The effects of a benzodiazepine on the hyperpolarizing and the depolarizing responses of hippocampal cells to GABA

Rats implanted with bilateral cannulas in the periaqueductal gray exhibited similar behavioral excitations following microinjections of morphine sulphate and ACTH_1–24. Injections were more effective when the sites were located within rather than below the periaqueductal gray. Analgesia was observed following morphine but not ACTH microinjection. These results confirm that morphine exerts a dual action, inhibitory (i.e. analgesic) and excitatory, with ACTH mimicking only the latter action.     

Antinociception produced by microinjection of morphine in the rat periaqueductal gray is enhanced in the foot, but not the tail, by intrathecal injection of α1-adrenoceptor antagonists

Antinociception produced by microinjection of morphine in the ventrolateral periaqueductal gray is mediated in part by α₂-adrenoceptors in the spinal cord dorsal horn. However, several recent reports demonstrate that microinjection of morphine in the ventrolateral periaqueductal gray inhibits nociceptive responses to noxious heating of the tail by activating descending neuronal systems that are different from those that inhibit the nociceptive responses to noxious heating of the feet. More specifically, α₂-adrenoceptors appear to mediate the antinociception produced by morphine using the tail-flick test, but not that using the foot-withdrawal or hot-plate tests. The present study extended these findings and determined the role of α₁-adrenoceptors in mediating the antinociceptive effects of morphine microinjected into the ventrolateral periaqueductal gray using both the foot-withdrawal and the tail-flick responses to noxious radiant heating in lightly anesthetized rats. Intrathecal injection of selective antagonists was used to determine whether the antinociceptive effects of morphine were modulated by α₁-adrenoceptors. Injection of the selective α₁-adrenoceptor antagonists prazosin or WB4101 potentiated the increase in the foot-withdrawal response latency produced by microinjection of morphine in the ventrolateral periaqueductal gray. In contrast, either prazosin or WB4101 partially reversed the increase in the tail-flick response latency produced by morphine. These results indicate that microinjection of morphine in the ventrolateral periaqueductal gray modulates nociceptive responses to noxious heating of the feet by activating descending neuronal systems that are different from those that inhibit the nociceptive responses to noxious heating of the tail. More specifically, α₁-adrenoceptors mediate a pro-nociceptive action of morphine using the foot-withdrawal response, but in contrast, α₁-adrenoceptors appear to mediate part of the antinociceptive effect of morphine determined using the tail-flick test.     

The periaqueductal gray: site of morphine analgesia and tolerance as shown by 2-way cross tolerance between systemic and intracerebral injections.

The periaqueductal gray was shown to be an important component of morphine analgesia and tolerance. Two-way analgesic cross tolerance was obtained between systemic and intracerebral morphine administrations when the intracerebral site was the periaqueductal gray. Rats were pretreated with intraperitoneal morphine and tested with intracerebral morphine in the periaqueductal gray. A dose-dependent reduction in analgesia as a function of morphine pretreatment level was obtained. Conversely, when rats were pretreated with intracerebral morphine in the periaqueductal gray and tested with intraperitoneal morphine, significant reductions in analgesia were obtained.     

Spinal cholinergic and monoamine receptors mediate the antinociceptive effect of morphine microinjected in the periaqueductal gray on the rat tail, but not the feet

The antinociceptive effects of morphine (5 μg) microinjected into the ventrolateral periaqueductal gray were determined using both the tail flick and the foot withdrawal responses to noxious radiant heating in lightly anesthetized rats. Intrathecal injection of appropriate antagonists was used to determine whether the antinociceptive effects of morphine were mediated byα₂-noradrenergic, serotonergic, opioid, or cholinergic muscarinic receptors. The increase in the foot withdrawal response latency produced by microinjection of morphine in the ventrolateral periaqueductal gray was reversed by intrathecal injection of the cholinergic muscarinic receptor antagonist atropine, but was not affected by the a2-adrenoceptor antagonist yohimbine, the serotonergic receptor antagonist methysergide, or the opioid receptor antagonist naloxone. In contrast, the increase in the tail flick response latency produced by morphine was reduced by either yohimbine, methysergide or atropine. These results indicate that microinjection of morphine in the ventrolateral periaqueductal gray inhibits nociceptive responses to noxious heating of the tail by activating descending neuronal systems that are different from those that inhibit the nociceptive responses to noxious heating of the feet. More specifically, serotonergic, muscarinic cholinergic andα₂-noradrenergic receptors appear to mediate the antinociception produced by morphine using the tail flick test. In contrast, muscarinic cholinergic, but not monoamine receptors appear to mediate the antinociceptive effects of morphine using the foot withdrawal response.     

Characterization of the spinal adrenergic receptors mediating the spinal effects produced by the microinjection of morphine into the periaqueductal gray

After the microinjection of morphine (5 micrograms/0.5 microliter) into the periaqueductal gray resulted in an increase in the hot-plate and tail-flick response latency of the unanesthetized rat, the alpha-adrenergic antagonists yohimbine, rauwolscine and corynanthine were given intrathecally. This treatment resulted in a dose-dependent reversal of the inhibition of the thermally evoked tail-flick reflex. The relative potency of these stereoisomers was: yohimbine = rauwolscine greater than corynanthine. Given the reported affinity of these agonists for the alpha 2 (yohimbine/rauwolscine) and alpha 1 (corynanthine) receptors, these observations suggest that the spinopetal noradrenergic systems are acting on alpha 2-adrenergic receptors. Prazosin, an agent with several orders of magnitude higher affinity for the alpha 1 than the alpha 2-receptor, was at best only equiactive with yohimbine. None of the intrathecal treatments produced a significant reversal of the effects of periaqueductal gray morphine on the hot-plate response. This suggests that the activation of spinopetal noradrenergic pathways alone cannot account for the suppression by morphine in the periaqueductal gray of this response which is organized at the supraspinal level.     

Contribution of acid sphingomyelinase in the periaqueductal gray region to morphine-induced analgesia in mice

Opioids are the most widely used drugs for long-term pain management, but their use is limited by the development of antinociceptive tolerance. The present study investigated the role of ceramide production through acid sphingomyelinase (ASM) activation in the periaqueductal gray region, a brain region implicated in opioid analgesia and tolerance. Morphine treatment was found, using immunohistochemistry, to increase ASM expression and intracellular ceramide in the periaqueductal gray 30 min after an acute injection (10 mg/kg). The effects of acute morphine treatment on ASM expression and ceramide generation in the periaqueductal gray region were completely blocked by pretreatment with naloxone and by silencing the ASM gene by plasmid-mediated transfection of ASM shRNA. In chronic morphine pellet-implanted mice, ASM expression and ceramide generation in the periaqueductal gray region were also significantly increased. Functionally, selective silencing of the ASM gene by local ASM shRNA transfection reduced the analgesic response to acute morphine, but the data on the effect of ASM shRNA on the development of antinociceptive tolerance were inconclusive. These data provide evidence that ASM activation and ceramide generation in the periaqueductal gray region play a major role in the antinociceptive mechanism of morphine.     

Prenatal morphine exposure decreases analgesia but not K+ channel activation

The present study has investigated the possible supraspinal adaptive changes induced by prenatal administration of morphine, including morphine-induced supraspinal antinociception in vivo, the density and binding affinity of mu-opioid receptors in the brain and the cellular action of morphine in brain slices in vitro. The cellular action of morphine was assessed by its activation of K+ channels in the ventrolateral periaqueductal gray (PAG), a crucial area for the supraspinal analgesic effect of morphine. Female rats were treated with morphine 7 days before mating at 2 mg/kg. The treatment was continued during pregnancy and after delivery at doses which increased by 1 mg/kg every 2 weeks. Experiments were conducted in the offspring at p14 days. Prenatal morphine exposure induced tolerance to supraspinal morphine-induced tail-flick response. The binding affinity and maximal binding of [(3)H]DAMGO in whole brain were not significant different between the morphine- or saline-treated dams. Autoradiographic analysis shows that the mu-opioid receptor density was decreased in the striatum, thalamus and amygdala but not in the midbrain, nucleus accumbens, hippocampus or cortex in morphine offspring. In ventrolateral PAG neurons, morphine activated inwardly rectifying K+ channels in 59% of recorded neurons of morphine offspring. Neither the magnitude of K channel activation nor the percentage of sensitive neurons was different between the saline- and morphine-treated offspring. It is concluded that prenatal morphine exposure induces tolerance to supraspinal analgesia and this tolerance is not attributed to a change in the mu-opioid receptor density or the receptor-function coupling efficiency in the midbrain periaqueductal gray.     

Effects of morphine on spontaneous multiple-unit activity: possible relation to mechanisms of analgesia and reward.

The effects of morphine (15 mg/kg) on multiple-unit activity in the awake rat were investigated at brain sites previously characterized by their ability to support stimulation-produced analgesia (SPA) and intracranial self-stimulation (ICSS). Of the SPA and SPA + ICSS sites, most of which were located in the periaqueductal gray matter, 91% showed increased multiple-unit activity after morphine administration (median increase = 80%). In contrast, only 50% of the ICSS-only sites, most of which were located in the lateral hypothalamus, and only 29% of sites supporting neither behavior showed this effect. All increases in multiple-unit activity were at least partly reversed by naloxone (1 mg/kg). Latencies to their onset and to analgesia measured by the tail-flick method were significantly correlated. A significant negative correlation was found between ICSS current thresholds and increases in multiple-unit activity after morphine at ICSS-only sites. These data lend further support to the suggestion that morphine exerts its analgesic action by activating an endogenous analgesic system and that the periaqueductal gray constitutes an important part of such a system. Furthermore, it is suggested that morphine's excitatory effect at self-stimulation loci may reflect its rewarding properties.     

Opioid receptor subtypes associated with ventral tegmental facilitation and periaqueductal gray inhibition of feeding

Eating was induced in sated animals by lateral hypothalamic electrical stimulation following central microinjections of mu- (morphine), delta-([D-Pen2,D-Pen5]enkephalin) or kappa-(U-50,488H) receptor agonists, or saline. With stimulation intensity fixed at a moderate level, time to eat 3 45-mg food pellets decreased with increases in stimulation frequency, approaching an asymptote near 7 s at ca. 70 Hz. Ventral tegmental injections (8 but not 0.8 nmol) of each of the 3 drugs reduced the minimum frequency required to produce eating of 3 pellets within 20 s and reduced the frequency at which asymptotic performance was produced; the drugs were equally effective at these doses. Naloxone (2 mg/kg) reversed the effects of each drug; naloxone was slightly more effective against morphine than against DPDPE or U-50,488H. These data suggest that all 3 receptor classes may contribute to the ventral tegmental facilitation of feeding. Periaqueductal gray injections (16 but not 1.6 nmol) of morphine had the opposite effect; they increased the stimulation frequency required to cause eating of 3 pellets in 20 s, and decreased the speed of eating across all stimulation frequencies. Periaqueductal gray injections of the delta- and kappa-agonists were each without effect. These data indicate that the periaqueductal gray inhibition of feeding is mediated solely by mu-receptors and their associated periaqueductal gray circuitry.     

Reinforcing effects of brain microinjections of morphine revealed by conditioned place preference

The previously documented place conditioning paradigm²⁵ was used to study the reinforcing effects of cerebral microinjections of morphine. Rats with implanted cannulae experienced place conditioning procedures, involving morphine administration into the IV (0.5 or 10 μg), III (10 μg) or lateral (0.5–10 μg) ventricles. Positive reinforcement, indicated by a significant preference for the place paired with morphine compared to the place similarly paired with control treatment, was seen in rats given 10 μg morphine into the lateral ventricle. The rats given 10 μg into the III ventricle also showed a preference, but the effect was not statistically significant. Positive reinforcement was subsequently demonstrated with morphine microinjections (10 μg) into the lateral hypothalamus, periaqueductal gray or nucleus accumbens. No clear preferences were produced by morphine injections into the caudate-putamen, amygdala or nucleus ambiguus. Following the final place conditioning test, rats were re-administered the treatment dose and analgesia and body temperature were measured. All three sites associated with reinforcement evidenced hyperthermia, but only the periaqueductal gray evidenced a short-latency analgesia. Sites with null place conditioning were not associated with any major behavioral effects. Using (+) and (−)-morphine (10 μg), it was demonstrated that only the active (−)-stereoisomer was effective in producing place preferences after injection into the periaqueductal gray. It was concluded that morphine administered directly into parts of the rat brain can produce place conditioning similar to that seen after systematically administered morphine²⁵. Morphine-produced place preference is not related to the acute depressant aspects of morphine, but may be related to the stimulant aspects.     

Synergistic analgesic interactions between the periaqueductal gray and the locus coeruleus.

Opiates modulate pain perception at a number of different levels within the central nervous system and the importance of synergistic spinal and supraspinal influences have been well documented. In the present study we demonstrate synergistic interactions between the periaqueductal gray and locus coeruleus. Administered either systemically or intracerebroventricularly (i.c.v.), ethylketocyclazocine elicits a potent naloxonazine-sensitive analgesia, indicating a mu 1 action. mu 1 Receptors also play a major role in opioid analgesic mechanisms in the periaqueductal gray and the locus coeruleus. However, microinjection of EKC into either the periaqueductal gray or locus coeruleus failed to elicit an analgesic response at any dose tested (0.1-20 micrograms) and, in additional studies, antagonized the analgesic actions of coadministered morphine or [D-Ser2,Leu5]enkephalin-Thr6 (DSLET). However, the simultaneous administration of EKC into both the periaqueductal gray (10 micrograms) and the locus coeruleus (10 micrograms; total combined dose 20 micrograms) produced a potent naloxonazine-sensitive analgesia greater than that observed with 50 micrograms i.c.v. These results suggest that EKC is a partial mu 1 agonist which lacks the efficacy to elicit analgesia when microinjected into either of the two brain regions alone. However, when exposed to several regions at once, either through simultaneous microinjections into the periaqueductal gray and locus coeruleus or by injection into the ventricle, EKC is a potent mu 1 analgesic. These results point out the existence of synergistic supraspinal interactions between the periaqueductal gray and the locus coeruleus, similar to the spinal/supraspinal interactions observed previously.     

Excitatory amino acids: role in morphine excitation in rat periaqueductal gray

Morphine was previously found to elicit an explosive excitatory behavior following its injection at a high dose in the rat periaqueductal gray (PAG). This non-naloxone reversible excitatory action of morphine was mimicked by the GABAA receptor antagonist, bicuculline, suggesting that morphine excitation was due in part to GABAA receptor blockade. In this paper, we report that injections of the excitatory amino acid (EAA) analogues, N-methyl-D-aspartate (NMDA), quisqualate (Q) or kainate (K) in the rat PAG resulted in similar (but not identical) behaviors. The excitatory actions of morphine or of NMDA (but not Q or K) were blocked or attenuated by the NMDA receptor antagonist, 2-amino-7-phosphonoheptanoate. These results show that both GABAA receptors as well as receptors for the EAAs may contribute to the excitatory actions of morphine in the PAG, and suggest that GABA may normally function to counterbalance a tonic excitatory influence of the EAAs.     

Opioid antagonists in the periaqueductal gray inhibit morphine and β-endorphin analgesia elicited from the amygdala of rats

In addition to brainstem sites of action, analgesia can be elicited following amygdala microinjections of morphine and μ-selective opioid agonists. The present study examined whether opioid analgesia elicited by either morphine or β-endorphin in the amygdala could be altered by either the general opioid antagonist, naltrexone, the μ-selective antagonist, β-funaltrexamine (BFNA) or theδ₂ antagonist, naltrindole isothiocyanate (Ntii) in the periaqueductal gray (PAG). Both morphine (2.5–5 μg) and β-endorphin (2.5–5 jig) microinjected into either the baso-lateral or central nuclei of the amygdala significantly increased tail-flick latencies and jump thresholds in rats. The increases were far more pronounced on the jump test than on the tail-flick test. Placements dorsal and medial to the amygdala were ineffective. Naltrexone (1–5 μg) in the PAG significantly reduced both morphine (tail-flick: 70–75%; jump: 60–81%) and β-endorphin (tail-flick: 100%; jump: 93%) analgesia elicited from the amygdala, indicating that an opioid synapse in the PAG was integral for the full expression of analgesia elicited from the amygdala by both agonists. Both BFNA (68%) and Ntii (100%) in the PAG significantly reduced morphine, but not β-endorphin analgesia in the amygdala on the tail-flick test. Ntii in the PAG was more effective in reducing morphine (60%) and β-endorphin (79%) analgesia in the amygdala on the jump test than BFNA (15–24%). Opioid agonist-induced analgesia in the amygdala was unaffected by opioid antagonists administered into control misplacements in the lateral mesencephalon, and the small hyperalgesia elicited by opioid antagonists in the PAG could not account for the reductions in opioid agonist effects in the amygdala. These data indicate that PAGδ₂ and to a lesser degree, μ opioid receptors are necessary for the full expression of morphine and β-endorphin analgesia elicited from the amygdala.     

II. Examination of spinal monoamine receptors through which brainstem opiate-sensitive systems act in the rat

Microinjection of morphine (5 μg) through stereotaxically implanted microinjection cannulas into the periaqueductal gray (104 sites), medial (n. raphe magnus; 26 sites) and paramedial (n. reticulogigantocellularis; 49 sites) medulla resulted in an increase in the latency of supraspinally (hot-plate) and spinally (tail-flick)-mediated responses evoked by thermal stimuli. This effect of intracerebral morphine on both hot-plate and tail-flick was dose-dependent, and reversed by systemically administered naloxone as well as by naloxone administered by microinjection into the same site. On the basis of frequency of occurrence, time of onset and magnitude of effect of the minimum effective dose, we could demonstrate no difference between the efficacy of morphine acting at sites in the periaqueductal gray, n. raphe magnus or n. reticulogigantocellularis on the supraspinally mediated response. In all areas examined, morphine was able to produce the maximum elevation in response latency. The microinjection of morphine into the periaqueductal gray frequently produced a total block of the thermally evoked spinally mediated tail-flick reflex. Unlike the periaqueductal gray, the systems through which opiates act in the n. raphe magnus or the n. reticulogigantocellularis to suppress spinal reflex activity displayed a clear plateau in their physiological effects. Microinjections of morphine into the n. raphe magnus or n. reticulogigantocellularis never produced a complete block of the spinal reflex. Further increases in inhibition could not be achieved by either a 3-fold increase in dose or bilateral injections into the paramedial medulla. The failure to block spinal reflex activity often occurred at sites where morphine would completely block the hot-plate response. These observations indicate that opiate receptor-linked systems in the mesencephalon and medulla can significantly attenuate the coordinated escape behavior otherwise evoked by a high-intensity thermal stimuli. We find there is no difference in the physiological efficacy of morphine acting in those regions on supraspinally mediated measures of pain responding. The differential effect on spinally mediated reflex function suggests that these several opiate linked systems produce their effect by discriminable mechanisms.     

Periaqueductal gray neurons response to microiontophoretically injected morphine in naive and morphine-dependent rats

The attempt of this study was to investigate the direct effects of increasing doses of morphine on the neuronal activity of the periaqueductal gray in morphine-naive and morphine-dependent rats. The microiontophoresis technique was used for this purpose. The four different responses induced by morphine exhibited dose-related patterns. Naloxone antagonized these responses in about 40% of the cases. Differences were found in the sensitivity of the neurons of morphine between naive and morphine-dependent rats. The phenomena of acute tolerance, chronic tolerance and dependence have been found. The results of this study indicate the presence of different neural populations in the periaqueductal gray in relation to their response to morphine, supporting the notion that subpopulations of opiate receptors exist within this brain area.     

GABAergic modulation of the analgesic effects of morphine microinjected in the ventral periaqueductal gray matter of the rat

The possibility that GABAergic neurons in the ventral periaqueductal gray matter modulate the analgesic effects of morphine microinjected into this brain area was investigated in the rat. Microinjection of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin 3-ol (THIP) (0.4 microgram in 0.2 microliter), a GABA agonist, in the ventral periaqueductal gray matter significantly reversed the increase of tail-flick latency induced by a prior injection of morphine sulfate (4 micrograms in 0.2 microliter) at the same site. Conversely, microinjection in the same region of picrotoxin (10 ng in 0.2 microliter), a GABA antagonist, significantly potentiated the analgesic effect of the same dose of morphine. These results suggest the existence of GABAergic neurons that tonically inhibit periaqueductal gray output neurons involved in centrifugal pain inhibition. The analgesic effects of opiates may, at least in part, result from disinhibition of these GABAergic neurons.     

Examination of spinal monoamine receptors through which brainstem opiate-sensitive systems act in the rat

The microinjection through stereotaxically implanted guide cannulae of morphine (5 micrograms/0.5 microliter) into the periaqueductal gray, the n. raphe magnus or the n. reticulogigantocellularis results in a significant elevation in the latency of a thermally evoked, spinally mediated reflex (tail-flick) and a supraspinally organized response (hot-plate). Spinal serotonin, noradrenalin, opiate and dopamine receptors were antagonized by the injection through chronically implanted intrathecal catheters of methysergide, phentolamine, naloxone and cis-flupenthixol, respectively. After a significant elevation of the tail-flick response latencies with intracerebral injections of morphine into the periaqueductal gray, the magnitude of the reversal produced by the intrathecally administered antagonists was phentolamine = methysergide much greater than naloxone = cis-flupenthixol = 0. After a significant elevation of the tail-flick response latency with intracerebral injections of morphine into the n. raphe magnus, the magnitude of the reversal produced by the intrathecally administered antagonist was methysergide greater than phentolamine greater than naloxone much greater than cis-flupenthixol = 0. After a significant elevation of the tail-flick response latency was produced by the microinjection of morphine into the n. reticulogigantocellularis, the magnitude of the reversal produced by intrathecal antagonists was phentolamine greater than naloxone much greater than methysergide = cis-flupenthixol = 0. None of the intrathecal antagonists reversed the elevation of the hot-plate response latencies produced by morphine injections into the n. raphe magnus and n. reticulogigantocellularis injections. A significant, but clearly subtotal reversal of the elevated hot-plate response latencies produced by periaqueductal gray morphine was produced by intrathecal phentolamine and methysergide. It is concluded that discrete populations of brain opiate receptors in the periaqueductal gray, n. raphe magnus and n. reticulogigantocellularis differentially activate spinal monoamine and opioid receptors to modulate thermally evoked spinally mediated reflexes. The general failure of treatments which reverse the segmental reflex inhibition to reverse the hot-plate effects suggests that: other spinopetal pathways are operative; that descending pathways activated by these manipulations do not contribute to the analgesic effects of brainstem morphine; and/or that in addition to spinopetal modulation, brainstem opiate receptors modulate nociceptive transmission at the brainstem level.     

Comparison of antinociceptive action of morphine in the periaqueductal gray, medial and paramedial medulla in rat

Microinjection of morphine (5 micrograms) through stereotaxically implanted microinjection cannulas into the periaqueductal gray (104 sites), medial (n. raphe magnus; 26 sites) and paramedial (n. reticulogigantocellularis; 49 sites) medulla resulted in an increase in the latency of supraspinally (hot-plate) and spinally (tail-flick)-mediated responses evoked by thermal stimuli. This effect of intracerebral morphine on both hot-plate and tail-flick was dose-dependent, and reversed by systemically administered naloxone as well as by naloxone administered by microinjection into the same site. On the basis of frequency of occurrence, time of onset and magnitude of effect of the minimum effective dose, we could demonstrate no difference between the efficacy of morphine acting at sites in the periaqueductal gray, n. raphe magnus or n. reticulogigantocellularis on the supraspinally mediated response. In all areas examined, morphine was able to produce the maximum elevation in response latency. The microinjection of morphine into the periaqueductal gray frequently produced a total block of the thermally evoked spinally mediated tail-flick reflex. Unlike the periaqueductal gray, the systems through which opiates act in the n. raphe magnus or the n. reticulogigantocellularis to suppress spinal reflex activity displayed a clear plateau in their physiological effects. Microinjections of morphine into the n. raphe magnus or n. reticulogigantocellularis never produced a complete block of the spinal reflex. Further increases in inhibition could not be achieved by either a 3-fold increase in dose or bilateral injections into the paramedial medulla. The failure to block spinal reflex activity often occurred at sites where morphine would completely block the hot-plate response. These observations indicate that opiate receptor-linked systems in the mesencephalon and medulla can significantly attenuate the coordinated escape behavior otherwise evoked by a high-intensity thermal stimuli. We find there is no difference in the physiological efficacy of morphine acting in those regions on supraspinally mediated measures of pain responding. The differential effect on spinally mediated reflex function suggests that these several opiate linked systems produce their effect by discriminable mechanisms.     

Distinct patterns of activated neurons throughout the rat midbrain periaqueductal gray induced by chemical stimulation within its subdivisions

This study provides a map of those neurons in the midbrain periaqueductal gray which are activated by chemical stimulation within different subdivisions of the periaqueductal gray. In pentobarbital anesthetized rats, the expression of the c-FOS protein was detected by immunocytochemistry and was used as a marker of neuronal activity. Microinjections of the γ-aminobutyric acid (GABA_A) receptor antagonist bicuculline (200 pmol in 50 nl) were used to increase selectively the firing rate of neurons originating from the injection site. The pattern of c-FOS immunoreactivity was highly specific for different injection sites. Dorsal injections were characterized by an extensive distribution of c-FOS immunoreactivity along the entire rostrocaudal extent of the periaqueductal gray, while ventral injections produced a much more restricted labeling. Following injection into the dorsal subdivision of the rostral periaqueductal gray, c-FOS immunoreactivity was present bilaterally in the dorsal and dorsolateral subdivisions of the rostral periaqueductal gray and was found in all subdivisions of the caudal periaqueductal gray. Dorsolateral injections at the level of the oculomotor nuclei produced strictly ipsilateral labeling in the dorsal and dorsolateral periaqueductal gray at the level of injection and throughout the ipsilateral half of the periaqueductal gray at more caudal levels. Stimulation in the ventrolateral periaqueductal gray induced FOS in the ventrolateral periaqueductal gray and the adjoining reticular formation. At rostral levels c-FOS immunoreactivity was also seen in the lateral periaqueductal gray but was absent caudal to the injection site. The identified patterns of activity in the periaqueductal gray provide a new basis for the interpretation of the diverse functional consequences of stimulation at periaqueductal gray sites. © 1995 Wiley-Liss, Inc.     

Further studies on interactions between periaqueductal gray, nucleus accumbens and habenula in antinociception.

Previous findings from this laboratory with the intracerebral microinjection technique suggested that the periaqueductal gray (PAG), nucleus accumbens, and habenula might constitute a unidirectional loop to play their roles in pain modulation. In the present study we demonstrate that intra-habenular injection of naloxone antagonizes the analgesia elicited by morphine injected into the periaqueductal gray (PAG) and that intra-accumbens injection of naloxone is capable of attenuating the analgesic effects of morphine injected into the habenula. These results indicate that the relationships between these nuclei may be more complex than the putative unidirectional loop.