Michael C. Koss

Pupillary dilation as an index of central nervous system α2-adrenoceptor activation

In recent years there has been increasing evidence that some antihypertensive drugs like clonidine and alpha-methyldopa (after conversion in the brain to alpha-methylnorepinephrine) may decrease sympathetic tone by stimulating central nervous system (CNS) alpha 2-adrenoceptors. These same drugs also produce pupillary dilation in cats and rats. In this review, evidence is presented supporting the hypothesis that clonidinelike drugs act either directly or indirectly on CNS postsynaptic alpha 2-adrenoceptors to cause pupillary dilation by reduction of parasympathetic neural tone to the iris. It is further suggested that the underlying physiologic mechanism for this mydriatic action is activation of an ascending pathway that provides tonic inhibitory input by releasing norepinephrine on neurons in the Edinger-Westphal complex. Yohimbine-sensitive pupillary dilation in these species may provide a simple and effective model for quantitatively accessing CNS alpha 2-adrenoceptor activity.

Evidence for a central postsynaptic action of clonidine

SummaryIntravenous administration of clonidine (1–100 μg/kg) produces a dose-dependent mydriasis in cats by inhibition of parasympathetic tone to the iris. The magnitude of CNS-induced pupillary dilation was similar in both normal anaesthetized cats and in araesthetized preparations pretreated with reserpine (5 mg/kg i.p.) and α-methyl-p-tyrosine (2×300 mg/kg i.p.). Pretreatment reduced the concentrations of noradrenaline, dopamine and serotonin to less than 3% of that control levels in most parts of the CNS in which these amines were measured. Clonidine produced bradycardia in control animals but not in pretreated cats. In amine depleted animals in which only one eye was innervated by the ciliary nerves (parasympathetic), clonidine produced mydriasis only on the innervated side.These experiments confirm our previous observations that clonidine produces mydriasis in the cat by means of inhibition of parasympathetic tone to the iris. It is concluded that if clonidine produces this effect by stimulating noradrenergic, dopaminergic or serotonergic receptors, then clonidine exerts its centrally-induced mydriatic effect by acting on post-synaptic mechanisms.

CNS adrenergic inhibition of parasympathetic oculomotor tone

Inhibition of parasympathetic neural tone to the iris was produced by electrical stimulation of the afferent sciatic nerve, medullary reticular formation, and posterior hypothalamus in anesthetized cats in which only the parasympathetic nerves to the eye were intact. Stimulation of all 3 sites of activation produced a graded pupillary dilation and reduction of tonic nerve activity in the short ciliary nerves. Intravenous administration of the alpha-2-adrenoceptor antagonist, yohimbine hydrochloride, (0.03-1.0 mg/kg) produced a dose-dependent antagonism of the mydriasis elicited by activation of the ascending (sciatic nerve and medullary) mechanisms but did not block the pupillary dilation evoked by stimulation of the system descending from the hypothalamus. This differential action of yohimbine was confirmed directly by means of nerve recordings taken from the parasympathetic nerve to the eye. Depletion of CNS monoamines with reserpine and alpha-m-p-tyrosine reduced the norepinephrine concentration of the medulla and midbrain by 95% and 97%, respectively. In these depleted preparations, stimulation of the hypothalamus still produced the characteristic mydriasis and inhibition of parasympathetic tonic activity whereas activation of ascending mechanisms (sciatic or medullary) were no longer effective in producing these effects. Taken together, these results suggest that ascending parasympatho -inhibition is mediated by a monoamine (probably norepinephrine) and that inhibition descending from the hypothalamus is mediated by a non-monoaminergic mechanism.

Studies on the mechanism of clonidine-induced mydriasis in the rat

Summary1.Intravenous administration of clonidine hydrochloride (3–100μg/kg) produced a dose-dependent pupillary dilation in anaesthetized rats. All experiments were carried out in rats in which vagosympathetic nerve trunks were sectioned bilaterally at the cervical level.2.Clonidine-induced mydriasis was present only in those preparations having intact parasympathetic neural tone to the iris.3.Depletion of CNS monoamines by more than 95% with reserpine (5mg/kg) and alpha-methyl-para-tyrosine (2×300mg/kg) failed to alter the dose-response relation to clonidine.4.Pretreatment with the alpha-2-adrenoceptor antagonist, yohimbine hydrochloride (1.5 mg/kg), produced about a 10-fold shift to the right in the pupillary dose-response curve to clonidine. Yohimbine administered after the highest dose of clonidine also antagonized the mydriatic response.5.The above results suggest that clonidine acts on CNS post-synaptic alpha-2-adrenoceptors to produce mydriasis by withdrawal of parasympathetic neural tone to the iris.6.In an attempt to assess the physiological substrate(s) involved, mydriatic responses, due to parasympatho-inhibition, were evoked by electrial stimulation of ascending (sciatic nerve and medullary) and descending (hypothalamic) pathways.7.Yohimbine (0.3 and 1.0mg/kg) produced a dose-dependent inhibition of the pupillary dilation evoked by stimulation of the sciatic nerve and medullary loci, whereas these doses of yohimbine failed to alter the dilation in response to hypothalamic stimulation.8.Similarly, monoamine depletion greatly antagonized the pupillary dilation elicited by sciatic nerve and medullary stimulation without significantly affecting mydriasis due to hypothalamic stimulation.8.These results suggest that pupillary dilation produced by activation of ascending inhibitory mechanisms may utilize a monoamine as an inhibitory neurotransmitter (perhaps noradrenaline) and that inhibition descending from the hypothalamus is not monoaminergic. It is further speculated that clonidine may produce mydriasis in this species by activation of CNS post-synaptic alpha-2-adrenoceptors (perhaps on neurons of the Edinger-Westphal nucleus) which normally subserve tonic inhibition arising from the periphery.

Topical clonidine produces mydriasis by a central nervous system action

One drop of clonidine solution (0.125, 0.25 or 0.5%) was administered topically to one eye in cats anesthetized with pentobarbital in which the vagosympathetic nerves had been sectioned. Clonidine caused a simultaneous dose-related mydriasis in both eyes along with a decrease in heart rate. The peak effects were observed in about 20--30 min. Topical administration of clonidine (0.5%) produced no effect on the parasympathectomized, eserinized iris but did dilate the opposite pupil. Epinephrine (0.1--30 microgram, i.a.) produced equal pupillary dilation in both eyes. In addition, topical clonidine caused a dramatic decrease in postganglionic ciliary nerve activity. All of the effects of clonidine were antagonized by yohimbine (0.5 mg/kg, i.v.). These results demonstrate that topical administration of clonidine causes my driasis in the cat and that this effect is mediated totally by means of CNS inhibition of parasympathetic tone to the iris.

Effects of clonidine and chlorpromazine on a sympathetic-cholinergic reflex

Intravenous administration of clonidine and chlorpromazine resulted in a dose-dependent inhibition of the amplitude of reflexly evoked electrodermal responses in intact and spinal cats. Yohimbine pretreatment (0.5 mg/kg, i.v.) antagonized the effects of clonidine but not chlorpromazine in both preparations. These findings confirm and expand previous observations that both clonidine and chlorpromazine inhibit the amplitude of centrally evoked responses in this sympathetic-cholinergic system. In addition, both drugs appeared to have a spinal site of action. The antagonism of the effects of clonidine by yohimbine suggests that the mechanism of the action of clonidine may be a result of activation of central inhibitory alpha-adrenergic receptors. The failure of yohimbine to antagonize the effects of chlorpromazine suggests that clonidine and chlorpromazine may depress these sympathetic reflexes by different mechanisms.

Role of nitric oxide in post-ischemic cerebral hyperemia in anesthetized rats

This study was undertaken to determine the extent to which nitric oxide (NO) mechanisms are involved in cerebral hyperemia following global brain ischemia. The vertebral arteries were cauterized through the first alar foramina in anesthetized male Sprague-Dawley rats and followed by 20-min occlusion of the common carotid arteries. Blood flow from the parietal cerebral cortex was measured using laser-Doppler flowmetry. In saline-treated animals, carotid occlusion reduced cerebral blood flow by approximately 95% with a maximal hyperemia of about 400% observed after 15 min of reperfusion. Pre-treatment with the nonspecific NO synthase inhibitor, L-NAME (NG-nitro-L-arginine methyl ester; 2, 10 and 50 mg kg(-1)), produced dose-related depression of post-ischemic hyperemia, whereas D-NAME (10 mg kg(-1)) was inactive. Pre-treatment with L-arginine (300 mg kg(-1), i.v.) prevented L-NAME attenuation of cerebral hyperemia. The selective neuronal NO synthase inhibitor, 7-nitroindazole (30 mg kg(-1)), was without significant depressant effect. These results suggest that NO (largely from vascular endothelium) is instrumental in development of post-ischemic cerebral hyperemia.

Role of nitric oxide in cutaneous blood flow increases in the rat hindpaw during dorsal column stimulation.

OBJECTIVE Dorsal column stimulation (DCS) increases blood flow to the extremities and may produce a limb-saving effect in addition to treatment of refractory chronic pain in patients with peripheral vascular disease. The purpose of this study was to examine the importance of nitric oxide in cutaneous vasodilation caused by DCS. METHODS Male Sprague-Dawley rats were anesthetized with pentobarbital (60 mg/kg, intraperitoneally). A unipolar ball electrode was placed on the left-side of the exposed spinal cord at approximately L1-L2. Blood flow was concurrently recorded from both hindpaw foot pads with laser doppler flowmeters. Blood flow responses were assessed during 1 minute of DCS (0.6 mA at 50 Hz, 0.2-ms pulse) at 10-minute intervals. Nitric oxide synthase was inhibited with NG-nitro-L-arginine methyl ester (L-NAME). Four groups of animals were examined. The first and second groups involved examination of the effects of DCS after 2 and 10 mg/kg L-NAME, respectively. In the third group, the effect of another nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (10 mg/kg), was examined on the responses to DCS. The fourth group of animals entailed comparison of the effects of DCS under control conditions, after the nicotinic receptor antagonist, hexamethonium (10 mg/kg), and during the combined presence of hexamethonium and L-NAME (10 mg/kg). RESULTS L-NAME markedly attenuated the cutaneous blood flow increases caused by DCS at doses of 2 or 10 mg/kg. Similarly, NG-monomethyl-L-arginine also attenuated the DCS response. Hexamethonium did not affect the cutaneous vasodilation caused by DCS. After hexamethonium, L-NAME no longer attenuated the DCS response. CONCLUSION Our results demonstrated that nitric oxide played a significant role in producing the DCS-induced increase in rat cutaneous hindpaw blood flow. The involvement of nitric oxide does not require the presence of autonomic efferent function; however, ganglionic blockade may unmask a mechanism for vasodilation during DCS that is independent of nitric oxide release.

Analysis of pupillary dilation produced by analogs of clonidine

Pupillary responses to intravenous administration of clonidine and five congeneric derivatives of clonidine were observed in anesthetized cats. All of the agents tested produced a dose-dependent mydriasis of long duration. The order of potency for these compounds was clonidine (St-155) greater than St.375 greater than St-606 greater than St-608 greater than St-91. Pretreatment with yohimbine hydrochloride (0.5 mg/kg i.v.) shifted the dose-response curve for all of the compounds tested to the right. A CNS inhibition of parasympathetic nerve activity was demonstrated for clonidine and St-375 by means of direct recordings from the postganglionic ciliary nerves. An effect of high doses of St-91 on nerve activity was also observed. These results suggest that all the analogs of clonidine tested (with the possible exception of St-91) act like clonidine to produce mydriasis by a central alpha-adrenergic inhibition of parasympathetic outflow to the eye.

Effect of NG-nitro-l-arginine methyl ester on functionally characterized muscarinic receptors in anesthetized cats

This study was undertaken to determine if the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), is a competitive antagonist of muscarinic receptors in vivo. Cats were anesthetized with pentobarbital (36 mg/kg, i.p.). Five peripheral muscarinic responses were characterized based on their sensitivity to intravenous administration of atropine (1-100 microg/kg), pirenzepine (1-100 microg/kg) or gallamine (30-3000 microg/kg) as follows: (1) muscarinic ganglionic transmission through the superior cervical ganglion to the nictitating membrane (M1), (2) electrically elicited vagal bradycardia (M2), (3) neurally evoked sudomotor responses (M3; non-endothelial), (4) basal pupil tone in sympathectomized cats (M3; non-endothelial) and (5) methacholine-induced depression of arterial blood pressure (M3; endothelial). Additional groups of animals were administered L-NAME (50 mg/kg, i.v.) to determine if this agent would alter activation of these muscarinic systems. L-NAME was devoid of effect on responses elicited by stimulation of muscarinic M1, M2 and M3 (non-endothelial) receptors. In contrast, L-NAME significantly reduced the depressor responses to i.v. methacholine (M3; endothelial), as did its non-alkyl ester congener, L-NA (NG-nitro-L-arginine; 25 mg/kg, i.v.). These results support the conclusion that although L-NAME inhibits synthesis of nitric oxide in vascular endothelial cells, it is not a generalized muscarinic receptor antagonist in vivo.