Microinjection Of Morphine Research Articles

Morphine injected into the periaqueductal gray attenuates brain stimulation-induced effects: An intensity discrimination study

A discrimination situation was used to study the effects of morphine microinjected into the periaqueductal gray (PAG) on the aversive effects induced by PAG or medial hypothalamic (MH) electrical stimulation. Rats were trained in a T-maze to interrupt a high intensity (HI) stimulation inducing a short escape latency by pressing the lever (HI lever) located in one arm of the maze and a low intensity (LI) stimulation inducing a longer escape latency by pressing the lever (LI lever) located in the other arm. Microinjections of 15 nmol (5 micrograms) or 40 nmol (13 micrograms) of morphine both lengthened the escape latencies and shifted towards the LI lever the animal's choice in order to interrupt HI stimulations. This effect of morphine showed a similar time course as regards both escape latency and lever choice; it was more marked on PAG than on MH stimulation-induced aversive effects. The data are discussed in terms of morphine microinjections into PAG lengthening the escape latency by decreasing the aversiveness of PAG or MH stimulation rather than by affecting the animals' ability to respond to such stimulations.

Morphine microinjected into the periaqueductal gray has differential effects on 3 classes of medullary neurons

The effects of microinjection of 5–10 μg of morphine into the midbrain periaqueductal gray (PAG) on the activity of neurons in the rostral ventral medulla (RVM) were studied in lightly anesthetized rats. Based on the relationship between changes in neuronal activity and the occurrence of the tail-flick reflex (TF), RVM neurons were divided into 3 groups: off-cells, on-cells and neutral cells. The off-cells exhibited an abrupt pause and the on-cells an acceleration beginning just prior to the occurrence of the TF. Neutral cell firing did not change at the time of the TF. Microinjections of morphine into the PAG which inhibited the TF had differential effects on the spontaneous activity of the 3 groups of neurons in RVM. Off-cells showed an increase and on-cells a decrease in spontaneous activity which preceded the inhibition of the TF. These microinjections also reduced the TF-related responses of off- and on-cells. The effects on cell activity were reversed by systemically administered naloxone and were not seen following microinjections which failed to block the TF. Neutral cell activity was unchanged following microinjection of morphine into the PAG. These results support the hypothesis that off- and on-cells in the RVM mediate the effects of microinjection of morphine into the PAG on spinal nociceptive reflexes.

Stimulation-produced spinal inhibition from the midbrain in the rat is mediated by an excitatory amino acid neurotransmitter in the medial medulla

It has been previously established that a bulbar relay plays an important role in descending inhibition of spinal dorsal horn nociceptive neurons and nociceptive reflexes produced by stimulation in the midbrain periaqueductal gray (PAG). In the present study, selected receptor antagonists were microinjected into the medial medullary nucleus raphe magnus (NRM) to determine whether descending inhibition of the tail flick (TF) reflex in the rat produced by focal electrical stimulation in the midbrain PAG was mediated by serotonin, opioid, or glutamate receptors on bulbospinal neurons in the NRM. It was determined in initial experiments that the serotonin receptor antagonist methysergide, the opioid receptor antagonist naloxone, the local anesthetic lidocaine, and the glutamate receptor antagonists gamma-D-glutamylglycine (DGG) and DL-2-amino-5-phosphonovalerate (APV) microinjected into the medulla all significantly increased the threshold of focal electrical stimulation in the medulla required to inhibit the TF reflex. The antinociceptive efficacy of agonists at opioid, serotonin, and glutamate receptors was also tested in other experiments. The microinjection of morphine (2.5-10 micrograms) into the NRM increased significantly TF latencies in a dose-dependent manner in rats in the awake or lightly anesthetized state; morphine was more potent in awake rats. Inhibition of the TF reflex produced by the microinjection of morphine was reversed by a subsequent microinjection of naloxone into the same site in the medulla. The microinjection of serotonin (5 and 10 micrograms), however, did not affect the latency of the TF reflex in either awake or lightly anesthetized rats. Glutamate (100 microM, 0.5 microliter) microinjected into the rostral ventral medulla produced an inhibition of the TF reflex of short duration that could be blocked or attenuated significantly by the glutamate receptor antagonists DGG or APV microinjected into the same site. In subsequent experiments, a nonspecific functional block was introduced adjacent to the NRM bilaterally in the medullary reticular formations (MRFs) by the microinjection of the local anesthetic lidocaine; receptor antagonists were then microinjected into the NRM and their effect on the threshold of focal electrical stimulation in the PAG to inhibit the TF reflex determined. No increase was seen in stimulation thresholds in the PAG following the microinjection of either methysergide or naloxone into the NRM. Following the microinjection of lidocaine, DGG or APV into the NRM, the stimulation threshold in the PAG for inhibition of the TF reflex was increased significantly.(ABSTRACT TRUNCATED AT 400 WORDS)

III. Comparison of the antinociceptive action of Mu and delta opioid receptor ligands in the periaqueductal gray matter, medial and paramedial ventral medulla in the rat as studied by the microinjection technique

In rats stereotaxically implanted with microinjection cannula in either the periaqueductal gray matter (PAG) or the medial/paramedial medullary reticular formation (MRF), microinjection of morphine, sufentanil, d-Ala 2- d-Leu 5-enkephalin (DADL) or d-Ser 2- Thr 6-leucine enkephalin (DSTLE) produced dose-dependent elevations in the response latency on tail-flick and hot plate tests. These effects were reversed by naloxone administered by microinjection into the same intracerebral site. Both mu (morphine and sufentanil) and delta (DADL and DSTLE) opioid receptor ligands produced a maximal elevation in the supraspinally mediated hot plate response when administered into either the PAG or the MRF. Similarly, mu and delta receptor ligands produced maximum elevations in the spinally mediated tail-flick response when microinjected into the PAG. In contrast, delta, but not mu, receptor agonists produced a total blockade of the tail-flick response following administraion into the MRF. Microinjection of mu (morphine) or delta (DADL) agonists into the PAG or the MRF also resulted in a naloxone-reversible inhibition of the visceral chemical evoked writhing response. These observations suggest that mu and delta opioid receptor linked systems within the MRF but not the PAG produce their antinociceptive effects by discriminable mechanisms with a differential action on spinopetal vs supraspinal modulatory systems.

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

A mesolimbic neuronal loop of analgesia: I. Activation by morphine of a serotonergic pathway from periaqueductal gray to nucleus accumbens.

A cerebral neuronal circuit implicated in morphine analgesia was studied in rabbits equipped with chronic intracerebral cannulae directed to periaqueductal grey (PAG) and to bilateral nuclei accumbens. Microinjection of morphine (10 micrograms) into PAG elicited a marked analgesic effect as measured by the latency of a radiant heat induced head-withdrawal reaction. This effect was dose-dependently attenuated by cinanserin, a serotonin (5-HT) blocker, and potentiated by 5-hydroxytryptophan (5-HTP), the direct precursor for 5-HT, administered into the nucleus accumbens. A significant analgesia was also shown when 5-HT (10 micrograms) was injected directly into the nucleus accumbens. The results suggest that morphine acting on PAG seems to activate an ascending serotonergic pathway and, by releasing 5-HT in nucleus accumbens, to exert an analgesic effect.

II. 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 μg/0.5 μl) 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≫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>phentolamine>naloxone≫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>naloxone≫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: (1) other spinopetal pathways are operative; (2) that descending pathways activated by these manipulations do not contribute to the analgesic effects of brainstem morphine; and/or (3) that in addition to spinopetal modulation, brainstem opiate receptors modulate nociceptive transmission at the brainstem level.

Intestinal effect and analgesia: Evidence for different involvement of opioid receptor subtypes in periaqueductal gray matter

Periaqueductal gray matter (PAG) has been shown to be one of the sites in the central nervous system where microinjections of morphine strongly inhibit intestinal transit. To investigate the nature of opioid receptor populations involved in this central effect, selective opioid agonists, FK33824 for μ, DALA for σ, dynorphin for κ and tentatively β-endorphin for ϵ, were microinjected in all PAG areas previously identified as morphine-sensitive for intestinal inhibition. The PAG-induced inhibition of intestinal transit appears to be mediated mainly by μ receptors and possibly by ϵ receptors. κ and σ receptors seem not to be involved.

Morphine injected into the habenula and dorsal posteromedial thalamus produces analgesia in the formalin test

Microinjection of morphine into the area of the habenula and dorsal posteromedial thalamus (H-PMT) produces analgesia for tonic pain as measured by the formalin test in the rat. Control injections of morphine into sites near the H-PMT result in less or no reduction in pain, indicating that the analgesia observed is probably due to a site of action within the H-PMT rather than at surrounding neural structures. The analgesia is fully developed by the first time of testing, 10-16 min following the microinjection, and is completely reversible by naloxone, an opiate antagonist. The analgesia recorded is most likely due to morphine's action on the habenula, parafascicular or paraventricular nucleus of the thalamus, or a combination of these structures.

Microinjection of morphine into nucleus reticularis paragigantocellularis of the rat: Suppression of noxious-evoked activity of nucleus raphe magnus neurons

Microinjection of 1 microgram of morphine into nucleus reticularis paragigantocellularis (Pgc) of anesthetized rats depressed both noxious-evoked and spontaneous activity of nociresponsive neurons in the nucleus raphe magnus (NRM). This effect was naloxone-reversible, and was not observed after control injections dorsal to Pgc. The percent change in spontaneous firing was significantly greater than the percent change in pinch-evoked firing. This reduction in NRM neuronal discharge may contribute to the antinociceptive effects produced by microinjection of morphine into Pgc.

Morphine applied to the ventral tegmentum differentially affects centrally and peripherally induced aversive effects

In the rat, a microinjection of 15 nmoles of morphine into the ventral tegmentum (VT) was found to suppress escape responding induced by electrical stimulation applied to the dorsal part of the mesencephalic central gray. This suppressant effect (1) could be reversed by a systemic injection of naloxone and (2) was unlikely to be due to gross motor impairment since morphine injected into the VT provoked a behavioral activation. A similar microinjection of morphine into the VT did not induce analgesic effects since it did not affect the reaction thresholds to a nociceptive stimulus. The mechanisms underlying these differential effects of morphine applied to the VT are discussed.

Naloxone injections into the periaqueductal grey area and arcuate nucleus block analgesia in defeated mice.

In a situation of social conflict, mice that are defeated by an opponent exhibit a marked analgesia. Microinjections of naloxone (1 or 10 micrograms) into the periaqueductal grey area (PAG) or into the region of the arcuate nucleus prior to the defeat prevented the emergence of analgesia. Microinjections of morphine (5 micrograms) into these sites had previously been shown to produce profound analgesia. Mice whose adrenals were removed rapidly developed analgesia when attacked by a stimulus animal. Injection of naloxone into PAG also antagonized defeat-induced analgesia in adrenalectomized mice. These observations indicate that sites and processes in the brain rather than in the periphery are responsible for the development of analgesia in mice that are subjected to social defeat.

Opposite pupillary size effects in the cat and dog after microinjections of morphine, normorphine and clonidine in the Edinger-Westphal nucleus

The Edinger-Westphal complex (EW) was explored as a possible site of action for the effects of morphine and clonidine to produce mydriasis in the cat and miosis in the dog. Morphine, normorphine and clonidine, dissolved in 0.5 μl 0.9% NaCl, were injected via chronic indwelling cannulae into or near the EW of the restrained cat and dog. In the cat, all 3 drugs produced a dose-dependent mydriasis. Clonidine (3–30 nmol) was 1.7 times more potent than normorphine (3–30 nmol) and 9.6 times more potent than morphine (10–60 nmol). Normorphine was 5.5 times more potent than morphine. Significant miosis resulted from single doses of morphine (17.5 nmol), normorphine (15.5 nmol) and clonidine (19 nmol) injected in the EW of the dog. Injections sites closest to the EW yielded the greatest changes in pupillary diameter. Naloxone antagonized the pupillary effects of normorphine in the cat and dog but had no effect on clonidine mydriasis in one cat. It was concluded that the EW region is an important site of action for the effects of morphine and clonidine on pupil diameter in both species. However, the neurocircuitry and neurochemistry of the EW probably differ between the cat and dog.

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 μg/0.5 μl) into the periaqueductal gray resulted in an increase in the hot-plate and tail-flick response latency of the unanesthetized rat, the α-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>corynanthine. Given the reported affinity of these agonists for the α 2 (yohimbine/rauwolscine) and α 1 (corynanthine) receptors, these observations suggest that the spinopetal noradrenergic systems are acting on α 2-adrenergic receptors. Prazosin, an agent with several orders of magnitude higher affinity for the α 1than theα 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.

Inhibition in spinal cord of nociceptive information by electrical stimulation and morphine microinjection at identical sites in midbrain of the cat : J. Neurophysiol., 51 (1984) 75–89

The descending inhibition of noxious heat-evoked spinal neuronal excitation produced by morphine (MOR) and electrical brain stimulation (EBS) given at identical sites in the midbrain was quantitatively studied in the anesthetized cat. Fifty-two dorsal horn units driven by electrical stimulation of the posterior tibial and/or superficial peroneal nerves at A- and C-fiber strength and responding to noxious radiant heating (50 degrees C) of the skin of the foot-or toepads were studied. All units also responded to mechanical skin stimuli and were located primarily in laminae IV-VI of the dorsal horn. MOR (10-20 micrograms) was administered at 23 different sites in the midbrain. MOR attenuated the heat-evoked responses of 14 of 18 dorsal horn units studied to a mean 43% of the control heat-evoked response when administered at 18 sites in and immediately surrounding the periaqueductal gray (PAG). MOR administered at four sites in the PAG failed to significantly attenuate spinal nociceptive responses. The 14 sites where MOR was efficacious were distributed throughout the PAG. The efficacy of the MOR-produced inhibition was not correlated significantly to the distance from the cerebral aqueduct for the 18 PAG sites examined nor was there any difference in the spinal inhibitory effects of MOR, whether administered dorsally or ventrally in the PAG. MOR also failed to affect spinal neuronal heat-evoked responses (n = 5) when administered at five sites in the reticular formation ipsilateral to the PAG. EBS at the same 23 sites where MOR was given and at 34 additional sites in the midbrain attenuated the heat-evoked responses of all but one dorsal horn unit studied (mean maximal inhibition to 35% of control). In the PAG, stimulation ventrally inhibited a significantly greater proportion (13/21, 62%) of heat-evoked spinal neuronal responses to less than or equal to 25% of the control heat-evoked response than did stimulation in the dorsal PAG (5/15, 33%). The efficacy of the stimulation-produced inhibition was not, however, correlated significantly to the distance from the cerebral aqueduct for the total 57 midbrain sites examined, the 23 sites at which MOR was also tested, or only those EBS sites in and surrounding the PAG (n = 44).(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacology of spinal adrenergic systems which modulate spinal nociceptive processing

Spinopetal pathways may be activated by a variety of brainstem manipulations including microinjections of morphine which are known to modulate spinal nociceptive processing. Based on the ability of these manipulations to release spinal noradrenalin; the ability to reverse the antinociceptive effects by intrathecal adrenergic antagonists and the fact that intrathecal injections of noradrenalin mimic the antinociceptive effect, it appears that the descending modulation may be mediated by descending noradrenergic systems. Examination of the spinal receptor systems with intrathecally administered agents indicates that spinal alpha, but not beta adrenergic receptor agonists produce a powerful analgesia as measured on a variety of reflex and operant measures in mouse, rat, cat, primate and man. On the basis of agonist and antagonist structure-activity relationships it appears that a significant effect can be produced in the absence of any detectable effect on motor function by the occupation of spinal receptors. Distinguishable alpha 1 receptors also appear “analgetically-coupled,” but their effects are uniformly contaminated by signs of cutaneous hyperreflexia at doses required to produce analgesia. The ordering of potency with which intrathecal adrenergic antagonists reverse the effects of intrathecal noradrenalin is indistinguishable from that of the reversal by these intrathecal agents of the antinociceptive effects evoked by brainstem morphine. This suggests that the population of spinal receptors acted upon by exogenously administered adrenergic agonists and endogenously released noradrenalin have indistinguishable characteristics.

Analgesic effects of cyclazocine and morphine microinjected into the rat dorsomedial hypothalamus demonstrated by the bradykinin-induced flexor reflex test

The analgesic effect of cyclazocine microinjected into the diencephalon of rats was studied by using the bradykinin-induced flexor reflex test. The dorsal portion of the medial hypothalamic area was sensitive to cyclazocine with respect to the production of analgesia (ED 50 2.6 μg/rat). Microinjections of morphine into the same portion also produced a dose-dependent analgesic effect (ED 50 2.5 μg/rat). As naloxone (0.9 mg/kg s.c.) antagonized both these analgesic effects, the findings suggest that the dorsal portion of the medial hypothalamic area of the rat is one of the primary target sites of analgesix drugs.

Specific opioid-amphetamine interactions in the caudate putamen.

Bilateral microinjection of morphine (0.003-3 micrograms/side) into the caudate putamen enhances the behavior induced by the IP injection of 1 mg/kg d-amphetamine phosphate in a dose-related manner. The duration of activity was prolonged and ambulation was changed to d-amphetamine stereotypy, a behavior normally associated with higher doses of d-amphetamine. The opioid activity was stereospecific in that levorphanol was active, whereas dextrorphan was not. The enhancement of d-amphetamine-induced behavior by the opioids was blocked by naloxone. D-ala2-met-Enkephalin also enhanced the amphetamine-induced behavior. This enhancement appears to be specific to the caudate putamen because the oral stereotypy observed appears to be a unique action of amphetamine in this region of the brain.

The biochemical and behavioral effects of phospholipase A 2 and morphine microinjections in the periaqueductal gray of the rat

In order to characterize the in vivo action of phospholipase A 2 (PLA 2) on opiate receptors and opiate-induced behaviors, the effects of injections of PLA 2 into the periaqueductal gray region (PAG) of the rat were assessed on free fatty acid (FFA) release, opiate-binding levels, and morphine-induced behaviors. Rats received bilateral PAG injections of 2 μg of PLA 2 while anesthetized. One hour later, regions around the cannulae tracts in PLA 2-treated rats contained over 2.5 times more FFA than saline-injected controls, and 3H-dihydromorphine binding was reduced on average more than 70%. In another series of experiments, conscious rats were given 2 μg of PLA 2 prior to 10 μg of morphine through cannulae chronically implanted into the PAG. PLA 2 did not significantly attenuate morphine-induced analgesia as measured by the tail-flick test to radiant heat, but did prevent the explosive motor behavior observed following morphine injections alone. PLA 2 by itself did not induce analgesia, but did cause explosive motor behavior 2 hr after the injections. Neither lysophosphatidylcholine nor trypsin resulted in motor seizures following PAG injections. It was concluded that the behavioral effects of PLA 2 result from the unique properties of the enzyme, rather than generalized membrane damage, and that the opioid sites and mechanisms that mediate analgesia are different from those associated with explosive motor behavior.

Effect of spinal cord lesions on convulsive activity induced by intrathecal morphine

Non-specific convulsive behavior induced by intrathecal (IT) morphine microinjection appears to be tonically modulated by centrifugal pathways originating within the brain since the frequency of IT morphine-induced hindlimb seizures and myoclonic twitches are both increased following spinal transection. These effects on convulsant activity are dissociable by selective neural lesions. Bilateral dorsolateral funiculus lesions potentiate seizure activity whereas ventral funiculus lesions and, to a lesser extent, interruption of cerebral cortical influences enhance myoclinic twitch activity. These initial studies indicate that distinct supraspinal centers differentially modulate myoclinic and seizure activity and suggest that an understanding of these systems may have clinical implications for the control of convulsant activity.