Involvement of cannabinoid (CB1)-receptors in the development and maintenance of opioid tolerance.

Sustained exposure to opioid agonists such as morphine increases levels of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn, a response implicated in the development of opioid tolerance and physical dependence. Recent evidence suggests that both the opioid-induced increase in CGRP and the development of opioid physical dependence are suppressed by blockade of spinal cannabinoid (CB1)-receptors. The present study examined whether CB1-receptor activity also has a role in the development of opioid tolerance. In rats implanted with spinal catheters, repeated acute injections of morphine (15 microg) delivered over 4 h resulted in a rapid decline of thermal and mechanical antinociception and a significant loss of analgesic potency, reflecting development of acute opioid tolerance. In another set of experiments, chronic administration of spinal morphine (15 microg) once daily for 5 days produced a similar loss of analgesic effect and a marked increase in CGRP-immunoreactivity in the superficial laminae of the dorsal horn. Consistent with the in vivo findings, primary cultures of adult dorsal root ganglion (DRG) neurons exposed to morphine for 5 days showed a significant increase in the number of CGRP-immunoreactive neurons. Co-administration of acute or chronic morphine with a CB1-receptor antagonist/inverse agonist, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM-251), inhibited the development of both acute and chronic analgesic tolerance. In animals already exhibiting tolerance to morphine, intervention with AM-251 restored morphine analgesic potency. Co-administration with AM-251 attenuated the morphine-induced increase in CGRP-immunoreactivity in the spinal cord and in DRG cultured neurons. Collectively, the results of this study suggest that activity of endocannabinoids, mediated via CB1-receptors, contributes to both the development and maintenance of opioid tolerance by influencing the opioid-induced increase in spinal CGRP.

Augmentation of spinal morphine analgesia and inhibition of tolerance by low doses of mu- and delta-opioid receptor antagonists.

BACKGROUND AND PURPOSE: Ultralow doses of naltrexone, a non-selective opioid antagonist, have previously been found to augment acute morphine analgesia and block the development of tolerance to this effect. Since morphine tolerance is dependent on the activity of micro and delta receptors, the present study investigated the effects of ultralow doses of antagonists selective for these receptor types on morphine analgesia and tolerance in tests of thermal and mechanical nociception. EXPERIMENTAL APPROACH: Effects of intrathecal administration of mu-receptor antagonists, CTOP (0.01 ng) or CTAP (0.001 ng), or a delta-receptor antagonist, naltrindole (0.01 ng), on spinal morphine analgesia and tolerance were evaluated using the tail-flick and paw-pressure tests in rats. KEY RESULTS: Both micro and delta antagonists augmented analgesia produced by a sub-maximal (5 microg) or maximal (15 microg) dose of morphine. Administration of the antagonists with morphine (15 microg) for 5 days inhibited the progressive decline of analgesia and prevented the loss of morphine potency. In animals exhibiting tolerance to morphine, administration of the antagonists with morphine produced a recovery of the analgesic response and restored morphine potency. CONCLUSIONS AND IMPLICATIONS: Combining ultralow doses of micro- or delta-receptor antagonists with spinal morphine augmented the acute analgesic effects, inhibited the induction of chronic tolerance and reversed established tolerance. The remarkably similar effects of micro- and delta-opioid receptor antagonists on morphine analgesia and tolerance are interpreted in terms of blockade of the latent excitatory effects of the agonist that limit expression of its full activity.

Facilitation of spinal morphine analgesia in normal and morphine tolerant animals by neuropeptide SF and related peptides.

Neuropeptide FF and related synthetic amidated peptides have been shown to elicit sustained anti-nociceptive responses and potently augment spinal anti-nociceptive actions of spinal morphine in tests of thermal and mechanical nociception. Recent studies have described the occurrence of another octapeptide, neuropeptide SF (NPSF) in the spinal cord and the cerebrospinal fluid and demonstrated its affinity for the NPFF receptors. This study examined the effects of NPSF and two putative precursor peptides, EFW-NPSF and NPAF, on the spinal actions of morphine in normal and opioid tolerant rats using the tailflick and pawpressure tests. In normal rats, NPSF demonstrated weak intrinsic activity but sub-effective doses of the peptide significantly increased the magnitude and duration of spinal morphine anti-nociception in both tests. A low-dose of NPSF also augmented the spinal actions of a delta receptor agonist, deltorphin. The morphine-potentiating effect of NPSF was shared by EFW-NPSF and the octadecapeptide NPAF. In animal rendered tolerant by continuous intrathecal infusion of morphine for 6 days, low dose NPSF itself elicited a significant anti-nociceptive response and potently increased morphine-induced response in both tests. In animals made tolerant by repeated injections of intrathecal morphine, administration of NPSF, EFW-NPSF, and NPAF with morphine reversed the loss of the anti-nociceptive effect and restored the agonist potency. The results demonstrate that in normal animals NPSF and related peptides exert strong potentiating effect on morphine anti-nociception at the spinal level and in tolerant animals these agents can reverse the loss of morphine potency.

Effects of chronic manipulations of dietary choline on locomotor activity, discrimination learning and cortical acetylcholine release in aging adult Fisher 344 rats.

Dietary levels of choline have been shown to influence central cholinergic neurotransmission. To further examine the behavioral and neurochemical effects of dietary choline, adult Fisher 344 rats were maintained on choline deficient (n = 6), enriched (n = 6) or usual lab chow (n = 6) diets for 38 weeks. During 14 weeks of free access to these diets, controls gained little weight whereas the deficient group increased and the enriched group decreased. For the remaining 24 weeks the weights of all groups were maintained at 90% of their original level. Locomotor activity did not differ significantly in a 90 min session but during a 20 hr session controls were most active, deficient rats least with the enriched group in between. The groups showed no significant differences in the acquisition or reversal of a discrimination. Spontaneous and evoked cortical release of acetylcholine was enhanced in the enriched group and decreased in the deficient group relative to controls. These data suggest that chronic manipulations of dietary choline may significantly influence locomotor and neurochemical activity but not discrimination learning.

Quinolinic acid neurotoxicity in the nucleus basalis antagonized by kynurenic acid.

Quinolinic acid, a metabolite of tryptophan, behaves as an excitotoxic amino acid. It has been proposed that quinolinic acid might be implicated in neurodegenerative diseases. The related metabolite, kynurenic acid, has been found to be a powerful antagonist of quinolinic acid. The ability of quinolinic acid, alone or in combination with kynurenic acid, to destroy cholinergic neurons projecting to the cortex was examined by morphological and biochemical criteria. The compounds were injected unilaterally into the nbm of the rat. Neuronal destruction of the basal forebrain occurred with quinolinic acid alone; however, no cell loss was observed when kynurenic and quinolinic acid were co-injected. Quinolinic acid lesions of the nucleus basalis caused significant decreases in cortical choline acetyltransferase, acetylcholinesterase, high affinity choline uptake and 3H-acetylcholine release. These reductions in cortical cholinergic markers were prevented by co-injecting kynurenic with quinolinic acid. A significant decrease in cortical choline acetyltransferase activity was observed three months following quinolinic acid lesions of the nucleus basalis. The results indicate that quinolinic acid can be used as an endogenous neurotoxin to produce lesions of the nbm resulting in impaired cortical cholinergic function similar to that seen in Alzheimer's disease.

Functional and neurochemical cortical cholinergic impairment following neurotoxic lesions of the nucleus basalis magnocellularis in the rat.

The effect of kainic and quinolinic acid on cortical cholinergic function was examined following injections of these agents into the nucleus basalis magnocellularis (nbm) or into the frontoparietal cortex. The release of cortical 3H-acetylcholine (3H-ACh), high affinity choline uptake (HACU) and acetylcholinesterase was measured 7 days following injections of saline (control), kainic acid (4.7 nmoles) and quinolinic acid (60, 150 and 300 nmoles) into the nbm. These cortical cholinergic parameters were also examined after injections of saline (control), kainic acid (9.4 nmoles) and quinolinic acid (300 nmoles) into the fronto-parietal cortex. The release of 3H-ACh, HACU and AChE was significantly reduced in animals injected with kainic or quinolinic acid into the nbm. Histological examination of stained sections showed a loss of cell bodies in the region of the nbm and the globus pallidus. The size of the lesion produced by quinolinic acid was proportional to the dose injected into the nbm. In animals injected with kainic acid or quinolinic acid into the cerebral cortex, the release of 3H-ACh, HACU and AChE was not significantly reduced when compared with control animals, although histological examination of stained cortical sections showed a marked loss of cortical neurons. The results show that quinolinic acid, an endogenous neuroexcitant, produces a deficit of cholinergic function similar to that described in the cortical tissue of patients with senile dementia of Alzheimer's type. The toxic effects of quinolinic acid on cortical cholinergic function are due to its action on cholinergic cell bodies in the nbm.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of neuropeptide FF neurons in the brainstem nucleus tractus solitarius following cardiovascular challenge and opiate withdrawal.

Neuropeptide FF (NPFF), a morphine modulatory peptide, is localized within discrete autonomic regions including the brainstem nucleus tractus solitarius (NTS) and the parabrachial nucleus (PBN). We investigated the activation of NPFF neurons in the NTS of rats induced by cardiovascular challenge and centrally generated opiate withdrawal. For hypotensive stimulation, we used systemic infusions of sodium nitroprusside (NP) or hemorrhage (HEM), and hypertension was achieved by intravenous phenylephrine (PHENYL) or angiotensin II (AII). In rats that received continuous intracerebroventricular injections of morphine, intraperitoneal injections of naloxone precipitated behavioural signs of opioid withdrawal. Activated NTS neurons were identified by using a combined immunohistochemistry for Fos and NPFF, and neurons projecting to the PBN were determined with a retrograde tracer. HEM, administration of vasoactive drugs, and opiate withdrawal produced a very robust activation of NTS neurons. In NP and HEM groups, 25.6+/-3.2% and 7.6+/-1.3% of NPFF neurons were activated, respectively. Lesser numbers of NPFF neurons were activated in the PHENYL (4.6+/-1.6%) and AII (2.4+/-0.8%) groups. However, following opiate withdrawal, virtually no Fos expression was observed in NPFF neurons. NPFF neurons activated during NP infusion constituted the largest number of cells projecting to the PBN. This study shows that NPFF neurons in NTS that project to the PBN respond selectively to NP as opposed to other cardiovascular challenges or opiate withdrawal. These data support an emerging and important role for NPFF in the context of central cardiovascular regulation.

Attenuation of malonate-induced degeneration of the nigrostriatal pathway by inhibitors of nitric oxide synthase.

Focal infusions of the succinate dehydrogenase inhibitor, malonate, into the substantia nigra pars compacta (SNc) of adult Sprague-Dawley rats resulted in a substantial depletion of ipsilateral striatal tyrosine hydroxylase (TH) activity. The percentage decrease in striatal TH activity following intranigral malonate (0.5 mumol/0.5 microliter) infusion was similar at 4 (58%) and 7 days (62%) post-infusion. To assess the role of N-methyl-D-aspartate (NMDA) receptor activation in malonate neurotoxicity, animals were pretreated with the NMDA receptor antagonist MK-801 (2 x 5 mg/kg, i.p.). Four days post-infusion of malonate (0.5 mumol/0.5 microliter) into the SNc, striatal TH activity was depleted by 58% in vehicle pretreated animals and 14% in the presence of MK-801 indicating a significant neuroprotective effect of MK-801 on malonate action. To determine the role of nitric oxide (NO) in malonate-induced nigral toxicity, the actions of malonate were evaluated in the presence of the nitric oxide synthase (NOS) inhibitors, 7-nitro indazole (7-NI) and N omega-nitro-L-arginine methyl ester (L- NAME). Systemic injections of 7-NI (20, 30, 40, 50 and 75 mg/kg, i.p.) produced a dose-related inhibition of nigral NOS activity which was maximal at a dose of 40 mg/kg. Intranigral infusion of malonate with 20 and 50 mg/kg 7-NI pretreatment produced a 46 and 31% decrease in striatal TH activity, respectively. Thus, a significant protective effect at the higher but not lower dose of 7-NI was observed. Pretreatment with a L- NAME regimen (2 x 250 mg/kg; i.p.), previously shown to inhibit brain NOS activity by greater than 86%, also produced a significant neuroprotective effect against malonate-induced neurotoxicity (30% decrease). The results of this study suggest that malonate-induced toxicity to the dopaminergic neurons of the nigrostriatal pathway is mediated, at least in part, by NMDA receptor activation and the formation of NO.

Potentiation of NMDA-mediated toxicity on nigrostriatal neurons by a low dose of 7-nitro indazole.

Recent evidence suggests that an excitotoxic mechanism may play a role in the etiology of Parkinson's disease. Previously, we have shown that the nigrostriatal dopaminergic neurons are sensitive to focal infusions of an N-methyl-D-aspartate (NMDA) receptor agonist; this toxicity was potentiated by systemic administration of the nitric oxide synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester. The present investigation was undertaken to assess the role of the selective neuronal NOS inhibitor, 7-nitro indazole (7-NI) on the neurotoxicity elicited by NMDA receptor activation in vivo. Single injections of 7-NI (0-125 mg/kg, i.p.) into male Sprague-Dawley rats resulted in a dose-dependent decrease in both nigral and cerebellar NOS activity measured 30 min post-injection. Maximal NOS inhibition was obtained with 20 mg/kg 7-NI (nigra: 90.2 +/- 3.7%, cerebellum: 86.7 +/- 6.3%). In addition, it was found that 7-NI (80 mg/kg, i.p.) did not cause an increase in mean arterial blood pressure over a 48 hr period. Vehicle pretreatment of animals prior to stereotaxic infusion of NMDA (15 nmol) into the substantia nigra compacta resulted in a 56.1 +/- 5.1% decrease in striatal tyrosine hydroxylase (TH) activity from the contralateral side. Pretreatment with 7-NI (5 and 80 mg/kg) produced a 76.9 +/- 3.2% and 49.8 +/- 5.6% decrease, respectively, in striatal TH activity. Thus, a significant increase in NMDA toxicity was observed at the lower but not higher dose of 7-NI. It was also observed that 7-NI (20 and 80 mg/kg) produced a decrease in locomotor activity over a 2 hr period.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection against quinolinic acid-mediated excitotoxicity in nigrostriatal dopaminergic neurons by endogenous kynurenic acid.

Endogenous excitotoxins have been implicated in the degeneration of dopaminergic neurons in the substantia nigra compacta of patients with Parkinson's disease. One such agent quinolinic acid is an endogenous excitatory amino acid receptor agonist. This study examined whether an increased level of endogenous kynurenic acid, an excitatory amino acid receptor antagonist, can protect nigrostriatal dopamine neurons against quinolinic acid-induced excitotoxic damage. Nigral infusion of quinolinic acid (60 nmoles) or N-methyl-D- aspartate (15 nmoles) produced a significant depletion in striatal tyrosine hydroxylase activity, a biochemical marker for dopaminergic neurons. Three hours following the intraventricular infusion of nicotinylalanine (5.6 nmoles), an agent that inhibits kynureninase and kynurenine hydroxylase activity, when combined with kynurenine (450 mg/kg i.p.), the precursor of kynurenic acid, and probenecid (200 mg/kg i.p.), an inhibitor of organic acid transport, the kynurenic acid in the whole brain and substantia nigra was increased 3.3-fold and 1.5-fold respectively when compared to rats that received saline, probenecid and kynurenine. This elevation in endogenous kynurenic acid prevented the quinolinic acid-induced reduction in striatal tyrosine hydroxylase. However, 9 h following the administration of nicotinylalanine with kynurenine and probenecid, a time when whole brain kynurenic acid levels had decreased 12-fold, quinolinic acid injections produced a significant depletion in striatal tyrosine hydroxylase. Intranigral infusion of quinolinic acid in rats that received saline with kynurenine and probenecid resulted in a significant depletion of ipsilateral striatal tyrosine hydroxylase. Administration of nicotinylalanine in combination with kynurenine and probenecid also blocked N-methyl-D-aspartate-induced depletion of tyrosine hydroxylase. Tyrosine hydroxylase immunohistochemical assessment of the substantia nigra confirmed quinolinic acid-induced neuronal cell loss and the ability of nicotinylalanine in combination with kynurenine and probenecid to protect neurons from quinolinic acid-induced toxicity. The present study demonstrates that increases in endogenous kynurenic acid can prevent the loss of nigrostriatal dopaminergic neurons resulting from a focal infusion of quinolinic acid or N-methyl-D-aspartate. The strategy of neuronal protection by increasing the brain kynurenic acid may be useful in retarding cell loss in Parkinson's disease and other neurodegenerative diseases where excitotoxic mechanisms have been implicated.

Excitotoxic lesions of rat basal forebrain: differential effects on choline acetyltransferase in the cortex and amygdala.

Previous studies have shown that basal forebrain lesions using different excitotoxins produce similar decreases in cortical choline acetyltransferase, but differential effects on memory. However, basal forebrain cholinergic neurons send efferents to the amygdala and cortex. The present studies compared the effects of several excitotoxins on choline acetyltransferase levels in both of these structures. Lesions of the basal forebrain were made in rats by infusing different doses of either alpha-amine-3-hydroxy-5-methyl-4-isoxazole propionic acid, ibotenic acid, quisqualic acid, quinolinic acid or N-methyl-D-aspartic acid and measuring choline acetyltransferase seven days later. All of the excitotoxins exerted a differential response on cholinergic neurons of the basal forebrain projecting to the cortex or amygdala. Quinolinic acid was a more potent neurotoxin to cholinergic neurons innervating the amygdala than those projecting to the cortex. In contrast, quisqualic acid and alpha-amine-3-hydroxy-5-methyl-4-isoxazole were more potent neurotoxins to the cortical projection. alpha-Amine-3-hydroxy-5-methyl-4-isoxazole propionic acid was the most potent excitotoxin for destroying cholinergic neurons innervating either the cortex or amygdala. A parallel neurotoxic response was obtained in the cortex and amygdala following infusion of ibotenic acid or N-methyl-D-aspartic acid with little selectivity for choline acetyltransferase depletion in the cortex or amygdala. Histological analysis of the injection site revealed that acetylcholinesterase-positive neurons were destroyed by the excitotoxins in a dose-dependent manner. Excitotoxins (ibotenic acid, quinolinic acid, N-methyl-D-aspartic acid) that produce the greatest impairments in memory were found to produce the greatest depletion of choline acetyltransferase in the amygdala.(ABSTRACT TRUNCATED AT 250 WORDS)

Picolinic acid blocks the neurotoxic but not the neuroexcitant properties of quinolinic acid in the rat brain: evidence from turning behaviour and tyrosine hydroxylase immunohistochemistry.

Previous results suggest that the tryptophan metabolite, picolinic acid may have the unusual properties of antagonizing the neurotoxic but not the neuroexcitant effects of another tryptophan metabolite, quinolinic acid in the central nervous system. The present experiments tested this possibility utilizing behavioural and tyrosine hydroxylase immunohistochemical techniques. In the first series of experiments, rats received injections of relatively high concentrations of 6-hydroxydopamine (12 micrograms in 1 or 2 microliters), quinolinic acid (120 nmol in 0.5 microliters), picolinic acid (480 nmol in 0.5 microliters) or co-treatments (0.5 microliters) with quinolinic (120 nmol) plus picolinic acid (480 nmol) into the region of the substantia nigra. Results revealed that 6-hydroxydopamine and quinolinic acid alone produced a large loss of tyrosine hydroxylase-positive cells in the pars compacta of the substantia nigra. Behavioural results for all 6-hydroxydopamine (n = 10) and for some quinolinate-treated rats (n = 5) revealed ipsi- and contraversive circling following amphetamine (1 mg/kg, i.p.) and apomorphine (0.5 mg/kg, s.c.), respectively, consistent with unilateral loss of dopamine cells in the substantia nigra. The remaining quinolinate-treated rats (n = 9) circled ipsiversively following either stimulant suggesting damage to the pars reticulata. Groups treated with picolinic acid alone (n = 6) or co-injected (n = 6) showed no loss of tyrosine hydroxylase-positive cells in the substantia nigra and no circling response to the stimulants. In the second series of experiments, low concentrations of quinolinic acid (2.5, 5.0, 7.5 nmol), picolinic acid (10, 20, 30 nmol), or the two together (7.5 plus 30 nmol, respectively) were microinjected (0.5 microliter) into the dorsal striatum and circling behaviour evaluated. These results revealed dose-dependent contralateral circling with either quinolinate or picolinate; co-injection of the two tryptophan metabolites also produced contralateral circling. It was concluded that picolinic acid blocks the neurotoxic but not the neuroexcitant effects of quinolinic acid.

Localization and modulation of calcitonin gene-related peptide-receptor component protein-immunoreactive cells in the rat central and peripheral nervous systems.

Calcitonin gene-related peptide (CGRP) is widely distributed in the central and peripheral nervous system. Its highly diverse biological activities are mediated via the G protein-coupled receptor that uniquely requires two accessory proteins for optimal function. CGRP receptor component protein (RCP) is a coupling protein necessary for CGRP-receptor signaling. In this study, we established the anatomical distribution of RCP in the rat central and peripheral nervous system and its relationship to CGRP immunoreactivity. RCP-immunoreactive (IR) perikarya are widely and selectively distributed in the cerebral cortex, septal nuclei, hippocampus, various hypothalamic nuclei, amygdala, nucleus colliculus, periaqueductal gray, parabrachial nuclei, locus coeruleus, cochlear nuclei, dorsal raphe nuclei, the solitary tractus nucleus and gracile nucleus, cerebellar cortex, various brainstem motor nuclei, the spinal dorsal and ventral horns. A sub-population of neurons in the dorsal root ganglia (DRG) and trigeminal ganglia were strongly RCP-IR. Overall, the localization of RCP-IR closely matched with that of CGRP-IR. We also determined whether RCP in DRG and dorsal horn neurons can be modulated by CGRP receptor blockade and pain-related pathological stimuli. The intrathecal injection of the antagonist CGRP(8-37) markedly increased RCP expression in the lumbar DRG and spinal dorsal horn. Carrageenan-induced plantar inflammation produced a dramatic bilateral increase in RCP expression in the dorsal horn while a partial sciatic nerve ligation reduced RCP expression in the ipsilateral superficial dorsal horn. Our data suggest that the distribution of RCP immunoreactivity is closely matched with CGRP immunoreactivity in most of central and peripheral nervous systems. The co-localization of RCP and CGRP in motoneurons and primary sensory neurons suggests that CGRP has an autocrine or paracrine effect on these neurons. Moreover, our data also suggest that RCP expression in DRG and spinal cord can be modulated during CGRP receptor blockade, inflammation or neuropathic pain and this CGRP receptor-associated protein is dynamically regulated.

Opioid and substance P receptor adaptations in the rat spinal cord following sub-chronic intrathecal treatment with morphine and naloxone.

The effect of continuous intrathecal infusion with morphine (5 mu/h) or naloxone (2 micrograms/h) was investigated with regard to analgesia and the apparent density of mu- and delta-opioid and neurokinin-I/substance P receptors in the rat spinal cord. Morphine infusion increased tail-flick and paw-pressure responses until day 4 after the mini-osmotic pump implant. A decline in antinociception, reflecting tolerance to morphine, was then apparent in both tests. Quantitative in vitro receptor autoradiography of [125I]FK-33824, [125I][D.Ala2]deltorphin-I and [125I] Bolton-Hunter substance P binding sites, as ligands of mu, delta and neurokinin-I/substance P receptors, respectively, was performed on lumbosacral spinal cord sections of seven-days tolerant animals. Treatments with morphine and naloxone induced a similar increase (37%) in the number of delta binding sites in the superficial laminae of the dorsal horn. In contrast, the density of mu-opioid receptors was only affected by naloxone (50% increase). Neurokinin-I/substance P binding parameters were not altered by these treatments. Thus, it appears that delta-opioid binding sites may be of special relevance with regard to the development of tolerance to opiates in the spinal cord.

Action of enkephalin analogues and morphine on brain acetylcholine release: differential reversal by naloxone and an opiate pentapeptide.

1 Methionine (Met)-enkephalin, leucine (Leu)-enkephalin and their synthetic analogues were tested for effects on the spontaneous release of cortical acetylcholine (ACh) in vivo. The ability of naloxone to reverse the action of enkephalins on ACh release was compared with its action against morphine. An enkephalin analogue, structurally related to Met-enkephalin, was tested for opiate antagonistic activity in ACh release experiments. 2 Intraventricular administration of Met-enkephalin, Leu-enkephalin, D-Ala2-Met5-enkephalinamide (DALA) and D-Ala2-D-Leu5-enkephalin (DALEU) produced a dose-related inhibition of cortical ACh release. Met- and Leu-enkephalin were very similar both in their potency and the time course of their action on ACh release. Both DALA and DALEU were more potent and had a longer duration of action than Leu-enkephalin. Systemic injections of two pentapeptides, D-Met2-Pro5-enkephalinamide and D-Ala2-MePhe4-Met5(O)-ol-enkephalin (33,824), produced a sustained inhibition of cortical ACh release. 3 Naloxone, administered systemically following the depression of ACh release induced by either intraventricular injections of enkephalins (DALA or DALEU), or systemic injections of enkephalins (D-Met2-Pro5-enkephalinamide or 33,824), reversed this depression and restored the release to baseline levels. The effect of D-Met2-Pro5-enkephalinamide on the release of ACh was reversed by naloxone with difficulty. Naloxone also reversed the inhibitory effect of systemic morphine and this reversal was associated with a large overshoot of ACh release. The latter was never observed in the enkephalin experiments. 4 Intraventricular injection of the pentapeptide, D-Ala2-D-Ala3-Met5-enkephalinamide (TAAPM), at doses that did not influence the basal ACh release, blocked or reversed the inhibitory effect of morphine on this release. This peptide did not block the effect of the non-opiate, chlorpromazine, under similar conditions. In two experiments TAAPM failed to reverse the inhibition of ACh release produced by systemically injected enkephalin, D-Met2-Pro5-enkephalinamide. 5 Effects of morphine and enkephalin on ACh release are discussed in terms of their action on difference opiate receptors.

Investigation of action of enkephalin on the spontaneous and evoked release of acetylcholine from rat cortical and striatal slices.

1 The effects of two synthetic enkephalins, D-Ala2-Met5-enkephalinamide (DALA) and D-Ala2-MePhe4-Met5-(0)-ol-enkephalin (33,824) were tested on the spontaneous and stimulated release of acetylcholine (ACh) from rat cortical and striatal slices. 2 DALA, but not 33,824, caused a modest increase in spontaneous cortical ACh release. Neither enkephalin affected the release of cortical ACh induced by high potassium (25 mM) or veratridine (20 microM). 3 DALA had no effect on the release of ACh from striatal slices occurring spontaneously or in the presence of potassium (25 mM), veratridine (5 microM) or ouabain (10 microM). The enkephalin 33,824 also had no significant action on the striatal ACh release except that it caused a slight enhancement of veratridine-evoked release. 4 It is suggested that enkephalins have no significant action on release of ACh from the cortex or striatum in vitro. Their action on ACh release from the cortex in vivo, seen in previous studies, may be exerted at a subcortical level.

Glutamate-evoked release of endogenous brain dopamine: inhibition by an excitatory amino acid antagonist and an enkephalin analogue.

The present study examined the effect of a selective delta-opioid receptor agonist [D-Ala2-D-Leu5] enkephalin (DADL) on the spontaneous and the L-glutamic acid (L-Glu)-evoked release of endogenous dopamine from superfused slices of rat caudate-putamen. The amount of dopamine in slice superfusates was measured by a sensitive method employing high-performance liquid chromatography with electrochemical detection (h.p.l.c.-e.d.) after a two-step separation procedure. The spontaneous release of endogenous dopamine was partially dependent on Ca2+, enhanced in Mg2+-free superfusion medium, partially reduced by tetrodotoxin (TTX, 0.3 microM), partially reduced by the putative excitatory amino acid receptor antagonist DL-2-amino-7-phosphonoheptanoic acid (DL-APH, 1 mM), and increased 10 fold by the dopamine uptake blocker, nomifensine (10 microM). DADL (5 and 50 nM) did not significantly affect spontaneous dopamine release. L-Glu (0.1-10 mM) produced a concentration-dependent release of endogenous dopamine from slices of caudate-putamen. This effect was Ca2+-dependent, strongly inhibited by 1.2 mM Mg2+, attenuated by DL-APH (1 mM), attenuated by TTX (0.3 microM), and enhanced by nomifensine (10 microM). In the presence of nomifensine DADL (50 nM) reduced significantly the L-Glu-evoked release of endogenous dopamine by 20%. The inhibitory effect of DADL was blocked by 10 microM naloxone. These results indicate that L-Glu stimulates the Ca2+-dependent release of endogenous dopamine in the caudate-putamen by activation of N-methy-D-aspartate-type of excitatory amino acid receptors. This release can be selectively modified by the delta-opioid agonist DADL in a naloxone-sensitive manner.

Modification of brain acetylcholine release by morphine and its antagonists in normal and morphine-dependent rats.

1 The spontaneous release of acetylcholine (ACh) from the cerebral cortex of control and morphine-dependent rats was investigated. The rate of resting output of ACh in morphine-dependent animals was lower than that in the control animals.2 Administration of naloxone and nalorphine to morphine-dependent rats was followed by a significant rise in the release of cortical ACh. In control rats no such increase in the release of ACh occurred after similar injections of narcotic antagonists.3 Injections of morphine produced a consistent decrease in the rate of spontaneous release of cortical ACh in the control rats, but similar injections in the dependent rats did not produce a decrease in the rate of cortical ACh release.4 The relevance of these results with regard to development of the narcotic abstinence syndrome is discussed.

Morphine-naloxone interaction in the central cholinergic system: the influence of subcortical lesioning and electrical stimulation.

1 The opiate antagonist naloxone, injected or topically applied to the cerebral cortex, had no significant effect on the spontaneous output of cortical acetylcholine (ACh) in rats. 2 Morphine (2.5 mg/kg) administered intravenously inhibited the release of cortical ACh. A subsequent injection of naloxone rapidly reversed morphine-induced inhibition, and produced a sustained increase in the release of ACh. Topical application of naloxone solutions, after morphine, produced a slow and weak reversal of its inhibitory action. 3 Destruction of the medial thalamus abolished both the inhibitory effects of morphine on the cortical ACh release, and its antagonism by naloxone administered after the agonist. 4 Injection of naloxone in a low dose (0.1 mg/kg) increased the release of cortical ACh provoked by electrical stimulation of either the medial thalamus or the reticular formation in normal rats. In the morphine-dependent rat, naloxone also facilitated the evoked release and its action was greater than in control animals. The facilitatory effect of naloxone on the cortical release evoked by stimulation of the medial thalamus was greater than its effect on the release evoked by stimulation of the reticular formation in both normal and morphine-dependent rats. 5 Naltrexone, a narcotic antagonist, also facilitated the electrically stimulated release of cortical ACh. 6 It is suggested that (a) morphine and naloxone act at a subcortical site, probably the medial thalamus, to modify the cortical ACh release and that (b) naloxone may facilitate the electrically-induced release of ACh in the CNS by antagonizing the effect of the endogenous morphine-like factor, enkephalin.

Stereospecific enhancement of evoked release of brain acetylcholine by narcotic antagonists.

1--Electrical stimulation of nerves in the forepaw of anaesthetized rats caused an increase in the release of acetylcholine (ACh) from the cerebral cortex in vivo. Actions of naloxone (Nal) enantiomers and naltrexone (Ntx) were tested on this release in normal animals and those lacking pituitary gland for 3 weeks. 2--In normal animals systemic administration of (-)-Nal or Ntx, but not (+)-Nal caused a significant increase in the evoked release of ACh. Spinally administered (-)-Nal did not produce this effect. Cortical application of (-)-Nal produced a smaller increase in the evoked release of ACh. 3--In hypophysectomized rats the stimulatory action of (-)-Nal or Ntx on ACh release was significantly reduced. The ability of (-)-Nal to reverse inhibitory action of morphine or the enkephalin (FK 33,824) was not affected by hypophysectomy. 4--It is suggested that (-)-Nal and Ntx increase the stimulated release of cortical ACh by blocking the inhibitory action of an endogenous opioid at a subcortical site. An intact pituitary appears essential for a full expression of the Nal effect on evoked ACh release.