Evidence for the involvement of presynaptic cholinergic functions in tolerance to diisopropylfluorophosphate

Rats were treated with diisopropylfluorophosphate (DFP) acutely or daily for 14 days. The involvement of various presynaptic and postsynaptic functions of the cholinergic system in the development of tolerance to DFP was studied. Receptor density and affinity of both muscarinic and nicotinic receptors, high-affinity choline uptake, and [K+]-evoked release of acetylcholine (ACh) by atropine were not changed after acute administration of 2 mg/kg DFP. Both muscarinic and nicotinic receptors were down-regulated to the same extent (40-50%) after subacute administration of DFP (1 mg/kg) without changes in their affinities. Binding sites of muscarinic receptors were maximally decreased after 7 days of DFP administration. Thereafter, they remained constant throughout 14 days of administration. One hour after the last injection of 2 mg/kg DFP to subacutely treated rats, the maximum velocity of high-affinity choline uptake was significantly decreased in the striatum (33%) and hippocampus (53%) without changes in Km values. Twenty-four hours after the last injection of DFP, only a higher dose of DFP (2 mg/kg) significantly inhibited choline uptake. Potassium-evoked release of ACh by slices of striatum was not different between acutely and subacutely treated rats. However, the release of ACh by slices of striatum and hippocampus was significantly increased by atropine in subacutely treated rats. It is suggested that along with the down-regulation of the postsynaptic receptors, subsensitivity of presynaptic functions of the cholinergic synapse also develops during subacute administration of DFP.     

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.     

Effect of the ''antidementia drug'' pantoyl-GABA on high affinity transport of choline and on the contents of choline and acetylcholine in rat brain.

1. Effect of pantoyl-gamma-aminobutyric acid (pantoyl-GABA) on high affinity transport of choline into synaptosomes and on the choline (Ch) and acetylcholine (ACh) concentrations of rat brain were studied. 2. Pantoyl-GABA was injected intraperitoneally four times at a dose of 500 mg kg-1 at intervals of 30 min. One hour after the last injection, rats were killed by decapitation for measurement of high affinity transport of Ch into synaptosomes or by microwave irradiation for the measurement of Ch and ACh concentrations. 3. Transport of Ch was increased into synaptosomes prepared from the cerebral cortex and hippocampus, but not into those from the striatum. 4. In the cerebral cortex and hippocampus, Ch concentration was increased and ACh concentration decreased. 5. Since treatments that enhance the activity of cholinergic neurones in vivo are reported to increase high affinity transport of Ch measured in vitro, the present results suggest that pantoyl-GABA may increase cholinergic activity in vivo. This action of the drug may be related to changes in the Ch and ACh concentrations.     

Brain acetylcholine in morphine pellet implanted rats given naloxone

Adult male rats were implanted with intraventricular (ivt.) brain cannulae for injection of 5 g of acetylseco-hemicholinium-3 (acetylseco HC-3) as a means of studying acetylcholine (ACh) utilization during morphine withdrawal. Animals were made dependent by implanting s.c. two 75 mg morphine base pellets 24 hrs apart. On the 4th day animals were given 10 mg/kg of naloxone i.p. and/or 5 g acetylseco HC-3 ivt. and sacrificed by decapitation at various times. The brains were removed and assayed for ACh using a pyrolysis gas Chromatographie procedure. Total brain ACh before or after acetylseco-HC-3 was not altered at 5, 30, 60 and 120 but was decreased at 10 min after naloxone. These results are in sharp contrast to our previous data of enhanced brain ACh utilization in withdrawn rats made dependent to morphine by several weeks of twice daily injections. It is apparent that short term morphine pellet administration does not produce the marked neurochemical and behavioral changes of long term morphine injections.Supported in part by grant DA 00830, USPHS.     

Effects of morphine on choline acetyltransferase levels in the caudate nucleus of the rat

1. Choline acetyltransferase (choline-o-acetyltransferase 2.3.1.6.) concentrations were determined in the caudate nucleus, thalamus, and cortex of control and morphine treated rats. The enzyme was assayed using a modified radiochemical method on a number of selected days, one hour after the last injection of 30 mg/kg of morphine and also during the subsequent phase of abstinence from morphine.2. Significant lowering of choline acetyltransferase activity in the caudate nucleus area was found in two cases, one hour after the first dose of morphine and upon subsequent abstinence from morphine.3. The enzyme activity in the two other parts of the brain remained at the normal levels.4. The presence of endogenous inhibitors formed during morphine administration was excluded.5. The relationship of a possible effect of morphine on the tissue binding of the enzyme and the subsequent lowering of its activity was tested by homogenization of the caudate nucleus area in different media. The decrease in enzyme activity occurred in all extraction media one hour after morphine administration.6. Inhibitory effects of in vitro addition of morphine to caudate nucleus homogenate, obtained from normal and morphine treated rats, were found to occur only at very high concentrations of the drug, negating the possibility of direct inhibitory effects of morphine.7. These experiments suggest two possible causes of the observed effects, which can be responsible for the lowering of enzyme activity, and can be operative simultaneously: (1) a negative feedback mechanism of accumulated acetylcholine, occurring after the first dose of morphine, and (2) the possible changes in enzyme configuration produced by morphine treatment.     

Morphine-induced opioid receptor down-regulation detected in intact adult rat brain cells

Intact brain cells dissociated from the brains of adult rats were used to study the regulation of opioid receptors by in vivo chronic morphine treatment. When the specific binding of [3H]naloxone in a physiological iso-osmotic buffer was compared in cells obtained from sham-operated rats and in those treated for 6 days with four (2 X 2) 75 mg morphine pellets, it was found that morphine treatment resulted in a significant reduction in the density of [3H]naloxone binding sites. This receptor down-regulation was not accompanied by a change in the receptor affinity for the ligand. This effect of morphine was reversed upon the removal of morphine pellets for 18 h after tolerance induction. When similar experiments were performed using brain homogenates prepared and assayed in the same physiological buffer, there was an increase in the number of [3H]naloxone binding sites in morphine-treated animals compared to controls. On the other hand, when the binding experiments were conducted in 50 mM Tris-HCl buffer, no difference in ligand binding was apparent between control and morphine-treated groups. The present results demonstrate opioid receptor down-regulation by chronic morphine treatment measured in intact brain cells, and suggest that different conclusions may be reached when other tissue preparations are used to assess the receptor density.     

The physical dependence liabilities of oxybutynin and morphine in rats

The anticholinergic/antispasmodic agent oxybutynin does not induce physical dependence in rats when administered by oral gavage twice daily for 40 days; nor did challenge with naloxone precipitate withdrawal signs in these (oxybutynin-treated) animals. In contrast, morphine treatment resulted in a high degree of physical dependence as evidenced by the withdrawal signs noted after treatment was halted. Challenge with naloxone also induced severe withdrawal signs in morphine-treated rats. Withdrawal signs characterised by squealing, teeth chattering and "wet-dog" shakes were seen in one from five morphine-treated rats challenged with oxybutynin.     

Discrete cues paired with naloxone-precipitated withdrawal from acute morphine dependence elicit conditioned withdrawal responses

Acute bolus doses of morphine induce a state of acute opioid dependence as measured by naloxone-precipitated withdrawal. Repeated morphine and precipitated withdrawal experience further enhances naloxone-induced withdrawal severity, partly because of direct neuroadaptation to repeated morphine, and partly because of conditioned associations of context and withdrawal experience. To determine whether a discrete tone/light conditioned stimulus could elicit conditioned withdrawal responses in acute dependence, rats trained on a fixed-ratio-15 operant schedule for food reward received morphine (5.6 mg/kg) 4x at daily or weekly intervals, with each morphine injection followed at 4 h by naloxone (1.0 mg/kg) and an operant session. The conditioned stimulus was presented to a Paired group after each naloxone injection. Separate control groups experienced the conditioned stimulus either at a different time of the day or on a different day of the week than naloxone (Unpaired), received naloxone without any conditioned stimulus exposure [Paired-no conditioned stimulus (Paired-NO CS)] or received vehicle instead of naloxone before conditioned stimulus presentation (NaI-Naive). On the test day, all rats received vehicle before conditioned stimulus exposure. The conditioned stimulus alone reliably suppressed responding in Paired groups relative to control conditions with either daily or weekly intervals between conditioning sessions. The administration of morphine 4 h before conditioned stimulus exposure on the test day was not necessary to observe conditioned withdrawal. Thus, conditioned withdrawal is reliably established to discrete cues associated with naloxone-precipitated withdrawal from acute, infrequent (weekly) opioid exposure.     

Primary sensory neurones and naloxone-precipitated morphine withdrawal.

1. The C-fibre-evoked depressor reflex following i.a. injection of capsaicin and the wiping movements following chemical irritation of the cornea by capsaicin were both found to be augmented in the naloxone-precipitated morphine withdrawal phase. 2. The in vitro capsaicin-evoked release of substance P from central terminals of C-fibre afferents in the spinal cord was decreased in morphine-treated rats. Following naloxone, the release in the morphine-treated group was as large as in the control group. 3. The C-fibre mediated plasma extravasation in the rat paw after naloxone was found to be the same in morphine-treated and control rats. 4. It is concluded that C-fibre-evoked reflex reactions are augmented during morphine withdrawal, but that the mechanisms responsible are either located postsynaptically to the primary sensory neurone or further centrally.     

Systemic morphine simultaneously decreases extracellular acetylcholine and increases dopamine in the nucleus accumbens of freely moving rats.

Microdialysis was used to measure extracellular levels of acetylcholine (ACh) and dopamine (DA) simultaneously in the nucleus accumbens (NAC) of freely moving rats. Systemic injection of morphine (20 mg/kg) significantly decreased ACh (30%, p less than .01) while it increased DA (55%, p less than .01). The effects of morphine were eliminated by naloxone. The results confirm that morphine increases DA and in addition, demonstrate an inhibitory influence of this opiate on extracellular levels of ACh in the NAC.     

Stimulation mechanism of guinea pig liver-mediated reduction of naloxone by morphine

The mechanism of the stimulatory effect of morphine on the reduction of naloxone has been elucidated using guinea pig liver naloxone reductase that is identical with morphine 6-dehydrogenase. The reaction products were quantitated by means of HPLC. When naloxone was incubated with the enzyme in the presence of NAD(P)H at pH 7.4 or pH optima, the production of 6 alpha-naloxol increased according to the added amount of morphine. The stimulation was predominant with NADH at pH 7.4. Under these conditions, the production of morphinone also increased in proportion to the amount of morphine. The enzymatic reduction of naloxone proceeded even if NAD(P)H was replaced by NAD(P)+ and morphine. At a fairly low concentration of NADH (0.01 mM), the enzyme produced 6 alpha-naloxol (0.3 mM), exceeding the stoichiometric amount in the presence of 16 mM morphine. Although the Vmax values for naloxone was increased by the addition of morphine, the Km values for naloxone remained unaltered. Besides other substrates for guinea pig liver morphine 6-dehydrogenase such as codeine, normorphine and ethylmorphine also enhanced the reduction of naloxone. From these results we concluded that the stimulation of guinea pig liver-mediated reduction of naloxone by morphine is caused by the acceleration of the redox of pyridine nucleotides conducted by the enzyme. These phenomena were further supported by the experiments with the liver cytosol. In addition, we confirmed that, in the guinea pig, the biliary excretion of the metabolites, naloxol and naloxol-3-glucuronide, increased after sc injection of naloxone with morphine.     

Cholinergic system of brain tissue in rats poisoned with the organophosphate, 0,0-dimethyl 0-(2,2-dichlorovinyl) phosphate

The cholinergic system of the brain was investigated in rats acutely poisoned with the organophosphate, 0,0-dimethyl 0-(2,2-dichlorovinyl) phosphate (DDVP), (6 mg/kg, sc, with saline as a control). The amounts of three fractions of acetylcholine (ACh)--free (extraterminal), labile-bound (intraterminal/cytoplasmic), and stable-bound (intraterminal/vesicular)--increased in the rats over a period of 5 to 60 min after injection of DDVP, showing peaks which were 2.45, 1.82, and 1.4 times as high as the respective control amounts. No difference was seen in the amount of any fraction of ACh between treated and control rats killed 3 and 24 hr after injection. Acetylcholinesterase (AChE) activity decreased to between 12 and 43% of the control over a period of 5 to 180 min and recovered almost completely within 24 hr after injection. No appreciable changes were seen in either spontaneous or potassium-induced ACh release in brain tissue slices obtained from rats treated with DDVP. ACh synthesis in slices was suppressed significantly 20 min, but not 24 hr, after injection of DDVP. In the brain crude synaptosomal preparation, high-affinity choline uptake, which is generally thought to be a rate-limiting step for ACh synthesis, was suppressed 20 min after DDVP. No appreciable changes were seen in high-affinity choline uptake at 24 hr low-affinity choline uptake, and choline acetyltransferase activity after injection of DDVP. These results suggest that ACh synthesis and high-affinity choline uptake may be in a suppressed state when ACh concentration, especially intraterminal ACh, is increased and AChE activity is decreased in the brain cholinergic system of rats poisoned with DDVP. The increase in the intraterminal ACh may be due to an inhibition of AChE activity at this site and/or a re-uptake of ACh in the synaptic cleft, not to an inhibition of ACh release or an increase in ACh synthesis.     

慢性给予吗啡诱导大鼠蓝斑核Fos蛋白表达

本文研究吗啡戒断大鼠蓝斑核Ｆｏｓ蛋白表达情况。采用连续５ｄｉｐ吗啡建立大鼠吗啡依赖模型，纳洛酮催促后对桥脑蓝斑核进行Ｆｏｓ蛋白的免疫细胞化学实验（ＩＣＣ）。结果发现吗啡组大鼠蓝斑核神经元Ｆｏｓ蛋白表达明显。这一结果从蛋白表达的角度证明蓝斑核在阿片类药物戒断中的作用机理。     

Actylcholine output from the cerebral cortex,choline uptake and muscarinic receptors in morphine-dependent,freely-moving rats

Spontaneous acetylcholine (ACh) output from the cerebral cortex, choline high affinity uptake and [³H]-QNB binding to muscarinic receptors in the cerebral cortex and caudate nucleus in freely moving rats made morphine-dependent by morphine pellet subcutaneous implantation were investigated before and during naloxone-induced withdrawal syndrome. The frequency and intensity of the withdrawal signs were also assessed.No significant change in ACh output was found in tolerant rats when compared with that of placebopellet implanted rats. During naloxone-induced withdrawal syndrome a 60% increase in ACh output occurred.In rats made dependent after a large septal lesion or treated for ten days with calcium gluconate (10 mg/kg i.m.) no increase in ACh output was found during the withdrawal syndrome. The intensity of some of its signs was also reduced.During the withdrawal syndrome a marked increase in choline high affinity uptake in the cerebral cortex and caudate nucleus was detected.The affinity of muscarinic receptors (K_D) for [³H]-QNB was significantly increased in the cerebral cortex and caudate nucleus of morphine-dependent rats before naloxone administration. It returned to normal during the withdrawal syndrome. In the caudate nucleus the number of binding sites (B_max) was decreased before and after the withdrawal syndrome.These findings emphasize the role of cholinergic mechanisms in opiate addiction.     

Effects of alpha4beta2 and alpha7 nicotinic acetylcholine receptor antagonists on place aversion induced by naloxone in single-dose morphine-treated rats

Acute dependence can be observed when naloxone is administered 24 h after even a single dose of morphine, and nicotine attenuates this naloxone-precipitated withdrawal syndrome. This acute dependence has been hypothesized to be associated with a dopaminergic mechanism. In the present study, the role of nicotinic acetylcholine receptor subtypes in the place aversion induced by naloxone in single-dose morphine-treated rats was investigated. Methyllycaconitine (1, 2 and 5 mg/kg), an alpha7 nicotinic acetylcholine receptor subtype inhibitor, significantly and dose dependently inhibited the attenuating effect of nicotine on naloxone-induced place aversion. In contrast, dihydroxy-beta-erithroidine (1, 2 and 5 mg/kg), an alpha4beta2 nicotinic acetylcholine receptor subtype inhibitor, did not have any effect on the attenuating effect of nicotine on naloxone-induced place aversion. These findings suggested that the alpha7 nicotinic acetylcholine receptor subtype is associated with the place aversion induced by naloxone in single-dose morphine-treated rats. Nicotinic acetylcholine receptor subtype inhibitors warrant further study as possible treatment for acute dependence.     

Action of 5-hydroxytryptamine on electrical activity of Auerbach's plexus in the ileum of the morphine dependent guinea pig

Guine pigs were made dependent to morphine by a daily s.c. injection of morphine hydrochloride. Half of them were given morphine hydrochloride (50 mg/kg) s.c. 30 min befor sacrifice; these animals were termed ‘morphine-treated’ guinea pigs. The animals in the abstinence syndrome were sacrificed 30 to 40 hr after the last morphine dose. Effective concentrations of 5-hydroxytryptmine on Auerbach's plexus of the guinea pig in the abstinence syndrome was significantly smaller than those on Auerbach's plexus of normal and morphine-treated animals, while the responses to nicotine and caerulein were unchanged. The results suggest that withdrawal of morphine excites the M-receptors in Auerbach's plexus.     

Chronopharmacological study of physical dependence on morphine in rats

Relationship between withdrawal time or naloxone injection time and withdrawal signs were examined in morphine-treated rats. Sixty-five rats were treated chronically with morphine-admixed food (1 mg/g food) for 7 days and were divided into 13 groups. The rats of 4 groups were abruptly withdrawn from morphine, and the rats of another 4 groups were given naloxone (3 mg/kg, s.c.) at 20:00 on the 8 th day and 2:00, 8:00 and 14:00 on the 9 th day after the morphine administration, respectively. Withdrawal signs were observed at intervals of 2 hr. After each naloxone injection, abnormal behaviors were observed for 60 min, and body weight was measured for 3 hr at intervals of 15 or 30 min. In the withdrawal test, weight loss at 24 hr after withdrawal in each group was approximately 10%, and there was no difference between each group. However, the body weight of non-treated rats and morphine-treated rats increased during the night period (20:00-8:00) and decreased during the daytime (8:00-20:00). Therefore, body weight reached the minimum at 20:00, and then this time is appropriated for withdrawal. In the naloxone test, withdrawal signs in the night period were more potent than that in the daytime. The withdrawal signs induced by naloxone at 8:00 showed the maximum magnitude. Plasma morphine levels in rats treated with morphine-admixed food were high in the night period and low in the daytime. These results suggest that the magnitude of naloxone-precipitated withdrawal signs depends on the amount of morphine in the plasma.     

Influence of hemicholinium (HC-3) on morphine analgesia, tolerance, physical dependence and on brain acetylcholine

Administration of HC-3 intracerebrally at a dose which reduced brain acetylcholine (ACh) without any change in choline (Ch) levels antagonized morphine antinociception slightly as measured by inhibition of the tail-flick response, in both naive mice and in mice rendered tolerant to and dependent on morphine by pellet implantation. However, the development of tolerance to morphine and of dependence on morphine were not affected by HC-3. Although naloxone precipitated withdrawal jumping was enhanced irrespective of whether the HC-3 was administered before or after the dependence on morphine had developed, body weight loss during abrupt withdrawal was unaffected by HC-3. The results indicate that although some of the acute and withdrawal effects of morphine may be associated either directly or indirectly with acetylcholine, these actions do not appear to be the primary process responsible for initiating the development of tolerance and dependence.     

Naloxone-precipitated withdrawal reveals sensitization to neurotransmitters in morphine tolerant/dependent rats

Summary Morphine tolerant/dependent rats were tested for their sensitivity to putative neurotransmitters or other receptor agonists injected intracerebroventricularly (i.c.v.) during naloxone-precipitated withdrawal. Dopamine, apomorphine, clonidine and serotonin were found to reinitiate withdrawal jumping behaviour when injected 30 min after naloxone. Dopamine and apomorphine also reinitiated jumping, but of a lesser intensity, when injected 3 h after naloxone-precipitated withdrawal. I.c.v. injection of acetylcholine or prostaglandin E₁ failed to reinitiate withdrawal jumping. In addition, all the above substances failed to induce jumping behaviour in naive rats or in morphine tolerant/dependent rats before naloxone-precipitated withdrawal. Morphine tolerance and dependence therefore appears to be associated with changes in the sensitivity of the CNS to putative neurotransmitter substances. These changes are best demonstrated during the sudden termination of opiate action that is caused by administration of naloxone.     

Sensitivity of morphine-tolerant rats to muscarinic and dopaminergic agonists: Relation to tolerance or withdrawal

Male hooded Wistar rats were treated chronically once daily with morphine sulfate (20 mg/kg SC) after completing a session in an operant chamber. Periodical challenges with morphine (8 mg/kg) prior to the operant session established that tolerance development was virtually complete within 10 days. The morphine-treated rats were more sensitive to the behavioral suppressing effects of apomorphine, a dopamine agonist, and pilocarpine, an acetylcholine agonist, when administered in the tolerant state (morphine given 30 min prior to operant session), regardless of whether they were administered peripherally or directly into the striatum. Conversely, the morphine-treated rats were less sensitive to both agonists when administered in the withdrawal state (morphine given 24 h prior to the operant session). In animals undergoing a similar regimen of chronic morphine treatment, receptor binding studies revealed a lowered affinity (Higher K _D apparent) for the dopamine receptor in the striatum of morphine-withdrawn rats, using ³H-spiroperidol as the ligand. The morphine-withdrawn rats also appeared to have fewer muscarinic cholinergic receptors (lower B _max), using ³H-QNB as the ligand. The also had a lower concentration of membrane-bound phosphodiesterase modulator protein. In general, no significant differences were observed for the above parameters in the morphine-tolerant rats. These behavioral and neurochemical studies are consistent with the view that morphine-tolerant rats are supersensitive to dopamine and acetylcholine agonists and morphine-withdrawn rats are subsensitive.