0270-6474/82/0211-1588$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 2, No. 11, pp. 1588-1595 Printed in U.S.A. November 1982 CHRONIC EFFECTS OF A MONOAMINE OXIDASE-INHIBITING ANTIDEPRESSANT: DECREASES IN FUNCTIONAL a-ADRENERGIC AUTORECEPTORSPRECEDETHEDECREASEIN NOREPINEPHRINE-STIMULATED CYCLIC ADENOSINE 3’:WMONOPHOSPHATE SYSTEMS IN RAT BRAIN1 ROBERT M. COHEN,*,” RICHARD P. EBSTEIN,*,3 JOHN W. DALY,* AND DENNIS L. MURPHY* *Clinical Neuropharmacology Branch, National Institute of Mental Health and ‘Laboratory of Bioorganic Chemistry, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland 20205 Received November 2, 1981; Revised April 9, 1982; Accepted May 7, 1982 Abstract Various antidepressant drugs (monoamine oxidase inhibitors and tricyclics) enhance norepineph- rine availability and lead to adaptive changes in brain noradrenergic systems, namely, decreases in the number of p receptors and in the responsiveness of adenylate cyclase to norepinephrine stimulation. After 21 days of treatment with 1 mg/kg/day of clorgyline, an A-type-selective monoamine oxidase inhibitor, but not after 3 days, there is an increase in norepinephrine release from rat brain microsacs in response to 43 mM KC1 stimulation. Microsacs prepared from 21-day clorgyline-treated animals also show a marked decrease in the inhibition of norepinephrine release caused by the az-selective agonist clonidine. These functional changes in norepinephrine release mechanisms are accompanied by a 53% reduction in brainstem (~2receptor density as measured by [3H]clonidine binding. At the same time, despite findings of a decrease in p receptor number as determined by [3H]dihydroalprenolo1 binding data, no significant decrease in the responses of cyclic adenosine 3’:5’-monophosphate (cyclic AMP) systems to norepinephrine stimulation is observed. Decreases in the cyclic AMP response are observed by day 35 of clorgyline treatment. The results provide direct physiological support for a change in the norepinephrine release mechanism and an effect on autoreceptors, specifically, preceding postsynaptic adaptive changes in the instance of one antidepressant, clorgyline. Difficulties in observing such changes with other antidepressants may result from the multiple nature of cu-adrenergic receptors, especially as measured by radioactive ligand techniques; the lack of a direct relationship between physiological changes and receptors as measured by radioligand techniques; the large doses of monoamine oxidase inhibitors used in some studies; and the possible multiplicity of antidepressant molecular mechanisms. Chronic administration of antidepressants to rats leads (Wolfe et al., 1978; Schweitzer et al., 1979). Several in- to decreases in P-adrenoreceptor numbers and to a de- vestigators have provided evidence that this decrease is crease in the norepinephrine-elicited accumulations of partially mediated through a change in the presynaptic cyclic adenosine 3’:5’-monophosphate (cyclic AMP) (Sul- release mechanisms involving a reduction in local nega- ser et al., 1978). This decrease in norepinephrine respon- tive feedback (Crews and Smith, 1978; Svensson and sivity is believed to be secondary to a preceding persist- Usdin, 1978; Tang et al., 1978; Spyraki and Fibiger, 1980). ence of increased norepinephrine levels in the synapse However, while radioligand binding data demonstrated clear decreases in ,& receptor numbers during antidepres- sant treatment, no analogous changes had been reported ’ We gratefully acknowledge the expert technical assistance of Cath- for az-adrenergic receptors (Bergstrom and Kellar, 1979; erine McLellan and Michelle Dauphin. Rosenblatt et al, 1979; Johnson et al., 1980). Recently, * To whom correspondence should be addressed at Clinical Neuro- pharmacology Branch, National Institute of Mental Health, National Cohen et al. (1982) provided data for a decrease in the Institutes of Health Clinical Center, Building 10, Room 3D41, Bethesda, number of a2-receptors which temporally preceded a MD 20205. reduction in j3 receptor density in the rat cortex during ’ Permanent address: Jerusalem Mental Health Center, Jerusalem, chronic administration of clorgyline, a selective inhibitor P.O.B. 140, Israel. of monoamine oxidase type A (MAO-A) which is a clin- 1588 The Journal of Neuroscience MAO Inhibitors Induce Autoreceptor Changes 1589 ically effective antidepressant agent. These apparent in- acid and unlabeled cyclic AMP (0.25 pmol), and homog- consistencies between reports using different antidepres- enized with a glass pestle. Homogenates were centrifuged sants may result from the existence of multiple Luz-adre- for 10 min at 3,000 rpm. Fifty-microliter aliquots were nergic receptors, some of which may play roles unrelated taken from the supernatant fraction and the radioactivity to presynaptic regulation. Therefore, it is important to was measured to determine the [3H]adenine nucleotide examine directly norepinephrine release and the (~2 re- pool in each sample. The cyclic [“HIAMP formed was ceptor-mediated regulation of this release in the central determined by filtering and washing the remaining su- nervous system during treatment with antidepressants pernatant fraction through Dowex and alumina columns such as clorgyline. A microsac preparation was chosen as described by Salomon et al. (1974). for these observations as the specific (~2receptor agonist Assay for norepinephrine release. Strips of rat cortex clonidine had been demonstrated previously to decrease obtained as described above were homogenized by glass potassium chloride-stimulated norepinephrine release in pestle in 10 vol of Krebs-Ringer buffer at 4°C. The a specific dose-dependent manner (De Langen et al., homogenate was centrifuged at 1,000 x g for 15 min. The 1979). To examine the functional physiology of the p pellet was suspended in 10 vol of buffer with 0.2 pM [3H] receptor, the effect of norepinephrine stimulation on cylic norepinephrine (60 &i) added, and uptake was allowed AMP formation was examined in brain slices. to proceed for 30 min at 37°C. The suspension was centrifuged at 1,000 x g for 5 min at 4°C and the pellet Materials and Methods was resuspended in 10 vol of buffer and centrifuged. The Animals. Six- to 12-week-old male Sprague-Dawley pellet was washed an additional two times in a similar rats (175 to 200 gm) were obtained from Taconic Farms, manner. The final suspension of microsacs from a single Germantown, NY. Half of the animals were implanted cortex was in 10 ml of calcium-free Krebs buffer. Three with Alzet mini osmotic pumps (Alza Corp., Palo Alto, hundred-microliter aliquots of the suspension were added CA) containing concentrations of clorgyline that were to 3 ml of buffer and gently mixed. Two milliliters then adjusted to provide for continuous I-mg/kg/day admin- were layered onto Sephadex GlO columns (polypropylene istration to each animal. This dose is sufficient to produce econo columns) for release experiments which were con- greater than 90% inhibition of MAO-A (Campbell et al., ducted at 25°C. Each column (46 columns from the 1979a, b). Animals were reimplanted every 2 weeks there- cortex of one rat) has a height of 0.75 to 1.0 cm and an after. approximate 0.2 ml/20 set flow rate. The columns were Assay of cyclic AMP generation. Cyclic AMP forma- washed with 5 ml of calcium-free Krebs buffer and the tion was measured by the adenine-prelabeling brain slice eluants were discarded. Columns then were washed with technique as originally described by Shimizu et al. (1969). 2 ml of the medium used for release, with the eluant This procedure results in data which are expressed as collected directly into scintillation vials. The releasing percentages of total radioactive adenine nucleotides pres- eluant consisted of KRB plus varying concentrations of ent as cyclic AMP (percentage of conversion) and are in KC1 (43 mM if not stated), CaC12 (0.05 InM if not stated), good agreement with those obtained from the measure- and various concentrations of drugs (e.g., clonidine (0.01 ments of endogenous levels of cyclic AMP in brain slices. to .lO PM)). Prior to column preparation, aliquots were Briefly, rats were sacrificed by decapitation and the obtained for Lowry protein determination (Lowry et al., brains obtained were placed in ice cold oxygenated 1951) and for initial norepinephrine accumulation. Krebs-Ringer buffer (KRB). The brains were placed onto Assay for a- and ,8-adrenergic receptor binding. For a chilled glass plate for dissection. The brainstem, cere- each treatment group, six frozen (-80°C) brainstem por- bellum, and hypothalamus were removed and stored at tions, approximating the region inclusive of medulla and -80°C for enzyme and receptor assays at a later date. pons, were weighed, thawed, and homogenized using a Small strips of cerebral cortical gray matter obtained Polytron in 10% (w/v) Tris-HCl (50 mM, pH 7.6) at 4°C. with a single edge razor blade from two or three rats were The homogenates were centrifuged at 18,500 x g for 10 placed into a second beaker of buffer. The strips then min at 4°C. The pellets were suspended in 50 vol of were sliced on a cooled block by a McIlwain tissue slicer buffer and centrifuged as before. The resulting pellets set at 260 pm, rotated X30’, and sliced again. The tissue (prepared membranes) were resuspended in 10 vol of slices then were incubated at 37°C for 15 min in 15 ml of buffer and used in the binding assay procedures. buffer. For Scatchard determination (Scatchard, 1949) of (YZ- The preincubated brain slices were transferred to a adrenergic receptor binding, 400~~1 aliquots of the pre- third beaker containing adenine (30 PM) and [ 3H]adenine pared membranes were added in triplicate to tubes con- (20 to 60 &i) and incubated at 37°C for 40 min. The taining the a2-adrenergic agonist [3H]clonidine (final con- buffer was decanted; the slices were washed twice with centrations ranged from 0.2 to 4 times KD) in a total buffer and then collected onto nylon mesh. Equal por- volume of 0.6 ml.