Anton M.L. Van Delft

ANTIAVERSIVE EFFECTS OF 5HT2C RECEPTOR AGONISTS AND FLUOXETINE IN A MODEL OF PANIC-LIKE ANXIETY IN RATS

Dose-dependent increases in threshold for operant fear/escape responses of rats submitted to aversive stimulation of the dorsolateral periaqueductal gray (dPAG) were recorded following intraperitoneal injection of three chemically unrelated but selective 5HT2C receptor agonists (Ro 60-0175, Org 12962 and Ro 60-0332) and fluoxetine. The decreased sensitivity of rats to the acute panic-like aversion elicited by stimulation of this limbic periventricular region was detected at dosages devoid of impairing effects on the latencies needed for operant brain stimulation interruption. In this paradigm which has been validated as a simulation of acute anxiety with relevance to panic disorder, the selective activation of 5HT2C receptors by Ro 60-0175, Org 12962 or Ro 60-0332 induces effects analogous to those observed following benzodiazepine receptor activation by antipanic agents such as clonazepam or alprazolam or following non-selective and indirect 5HT receptor activation by fluoxetine. Potency and efficacy of 5HT2C receptor agonists were intermediate between those of clonazepam and fluoxetine, indicating authentic antiaversive properties and suggesting antipanic potential for these 5HT2C receptor agonists. In addition, these data suggest that the 5HT2C receptor subtype may play a major role in the therapeutic properties of selective serotonin reuptake inhibitors. It is also speculated that serotonin/benzodiazepine interactions existing in the brain may functionally involve the 5HT2C receptor subtypes and that the anxiogenic action reported under certain circumstances for 5HT mimetics are not mediated by 5HT2C receptor subtypes.

Depletion of brain serotonin differently affects behaviors induced by 5HT1A, 5HT1C, and 5HT2 receptor activation in rats

5-hydroxytryptamine (5HT)-depleted rats were subjected to behavioral experiments in which the response to activation of 5HT1A, 5HT1c, and 5HT2 receptor subtypes was measured. Depletion of 5HT was produced by unilateral intracerebroventricular injection of 5,7-dihydroxytryptamine (100 micrograms/rat) or by systemic injection of p-chlorophenylalanine (150 mg/kg injected intraperitoneally 72, 48, and 24 h before the test). The dose-response curve of the 5HT1A-mediated, 8-hydroxy-2-(di-n-propylamino)tetralin (0.022-0.46 mg/kg)-induced lower lip retraction was not changed after depletion, nor was the dose-response curve of the 5HT2 receptor-mediated (+-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (0.046-1.0 mg/kg)-induced head shake response. The dose-response curve for penile erections, a 5HT1c receptor-mediated response after mCPP (0.1-1.0 mg/kg), a direct 5HT1c agonist, is shifted to the left after 5HT depletion, whereas the response to indirect activation of the 5HT1c receptor with the 5HT reuptake inhibitors citalopram (2.2-4.6 mg/kg) and paroxetine (0.22-2.2 mg/kg) was inhibited after 5HT depletion. These results suggest that 5HT1c receptors are more subject to denervation supersensitivity than 5HT1A and 5HT2 receptors. This lesion model may be useful to discriminate behaviorally between direct and indirect activation of the 5HT1c receptor.

Reversal by NGF of cytostatic drug-induced reduction of neurite outgrowth in rat dorsal root ganglia in vitro.

Cytostatic drugs, like cisplatin, vincristine and taxol, when given to cancer patients may cause peripheral neuropathies. We were interested in the potential neuroprotective effects of neurotrophic factors against such neuropathies. To this aim we studied the effects of these cytostatic agents on sensory fibers located in the dorsal root ganglia (DRG) in vitro and studied whether nerve growth factor (NGF) could reverse the cytostatic induced morphological changes on intact DRG (1 DRG/well, n = 10 per dose). Neuritogenesis from DRG was measured with an image analysis system following exposure to different concentrations of cytostatic drugs in the presence of 3 ng NGF/ml and cytosine arabinoside (Ara-C, 10(-6) M). Relative neurite outgrowth in intact DRG in culture was reduced dose-dependently, (a) by vincristine starting at a dose of 0.4 ng/ml for 2 days (-33% as compared to control; P < 0.001, Students t-test); (b) by taxol 10 ng/ml (-60%; P < 0.001), and (c) by cisplatin 3 micrograms/ml (-47%, P < 0.001). Cisplatin also prevented the migration of satellite cells away from the intact DRG along the extending neurites into the well in contrast to control, vincristine, or taxol. To evaluate the neuroprotective potential of NGF in this in vitro cytostatic neuropathy model, we incubated intact DRG with cytostatic agents in combination with increasing amounts of NGF. Neurite outgrowth from DRG treated with vincristine (0.5 ng/ml)+NGF (3 ng/ml) for 2 days was significantly higher (+87%) than after treatment with vincristine + 1 ng NGF/ml (P < 0.001). Neurite outgrowth from DRG treated with taxol (20 ng/ml)+NGF (3 ng/ml) for 2 days was significantly higher (+228%) than after taxol + 1 ng NGF/ml (P < 0.05). Neuritogenesis from DRG treated with cisplatin (2.5 micrograms/ml)+NGF (3 ng/ml) for 2 days was significantly increased (+105%) compared to treatment with cisplatin + 1 ng NGF/ml (P < 0.001). DRG thus appear to be a very suitable model for studying cytostatic drug-induced neuropathies in vitro and NGF has a clear neuroprotective effect on the vincristine-, taxol-, and cisplatin-induced neuropathies in this in vitro model.

Effects of serotonin receptor antagonists on PAG stimulation induced aversion: different contributions of 5HT1, 5HT2 and 5HT3 receptors

The effects of serotonin receptor antagonists with differential selectivity for the various classes of 5HT receptors (5HT1, 5HT2 and 5HT3) were tested for their effects on the response to aversive brain stimulation. Electrical stimulation was administered to the dorsal part of the periaqueductal gray matter (PAG), one of the main cerebral structures subserving negative reinforcement. Stimulation frequency thresholds for escape responses were recorded before and following administration of the compounds. Ketanserin (0.32–32 mg/kg IP), trazodone (1.0–22 mg/kg), pirenperone (0.032–1.0 mg/kg) and spiperone (0.1–0.2 mg/kg) dose-dependently increased stimulation frequency thresholds necessary to induce escape responses. Opposite effects were observed with mianserin (0.01–32 mg/kg) and metergoline (0.032–10 mg/kg) which decreased threshold for escape. ICS 205-930 (0.01–10 mg/kg), MDL 72222 (0.1 22 mg/kg) and GR 38032 F (0.1–10 mg/kg) did not affect the stimulation frequency threshold for escape. Prazosin (0.1–2.2 mg/kg) did not specifically affect aversive brain stimulation. Haloperidol (0.02–1.0 mg/kg) increased the frequency threshold for escape responses but with some motoric side effects. These data show that the various types of 5HT receptors differentially contribute to the control of central aversive systems in rats. It is suggested that blockade of 5HT2 receptors suppresses the central aversive system, whereas blockade of some 5HT1 receptors enhances aversion and overcomes the 5HT2-mediated suppression. Blockade of 5HT3 receptors has no effects. Dopamine receptor blockade further contributes to the suppression of the central aversive system. The relevance of these findings to some pathophysiological mechanisms of anxiety and depressive disorders is discussed.

Comparative pharmacology of mianserin, its main metabolites and 6-azamianserin

SummaryMianserin, its main metabolites (8-hydroxymianserin, desmethylmianserin and mianserin-N-oxide) and a mianserin analogue, 6-azamianserin (ORG 3770), were compared with regard to effects on monoamine uptake systems, α-adrenoceptors, rat exploratory activity and rat muricidal behaviour. Mianserin and desmethylmianserin inhibited noradrenaline uptake into synaptosomes (IC50s 30 and 60 nM, respectively) whereas the other compounds were much less active. Synaptosomal serotonin uptake was only inhibited to a small extent by desmethylmianserin (IC50 6 μM) and 8-hydroxymianserin (IC50 9 μM). Dopamine uptake was not affected by any of the compounds tested. All compounds except mianserin-N-oxide blocked presynaptic α-receptors as shown by the potentiation of high-K-induced release of noradrenaline from rat cerebral cortex slices. For mianserin and 6-azamianserin this blockade was shown to be stereoselective. Desmethylmianserin was less potent than mianserin. Binding of 3H-dihydroergocryptine to rat cerebral cortex membranes was inhibited by all compounds except mianserin-N-oxide. Again, desmethylmianserin was less active than mianserin. None of the compounds appeared to block presynaptic α-receptors in preference to postsynaptic α-adrenoceptors. This was confirmed by the fact that the compounds studied failed to antagonize clonidine-induced sedation in the open field. Clonidine-induced diuresis, however, was stereoselectively inhibited by 6-azamianserin, but the involvement of α2-receptors in this phenomenon is not firmly established.Antihistamine properties as determined by 3H-mepyramine binding to rat brain membranes were most pronounced for 6-azamianserin. Mianserin was slightly less potent and desmethylmianserin and 8-hydroxymianserin were 10 and 30 times less potent than mianserin, respectively.Muricidal behaviour was inhibited by all compounds except mianserin-N-oxide. The least active was 8-hydroxymianserin. In contrast to mianserin and desmethylmianserin, the blockade of muricidal behaviour by 6-azamianserin was non-specific since it occurred at doses which caused a strong depression of rat open field behaviour. Mianserin was less sedative than 6-azamianserin whereas the metabolites showed no sedative effects at doses up to 32 mg/kg in the open field.It is concluded that the main metabolites of mianserin possess pharmacological properties which may add to the therapeutic potential of mianserin. Clinical testing of 8-hydroxymianserin and 6-azamianserin may give an answer to the question whether the antidepressant effect of mianserin is solely based upon its interaction with presynaptic α-adrenoceptors or is due to a concomitant blockade of noradrenaline reuptake.

Opposite control mediated by central 5-HT1A and non-5-HT1A (5-HT1B or 5-HT1C) receptors on periaqueductal gray aversion

8-OH-DPAT, a selective 5-HT1A agonist, and mCPP, which has preferential affinity for 5-HT1B and 5-HT1C receptors, were studied for their effects on aversive brain stimulation in rats. Opposite effects were found with these two agonists: D, L-8-hydroxy-N,N-dipropyl-2-aminotetralin HBr (8-OH-DPAT; 0.1-1.0 mg/kg i.p.) dose dependently decreased the threshold for neurostimulation-induced escape behaviour while mCPP (0.1-1.0 mg/kg i.p.) dose dependently increased the threshold. The proaversive effect of 8-OH-DPAT and the antiaversive effect of mCPP suggest that 5-HT1A and non-5-HT1a (5-HT1B or 5-HT1C) receptors play distinct roles in mechanisms of aversion, perhaps at different locations in the CNS.

5-HT1C receptors in the serotonergic control of periaqueductal gray induced aversion in rats.

The functional role of brain 5-HT and 5-HT receptor subtypes in periaqueductal gray (PAG) induced aversion has been investigated in rats. Antiaversive effects were found with the serotonin agonists TFMPP, mCPP and DOI but not with RU 24969 which was found to facilitate PAG aversion. The first three serotonin agonists share potent 5-HT1C activity while RU 24969 differs with a high 5-HT1A activity. Proaversive effects were found with the mixed 5-HT1C/5-HT2 antagonists cyproheptadine and ritanserin; this effect was already reported for the mixed 5-HT1C/5-HT2 antagonists metergoline and mianserin and is opposite to the effects of the selective 5-HT2 antagonists ketanserin, pirenperone, trazodone and spiperone. The antiaversive effects of mCPP (1 mg/kg) could be prevented by pretreatment of the animals with mianserin (1 and 10 mg/kg). These results suggest that 5-HT1C receptors play an important role in the serotonergic control of PAG aversion. 5-HT1C receptor activation seems to mediate antiaversive effects whereas acute 5-HT1C receptor blockade appears to facilitate PAG aversion. Functional interactions take place between several receptor types in the in vivo control of PAG aversion, where 5-HT1C receptors appear to play a predominant function.

Genomic organisation and functional expression of the gene encoding the human serotonin 5-HT2C receptor

The 5-HT2C receptor gene is unique among the members of the 5-HT receptor family by virtue of its genomic organisation. The human 5-HT2C receptor gene, unlike many other genes for guanine nucleotide binding (G)-proteins, contains three introns which interrupt the coding sequence into four exons. The first two introns are at equivalent positions as compared to the intervening sequences previously found in the 5-HT2(A) receptor gene, suggesting a close evolutionary relationship between both genes. Southern blot analysis shows that the 5-HT2C receptor gene is a single copy gene. Furthermore, we report the functional expression of a complementary DNA for the 5-HT2C receptor, cloned from hippocampal RNA. Membranes prepared from NIH 3T3 cells stably expressing the 5-HT2C receptor cDNA, displayed a single population of high affinity sites for the antagonist [3H]mesulergine (Kd = 2.9 +/- 0.4 nM, Bmax = 44.3 +/- 7.2 pmol/mg protein) as well as for [3H]5-HT (Kd = 9.9 +/- 0.7 nM, Bmax = 13.6 +/- 1.0 pmol/mg protein). Displacement of [3H]mesulergine and [3H]5HT binding by ligands indicated a pharmacological similarity of these binding sites with porcine and rat choroid plexus 5-HT2C receptors. Furthermore, activation of the 5-HT2C receptor with 5-HT results in an increased phospholipase C activity.

Antagonism of 8-OH-DPAT-induced behaviour in rats.

Selective activation of the 5-HT1A receptor induces lower lip retraction (LLR) in rats. 8-Hydroxy-dipropylamino tetralin (8-OH-DPAT)-induced LLR could not be antagonised by the 5-HT antagonists methysergide, metergoline or mesulergine. In fact, some 5-HT antagonists induced LLR. However, 8-OH-DPAT-induced LLR could be antagonised by pindolol, spiperone, spiroxatrine and NAN-190, but not by the beta 1-adrenoceptor antagonist metoprolol, the beta 2-adrenoceptor antagonist butoxamine or the dopamine antagonist haloperidol. This antagonism was competitive as the dose-response curve of 8-OH-DPAT was shifted to the right. Pindolol, spiperone, spiroxatrine and NAN-190 all have a high affinity for the 5-HT1A receptor. This indicates that blockade of 8-OH-DPAT-induced LLR is only possible by selective blockade of 5-HT1A receptors. A possible mechanism of action is discussed. The increased defecation induced by 8-OH-DPAT could be antagonised by pindolol and NAN-190. The effect of spiroxatrine and haloperidol on the 8-OH-DPAT-induced increase in defecation was bimodal: an increase after a low and a decrease after a high dose of 8-OH-DPAT. Metoprolol and butoxamine had no effect on the 8-OH-DPAT-induced increase in defecation, thereby excluding an influence of beta-adrenoceptors.

The effect of antidepressants on aversive periaqueductal gray stimulation in rats

The selective serotonin (5-HT) uptake inhibitors fluvoxamine and sertraline had anti-aversive effects when administered acutely. Imipramine and clomipramine, which combine noradrenaline (NA) and 5-HT uptake blocking properties, did not have significant effects, whereas the mixed dopamine (DA)/NA uptake blocker nomifensine decreased the thresholds for escape from aversive periaqueductal gray stimulation. These results suggest that indirect 5-HT receptor activation suppresses periaqueductal gray aversion. Conversely, indirect DA and perhaps NA receptor activation appears to enhance periaqueductal gray aversion in rats.