Brain Research, 592 (1992) 163-169

163

© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 18134

Stereoselective effects of central a2-adrenergic agonist medetomidine on in vivo catechol activity in the rat rostral ventrolateral medulla (RVLM) M. H u n g a B. M i l n e b, C. Loomis c and K. J h a m a n d a s a Departments of ~ Pharmacology and Toxicology, and b Anaesthesia, Faculty of Medicine, Queen's University, Kingston, Ontario K7L 3N6 (Canada), and c School of Pharmacy, Faculty of Medicine, Memorial Unirersity, St. John's, Newfoundland AIB 31/6 (Canada) (Accepted 12 May 1992)

Key words: Dexmedetomidine; Levomedetomidine; Atipamezole; Voltammetry (in vivo); a2-Adrenoceptor

The stereoselective central effects of a novel, highly potent and selective a2-agonist medetomidine on adrenergic neuronal activity, reflected by changes in catechol oxidation current, in the rostral ventrolatcral medulla of the halothane-anesthetized rat were examined using in vivo differential normal pulse voltammetry. Dexmedetomidine, the active isomer, significantly decreased catechol oxidation current to 33.4 + 4.5% of baseline when given centrally (! ~g, i.c.v.) and to 10.3+3.9% of baseline when given systemically (50 /~g/kg, i.e.). Dexmedetomidine also significantly reduced mean arterial blood pressure by 19.9% following central administration hut significantly increased mean arterial blood pressure by 59.9% following systemic administration. Levomedetomidine, the inactive isomer, had no effect on catechol oxidation current or blood pressure. The depressant effects of dexmedetomid~ne on catechol oxidation current were reversed by the selective a2-adrenoceptor antagonist atipamezole (2 p.g, i.c.v, or 200/~g/kg, i.e.). The results of the present study demonstrate, to our knowledge, for the first time the central stereoselective effects of medetomidine and antagonism by atipamezole on rostral ventrolateral medulla activity in the anesthetized rat.

INTRODUCTION ¢2.Adrcnoceptor agonists, such as clonidine, produce a number of pharmacological effects including hypotension 4'~, suppression of symptoms of opiate withdrawal 2°, and reduction in anesthetic requiremerits 3a6'17'2~. The most widely used clinical application of an-agents is in the treatment of hypertension I't1°42. However, there is increasing use of these agents as sedative-analgesics in veterinary medicine 39 and as anesthetic adjuncts t6.tT, ot2-Agonists are beneficial in providing perioperative hemodynamie stability and contribute to a reduction in sympathetic responses to surgery1:"tT'es. The precise mechanism of action of ~2-agonists o n hemodynamics has been attributed to an interactio~t of these agents with central az-adrenoceptors reducing sympathetic neuronal activity 25. It is well documented that the rostral ventrolateral medulla (RVLM), an important centre in the control of sympathetic outflow, plays a crucial role in the control of blood pressure (for

review, see ref. 38). Accumulating evidence suggests that within a restricted region of the RVLM, the CI neuronal group 24 serves as the major site of af,tion mediating the hypotensive effects of t~2-agonists 14'35'47. Injections of clonidine or a-methyl noradrenaline into the C1 area lowers arterial pressure 4'22'35. Bilateral electrolytic lesions of this brain region have also been shown to prevent the hypotensive effects of systemic clonidine 5,6 and abolish baroreceptor reflex mechanisms 22,23. Until recently, few studies have demonstrated a correlation between changes in blood pressure and changes in RVLM neuronal activity. Hypotension induced by halothane or nitroprusside has been shown to increase RVLM catecholaminergi¢ r.euronal activity measured by catechol oxidation current using in vivo voltammetry 2,32. On the other hand, hypertension induced by phenylephrine has been shown to decrease RVLM neuronal activity 2. These studies suggest that changes in the RVLM neuronal activity are opposite to the changes in blood prgssure. However, hypotension

Correspondence: B. Milne, Department of Anaesthesia, Kingston Gerleral Hospital, 76 Stuart St. Kingston, Ontario, Canada. Fax: (1) (613) 545 6412.

164 induced by clonidine given systemically in low doses 4s or intraperitoneaily in high doses 46 has been shown to correlate with decreased neuronal activity within the ventrolateral medulla in electrochemical studies using in rive voltammetry. This reduction in neuronal activity within the RVLM following cionidine is consistent with the mechanism of action by which a2-agonists mediate their effects, acting either presynaptically or postsynaptically to decrease catecholamine release or alter neuronal firing thus decreasing sympathetic activity. However, stereoselectivity and antagonism of centrally administered ot2-agonists on RVLM activity usin~ in vivo voltammetry has not been examined. in vivo veltammetry is an electrochemical technique for monitoring catechol metabolism in brain regions containing catecholaminergic cell bodies (for review, see ref. 8). By this method, changes in catechol oxidation current serve as an index of the activity of catecholaminergic neurons 7'%1s'36"46. The aim of this work was to examine the stereoselective central effects of a novel, highly potent and selective az-ogonist medetomidine on adrenergic neuronal activity, reflected by changes in catechol oxidation current, in the rat rostral ventrolateral medulla using the in rive voltammetric technique of differential normal pulse voltammetry (DNPV) combined with electrochemically treated carbon fiber electrodes, and to determine whether changes in RVLM activity correlate with changes in blood pressure. The antagonism of the effects of medetomidine were also examined using the selective a,-antagonist atipamezolc. Medetomidine has been described as the most potent and selective a,-agonist available and demonstrates activity at bc,th pre- and postsynaptic a2-adrenoceptor# ° to produce similar pharmacological effects as other ot2-agonists including hypotension, bradycardia, sedation and analgesia (for review see ref. 49). Dexrnedetomidine, the dextro-isomer of medetomidine, is mere efficacious than clonidine and appears to be 10-100 times more selective for the c~,-adrenoceptor titan clonidine 3°,5°, MATER:ALS AND METHGDS Male Sprague-Dawley rats (Charles River, 350-400 g) were anesthetized using halothane and oxygen (induction - 4% halothane, maintenance = 1,5-2% halothane), Animals were given metocurine (200 p.g/kg i,v.) to produce muscle relaxation and were mechanically ventilated using a Harvard rodent respirator ( f = 50 strokes/rain; oxygen; inspired halothane, !.5-2%) to m:~intainPaCe: in the range of 35-45 mm Hg, Rectal temperature was maintained at 37~:0,5°C with a warming blanket connected to a YSI temperature cc.ltroller, All animals received a continuous saline (0,9%) infusion using a Harvard minipump (5 m l / k g / h ) via a femoral venous catheter and arterial blood pressure was monitored on a Grass Model ? physiograph via a femoral arterial catheter coupled to a Statham blood pressure transducer. Arterial blood samples (120 .u.I), drawn follow-

ing instrumentation, and both during and at the end of the exper; taunt, were analyzed (Instrumentation Laboratory System IL 1301 blood gas analyzer) to ensure that normal physicMgical pH and P a C e 2 were maintained. Treated carbon fiber microelectrodes were implanted as previously described "~2. Briefly, rats were placed prone in a stereotaxis frame, incisor bar 10 mm below the interaural line. The dorsal surface of the medulla oblongata was exposed by minimally resecting the occipital bone to create a small surgical window through the skull. A small incision was made through the membrane covering the cisterna magna and the treated carbon fiber was lowered through a small opening in the pia towards the Cl group of the RVLM following the co-ordinates: 30° angle with vertical zero; 1.0 mm anterior and 1.8 mm lateral from calamus scriptorius; and a depth of 3.0-3.5 mm from the medullary surface. Microelectrode placement in tl~ RVLM was identified by lowering the electrode through the R'vC:vl until the catechol oxidation current peak diminished in height and then raising the electrode to the depth at which the catechol oxidation peak height was maximum. An auxiliary electrode and a A3/AgCI reference electrode were placed on the skull surface by means of a semi-liquid contact. Differential normal pulse voltammetry (DNPV) (Biopulse, Tacu:0~el, Villeurbanne, France) was performed as previous!y described 46 every 3 rain. Carbon fiber electrodes (MCF 1, Tacussel) were electrically treated in vitro as previously described zl and tested in a standard phosphate buffered saline (PBS, pH 7.4) solution containing ascorbic acid (200 /zM) (Sigma Chemical Co., St. Louis, M e ) and 3,4-dihydroxyphenylacetic acid (DOPAC) (20 #M) (Sigma Chemical Co.). Catechol oxidation current was identified as a voltammetric peak occurring at +55 mV both in rive and in vitro. Fofiowing instrumentation and electrode implantation, steady state halothane anesthesia was maintained and a i h period was allowed for stabilization such that the catechol oxidation current peak height did not vary by more than 10% since changes in blood pressure,, pH. Pace z, and level of anesthesia may affect brain catecholamine function 1~'3~, To examine the central effect of different drugs on catechol oxidation current (CA. OC) in the RVLM, an intracerehroventrieular (i.c.v.) canlmla (25-gauge) was implanted into the lateral ventricle: 0.2 mm poslerior and 1,5 mm lateral from bregma~al a depth of 3.0 mm from the brain surface; and separate groups of animals were treLited as follows: (i) saline (5/~1, i.e.v., n - 4); (it) dexmedetomidine (I /~g, i.c.v., n - 4): or (iii) levomodetomidine (I lift, i.c.v,, n ~. 4). Following u 45 rain recording perk)d after initial drug treatment, each group received atipamezole (2/,tg, Lea.). in a separate group of :mimals treated with dexmedetomidine (l #g. i.e.v., n ,, 4), saline (5 /,tl, i.e.v.) was administered 45 rain following initial drug treatment. For comparison between the central and systemic effects of dexmedetomidine, separate groups of animals were treated with either: (1) saline (0.3 ml, i.v., n = 4); (2) dexmedetomidine (50 p,g/kg, i,v., n = 4); or (3) levomedetomidine (50 p,g/kg, i.v., n = 3), followed by atipamezole (200 p,g/kg, i,v,) 45 rain later. Catechol oxidation current was measured for a total of 90 rain at 3 rain intervals following the initial saline or drug administration, Mean arterial pressure (MAP, mm Hg) was monitored throughout the experiment and recorded before and I, 5, 15, 30 and 45 rain following each drug treatment. Dexmedetomidine, levomedetomidine and atipamezole (Farmos Group Ltd, Reseorch Center. Turku, Finland) were dissolved in saline (0,9%) and administered in volumes of 5/zl for i.c.v. administration and 0,3 ml for i,v, administration. At the end of each experiment, pargyline (75 mg/kg, i.p.), a monoamine oxidase (MAC)) inhibitor, was administered to ascertain the identity and decay of the catechol oxidation current 7, and an electrolytic lesion ( + 5 V De, 5 s) was made with the carbon fiber electrode and the brain was rapidly removed for histological examination with Cresyl violet staining as reported previously 32. Results of the catechol oxidation current measurements were expressed as a percentage of the mean baseline value calculated by averaging the four absolute values of the cateehol oxidation current peak heights measured prior to initial drug treatment. Mean arterial pressure was ~:alculatedas (diastolic pressure + 1/3 pulse pressure) where pulse pressure = (systolic-diastolic pressure). Results are ex-

165 pressed as the mean + S.E.M. Statistical analysis of data were performed using repeated measures analysis of variance (ANOVA) followed by the New, aan-Keuls test to determine significance at the P < 0.05 level (BMDP4V - General Univariate and Multivariate ANOVA, BMDP Statistical Software, Inc.).

ment: CA. OC range 35.7 :t: 4.2% - 41.5 _+3.2% of baseline. Intracerebroventricular levomedetomidine (1 /~g in 5/~l saline), the pharmacologically inactive levoisomer of medetomidine, or saline vehicle (5 ~l, i.c.v.) had no effect on CA. OC or MAP (Fig. 1). The administration of atipamezole (2 /~g, i.c.v.) 45 min following levomedetomidine or saline had no effect on CA. OC or MAP. For comparison between the central and systemic effects of dexmedetomidine, intravenous dexmedetomidine (50/~g/kg in 0.3 ml saline) resulted in a marked and significant decrease in CA. OC in the RVLM. A significant decrease in CA. OC was observed within 15 min following i.v. administration, peak CA- OC reduction 10.3 + 3.9% of baseline 45 rain post injection (Fig. 2a). In contrast to the central effects, systemic dexmedetomidine (50/zg/kg, i.v.) produced a significant increase in MAP. MAP was significantly increased by 59.9% (from 83.3 ± 9.4 to 133.2 _+9.3 mm Hg) within 1 rain following drug administration and then slowly returned to baseline values (Fig. 2b). Administration of the antagonist atipamezole (200 p.g/kg, i.v.) 45 rain after dexmedetomidine quickly reversed the depressant effects of dexmedetomidine on CA. OC (Fig. 2a) and produced a decrease in MAP (from 84.5 + 9.2 to 62.1 + 8.2 mm Hg) within 1 min following administration but the blood pressure quickly returned to baseline values within 5 rain (80.4 + 7.9 mm Hg) (Fig. 2b). Similar to the central administration of dexmedetomidine, CA. OC remained reduced for the duration of the experiment when saline (0.3 ml, i.v.) was administered 45 rain following dexmedetomidine in a separate

RESULTS lntracerebroventricular administration of the a zagonist dexmedetomidine (1 /zg in 5 /zl saline), the pharmacologically active dextro-isomer of medetomidine, resulted in a decrease in RVLM neuronal activity as reflected by a decrease in catechol oxidation current (CA" OC). A significant decrease in CA. OC was observed within 15 rain following i.c.v, administration, peak C A - O C reduction 33.4 + 4.5% of baseline 45 min post injection (Fig. la). A significant decrease in mean arterial pressure (MAP) was also observed following the i.c.v, administration of dexmedetomidine. MAP was significantly reduced by 19.9% (from 84.3 + 1.7 to 67.5 + 1.0 mm Hg) within 1 min following drug administration and returned to baseline values within 30 min (Fig. lb). The administration of the selective a2-antagonist atipamezole (2/zg in 5/zl saline i.c.v.) 45 rain after dexmedetomidine reversed the depressant effects of dexmedetomidine on CA. OC (Fig. la) and had no significant effect on MAP (Fig. lb). The administration of saline (5/zl, i.c.v.) 45 rain after dexmedetomidine had no effect on the depressant effects of the agonist on CA. OC (Fig. la) or on MAP (Fig. lb). In this group, C A . O C was reduced to 35.7 + 4.2% of baseline 45 rain following dexmedetomidine injection and remained depressed for the duration of the experi-

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Fig. I. The effects of central dexmedetomldine (1 /~g in 5 #1 saline), levomedctomidine (| /~g in 5 p,I saline), and saline (5 t~1) on (a) catcchol oxidation current ( C A . O C ) in the R V L M and (b) mean arterial blood pressure (MAP). Atipamezole (2 /~g in 5 p,I saline), the selective az-antagonist, or ~aline (5 ~1)was administered 45 rain following initial drug treatment. Results represent the mean-I-S.E.M., n = 4 in each group. Catechol oxidation current is expressed as a percent (%) baseline, mean arterial pressure ( M A P ) is expressed as mr, Hg. * Represents significance at P < 0.05 of the dexmedetomidine + atipamezole group (filled triangles) compared to the dexmedetomidine + saline group (open triangles). * * Represents significance at P < 0.05 of the dexmedetomidine + saline group (open triangles) compared to the saline + atipamezole group (open boxes) and the levomedetomidine + atipamezole group (filled circles).

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Fig. 2. The effect of systemic dexmedetomidine (50 p,g/kg in 0.3 ml saline), levomedetomidine (50 pg/kg in 0.3 ml saline), and saline (0.3 ml, i.v.) on (a) catechol oxidation current (CA.OC) in the RVLM and (b) mean arterial blood pressure (MAP). Atipnmezole (200 p,g/kg in 0.3 ml saline) was administered 45 min following initial drug treatment. Results represent the mean ~. S.E.M., n = 4 for dexmedetomidine and saline groups, n = 3 for levomedetomidine group. Catechol oxidation current is expressed as a percent (%) baseline, mean arterial pressure (MAP) is expressed as mm Hg. * Represents significance at P < 0.05 of the dexmedetomidine + atipamezole group (filled triangles) compared to peak dexmedetomidine effect. * * Represents significance at P < 0.05 of the dexmedeiomidine + atipamezole group (open triangles) compared to the saline + atipamezole group (open boxes) and the levomedetomidine + atipamezole group (filled circles).

group of animals (n = 2, data not shown). Administration of saline (0.3 ml, i.v.) or levomedetomidine (50 Izg/kg, i.v.) had no effect on CA. OC or MAP (Fig. 2). Atipamezole (200 ~ g / k g , i.v.) administration 45 rain following saline or levomedetomidine injection also had no effect on CA. OC or MAP. In all experiments, no significant changes in pH or PaCO2 values were observed between control and post-treatment values. All values were within normal limits and PaO 2 values were greater than 100 mm Hg. Administration of the MAO inhibitor par~line at the end of each experiment resulted in a decay of the catechol oxidation current peak height and the catechol oxidation current signal completely disappeared within 20 min of pargyline injection. The loss of this signal further ascertains the identity of the catechol oxidation current 7. Histological examination with Cresyl ~.iolet staining as described and reported 32 previously confirmed the placement of the electrode tip within the RVLM. Briefly, lesions were located in the coronal plane 1,100-1,600 ~tm rostral to the obex, where the obex is defined as the closing of the 4th ventricle, next to the inferior olive corresponding to th~ nucleus paragigantocelhtlaris (PGi) where the eatecholaminergic cells bodies are adrenergic. DISCUSSION This study examines the effects of the stereoisomers of the highly potent and selective a2-agonist medeto-

midine on adrenergic activity in the rat RVLM using a neurochemical index measured by electrochemical methods. By using differential normal pulse voltammetry, changes in adrenergic neuronal activity - reflected by changes in catechol oxidation current - have been monitored on-line in a bioehemically specific manner in the RVLM, Changes in catechol oxidation current, mainly due to the oxidation of the deaminated metabolite of dopamine 3,4-dihydro~;yphenylacetic acid (DOPAC), have been reliably monitored in the brain including the RVLM using DNPV in previous studios ~'2~''~2.-~TA6'4s,Such cha.ges serve as an index of neuronal activity of catecholaminergie neurons 1"'~7'46. The DOPAC oxidation current peak observed in the present study satisfied criteria established previously 7'32'37'4~.Briefly, it was identified as a peak appearing at a potential of +55 mV, corresponding to the oxidation potential of DOPAC in vitro, in a restricted orca of the ventrolateral medulla between 3.0 and 3.5 mm below the medullary surface and decayed following the administration of the MAO inhibitor pargyline. Following histological examination, a lesion at the recording site of the microelectrode was observed between 1,100-1,600 tzm rostral to the obex, next to the inferior olive corresponding to the area of the nucleus paragigantocellularis ia the C1 region as reported previously 32. This area of the RVLM (C1 region) contains adrenergic cell bodies and previous studies suggest that the CA" OC recorded from this region reflects the catecholaminergic activity of these adrenergic neurons Is,z6,32. However, the possibility of a

167 contribution to the catechol oxidation signal from noradrenergic terminals to this region cannot be excluded. In the present study, dexmedetomidine, the pharmacologically active dextro-isomer of medetomidine, reduced RVLM adrenergic neuronal activity, reflected by a decrease in catechol oxidation current peak height, following both central and systemic administration. Levomedetomidine, the pharmacologically inactive levo-isomer of medetomidine, demonstrated no activity on catechol oxidation current in the present study. The decrease in CA. OC peak height observed following dexmedetomidine is consistent with the findings of previous studies following the systemic administration of the prototype ot2-agonist clonidine 37'46'4s but demonstrates, to our knowledge, for the first time the stereoselective actions of such agents administered directly into the cerebroventricular space. The obsel~,ed decrease in adrenergic activity following a2-agonist administration is consistent with the action of a2-agonist'.~ decreasing central sympathetic outflow. Although it has been postulated that a2-agonists act presynaptically via autoinhibitory mechanisms to decrease ¢atecholamine release from central catecholaminergic neurons 25'27, the possibility of a2-agonists acting postsynaptically to decrease central catecholamine~gic neuronal firing directly or indirectly via non-adrenergic neurons can not be ruled out. Reversal of the depressant effects on dexmedetomidine on catechol oxidation current by the selective az-antagonist atipamezole further suggests involvement of a2-adrenoceptors. Interestingly, administration of the selective a2-antagonist atipamezole following saline, dexmedetomidine or levomedetomidine did not result in increased catecholaminergic neuronal activity, as reflected by changes in catechol oxidation current, above pretreatment levels in the present study. Previous studies have shown increased catechol turnover following blockade of presynaptic a2-adrenoceptors using other a2-antagonists such as yohimbine and idazoxan 33'34'5~, ~'esuits consistent with presynaptic autoinhibitory mechanisms which regulate neurotransmitter release 27'45. Although dexmedetomidine and atipamezole have been described as a2-selective agents, dexmedetomidine is thought to be 10-100 times more selective for the a2-adrenoceptor than clonidine 3°'5° and atipamezole is at least a 100 times more selective than idazoxan 49'5°, these agents may interact with other sites including postsynaptic sites in the RVLM. Clonidine is known to interact with the recently proposed imidazoline-sensitire receptors in the RVLM ~s and activation of these sites may be responsible for mediating the actions of dexmedetomidine. However, other regulatory influ-

ences from other brain regions on RVLM cannot be ruled out in the failure to observe increased neuronal activity following atipamezole in the present study even if disinhibition of the presynaptic a2 autoregulatory mechanism of adrenergic neurons does indeed occur. Since it is well documented that the RVLM serves an important site in mediating the hypotensive effects of a2-agonists 14'35'4~,changes in catecholaminergic neuronal activity would be expected to correlate with changes in blood pressure. A decrease in mean arterial pressure was observed following the central administration of dexmedetomidine (Fig. lb). However, this decrease in MAP was of short duration in comparison to the long-lasting depressant effects of the drug on CA" OC. Although injections of clonidine or a-methyl noradrenaline into the RVLM also produce decreases in arterial pressure 4'23'35, the effects observed here suggest that other compensatory mechanisms are activated to maintain resting blood pressure levels. It has been previously demonstrated that peripherally mediated decreases in blood pressure increase catechol oxidation peak height through baroreflex mechanisms 37 and similar mechanisms might exist following prolonged central neuronal depression of hemodynamic control centers like the RVLM. In contrast to the blood pressure lowering effects of central dexmedetomidine, a sharp increase in blood pressure was observed following systemic dexmedetomidine (Fig. 2b). This observation is consistent with the previous finding of an initial increase in arterial pressure following systemic dexmedetomidine in dogs4~. At a dose of dexmedetomidine similar to that administered in the present study, vasopressor effects of medetomidine (30 /.~g/kg, i.e.) have also been described in the rat 39 and it is likely that such increases in MAP involve the activation of peripheral a2-adrenoceptors located postsynaptically on blood vessels to produce vasoconstriction 1°'~3'3~. Increases in blood pressure following phenylephrine have been shown to decrease RVLM neuronal activity 2 and the observed increase in MAP in the present study may contribute to the greater decrease in CA. OC observed following systemic administration compared to central administration of the dexmedetomidine. However, differences in drug concentrations at the site of action cannot be excluded when comparing drug effects following central versus systemic routes of drug administration. It is unlikely that the reported sedative-analgesic effects of dexmedetomidine t2,3°,44, thus reducing the anesthetic requirements, significantly influenced CA" OC in the RVLM in the present model since previous work found an increase in catechol activity following increasing concentrations of halothane 32.

168

The present study demonstrates, to our knowledge, for the first time the central stereoselective effects of the novel, highly potent and selective a2-adrenoceptor agonist medetomidine on catechol neuronal activity in the RVLM in the anesthetized rat. Dexmedetomidine, when administered centrally, decreases in RVLM activity and arterial blood pressure. These data suggesting that a correlation exists between a decrease in RVLM activity and a decrease in blood pressure following central administration of the drug may further implicate the RVLM in the central control of blood pressure. Acknowledgemems. This work was supported by the Medical Research Council of Canada and presented in part at the 21st Annual Meeting of the Canadian Anaesthetists' Society, June 1991, Quebec City. Quebec, Canada. The authors wish to thank Dr. Luc Quintin (Lyon, France) for his helpful comments in developing the technique of in vivo voltammetry in the RVLM and acknowledge Dr. Risto Lammintausta, Farmos Group Ltd. Research Center (Turku, Finland) for the generous gift of dexmedetomidine, levomedetomidine, and atipamezole. The authors also wish to thank Mrs. J. LeSarge for assistance in the preparation of this manuscript.

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