Nicotine withdrawal leads to increased sensitivity of serotonergic neurons to the 5-HT1A agonist 8-OH-DPAT

In order to explore the neurophysiology of nicotine withdrawal, we examined the activity of serotonergic neurons in the dorsal raphe nucleus in rats undergoing withdrawal from chronic exposure to nicotine. Animals were exposed to nicotine (6 mg/kg per day base) via SC implanted osmotic minipumps. After 12 days the pumps were removed and the animals allowed to go through spontaneous withdrawal. Rats were anesthetized on various days of the procedure and the effect of the 5-hydroxytryptamine (5-HT)-1A agonist 8-OH-DPAT on the single-unit activity of serotonergic neurons in the dorsal raphe nucleus was examined. The sensitivity of serotonergic neurons to 8-OH-DPAT was not changed by the chronic administration of nicotine or saline; slightly increased on day 2 of withdrawal; significantly increased on days 3 and 4 of withdrawal; and no longer significantly increased by day 7 of withdrawal. These results indicate that serotonergic neurons in the dorsal raphe nucleus have an increased sensitivity to systemically administered 8-OH-DPAT in rats undergoing nicotine withdrawal and that the serotonergic system may play a role in the symptoms of nicotine withdrawal. Received: 13 September 1996/Final version: 29 April 1997     

Orexin A excites serotonergic neurons in the dorsal raphe nucleus of the rat

Orexin A (10-300 nM) strongly excited dorsal raphe serotonergic neurons maintained in vitro. The depolarization persisted in the presence of tetrodotoxin (TTX, 0.5 microM) and was associated with an increase in input resistance. These results have relevance in the context of food intake regulation and the disease, narcolepsy.     

Effect of repeated amiflamine administration on serotonergic and noradrenergic neurotransmission: Electrophysiological studies in the rat CNS

Summary Amiflamine is a selective and reversible inhibitor of monoamine oxidase (MAO) type A which exerts a preferential effect on serotonin (5-HT) catabolism. The present studies were undertaken to compare the effects of repeated administration of amiflamine (2 mg/kg, twice daily) on several aspects of the functioning of the 5-HT and norepinephrine (NE) systems in the rat CNS. The activity of MAO-A and B was assessed in forebrain slices and the whole brain contents of the neurotransmitters and their metabolites were determined by HPLC after 2-, 7- and 21-day treatments. MAO-A was inhibited by about 50% 2 h after the last dose and its activity was back to normal in rats sacrificed 12 h after the last dose. The activity of MAO-B was unaffected two or 12 h after the last dose. Whole brain concentration of 5-HT was increased to a greater degree than that of NE following repeated administration of amiflamine. These increases in 5-HT and NE were accompanied by decreased levels of their respective metabolites 5-hydroxy-indoleacetic acid and 3-methoxy-4-hydroxyphenylethyleneglycol. The firing activity of dorsal raphe 5-HT neurons, but not that of NE neurons, was markedl decreased 2–6 h after the last dose of a 2-day treatment. However, 2–6 h after a 21-day treatment, the firing activity of 5-HT neurons was back to normal, whereas that of NE neurons was decreased by 30%. The recovery of firing activity of 5-HT neurons following long-term amiflamine is attributable to a desensitization of the somatic 5-HT autoreceptor as indicated by the decreased responsiveness of 5-HT neurons to intravenous LSD. In contrast, the sensitivity of the somatic NE autoreceptor was not modified, as verified by the unchanged responsiveness of NE neurons to clonidine, an ₂ agonist. The duration of the suppression of firing activity of hippocampus pyramidal neurons produced by the electrical activation of the ascending 5-HT pathway was prolonged in amiflamine-treated rats as compared to controls. This increased synaptic efficacy was attributable to a presynaptic modification since the responsiveness of the same neurons to microiontophoretically-applied 5-HT was not changed. The efficacy of the stimulation of the dorsal NE bundle, as well as that of the microiontophoretic applied of NE, were unchanged in the same rats. Since the efficacy of the stimulation of the 5-HT pathway is increased at the time 5-HT neurons have regained their normal firing rate, it is concluded that 5-HT neurotransmission is enhanced following the long-term treatment with amiflamine.     

Monoamine oxidase inhibitors increase preferentially extracellular 5-hydroxytryptamine in the midbrain raphe nuclei. A brain microdialysis study in the awake rat

Summary We have examined the local and systemic effects of clorgyline, tranylcypromine and deprenyl on extracellular serotonin (5-HT) and 5-hydroxyindoleacetic acid in the raphe nuclei and in frontal cortex of awake, freely-moving rats using microdialysis. When administered through the dialysis probe, monoamine oxidase (monoamine: oxygen oxidoreductase (deaminating), E.C. 1.4.3.4., MAO) inhibitors increased 5-HT output in a dose-dependent manner in both brain areas. The effects were more pronounced in the raphe nuclei for the three MAO inhibitors at all doses assayed.When the monoamine oxidase inhibitors were given i.p., dialysate 5-HT increased dramatically, after tranylcypromine (15 mg/kg), in raphe nuclei and frontal cortex (area under the curve (AUC) to 4 h post-treatment: 63-fold and 11-fold, respectively) whereas the effects of clorgyline (10 mg/kg) were much less pronounced (+ 47% increase in the AUC for raphe nuclei, P < 0.09; + 18% increase in the AUC for frontal cortex, n.s.). Deprenyl (2.5 mg/kg, i.p.) induced a moderate (+ 22%) increase of dialysate 5-HT from the raphe nuclei but did not cause a change in dialysate 5-HT from the frontal cortex (+ 4%). However, clorgyline, or deprenyl, dramatically increased dialysate 5-HT in animals which had been pre-treated with the above dose of deprenyl, or clorgyline, respectively, showing that the blockade of both forms of MAO results in much larger increases of extracellular 5-HT than does the blockade of either form alone.These results indicate that: (a) deamination by MAO participates actively in the control of the extracellular concentration of 5-HT in those areas of the brain that are rich in serotoninergic nerve terminals as well as in cell bodies, (b) in vivo, brain 5-HT is deaminated preferentially by MAO-A but its full inhibition does not result in an increased release of 5-HT, in spite of a large accumulation of 5-HT in the brain tissue, (c) MAO-B deaminates 5-HT when the A-form is inhibited (in this situation, MAO-B participates actively in the control of a releasable pool of 5-HT), (d) the raphe nuclei appears to be a preferential site of action of MAO inhibitors, administered either locally or systemically. These results may help to understand the model of action of MAO inhibitors as antidepressant drugs.Correspondence to F. Artigas at the above address     

Pharmacological properties of the anti-Parkinson drug rasagiline; modification of endogenous brain amines,reserpine reversal,serotonergic and dopaminergic behaviours

Rasagiline [N-propargyl-1R(+)-aminoindan; TVP1012] is a potent irreversible monoamine oxidase (MAO) inhibitor with selectivity for type B of the enzyme, which is being developed for treatment of Parkinson's disease. In this study we examined effects of rasagiline on CNS monoamine levels, modification of behavioural response to L-tryptophan, fluoxetine and L-DOPA, and reversal of reserpine syndrome. Reserpine-induced ptosis was reversed by rasagiline at doses above 2 mg x kg(-1) i.p., which inhibit MAO-A as well as MAO-B, but not at MAO-B-selective doses. However, combination of rasagiline (10 mg x kg(-1) i.p.) with L-DOPA or L-tryptophan (50 mg x kg(-1) i.p.), or rasagiline (10 mg x kg(-1) p.o.) with fluoxetine (10 mg x kg(-1) p.o.), did not induce the behavioural hyperactivity syndrome which is seen following inhibition of both MAO-A and MAO-B by tranylcypromine together with the monoamine precursors. Following oral administration, levels of noradrenaline (NA), 5-hydroxytryptamine (5-HT) and dopamine (DA) were unaffected in hippocampus and striatum after single doses of rasagiline up to 2 mg x kg(-1). Following chronic oral administration (21 days, one dose daily), levels of NA, 5-HT and DA in hippocampus and striatum were unaffected by rasagiline at doses up to 1 mg x kg(-1). Rasagiline does not modify CNS monoamine tissue levels or monoamine-induced behavioural syndromes at doses which selectively inhibit MAO-B but not MAO-A.     

Gender and gonadal status modulation of dorsal raphe nucleus serotonergic neurons. Part I: effects of gender and pregnancy

Gender differences in susceptibility to affective disorders are well documented. The ovarian steroids, estrogen (E) and progesterone (P), may modulate the function of the serotonergic (5-HT) system, implicated in the etiology and treatment of affective disorders. We tested the hypothesis that ovarian steroid modulation of 5-HT function could result in a modification of the 5-HT neuronal firing activity. Extracellular unitary recordings of dorsal raphe nucleus 5-HT neurons were obtained in male rats and in female rats during natural E and P fluctuations. The average firing activity of 5-HT neurons was significantly higher in males (41%) than in freely cycling (CF) and in ovariectomized (OVX) females. During pregnancy, it increased gradually and by up to 136% on gestational day 17, then declined before parturition. In the postpartum period (PP), the firing rate decreased markedly compared to P17 but remained 63% higher than in CF. During pregnancy, the firing rate variations were closely correlated with P plasmatic levels. Finally no modification of the basal firing activity of locus coeruleus noradrenergic neurons was found in any group tested. Our results thus reveal a gender and pregnancy-dependent modulation of 5-HT firing rate that would impact 5-HT-mediated neurotransmission.     

Peripheral serotonergic markers in patients with suicidal behavior

Peripheral serotonergic parameters were studied in 17 patients with a history of suicidal behavior and in 17 age- and sex-matched healthy controls. Serotonin₂ receptor function in platelets, measured as serotonin-induced [³²P]phosphatidylinositol 4,5-biphosphate hydrolysis, was significantly higher in patients than in age- and sex-matched controls. Increased serotonin₂ receptor transduction was not correlated to psychiatric diagnosis, drug treatment or a history of violent suicide attempt. Monoamine oxidase activity was lower in platelets from patients with a history of suocidal behavior than in controls. In addition, whole blood serotonin was analyzed but no significant alterations were found in blood from patients.     

The role of serotonergic neurons in dorsal raphe, median raphe and anterior hypothalamic pressor mechanisms

The role of serotonergic neurons in the dorsal raphe and median raphe in the pressor response to electrical stimulation of these areas, and the contribution of these neurons to the pressor response to serotonin (5-HT) in the anterior hypothalamus-preoptic area (AH/PO) have been studied by the use of local injections of 5,7-dihydroxytryptamine (5,7-DHT), a neurotoxin selective for 5-hydroxytryptamine (5-HT). When blood pressure was recorded in urethane-anesthetized rats, selective lesions of 5-HT-containing neurons in the dorsal raphe nucleus reduced by 60% the pressor response to electrical stimulation (50 Hz, 100-150 microA, 0.3 msec pulse duration) of this nucleus. On the other hand, selective lesion of 5-HT-containing neurons in the median raphe nucleus had no effect on the pressor response to electrical stimulation of this area. Injection of 5,7-dihydroxytryptamine into the anterior hypothalamus/preoptic area resulted in an increased pressor response to the injection of 5-HT (5 nmol) into the lesioned area 10 days later. Furthermore, the destruction of 5-HT-containing neurons in the dorsal raphe nucleus resulted in an enhanced pressor response to the injection of 5-HT (5 nmol) into the anterior hypothalamus/preoptic area, while the destruction of 5-HT-containing neurons in the median raphe nucleus had no effect on the pressor response to the injection of 5-HT (5 nmol) into the anterior hypothalamus/preoptic area. Therefore, it appears that 5-HT neurons in the dorsal raphe nucleus are important in the pressor response to electrical stimulation and are involved in a pressor mechanism in the anterior hypothalamus/preoptic area.(ABSTRACT TRUNCATED AT 250 WORDS)     

5,7-Dihydroxitryptamine toxicity to serotonergic neurons in serum free raphe cultures

5,7-Dihydroxytryptamine (5,7-DHT), is an experimentally widely used selective serotonergic neurotoxin, though the mechanisms of toxicity remain to be fully elucidated. In the present study, we evaluated 5,7-dihydroxitryptamine (5,7-DHT) induced serotonergic neurotoxicity in foetal raphe serum free cultures from the rat. For this purpose, a model of foetal raphe serum free neuronal cultures from the rat was established, containing about 16% serotonergic neurons and studied up to 3 months. Two weeks old raphe cultures were exposed to the serotonergic neurotoxin 5,7-DHT (concentration range 10-100 microM) for 72 h, after which the medium was replaced and neurotoxicity was evaluated by immunocitochemistry 1 week later. Lactate dehydrogenase release into the medium, 72 h after exposure to 5,7-DHT, showed a concentration-dependent neurotoxicity. To access morphologically the serotonergic toxicity tryptophan hydroxylase (TPH) was used as a specific marker of these neurons. Immunocitochemistry using TPH antisera showed a concentration-dependent serotonergic neurotoxicity induced by 5,7-DHT. Serotonergic neurons showed the typical pattern of "pruning" accompanied by axon terminals and dendrites loss, which were either partial or total. The axotomy induced by the neurotoxin was morphologically characteristic of retrograde axonal degeneration. Fluoxetine (0.1 microM) pre-treatment reduced 5,7-DHT-induced serotonergic neurotoxicity. These results indicate that the mechanism by which 5,7-DHT-induces serotonergic neurotoxicity is, at least partially, dependent on the toxin uptake by the serotonin transporter. Finally, we have established a robust model of primary raphe neuronal culture to evaluate serotonergic neurons development and the mechanisms of toxicity involving this neuronal population.     

Serotonin uptake blockers inhibit the firing of presumed serotonergic dorsal raphe neurons in vitro

Some antidepressant drugs are potent inhibitors of neuronal uptake of serotonin. In vivo, these compounds inhibit serotonergic dorsal raphe neuronal firing rates, presumably through increased stimulation of 5-HT1a autoreceptors. We recorded from electrophysiologically identified serotonergic dorsal raphe neurons in rat brain slices and determined the effects of five serotonin uptake blockers on the firing rates of these units in vitro. Each drug decreased the neuronal firing rates in a concentration-dependent manner. IC50 values derived from concentration-response curves are: fluvoxamine, 0.8 μM; sertraline, 1.1 μM; imipramine, 2.7 μM; chlorimipramine, 2.8 μM; and fluoxetine, 4.2 μM. Exposure of brain slices to 10 μM tetrabenazine, a serotonin depleting agent, prior to treatment with serotonin uptake blockers resulted in a rightward shift of the concentration-response curve. In vitro single unit recording allows: (1) direct comparison with neurochemical data obtained in vitro; (2) access to tissue bypassing blood brain barrier and liver enzymes; (3) quick wash out of drug from tissue; and (4) ability to record from single unitsover long periods (hours). This in vitro test provides a fast, simple means of determining neurophysiological effects of potential antidepressant drugs on the serotonin system.     

Cotinine inhibits catecholamine release evoked by cholinergic stimulation from the rat adrenal medulla

The aim of the present study was to clarify whether cotinine affects the release of catecholamines (CA) from the isolated perfused rat adrenal gland, and to establish the mechanism of its action, in comparison with the response of nicotine. Cotinine (0.3-3 mM), when perfused into an adrenal vein for 60 min, inhibited CA secretory responses evoked by ACh (5.32 mM), DMPP (a selective neuronal nicotinic agonist, 100 microM for 2 min) and McN-A-343 (a selective muscarinic M1-agonist, 100 microM for 2 min) in dose- and time-dependent manners. However, cotinine did not affect CA secretion by high K+ (56 mM). Cotinine itself also failed to affect basal CA output. Furthermore, in the presence of cotinine (1 mM), CA secretory responses evoked by Bay-K-8644 (an activator of L-type Ca2+ channels, 10 microM) and cyclopiazonic acid (an inhibitor of cytoplasmic Ca2+-ATPase, 10 microM) were relative time-dependently attenuated. However, nicotine (30 microM), given into the adrenal gland for 60 min, initially rather enhanced CA secretory responses evoked by ACh and high K+, followed by the inhibition later, while it time-dependently depressed the CA release evoked by McN-A-343 and DMPP. Taken together, these results suggest that cotinine inhibits greatly CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors, but does fail to affect that by the direct membrane-depolarization. It seems that this inhibitory effect of cotinine may be exerted by the cholinergic blockade, which is associated with blocking both the calcium influx into the rat adrenal medullary chromaffin cells and Ca2+ release from the cytoplasmic calcium store. It also seems that there is a big difference in the mode of action between cotinine and nicotine in the rat adrenomedullary CA secretion.     

Allopregnanolone and ganaxolone increase the firing activity of dorsal raphe nucleus serotonergic neurons in female rats

Accumulating evidence suggest a reciprocal interaction between neurosteroids, especially 5alpha-pregnan-3alpha-ol,20-one (3alpha,5alpha-THP, allopregnanolone), and the serotonergic (5-HT) system. Both 5-HT and neurosteroids seem to play an important role in the pathophysiology of major depression. We have previously shown that a 7-d treatment with 3alpha,5alpha-THP drastically increases the spontaneous firing activity of dorsal raphe nucleus (DRN) 5-HT neurons in female rats. This study was thus undertaken to better characterize this modulation and to assess the effects of ganaxolone, a synthetic analogue of 3alpha,5alpha-THP. Female rats received 50 microg/kg.d of 3alpha,5alpha-THP or ganaxolone for 3 and 7 days. Others received 3alpha,5alpha-THP concomitantly with the antiprogestin RU486 (50 microg/kg.d, each), which was also administered alone. Acute experiments were also carried out with a single injection of 3alpha,5alpha-THP (1 microg/kg). Finally, both 3alpha,5alpha-THP and ganaxolone (50 microg/kg.d) were administered along with the selective serotonin reuptake inhibitor (SSRI) citalopram (10 mg/kg.d). In-vivo extracellular unitary recordings of 5-HT neurons from the DRN, revealed that 3alpha,5alpha-THP and ganaxolone increased their firing activity after 3 and 7 d of treatment. A 7-d treatment with RU486 had the same effect. Furthermore, an increase could be seen as soon as after 30-60 min following a single injection with 3alpha,5alpha-THP. Interestingly, both 3alpha,5alpha-THP and ganaxolone prevented the citalopram-induced reduction in firing activity after 3-d treatments. These data demonstrate the ability of 3alpha,5alpha-THP and ganaxolone to positively modulate the firing activity of DRN 5-HT neurons in female rats. Moreover, these results suggest that these neuroactive steroids might represent interesting adjuvants in the treatment of mood disorders in female patients.     

Receptor-mediated regulation of serotonin output in the rat dorsal raphe nucleus: effects of risperidone

OBJECTIVES: The present study was undertaken to characterize the regulation of serotonin (5-HT) efflux and neuronal activity in the dorsal raphe nucleus (DRN) as well as to examine the potential ability of the antipsychotic drug risperidone to interfere with these mechanisms. METHODS AND RESULTS: By using microdialysis in freely moving rats, it was found that administration of the alpha2 adrenoceptor antagonist idazoxan (0.25 mg/kg, SC), the 5-HT1B/D receptor antagonist GR 127,935 (1.0 mg/kg, SC) and risperidone (0.6 or 2.0 mg/kg, SC) increased 5-HT output in the DRN. Local DRN perfusion with GR 127,935 or risperidone via reversed dialysis (100 or 10-100 microM, respectively) enhanced 5-HT efflux in this area, whereas idazoxan (10-100 microM) failed to affect this parameter. Both systemic administration and reversed DRN dialysis of the D2/3 and 5-HT2A receptor antagonists raclopride (2.0 mg/kg, SC or 10-100 microM) and MDL 100,907 (1.0 mg/kg, SC or 10-100 microM), respectively, were without effect. Intraraphe dialysis of the 5-HT1B/D receptor agonist CP 135,807 (0.2 microM) decreased the efflux of 5-HT in the DRN, an effect which was antagonized by co-administration of either GR 127,935 or risperidone (10 and 3.3 microM, respectively). By using single-cell recording, it was found that administration of GR 127,935 (50-400 microg/kg, IV) decreased, whereas CP 135,807 (2.5-20 microg/kg, IV) increased firing of 5-HT cells in the DRN. CONCLUSIONS: Our findings suggest a regulatory role of local 5-HT1B/D receptors on 5-HT efflux as well as cell firing in the DRN and indicate that risperidone may interfere with the regulation of 5-HT availability in this area primarily via blockade of 5-HT1D receptors.     

Differential effects of clomipramine and clorgyline on the sensitivity of cortical neurons to serotonin: Effect of chronic treatment

The sensitivity of rostral and cingulate cortical neurons to microiontophoretically administered serotonin (5-HT) was compared in groups of rats treated either acutely or chronically with clomipramine (10 mg/kg i.p.) or with clorgyline (0.3 mg/kg i.p.). The sensitivity of cortical neurons to 5-HT was significantly reduced in both cortical areas of animals treated chronically with clorgyline. By contrast, no change of sensitivity was observed in the clomipramine-treated animals. These findings are consistent with biochemical studies showing a decrease in 5-HT receptors following the chronic treatment with clorgyline and an absence of effect on 5-HT receptors following the chronic treatment with clomipramine.     

Effects of acute and chronic administration of fluoxetine on the activity of serotonergic neurons in the dorsal raphe nucleus of the rat

The gradual recovery of activity of serotonergic neurons following an initial inhibition has been hypothesized to play an important role in the delayed onset of efficacy of selective serotonin reuptake inhibitors. This study explored the clinical relevance of this hypothesis by examining the effects of different doses and routes of administration of fluoxetine on the recovery of activity of serotonergic neurons over the course of a 21-day exposure. Single-unit, extracellular recordings of serotonergic neurons were made in the dorsal raphe nucleus of anesthetized male rats. Acute i.v., s.c. and i.p. administration of fluoxetine inhibited the activity of serotonergic neurons. With chronic administration of fluoxetine, at clinically relevant doses, the activity of serotonergic neurons gradually recovered to baseline levels over the course of 14-21 days. The dose of fluoxetine (5, 10 or 20 mg/kg per day) did not make a significant difference in the time course of the recovery of activity of serotonergic neurons. A significant, non-parallel shift in the dose-response curve of serotonergic neurons to the serotonin-1A (5-HT1A) agonist 8-OH-DPAT occurred over the 21 days of treatment with fluoxetine, indicating a desensitization of the 5-HT1A receptor during this period. The recovery of firing did not correlate with either plasma or cerebrospinal fluid levels of fluoxetine or norfluoxetine. These results indicate that, similar to the effects of dose on the speed of onset of the clinical effects of SSRIs, increasing the dose of fluoxetine does not hasten the recovery of firing of serotonergic neurons during chronic administration. These results support the hypothesis that desensitization of the 5-HT1A receptor and consequent recovery of firing of 5-HT cells in the dorsal raphe nucleus plays a role in the delayed therapeutic onset of fluoxetine.     

5-HT<Subscript>1A</Subscript> and 5-HT<Subscript>2A</Subscript> receptors in the rat dorsal periaqueductal gray mediate the antipanic-like effect induced by the stimulation of serotonergic neurons in the dorsal raphe nucleus

Rationale: It has been proposed that the serotonergic pathway that connects the dorsal raphe nucleus (DRN) to the dorsal periaqueductal gray (DPAG) is implicated in the regulation of escape, a behavior that has been related to panic. Objectives: We further evaluated this hypothesis by investigating whether intra-DRN injection of the 5-HT_1A receptor antagonist WAY-100635 changes the escape response of rats submitted to the elevated T-maze. This test also measures inhibitory avoidance, which has been associated with generalized anxiety disorder. We also investigated whether the 5-HT_1A and 5-HT_2A receptors in the DPAG mediate the behavioral consequences induced by the injection of WAY-100635 into the DRN. Results: Intra-DRN injection of WAY-100635 facilitated inhibitory avoidance, while impairing escape. The same effect was obtained after intra-DRN injection of the glutamate receptor agonist kainic acid. Preadministration of WAY-100635 into the DPAG counteracted the effect induced by intra-DRN injection of WAY-100635 and of kainic acid on escape, but not on inhibitory avoidance. Preadministration of the preferential 5-HT_2A receptor antagonist ketanserin into the DPAG abolished the effects of intra-DRN injection of WAY-100635 on both elevated T-maze tasks. Conclusion: The results are indicative that 5-HT_1A autoreceptors in the DRN are under tonic inhibitory influence by endogenous 5-HT. The effects of 5-HT release in the DPAG after intra-DRN injection of WAY-100635 and kainic acid on inhibitory avoidance and escape involve different 5-HT receptor subtypes. Whereas 5-HT_2A receptors in the DPAG seem to mediate the effect of 5-HT on both behaviors, 5-HT_1A receptors are only involved in the regulation of escape.     

Collateral inhibition of serotonergic neurones in the rat dorsal raphe nucleus: pharmacological evidence.

Previous studies have shown that serotonergic (5-HT) neurones in the dorsal raphe are inhibited following electrical stimulation of the major ascending 5-HT fibre pathway in the ventromedial tegmentum (VMT); this effect appears to be mediated directly through 5-HT axon collaterals. This possibility was tested further in the present study by pharmacological manipulations of the 5-HT system. When rats were pretreated with either para-chlorophenylalanine (P-CPA, which inhibits trypto phan hydroxylase, the rate-limiting enzyme for the 5-HT synthesis) or reserpine (which is believed to block storage of biogenic amines) or both, the inhibitory responses of antidromically identified 5-HT neurones to VMT stimulation were either totally prevented or markedly reduced in the great majority of cells tested. Furthermore, the P-CPA effects were reversed by the injection of 5-hydroxytryptophan (the immediate precursor of 5-HT which bypasses the synthesis step inhibited by P-CPA); low doses of l-dihydroxyphenylalanine (a precursor of catecholamines) were not effective. In some cells after the drug treatment, prolonged periods of suppression of firing of 5-HT cells still occurred following VMT stimulation. In such cells, continuous stimulation of the VMT (at 10 Hz for 3 min). which presumably depleted residual transmitter, diminished the VMT-induced effect. Of some possible 5-HT antagonists tested, only metergoline (1–2 mg/kg, i.v.) was effective in antagonizing the VMT induced depressant effect on 5-HT neurones; however, the development of the effect was extremely delayed. The present results show that the VMT-induced depression of firing of 5-HT neurones depends on the availability of 5-HT. This finding is consistent with the view that the inhibitory responses of 5-HT neurones to VMT stimulation are mediated directly through 5-HT axon collaterals (or dendo-dendritic junctions).     

Pharmacological characterisation of the orexin receptor subtype mediating postsynaptic excitation in the rat dorsal raphe nucleus

Electrophysiological recordings from dorsal raphe nucleus (DRN) neurones in rat brain slices have revealed that the orexins can cause direct and reversible depolarisation of the postsynaptic membrane. Whilst it is known that the membrane depolarisation produced by orexin-A can dramatically increase the firing rate of DRN neurones, quantitative pharmacological analysis that determines the receptor subtype mediating the orexinergic response has not yet been performed. Here, we demonstrate that the rank order of potencies of orexin receptor agonists to excite serotonergic DRN neurones is orexin-A=orexin-B>SB-668875-DM. In contrast, the rank order of potency of these agonists to excite noradrenergic locus coreleus (LC) neurones is orexin-A>orexin-B>SB-668875-DM. We show further that the orexin receptor antagonist, SB-334867-A, inhibits the effects of orexin-A in the LC and DRN with pK_B values of 6.93 and 5.84, respectively, values similar to those calculated for human OX₁ (7.27) and OX₂ (5.60) receptors expressed in CHO cells. These data suggest a differential role for OX₁ and OX₂ receptors in stimulating distinct populations of monoaminergic neurones in the rat CNS with OX₂ receptors exhibiting a more pronounced functional significance in serotonergic neurones and OX₁ in noradrenergic neurones.     

The effect of amantadine on radiolabeled biogenic amines in the rat brain

Intracisternally (i.ci.) administered radiolabeled dopamine, l-dopa or norepinephrine were used to study the mode of oction of amantadine as an anti-Parkinson agent. The data show thatpretreatment with amantadine resulted in higher radioactivity concentration in all brain areas investigated when radiolabeled dopamine was administered i.ci. This increase in the brain radioactivity was reflected in higher levels of dopamine and its metabolites. Amantadine had no effect on the brain radioactivity concentration in experiments in which radiolabeled l-dopa or norepinephrine were administered i.ci. Amantadine also did not affect MAO activity in rat brain and liver, and was shown to be a very weak inhibitor of dopamine or serotonin uptake into synaptosomes of rat corpus striatum. Possible modes of action of amantadine are discussed.