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

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.

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-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

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

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

1 Animals

Male wild-type (WT) and 5-HT1A

(-/-)/5-HT2A (-/-) receptors deficient mice, 6 - 8

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 3 - 6 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

2 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

4 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-4-iodoamphetamine (DOI) is known as a

selective 5-HT2A receptor agonist;

alpha-(2.3-dimethoxyphenyl)-1-(2-(4-fluorophenylethyl))-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 tri-hydrochloride (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

subcutaneously (s.c) at the doses of 1 -

10 mg/kg (DOI); 2 mg/kg (MDL100907);

0.1 mg/kg (8-OHDPAT) and 0.5 mg/kg (WAY100635) N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine) (DSP-4) was dissolved in NaCl (0.9%) and injected intraperitoneally (i.p) at the dose 25 and

50 mg/kg

3 In vivo electrophysiological recordings

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 2 M NaCl solution Single glass micropipettes pulled on a pipette puller

(Narishige, Japan) with impedances ranging

from 2.5 to 5 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

4 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

two-way 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

1 Effects of the genetic inactivation

of the 5-HT 2A 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]

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-HT 2A +/+ wild-types (WT, n = 5 mice per group)

Figure 2 showed that 5-HT2A-/- displayed a higher spontaneous firing rate of DR

5-HT neurons than their wild-type littermates, raising the possibility that 5-5-HT2A 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-HT2A+/+ and 5-HT2A-/- 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)

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 pre-treatment 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

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

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-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-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

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

5-HT1A 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

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

5-HT1A -/- mice indicating 5-HT2A receptor hypersensitivity in these mutants This observation was corroborated by data, showing an increased expression of

5-HT2A 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

5-HT1A -/- 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

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 HT neurons in

5-HT1A -/- 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

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