Spinal 5-HT2A receptors regulate cutaneous sympathetic vasomotor outflow in rabbits and rats; relevance for cutaneous vasoconstriction elicited by MDMA (3,4-methylenedioxymethamphetamine, “Ecstasy”) and its reversal by clozapine

Abstract

We determined whether spinal 5-hydroxytryptamine 2A (5-HT2A) receptors contribute to resting cutaneous sympathetic vasomotor activity, and to increases in activity elicited by electrical stimulation of the medullary raphe/parapyramidal region, and whether these receptors are involved in the cutaneous vasoconstricting action of systemically administered MDMA (3,4-methylenedioxymethamphetamine, “Ecstasy”) and its reversal by clozapine. Experiments were conducted in urethane-anesthetized rabbits and rats.

Administration of the 5-HT2A antagonist, trans-4-((3Z)3-[(2-Dimethylaminoethyl)oxyimino]-3-(2-fluorophenyl)propen-1-yl)-phenol, hemifumarate (SR 46349B, 0.1 mg/kg, i.v.) inhibited resting ear pinna sympathetic vasomotor nerve discharge and reduced the extent to which raphe/parapyramidal electrical stimulation caused ear pinna (rabbit) and tail (rat) artery blood flow to fall. Clozapine (0.125–0.5 mg/kg, i.v.) also reduced the fall in ear pinna blood flow elicited by raphe/parapyramidal stimulation. In rabbits, after inactivation of raphe/parapyramidal function by local microinjection of muscimol (1 nmol in 100 nl), the 5-HT2A agonist R(−)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI, 50 μg/kg, i.v.) increased ear pinna sympathetic nerve activity from 3±2% to 129±5% of pre-muscimol levels (P<0.01, n=6), and this increase was abolished by section of the ipsilateral cervical sympathetic nerve trunk. MDMA (2 mg/kg, i.v.) after muscimol decreased ear pinna blood flow from 33±10 to 2±1 cm/s (P<0.01, n=5) and increased ear pinna sympathetic nerve activity from 8±4% to 120±41% of pre-muscimol levels (P<0.01, n=6). The MDMA-elicited increase in nerve activity was abolished by SR 46349B. Data suggest that spinal 5-HT2A receptors contribute to sympathetically induced cutaneous vasoconstriction regulated by raphe/parapyramidal neurons in the brainstem, and that these receptors contribute to the cutaneous vasoconstricting action of MDMA and its reversal by clozapine.

Introduction

Stimulation of 5-hydroxytryptamine 2A (5-HT2A) receptors within the central nervous system (CNS) in rabbits and rats with R(−)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) results in activation of the cutaneous sympathetic vasomotor outflow, leading to constriction of thermoregulatory cutaneous vascular beds [4]. Histological studies have demonstrated that 5-HT2A receptors are widely distributed within the central nervous system, including the spinal cord [7]. At present, there is no information as to the location of subgroups of these receptors relevant to CNS control of the sympathetic cutaneous vasomotor outflow.

The rostral medullary raphe/parapyramidal region is an important brainstem-spinal relay for regulation of sympathetic vasomotor control of the cutaneous vascular bed [2], [3], [19], [23]. The population of sympathetic premotor neurons in the raphe region includes neurons that synthesize 5-HT [11], [12], [26]. Thus, it is possible that a proportion of the raphe-spinal neurons controlling sympathetic cutaneous vasomotor preganglionic neurons utilize 5-HT as a neurotransmitter, and that actions of 5-HT released from spinal terminal boutons includes stimulation of 5-HT2A receptors located on the relevant spinal sympathetic preganglionic neurons.

Sympathetically mediated cutaneous vasoconstriction contributes to hyperthermia elicited by MDMA (3,4-methylenedioxymethamphetamine, “Ecstasy”) [5], [22] and clozapine, an atypical antipsychotic agent, reverses these changes [5]. MDMA stimulates 5-HT release [9], [10] and clozapine has antagonist actions at 5-HT2A receptors [1], [15]. Thus, given that the excitatory actions of bulbospinal 5-HT neurons on cutaneous vasomotor sympathetic preganglionic neurons may involve 5-HT2A receptors, we hypothesised that MDMA-elicited cutaneous vasoconstriction and its reversal by clozapine are at least partially mediated by stimulation and blockade, respectively, of spinal 5-HT2A receptors. The present study investigates this hypothesis in anesthetized rabbits and rats. We recorded ear pinna blood flow in rabbits and tail blood flow in rats, as well as, in rabbits, postganglionic cutaneous vasomotor sympathetic nerve activity in the ear pinna.

As a first experimental strategy (in rabbits and rats), we activated cutaneous sympathetic outflow and vasoconstricted the cutaneous vascular bed by electrically stimulating the raphe/parapyramidal region. We determined whether intravenous administration of SR 46349B (trans-4-((3Z)3-[(2-Dimethylaminoethyl)oxyimino]-3-(2-fluorophenyl)propen-1-yl)-phenol, hemifumarate), a 5-HT2A receptor antagonist [24], affects resting cutaneous sympathetic vasomotor nerve activity, and whether the drug reduces cutaneous sympathetic vasomotor activation elicited by electrical stimulation of the raphe/parapyramidal region. To investigate possible CNS sites of action for the cutaneous vasomotor effects of clozapine, we also tested whether intravenous administration of clozapine reduces cutaneous sympathetic vasomotor activation elicited by electrical stimulation of the raphe/parapyramidal region. We controlled for the possibility of peripheral actions of SR 46349B and clozapine by assessing the effects of intravenous administration of these agents on ear pinna vasoconstriction elicited by electrical stimulation of the cervical sympathetic trunk in rabbits. Because SR 46349B has not previously been shown to reverse the cutaneous vasoconstricting actions of DOI, we first demonstrated that this is the case, carrying out the experiments in conscious rabbits.

As a second experimental strategy (in rabbits), we chemically inhibited neuronal function in the raphe/parapyramidal region by focal microinjections of muscimol, confirming the effectiveness of the inhibition by demonstrating loss of the cutaneous vasomotor sympathoexcitatory response normally elicited by stimulation of the spinal tract of the trigeminal nerve [2]. We then determined whether intravenous administration of DOI and similar administration of MDMA, activates cutaneous sympathetic nerve discharge in animals with raphe/parapyramidal inhibition, and whether any increase in nerve discharge is reversed by subsequent intravenous administration of SR 46349B.