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

Nội dung text: 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

1. Journal of military pharmaco-medicine n o7-2017 THE THERAPEUTIC ROLE OF 5-HT 2A 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-HT 2A receptor in the regulation of the serotonergic system, its functional interactions with 5-HT 1A 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-HT 2A -/- 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-HT 2A receptor agonist DOI reduced the firing activity of 5-HT neurons in wild-type mice while these effects were completely blunted in 5-HT 2A -/- 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-HT 1A -/- KO mice, probably in relation with hypersensitivity of 5-HT 2A receptor in these mutants. Conclusion: This study clearly indicates that 5-HT 2A 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-HT 2A receptor; 5-HT neurons; Escitalopram; Central nervous system. INTRODUCTION [7] . Both receptors draw particular attention Selective serotonin (5-HT) reuptake because of their close association with inhibitors (SSRIs) are indirect-acting 5-HT negative feedback on serotonergic neurons receptor agonists since they block the in response to SSRIs. 5-HT 1A autoreceptor reuptake of 5-HT, increase 5-HT extracellular is known to play a role in mood disorders concentrations in various brain regions and their treatments [10]. However, and finally activate multiple 5-HT receptor studies on the therapeutic role of 5-HT 2A types. In the central nervous system (CNS), receptor that activation of 5-HT 2A receptors 5-HT 1A and 5-HT 2A receptors are the target may attenuate the therapeutic effects for effective drugs in the treatment of of SSRIs even in absence of 5-HT 1A psychiatric disorders such as major autoreceptors, have not been clarified. depression and schizophrenia, respectively 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
2. Journal of military pharmaco-medicine N o7-2017 experiment to further examine the reciprocal selective 5-HT 2A receptor agonist; interactions between 5-HT 1A and 5-HT 2A alpha-(2.3-dimethoxyphenyl)-1-(2-(4- receptors and combined escitalopram fluorophenylethyl))-4-piperidine methanol (SSRI) by using deficient mice for these (MDL 100907) is known as a selective receptors or wild-type mice treated either 5-HT 2A receptor antagonist; 8-hydroxy-2- with the selective agonist/antagonist of (di-n-propylamino) tetralin hydrobromide 5-HT 1A (8-OH-DPAT/WAY100635) or (8-OHDPAT) is known as a selective 5-HT 2A (DOI/MDL100907) receptors. 5-HT 1A receptor agonist and (N-{2-[4 (2-methoxyphenyl)-1-piperazinyl]ethyl}-N- MATERIALS AND METHODS (2-pyridinyl) cyclohexanecarboxamide tri- 1. Animals. hydrochloride (WAY100635) is known as Male wild-type (WT) and 5-HT 1A (-/-)/5- a selective 5-HT 1A receptor antagonist, were HT 2A (-/-) receptors deficient mice, 6 - 8 obtained from Sigma-Aldrich (L’Isle d’Abeau, weeks old, weight 24 - 32 g, were used in France) and dissolved in distilled water or this study. Mice that were maintained at beta-cyclodextrin. These pharmacological Weill Medical College of Cornell University, compounds were administered were transferred to our laboratory in order subcutaneously (s.c) at the doses of 1 - to grow a stable colony in the animal facility 10 mg/kg (DOI); 2 mg/kg (MDL100907); of the Faculté de Pharmacie, University of 0.1 mg/kg (8-OHDPAT) and 0.5 mg/kg Paris 11 (France). Experimental animals (WAY100635). N-(2-Chloroethyl)-N-ethyl- were housed in our animal care facility in 2-bromobenzylamine) (DSP-4) was groups of 3 - 6 and kept under standard dissolved in NaCl (0.9%) and injected conditions (room temperature of 22 - 23°C, intraperitoneally (i.p) at the dose 25 and 12:12 light - dark cycle, free access to 50 mg/kg. food and water). Mice were tested between 3. In vivo electrophysiological 9.00 a.m and 5.00 p.m during the light recordings. phase. Mice were anaesthetized with chloral 2. Drugs and administration. hydrate (400 mg/kg i.p) and placed in a Escitalopram oxalate was provided by stereotaxic frame (using the David Kopf Lundbeck (Denmark) and dissolved in mouse adaptor) with the horizontally NaCl 0.9% NaCl/NaCl/dimethylsulfoxide positioned skull. The extracellular recordings (80/20). It was administered by the were performed using single glass subcutaneous (s.c.) route at a dose of micropipettes (R&D Scientific Glass, USA) 4 mg/kg, which was known to produce an increase in extracellular levels of 5-HT, for recordings in the dorsal raphe (DR). without affecting dopaminergic and Micropipettes were preloaded with fibre noradrenergic neurotransmissions in the glass strands to promote capillary filling frontal cortex [8]. 1.2,5-dimethoxy-4- with a 2 M NaCl solution. Single glass iodoamphetamine (DOI) is known as a micropipettes pulled on a pipette puller 28
3. Journal of military pharmaco-medicine n o7-2017 (Narishige, Japan) with impedances ranging potential positive action by the from 2.5 to 5 mV, were positioned 0.2 - electrophysiological signal recording 0.5 mm posterior to the interaural line on system (R&D Scientific, USA). Identity of the midline and lowered into the DR, usually all recorded cells was confirmed by attained at a depth of 2.5 - 3.5 mm from visualization of the biocytin-filled cells. the brain surface. The DR 5-HT neurons Immunohistochemical identification of each were then identified according to the neuron recorded in patch-clamp configuration following criteria: a slow (0.5 - 2.5 Hz) and was completed as previously described regular firing rate and a long duration, [10] (figure 1) . Figure 1: Model of in vivo electrophysiological signal recording system. 4. Data statistical analysis. with the computer software StatView Electrophysiological data obtained 4.02 when they were appropriate. The from recordings in the DR were level of statistical significance was set ± (p < 0.05). expressed as means S.E.M. of the firing rate of 5-HT neurons. A one- or two- RESULTS way analysis of variance (ANOVA) with 1. Effects of the genetic inactivation repeated measures (when appropriate) on of the 5-HT 2A receptor on the pre-treatment, treatment and/or genotype spontaneous firing rate of 5-HT (5-HT +/+ vs. 5-HT -/-) factors was 2A 2A neurons in the dorsal raphe. performed to compare the experimental groups. When ANOVA were statistically One-way ANOVA on the basal firing significant, pair wise comparisons were rate of DR 5-HT neurons revealed a performed using Fisher protected least significant effect of genotype factor significance difference post hoc test [F(1.92) = 5.6, p < 0.05]. 29
4. Journal of military pharmaco-medicine N o7-2017 Figure 2: Effects of the genetic inactivation of the 5-HT 2A receptor on the spontaneous firing rate of 5-HT neurons in the dorsal raphe. (*p < 0.05: significantly different from 5-HT 2A +/+ wild-types (WT, n = 5 mice per group) Figure 2 showed that 5-HT 2A -/- displayed a higher spontaneous firing rate of DR 5- HT neurons than their wild-type littermates, raising the possibility that 5-HT 2A receptor exerted a tonic receptor inhibitory influence on serotonergic neuronal activity. 2. Effects of DOI, a selective 5-HT 2A receptor agonist on the firing rate of 5-HT neurons in the dorsal raphe of 5-HT 2A +/+ and 5-HT 2A -/- mice. Figure 3: Effects of DOI on the firing rate of 5-HT neurons in the dorsal raphe of 5-HT 2A +/+ and 5-HT 2A -/- mice. (A. Percent of inhibition of basal DR 5-HT firing rate in 5-HT 2A +/+ and 5-HT 2A -/- 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 7 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 = 5 mice per group) 30
5. Journal of military pharmaco-medicine n o7-2017 5-HT 2A and 5-HT 2A -/- mice were then 5-HT 2A receptor agonist in 5-HT 2A +/+ mice administered with increasing doses of pre-treated with the neurotoxin DSP-4 DOI (1-10 mg/kg s.c.). A two-way ANOVA at the doses of 25 and 50 mg/kg i.p. with repeated measures on the firing rate One-way ANOVA with repeated measures of DR 5-HT neurons indicated a on the firing rate of DR 5-HT neurons significant effect of treatment [F(1.36) = indicated a significant effect of pre- 11.6, p < 0.01] and genotype factors treatment factor [F(2, 48) = 7.06, p < 0.05). [F(1.36) = 25.3, p < 0.001). DOI induced a The inhibitory effect of DOI on DR 5-HT dose-dependent inhibition of DR 5-HT neuronal activity was significantly attenuated in DSP-4 (25 mg/kg; i.p.) pre-treated mice neuronal activity in 5-HT 2A +/+ with a whereas no greater electrophysiological maximal decreased observation at the effects were observed by increasing the highest dose test (10 mg/kg; s.c.). In a dose of DSP-4 at 50 mg/kg; i.p (figure 3B) . marked contrast, this inhibitory effect was Since, it was previously reported that completely blunted in 5-HT -/- mice thus 2A DSP-4 (25 mg/kg; i.p.) resulted in a selective confirming the inhibitory role of 5-HT 2A lesion of the noradrenergic system whereas receptor on the serotonergic system and DSP-4 (50 mg/kg; i.p.) produced the demonstrating the selectivity of DOI ablation of both the noradrenergic and towards 5-HT 2A receptor in this response dopaminergic systems (Cassano et al, (figure 3A) . 2009) [5], the present data strongly In order to identify the brain region suggested that the inhibitory effect of DOI involved in the inhibitory effect of DOI, we on DR 5-HT neuronal activity involved, at then examined the effect of this selective least in part, NE neurons. 3. Effects of escitalopram (SSRI) on the firing rate of 5-HT neurons in the dorsal raphe of 5-HT 1A +/+ and 5-HT 1A -/- mice. 31
6. Journal of military pharmaco-medicine N o7-2017 Figure 4: Effects of the SSRI escitalopram on the firing rate of 5-HT neurons in the dorsal raphe of 5-HT 1A +/+ and 5-HT 1A -/- mice. (A. Firing rate of DR 5-HT neurons in 5-HT 1A +/+ mice injected with vehicle (baseline), escitalopram (ESC 4 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-HT 1A -/- mice injected with vehicle (baseline), 8-OHDPAT (0.2 mg/kg; s.c.), escitalopram (ESC 4 mg/kg s.c), escitalopram + MDL100907 (2 mg/kg; s.c.) or escitalopram + WAY 100635 (0.5 mg/kg; s.c.), when administrating esc 4 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 = 5 mice per group) Having established that 5-HT 2A receptor WAY 100635 (figure 4A) . In 5-HT 1A -/- mice, exerted an inhibitory effect on DR 5-HT the inhibitory effect of escitalopram was neuronal activity, we hypothesized that reversed by MDL100907 but not by the inhibitory action of SSRIs might persist WAY100635 (figures 4B-C) . in mice lack of the 5-HT 1A autoreceptor. In 5-HT 1A +/+ and 5-HT 1A -/- mice, separate DISCUSSION one-way ANOVA on the firing rate of DR 5-HT 1A autoreceptor plays a role in 5-HT neurons indicated a significant effect mood disorders and their treatments. of treatment factor [F(2,15) = 9.1, p < 0.01], Indeed, an increase in somatodendritic 5- [F(2,17) = 14.6, p < 0.01] and [F(3,21) HT 1A autoreceptor density in the dorsal = 6.2, p < 0.05]. All dorsal raphe 5-HT raphe (DR) attenuates the therapeutic neurons tested in 5-HT 1A +/+ and 5-HT 1A -/- activity of selective serotonin reuptake mice were inhibited by escitalopram inhibitors (SSRIs), whereas their functional (4 mg/kg s.c.). In 5-HT 1A +/+ mice, the desensitization promotes activation of inhibitory effect of escitalopram was reversed brain serotonergic transmission, thereby by the subcutaneous administration of representing an adaptive change relevant 32
7. Journal of military pharmaco-medicine n o7-2017 to their therapeutic effect [10]. Nevertheless, trigger one or both systems [5]. Interestingly, multiple source of evidence suggests that we showed that the loss of noradrenergic other serotonegic receptors including neurons attenuated the inhibitory effects 5-HT 2A type may also exert inhibitory of DOI on the activity of DR 5-HT neurons effects on the DR 5-HT neurons raising while the combined lesion of the the possibility that the functional inactivation noradrenergic and the dopaminergic of 5-HT 1A autoreceptor would be necessary, systems failed to produce additional but not sufficient for SSRIs to exert their attenuation. These results strongly optimal antidepressant activity. The present suggested that the control of serotonergic study examined the role of 5-HT 2A receptor neurons by 5-HT 2A receptor specifically in the regulation of the serotonergic involved noradrenergic neurons, which are system and more particularly its functional anatomically and functionally connected interaction with 5-HT 1A autoreceptor. to 5-HT neurons in the DR [6]. This Our electrophysiological data demonstrated conclusion is consistent with previous that the inactivation or the stimulation of data from Dr Blier’s group reporting that 5-HT 2A receptor increased or reduced SSRI treatment enhances a tonic inhibitory respectively the firing activity of DR 5-HT influence by 5-HT on Locus Coeruleus neurons. These findings concur with neurons through postsynaptic 5-HT 2A previous studies in rats showing that the receptors that are located on local administration of the 5-HT 2A receptor GABAergic interneurons [3]. Having agonist DOI produced an inhibitory impact shown that 5-HT 2A receptor exerts a tonic upon the serotonergic system [9]. inhibitory effect in the DR of wild-type Nevertheless, since the selectivity of DOI mice, we next asked whether this receptor for 5-HT 2A receptor remains somewhat could maintain or enhance his negative equivocal [2], our study examined whether influence in mice lacking the 5-HT 1A its electrophysiological effects were receptor. Interestingly, we found that the altered in 5-HT 2A -/- mice or not. The ability of DOI to decrease DR 5-HT observation that DOI-induced decrease in neuronal activity was potentiated in 5- DR 5-HT neuronal activity was completely HT 1A -/- mice indicating 5-HT 2A receptor blunted in 5-HT 2A -/- mice confirmed the hypersensitivity in these mutants. This involvement of 5-HT 2A in this response. observation was corroborated by data, The possibility that DOI might have acted showing an increased expression of 5- through another receptor such as 5-HT 2C HT 2A receptor in 5-HT 1A -/- mice. In this could not be ruled out. In order to identify context, we tested the effect of the the brain region involved in the inhibitory escitalopram (SSRI) on the firing rate of effect of DOI, we tested the effect of this DR 5-HT neurons in 5-HT 1A +/+ and 5- selective 5-HT 2A receptor agonist in mice HT 1A -/- mice. As expected and previously displaying a lesion of the noradrenergic shown, escitalopram decreased the firing or noradrenergic/dopaminergic systems. rate of serotonergic neurons in wild-type Indeed, it was reported that depending on mice. More surprisingly, this response the dose, the neurotoxin DSP-4 could persisted in 5-HT 1A -/- and was reversed 33
8. Journal of military pharmaco-medicine N o7-2017 by the 5-HT 2A receptor antagonist for dexfenfluramine anorectic effects. MDL100907. This stands in contrast with Neuropsychopharmacology. 2010, 36 (2), a previous electrophysiological study pp.423-33. showing that fluoxetine failed to decrease 3. Blier P, Szabo S.T. Potential to neuronal activity of 5-HT neurons in 5- mechanisms of action of atypical antipsychotic HT -/- mice [1] but this lack of response medications in treatment-resistant depression 1A and anxiety. J Clin Psychiatry. 2005, 66 (8), can result from the 5-HT antagonistic 2A/2C pp.30-40. activity of the SSRI. Thus, one of the most 4. Cadogan A.K, Marsden C.A, Tulloch I et remarkable result obtained in this study al. Evidence that chronic administration of was the observation that 5-HT 2A receptors paroxetine or fluoxetine enhances 5-HT2 maintain an inhibitory influence on the receptor function in the brain of the guinea serotonergic system, particularly in pig. Neuropharmacology. 1993, 3, pp.249-56. response to increased levels of endogenous 5. Cassano T, Gaetani S, Morgese M.G et 5-HT induced by SSRI. The genetic al. Monoaminergic changes in locus coeruleus inactivation of 5-HT 1A was compensated and dorsal raphe nucleus following noradrenaline by an over-expression of 5-HT 2A receptor depletion. Neurochem Res. 2009, 34 (8), thereby maintaining an inhibitory effect on pp.1417-26. the serotonergic system. Such functional 6. Guiard B.P, El Mansari M, Blier P. interaction between both receptors has Prospect of a dopamine contribution in the been reported previously. In particular, it next generation of antidepressant drugs: the triple reuptake inhibitors. Curr Drug Targets. has been shown that sustained administration 2009, 10 (11), pp.1069-84. of SSRI produced a functional desensitization 7. Li J.X, Crocker C, Koek W et al. Effects of 5-HT receptor while increasing that of 1A of serotonin (5-HT)1A and 5-HT2A receptor 5-HT 2A receptor [4]. agonists on schedule-controlled responding in CONCLUSION rats: drug combination studies. Psychopharmacology (Berl). 2011, 213 (2-3), pp.489-97. This study clearly indicates that 5-HT 2A 8. Nguyen H.T, Guiard B.P, Bacq A et al. receptor acts in concert to maintain an Blockade of the high-affinity noradrenaline inhibitory influence on the serotonergic transporter (NET) by the selective 5-HT system, particularly in response of effects reuptake inhibitor escitalopram: an in vivo of the escitalopram to increased levels of microdialysis study in mice. Br J Pharmacol. endogenous 5-HT. 2013, 168 (1), pp.103-16. 9. Rahman W, Bannister K, Bee LA et al. A REFERENCES pronociceptive role for the 5-HT2 receptor on 1. Amargós-Bosch M, Bortolozzi A, Puig spinal nociceptive transmission: an in vivo MV et al. Co-expression and in vivo interaction electrophysiological study in the rat. 2011, of serotonin1A and serotonin2A receptors in 1382, pp.29-36. pyramidal neurons of prefrontal cortex. Cereb 10. Richardson-Jones J.W, Craige C.P, Cortex. 2004, 14 (3), pp.281-99. Guiard B.P et al. 5-HT1A autoreceptor levels 2. Banas S.M, Doly S, Boutourlinsky K et determine vulnerability to stress and response al. Deconstructing antiobesity compound action: to antidepressants. Neuron. 2010, 65 (1), requirement of serotonin 5-HT2B receptors pp.40-52. 34