Feeding induced by opiates injected into the paraventricular hypothalamus

Injection of morphine or d-ala-2-met-5-enkephalinamide (DALA) into the paraventricular nucleus of the hypothalamus (PVN) produced a dose dependent increase in feeding in rats. DALA increased feeding within 45 minutes; morphine within 90 minutes. Naloxone injected into the PVN diminished the effect. DALA increased water intake only when food was available, suggesting the primary effect was on feeding. In summary, an enkephalin analogue induced feeding, and an opiate receptor blocker attenuated it; therefore the PVN may contain opiate receptors that facilitate feeding.     

Interactions of morphine with putative neurotransmitters in the mesencephalic reticular formation

Acetylcholine (ACh) and norepinephrine (NE) have been identified previously as putative nociceptive neurotransmitters in the mesencephalic reticular formation (MRF) of the rat because they frequently mimic the change in neuronal firing (usually an increase) evoked by a noxious stimulus (NS). The purpose of this study was to determine if 1.) morphine (M) acts to prevent the increase in firing evoked by a NS by blocking the effects of either of these two neurotransmitters and 2.) if this effect is a specific narcotic effect. Using the technique of microiontophoresis in conjunction with extracellular recording, we located single units in the MRF in which 1.) neuronal firing was accelerated by a NS: 2.) M blocked this response; and 3.) either ACh or NE mimicked the effect of the NS. Neurons meeting these three criteria were studied further to determined if morphine would also block the response to either of the neurotransmitters and if this was a specific narcotic effect. We found that morphine blocked the increase in neuronal firing evoked by the NS and ACh or the NS and NE in over 50% of the cells meeting the above criteria. Some neurons were found in which both ACh and NE mimicked the NS and M blocked all three responses. This blockade of these neurotransmitters was a specific narcotic effect because it could be reversed by the systematic administration of naloxone. These data lead to the tentative hypothesis that M, acting via an opiate receptor, blocks the increase in neuronal firing evoked by a NS by blocking the postsynaptic effects of either ACh or NE. This may be one of the mechanisms by which morphine acts to produce analgesia.     

Morphine: Ability to block neuronal activity evoked by a nociceptive stimulus

The effect of morphine on the neuronal activity evoked by a nociceptive stimulus, a foot pinch, was studied in the dorsal raphe nucleus (DR) and in the mesencephalic reticular formation (MRF) of the rat. In the MRF and adjacent areas, neuronal firing was accelerated by the nociceptive stimulus. Morphine blocked this acceleration when administered either microintophoretically or i.v. Three lines of evidence indicate that this is a specific narcotic effect. First, naloxone, a specific narcotic antagonist, antagonized the effect of morphine. Secondly, two morphine agonists, oxymorphone and methadone, blocked the evoked neuronal acceleration like morphine when administered either microiontophoretically or i.v.; naloxone also blocked the effects of the two agonists. Finally, two non-opioid CNS depressants did not block the acceleration in neuronal firing even though microintophoretic ejection currents 2–5 times greater than those for morphine were used. In contrast, neuronal firing in the DR was rarely altered by the nociceptive stimulus or by morphine, administered either microiontophoretically or i.v. Furthermore, morphine did not affect the inhibition produced by 5-HT on neurons in the DR.It is concluded from this study that the MRF is a possible site of action for the antinociceptive effects of morphine. It is also concluded that morphine does not affect the spontaneous neuronal firing rate in the DR and that the DR is not a site of action of the antinociceptive effects of morphine when a foot pinch is used as the nociceptive stimulus.     

Significance of the hypothalamus opioid system in the mechanism of morphine analgesia

It has been demonstrated in experiments on conscious rabbits that microinjections of nalorphine to the paraventricular area of the hypothalamus blocked morphine analgesia assessed from the tail-flick test and evoked potential variation in the sensorimotor area of the brain cortex in response to nociceptive electrocutaneous stimulation. An analogous but less powerful effect was produced by nalorphine injected into the large raphe nucleus. It is assumed that morphine analgesia is primarily mediated by opioid structures of the hypothalamus.     

Behavioral effects and in vivo degradation of intraventricularly administered dynorphin-(1-13) and D-Ala2-dynorphin-(1-11) in rats

Lateral intraventricular (LV) or cerebral aqueduct (CA) administration of the opioid peptide, dynorphin-(1-13), induced catalepsy and analgesia in rats. Onset was earlier and duration shorter than with morphine or β_c-endorphin. The dose required to induce analgesia was reduced at least tenfold when dynorphin-(1-13) was administered into CA rather than LV. An analogue, D-Ala²-dynorphin-(1-11), was more stable than dynorphin-(1-13) in brain, and produced a comparable degree of catalepsy and even more profound analgesia at one-tenth the dose. These effects of dynorphin-(1-13) and D-Ala²-dynorphin-(1-11) were significantly antagonized by naloxone pretreatment. Rats treated with dynorphin-(1-13) or a high dose of D-Ala²-dynorphin-(1-11) exhibited bizarre postures immediately following LV administration, with limb rigidity and “barrel-rolling”. These effects were not blocked by naloxone.     

A spinal site mediates opiate analgesia in frogs

The application of acetic acid to the hind leg of a frog will induce a spinally mediated wiping reflex only if the acetic acid concentration is above a certain threshold. By using this reflex as the basis of a test for nociception, we show that morphine sulfate is a potent analgesic in the frog when injected into the lumbar area of the spinal cord. Significant analgesia is induced within 5 min after injection of as little as 0.0316 μg of morphine sulfate. Low doses of morphine sulfate (0.0316 or 0.1 μg) induce analgesia which dissipates within 1 h while for higher doses (0.316, 1.0 or 3.16 μg) the analgesia persists for at least 3 h. The analgesic effect of 0.316 μg of morphine sulfate is completely blocked by naloxone HCl at either 0.158 or 0.316 μg. Animals receiving naloxone alone (0.316 μg) appear to be slightly hyperalgesic compared to saline injected controls but this effect is not significant.     

Regional release of [3H]dopamine from rat brain in vitro: effects of opioids on release induced by potassium, nicotine, and L-glutamic acid

Previous studies have suggested that the release of dopamine (DA) in the rat brain may be sensitive to modulation by opioid agents, including the endogenous opioid peptides (enkephalins and endorphins). The present study examined the effects of morphine and the enkephalin analogue D-Ala2-Met5-enkephalinamide (DALA) on the release of radiolabeled DA from superfused slices of rat brain regions. The release of preloaded [3H]DA was evoked from slices of the caudate-putamen (CP) by application of potassium (K+), nicotine (NIC), or L-glutamic acid (L-GLU). The release of [3H]DA from slices of the nucleus accumbens (NA), olfactory tubercle (OT), and substantia nigra (SN) was evoked by L-GLU. Both K+ and NIC evoked a concentration-related release of [3H]DA from CP slices. K+-induced release was only partially dependent on calcium (Ca2+), while NIC-evoked release was completely Ca2+ independent. Neither morphine nor DALA influenced the release of [3H]DA evoked by K+ or NIC. L-GLU produced a concentration-dependent release of [3H]DA from slices of CP, NA, OT, and SN. In all four brain regions, this release was (a) Ca2+-dependent, (b) strongly inhibited by low concentrations of magnesium (Mg2+), (c) greater than the release evoked by D-GLU, (d) attenuated by the putative L-GLU receptor antagonist glutamic acid diethylester (GDEE), and (e) insensitive to tetrodotoxin (TTX) except in the SN. Morphine produced a significant inhibition of L-GLU-evoked [3H]DA release from all four regions. Naloxone, which by itself had no significant effect on the L-GLU-evoked release of [3H]DA, blocked the inhibitory effect of morphine on this release in the CP but not in the other regions. Levorphanol and dextrorphan were equipotent in reducing the glutamate-stimulated release of [3H]DA from CP slices. DALA had no effect on L-GLU-induced release in any of the brain regions examined. The results indicate that L-GLU provokes regional release of DA by acting at a Mg2+-sensitive glutamate receptor. This release is selectively modified by morphine through a mechanism which is insensitive to naloxone.     

Analgesia induced by intrathecal injection of dynorphin B in the rat

A dose-dependent analgesic effect of intrathecally injected dynorphin B was observed in rats using the tail flick as nociceptive test. Intrathecal injection of 20 nmol of dynorphin B increased the tail flick latency by 90 +/- 23%, an effect that lasted about 90 min. For the same degree of analgesia, dynorphin B was 50% more potent than morphine on a molar basis. The analgesic effect of this dose of dynorphin B was partially blocked by 10 mg/kg, but not by 1 mg/kg, of subcutaneous naloxone, showing a relative resistance to naloxone reversal as compared with morphine analgesia. The analgesia produced by dynorphin B was unchanged in morphine-tolerant rats but was significantly decreased in rats tolerant to ethylketazocine. These results suggest that dynorphin B produces its potent analgesic effect by activation of kappa rather than mu opioid receptors in the rat spinal cord.     

DMSO (dimetyl sulfoxide), morphine and analgesia

DMSO was compared to morphine in rats to determine its relative analgesic effects. DMSO produces analgesia that is comparable in magnitude to morphine although its duration (6–7 hrs) is longer than that of morphine (≤ 2 hrs). DMSO apparently produced analgesia both by an action at the site at which it was administered as well as at a site that was remote to the site of administration. The mechanism of action of DMSO is apparently different from that of morphine because naloxone, a specific narcotic antagonist, does not block the analgesic effect of DMSO. However, DMSO has toxic effects such as hematuria (bloody urine). Therefore, the toxicity observed may restrict the clinical usefulness of DMSO as an analgesic drug.     

Contributions of spinal d-amino acid oxidase to bone cancer pain

d-Amino acid oxidase (DAAO), a FAD-dependent peroxisomal flavoenzyme that catalyzes oxidation of d-amino acids to hydrogen peroxide, is distributed in the spinal cord almost exclusively expressed within astrocytes. The present study aims to explore potential contributions of spinal DAAO to the development of bone cancer pain and morphine tolerance to analgesia. Tibia inoculation of carcinoma cells produced mechanical allodynia (but not heat hyperalgesia), in synchronous with induction of DAAO expression and DAAO enzymatic activity, as well as activation of spinal astrocytes marked by GFAP. Subcutaneous and intrathecal injection of the specific DAAO inhibitor CBIO (5-chloro-benzo[d]isoxazol-3-ol) blocked mechanical allodynia in a dose- and time-dependent manner in tumor-bearing rats, with maximum inhibition of 40–50?%. Multi-daily intrathecal injections of the DAAO gene silencer siRNA/DAAO also yielded anti-allodynic effects by approximately 40?% and the analgesia remained for at least 6?days. Subcutaneous injection of CBIO suppressed the production of spinal hydrogen peroxide and GFAP expression.?7-Day multiple bi-daily injections of CBIO produced anti-allodynia without inducing self-tolerance to analgesia or cross-tolerance to morphine, and concurrent injections of CBIO with morphine produced apparent additive anti-allodynia and completely prevented morphine tolerance in behaviors and spinal expression of μ-opioid receptors. Our results provide the first evidence that spinal DAAO contributes to the development of morphine tolerance to analgesia and bone cancer pain accounting for 40–50?% pain status, probably via production of hydrogen peroxide leading to activation of astrocytes. The unique characterizations of DAAO inhibitors make them a potential for the treatment of cancer pain when they are administered alone or in combination with morphine.     

Discriminative effects of morphine administered intracerabrally in the rat

Rats were trained in a two-choice discrete trial avoidance paradigm to discriminate between saline and 3.0 mg/kg of morphine administered S.C. The microinjection of 0.3–3.0 μg of morphine into the lateral ventricle produced discriminative effects equivalent to those of the systemic training dose as measured by responding on the morphine-appropriate choice lever. Discriminative effects equivalent to those of the morphine training dose were not consistently produced by administration of morphine into the periaqueductal gray, lateral septum or dorsomedial thalamus in doses as high as 10 μg. However, the discriminative effects of systematically administered morphine were blocked by 10–30 μg of naloxone administered intracerebrally at all of the brain sites tested. Thus, the primary site at which morphine acts to produce discriminative effects in the rat is central, although the specific brain areas mediating these effects remain unidentified. The actions of naloxone could be the result of diffusion of the drug into the ventricular system or into the systemic circulation.     

The biochemical and behavioral effects of phospholipase A2 and morphine microinjections in the periaqueductal gray of the rat

In order to characterize the in vivo action of phospholipase A2 (PLA2) on opiate receptors and opiate-induced behaviors, the effects of injections of PLA2 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 micrograms of PLA2 while anesthetized. One hour later, regions around the cannulae tracts in PLA2-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 micrograms of PLA2 prior to 10 micrograms of morphine through cannulae chronically implanted into the PAG. PLA2 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. PLA2 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 PLA2 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.     

Analgesic effects of ethylketocyclazocine and morphine in rat and toad

We have previously found rat and toad (Bufo marinus) brain to contain inverse ratios of benzomorphan-preferring (kappa/sigma) and morphine-preferring (mu) opioid receptor types. The aim of the present study was to compare in vivo pharmacologic activity of a benzomorphan, ethylketocyclazocine (EKC) and morphine sulfate (MS) in rat and toad. Footshock intensity thresholds for eliciting locomotion were determined and dose-response curves for EKC and MS analgesia were obtained. Drugs were injected subcutaneously. In rats (high mu, low kappa in brain), both compounds produced analgesia and displayed similar sensitivity to naloxone antagonism. The analgesic effects of EKC and MS may, therefore, be mediated by a common receptor type (mu) in this pain test in rats. In toads (high kappa, low mu in brain), MS produced naloxone-reversible analgesia at doses 20-fold higher than were effective in rats. Toads did not display EKC analgesia at doses below those producing motor impairment. Moreover, 50-fold higher doses were required to produce such impairment in toads. Thirty minutes following subcutaneous injection of 3H-EKC, similar concentrations were found in rat and toad brain. Uptake into brain is probably not a factor in the behavioral resistance of toads to EKC.     

Systemic morphine-induced release of serotonin in the rostroventral medulla is not mimicked by morphine microinjection into the periaqueductal gray

We used in vivo microdialysis in awake rats to test the hypothesis that intravenous morphine increases serotonin (5-HT) release within the rostral ventromedial medulla (RVM). We also injected morphine into various sites along the rostrocaudal extent of the periaqueductal gray (PAG), and examined the extent of its diffusion to the RVM. Intravenous morphine (3.0 mg/kg) produced thermal antinociception and increased RVM dialysate 5-HT, 5-hydroxyindole acetic acid (5-HIAA), and homovanillic acid (HVA) in a naloxone-reversible manner. As neither PAG microinjection of morphine (5 micro g/0.5 micro L) nor RVM administration of fentanyl or d-Ala(2),NMePhe(4),Gly-ol(5)]enkephalin (DAMGO) increased RVM 5-HT, we were unable to determine the precise site of action of morphine. Surprisingly, peak morphine levels in the RVM were higher after microinjection into the caudal PAG as compared to either intravenous injection or microinjection into more rostral sites within the PAG. Naloxone-precipitated withdrawal in morphine-tolerant rats not only increased extracellular 5-HT in the RVM, but also dopamine (DA) and HVA. We conclude that substantial amounts of morphine diffuse from the PAG to the RVM, and speculate that opioid receptor interactions at multiple brain sites mediate the analgesic effects of PAG morphine. Further studies will be required to elucidate the contribution of 5-HT and DA release in the RVM to opioid analgesia and opioid withdrawal.     

Naloxone blocks the effect of 5-hydroxy-tryptophan on passive avoidance learning in rats: Implication of endogenous opioid peptides

The synthetic opioid peptide D-Ala²-met-enkephalinamide (DALA) impairs retention performance of rats on a one-trial passive avoidance task. The same effect is observed after administration of 5-hydroxy-tryptophan (5-HTP). Both drugs appear to impair learning by disrupting memory consolidation since similar effects are observed when they are given either peripherally before the acquisition test or intracerebroventricularly immediately after. A moderate dose of naloxone prevents the amnesia produced by DALA or 5-HTP. However, the opiate antagonist does not block the behavioral syndrome induced by clorgyline and 5-HTP. Moreover, bacitracin, a peptidase inhibitor, potentiates the effect of 5-HTP on the passive avoidance task. The results suggest that endogenous opioid peptides are involved in the amnesic effect of 5-HTP.     

Non-opioid analgesia of the neuropeptide, neo-kyotorphin and possible mediation by inhibition of GABA release in the mouse brain

The novel neuropeptide, neo-kyotorphin, produced a naloxone-resistant analgesia in the tail pinch test when given (IC) to mice. Pretreatments with implantation of a morphine pellet or with phentolamine (10 micrograms IT) or with reserpine (10 mg/kg SC) did not attenuate this analgesia, yet the analgesia was antagonized by GABA mimetics, such as muscimol (0.1 microgram IC), nipecotic acid (100 mg/kg IP). Neo-kyotorphin inhibited the Ca2+-dependent and depolarization-evoked release of 3H-GABA, from crude synaptosomes of the lower brain stem of rats. These findings suggest that inhibition of GABA in the brain may in part be involved in neo-kyotorphin-induced analgesia.     

Inhibition of copulatory behavior in male rats by D-ALA2-Met-Enkephalinamide

D-Ala²-Met-Enkephalinamide (DALA), a synthetic analog of met-enkephaline resistant to enzymatic degradation, was injected intraventricularly to sexually experienced male rats paired with receptive females. A dose of DALA of 6 μg which did not influence spontaneous motor activity, completely suppressed the copulatory behavior of all animals tested. A dose of 3 μg significantly increase mounting and intromission latencies, but did not influence other measures of the copulatory behavior. The effect of DALA was prevented by naloxone (1 mg/Kg), a specific inhibitor of opioid receptors. The results suggest that enkephalins may play a role in the regulation of copulatory behavior.     

Attenuation of systemic morphine-induced analgesia by central administration of ghrelin and related peptides in mice

Ghrelin, an acylated 28-amino peptide secreted in the gastric endocrine cells, has been demonstrated to stimulate the release of growth hormone, increase food intake, and inhibit pro-inflammatory cascade, etc. Ghrelin mainly combines with its receptor (GHS-R1α) to play the role in physiological and pathological functions. It has been reported that ghrelin plays important roles in the control of pain through interaction with the opioid system in inflammatory pain and acute pain. However, very few studies show the effect of supraspinal ghrelin system on antinociception induced by intraperitoneal (i.p.) administration of morphine. In the present study, intracerebroventricular (i.c.v.) injection of ghrelin (0.1, 1, 10 and 100 nmol/L) produced inhibition of systemic morphine (6 mg/kg, i.p.) analgesia in the tail withdrawal test. Similarly, i.c.v. injection GHRP-6 and GHRP-2 which are the agonists of GHS-R1α, also decreased analgesia effect induced by morphine injected intraperitoneally in mice. Furthermore, these anti-opioid activities of ghrelin and related peptides were not blocked by pretreatment with the GHS-R1α selective antagonist [d-Lys³]-GHRP-6 (100 nmol/L, i.c.v.). These results demonstrated that central ghrelin and related peptides could inhibit the analgesia effect induced by intraperitoneal (i.p.) administration of morphine. The anti-opioid effects of ghrelin and related peptides do not interact with GHS-R1a. These findings may pave the way for a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation.     

Decrease of cyclic GMP in cerebellum by intrastriatal D-Ala2-met-enkephalinamide.

The bilateral intrastriatal injection of D-Ala²-Met-Enkephalinamide (DALA) at doses of ranging from 12 to 50 μg decreased cyclic-GMP content in the cerebellum and produced catalepsy. These effects were prevented by naltrexone, an opiate receptor antagonist but not by apomorphine, a dopamine agonist. The bilateral injection of DALA in the cerebellum, and substantia nigra neither decreased cerebellar cyclic GMP content nor produced catalepsy. The bilateral injection of DALA (20 ug) into the ventromedial thalamic nuclei caused marked catalepsy but failed to decrease cerebellar cyclic GMP. The results suggest that the effect of DALA on cGMP can be differentiated from the cataleptic response and that it is mediated by an action on opioid receptors located in the striatum, beyond DA receptors.     

Methionine oxidation enhances opioid activity of an enkephalin analog

D-ala2-met-sulfoxide5-enkephalinamide, DALA(0), was synthesized by oxidizing the 5-methionine residue of D-ala2-met5-enkephalinamide (DALA). Antinociception was assessed on the hot-plate and catalepsy estimated using an immobility test in rats administered DALA, DALA(0) and morphine intraventricularly. By comparing areas under time-effect curves, DALA(0) was 30 times more antinociceptive and up to 40 times more cataleptogenic than DALA. For comparison, morphine induced one-tenth the antinociception and one-fortieth the immobility caused by DALA(0). These results demonstrate that the opiate activity of DALA is clearly enhanced by oxidation of its terminal methionine.