Microinjection Of Morphine Research Articles

Wild running elicited by microinjections of bicuculline or morphine into the inferior colliculus of rats: Lack of effect of periaqueductal gray lesions

Bicuculline methiodide, a GABA A receptor antagonist, or a high dose of morphine was injected at the same site within the inferior colliculus (IC) of rats. Both drugs elicited the same behavioral activity (wild running). However, the time course and magnitude of the effects of the two drugs differed. Since the behavioral activation elicited was reminiscent of what was found with microinjections of bicuculline methiodide or morphine into the periaqueductal gray (PAG), we lesioned the PAG in another group of rats. It was found that extensive lesions of the PAG including those extending to the medial part of the superior colliculus did not significantly reduce the wild running.

Circuitry linking opioid-sensitive nociceptive modulatory systems in periaqueductal gray and spinal cord with rostral ventromedial medulla

The interactions among opioid-sensitive nociceptive modulatory systems, which include the midbrain periaqueductal gray, rostral ventromedial medulla and spinal cord, are likely to play a central role in the potent antinociception that results when morphine is administered systemically. The aim of the present study was to investigate the mechanisms through which local application of morphine, either in the periaqueductal gray or at the lumbar spinal cord in the rat, influences the activity of one population of putative nociceptive modulatory neurons in rostral ventromedial medulla, i.e. “on-cells”. Previous studies have shown that the spontaneous and tail-flick-related firing of on-cells is invariably depressed when morphine is given systemically in doses demonstrated to inhibit the tail-flick reflex, and that a similar depression of this activity is produced when morphine is applied directly in the periaqueductal gray or intrathecal space. In the present experiments, on-cells were activated pharmacologically using iontophoretically applied glutamate to provide an indication of whether morphine-induced suppression of on-cell firing reflected a postsynaptic inhibition or a disfacilitation resulting from blockade of an excitatory input to the on-cell. Microinjection of morphine into the periaqueductal gray blocked glutamate-evoked activity of on-cells in parallel with its suppression of the tail-flick reflex, suggesting activation of an inhibitory input to these cells. No change in glutamate-evoked activity occurred in rats in which morphine did not produce antinociception. Intrathecal administration of morphine did not alter the glutamate-evoked activity of these neurons despite blocking the tail-flick reflex, suggesting that morphine acting in the spinal cord removes an excitatory input to on-cells. Thus, in addition to possible direct effects of morphine on rostral ventromedial medulla on-cells, morphine administered systemically will simultaneously engage inhibitory influences from the periaqueductal gray and remove facilitating inputs from the spinal cord. Each of these mechanisms is likely to contribute to the potent antinociception that results when morphine is administered systemically.

Naloxone blocks opioid peptide release in periaqueductal gray and amygdala elicited by morphine injected into N. accumbens

Previous studies from this laboratory suggested that the periaqueductal gray (PAG), nucleus accumbens, and amygdala might take part in a serial, unidirectional mesolimbic loop to play their roles in pain modulation. It has been proposed that morphine injected into one of these nuclei would cause the release of opioid peptides in one nucleus after another. This working hypothesis was examined in the present study by perfusing simultaneously the PAG and the amygdala after microinjection of morphine into the N. accumbens. It was found that microinjection of morphine increased the content of immunoreactive enkephalins (ir-ENK) and immunoreactive β-endorphin (ir-β-EP) in the perfusate of the PAG and the amygdala. When the perfusion fluid contained 3 μM of naloxone, the increase of ir-ENK and ir-β-EP was reduced significantly. These results indicate that the three nuclei were not serially connected in a unidirectional loop.

Naloxone blocks opioid peptide release in N. Accumbens and amygdala elicited by morphine injected into periaqueductal gray

It has been proposed that a serial, unidirectional circuit from the PAG to the N. accumbens and the amygdala are involved in antinociception and that enkephalins (ENK) and β-endorphin (β-EP) act as neurotansmitters in this circuitry. In the present study, we measured the release of ENK and β-EP by simultaneous push-pull perfusion of the N. accumbens and amygdala after microinjection of morphine into the PAG. Morphine administration elicited an increase in immunoreactive ENK and β-EP in both the N. accumbens and the amygdala, which was antagonized in each nucleus by perfusion with naloxone. These data suggest that the three nuclei were not serially connected and that they may take part in one and the same antinociceptive system with an “all or none” character.

Inhibition of mesencephalic morphine analgesia by methysergide in the medial ventral medulla of rats

The neural substrates of endogenous supraspinal opioid pain inhibition are mediated in part by connections between the midbrain periaqueductal gray (PAG) and the ventral-medial medulla, including the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis (NRGC). To ascertain whether a serotonergic synapse participated in this pathway, the present study determined whether microinjections of methysergide into the NRM or NRGC would alter analgesia elicited by morphine microinjections into the PAG. Morphine (2.5 μg) in the PAG and immediately adjacent areas produced significant analgesia on the tail-flick and jump tests in rats. Pretreatment with the serotonin receptor antagonist methysergide (0.5–5 μg) in either the NRM or NRGC significantly reduced morphine analgesia elicited from the PAG by 69% on the tail-flick and by 50% on the jump tests without altering basal nociceptive thresholds. Medullary placements ventral or lateral to the NRM/NRGC failed to support this antagonistic effect. These data indicate that a ventro-medial medullary serotonergic synapse participates in the transmission of opioid pain-inhibitory signals from the PAG.

Neurochemical studies on the mesolimbic circuitry of antinociception

Previous studies using the technique of microinjection into brain nuclei indicated that the periaqueductal gray (PAG), nucleus accumbens, habenula and amygdala play an essential role in pain modulation and that these nuclei possibly act through a ‘mesolimbic neural loop‘ to exert an analgesic effect, in which Met-enkephalin (MEK) and β-endorphin (β-EP) have been implicated as the two major opioid peptides involved in antinociception. In the present study performed in rabbits, intracranial microinjection was supplemented with push-pull perfusion and radioimmunoassay to determine whether the release of enkephalins (ENK) and β-EP was increased in these nuclei when the putative neural circuit was activated by morphine administered into one of the nuclei. The results showed: (1) microinjection of morphine into the PAG increased the release of ENK and β-EP in the N. accumbens, and vice versa; (2) microinjection of morphine into the N. accumbens increased the release of ENK and β-EP in the amygdala, and vice versa; (3) morphine microinjected into the PAG caused an increase in the release of ENK and β-EP in the amygdala and vice versa, although the release of ENK in PAG was statistically not significant. These results indicate that PAG, N. accumbens and amygdala are connected in a network served by a positive feedback circuitry.

Naloxone blocks the release of opioid peptides in periaqueductal gray and N. accumbens induced by intra-amygdaloid injection of morphine

The working hypothesis that the periaqueductal gray (PAG), N. accumbens and amygdala were connected serially in a unidirectional loop for antinociception, in which Met-enkephalin and β-endorphin were considered to be two important analgesic neurotransmitters, was examined by simultaneously perfusing the PAG and N. accumbens after microinjection of morphine into the amygdala. Intra-amygdaloid injection of morphine increased the release of enkephalins and β-endorphin in the PAG and N. accumbens. When the perfusion fluid contained 3 μM of naloxone, the release of enkephalins and β-endorphin was reduced in both the PAG and the N. accumbens. These results do not support the hypothesis of a unidirectional loop and its putative sequence.

Dopamine and conditioned reinforcement

It has been shown that infusion of certain neuropeptides into the ventral tegmental area (VTA) results in increased motor activity and enhanced dopamine turnover in the nucleus accumbens. In the present experiments, substance P (SP), neurotensin (NT), d-ala-metenkephalin (DALA) and morphine sulfate (MS) were injected bilaterally into the VTA and their effects on conditioned reinforcement were assessed. These effects were compared with infusion of amphetamine into the nucleus accumbens, which has previously been shown to strongly enhance responding for conditioned reinforcers. For these experiments, hungry rats were trained to associate a compound stimulus (light and click) with the presentation of food. In the test phase, responding on one lever (CR lever) resulted in the presentation of the stimulus but no food. Responding on the other (NCR lever) had no consequences. Different groups of animals received microinjections (0.5 microliter, bilaterally) of SP (0, 0.03, 0.3, 3.0 micrograms), NT (0, 0.025, 0.25, 0.5 microgram), DALA (0, 0.01, 0.1, 1.0 microgram) or morphine (0, 0.025, 0.25, 2.5 micrograms) into the VTA. SP infusion into the VTA resulted in a small increase in responding which was not selective for the CR lever. NT, DALA and morphine had no effect on responding for conditioned reward. In contrast, amphetamine (0, 0.2, 2.0, 20 micrograms) injected into the nucleus accumbens markedly enhanced responding for conditioned reward. These findings suggest that stimulation of the mesolimbic system at the level of the DA cell bodies, which induces a small increase in DA turnover, is not sufficient to potentiate responding for conditioned reward. On the other hand, an important requirement for potentiation may be excessive release of dopamine in the nucleus accumbens.

Effect of microinjection of morphine and tramadol into the locus coeruleus on nociceptive responses of spinal neurons and arterial pressure changes

Effect of microinjection of morphine and tramadol into the locus coeruleus on nociceptive responses of spinal neurons and arterial pressure changes

Effects of morphine and midazolam on reactivity to peripheral noxious and central aversive stimuli

Electrical stimulation of the mesencephalic tectum elicits behavioral and autonomic responses similar to those following peripheral noxious stimulation. Benzodiazepine and opioid compounds attenuate escape behavior induced by electrical stimulation of the dorsal periaqueductal gray (PAG) and deep layers of the superior colliculus (SC). The present study determines if microinjections of midazolam and morphine applied to these PAG-SC sites affect both responsiveness to peripheral noxious stimulation and to aversive PAG-SC stimulation. Both aversive brain stimulation or foot-shocks applied at threshold intensities caused running or jumps concomitant with increases in mean arterial blood pressure (MBP) and heart rate (HR). Microinjection of both drugs attenuated the behavioral reaction and increases in MBP and HR induced by mesencephalic tectum stimulation, while attenuating only the increase in heart rate induced by peripheral painful stimulation. These results suggest that the neural substrates of the behavioral and autonomic effects of stimulating the mesencephalic tectum and peripheral nociceptors are different although they may partially overlap.

Suppression of a hind limb flexion withdrawal reflex by microinjection of glutamate or morphine into the periaqueductal gray in the rat

Microinjection into the midbrain periaqueductal gray (PAG) or lateral reticular formation (LRF) of the neuronal excitant glutamate produces analgesia, and suppresses the responses of a fraction of spinal dorsal horn neurons to noxious heat applied to ventral hind paw skin. Microinjection of morphine into the PAG also produces analgesia, but has been reported to frequently facilitate, as well as to suppress or have no effect, on nociceptive spinal neurons. In anesthetized rats, we tested whether (a) glutamate microinjections into PAG or LRF, and (b) morphine microinjections into PAG, affected the isometric force of hind limb withdrawal elicited by the same noxious heat stimuli on the hind paw as used in single-unit studies of dorsal horn neurons. Glutamate (0.5 M; 0.1–0.5 μl) microinjected at 9 12 PAG and 8 10 LRF sites suppressed the reflex, and had no effect or facilitated th from the remaining sites. Morphine (5 μg in 0.5 μl) microinjected at each of 10 PAG sites suppressed the reflex in a naloxone-reversible manner. Suppression usually began shortly after morphine, peaked at 20–40 min, and lasted > 60 min. The integrated flexion reflex thus appears to be more susceptible to chemical midbrain stimulation under these experimental conditions, compared to previous studies of single dorsal horn neurons.

Blockade of morphine analgesia by both pertussis and cholera toxins in the periaqueductal gray and locus coeruleus

Rats demonstrating analgesia following microinjection of morphine into the periaqueductal gray (PAG) or locus coeruleus (LC) were injected with either pertussis toxin, cholera toxin or saline into the same brain region. Both pertussis and cholera toxin blocked the analgesic effect of morphine at both injection sites for up to 7 days after toxin treatment. These results indicate that morphine analgesia is a complex response involving systems dependent upon G s as well as G i of G o proteins.

Further studies of the effects of intranigral morphine on behavioral responses to noxious stimuli

Bilateral intranigral microinjection of morphine produces dose-related and naloxone reversible analgesic-like effects on the hot-plate and tail-flick tests. The main objectives of the present studies were to further characterize the analgesic-like effects of intranigral morphine, to determine whether these effects were related to a general impairment of sensory or motor function, and to assess their anatomical specificity. The principal findings are: (1) intranigral morphine (10 μg) suppresses pain-related behavior without altering responses to a variety of non-noxious auditory, visual, and somatic stimuli, and without producing motor impairment; (2) movement of injector needles approximately 1 mm rostral, dorsal, or medial to the active nigral site significantly reduces the analgesic-like effect of morphine on the tail-flick test; and (3) electrolytic lesions confined to the nigra significantly reduced the analgesic-like effect of morphine on the hot-plate test. It is concluded that the analgesic-like effects of intranigral morphine are mediated by the substantia nigra and that these effects are specifically related to pain.

The role of glutamate in opiate descending inhibition of nociceptive spinal reflexes

The present experiment examined descending inhibition of the noniceptive tail-flick reflex produced by microinjection of morphine and glutamate into the periaqueductal gray (PAG) matter and the neurotrasmitters mediating the inhibition at the level of the nucleus raphe magnus (NRM). The longlasting opiate analgesia was significantly reduced by microinjection of excitatory amino acid antagonists 1-( p-chlorobenzoyl)-piperazine-2,3-dicar☐ylate (PCB, 3.25 μmol) or dl-2-amino-5-phosphono-valerate (APV, 25.38 μmol) into the NRM, whereas the short-lived glutamate analgesia was not. This indicates that although both opiate and non-opiate analgesia may originate in the PAG, the former is relayed through the NRM, whereas the latter is relayed by additional or different nuclei in the medulla. Two observations shed light on the question which receptors mediate the above effect in the NRM. First, PCB blocked morphine analgesia at doses that were 8 times lower than doses of APV that were effective. Second, analgesia produced by injection of glutamate into the NRM was antagonized by PCB (3.25 μmol), whereas APV (25.38 μmol) failed to do so. Together these results indicate that kainate/quisqualate, but not N-methyl- d-aspartate (NMDA), receptors are implicated in the NRM as a relay station in opiate descending inhibition.

Intracerebroventricular morphine decreases descending inhibitions acting on lumbar dorsal horn neuronal activities related to pain in the rat : D. Bouhassira, L. Villanueva and D. Le Bars, J. Pharmacol. Exp. Ther., 247 (1988) 332–342

Recordings were made from convergent neurons in the lumbar dorsal horn of the rat. These neurons were activated by both innocuous and noxious stimuli applied to their excitatory receptive fields located on the extremity of the ipsilateral hindpaw. Transcutaneous application of suprathreshold 2-msec square-wave pulses to the center of the receptive field resulted in responses to A- and C-fiber activation being observed: 27.2 +/- 2.2 (mean +/- S.E.M.) C-fiber latency spikes were evoked per stimulus. This type of response was inhibited by applying noxious conditioning stimuli to heterotopic areas of the body; in particular, immersing the tail in a 52 degrees C waterbath caused a 74.2 +/- 2.0% inhibition of the C-fiber evoked responses; such inhibitory processes have been termed diffuse noxious inhibitory controls (DNIC). The effects of microinjections of morphine (0.6-40 micrograms; 2 microliter) within the 3rd ventricle on both the unconditioned C-fiber-evoked responses and the inhibitory processes triggered from the tail were investigated in an attempt to answer two questions: 1) does i.c.v. morphine increase tonic descending inhibitory processes? and 2) what are the effects of i.c.v. morphine on descending inhibitory processes triggered phasically by noxious stimuli? The predominant effect of i.c.v. morphine on the C-fiber-evoked responses was a facilitation (17 of 26 cases). Such a facilitation was dose-related in the 0.6 to 40 microgram range and naloxone reversible; it plateaued from 20 min after the microinjection. No clear relationship was found between the number of C-fiber evoked responses in the control sequences and the subsequent effect of i.c.v. morphine. Intracerebroventricular morphine clearly reduced DNIC in the majority of cases (21 of 26). Such a reduction was dose-related in the 0.6 to 2.5 microgram range and naloxone reversible; it plateaued within 90 min of microinjection. No clear relationship was found between the changes in DNIC and either the number of C-fiber-evoked spikes in the control sequences or the changes in the C-fiber responses induced by i.c.v. morphine. Autoradiographic controls using [3H]morphine showed a labeling along the ventricle wall including the hypothalamus, the periaqueductal gray matter and the floor of the 4th ventricle, three regions which have been implicated in the control of nociceptive transmission at the spinal level. Diffusion from the ventricle wall was over a distance of 0.5 mm and was identical whether observed 20 or 95 min after the microinjections.(ABSTRACT TRUNCATED AT 400 WORDS)

The periaqueductal gray matter mediates opiate-induced immunosuppression.

The periaqueductal gray matter of the mesencephalon (PAG) subserves a variety of diverse autonomic functions and also appears to be a site for opiate action in the induction of immunosuppression. Microinjections of morphine into the PAG, but not into other opiate receptor-containing neuroanatomical sites, result in a rapid suppression of natural killer (NK) cell activity. The NK cell suppression can be blocked by prior peripheral administration of the opiate antagonist naltrexone. These findings demonstrate that certain central actions of opiates that produce changes in NK cell function are mediated through opiate receptors in the PAG and identify a brain region involved in opiate regulation of immune function.

Electrophysiological analysis of the neural circuitry underlying opiate effects in the nucleus accumbens septi

Systemically administered opiates inhibit or excite spontaneously active single units of the nucleus accumbens septi (NAS) but do not affect fimbria-evoked NAS responses. However, microinjections of morphine to the dopamine cell bodies of the ventral tegmental area (VTA-M) do inhibit these evoked responses. Since systemic heroin can reverse the inhibitory effect of VTA-M on the fimbria-evoked NAS responses, systemic opiates might exert actions on other brain regions that oppose the inhibitory effect of VTA-M on the NAS. Evidence is provided indicating that this opposing action can arise from opiate actions at the ventral subiculum of the hippocampus.

Characterization of stimulation-produced analgesia from the nucleus tractus solitarius in the rat

Electrical stimulation of the commissural region of the nucleus tractus solitarius (NTS) inhibits the tail-flick reflex evoked by noxious heat. This antinociception can be measured in the awake or pentobarbital anesthetized rat at current intensities that do not induce overt behavioral side effects. Glutamate microinjections into the NTS, but not immediately surrounding the NTS, also inhibit the tail-flick reflex, demonstrating that activation of NTS cell bodies, and not fibers of passage, mediates antinociception from this region. In contrast, morphine microinjections into the NTS have no effect on the tail-flick reflex in anesthetized rats. These findings provide further evidence that the NTS is involved in the modulation of nociception.

Comparison of the antinociceptive effect of morphine and glutamate at coincidental sites in the periaqueductal gray and medial medulla in rats

In rats, microinjection of the opioid agonist morphine (15–45 nmol/0.5 μl) and the excitatory amino acid monosodium glutamate (30–60 nmol/0.5 μl) into identical brainstem sites within the mesecephalic perialueductal gray matter (PAG; 23 sites) and the rostral ventromedial medulla (RVM; 22 sites) produced an increase of tail flick and hot plate response latencies. At both PAG and RVM sites, there was a statistically significant relationship between the effect obtained with morphine and with glutamate on the two nociceptive responses. While morphine and glutamate procedured indistinguishable inhibition of tail flick and hot plate response latencies in the PAG, the tail flick inhibition following RVM morphine, but not RVM glutamate, displayed a clear plateau. One parsimonious interpretation of these data is that (1) glutamate directly increases the activity in the bulbospinal pathway, and (2) morphine inhibits an evoked or tonic suppression of a bulbospinal projection.

Microinjections of glutamate or morphine at coincident midbrain sites have different effects on nociceptive dorsal horn neurons in the rat

Responses of single lumbar spinal neurons to noxious skin heating (50°C, 10 s) were electrophysiologically recorded in barbiturate-anesthetized rats. Responses of all neurons were suppressed by electrical stimulation in the midbrain periaqueductal gray (PAG) or lateral reticular formation (LRF). Microinjection of glutamate (GLU, 0.1–0.3 μl, 0.5 M) into the PAG rapidly (within 15 s) suppressed (to 13–55% of control) the responses of 6 16 neurons with recovery within 8 min. The remainder were affected less at even higher doses (0.5–1 μl). Responses of 4 10 neurons were suppressed following GLU microinjected into the LRF. We also tested effects of microinjection of morphine (MOR, 5 μg/0.5 μl) into GLU-sensitive and insensitive PAG sites. Responses of 4 neurons were unaffected, 4 were enhanced (to 130–155%), and 2 suppressed (to 43 and 57%) following MOR in PAG, with enhancement or suppression beginning within 12–20 min and lasting 40 to over 70 min. The differing effects of GLU and MOR may reflect different mechanisms for the descending modulation of spinal nociceptive transmission.