Periaqueductal Gray Region Research Articles

Effects of Herbal Bath "HAC" on Functional Recovery and c-Fos Expression in the Ventrolateral Periaqueductal Gray Region after Sciatic Crushed Nerve Injury in Rats

Peripheral nerve injuries are a commonly encountered clinical problem and often result in a chronic pain and severe functional deficits. c-Fos expression is sometimes used as a marker of increased neuronal activity. We have developed herbal bath “HAC” for pain control using the following herbs: Harpagophytum procumbens, Atractylodes japonica, and Corydalis tuber. In the present study, we investigated the effects of herbal bath “HAC” on the recovery rate of the locomotor function and the expression of c-Fos in the ventrolateral periaqueductal gray (vlPAG) region of brain following sciatic crushed nerve injury in rats. Walking track analysis for the evaluation of functional recovery and immunohistochemistry for the c-Fos expression were used for this study. In the present results, characteristic gait change with dropping of the sciatic function index (SFI) was observed and c-Fos expression in the vlPAG was suppressed following sciatic crushed nerve injury in rats. Immersion into herbal bath “HAC” enhanced SFI value and restored c-Fos expression in the vlPAG to the control value. These results suggest that herbal bath “HAC” might activate neurons in the vlPAG, and it facilitates functional recovery from peripheral nerve injury. Here we showed that herbal bath “HAC” could be used as a new therapeutic intervention for pain control and functional recovery from peripheral nerve injury.

Effects of amygdalin on the functional recovery and c-Fos expression in the ventrolateral periaqueductal gray region after sciatic crushed nerve injury in rats

Peripheral nerve injuries are a commonly encountered clinical problem and often result in a chronic pain and severe functional deficits. c-Fos expression is sometimes used as a marker of increased neuronal activity. We have developed herbal bath “HAC” for pain control using the following herbs: Harpagophytum procumbens, Atractylodes japonica, and Corydalis tuber. In the present study, we investigated the effects of herbal bath “HAC” on the recovery rate of the locomotor function and the expression of c-Fos in the ventrolateral periaqueductal gray (vlPAG) region of brain following sciatic crushed nerve injury in rats. Walking track analysis for the evaluation of functional recovery and immunohistochemistry for the c-Fos expression were used for this study. In the present results, characteristic gait change with dropping of the sciatic function index (SFI) was observed and c-Fos expression in the vlPAG was suppressed following sciatic crushed nerve injury in rats. Immersion into herbal bath “HAC” enhanced SFI value and restored c-Fos expression in the vlPAG to the control value. These results suggest that herbal bath “HAC” might activate neurons in the vlPAG, and it facilitates functional recovery from peripheral nerve injury. Here we showed that herbal bath “HAC” could be used as a new therapeutic intervention for pain control and functional recovery from peripheral nerve injury.

Nitrous oxide-antinociception is mediated by opioid receptors and nitric oxide in the periaqueductal gray region of the midbrain

Previous studies have shown that nitrous oxide (N2O)-induced antinociception is sensitive to antagonism by blockade of opioid receptors and also by inhibition of nitric oxide (NO) production. The present study was conducted to determine whether these occur within the same brain site. Mice were stereotaxically implanted with microinjection cannulae in the periaqueductal gray (PAG) area of the midbrain. In saline-pretreated mice, exposure to 70% N2O resulted in a concentration-dependent antinociceptive effect in the mouse abdominal constriction test. Pretreatment with an opioid antagonist in the PAG significantly antagonized the antinociceptive effect. Pretreatment with an inhibitor of NO production in the PAG also significantly antagonized the antinociceptive effect. These findings suggest that N2O acts in the PAG via an NO-dependent, opioid receptor-mediated mechanism to induce antinociception.

Index of suspicion in the nursery

A set of nonidentical twins is born at 24 weeks gestational age by normal spontaneous vaginal delivery to a 25-year-old gravida 3 para 2 female. The infants are appropriate for gestational age, weighing 525 and 520 g. Both twins are intubated in the delivery room, given surfactant via endotracheal tubes, and placed on high-frequency oscillators in the neonatal intensive care unit (NICU). Results of clinical examination on admission to the NICU are normal for both twins. On the fourth day after birth, each infant is started on continuous fentanyl infusions at 3 mcg/kg per hour. Urinary output begins to decline 24 hours after initiation of the fentanyl infusions, with a simultaneous decline in renal function (TableT1). On postnatal day 7, marked abdominal distention is noted in both twins.Renal ultrasonography on both infants reveals bilateral hydronephrosis and very distended, urine-filled bladders extending under the hepatic border (Figs. 1 and 2). Fentanyl infusions are discontinued, and indwelling catheters are inserted, allowing bladder drainage in each infant. After catheterization, both twins experience polyuria and improvement in renal function. Repeat renal ultrasonography on postnatal day 9 demonstrates resolution of hydronephrosis and normal bladder size (Figs. 3 and 4)The association between fentanyl infusion and bladder urinary retention remains unknown. The first step in elucidation begins with the differential diagnosis of urinary tract obstruction and hydronephrosis in neonates. The clinician must rule out transient in utero hydronephrosis, tumors, urologic anomalies, and bladder asphyxia. Renal ultrasonography can be performed to rule out ascites or renal vascular diseases as the cause for acute renal failure. The next step involves understanding the mechanism of bladder retention with fentanyl use.There are numerous possible mechanisms for fentanyl-induced bladder retention. The causative mechanism is relaxation of the detrusor muscle and increased urinary bladder compliance. Mechanisms that have been gleaned from animal studies indicate that morphine and other opiates cause urinary retention by activating mu- and delta-opioid receptors in the spinal cord and midbrain periaqueductal gray (PAG) region. Terris and Merguerian found urinary retention to be induced by an increase in urethral sphincter pressures.These infants presented with deteriorating renal function and acute urinary retention requiring urethral catheter placement. Each patient’s prenatal ultrasonography image was normal, which ruled out posterior uretheral valves. Each patient had normal urine output and displayed normal renal function prior to starting fentanyl infusions. The absence of bladder trabeculation signifies a lack of long-standing outflow obstruction. In these twin infants, infusions of fentanyl resulted in bladder outflow obstruction and bilateral ureteral obstruction. The insertion of indwelling catheters allowed drainage of 30 and 25 mL of urine from each bladder. Cessation of the fentanyl infusions resulted in resolution of acute renal failure and hydronephrosis. Follow-up renal ultrasonography showed resolution of urinary bladder distention and hydronephrosis, which strongly suggests that fentanyl infusions were associated with the bladder obstructions.Utpala and associates reported two cases of preterm infants (unrelated) who had urinary retention and renal failure due to continuous fentanyl infusions, both of whom had mild swelling of the suprapubic area. In the twins in this case, marked bladder distention extended beyond the hepatic border.After confirmation of urinary retention through physical examination and renal ultrasonography, discontinuing the causative agent and helping the body eliminate excess fluid should prove beneficial in reversing urine retention. Discontinuing fentanyl should reverse the process causing the urine retention, and catheter placement provides a conduit for fluid to escape from the bladder.Long-term complications with continued use of fentanyl infusions after the development of anuria remain largely undefined, but they may involve organ damage, loss of function, or the need for transplantation.Neonatal urinary obstruction is rare, but must be considered in infants who are receiving fentanyl infusions. Infants may present with decreased urine output and abdominal distention after initiation of such infusions. Discontinuing fentanyl infusions and inserting indwelling urinary catheters proved beneficial in evacuating the bladders and improving renal function in this case.

Rapid heterologous desensitization of antinociceptive activity between mu or delta opioid receptors and chemokine receptors in rats

Previous studies have shown pretreatment with chemokines CCL5/RANTES (100 ng) or CXCL12/SDF-1alpha (100 ng) injected into the periaqueductal grey (PAG) region of the brain, 30 min before the mu opioid agonist DAMGO (400 ng), blocked the antinociception induced by DAMGO in the in vivo cold water tail-flick (CWT) antinociceptive test in rats. In the present experiments, we tested whether the action of other agonists at mu and delta opioid receptors is blocked when CCL5/RANTES or CXCL12/SDF-1alpha is administered into the PAG 30 min before, or co-administered with, opioid agonists in the CWT assay. The results showed that: (1) CXCL12/SDF-1alpha (100 ng, PAG) or CCL5/RANTES (100 ng, PAG), given 30 min before the opioid agonist morphine, or selective delta opioid receptor agonist DPDPE, blocked the antinociceptive effect of these drugs; (2) CXCL12/SDF-1alpha (100 ng, PAG) or CCL5/RANTES (100 ng, PAG), injected at the same time as DAMGO or DPDPE, significantly reduced the antinociceptive effect induced by these drugs. These results demonstrate that the heterologous desensitization is rapid between the mu or delta opioid receptors and either CCL5/RANTES receptor CCR5 or CXCL12/SDF-1alpha receptor CXCR4 in vivo, but the effect is greater if the chemokine is administered before the opioid.

Chemical stimulation of visceral afferents activates medullary neurones projecting to the central amygdala and periaqueductal grey

Cholecystokinin (CCK) stimulates gastrointestinal vagal afferent neurones that signal visceral sensations. We wished to determine whether neurones of the nucleus of the solitary tract (NTS) or ventrolateral medulla (VLM) convey visceral afferent information to the central nucleus of the amygdala (CeA) or periaqueductal grey region (PAG), structures that play a key role in adaptive autonomic responses triggered by stress or fear. Male Sprague–Dawley rats received a unilateral microinjection of the tracer cholera toxin subunit B (CTB, 1%) into the CeA or PAG followed, 7 days later, by an injection of CCK (100 μg/kg, i.p.) or saline. Brains were processed for detection of Fos protein (Fos-IR) and CTB. CCK induced increased expression of Fos-IR in the NTS and the VLM, relative to control. When CTB was injected into the CeA, CTB-immunoreactive (CTB-IR) neurones were more numerous in the rostral NTS ipsilateral to the injection site, whereas they were homogeneously distributed throughout the VLM. Double-labelled neurones (Fos-IR + CTB-IR) were most numerous in the ipsilateral NTS and caudal VLM. The NTS contained the higher percentage of CTB-IR neurones activated by CCK. When CTB was injected into the PAG, CTB-IR neurones were more numerous in the ipsilateral NTS whereas they were distributed relatively evenly bilaterally in the rostral VLM. Double-labelled neurones were not differentially distributed along the rostrocaudal axis of the NTS but were more numerous in this structure when compared with the VLM. NTS and VLM neurones may convey visceral afferent information to the CeA and the PAG.

Methodological aspects of rat β-endorphin analysis—influence of diurnal variation

β-Endorphin radioimmunoassays (RIAs) are widely performed following physical, emotional and environmental challenges in the rat. In the literature, a wide range of techniques have been described, but in the present study, we have focused on methodological aspects of β-endorphin RIAs, investigating various characteristics of human and rat specific antibodies. Initial studies verified that the RIA outcome was not appropriate when using non-species compatible components. Novel rat β-endorphin antibodies, r 4114 and r 4268, were raised in rabbits and characterised in terms of specificity, avidity and titer. Both of the new antisera showed 68.1% cross-reactivity with human β-endorphin. The ED 50 was 50 ± 8 pmol/l, and the mean ED 80 was 17 pmol/l for r 4268 but three-fold higher for r 4114. The intra-assay coefficient of variation (CV) was 7% at 100 pmol/l and the inter-assay CV was 10% at the same level for r 4268 and similar for r 4114. Using this novel rat β-endorphin RIA for analyses of diurnal influence and removal from the Animal House cage, no significant changes were observed in either the hypothalamus or peri-aqueductal grey regions. These results suggest that rat β-endorphin concentrations in these brain areas are not affected by order of removal or diurnal variation.

Comparison of opioid receptor distributions in the rat central nervous system

The opioid receptors, μ, δ and κ, conduct the major pharmacological effects of opioid drugs, and exhibit intriguing functional relationships and interactions in the CNS. Previously established hypotheses regarding the mechanisms underlying these phenomena specify theoretical patterns of relative cellular localisation for the different receptor types. In this study, we have used double-label immunohistochemistry to compare the cellular distributions of δ and κ receptors with those of μ receptors in the rat CNS. Regions of established significance in opioid addiction were examined. Extensive μ/δ co-localisation was observed in neuron-like cells in several regions. μ and κ receptors were also often co-localised in neuron-like cell bodies in several regions. However, intense κ immunoreactivity (ir) also appeared in a separate, morphologically distinct population of cells that did not express μ receptors. These small, ovoid cells were often closely apposed against the larger, μ-ir cell bodies. Such cellular appositions were seen in several regions, but were particularly common in the medial thalamus, the periaqueductal grey and brainstem regions. These findings support proposals that functional similarities, synergy and cooperativity between μ and δ receptors arise from widespread co-expression by cells and intracellular molecular interactions. Although co-expression of μ and κ receptors was also detected, the appearance of a separate population of κ-expressing cells supports proposals that the contrasting and functionally antagonistic properties of μ and κ receptors are due to expression in physiologically distinct cell types. Greater understanding of opioid receptor interaction mechanisms may provide possibilities for therapeutic intervention in opioid addiction and other conditions.

A Role for the Periaqueductal Gray in Switching Adaptive Behavioral Responses

Previous studies suggested a role for the rostral lateral periaqueductal gray (PAG) in the inhibition of maternal behavior induced by low doses of morphine in dams with previous morphine experience. In the present study, we first showed that unilateral NMDA lesions placed in this particular PAG region prevented the morphine-induced inhibition of maternal behavior in previously morphine-sensitized dams. As suggested by previous Fos data on the PAG, predatory hunting appears as a likely candidate to replace maternal behavior in the morphine-treated dams. By testing saline- and morphine-treated dams with live cockroaches only, we have presently shown that morphine challenge increased insect hunting. Moreover, morphine- and saline-treated dams were also observed in an environment containing pups and roaches. Although most of the saline-treated animals displayed active nursing and only occasionally presented insect hunting, all of the morphine-treated animals ignored the pups and avidly pursued and caught the roaches. We next questioned whether the rostral lateral PAG would be involved in this behavioral switch. Our results showed that unilateral lesions of the rostral lateral PAG, but not other parts of the PAG, partially impaired predatory hunting and restored part of the maternal response. Moreover, bilateral lesions of the rostral lateral PAG produced even more dramatic effects in inhibiting insect hunting and restoring maternal behavior. The present findings indisputably show that the rostral lateral PAG influences switching from maternal to hunting behavior in morphine-treated dams, thus supporting a previously unsuspected role for the PAG in selecting adaptive behavioral responses.

Identification of neural circuits involved in female genital responses in the rat: a dual virus and anterograde tracing study

The spinal and peripheral innervation of the clitoris and vagina are fairly well understood. However, little is known regarding supraspinal control of these pelvic structures. The multisynaptic tracer pseudorabies virus (PRV) was used to map the brain neurons that innervate the clitoris and vagina. To delineate forebrain input on PRV-labeled cells, the anterograde tracer biotinylated dextran amine was injected in the medial preoptic area (MPO), ventromedial nucleus of the hypothalamus (VMN), or the midbrain periaqueductal gray (PAG) 10 days before viral injections. These brain regions have been intimately linked to various aspects of female reproductive behavior. After viral injections (4 days) in the vagina and clitoris, PRV-labeled cells were observed in the paraventricular nucleus (PVN), Barrington's nucleus, the A5 region, and the nucleus paragigantocellularis (nPGi). At 5 days postviral administration, additional PRV-labeled cells were observed within the preoptic region, VMN, PAG, and lateral hypothalamus. Anterograde labeling from the MPO terminated among PRV-positive cells primarily within the dorsal PVN of the hypothalamus, ventrolateral VMN (VMNvl), caudal PAG, and nPGi. Anterograde labeling from the VMN terminated among PRV-positive cells in the MPO and lateral/ventrolateral PAG. Anterograde labeling from the PAG terminated among PRV-positive cells in the PVN, ventral hypothalamus, and nPGi. Transynaptically labeled cells in the lateral hypothalamus, Barrington's nucleus, and ventromedial medulla received innervation from all three sources. These studies, together, identify several central nervous system (CNS) sites participating in the neural control of female sexual responses. They also provide the first data demonstrating a link between the MPO, VMNvl, and PAG and CNS regions innervating the clitoris and vagina, providing support that these areas play a major role in female genital responses.

Excitatory amino acid receptors in the periaqueductal gray mediate the cardiovascular response evoked by activation of dorsomedial hypothalamic neurons

Neurons in the region of dorsomedial hypothalamus are involved in the organization of the physiological responses to emotional stress. We have recently shown that the cardiovascular response evoked by activation of dorsomedial hypothalamus neurons is largely dependent on a synaptic relay with the lateral/dorsolateral periaqueductal gray region. In this study, we aimed to investigate whether excitatory amino acid receptors at the lateral/dorsolateral periaqueductal gray region are involved in mediating the response evoked by activation of dorsomedial hypothalamus neurons. In conscious rats, the cardiovascular effects produced by microinjection of GABA A receptor antagonist, bicuculline methiodide into the dorsomedial hypothalamus were evaluated before and after injection of different excitatory amino acid antagonists into lateral/dorsolateral periaqueductal gray region. Pretreatment of lateral/dorsolateral periaqueductal gray region with the non-selective ionotropic excitatory amino acid receptor antagonist kynurenic acid or with the N-methyl- d-aspartate receptor-selective antagonist, MK-801, largely reduced the tachycardic and pressor effects evoked by activation of dorsomedial hypothalamus neurons by bicuculline methiodide microinjection (heart rate 90 and 74%; blood pressure 81 and 84%, respectively). The non- N-methyl- d-aspartate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, did not alter the cardiovascular response evoked by dorsomedial hypothalamus activation. In an additional series of experiments, microinjection of the N-methyl- d-aspartate receptor agonist, N-methyl- d-aspartate, into the lateral/dorsolateral periaqueductal gray region, evoked an increase in heart rate and a pressor response that was accompanied by an increase in locomotor activity. These effects were not altered by pretreatment of lateral/dorsolateral periaqueductal gray region neurons with 6-cyano-7-nitroquinoxaline-2,3-dione but were completely abolished by MK-801. Altogether, these findings indicate that the cardiovascular response evoked by dorsomedial hypothalamus activation involves a synaptic relay at the lateral/dorsolateral periaqueductal gray region that is mediated at least in large part by excitatory amino acid receptors, possibly N-methyl- d-aspartate receptors.

Reply: Listening to the Past: History, Psychiatry, and Anxiety

Dear Editor: Andrea Tone explores the history of psychiatry and the rise of biological psychiatry and suggests ways in which the study of history can shed light on current psychiatric practice (1). Focusing on anxiolytics as a case study in the development of psychopharmacology, she shows how social and political factors converged to popularize, and later stigmatize, outpatient treatments for anxiety. However, the development and understanding of anxiety disorders, especially panic disorder, have been far from only a psychopharmacological evolution. Psychopharmacology for panic disorder became especially developed during the 20th-century. The evolution of psychopharmacology in relation to panic disorder can be divided into 3 main moments: first, Klein's observation of the effectiveness of the tricyclic antidepressants (2); second, the perception of the effectiveness of the benzodiazepines; and finally, the noted effectiveness of the selective serotonin reuptake inhibitors (3). The biological perspective also entails putting anxiety in the frame of the evolutionist paradigm. Charles Darwin, in The Expression of Emotion in Man and in Animals (4), pointed the way to the search for the adaptive value of the behavioural and psychological processes. Anxiety and fear have their roots in the defensive reactions of animals observed in response to dangers normally found in the environment. The interpretation of a stimulus or a situation as dangerous depends on cognitive operations. In humans, cognitive factors acquire importance because of the intervention of socially codified verbal or nonverbal symbols. Behavioural responses to fear are accompanied by intense physiological alterations-physical symptoms-and alterations in the emotional state. The physiological alterations comprise objective measures of anxiety. Thus cardiac frequency, arterial pressure, respiratory frequency, and increased electrical conductivity of the skin brought on by sweating are the measures most frequently used to determine the degree of anxiety. The 21st-century will probably be marked by the development of genetic and neuroimaging data about panic and other anxiety disorders. Panic attacks seem to originate from a network of fear with altered sensibility. This network includes the prefrontal cortex, insula, thalamus, tonsils, and tonsil projections toward the brain stem and hypothalamus (5). Recent research on physiological consequences during a panic attack has shown remarkable findings in regional brain activity in different neuroimaging studies (5). The studies have shown significant increases in cerebral blood flow in panic disorder in several subcortical structures, including the thalamus, hypothalamus, and periaqueductal gray region, in addition to the somatosensory and associative cortices, and the cingulate. Advances in the neuroimaging techniques and technologies available to study key areas involved in panic attacks, and the ability to induce laboratorial panic attacks, thus further detailing brain function during a panic attack, will greatly increase our understanding of the exact neuroanatomical substrates for panic and phobic avoidance. It will also further our understanding of the sites of action of effective therapies. …

Enhanced Fos expression in glutamic acid decarboxylase immunoreactive neurons of the mouse periaqueductal grey during opioid withdrawal

Previous studies using c-Fos immunohistochemistry suggest that a sub-population of neurons in the midbrain periaqueductal gray region is activated during opioid withdrawal. The neurochemical identity of these cells is unknown but cellular physiological studies have implicated GABAergic neurons. The present study investigated whether GABAergic neurons are activated in the mouse periaqueductal gray during opioid withdrawal using dual-antibody immunohistochemistry for Fos and glutamic acid decarboxylase. Both chronic opioid treatment and naloxone-precipitated opioid withdrawal increased Fos expression in the periaqueductal gray, with the greatest increase being four-fold in the caudal ventrolateral subdivision following withdrawal. Neurons stained for both Fos and glutamic acid decarboxylase were greatly enhanced in all subdivisions of the periaqueductal gray following withdrawal, particularly in the lateral and ventrolateral divisions where the increase was up to 70-fold. These results suggest that activation of a subpopulation of GABAergic interneurons in the periaqueductal gray plays a role in opioid withdrawal.

Connectivity of the human periventricular—periaqueductal gray region

The periventricular gray (PVG) zone and its continuation, the periaqueductal gray (PAG) substance, have been targets for deep brain stimulation (DBS) in the alleviation of intractable pain for longer than two decades. Nevertheless, the anatomical connectivity of this region has been fairly poorly defined. The effects of DBS in this region are probably related to the release of endogenous endorphins, but until the connectivity of this region is better understood the mechanisms will remain unclear. Diffusion tractography was used to trace the pathways of the PVG-PAG region in seven healthy human volunteers. Images were acquired with the aid of a 1.5-tesla magnetic resonance imaging system. The region of interest was located just lateral to the posterior commissure and extended caudally to the level of the superior colliculus. Probabilistic diffusion tractography was performed from each voxel in each patient's PVG-PAG region. The PVG-PAG region was found to yield descending projections to the spinal cord and cerebellum. Ascending projections to the thalamus and frontal lobes were also observed. These findings suggest that the PVG-PAG region may modulate pain by two mechanisms: one involving the antinociceptive system in the spinal cord and the other involving influences on the central pain network.

Endocannabinoids Mediate Stress-Induced Analgesia

Acute stress decreases pain perception. Hohmann et al. show that, in rats, endocannabinoid signaling is involved in mediating stress-induced analgesia (SIA): Antagonists of the CB 1 endocannabinoid receptor block SIA, as does chronic exposure to CB1 agonists (which produces tolerance to cannabinoids), but opiate antagonists do not. Rats sacrificed at various times after exposure to foot shock (an acute stress) showed increased concentration of 2-arachidonoylglycerol (2-AG) in the dorsal midbrain after 2 minutes and a return to baseline for this endocannabinoid by 15 minutes. The concentration of anandamide peaked approximately 15 minutes after shock. The authors developed a selective inhibitor (an N -biphenyl carbamate) of the 2-AG-hydrolyzing enzyme monoacylglycerol lipase (MGL) and showed that microinjection of this inhibitor into the periaqueductal gray region enhanced SIA and that this effect was blocked by administration of the CB 1 antagonist rimonabant. Inhibition of the anandamide-metabolizing enzyme also enhanced SIA, and this was also blocked by rimonabant. Thus, endocannabinoid-metabolizing enzymes may serve as targets for treatment of pain and stress disorders. A. G. Hohmann, R. L. Suplita, N. M. Bolton, M. H. Neely, D. Fegley, R. Mangieri, J. F. Krey, J. M. Walker, P. V. Holmes, J. D. Crystal, A. Duranti, A. Tontini, M. Mor, G. Tarzia, D. Piomelli, An endocannabinoid mechanism for stress-induced analgesia. Nature 435 , 1108-1112 (2005). [PubMed]

The hypotension evoked by visceral nociception is mediated by delta opioid receptors in the periaqueductal gray

This study tested the hypothesis that the ventrolateral column of the midbrain periaqueductal gray (vlPAG) region mediates the hypotension and bradycardia evoked by visceral nociception. To test this, the local anesthetic lidocaine (2%; 0.5 μl) was microinjected into the vlPAG of halothane-anesthetized rats bilaterally and visceral nociception was induced 2 min later by injecting 5% acetic acid (0.5 ml) intraperitoneally. Acetic acid injection caused an abrupt fall in arterial pressure (−12.2±2.1 mm Hg) and heart rate (−37±93 bpm) lasting approximately 15 min. Lidocaine injection into the vlPAG prevented the fall in arterial pressure and heart rate completely. Cobalt chloride (5 mM; 0.2 or 0.5 μl) injection into the vlPAG also prevented nociceptive hypotension but it did not affect the fall in heart rate significantly. Lidocaine pretreatment also inhibited the depressor response caused by intramuscular formalin (5%; 0.2 ml) administration, a model of deep somatic nociception, although it did not prevent the response completely. To determine if opioid receptors mediate the response, selective mu, delta or kappa opioid receptor antagonists were microinjected into the vlPAG 5 min before intraperitoneal (ip) acetic acid administration. Naltrindole, a delta receptor antagonist, inhibited the response significantly but mu and kappa antagonists were completely ineffective. Lidocaine and naltrindole had no effect when injected into the dorsolateral PAG and did not influence cardiovascular function when injected into the vlPAG of saline treated control animals. These data support the hypothesis that the vlPAG mediates the depressor response evoked by visceral nociception and indicate that delta opioid receptors participate in the response.

Activation of mu opioid receptors in the ventrolateral periaqueductal gray inhibits reflex micturition in anesthetized rats

This study tested the hypothesis that morphine and other opiates cause urinary retention by activating mu opioid receptors in the midbrain periaqueductal gray (PAG) region. Selective mu, delta and kappa receptor agonists were microinjected into the PAG of urethane-anesthetized rats and the amplitude and incidence of bladder contractions were recorded during continuous saline infusion. Arterial pressure was monitored through a femoral artery catheter. Microinjection of the mu receptor agonist DAMGO into the ventrolateral PAG (vlPAG) suppressed volume-evoked bladder contractions completely. Bladder contractions ceased within 5 min of DAMGO injection and remained essentially undetectable for the rest of the 20 min recording period. Microinjection of the delta receptor agonist DPDPE into the vlPAG did not significantly affect either the amplitude of bladder contractions or the time interval separating contractions. The kappa receptor agonist U-69593 caused no discernible change in amplitude but increased the interval between bladder contractions significantly. Microinjection of DAMGO, DPDPE or U-69593 into the lateral or dorsolateral PAG columns was ineffective. DAMGO injection into the vlPAG increased arterial pressure whereas DPDPE and U-69593 produced a small but significant depressor response. These data support the hypothesis that mu and kappa receptors in the vlPAG participate in the micturition reflex.

GABAergic projection from the ventral tegmental area and substantia nigra to the periaqueductal gray region and the dorsal raphe nucleus.

Previous studies have shown that neurons in the ventral tegmental area (VTA) and substantia nigra (SN) project to the ventrolateral periaqueductal gray (PAGvl) and dorsal raphe nucleus (DR). Research has also shown that stimulation of neurons in the VTA/SN elicits cardiovascular depressor responses that are mediated by a projection to the PAGvl/DR. Anatomic and physiological experiments were done in the present study to determine the neurochemical identity of the VTA/SN projection to the PAGvl/DR. Experiments were done to characterize the origin and chemical nature of this projection by combining cholera toxin B tracing with immunofluorescence for the 67K isoform of glutamic acid decarboxylase (GAD) and tyrosine hydroxylase. The PAGvl/DR region was found to receive a substantial input from neurons in the VTA, SN, and deep mesencephalic nucleus. The DR was preferentially innervated by neurons in the VTA, whereas the PAGvl was preferentially innervated by neurons in the SN. A proportion of neurons in the VTA and the reticular portion of the SN found to project to the PAGvl/DR were GAD positive. In addition, experiments were done in urethane-anesthetized rats to determine whether injections of a gamma-aminobutyric acid (GABA) antagonist in the region of the PAGvl/DR attenuated the cardiovascular depressor responses produced by glutamate stimulation of the VTA/SN. Injections of the GABA-blocking agent picrotoxin (2.5 nmol, 500 nl) into the PAGvl/DR eliminated the cardiovascular responses from stimulation of the VTA/SN (0.01 M, 50 nl). The results of the present investigation provide evidence for a GABAergic projection from the VTA/SN to the PAGvl/DR. This projection may be an important regulator of the PAGvl/DR, an area of the midbrain involved in the production of behavioral and physiological responses to pain and stress.

Role of periaqueductal gray on the cardiovascular response evoked by disinhibition of the dorsomedial hypothalamus

Activation of neurons in the region of the dorsomedial hypothalamus (DMH), by microinjection of the GABA A receptor antagonist bicuculline methiodide (BMI) results in increases in arterial pressure, heart rate as well as behavioral changes similar to those evoked by acute emotional stress. Previous anatomic studies clearly demonstrated projections from the DMH to the midbrain periaqueductal gray (PAG), a brain region implicated in the organization of behavioral strategies associated with specific cardiovascular responses. In this study, physiological experiments in conscious rats were used to investigate the functional significance of this pathway. Unilateral inhibition of the lateral dorsolateral region of the PAG (l/dlPAG) with the GABA A receptor agonist, muscimol (1 nmol/100 nl) largely reduced the tachycardia and the pressor response produced by microinjection of BMI (10 pmol/100 nl) into the ipsilateral DMH. In contrast, inhibition of the ventrolateral PAG (vlPAG) region had no significant effect on the cardiovascular response evoked from disinhibition of the ipsilateral DMH. Our present results indicate that the l/dlPAG region is an important synaptic relay in the descending cardiovascular pathways from the DMH.

Heterologous µ‐opioid receptor adaptation by repeated stimulation of κ‐opioid receptor: up‐regulation of G‐protein activation and antinociception

The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride [(-)U-50,488H] on antinociception and G-protein activation induced by mu-opioid receptor agonists in mice. A single s.c. injection of (-)U-50,488H produced a dose-dependent antinociception, and this effect was reversed by a selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Furthermore, a single s.c. pre-treatment with (-)U-50,488H had no effect on the mu-opioid receptor agonist-induced antinociception. In contrast, repeated s.c. administration of (-)U-50,488H resulted in the development of tolerance to (-)U-50,488H-induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (-)U-50,488H significantly enhanced the antinociceptive effect of selective mu-opioid receptor agonists endomorphin-1, endomorphin-2 and [d-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAMGO). Using the guanosine-5'-o-(3-[35S]thio) triphosphate ([35S]GTP gamma S) binding assay, we found that (-)U-50,488H was able to produce a nor-BNI-reversible increase in [35S]GTP gamma S binding to membranes of the mouse thalamus, which has a high level of kappa-opioid receptors. Repeated administration of (-)U-50,488H caused a significant reduction in the (-)U-50,488H-stimulated [35S]GTP gamma S binding in this region, whereas chronic treatment with (-)U-50,488H exhibited the increase in the endomorphin-1-, endomorphin-2- and DAMGO-stimulated [35S]GTP gamma S bindings in membranes of the thalamus and periaqueductal gray. These results suggest that repeated stimulation of kappa-opioid receptors leads to the heterologous up-regulation of mu-opioid receptor functions in the thalamus and periaqueductal gray regions, which may be associated with the supersensitivity of mu-opioid receptor-mediated antinociception.