral periaqueductal gray mediated by 5-HT(1A) receptors in the RVLM

The role of 5-hydroxytryptamine 1A (5-HT(1A)) receptors located in the rostral ventrolateral medulla (RVLM) in the mediation of a sympathoinhibitory and depressor response elicited from the ventrolateral periaqueductal gray (vlPAG) matter of the midbrain was examined in pentobarbital sodium-anesthetized rats. Activation of neurons in the vlPAG evoked a decrease in renal and lumbar sympathetic nerve activities and a decrease in arterial blood pressure. After microinjection of the specific 5-HT(1A)-receptor antagonist WAY-100635 into the pressor area of the RVLM, the vlPAG-evoked sympathoinhibition and hypotension was attenuated to control levels (7 of 15 animals) or converted into a sympathoexcitation and pressor response (8 of 15 animals). Baroreflex inhibition of sympathetic nerve activity was not impaired by microinjection of WAY into the sympathoexcitatory region of the RVLM. These data suggest that sympathoinhibition and hypotension elicited by activation of neurons in the vlPAG are mediated by 5-HT(1A) receptors in the RVLM.     

Influence of rostral ventrolateral medulla on renal sympathetic baroreflex in conscious rabbits

Previous studies with anesthetized animals have shown that the pressor region of the rostral ventrolateral medulla (RVLM) is a critical site in vasomotor control. The aim of this study was to develop, in conscious rabbits, a technique for microinjecting into the RVLM and to determine the influence of this area on renal sympathetic nerve activity (RSNA) and arterial pressure (AP) using local injections of glutamate, rilmenidine, ANG II and sarile. Rabbits were implanted with guide cannulas for bilateral microinjections into the RVLM (n = 7) or into the intermediate ventrolateral medulla (IVLM, n = 6) and an electrode for measuring RSNA. After 7 days of recovery, injections of glutamate (10 and 20 nmol) into the RVLM increased RSNA by 81 and 88% and AP by 17 and 25 mmHg, respectively. Infusion of glutamate (2 nmol/min) into the RVLM increased AP by 15 mmHg and the RSNA baroreflex range by 38%. By contrast, injection of the imidazoline receptor agonist rilmenidine (4 nmol) into the RVLM decreased AP by 8 mmHg and the RSNA baroreflex range by 37%. Injections of rilmenidine into the IVLM did not alter AP or RSNA. Surprisingly, treatments with ANG II (4 pmol/min) or the ANG II receptor antagonist sarile (500 pmol) into the RVLM did not affect the resting or baroreflex parameters. Infusion of ANG II (4 pmol/min) into the fourth ventricle increased AP and facilitated the RSNA baroreflex. Our results show that agents administered via a novel microinjecting system for conscious rabbits can selectively modulate neuronal activity in circumscribed regions of the ventrolateral medulla. We conclude that the RVLM plays a key role in circulatory control in conscious rabbits. However, we find no evidence for the role of ANG II receptors in the RVLM in the moment-to-moment regulation of AP and RSNA.     

Opioid peptides within the midbrain periaqueductal gray suppress affective defense behavior in the cat

The effects of the methionine-enkephalin analog [D-Ala2-Met5]-enkephalinamide (DAME) upon the threshold for affective defense behavior were determined following microinjections placed into midbrain periaqueductal gray sites from which this response was elicited. Affective defense behavior was elicited by electrical stimulation through a cannula electrode situated in the dorsal aspect of the midbrain periaqueductal gray. Dose-response curves characterizing the effects of DAME upon affective defense behavior were determined utilizing the following doses: 0.25, 0.5 and 1.0 microgram in 0.5 microliter saline, pH = 7.4 or vehicle control (saline). Response thresholds were tested 10-30, 30-60, 60-90, 120-150, 180-210, 1440-1470 and 2880-2910 min postinjection. The results obtained indicated that injections of DAME at a dose of 1.0 microgram/0.5 microliter produced significant, long duration elevations in affective defense thresholds, lasting up to 1440-1470 min postinjection. Lower doses of DAME (0.25 and 0.5 microgram/0.5 microliter) also resulted in significant increases in affective defense thresholds, but these effects were of shorter durations (60-90 and 120-150 min) postinjection, respectively. The suppressive effects of DAME were blocked when animals were pretreated with naloxone (10 micrograms/0.5 microliter) microinjected into the same midbrain periaqueductal gray site into which 0.25 microgram DAME was injected and affective defense behavior was elicited.     

c-Fos expression in the midbrain periaqueductal gray during static muscle contraction

The periaqueductal gray (PAG) of the midbrain is involved in the autonomic regulation of the cardiovascular system. The purpose of this study was to determine if static contraction of the skeletal muscle, which increases arterial blood pressure and heart rate, activates neuronal cells in the PAG by examining Fos-like immunoreactivity (FLI). Muscle contraction was induced by electrical stimulation of the L7 and S1 ventral roots of the spinal cord in anesthetized cats. An intravenous infusion of phenylephrine (PE) was used to selectively activate arterial baroreceptors. Extensive FLI was observed within the ventromedial region (VM) of the rostral PAG, the dorsolateral (DL), lateral (L), and ventrolateral (VL) regions of the middle and caudal PAG in barointact animals with muscle contractions, and in barointact animals with PE infusion. However, muscle contraction caused a lesser number of FLI in the VM region of the rostral PAG, the DL, L, and VL regions of the middle PAG and the L and VL regions of the caudal PAG after barodenervation compared with barointact animals. Additionally, the number of FLI in the DL and L regions of the middle PAG was greater in barodenervated animals with muscle contraction than in barodenervated control animals. Thus these results indicated that both muscle receptor and baroreceptor afferent inputs activate neuronal cells in regions of the PAG during muscle contraction. Furthermore, afferents from skeletal muscle activate neurons in specific regions of the PAG independent of arterial baroreceptor input. Therefore, neuronal cells in the PAG may play a role in determining the cardiovascular responses during the exercise pressor reflex.     

Pregnancy alters hemodynamic responses to hemorrhage in conscious rabbits

Pregnant animals are less able to maintain mean arterial pressure (MAP) during hemorrhage compared with nonpregnant animals, but the hemodynamic basis of this difference is unknown. The hypothesis that pregnancy attenuates responses of cardiac output, as well as total peripheral resistance (TPR) and femoral conductance, to hemorrhage was tested in conscious rabbits in both the pregnant and nonpregnant state (n = 10). During continuous slow blood loss (2% of the initial blood volume per minute), MAP was maintained initially in both groups. However, MAP then abruptly decreased to <45 mmHg in all animals after a smaller percentage of the initial blood volume was removed in pregnant compared with nonpregnant rabbits (43.6 +/- 1.7%, nonpregnant; 29.6 +/- 2.2%, pregnant; P < 0.005). The more rapid transition to hypotension exhibited by pregnant rabbits was associated with greater initial falls in cardiac output (-56 +/- 10 ml/min, nonpregnant; -216 +/- 33 ml/min, pregnant; P < 0.005) and stroke volume (0.8 +/- 0.1 ml/beat, nonpregnant; -1.3 +/- 0.1 ml/beat, pregnant; P < 0.05). In addition, the increase in TPR as a function of the decrease in cardiac output was markedly attenuated (P < 0.0001) during pregnancy. Whereas femoral conductance decreased in nonpregnant rabbits, it did not change significantly in pregnant animals. In conclusion, the lesser ability of conscious pregnant rabbits to maintain MAP during hemorrhage is due largely to a greater decrease in cardiac output but also to inadequate reflex increases in TPR, possibly in part in the femoral vascular bed.     

Pre- and postsynaptic inhibition mediated by GABA(B) receptors in rat ventrolateral periaqueductal gray neurons

The present study examined the actions of a GABA(B)-receptor agonist, baclofen, on synaptic transmission in rat ventrolateral periaqueductal gray (PAG) neurons of brainstem slices by using whole-cell voltage-clamp recordings. Baclofen (10 microM) induced a slow outward current (peak amplitude: 30.1+/-3.1pA, n=13) at -70mV, which persisted in the presence of tetrodotoxin (0.5 microM) and was diminished in the presence of postsynaptic intracellular K(+)-channel blockers (Cs(+) and TEA) and GDP-beta-S, indicating a direct postsynaptic depression mediated by K(+) channels and G proteins. Baclofen (10 microM) also decreased the frequency of both glutamatergic spontaneous EPSC (by 36+/-7%, n=11) and GABAergic spontaneous IPSC (by 37+/-12%, n=6) without changes in their amplitudes, indicating its presynaptic inhibitions. Taken together, the activation of postsynaptic GABA(B) receptors inhibits ventrolateral PAG neurons directly. At the same time, activating presynaptic GABA(B) receptors on glutamatergic and GABAergic nerve terminals inhibits glutamate and GABA release, respectively. The overall effects might influence an output of ventrolateral PAG neurons that build up the descending pain control system to the spinal dorsal horn.     

Blockade of the nociceptin/orphanin FQ/NOP receptor system in the rat ventrolateral periaqueductal gray potentiates DAMGO analgesia

Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are involved in various biological functions including pain. High density of NOP receptor has been found in the ventrolateral periaqueductal gray (vlPAG), the main output pathway involved in descending pain-control system. The aim of our work was to evaluate the involvement of the N/OFQ/NOP system in the modulation of MOP analgesia in the rat vlPAG using UFP-101, a selective NOP antagonist. N/OFQ significantly blocked DAMGO (a selective MOP agonist) analgesia, while UFP-101 enhanced the effect of the opioid given at a subanalgesic dose. These results confirm our hypothesis of an antiopioid role for N/OFQ in the vlPAG.     

Microinjection of muscimol into the periaqueductal gray suppresses cardiovascular and neuroendocrine response to air jet stress in conscious rats

Microinjection of the neuronal inhibitor muscimol into the dorsomedial hypothalamus (DMH) suppresses increases in heart rate (HR), mean arterial pressure (MAP), and circulating levels of adrenocorticotropic hormone (ACTH) evoked in air jet stress in conscious rats. Similar injection of muscimol into the caudal region of the lateral/dorsolateral periaqueductal gray (l/dlPAG) reduces autonomic responses evoked from the DMH, leading to the suggestion that neurons in the l/dlPAG may represent a descending relay for DMH-induced increases in HR and MAP. Here, we examined the role of neuronal activity in the caudal l/dlPAG on the increases in MAP, HR, and plasma ACTH seen in air jet stress in rats. Microinjection of muscimol into the caudal l/dlPAG reduced stress-induced increases in HR and MAP, while identical injections into sites just dorsal or into the rostral l/dlPAG had no effect. Microinjection of a combination of the glutamate receptor antagonists 2-amino-5-phosphonopentanoate (AP5) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) into the caudal l/dlPAG decreased stress-induced increases in HR alone only at the end of the 20-min stress period but significantly accelerated return to baseline. Surprisingly, microinjection of muscimol into the caudal l/dlPAG also reduced the stress-induced increase in plasma ACTH by 51%. Compared with unstressed control rats, rats exposed to air jet stress exhibited approximately 3 times the number of Fos-positive neurons in the l/dlPAG. These findings suggest that neurons in the l/dlPAG are activated in air jet stress and that this activity contributes to increases in HR, MAP, and plasma ACTH.     

Effects of lordosis-relevant neuropeptides on midbrain periaqueductal gray neuronal activity in vitro.

Certain neuropeptides can facilitate lordosis by acting on midbrain periaqueductal gray (PAG) in estrogen-primed female rats. Here, we investigated responses of individual PAG neurons in vitro, to five neuropeptides: substance P (SP), luteinizing hormone-releasing hormone (LHRH), prolactin (PRL), oxytocin (OT), and thyrotropin-releasing hormone (TRH). Substance P, OT, and TRH excited spontaneous activity of PAG neurons through neurotransmitter-like actions in a dose-dependent manner, whereas LHRH and PRL virtually never affected PAG neurons this way. Oxytocin acted through oxytocin receptors located on the recorded PAG neurons, since excitatory actions of OT were 1) not abolished by synaptic blockade, 2) mimicked by the OT-specific agonist [Thr4, Gly7]OT but not by arginine vasopressin, and 3) blocked by the OT-specific antagonist [d(CH2)5,Tyr(Me)2,Orn8]vasotocin. Although LHRH had no neurotransmitter-like action on spontaneous activity of PAG neurons, it, as well as SP, could modulate responses of some dorsal PAG neurons to GABAA and GABAB agonists or norepinephrine. Neuromodulatory actions of LHRH and SP could help facilitate lordosis through PAG neurons.     

Effect of precipitated morphine withdrawal on post-translational processing of prothyrotropin releasing hormone (proTRH) in the ventrolateral column of the midbrain periaqueductal gray

We have demonstrated that during opiate withdrawal, preprothyrotropin releasing hormone (preproTRH) mRNA is increased in neurons of the midbrain periaqueductal gray matter (PAG) while the concentration of TRH remained unaltered, suggesting that the processing of proTRH may be different in this region of the brain. The aim of the present study was to determine which of the proTRH-derived peptides are affected by opiate withdrawal in the PAG. These changes were compared to other TRH-containing areas such as the hypothalamic paraventricular nucleus (PVN), median eminence (ME) and the lateral hypothalamus (LH). Control and morphine-treated rats 24 h following naltrexone-precipitated withdrawal were decapitated and the brain microdissected. Pooled samples from each animal group were acid extracted, and peptides were electrophoretically separated then analyzed by specific radioimmunoassay. Opiate withdrawal caused a significant change in the level of some post-translational processing products derived from the TRH precursor. In the PAG, opiate withdrawal resulted in an accumulation of the intervening preproTRH(83-106) peptide from the N-terminal side of the prohormone, while the levels of the C-terminal preproTRH(208-285) peptide were reduced, with no change in preproTRH(25-50) or TRH, itself, as compared to control animals. Immunohistochemical analysis also showed significant increases in cellular preproTRH(83-106) peptide immunolabeling in the PAG. Opiate withdrawal in the lateral hypothalamus, unlike from the PAG, was accompanied by an increase in the concentration of TRH. In addition, western blot analysis showed that during opiate withdrawal, the mature form of the prohormone convertase 2 (PC2) increased only in PAG as compared with their respective controls. Thus, these results demonstrate a region-specific regulation of TRH prohormone processing in the brain, which may engage PC2, further suggesting a role for specific proTRH-derived peptides in the manifestations of opiate withdrawal.     

Role of the midbrain periaqueductal gray matter in conditioned reflexes

Unit activity in the midbrain periaqueductal gray matter (PGM) during an instrumental placing reflex, its extinction, differentiation, and conditioned inhibition, was studied in chronic experiments on cats. Spike responses 1–2 sec in duration in 69 (36.7%) of 182 neurons preceded by 400–800 msec the beginning of conditioned-reflex and voluntary intertrial movements. These advanced responses appeared 200 msec before the corresponding advance responses of motor cortical neurons. Fifty-eight neurons (30.9%) responded directly to acoustic stimulation with a latent period of 10–50 msec for 2–6 sec, 19 neurons (10.1%) generated double responses, linked with both the acoustic stimulus and subsequent conditioned-reflex movement, and 42 neurons (22.3%) did not respond to acoustic stimulation, although individual neurons of this group changed the level of their spontaneous activity in response to repeated conditioned stimulation, and this change was maintained for some tens of minutes. Extinction, differentiation, and conditioned inhibition all abolished conditioned-reflex movements, but each type of internal inhibition was accompanied by its own characteristic changes in the firing pattern of PGM neurons. Functional independence of neurons of the first and second groups was demonstrated during extinction and recovery of the conditioned-reflex. The results indicate the important role of PGM not only in the mechanism of the conditioned reflex, but also in the development of its internal inhibition.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 3, pp. 403–419, May–June, 1984.     

Modulation of neuron activity of the midbrain periaqueductal gray matter influenced by monoaminergic brainstem structures

A study was done on base activity and changes in base activity (BA) of neurons in periaqueductal gray matter (PAG) during stimulation of monoaminergic structures of the brainstem: the nucleus raphe magnus (NRM), the locus coeruleus (LC), and the substantia nigra (SN), in rats anesthetized with hexenal (200 mg/kg). Three types of PAG neurons that differed in BA structure were identified. NRM, LC and SN stimulation changed BA only in type III neurons. Stimulation of these structures evoked an increase in BA frequency in 11.0–14.5%, and inhibition in 31.0–47.5% of type III neurons. Simultaneous stimulation of two structures led to a marked drop in intensity of effects. A depressing effect on BA was always detected if stimulation of one of the structures suppressed BA. Stimulation of two structures, with one being the NRM, was most effective. The role of PAG in the organization of the brain-stem component of the antinociceptive mechanism is discussed.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 24, No. 1, pp. 52–60, January–February, 1992.