Modulation of catecholamine release by α2-adrenoceptors and I1-imidazoline receptors in rat brain

Brain Research 744 Ž1997. 216–226

Research report

Modulation of catecholamine release by a 2-adrenoceptors and I 1-imidazoline receptors in rat brain J. Javier Meana b

a,b,)

, Mario Herrera-Marschitz a , Michel Goiny a , Rodolfo Silveira

a

a Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden Department of Pharmacology, UniÕersity of the Basque Country, 48940 Leioa, Bizkaia, Spain

Accepted 27 August 1996

Abstract The physiological and pharmacological effects of imidazoliŽdi.ne derivatives, such as clonidine, have been related not only to the interaction with a 2-adrenoceptors but also to their activity on non-adrenoceptor sites termed imidazoline receptors. The modulation of catecholamine release by imidazoline drugs was studied by monitoring extracellular levels of norepinephrine ŽNE., dopamine ŽDA., 3,4-dihydroxyphenylacetic acid ŽDOPAC. and homovanillic acid ŽHVA. with microdialysis in cingulate cortex of rats, with or without irreversible a 2-adrenoceptor blockade. NE and DA levels were in the 1 nM range whereas DOPAC and HVA levels were ( 100 nM. NE and DA levels were increased when the uptake blocker desipramine Ž1 mM. or KCl Ž100 mM. were added to the perfusion medium. Clonidine induced a dose-dependent Ž0.3–1.2 mgrkg i.p.. decrease in NE Žmax 61%. and DA Žmax 40%. levels that was reversed by the a 2-adrenoceptor antagonist RX821002. After a 2-adrenoceptor irreversible blockade with the alkylating agent N-ethoxycarbonyl-2ethoxy-1,2-dihydroquinoline ŽEEDQ., w 3 Hxclonidine binding to a 2-adrenoceptors was reduced by 94 " 1%. Under such conditions, clonidine elicited a paradoxical dose-dependent Ž0.6–2.4 mgrkg i.p.. increase of NE Žmax 56%. without modifications in DA, DOPAC and HVA levels. The stimulatory effect of clonidine was prevented by the imidazoline receptor antagonist idazoxan Ž10 mgrkg i.p.. but not by RX821002 Ž5 mgrkg i.p... In rats pretreated with EEDQ, cirazoline ŽI 1rI 2-imidazoline receptor agonist., moxonidine ŽI 1-imidazoline receptor agonist., but not guanabenz ŽI 2-imidazoline receptor agonist. Ž1.2–2.4 mgrkg i.p.. elicited an increase of NE levels in a similar manner to clonidine Ž11–82%.. Idazoxan also abolished these responses to cirazoline or moxonidine. In contrast to systemic administration, local perfusion of clonidine Ž10–100 mM. through the microdialysis probe under a 2-adrenoceptor alkylating conditions, did not modify extracellular levels of NE and DA suggesting an indirect mechanism. The results demonstrate that clonidine and related imidazoliŽdi.ne drugs are able not only to inhibit NE release in rat cerebral cortex involving an a 2-adrenoceptor mechanism, but also to induce a paradoxical NE release through an indirect extracortical mechanism. The findings evidence that the indirect modulation of NE levels by imidazoline drugs is mainly due to a functional activity on I 1-imidazoline receptors. Keywords: Imidazoline receptor; a 2 -Adrenoceptor; Microdialysis; Norepinephrine; Dopamine; Clonidine; Rat

1. Introduction Several in vitro evidences suggest that central norepinephrine ŽNE. release is modulated by presynaptic a 2adrenoceptors w32,58x. In vivo studies using microdialysis have shown that a 2-adrenoceptor agonists, such as clonidine, induces a decrease in extracellular levels of NE in rat cortex w33,69x, whereas blockade of a 2-adrenoceptors enhances these levels w15,69x. In the same area, a tonic inhibitory modulation of NE release by D 2-dopamine re-

) Corresponding author at address ‘b’. Fax: q34 Ž4. 464-8152; E-mail: [email protected]

ceptors has also been shown w55x, suggesting a local interaction between NE and dopamine ŽDA. neurotransmission systems. V a rio u s a d re n e rg ic c o m p o u n d s w ith a n imidazoliŽdi.nerguanidine chemical structure, such as the a 2-adrenoceptor antagonist idazoxan and the a 2-adrenoceptor agonists clonidine, moxonidine or guanabenz also recognize with high affinity non-adrenergic binding sites w11,16,18,21,40,42,72x. Based on competition binding experiments and according to anatomical and cellular distribution, the non-adrenergic binding site that is identified by w 3 Hxidazoxan has been termed I 2-imidazoline receptor w37,42x and the other non-adrenoceptor site labeled by p-w 3 Hxaminoclonidine has been termed I 1-imidazoline re-

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 0 8 0 - 3

J. JaÕier Meana et al.r Brain Research 744 (1997) 216–226

ceptor w20x. In the brain, the I 1-imidazoline receptor has been found in the ventrolateral medulla w11,18x at the level of the nucleus reticularis lateralis w10,16x. Among other functions, this putative receptor has been implicated in the hypotensive effect of clonidine and other agents with similar structures w8,12,21,22,65,66x. One or probably several compounds have been isolated and proposed as the imidazoline endogenous ligand w6,19x. More recently, agmatine Ždecarboxylated arginine. has been purified and proposed to be a new endogenous ligand for imidazoline receptors w34x. These endogenous ligands stimulate the release of catecholamines from adrenal chromaffin cells w34x and appear to be implicated in the central control of blood pressure w9,38x. However, at present, the functional role of agmatine is unclear w49,51,61x. It is well known that clonidine inhibits the firing of locus coeruleus neurons by acting on a 2-adrenoceptors w14x. However, a paradoxical stimulatory effect of clonidine and other imidazolineroxazoline drugs on locus coeruleus activity has been demonstrated after irreversible inactivation of these receptors with the alkylating agent EEDQ w50,56x. Thus, in vivo inactivation of central a 2adrenoceptors with EEDQ could be a useful tool to investigate the modulation of NE release by imidazoline receptors in the central nervous system ŽCNS.. The present study was designed to analyse the role of imidazoline receptors modulating catecholamine release using in vivo microdialysis w68x. Extracellular NE, DA and their metabolite levels were monitored in rat cingulate cortex after systemic administration of clonidine. Similar monitoring was performed in a 2-adrenoceptor alkylating conditions with the agent EEDQ. RX821002, the methoxy analog of idazoxan, was used as a selective a 2-adrenoceptor antagonist lacking activity at cortical imidazoline receptors w30,71x. To further elucidate the pharmacological profile of this modulation, the effects of systemic administration of I 1-imidazoline receptor agonists Žclonidine, cirazoline, moxonidine., I 2-imidazoline receptor agonists Žguanabenz, cirazoline. and an imidazoline receptor antagonist Židazoxan. were examined. The possibility of an indirect activity of imidazoline receptors on cortical levels of catecholamines was also considered by examining the effects of local perfusion of imidazoline drugs in cingulate cortex after a 2-adrenoceptor inactivation. The degree of a 2-adrenoceptor inactivation was verified by studying w 3 Hxclonidine specific binding to homogenates of cortical membranes of rat brain.

2. Materials and methods 2.1. Materials w 3 HxClonidine Žspecific activity 60 Cirmmol. was purchased from New England NuclearrDuPont ŽUSA. and stored at y208C. For the binding assays, appropriate

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amounts of the stock solution were diluted with distilled and purified water ŽMilli-ROrMilli-Q-plus. containing 2.5 mM HCl and 6% ethanol. Clonidine hydrochloride, benextramine hydrochloride, EEDQ Ž N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline., Žy.-norepinephrine hydrochloride, Žy.-dihydroxyphenylacetic acid ŽDOPAC., homovanillic acid ŽHVA., dopamine hydrochloride, desipramine hydrochloride and guanabenz were purchased from Sigma ŽUSA.. Cirazoline, idazoxan hydrochloride and moxonidine were gifts from Synthelabo Recherche ŽFrance., Reckitt & Colman ŽUK. and Solvay Pharma Deutschland ŽGermany., respectively. RX821002 Ž2-w2-Ž2-methoxy-1,4-benzodioxanyl.ximidazoline. hydrochloride was synthesized at S.A. LASA Laboratorios ŽSpain.. Microdialysis perfusion fluid was supplied by Karolinska Apoteket ŽSweden.. Salts and reagents for the preparation of HPLC eluent were of the highest purity available at commercial sources and were diluted with distilled-UV irradiated water. 2.2. Animals Male Sprague–Dawley rats ŽALAB, Stockholm, Sweden. weighing 450–550 g, with food and water ad libitum, were used. Animals were kept in a temperature-controlled environment on a 12-h lightrdark cycle. Animal care and experimental protocols were performed in agreement with the EU regulations and were approved by the Swedish Committee for ethical experiments on laboratory animals. 2.3. Intracerebral microdialysis The animals were anesthetized using a mixture of air and halothane and placed in a Kopf stereotaxic frame. Two microdialysis probes Ž4 mm long = 0.5 mm diameter; CMArMicrodialysis AB, Sweden. were bilaterally and simultaneously implanted in the cingulate cortex Žcoordinates: B q2.8, L "1.0, V y4.5. according to the atlas of Paxinos and Watson w48x. Halothane anesthesia was maintained throughout the microdialysis experiment by free breathing into a mask fitted over the nose of the rat Ž1.5% halothane in air flow of 1.5 lrmin.. Body temperature was kept at 378C using a temperature control system. The microdialysis probes were perfused, at a flow rate of 1 mlrmin, with a modified CSF solution Ž148 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl 2 , 0.85 mM MgCl 2 , pH 7.4.. The NE reuptake blocker desipramine Ž1 mM., KCl Ž100 mM. or drugs Žwhen administered locally through the microdialysis probe. were dissolved and included in the perfusion medium. Changes in the perfusion fluid were performed using a syringe selector coupled to a microfraction collector. Samples Ž35 ml. were collected from right and left cingulate cortex every 35 min and analyzed by HPLC with electrochemical detection. Mean in vitro recoveries of NE, DA, DOPAC and HVA, at room temperature, were 45% approximately Žrange 32–60%; n s 10..

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After microdialysis experiments, the animals were killed with an overdose of halothane and the brains were rapidly dissected out, frozen on the stage of a CO 2-cryomicrotome and cut into 30-mm sections. Histological examination at low magnification showed that the microdialysis probes were implanted correctly in all the rats.

delivered bilaterally in the cingulate cortex via the perfusion medium. EEDQ had to be selected as the irreversible antagonist for systemic treatments since benextramine, considered as the most specific irreversible ligand for a 2-adrenoceptor, does not cross the blood–brain barrier w63x.

2.4. Biochemical analyses

2.6. Receptor binding assays

20 ml of the dialysate samples were analyzed by reverse phase HPLC at room temperature to determinate the contents of NE, DA, DOPAC and HVA with reference to freshly diluted standards. The system consisted of a LKB 2150 pump ŽLKB Pharmacia, Sweden., a CMAr200 refrigerated microsampler ŽCMArMicrodialysis AB., a CMAr260 degasser, a LC4C amperometric detector ŽBioanalytical Systems, BAS, USA. with the working electrode set at q780 mV with respect to an AgrAgCl reference electrode ŽBAS. and a SP4600 integrator ŽSpectra-Physics, USA.. The mobile phase, a NaH 2 PO4 Ž0.12 M. buffer ŽpH 4.26. containing 1-octanesulphonic acid Ž1 mM., EDTA Ž0.3 mM. and methanol Ž12% vrv., was delivered at a flow rate of 0.75 mlrmin onto a Biophase ODS 5 mm column Ž250 = 4.6 mm, BAS.. The detection limits for NE and DA were between 0.2 and 0.5 nM.

Control and EEDQ-treated rats were anesthetized, killed and the brains dissected out and stored at y808C for further receptor binding assays. Neural membranes ŽP2 fractions. from fronto-parietal cortex were prepared as described previously w7x. Briefly, the thawed tissue samples were homogenized in Tris-sucrose buffer ŽTris-HCl 5 mM; sucrose 0.25 M; MgCl 2 1 mM; pH 7.4. and centrifuged at 1,100 = g for 10 min. The supernatants were centrifuged at 40,000 = g for 10 min and the pellet was washed and recentrifuged twice with Tris-incubation buffer ŽTris-HCl 50 mM; MgCl 2 10 mM; pH 7.5. before the final preparation of neural membranes to a protein content of 700–1000 mgrml w35x. w 3 HxClonidine binding Ž0.25–16 nM, seven concentrations. to a 2-adrenoceptors was measured, at equilibrium, in 550 ml aliquots of neural membranes incubated 30 min at 258C. Non-specific binding was defined in the presence of the endogenous neurotransmitter Žy.-norepinephrine Ž10 mM.. Incubations were terminated by dilution with 5 ml ice-cold incubation buffer Ž48C. and free and bound ligands were separated by rapid filtration under vacuum through GFrC glass filters using a Cell harvester ŽBrandel 30R, USA.. The filters were rinsed two times with 5 ml of ice-cold incubation buffer, air-dried and counted for radioactivity by liquid scintillation spectrometry using ‘OptiPhase HiSafe II’ cocktail ŽLKB, UK. with a counting efficiency of approximately 50% ŽPackard model 2200 CA..

2.5. Drug treatment Administration of drugs or modification of the microdialysis perfusion fluid was performed 210 min after microdialysis implantation, when dialysate levels showed stable values. When given systemically, clonidine Ž0.3–1.2 mgrkg i.p.., RX821002 Ž2–5 mgrkg i.p.. and idazoxan Ž10 mgrkg i.p.. were dissolved in 0.9% NaCl Žsaline. and their effects were compared to preinjection values or to values in saline-treated animals. In some experiments the irreversible a 2-adrenoceptor antagonist EEDQ Ž6 mgrkg i.p., dissolved in ethanol and then diluted with propylene glycolrwater in a final volume ratio of 1:1:2. was administered 6 h before starting the collection of dialysates. The dose of EEDQ and time for evaluation were those reported as the most suitable for complete a 2-adrenoceptor irreversible blockade w43,50x. Control groups for these experiments were pretreated with the EEDQ-vehicle solution Žethanolrpropylene glycolrwater, 1:1:2. 6 h before the assay. In the microdialysis experiments performed in EEDQ-treated rats, samples were collected for 70 min Žtwo basal samples. before clonidine Ž0.6–2.4 mgrkg i.p.., cirazoline Ž1.2–2.4 mgrkg i.p.., moxonidine Ž1.2–2.4 mgrkg i.p.. or guanabenz Ž1.2–2.4 mgrkg i.p.. were administered. For local administration, clonidine Ž10–100 mM. was perfused through the probe implanted on the left cingulate cortex. Extracellular levels obtained after drug administration were compared to preinjection levels or to levels in the contralateral drug-free cortex. The irreversible a 2-adrenoceptor antagonist benextramine Ž100 mM. was

2.7. Data analysis NE, DA, DOPAC and HVA values are expressed as the concentrations Žmean " S.E.M.. found in the microdialysis perfusates or as the percentage of baseline values determined from samples collected immediately before any drug application. Levels are not corrected for recovery values. The statistical analysis was carried out with absolute values by one or two-way analysis of variance ŽANOVA. and corresponding post-hoc tests. Experimental data of each animal were pooled and analysed for the best, simple dose-effect curve using the non-linear least squares fitting program GraFit. Analysis of binding saturation isotherms Ž K d , dissociation constant; Bmax , maximal density of binding sites. were performed by computer-assisted non-linear regression using the EBDA-LIGAND programs w47x. All experiments were analysed assuming a one-site model of radioligand binding. Student’s test was used for statistical evaluation.

J. JaÕier Meana et al.r Brain Research 744 (1997) 216–226

Results in all the experiments were considered statistically significant at P - 0.05 for two-tail tests.

3. Results 3.1. Cortical extracellular leÕels of norepinephrine, dopamine and metabolites Initial studies were carried out to validate the experimental procedure. Under basal conditions and in the absence of the uptake blocker desipramine the dialysate levels were 0.64 " 0.47 nM for NE and 1.5 " 0.5 nM for DA. In similar conditions, DOPAC and HVA levels were 86 " 25 nM and 145 " 53 nM respectively Žaveraged for 4–6 experiments.. In agreement with the idea that activation of a 2-adrenoceptors results in inhibition of NE release w32,58x the agonist clonidine reduced NE levels to values that ranged the limit of detection Ž0.2 nM in our standard conditions.. In all subsequent experiments, in order to enhance the detectability of NE, the selective NE uptake blocker desipramine Ž1 mM. was included in the perfusion solution since the beginning of the experiments. Desipramine caused an increase in basal extracellular levels of NE Ž4.8 " 0.6 nM; n s 7. and DA Ž3.3 " 0.9 nM; n s 7., but DOPAC and HVA levels were not modified. Under these conditions, local infusion of KCl Ž100 mM. through the microdialysis probe induced a significant elevation of extracellular levels of NE and DA Ž425 " 105% and 326 " 65% of the basal values, respectively, n s 6; P - 0.05.. After KCl addition, DOPAC and HVA levels were decreased to 58 " 14% and 80 " 8% of the basal values, respectively Ž n s 6.. Systemic administration of the selective a 2-adrenoceptor antagonist RX821002 Ž2 mgrkg i.p.. produced an increase in NE levels, reaching a maximum Ž299 " 36% of the basal levels, n s 8; P - 0.01. within the first 35 min after drug injection.

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3.2. Effects of systemic clonidine and RX821002 in naiÕe animals Administration of clonidine Ž0.3–1.2 mgrkg i.p.. caused a dose-dependent decrease in cortical NE levels Ž F3,28 s 4.42; P - 0.05. ŽTable 1.. No significant differences were found between effects on left and right cortex. The maximal inhibition by clonidine was achieved at the third sampling period after drug administration Ž F3,28 s 45.49; P - 0.05. ŽFig. 1.. The half-maximal effect dose estimated from the analysis of the dose-effect curve was 0.49 mgrkg i.p. ŽFig. 2A.. The administration of the selective a 2adrenoceptor antagonist RX821002 Ž2 mgrkg i.p.. 210 min after clonidine increased NE levels by 2–3-fold over basal values ŽTable 1 and Fig. 1.. DA levels were also significantly decreased after clonidine injection Ž F5,122 s 3.06; P - 0.05.. The decline was maximal at the third sampling period although it was less marked ŽTable 1. and more persistent Ždata not shown. than that observed in NE levels. Following RX821002 administration, DA levels in clonidine-treated groups were not significantly different from their basal values but they were significantly lower than the corresponding values in the saline-treated group ŽTable 1.. Clonidine treatment tended to increase DOPAC and to decrease HVA levels, although these changes were not significant ŽTable 1.. 3.3. Effects of systemic clonidine, RX821002 and idazoxan after a 2-adrenoceptor inactiÕation with N-ethoxycarbonyl2-ethoxy-1,2-dihydroquinoline In order to provide clear evidence that a 2-adrenoceptors are inactivated after in vivo EEDQ treatment Ž6 mgrkg injected i.p. 6 h before the assay., the specific binding of w 3 Hxclonidine to a 2-adrenoceptors was evaluated in cortical membranes. Treatment of rats with a single

Fig. 1. Effects of clonidine Ž1.2 mgrkg i.p.. and RX821002 Ž2 and 5 mgrkg i.p.. on extracellular NE levels of control ŽB. and EEDQ-pretreated ŽI. rats. Basal levels ŽB. were obtained 6 h after EEDQ treatment of the rats. Results are absolute mean values " S.E.M. from 4–5 rats in each group.

J. JaÕier Meana et al.r Brain Research 744 (1997) 216–226

220

Table 1 Maximal effect induced by clonidine and RX821002 on cortical extracellular levels of NE, DA, DOPAC and HVA in the left cingulate cortex of n rats

Basal Saline Clonidine Clonidine Clonidine Saline qRX821002 Clonidine qRX821002 Clonidine qRX821002 Clonidine qRX821002

DoseŽmgrkg i.p..

NEŽ%.

DAŽ%.

DOPACŽ%.

HVAŽ%.

n

0.3 0.6 1.2

100 93 " 4 65 " 7 a 54 " 16 a 39 " 13 a

100 97 " 4 80 " 12 60 " 16 61 " 17

100 112 " 10 145 " 47 290 " 123 376 " 192

100 94 " 4 76 " 8 64 " 15 y

4 4 6 5

213 " 49

249 " 39

87 " 7

3

2 0.3 2 0.6 2 1.2 2

a

a a a

b

115 " 2

163 " 28

90 " 34

71 " 24

62 " 11

4

162 " 31

104 " 11

76 " 68

78 " 29

6

254 " 38

81 " 14

100 " 9

y

5

Clonidine or saline administration was performed 210 min after implantation and RX821002 was administered 420 min after implantation Ž210 min after clonidine or saline.. Basal levels from all the experiments were 5.4 " 0.5 nM for NE, 4.6 " 1.1 nM for DA, 63 " 14 nM for DOPAC and 116 " 4 nM for HVA Ž n s 37.. Values are expressed as percentage mean " S.E.M. of the corresponding basal levels. a b , Significant differences from the corresponding saline values Ž a P - 0.05, b P - 0.01; Student’s t-test..

dose of EEDQ reduced the specific binding of w 3 Hxclonidine to a 2-adrenoceptors by 94 " 1% in relation to vehicle-treated groups, but it did not change the affinity ŽTable 2.. In basal conditions, NE, DA, DOPAC and HVA levels after pretreatment with the vehicle solution used to dilute EEDQ were not different from those obtained in naive rats Ždata not shown.. Furthermore, clonidine Ž1.2 mgrkg i.p.. administered in a group of vehicle-treated rats elicited a pattern of effect on NE and DA levels overtly similar to that described in naive Žnon-pretreated. rats Žsee Fig. 1.. Basal extracellular levels, evaluated with microdialysis after EEDQ treatment, were 6.4 " 0.4 nM for NE and 3.4 " 0.3 nM for DA Žmean " SEM in 24 animals.. In

EEDQ-pretreated rats, systemic administration of clonidine Ž0.6–2.4 mgrkg i.p.. resulted in a significant dose-dependent increase of NE levels Ž F2,48 s 5.07; P - 0.05. in both left and right cingulate cortex ŽTable 3.. Under these experimental conditions, the increase in NE concentration reached a peak in the second sampling fraction Ž35–70 min. after the administration of clonidine Ž F3,48 s 8.28; P - 0.01. and returned to basal values within 70–105 min ŽFig. 1.. The half-maximal effect dose for the stimulatory effect of clonidine, estimated from the dose-effect relations for the individual maximal effects ŽFig. 2B. was 1.03 " 0.58 mgrkg. Systemic administration of the selective a 2-adrenoceptor antagonist RX821002 Ž5 mgrkg i.p.. administered 210 min after clonidine, failed to induce the opposite effect to clonidine and even produced a small increase in NE concentration ŽFig. 1.. In additional experiments the administration of RX821002 Ž5 mgrkg i.p.. 35 min before clonidine in EEDQ-pretreated rats did not prevent the

Table 2 Effect of treatment with EEDQ on biochemical parameters of a 2 -adrenoceptors evaluated by specific binding of w 3 Hxclonidine to rat cortical membranes

ControlŽvehicle-treated rats. EEDQŽ6 mgrkg i.p.. Fig. 2. Dose–effect curves of i.p. clonidine on cortical NE levels in naive ŽA. and EEDQ-pretreated ŽB. rats. The points represent means"S.E.M. of the experimental maximal inhibitory ŽA. or stimulatory ŽB. effect of clonidine measured bilaterally as a percentage of the corresponding individual basal value. Five or six animals were included in each experimental group. The lines represent the theoretical dose-response lines generated by a simultaneous non-linear fit of individual percentage values.

K d ŽnM.

Bmax Žfmolrmg protein.

n

5.06"0.73 3.93"1.88

154"23 10"1 a

8 9

The specific binding of w 3 Hxclonidine Ž0.25–16 nM; seven concentrations. to a 2 -adrenoceptors was calculated by the non-linear fitting program LIGAND. Non-specific binding was defined in the presence of Žy.-norepinephrine Ž10 mM.. K d is the apparent dissociation constant and Bma x is the maximum number of binding sites. Values are mean"S.E.M. of n rats. a P - 0.001 when compared with the control values ŽStudent’s t-test..

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221

Table 3 Maximal effect induced by clonidine and RX821002 on cortical extracellular levels of NE, DA, DOPAC and HVA in the left cingulate cortex of n rats after inactivation of a 2-adrenoceptors with EEDQ Basal Saline Clonidine Clonidine Clonidine

DoseŽmgrkg i.p..

NEŽ%.

DAŽ%.

DOPACŽ%.

HVAŽ%.

n

0.6 1.2 2.4

100 95 " 8 129 " 7 a 144 " 17 a 156 " 42 a

100 103 " 13 106 " 9 115 " 10 109 " 10

100 98 " 12 113 " 26 145 " 36 108 " 4

100 83 " 13 97 " 7 102 " 2 111 " 6

9 6 5 6

Clonidine or saline administration was performed 210 min after implantation. EEDQ Ž6 mgrkg i.p.. administered 6 h before starting the collection of dialysates. Basal values were: 6.9 " 0.6 nM for NE, 3.6 " 0.4 nM for DA, 58 " 9 nM for DOPAC and 180 " 26 nM for HVA Ž n s 26.. Values are expressed as percentage mean " S.E.M. of the corresponding basal levels. a Significant differences from the corresponding saline values Ž a P - 0.05; Student’s t-test..

stimulatory effects of clonidine Ž130 " 16% and 142 " 16% of basal values in the absence and presence of RX821002, respectively; n s 6.. Extracellular DA levels were not modified after clonidine injection in EEDQ-pretreated rats ŽTable 3.. At this point, new experiments were designed to assess whether the stimulatory effect of clonidine on NE levels could be derived from activity through imidazoline receptors. Thus, the effects of idazoxan, which in addition to an a 2-adrenoceptor antagonist has also been proposed to be an I 1-imidazoline receptor antagonist w22,23,66x, were evaluated in EEDQ-pretreated rats ŽFig. 3.. Idazoxan Ž10 mgrkg i.p.. failed to change NE levels, but when administered 35 min before clonidine abolished the stimulatory activity of this drug in EEDQ-pretreated rats ŽFig. 3..

3.4. Effects of imidazoline drugs after a 2-adrenoceptor inactiÕation with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline Since most of the imidazolinerguanidine drugs also act at a 2-adrenoceptors, their potential imidazoline receptormediated effects were evaluated after pretreatment with EEDQ Ž6 mgrkg i.p.. 6 h before the beginning of the collection of samples. The non-selective I 1rI 2-imidazoline receptor agonist cirazoline and the selective I 1-imidazoline receptor agonist moxonidine increased the levels of NE in a dose-dependent manner ŽTable 4.. In contrast, the guanidine derivative guanabenz which has been proposed as a selective I 2-imidazoline receptor agonist w29,64x failed to modify NE concentration ŽTable 4.. Idazoxan Ž10 mgrkg i.p.., when injected 35 min before the administration of the agonists blocked the stimulatory effects induced by cirazoline or moxonidine on NE extracellular levels ŽFig. 3..

Table 4 Maximal effect induced by several imidazoline drugs on cortical extracellular levels of NE in the left cingulate cortex of n rats after inactivation of a 2-adrenoceptors with EEDQ

Fig. 3. Effect of i.p. administration of the agonists clonidine Ž1.2 mgrkg i.p.., cirazoline Ž2.4 mgrkg i.p.. or moxonidine Ž2.4 mgrkg i.p.. in the presence Žopen columns. or absence Žblack columns. of previous treatment with the antagonist idazoxan Ž10 mgrkg i.p., 35 min before agonist. on extracellular NE levels. Columns represent means"S.E.M. Ž ns 3–4 rats. expressed as the percentage of NE levels observed before drug administration. All the rats had been pretreated with EEDQ Ž6 mgrkg i.p., 6 h before. to mask activity on a 2-adrenoceptors Žsee text for details.. Basal levels were 6.1"1.1 nM Žrange 2.5–7.8 nM.. ) , ) ) Significant differences from the corresponding values in the presence of idazoxan Ž ) P - 0.05, ) ) P - 0.01; Student’s t-test..

Drug Žmgrkg i.p..

Dose Ž%.

NE Ž%.

n

Basal Cirazoline Cirazoline Moxonidine Moxonidine Guanabenz Guanabenz

1.2 2.4 1.2 2.4 1.2 2.4

100 124"8 a 182"26 a 111"10 120"7 a 97"14 104"9

4 4 3 4 3 4

Drug administration were performed 210 min after implantation. EEDQ Ž6 mgrkg i.p.. was administered 6 h before starting the collection of dialysates. NE basal levels from all the experiments were 7.6"1.5 nM Ž ns 22.. Values are expressed as percentage mean"S.E.M. of the corresponding basal levels. a Significant differences from the corresponding basal values Ž a P - 0.05; Student’s t-test..

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3.5. Effect of local administration of clonidine and RX821002 after a 2-adrenoceptor inactiÕation with Nethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline or benextramine To determine whether the stimulatory effect on the NE levels mediated by imidazoline receptors is due to local effects in cingulate cortex, clonidine Ž10–100 mM. was perfused through the microdialysis probe after pretreatment with EEDQ Ž6 mgrkg i.p., 6 h before the assay.. Clonidine perfused up to 100 mM concentration into the left hemisphere failed to alter NE levels Ž6.1 " 1.7 nM at 70 min; n s 3. compared with concentration values simultaneously obtained in the right hemisphere which was used as control Ž6.0 " 0.6 nM at 70 min; n s 3.. No effect was observed on DA, DOPAC or HVA levels. In an attempt to further evaluate the effects of clonidine after a 2-adrenoceptor local inactivation, the more selective irreversible a 2-adrenoceptor antagonist benextramine Ž100 mM. was included in the perfusion medium from the beginning of the microdialysis experiments in naive Žnontreated with EEDQ. rats. Benextramine increased extracellular NE levels Ž157 " 13% of basal values in normal conditions, n s 24; P - 0.05., but not DA levels Ž113 " 15%; n s 24.. In the continuos presence of local benextramine, inclusion of clonidine Ž10 mM. in the medium perfusing the left cingulate cortex did not modify NE levels Ž F4,27 s 1.99; P s 0.12.. Two sampling periods after changing the perfusion medium, NE levels in the left cortex Žclonidine side. were similar to basal values Ž95 " 9% of their own basal values; n s 8. and were not different from NE values observed in the right cortex Žcontrol side. Ž107 " 17% of their own basal values; n s 8. Ž F1,27 s 0.30; P s 0.59.. At 100 mM concentration in the medium, clonidine did not change NE levels, either Ždata not shown.. Neither DA nor DOPAC or HVA levels were modified after clonidine administration Ždata not shown.. The systemic injection of RX821002 Ž5 mgrkg i.p.. 140 min after the start of the clonidine perfusion induced a bilateral increase in NE levels to approx. 130% of basal values but did not modify DA levels Ž92 " 7% of basal values; n s 2..

4. Discussion The present study reports on a new neurochemical role for I 1-imidazoline receptors in the CNS. The functional characterization was performed by monitoring extracellular levels of NE, DA, DOPAC and HVA with in vivo microdialysis in the cingulate cortex of halothane-anesthetized rats. The cingulate cortex receives noradrenergic projections from the locus coeruleus through the dorsal tegmental bundle w67x. Herein, NE, DA and metabolite levels measured in the extracellular space of the cingulate cortex

were in the nanomolar range which is in general agreement with previous reports w57,70x. NE levels were responsive to manipulations of noradrenergic neuronal activity. Thus, cortical NE levels were increased after inclusion of desipramine, a selective uptake blocker, or KCl, a depolarizing agent, in the perfusion medium. Clonidine, a drug known to decrease central noradrenergic activity as a result of its a 2-adrenoceptor agonist properties w32,58x, decreased NE levels whereas RX821002, which increases noradrenergic neuronal activity via a 2-adrenoceptor antagonism w59,73x, enhanced the release of the amine. Therefore, these results confirm that NE monitored by in vivo microdialysis in the cingulate cortex represents, like in other brain areas, neuronal noradrenergic activity w1,15,33,69x. The concentration-dependent reduction in NE overflow induced by systemic clonidine ŽFig. 2A. was less marked than in previous reports when equivalent doses Ž0.2–0.3 mgrkg i.p.. are considered w1,31,33x. However, the maximal inhibitory effect reached after the highest dose of clonidine used in the present study Ž1.2 mgrkg i.p.. was similar Žapprox. 60%. to values previously reported. Such a shift to the right of the dose-effect curve for clonidine is probably due to the enhanced levels of the endogenous NE during desipramine perfusion w33,69x leading to an enhanced competition effect between clonidine and NE at the a 2-adrenoceptors w24x. RX821002, the methoxy analog of idazoxan, is a new a 2-adrenoceptor antagonist w30x that crosses the blood– brain barrier w41x; its pharmacological properties have not been assessed with microdialysis. RX821002 has 2500– 3200-times lower affinity at cortical imidazoline receptors w37,42x and 10 times higher affinity at a 2-adrenoceptors w30,37,42,71x than idazoxan. Following systemic administration, a 2-adrenoceptor antagonists would act to increase the neuronal firing rate at the level of the locus coeruleus and also to enhance the neurotransmitter release in projecting areas by acting on presynaptic autoreceptors located on noradrenergic terminals. The increase of NE levels produced by RX821002 is in agreement with this hypothesis and is similar Ž2–3-fold the basal levels. to that seen in other microdialysis studies, in which less selective antagonists such as yohimbine or idazoxan were administered w1,33,69x. A number of microdialysis studies have documented the presence of DA in the rat cortex w28,36,44,57x. Extracellular DA levels were increased after desipramine, a potent inhibitor of NE uptake but a weak inhibitor of DA uptake, indicating that in cerebral cortex extracellular DA may be taken up by noradrenergic terminals w13,52x. On other hand, the administration of the a 2-adrenoceptor agonist clonidine induced a decrease in DA levels, whereas the a 2-adrenoceptor antagonist RX821002 increased DA in dialysate samples ŽTable 1.. These results could be compatible with a noradrenergic origin of DA in cingulate cortex, where it serves as precursor in NE synthesis w4x. In this context, the a 2-adrenoceptor-mediated modulation of

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the extracellular levels of DA could reflect an indirect action through the modulation of the synthesis w3x and release of NE w32,58x. Furthermore, the fact that the levels of NE and DA after clonidine treatment showed different time-course and dose–response relations together with the finding that RX821002, was unable to reverse the clonidine-induced decreases in DA ŽTable 1., might suggest an indirect modulation by noradrenergic agents of the DA release from noradrenergic terminals. However, a separate release of DA and NE from the corresponding terminals could also be compatible with the previous data w27,74x. The reduction in DA cortical levels observed following deafferentation from A9 and A10 dopaminergic cells groups w28x supports the hypothesis of a specific dopaminergic innervation of cingulate cortex. This possibility is also in agreement with earlier studies where the a 2-adrenoceptor antagonist yohimbine increased brain DA turnover by acting on NE neurons w3x. Thus, based in these data, a tonic regulation of the dopaminergic system by noradrenergic terminals in the cingulate cortex, as in the prefrontal cortex w27x, could be proposed as an alternative hypothesis. Imidazoline derivatives, such as clonidine, have been shown to exert stimulatory effects on firing of locus coeruleus noradrenergic neurons w50,56x. Since this effect is normally masked by the more prominent a 2-adrenoceptor-mediated inhibition of noradrenergic neurons, an irreversible blockade of a 2-adrenoceptors is necessary. Although such a blockade can be achieved by administration of benextramine w63x, this drug does not cross the blood– brain barrier w63x. The peptide-coupling agent EEDQ appears to be a satisfying alternative because it is able to inactivate a 2-adrenoceptors in vivo w2,7x and does not have effect on imidazoline receptors w43x. It has been reported that EEDQ, at 6 mgrkg subcutaneously, is able to also block several neurotransmitter receptors with the following order of sensitivity and maximal degree of inactivation: a 2 Ž95%. ) a 1 Ž80%. ) D2 ( D1 Ž70%. ) 5-HT2 ( 5-HT1 Ž60%. ) b Ž25%. ) muscarinic Ž10%. w39x. Despite the moderate affinity of clonidine and moxonidine for a 1adrenoceptors and the a 1-agonist properties of cirazoline, the dosis of EEDQ used in the present study appears to be rather selective for a 2-adrenoceptors. The specific binding of w 3 Hxclonidine to a 2-adrenoceptors in rat cortex using Žy.-norepinephrine to define the non-specific binding confirmed the almost full alkylating properties of EEDQ on a 2-adrenoceptors ŽTable 2.. Under irreversible a 2-adrenoceptor blockade, clonidine induced a dose-dependent increase in NE levels ŽFig. 2B, Table 3. which was not abolished or reversed by RX821002 administration but could be blocked by the imidazoline receptor antagonist idazoxan w22,23,66x ŽFig. 3.. Thus, the present results suggest a non-adrenergic mechanism of action for clonidine that should be related to its properties as an imidazoline drug. However, since EEDQ shows ability to also interact with a 1-adrenoceptors, dopaminergic and serotonergic receptors, the possibility of a cross-talk between

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these receptors and imidazoline receptors cannot be absolutely dismissed. Anatomical, cellular, biochemical and pharmacological evidences for the existence of several imidazoline receptors have been provided w53x. In the CNS, clonidine and moxonidine show high affinity properties as I 1-imidazoline receptor agonists w12,65,66x whereas possess low affinity for the I 2-imidazoline receptor w16,42,54x. Conversely, the guanidine derivative guanabenz binds with high affinity to the I 2-imidazoline receptor w20,42,54x and is relatively inactive at the I 1-imidazoline receptor w20,21x. Cirazoline, considered as an imidazoline receptor agonist, binds with similar high affinity to the I 1- w11,20x and the I 2-imidazoline receptors w16,20,42,54x. In the present study, clonidine, cirazoline and moxonidine but not guanabenz increased NE extracellular levels after alkylation of a 2adrenoceptors ŽTable 4.. These findings, taken together with the blockade elicited by the imidazoline receptor antagonist idazoxan ŽFig. 3., strongly suggest that I 1-imidazoline receptors mediate the rise of NE levels in the rat cingulate cortex. In the rat CNS, clonidine and other I 1-imidazoline receptor agonists induce stimulatory effects of locus coeruleus neurons, in the presence of a 2-adrenoceptor alkylating agents, in contrast with their habitual inhibitory properties w50,56x. The present results in rat cortex demonstrate similar in vivo effects on NE extracellular levels. By contrast, a clonidine-inhibitory presynaptic effect on NE release mediated by imidazoline receptors on postganglionic sympathetic nerve terminals has been evidenced w25,45x. The apparent discrepancy between these studies and our results might be explained by the location w26x and the different pharmacological properties w46x of the imidazoline receptors evaluated in the peripheral nervous system. To verify the stimulatory effect of clonidine and related drugs higher doses than those used for the inhibitory effect are required ŽFig. 2.. This result fits well with results previously obtained with clonidine and rilmenidine w50,60x and could reflect the density ratio of I 1-imidazoline receptors to a 2-adrenoceptors w21,23x. Although systemic administration of clonidine after a 2-adrenoceptor alkylation stimulated NE release, the drug failed to induce a similar response following local administration via the microdialysis probe. Analogously and by using an in vitro approach, imidazoline receptors in cortex appear not to mediate a local effect on NE levels w26x. The observation that local perfusion of clonidine does not modify extracellular NE levels suggests that the stimulatory effect may be mediated by an indirect mechanism unrelated to the presynaptic action on noradrenergic terminals. Thus, according to previous results w50,56,62x, an increase in the firing activity of noradrenergic neurons could be responsible for an enhanced release of the neurotransmitter in the target areas w33x. An important afferent pathway to the locus coeruleus that releases excitatory amino acids arises from the ventrolateral medulla w5,17x. In

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this context, recent results suggest that the non-selective excitatory amino acid antagonist kynurenic acid is able to block the stimulatory effect of clonidine on the firing of locus coeruleus noradrenergic neurons w56x. Since the I 1imidazoline receptors in the CNS seem to be mainly located in the ventrolateral medulla at the level of the nucleus reticularis lateralis w10,11,16,18x, it is tempting to suggest that the noradrenergic stimulation elicited by imidazoline drugs could be dependent on a neural pathway originating from or projecting through the ventrolateral medulla. In contrast to NE overflow, the DA and metabolite levels did not respond to systemic or local administration of clonidine after blockade of a 2-adrenoceptors. It seems that only noradrenergic transmission is modulated by imidazoline receptors. A better knowledge of imidazoline receptor location and functions should bring valuable information about noradrenergic and dopaminergic interactions in the cortex. In conclusion, clonidine and related imidazoliŽdi.ne drugs are able not only to inhibit NE release in rat cerebral cortex involving a 2-adrenoceptor mechanisms, but also to induce a paradoxical NE release through an indirect mechanism that is not located on NE terminals. This latter effect does not appear to be related to a 2-adrenoceptors, but involves I 1-imidazoline receptors.

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Acknowledgements w16x

This study was supported by the Swedish Medical Research Council Ž8669., Basque Government ŽPI 95r55. and Spanish CICYT ŽSAF 93-0459. and CDTI Ž92-0178.. J.J.M. is a recipient of a fellowship from the Foundation Wenner-Grenska Samfundet.

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