Tricyclic Imipramine Modification of the Circadian Rhythms of Hypothalamic Serotonin, its Precursors and Acid Catabolite in Individually Housed Rats

Circadian rhythms of serotonin (5HT), its precursors tryptophan (TP) and 5-hydroxy-tryptophan (5HTP) and its acid catabolite 5-hydroxy-indoleacetic acid (5HIAA), were determined in the hypothalamus of control rats and rats which had been treated continuously with subcutaneous imipramine (10 mg/kg/day) for 2 weeks.

Rats were individually housed and entrained to LD12:12. Controls showed the 5HT and TP peaks in the light and dark periods respectively, as reported in the literature, but no inverted correlation (antiphase) between SHT and 5HIAA rhythms.

Imipramine significantly modified circadian rhythm characteristics: the 5HT acrophase was advanced, that of TP and 5HIAA was delayed. Imipramine also significantly increased hypothalamic SHT and TP concentrations.     

Chronic administration of clomipramine inhibits morphine hot plate analgesia

Clomipramine, chronically administered in mice, for 3 days, inhibits partially but significantly morphine analgesia in the hot plate test, when used at dose of 10 mg/kg/day, i.p.; 2.5 and 5 mg/kg/day were ineffective. Neither higher doses (20 and 40 mg/kg/day) nor longer duration of pretreatment (8 and 16 days) modified the intensity of this inhibition. Reduction in morphine analgesia was obtained after a 24h delay between the last injection of clomipramine and that of morphine (30 min before testing), while clomipramine did not induce any antinociceptive effect and clomipramine and desmethylclomipramine plasma and brain levels were low or undetectable. These results provide new evidence for the interaction between clomipramine and the endogenous opiate system. A pharmacokinetic interaction between clomipramine and morphine was excluded; involvement of change in opiate and 5 HT2 receptors by chronic administration of clomipramine is discussed.     

Effect of buspirone and its metabolite 1-(2-pyrimidinyl)-piperazine on hippocampal serotoninergic system, studied in freely moving rats.

The effects of the anxiolytic drug buspirone and its metabolite 1-(2-pyrimidinyl)-piperazine (1PP) were studied on the serotoninergic system in the hippocampus of freely moving rats. Pulse voltammetry was used in association with chronically implanted carbon fiber microelectrodes to record 5HIAA, the serotonin metabolite in the extracellular space, almost continuously. Buspirone, 2.5 mg/kg i.p. was ineffective, but the dose of 10 mg/kg lowered 5HIAA between about 45 and 150 min; the same decrease was obtained with 40 mg/kg. This effect can be explained by an agonistic action on 5HT1 A receptors. The metabolite 1PP, which displays alpha 2 adrenoceptor blocking properties, either had no effect or raised extracellular 5HIAA, depending on the dose (1.5 or 6 mg/kg). The rapid metabolization of buspirone to 1PP can thus explain the short time course of the drug effect. Pretreatment with 1PP could only partially prevent buspirone's effect on the serotoninergic system.     

Substance P levels in various regions of the rat central nervous system after acute and chronic morphine treatment

Substance P levels were measured in various CNS regions from rats treated acutely and chronically with morphine. There was no observable effect in the group treated with an acute dose of morphine (10 mg/kg) and sacrificed after 2 h. After 35 days chronic treatment with increasing doses of the drug, the rats were divided into three groups and sacrificed 2 h, 24 h and 7 days after the last injection. The substance P level was increased in the corpus striatum 2 h and 24 h and in the medulla oblongata and dorsal part of the spinal cord 2 h after withdrawal. Seven days after the last injection the levels had returned to normal in these areas. No effects were observed in the cerebral cortex, the hypothalamus or the ventral spinal cord at any time of measurement. Earlier studies have demonstrated that morphine inhibits release of substance P. The observed increase in tissue levels after long-term treatment is therefore interpreted as an accumulation of substance P in the neurons.     

Monoamine Metabolism in the Locus Coeruleus Measured Concurrently with Behavior During Opiate Withdrawal: An In Vivo Microdialysis Study in Freely Moving Rats

Abstract: Using microdialysis, changes in monoamine metabolism were monitored in the locus coeruleus of freely moving rats during opiate withdrawal concomitantly with behavioral symptoms. Rats were infused with morphine (2 mg/kg/h, s.c.) or saline for 5 days and challenged with naltrexone (100 mg/kg, s.c.) on day 6. Following naltrexone challenge, the classic behavioral symptoms of morphine withdrawal were observed in rats treated with morphine but not in saline-infused rats. In morphine-dependent rats, naltrexone induced a marked increase (280%) in dialysate concentrations of 3,4-dihydroxyphenylacetic acid, an index of the functional activity of the noradrenergic neurons in the locus coeruleus. The local concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were also increased (70%) during morphine withdrawal. Taken together, these results (a) confirm in unanesthetized rats the hypothesis of an activation by opiate withdrawal of noradrenergic neurons in the locus coeruleus and (b) suggest an increase in serotonergic transmission in the same nucleus during morphine withdrawal.     

Hypothalamic self-stimulation during the abstinence syndrome in morphine-dependent rats

Rats with lateral hypothalamic self-stimulation were treated chronically with morphine (30 injections in the course of 15 days) in doses increasing stepwise from 20 to 120 mg/kg per injection. Morphine facilitated self-stimulation from the 9th injection. Both short-term abstinence (16-18 hours) and cessation of the narcotic resulted in inhibition of the response. Full suppression of self-stimulation occurred under the administration of nalorphine, morphine antagonist, in a dose of 5 mg/kg.     

Tolerance develops to the haloperidol-induced increase in the efflux of dopamine metabolites from the brains of unanesthetized, freely-moving rats

The lateral cerebral ventricles of freely moving rats were perfused by means of chronically implanted push-pull cannulae every second day for 2 weeks. Perfusates were analyzed for metabolites of dopamine [dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] and of 5-hydroxytryptamine [5-hydroxyindoleacetic acid (5HIAA)] using high performance liquid chromatography and an amperometric detector. Rats received daily subcutaneous injections of haloperidol (1 mg/kg) or its vehicle. After the first injection of haloperidol the concentrations of DOPAC and HVA were markedly increased while that of 5HIAA was unchanged. Complete tolerance developed to the haloperidol-induced increased efflux of dopamine metabolites by day 9, although a higher dose of haloperidol (2 mgf/kg) on day 15 was still capable of eliciting a modest increase in the efflux of DOPAC and HVA.     

Prostaglandin synthesis inhibition and monoamine metabolites in the rat brain.

The effect of indomethacin 3 mg/kg on levels of homovanillic acid (HVA), 4-hydroxy-3-methoxy phenyl ethylene glycol (HMPG) and 5-hydroxy indol acetic acid (5HIAA) was studied in rat striatum and olfactory tubercle with and without pretreatment with morphine 10 mg/kg. Indomethacin caused a small decrease in resting levels of HVA in striatum but not in olfactory tubercle. No effects were seen on resting or morphine induced changes in the levels of these monoamine metabolites. Likewise indomethacin 20 mg/kg failed to alter the elevation of HVA induced by chlorpromazine 15 mg/kg or the decrease of HVA induced by apomorphine (1--10 mg/kg) in the rat striatum. Our results do not support a major role for endogenous prostaglandins in the modulation of monoamine neurotransmission in the rat brain.     

Chronic treatment with high doses of haloperidol fails to decrease the time course for the development of depolarization inactivation of midbrain dopamine neurons

Using extracellular single unit recording techniques, we investigated the effects produced by chronic treatment with high doses of haloperidol (CHAL, 5 mg/kg/day, s.c.) on midbrain dopamine (DA) neuronal activity. This regimen of HAL treatment produced a time-dependent reduction in the number of spontaneously active DA neurons. Additionally, this dose regimen induced an irregular firing pattern in many of the remaining active DA neurons in both the ventral tegmental area (A10) and substantia nigra pars compacta (A9) regions. These effects were comparable to those obtained previously in rats treated chronically with lower doses of HAL (0.5 mg/kg/day, s.c.). However, there was a greater decrease in the number of spontaneously active DA cells detected in rats treated with high doses of HAL for three weeks compared to those receiving the low doses. On the other hand, higher doses of apomorphine (200 micrograms/kg, i.v.) were required to reverse both the reduction of DA activity and irregular discharge pattern in rats treated chronically with high doses of HAL. In conclusion, the results of the present study substantiate the view that CHAL-induced depolarization inactivation (DI) of DA neurons is a time-dependent process and chronic treatment with high doses of HAL did not shorten the time course required for the development of DI on the majority of midbrain DA neurons.     

In vivo characterization of the opioid antagonist nalmefene in mice

AimsThe current study assessed the in vivo antagonist properties of nalmefene using procedures previously used to characterize the opioid antagonists naloxone, naltrexone, 6β-naltrexol and nalbuphine.Main methodsICR mice were used to generate antagonist dose–response curves with intraperitoneal (i.p.) nalmefene against fixed A₉₀ doses of morphine in models of morphine-stimulated hyperlocomotion and antinociception. Additional dose–response curves for antagonist precipitated opioid withdrawal were run in mice treated acutely (100 mg/kg, s.c., ? 4 h) or chronically (75 mg pellet, s.c., ? 72 h) with morphine. Comparisons were made between antagonist potency and degree of precipitated withdrawal.Key findingsNalmefene produced dose- and time-related antagonism of morphine-induced increases in locomotor activity with a calculated ID₅₀ (and 95% confidence interval) of 0.014 (0.007–0.027) mg/kg. Nalmefene produced rapid reversal of morphine-induced locomotor activity (5.1 min for 50% reduction in morphine effect). A 0.32 mg/kg dose of nalmefene produced blockade of morphine-induced antinociception in the 55 °C tail-flick test that lasted approximately 2 h. Nalmefene was able to potently precipitate withdrawal in mice treated acutely or chronically with morphine.SignificanceThese results demonstrate that nalmefene is similar to naloxone and naltrexone with respect to its in vivo pharmacology in mice. Specifically, nalmefene produces potent antagonism of morphine agonist effects while precipitating severe withdrawal. The compound has a slower onset and longer duration of action compared to naloxone and naltrexone. The data allows for a more complete preclinical comparison of nalmefene against other opioid antagonists including the putative opioid neutral antagonist 6β-naltrexol.     

Stereospecific aversive property of narcotic antagonists in morphine-free rats

If endogenous, morphine-like substances have physiological functions, narcotic antagonists should have effects $\text{in vivo}$ even in the absence of exogenous, narcotic agonists. This hypothesis was supported by studies of taste aversions conditioned with narcotic antagonists; rats drank smaller amounts of distinctively flavoured solutions when their consumption on previous occasions preceded injections of naloxone (1–10 mg/kg), naltrexone (3.2 mg/kg), Mr 1452 (10 mg/kg) or (-)-BC-2860 (10 mg/kg). Stereoisomers (i.e. Mr 1453, (+)-BC-2860) which were inactive as narcotic antagonists did not induce significant taste aversions. It was suggested that the consistency and stereospecificity of aversion with the antagonists gave some support to interpretations in terms of antagonist actions at receptors for endogenous opioids.     

Diazepam increases 5-hydroxyindole concentrations in rat brain and spinal cord

Diazepam elevates serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) concentrations in rat brain and spinal cord. The maximal effect occurs 1–2 hrs after drug injection and is dose related between 5–20 mg/kg (intraperitoneal). The action of diazepam on brain 5HT and 5HIAA concentrations is modified by previous food consumption: the ingestion of a diet that raises brain 5HT and 5HIAA one hour before drug injection enhances the diazepam-induced increase in brain indoles; consumption of a diet that lowers brain 5HT and 5HIAA partially blocks the elevation in brain indoles that follows diazepam injection.     

The effect of pentobarbital on the antinociceptive action of morphine in morphine-tolerant and non-tolerant rats

The interaction of sodium pentobarbital with morphine sulfate in both morphine-tolerant and non-tolerant rats was investigated using the tail-compression test for analgesia. Male Sprague-Dawley rats (300–350 g) were given pentobarbital (4, 8, or 16 mg/kg) 5 min before morphine (2, 4, 6, or 8 mg/kg). Control animals received two saline injections, or pentobarbital plus saline, or saline plus morphine. All injections were subcutaneous. Prior to the first injection, a baseline nociceptive threshold was determined for each rat by applying a modified micrometer to its tail and increasing the pressure until a squeak was elicited. Test readings were taken every half-hour for 2 hr beginning 30 min after the second injection. For the chronic studies, animals were first made tolerant to morphine by the administration of the narcotic twice a day for 3 days, increasing the dose from 10 to 50 mg/kg/injection. Identical testing procedures were then followed with these rats except that the test dose of morphine given on day 4 was in the range 8–128 mg/kg. It was found that Na pentobarbital, in the subanesthetic doses used, had neither antinociceptive nor hyperalgesic properties. Furthermore, the barbiturate had no effect on the antinociceptive action of morphine in either morphine-tolerant or non-tolerant rats.     

Effects of exogenous cholecystokinin octapeptide on acquisition of naloxone precipitated withdrawal induced conditioned place aversion in rats

Cholecystokinin octapeptide (CCK-8), a gut-brain peptide, regulates a variety of physiological behavioral processes. Previously, we reported that exogenous CCK-8 attenuated morphine-induced conditioned place preference, but the possible effects of CCK-8 on aversively motivated drug seeking remained unclear. To investigate the effects of endogenous and exogenous CCK on negative components of morphine withdrawal, we evaluated the effects of CCK receptor antagonists and CCK-8 on the naloxone-precipitated withdrawal-induced conditioned place aversion (CPA). The results showed that CCK2 receptor antagonist (LY-288,513, 10 μg, i.c.v.), but not CCK1 receptor antagonist (L-364,718, 10 μg, i.c.v.), inhibited the acquisition of CPA when given prior to naloxone (0.3 mg/kg) administration in morphine-dependent rats. Similarly, CCK-8 (0.1-1 μg, i.c.v.) significantly attenuated naloxone-precipitated withdrawal-induced CPA, and this inhibitory function was blocked by co-injection with L-364,718. Microinjection of L-364,718, LY-288,513 or CCK-8 to saline pretreated rats produced neither a conditioned preference nor aversion, and the induction of CPA by CCK-8 itself after morphine pretreatments was not significant. Our study identifies a different role of CCK1 and CCK2 receptors in negative affective components of morphine abstinence and an inhibitory effect of exogenous CCK-8 on naloxone-precipitated withdrawal-induced CPA via CCK1 receptor.     

Prostaglandin synthesis inhibition and monoamine metabolites in the rat brain

The effect of indomethacin 3 mg/kg on levels of homovanillic acid (HVA), 4-hydroxy-3-methoxy phenyl ethylene glycol (HMPG) and 5-hydroxy indol acetic acid (5HIAA) was studied in rat striatum and olfactory tubercle with and without pretreatment with morphine 10 mg/kg. Indomethacin caused a small decrease in resting levels of HVA in striatum but not in olfactory tubercle. No effects were seen on resting or morphine induced changes in the levels of these monoamine metabolites. Likewise indomethacin 20 mg/kg failed to alter the elevation of HVA induced by chlorpromazine 15 mg/kg or the decrease of HVA induced by apomorphine (1–10 mg/kg) in the rat striatum. Our results do not support a major role for endogenous prostaglandins in the modulation of monoamine neurotransmission in the rat brain.     

Dietary serotonin and alcohol combined may provoke adverse physiological symptoms due to 5-hydroxytryptophol

The urinary excretion products of serotonin (5-hydroxytryptamine, 5HT) are 5-hydroxyindole-3-acetic acid (5HIAA) and 5-hydroxytryptophol (5HTOL), and the ratio of 5HTOL to 5HIAA is normally very low (< 0.01 ) in man. Intake of foods rich in 5HT (high amounts in banana, pineapple, and walnuts) induces a general increase in the output of 5HT metabolites, without affecting the 5HTOL/5HIAA ratio. In contrast, during metabolism of ethanol there is a shift in the catabolic pattern of 5HT, and the formation of 5HTOL increases appreciably at the expense of 5HIAA. Accordingly, the urinary 5HTOL/ 5HIAA ratio increases and does not recover to baseline levels until several hours after ethanol has been cleared from the body. When 10 healthy subjects ingested a moderate dose of ethanol (0.5 g/kg), the urinary 5HTOL/SHIAA ratio was increased approximately 70-fold on average at 4 h after intake. When the same amount of ethanol was ingested together with 3 bananas (approximately 10 mg 5HT), this ratio was increased approximately 100-fold at 4 h and still significantly higher than baseline levels at 24 h. Starting at 3-4 h after the combined intake of ethanol and banana, 7 subjects experienced one or more unpleasant symptoms (diarrhea, headache, and fatigue) which are associated with the 5HT system. The events were transient but typically lasted for several hours, and the duration correlated with the time period during which 5HTOL levels were raised. Intake of ethanol and banana separately produced much lower increases in 5HTOL output and caused no corresponding effects. This observation indicate that dietary 5HT intake together with even a moderate dose of ethanol can provoke unpleasant physiological symptoms. The symptoms may be attributed to the high concentration of 5HTOL.     

Roles of 5-HT3 and opioid receptors in the ethanol-induced place preference in rats exposed to conditioned fear stress.

The effect of the selective 5-HT3 receptor antagonist ondansetron on the ethanol-induced place preference in rats exposed to conditioned fear stress, which stimulates the release of endogenous opioid peptides (beta-endorphin and enkephalins), was investigated using the conditioned place preference paradigm. In addition, we also examined the effect of ondansetron on the ethanol-induced place preference enhanced by the administration of mu- and delta-opioid receptor agonists (exogenous opioids). The administration of ethanol (300 mg/kg, i.p.) induced a significant place preference in rats exposed to conditioned fear stress. Pretreatment with ondansetron (0.01 and 0.1 mg/kg, s.c.) effectively attenuated this ethanol-induced place preference. When the mu-opioid receptor agonist morphine (0.1 mg/kg, s.c.) or the selective delta-opioid receptor agonist 2-methyl-4a(alpha)-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a(alpha)-octah ydroquinolino [2,3,3-g] isoquinoline (TAN-67; 20 mg/kg, s.c.) was administered in combination with 75 mg/kg ethanol (which tended to produce a place preference), the ethanol-induced place preference was significantly enhanced. The selective mu-opioid receptor antagonist beta-funaltrexamine at a dose of 10 mg/kg significantly attenuated the enhancement of the ethanol-induced place preference produced by morphine. Ondansetron (0.1 mg/kg, s.c.) also significantly attenuated the enhancement of the ethanol-induced place preference produced by morphine. Furthermore, the selective delta-opioid receptor antagonist naltrindole at a dose of 3 mg/kg significantly attenuated the enhancement of the ethanol-induced place preference produced by TAN-67. Ondansetron (0.1 mg/kg, s.c.) slightly, but significantly, attenuated the enhancement of the ethanol-induced place preference produced by TAN-67. These results suggest that 5-HT3 receptors may be involved in the rewarding mechanism of ethanol under psychological stress, and may play an important role in the rewarding effect of ethanol through the activation of mu- and delta-opioid receptors.     

Activation of bulbospinal serotonergic neurons during cold exposure.

In a four-part study, we expand on our previous report that bulbospinal serotonin (5HT) neuronal activation occurs with 24 h of cold exposure. To characterize temporal aspects, rats were exposed to 3 degrees C or were maintained at 22 degrees C for 2, 8, 48, or 96 h (experiment 1) or for 15, 30, or 60 min (experiment 2). To ensure that cold-induced changes in 5HT activity were not due to disturbances in diurnal pattern, rats in experiment 3 were exposed to cold (8 h) during the dark cycle. To explore the hypothesis that cold-induced 5HT activation is part of a broad metabolic response that includes activation of the sympathetic nervous system, metabolically impaired (hypothyroid) rats were exposed to 8 degrees C in experiment 4. Significant increments in 5-hydroxyindoleacetic acid (SHIAA) concentration were evident by 60 min of cold exposure and existed at all later time points measured. These findings were most robust in spinal cord and rostral brainstem. Activation in spinal cord was also found when rats were exposed to 8 h of cold during the dark cycle, the active period for rats. In experiment 4, hypothyroid rats exhibited significantly greater norepinephrine excretion compared with control rats exposed to the same cold stimulus; this finding was accompanied by significantly greater increments in 5HIAA concentration in rostral brainstem and spinal cord of hypothyroid rats. In addition, significant elevations in tryptophan concentration were noted throughout the brainstem and spinal cord of cold-exposed, hypothyroid rats relative to room temperature, hypothyroid rats. This finding suggested that elevations in 5HIAA concentration in these rats were due to increases in precursor availability. The implications of these findings relative to autonomic and metabolic control are discussed.     

Investigations on drug produced and subjectively experienced discriminative stimuli. 2. Loperamide, an antidiarrheal devoid of narcotic cue producing actions.

By means of a new behavioral procedure effectively controlling the narcotic stimulus properties of drugs, it was found that the oral administration of fentanyl, morphine, codeine and diphenoxylate produced the narcotic cue. The new antidiarrheal loperamide did not produce this cue in rats as sensitive as being able to detect 0.005 mg/kg s.c. fentanyl. The results are discussed in terms of drug abuse liability.