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Objectives: To study the therapeutic role of 5-HT2A receptor in the regulation of the serotonergic system, its functional interactions with 5-HT1A autoreceptor and effects of escitalopram (a selective serotonin (5-HT) reuptake inhibitor, SSRI) on the firing rate of 5-HT neurons in the central nervous system.
Journal of military pharmaco-medicine no7-2017 THE THERAPEUTIC ROLE OF 5-HT2A RECEPTOR IN DEPRESSION AND EFFECTS OF ESCITALOPRAM ON THE FIRING RATE OF 5-HT NEURONS IN THE CENTRAL NERVOUS SYSTEM Nguyen Thanh Hai*; Bruno P Guiard** SUMMARY Objectives: To study the therapeutic role of 5-HT2A receptor in the regulation of the serotonergic system, its functional interactions with 5-HT1A autoreceptor and effects of escitalopram (a selective serotonin (5-HT) reuptake inhibitor, SSRI) on the firing rate of 5-HT neurons in the central nervous system Methods: The present study was examined by using in vivo electrophysiological experiments Results: In 5-HT2A-/- knock-out (KO) mice, a significant increase in the firing activity of dorsal raphe 5-HT neurons was observed to suggest a tonic inhibitory influence of this receptor upon serotonergic neurons Consistent with this hypothesis, the preferential 5-HT2A receptor agonist DOI reduced the firing activity of 5-HT neurons in wild-type mice while these effects were completely blunted in 5-HT2A-/- mutants Interestingly, in wild-type mice the lesion of the noradrenergic system attenuated the inhibitory effects of DOI on 5-HT neurons The study showed that the inhibitory effects of the escitalopram (SSRI) currently described in wild-type mice, persisted in 5-HT1A-/- KO mice, probably in relation with hypersensitivity of 5-HT2A receptor in these mutants Conclusion: This study clearly indicates that 5-HT2A receptor acts in concert to maintain an inhibitory influence on the serotonergic system, particularly in response of effects of the escitalopram on the increased levels of endogenous 5-HT * Keywords: 5-HT2A receptor; 5-HT neurons; Escitalopram; Central nervous system INTRODUCTION Selective serotonin (5-HT) reuptake inhibitors (SSRIs) are indirect-acting 5-HT receptor agonists since they block the reuptake of 5-HT, increase 5-HT extracellular concentrations in various brain regions and finally activate multiple 5-HT receptor types In the central nervous system (CNS), 5-HT1A and 5-HT2A receptors are the target for effective drugs in the treatment of psychiatric disorders such as major depression and schizophrenia, respectively [7] Both receptors draw particular attention because of their close association with negative feedback on serotonergic neurons in response to SSRIs 5-HT1A autoreceptor is known to play a role in mood disorders and their treatments [10] However, studies on the therapeutic role of 5-HT2A receptor that activation of 5-HT2A receptors may attenuate the therapeutic effects of SSRIs even in absence of 5-HT1A autoreceptors, have not been clarified This study used electrophysiological * Hanoi University of Pharmacy **University of Paris Sud 11 Corresponding author: Nguyen Thanh Hai (haint@hup.edu.vn) Date received: 12/06/2017 Date accepted: 07/08/2017 27 Journal of military pharmaco-medicine No7-2017 experiment to further examine the reciprocal interactions between 5-HT1A and 5-HT2A receptors and combined escitalopram (SSRI) by using deficient mice for these receptors or wild-type mice treated either with the selective agonist/antagonist of 5-HT1A (8-OH-DPAT/WAY100635) or 5-HT2A (DOI/MDL100907) receptors MATERIALS AND METHODS Animals Male wild-type (WT) and 5-HT1A (-/-)/5HT2A (-/-) receptors deficient mice, - weeks old, weight 24 - 32 g, were used in this study Mice that were maintained at Weill Medical College of Cornell University, were transferred to our laboratory in order to grow a stable colony in the animal facility of the Faculté de Pharmacie, University of Paris 11 (France) Experimental animals were housed in our animal care facility in groups of - and kept under standard conditions (room temperature of 22 - 23°C, 12:12 light - dark cycle, free access to food and water) Mice were tested between 9.00 a.m and 5.00 p.m during the light phase Drugs and administration Escitalopram oxalate was provided by Lundbeck (Denmark) and dissolved in NaCl 0.9% NaCl/NaCl/dimethylsulfoxide (80/20) It was administered by the subcutaneous (s.c.) route at a dose of mg/kg, which was known to produce an increase in extracellular levels of 5-HT, without affecting dopaminergic and noradrenergic neurotransmissions in the frontal cortex [8] 1.2,5-dimethoxy-4iodoamphetamine (DOI) is known as a 28 selective 5-HT2A receptor agonist; alpha-(2.3-dimethoxyphenyl)-1-(2-(4fluorophenylethyl))-4-piperidine methanol (MDL 100907) is known as a selective 5-HT2A receptor antagonist; 8-hydroxy-2(di-n-propylamino) tetralin hydrobromide (8-OHDPAT) is known as a selective 5-HT1A receptor agonist and (N-{2-[4 (2-methoxyphenyl)-1-piperazinyl]ethyl}-N(2-pyridinyl) cyclohexanecarboxamide trihydrochloride (WAY100635) is known as a selective 5-HT1A receptor antagonist, were obtained from Sigma-Aldrich (L’Isle d’Abeau, France) and dissolved in distilled water or beta-cyclodextrin These pharmacological compounds were administered subcutaneously (s.c) at the doses of 10 mg/kg (DOI); mg/kg (MDL100907); 0.1 mg/kg (8-OHDPAT) and 0.5 mg/kg (WAY100635) N-(2-Chloroethyl)-N-ethyl2-bromobenzylamine) (DSP-4) was dissolved in NaCl (0.9%) and injected intraperitoneally (i.p) at the dose 25 and 50 mg/kg In vivo recordings electrophysiological Mice were anaesthetized with chloral hydrate (400 mg/kg i.p) and placed in a stereotaxic frame (using the David Kopf mouse adaptor) with the horizontally positioned skull The extracellular recordings were performed using single glass micropipettes (R&D Scientific Glass, USA) for recordings in the dorsal raphe (DR) Micropipettes were preloaded with fibre glass strands to promote capillary filling with a M NaCl solution Single glass micropipettes pulled on a pipette puller Journal of military pharmaco-medicine no7-2017 (Narishige, Japan) with impedances ranging from 2.5 to mV, were positioned 0.2 0.5 mm posterior to the interaural line on the midline and lowered into the DR, usually attained at a depth of 2.5 - 3.5 mm from the brain surface The DR 5-HT neurons were then identified according to the following criteria: a slow (0.5 - 2.5 Hz) and regular firing rate and a long duration, potential positive action by the electrophysiological signal recording system (R&D Scientific, USA) Identity of all recorded cells was confirmed by visualization of the biocytin-filled cells Immunohistochemical identification of each neuron recorded in patch-clamp configuration was completed as previously described [10] (figure 1) Figure 1: Model of in vivo electrophysiological signal recording system Data statistical analysis Electrophysiological data obtained from recordings in the DR were expressed as means ± S.E.M of the firing rate of 5-HT neurons A one- or twoway analysis of variance (ANOVA) with repeated measures (when appropriate) on pre-treatment, treatment and/or genotype (5-HT2A+/+ vs 5-HT2A-/-) factors was performed to compare the experimental groups When ANOVA were statistically significant, pair wise comparisons were performed using Fisher protected least significance difference post hoc test with the computer software StatView 4.02 when they were appropriate The level of statistical significance was set (p < 0.05) RESULTS Effects of the genetic inactivation of the 5-HT2A receptor on the spontaneous firing rate of 5-HT neurons in the dorsal raphe One-way ANOVA on the basal firing rate of DR 5-HT neurons revealed a significant effect of genotype factor [F(1.92) = 5.6, p < 0.05] 29 Journal of military pharmaco-medicine No7-2017 Figure 2: Effects of the genetic inactivation of the 5-HT2A receptor on the spontaneous firing rate of 5-HT neurons in the dorsal raphe (*p < 0.05: significantly different from 5-HT2A +/+ wild-types (WT, n = mice per group) Figure showed that 5-HT2A-/- displayed a higher spontaneous firing rate of DR 5HT neurons than their wild-type littermates, raising the possibility that 5-HT2A receptor exerted a tonic receptor inhibitory influence on serotonergic neuronal activity Effects of DOI, a selective 5-HT2A receptor agonist on the firing rate of 5-HT neurons in the dorsal raphe of 5-HT2A+/+ and 5-HT2A-/- mice Figure 3: Effects of DOI on the firing rate of 5-HT neurons in the dorsal raphe of 5-HT2A+/+ and 5-HT2A-/- mice (A Percent of inhibition of basal DR 5-HT firing rate in 5-HT2A+/+ and 5-HT2A-/- mice administrated with DOI (1-10 mg/kg s.c.) B Percentage of inhibition of basal DR 5-HT firing rate in WT induced by DOI in DSP-4 (25; 50 mg/kg i.p.) pre-treated mice In these experiments, DSP-4 was injected days before the electrophysiological recordings) (*p < 0.05; **p < 0.01 and ***p < 0.001: significantly different from baseline of the corresponding group *p < 0.05: significantly different from and DSP-4 pre-treated mice (n = mice per group) 30 Journal of military pharmaco-medicine no7-2017 5-HT2A and 5-HT2A-/- mice were then administered with increasing doses of DOI (1-10 mg/kg s.c.) A two-way ANOVA with repeated measures on the firing rate of DR 5-HT neurons indicated a significant effect of treatment [F(1.36) = 11.6, p < 0.01] and genotype factors [F(1.36) = 25.3, p < 0.001) DOI induced a dose-dependent inhibition of DR 5-HT neuronal activity in 5-HT2A+/+ with a maximal decreased observation at the highest dose test (10 mg/kg; s.c.) In a marked contrast, this inhibitory effect was completely blunted in 5-HT2A-/- mice thus confirming the inhibitory role of 5-HT2A receptor on the serotonergic system and demonstrating the selectivity of DOI towards 5-HT2A receptor in this response (figure 3A) In order to identify the brain region involved in the inhibitory effect of DOI, we then examined the effect of this selective 5-HT2A receptor agonist in 5-HT2A+/+ mice pre-treated with the neurotoxin DSP-4 at the doses of 25 and 50 mg/kg i.p One-way ANOVA with repeated measures on the firing rate of DR 5-HT neurons indicated a significant effect of pretreatment factor [F(2, 48) = 7.06, p < 0.05) The inhibitory effect of DOI on DR 5-HT neuronal activity was significantly attenuated in DSP-4 (25 mg/kg; i.p.) pre-treated mice whereas no greater electrophysiological effects were observed by increasing the dose of DSP-4 at 50 mg/kg; i.p (figure 3B) Since, it was previously reported that DSP-4 (25 mg/kg; i.p.) resulted in a selective lesion of the noradrenergic system whereas DSP-4 (50 mg/kg; i.p.) produced the ablation of both the noradrenergic and dopaminergic systems (Cassano et al, 2009) [5], the present data strongly suggested that the inhibitory effect of DOI on DR 5-HT neuronal activity involved, at least in part, NE neurons Effects of escitalopram (SSRI) on the firing rate of 5-HT neurons in the dorsal raphe of 5-HT1A+/+ and 5-HT1A-/- mice 31 Journal of military pharmaco-medicine No7-2017 Figure 4: Effects of the SSRI escitalopram on the firing rate of 5-HT neurons in the dorsal raphe of 5-HT1A+/+ and 5-HT1A-/- mice (A Firing rate of DR 5-HT neurons in 5-HT1A+/+ mice injected with vehicle (baseline), escitalopram (ESC mg/kg s.c) or escitalopram + WAY100635 (0.5 mg/kg; s.c.) B, C Firing rate of DR 5-HT neurons in the DR 5-HT1A-/- mice injected with vehicle (baseline), 8-OHDPAT (0.2 mg/kg; s.c.), escitalopram (ESC mg/kg s.c), escitalopram + MDL100907 (2 mg/kg; s.c.) or escitalopram + WAY 100635 (0.5 mg/kg; s.c.), when administrating esc mg/kg s.c or esc + WAY 100635 or esc + MDL 100907) (**p < 0.01; ***p < 0.001: significantly different from baseline *p < 0.05; **p < 0.01: significantly different escitalpram injected mice; ns: not significant (n = mice per group) Having established that 5-HT2A receptor exerted an inhibitory effect on DR 5-HT neuronal activity, we hypothesized that the inhibitory action of SSRIs might persist in mice lack of the 5-HT1A autoreceptor In 5-HT1A+/+ and 5-HT1A-/- mice, separate one-way ANOVA on the firing rate of DR 5-HT neurons indicated a significant effect of treatment factor [F(2,15) = 9.1, p < 0.01], [F(2,17) = 14.6, p < 0.01] and [F(3,21) = 6.2, p < 0.05] All dorsal raphe 5-HT neurons tested in 5-HT1A+/+ and 5-HT1A-/mice were inhibited by escitalopram (4 mg/kg s.c.) In 5-HT1A+/+ mice, the inhibitory effect of escitalopram was reversed by the subcutaneous administration of 32 WAY 100635 (figure 4A) In 5-HT1A-/- mice, the inhibitory effect of escitalopram was reversed by MDL100907 but not by WAY100635 (figures 4B-C) DISCUSSION 5-HT1A autoreceptor plays a role in mood disorders and their treatments Indeed, an increase in somatodendritic 5HT1A autoreceptor density in the dorsal raphe (DR) attenuates the therapeutic activity of selective serotonin reuptake inhibitors (SSRIs), whereas their functional desensitization promotes activation of brain serotonergic transmission, thereby representing an adaptive change relevant Journal of military pharmaco-medicine no7-2017 to their therapeutic effect [10] Nevertheless, multiple source of evidence suggests that other serotonegic receptors including 5-HT2A type may also exert inhibitory effects on the DR 5-HT neurons raising the possibility that the functional inactivation of 5-HT1A autoreceptor would be necessary, but not sufficient for SSRIs to exert their optimal antidepressant activity The present study examined the role of 5-HT2A receptor in the regulation of the serotonergic system and more particularly its functional interaction with 5-HT1A autoreceptor Our electrophysiological data demonstrated that the inactivation or the stimulation of 5-HT2A receptor increased or reduced respectively the firing activity of DR 5-HT neurons These findings concur with previous studies in rats showing that the administration of the 5-HT2A receptor agonist DOI produced an inhibitory impact upon the serotonergic system [9] Nevertheless, since the selectivity of DOI for 5-HT2A receptor remains somewhat equivocal [2], our study examined whether its electrophysiological effects were altered in 5-HT2A-/- mice or not The observation that DOI-induced decrease in DR 5-HT neuronal activity was completely blunted in 5-HT2A-/- mice confirmed the involvement of 5-HT2A in this response The possibility that DOI might have acted through another receptor such as 5-HT2C could not be ruled out In order to identify the brain region involved in the inhibitory effect of DOI, we tested the effect of this selective 5-HT2A receptor agonist in mice displaying a lesion of the noradrenergic or noradrenergic/dopaminergic systems Indeed, it was reported that depending on the dose, the neurotoxin DSP-4 could trigger one or both systems [5] Interestingly, we showed that the loss of noradrenergic neurons attenuated the inhibitory effects of DOI on the activity of DR 5-HT neurons while the combined lesion of the noradrenergic and the dopaminergic systems failed to produce additional attenuation These results strongly suggested that the control of serotonergic neurons by 5-HT2A receptor specifically involved noradrenergic neurons, which are anatomically and functionally connected to 5-HT neurons in the DR [6] This conclusion is consistent with previous data from Dr Blier’s group reporting that SSRI treatment enhances a tonic inhibitory influence by 5-HT on Locus Coeruleus neurons through postsynaptic 5-HT2A receptors that are located on local GABAergic interneurons [3] Having shown that 5-HT2A receptor exerts a tonic inhibitory effect in the DR of wild-type mice, we next asked whether this receptor could maintain or enhance his negative influence in mice lacking the 5-HT1A receptor Interestingly, we found that the ability of DOI to decrease DR 5-HT neuronal activity was potentiated in 5HT1A -/- mice indicating 5-HT2A receptor hypersensitivity in these mutants This observation was corroborated by data, showing an increased expression of 5HT2A receptor in 5-HT1A -/- mice In this context, we tested the effect of the escitalopram (SSRI) on the firing rate of DR 5-HT neurons in 5-HT1A +/+ and 5HT1A -/- mice As expected and previously shown, escitalopram decreased the firing rate of serotonergic neurons in wild-type mice More surprisingly, this response persisted in 5-HT1A -/- and was reversed 33 Journal of military pharmaco-medicine No7-2017 by the 5-HT2A receptor antagonist MDL100907 This stands in contrast with a previous electrophysiological study showing that fluoxetine failed to decrease to neuronal activity of 5-HT neurons in 5HT1A -/- mice [1] but this lack of response can result from the 5-HT2A/2C antagonistic activity of the SSRI Thus, one of the most remarkable result obtained in this study was the observation that 5-HT2A receptors maintain an inhibitory influence on the serotonergic system, particularly in response to increased levels of endogenous 5-HT induced by SSRI The genetic inactivation of 5-HT1A was compensated by an over-expression of 5-HT2A receptor thereby maintaining an inhibitory effect on the serotonergic system Such functional interaction between both receptors has been reported previously In particular, it has been shown that sustained administration of SSRI produced a functional desensitization of 5-HT1A receptor while increasing that of 5-HT2A receptor [4] CONCLUSION This study clearly indicates that 5-HT2A receptor acts in concert to maintain an inhibitory influence on the serotonergic system, particularly in response of effects of the escitalopram to increased levels of endogenous 5-HT REFERENCES Amargós-Bosch M, Bortolozzi A, Puig MV et al Co-expression and in vivo interaction of serotonin1A and serotonin2A receptors in pyramidal neurons of prefrontal cortex Cereb Cortex 2004, 14 (3), pp.281-99 Banas S.M, Doly S, Boutourlinsky K et al Deconstructing antiobesity compound action: requirement of serotonin 5-HT2B receptors 34 for dexfenfluramine anorectic effects Neuropsychopharmacology 2010, 36 (2), pp.423-33 Blier P, Szabo S.T Potential mechanisms of action of atypical antipsychotic medications in treatment-resistant depression and anxiety J Clin Psychiatry 2005, 66 (8), pp.30-40 Cadogan A.K, Marsden C.A, Tulloch I et al Evidence that chronic administration of paroxetine or fluoxetine enhances 5-HT2 receptor function in the brain of the guinea pig Neuropharmacology 1993, 3, pp.249-56 Cassano T, Gaetani S, Morgese M.G et al Monoaminergic changes in locus coeruleus and dorsal raphe nucleus following noradrenaline depletion Neurochem Res 2009, 34 (8), pp.1417-26 Guiard B.P, El Mansari M, Blier P Prospect of a dopamine contribution in the next generation of antidepressant drugs: the triple reuptake inhibitors Curr Drug Targets 2009, 10 (11), pp.1069-84 Li J.X, Crocker C, Koek W et al Effects of serotonin (5-HT)1A and 5-HT2A receptor agonists on schedule-controlled responding in rats: drug combination studies Psychopharmacology (Berl) 2011, 213 (2-3), pp.489-97 Nguyen H.T, Guiard B.P, Bacq A et al Blockade of the high-affinity noradrenaline transporter (NET) by the selective 5-HT reuptake inhibitor escitalopram: an in vivo microdialysis study in mice Br J Pharmacol 2013, 168 (1), pp.103-16 Rahman W, Bannister K, Bee LA et al A pronociceptive role for the 5-HT2 receptor on spinal nociceptive transmission: an in vivo electrophysiological study in the rat 2011, 1382, pp.29-36 10 Richardson-Jones J.W, Craige C.P, Guiard B.P et al 5-HT1A autoreceptor levels determine vulnerability to stress and response to antidepressants Neuron 2010, 65 (1), pp.40-52 ... raphe of 5-HT2 A+ /+ and 5-HT2 A- /- mice Figure 3: Effects of DOI on the firing rate of 5-HT neurons in the dorsal raphe of 5-HT2 A+ /+ and 5-HT2 A- /- mice (A Percent of inhibition of basal DR 5-HT firing. .. Figure 4: Effects of the SSRI escitalopram on the firing rate of 5-HT neurons in the dorsal raphe of 5-HT1 A+/+ and 5-HT1 A-/- mice (A Firing rate of DR 5-HT neurons in 5-HT1 A+/+ mice injected with... that 5-HT2 A receptor exerted a tonic receptor inhibitory influence on serotonergic neuronal activity Effects of DOI, a selective 5-HT2 A receptor agonist on the firing rate of 5-HT neurons in the