The effect of different lesions of the median raphe on morphine analgesia

The effect of different manipulations of the nucleus medianus raphe (MR) on morphine analgesia was investigated in rats using the tail-immersion test. Electrolytic lesions of this structure antagonized morphine analgesia, while injections of 5,7-dihydroxytryptamine (to destroy serotonergic neurons) or ibotenic acid (to destroy cell bodies) in the medianus raphe did not alter the effect of morphine. Injection of naloxone (0.5 and 0.1 micrograms) in the MR antagonized morphine analgesia. These results suggest the importance of this structure for morphine analgesia in this test, although the substrates within the nucleus that mediate this action are still unknown.     

The periaqueductal gray-raphe magnus projection contains somatostatin, neurotensin and serotonin but not cholecystokinin

The retrograde transport-HRP-immunocytochemical technique was employed to ascertain if the periaqueductal gray-raphe magnus projection arises from neurons containing somatostatin, neurotensin, serotonin or cholecystokinin. Following HRP injections into the raphe magnus (NRM) double-labeled cells containing HRP reaction product and somatostatin-, neurotensin- or serotonin-like immunoreactivity were identified in the midbrain periaqueductal gray (PAG). No cholecystokinin-like immunoreactive double-labeled neurons were found in the PAG. These results indicate that the PAG-NRM pathway contains somatostatin, neurotensin and serotonin but not cholecystokinin.     

Separate involvement of the spinal noradrenergic and serotonergic systems in morphine analgesia: the differences in mechanical and thermal algesic tests

Experiments using 3 analgesic tests, the tail-pinch, hot-plate and tail-flick methods, were done to evaluate the roles of the spinal noradrenergic and serotonergic systems in the production of morphine analgesia in rats. To deplete noradrenaline or serotonin in the spinal cord, 6-hydroxydopamine or 5,6-dihydroxytryptamine was given intrathecally. 6-Hydroxydopamine suppressed the antinociceptive effects of morphine injected systemically or intracerebrally (into the nuclei reticularis gigantocellularis and paragigantocellularis or into the periaqueductal gray matter) in the tail-pinch test, but not significantly in the hot-plate and tail-flick tests. Conversely, 5,6-dihydroxytryptamine suppressed the antinociceptive effects of systemically given morphine in the hot-plate test, but not significantly in the tail-pinch and the tail-flick tests. The results not only provide further evidence for the involvement of the descending inhibitory systems in morphine antinociception, but also show that the extent of participation of the spinal noradrenergic and serotonergic systems in the effects of morphine has to be carefully assessed as different analgesic tests (tail-pinch, tail-flick and hot-plate) yield different results.     

Opiate-mediated inhibition of the release of cholecystokinin and substance P, but not neurotensin from cat hypothalamic slices

The neuroactive peptides neurotensin (NT), substance P (SP) and cholecystokinin (CCK) have been shown to be distributed in the hypothalamus. These peptides may be part of hypothalamic mechanisms which regulate the release of pituitary hormones and feeding behavior. Numerous experiments have demonstrated opiate modulation of anterior pituitary hormone release. These effects have been reported to be mediated via a hypothalamic mechanism, which modulates the secretion of releasing, release inhibiting factors or other neuroactive peptides such as SP, CCK and NT. We have examined the effects of morphine on the potassium-stimulated, calcium-dependent release of SP, CCK and NT from cat hypothalamic slices. The potassium-stimulated release of SP and CCK was profoundly depressed by the addition of morphine (10⁻⁵ M) in a naloxone-reversible manner. This morphine inhibition was shown to be stereospecific, levorphanol (10⁻⁷ M) depressed the release, while dextrophan (10⁻⁷ M) was inactive. Gel filtration chromatography of the potassium-stimulated release was determined to be isographic with authentic NT, SP and CCK-8, respectively. There was no indication of any gastrin-like activity. These data may suggest a regulatory mechanism through which opiates exert some of their neuroendocrine or feeding regulatory effects.     

The relationship between morphine analgesia and the activity of bulbo-spinal serotonergic system as studied by tolerance phenomenon

The effect of various doses of acute morphine on both analgesia and 5-hydroxytryptamine (5-HT) synthesis in the brain and the spinal cord has been studied in rats rendered tolerant by chronic administration of the analgesic. In morphine-tolerant rats, the incorporation of tritiated-L-tryptophan (TRP) in the brain and the spinal cord was higher than in non-tolerant rats, but there was no significant difference in the synthesis rate of the newly formed 5-HT between the two groups. An acute dose of morphine (10 mg/kg) which induced a powerful analgesia and a large increase in 5-HT synthesis in non-tolerant rats, did not produce analgesia nor changes in 5-HT synthesis in tolerant rats. Higher acute doses of morphine which restored analgesia in tolerant rats, induced a discrete increase in [3H]TRP incorporation and a marked increase in 5-HT synthesis in the spinal cord of these animals. The same doses significantly increased [3H]TRP incorporation in the forebrain but did not modify 5-HT synthesis. These results show that tolerance to morphine is associated with a decrease in the effects of the drug on 5-HT synthesis in the spinal cord and the brain and tend further support to the hypothesis that an enhancement of 5-HT synthesis in the spinal cord, induced independently of modifications of the availability of TRP, is associated with the analgesic effect of morphine.     

Possible involvement of the amydaloid complex in morphine analgesia as studied by electrolytic lesions in rats

The analgesic effects of morphine (5 mg/kg i.p.) were studied in biamygalectomized rats. (1) Using the tail-flick test neither withdrawal latencies nor morphine time-course and efficacy were affected by the lesions. (2) The threshold for vocalization to electrical stimulation of the tail was greatly increased in lesioned rats; however, statistical analysis revealed no significant change in the analgesic efficacy of morphine.     

Potentiation of morphine analgesia by the cholecystokinin antagonist proglumide

Recent evidence has suggested that cholecystokinin (CCK) may act as a physiological opiate antagonist. Both the overlap of CCK and opiate systems within the central nervous system and the fact that exogenous CCK can antagonize opiate analgesia suggest that endogenous CCK systems interact with opiate-mediated pain inhibitory systems. In the present series of experiments, we examined the effect of the CCK receptor antagonist proglumide on various forms of morphine analgesia. We have observed that proglumide can potentiate morphine analgesia following systemic, intrathecal or intracerebral administration of these drugs. Endogenous CCK systems do not appear to be tonically active since neither systemic, intrathecal nor intracerebral proglumide typically produced measurable analgesia in the absence of morphine. These data suggest that CCK may be released in response to opiate administration and acts to return the organism toward its basal level of pain sensitivity. If such a hypothesis is in fact true, then CCK blockade may be of clinical value in the treatment of pain.     

Unilateral analgesia produced by intraventricular morphine

Morphine injected into the lateral ventricle of the rat produced unilateral analgesia in the formalin test, which involves continuous, moderated pain. In contrast, analgesia was produced bilaterally in the foot-flick test which involves brief, rapidly rising pain. In the formalin test, intraventricular morphine produced analgesia in the ipsilateral but not the contralateral hindpaw. Analgesia was achieved with relatively low doses of morphine (2.5–10.0 μg) in the formalin test while very high doses (50–200 μg) were necessary to produce analgesia in the foot-flick test. These results add to other data indicating that different neural mechanisms underlie opiate analgesia in different types of pain. Moreover, they indicate that, in the formalin test, the neural mechanisms of morphine analgesia are somatotopically organized and that forebrain structures are likely to be involved.     

Comparison of the effects of ventral medullary lesions on systemic and microinjection morphine analgesia

The effects of electrolytic lesions of the nucleus raphe magnus (NRM), nucleus reticularis paragigantocellularis (PGC) and nucleus raphe alatus (NRA) on analgesia elicited in the rat from systemic morphine and morphine microinjection into the periaqueductal gray (PAG) were evaluated using the tail flick test. No consistent change in baseline pain sensitivity was observed following lesions of the NRM, PGC or NRA. To determine the effect of ventral medullary lesions on systemic porphine analgesia, pain sensitivity was assessed prior to and 40 min after 6 mg/kg morphine administration (i.p.) at 2 days preceding lesioning and 5, 12 and 19 days post-lesion. NRM and PGC lesions produced only slight reductions in analgesia at 5 days after surgery. It was observed that large NRM, large PGC, and NRA lesions significantly attenuated analgesia evaluated at 12 days post-lesion. Smaller lesions confined within the NRM or PGC were reliably less effective than the larger lesions in reducing analgesia. In a subsequent study, 5 μg morphine in 0.5 μl saline was microinjected into the ventral PAG at the level of the dorsal raphe. Identical testing procedures were used and the analgesia was assessed at 2 days before lesioning and 5 and 12 days post-lesion. In contrast to the previous study, large NRM lesions abolished analgesia as early as 5 days following lesioning. Small NRM lesions were less effective and PGC lesions were generally ineffective in attenuating analgesia induced by morphine microinjection. We conclude that the NRA may act as a functional unit in the mediation of systemic morphine analgesia. In contrast, analgesia elicited from intracerebral (PAG) morphine microinjection is mediated via the NRM.     

Stress-induced analgesia in frogs: evidence for the involvement of an opioid system

Immobilization of frogs for 1 h induces analgesia which is blocked but not reversed by low doses of naloxone. After 9 days of daily immobilization for 1 h, the treatment fails to cause analgesia thus indicating that tolerance has developed. Animals tolerant to i immobilization-induced analgesia do not show cross-tolerance to the analgesic action of morphine. The development of tolerance to this form of stress-induced analgesia and the ability of naloxone to prevent its occurrence indicate the involvement of opioid pathways. The lack of cross tolerance to morphine suggests that μ receptors are not involved.     

Effect of DLF lesions at different spinal levels on morphine induced analgesia

The effect of morphine on tail flick latency was tested in 3 groups of rats. It is shown that in normal rats and in rats with a bilateral DLF lesion at Th10 or lower spinal root segments, morphine increased the tail flick latency. After a bilateral DLF lesion at Th9 or higher segments, morphine did not change the tail flick latency.     

Brainstem lesions dissociate neural mechanisms of morphine analgesia in different kinds of pain

The effects of brainstem lesions on morphine analgesia were examined using the formalin test which produces moderate pain that lasts about 2 h, and the tail-flick test which measures brief threshold-level pain. Lesions of the nucleus raphe magnus attenuated and small lesions of the central tegmental nucleus potentiated the effects of morphine in the tail-flick test. Lesions of the median raphe nucleus potentiated the effects of morphine in the formalin test. Large lesins of the pontine reticular formation had no effect in either pain test. These results indicate that the neural mechanisms underlying morphine analgesia are different in different kinds of pain.     

Evidence for the independence of brainstem mechanisms mediating analgesia induced by morphine and two forms of stress

Exposure to inescapable footshock stress causes potent analgesia in the rat. According to several criteria, prolonged, intermitent footshock elicits analgesia mediated by opioid peptides, whereas brief, continuous footshock produces non-opioid analgesia. We now report that these neurochemically discrete forms of stress analgesia also have different neuroanatomical bases. Electrolytic lesions damaging greater than 85% of the n. raphe magnus (‘complete’ NRM lesions), but not lesions of the same size causing less NRM damage (partial NRM lesions) significantly reduce only the non-opioid form of stress analgesia. In the same animals, complete and partial NRM lesions disrupt morphine analgesia; however, our analyses indicate that this effect is not mediated b by the same substrate involved in either form of stress analgesia. These results support the existence of multiple endogenous analgesia mechanisms and indicate a complex role of the NRM in these systems.     

Blocking the R-type (Cav2.3) Ca2+ channel enhanced morphine analgesia and reduced morphine tolerance

Morphine is the drug of choice to treat intractable pain, although prolonged administration often causes undesirable side-effects including analgesic tolerance. It is speculated that voltage-dependent Ca(2+) channels (VDCCs) play a key role in morphine analgesia and tolerance. To examine the subtype specificity of VDCCs in these processes, we analysed mice lacking N-type (Ca(v)2.2) or R-type (Ca(v)2.3) VDCCs. Systemic morphine administration or exposure to warm water swim-stress, known to induce endogenous opioid release, resulted in greater analgesia in Ca(v)2.3(-/-) mice than in controls. Moreover, Ca(v)2.3(-/-) mice showed resistance to morphine tolerance. In contrast, Ca(v)2.2(-/-) mice showed similar levels of analgesia and tolerance to control mice. Intracerebroventricular (i.c.v.) but not intrathecal (i.t.) administration of morphine reproduced the result of systemic morphine in Ca(v)2.3(-/-) mice. Furthermore, i.c.v. administration of an R-type channel blocker potentiated morphine analgesia in wild-type mice. Thus, the inhibition of R-type Ca(2+) current could lead to high-efficiency opioid therapy without tolerance.     

Magnetic field inhibition of morphine-induced analgesia and behavioral activity in mice: Evidence for involvement of calcium ions

An exposure for 60 min to a 0.5 Hz rotating magnetic field (1.5-90 G) significantly reduced the day-time analgesic (in CF-1 mice) and locomotory (in C-57BL mice) effects of morphine (10 mg/kg). Intracerebroventricular (i.c.v.) injections of a calcium chelator, EGTA, blocked these effects, while administration of the calcium ionophore, A23187, potentiated the inhibitory actions. In a parallel fashion, i.c.v. administration of Ca2+ reduced, in a dose-related manner, the analgesic and locomotory effects of morphine in control CF-1 and C57 mice. These latter inhibitory effects could also be blocked by EGTA and augmented by A23187, indicating that opiate effects on activity and nociception are both sensitive to antagonism by calcium. Taken together these results suggest that exposure to magnetic stimuli may alter morphine-induced responses in mice, in a manner compatible and consistent with effects on Ca2+ and possibly other divalent ions.     

Post-stress facilitation of serotonergic, but not noradrenergic, neurotransmission in the dorsal hippocampus prevents learned helplessness development in rats

Recent pieces of evidence suggest that the dorsal hippocampus may mediate adaptation to severe and inescapable stress, possibly by the facilitation of serotonergic and/or noradrenergic neurotransmission. Chronic social stress and high corticosteroid levels would impair this coping mechanism, predisposing animals to learned helplessness. To test the hypothesis that increasing serotonin or noradrenaline levels in the dorsal hippocampus would attenuate the development of learned helplessness (LH), rats received inescapable foot shock (IS) and were tested in a shuttle box 24 h latter. Prestressed animals showed impairment of escape responses. This effect was prevented by bilateral intrahippocampal injections of zimelidine (100 nmol/0.5 microl), a serotonin reuptake blocker, immediately after IS. This effect was not observed when zimelidine was administered before or 2 h after IS. Bilateral intrahippocampal injections of desipramine (3 or 30 nmol/0.5 microl), a noradrenaline reuptake blocker, before IS or immediately after it did not prevent LH development. Desipramine (30 nmol) enhanced LH development when injected before IS. These data suggest that poststress facilitation of hippocampal serotonergic, but not noradrenergic, neurotransmission in the dorsal hippocampus facilitates adaptation to severe inescapable stress. Antidepressant effects of noradrenaline-selective drugs seem to depend on other structures than the dorsal hippocampus.     

Single nerve capsaicin: Effects on pain and morphine analgesia in the formalin and foot-flick tests

Application of capsaicin to the sciatic nerve reduces responsiveness to pain in the foot-flick test which examines bried, threshold-level pain. The purpose of the present study was to determine if a similar reduction occurs in the formalin test which examines supra-threshold, deep pain that persists for several hours.The sciatic nerve on one side in the rat was exposed and soaked for 15 min in a solution of capsaicin and the saphenous nerve was ligated and cut. The operated foot was tested for sensitivity to pain in the formalin and foot-flick tests 2 days to 12 weeks later both with and without morphine. The capsaicin treatment produced a substantial reduction in sensitivity to foot-flick heat pain at all times after surgery. In the formalin test, the effects were small and tended to suggest that the rats felt more rather than less pain. The capsaicin treatment markedly reduced the sensitivity of formalin test pain to morphine. This effect appeared about one week after surgery and persisted for 12 weeks. The results suggest that capsaicin-sensitive unmyelinated afferents play a role in the threshold-level, non-damaging heat pain, but are not involved in pain resulting from tissue damage. However, these afferents appear to be important for the spinal action of morphine on this type of pain.     

Possible mechanisms of morphine analgesia.

The body has an endogenous analgesic system that prevents excess pain from interfering with the normal body functions. Depression of pain sensations occurs within the dorsal horn of the spinal cord where the primary pain fibers, which transmit pain sensations from the periphery, synapse with neurons that transmit pain to the higher centers. There appear to be two mechanisms by which the transmission of pain sensations are depressed; these include hyperpolarization of interneurons within the dorsal cord and depressing the release of the neurotransmitters associated with pain transmission. Activation of the analgesic mechanisms results from an interaction between specific neurotransmitters, such as enkephalin, serotonin, or norepinephrine, and specific receptors located on the neurons that transmit pain. The spinal analgesic mechanisms can be activated by either pain or nonpainful sensations arriving from the periphery or by supraspinal mechanisms. The supraspinal mechanisms originate in specific structures within the brainstem that include the periaqueductal gray matter, locus ceruleus, and nuclei in the medulla. These systems are activated either by ascending pain impulses or by higher centers such as the cortex or hypothalamus that, in turn, activate the spinal analgesic systems. There are three systems associated with activation of the supraspinal mechanisms. These include the opioid system associated with the release of the endorphins, the adrenergic system associated with the release of norepinephrine, and the serotonergic system associated with the release of serotonin. The interaction between these systems activates the spinal analgesic system. When the endogenous analgesic systems fail to control pain, analgesic drugs can be used to enhance the endogenous systems. Opiate drugs, such as morphine, interact with opioid receptors and produce analgesia by the same mechanisms as enkephalin, i.e., hyperpolarization of interneurons and depressing the release of transmitters associated with transmission of pain. In addition, morphine can interact with opioid receptors located in the supraspinal structures and activate the supraspinal system. Adrenergic drugs that interact with specific receptors also produce analgesia and it has been suggested that morphine interacts with the adrenergic system to produce analgesia.     

Activation of the central serotonergic system in response to delayed but not omitted rewards

The forebrain serotonergic system is a crucial component in the control of impulsive behaviours. However, there is no direct evidence for natural serotonin activity during behaviours for delayed rewards as opposed to immediate rewards. Herein we show that serotonin efflux is enhanced while rats perform a task that requires waiting for a delayed reward. We simultaneously measured the levels of serotonin and dopamine in the dorsal raphe nucleus using in vivo microdialysis. Rats performed a sequential food-water navigation task under three reward conditions: immediate, delayed and intermittent. During the delayed reward condition, in which the rat had to wait for up to 4 s at the reward sites, the level of serotonin was significantly higher than that during the immediate reward condition, whereas the level of dopamine did not change significantly. By contrast, during the intermittent reward condition, in which food was given on only about one-third of the site visits, the level of dopamine was lower than that during the immediate reward condition, whereas the level of serotonin did not change significantly. Dopamine efflux, but not serotonin efflux, was positively correlated with reward consumption during the task. There was no reciprocal relationship between serotonin and dopamine. This is the first direct evidence that activation of the serotonergic system occurs specifically in relation to waiting for a delayed reward.