Reduced sensitivity of neurons to noradrenamine after chronic treatment with antidepressant drugs

European Journal of Pharmacology, 63 (1980) 7--13

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© Elsevier/North-Holland Biomedical Press

R E D U C E D SENSITIVITY O F N E U R O N S TO N O R A D R E N A L I N E A F T E R CHRONIC T R E A T M E N T WITH ANTIDEPRESSANT D R U G S H.-R. OLPE and A. SCHELLENBERG

Biology Research Laboratory, Pharmaceuticals Division, Ciba-Geigy Ltd. Basle, Switzerland Received 4 December 1979, accepted 16 January 1980

H.-R. OLPE and A. SCHELLENBERG, Reduced sensitivity of neurons to noradrenaline after chronic treatment with antidepressant drugs, European J. Pharmacol. 63 (1980) 7--13. The sensitivity of cingulate cortical neurons to microiontophoretically administered noradrenaline (NE) and GABA was investigated in groups of rats treated either chronically or acutely with various antidepressant drugs or with the tricyclic antiepileptic carbamazepine. The chronically treated animals received one daily intraperitoneal injection of either desipramine (10 mg/kg), clomipramine (10 mg/kg), maprotiline (25 mg/kg), tranylcypromine (1 mg/kg) or carbamazepine (30 mg/kg p.o.) for 4 weeks. The acutely treated rats received one daily injection of the vehicle for 4 weeks, followed by one single injection of one of the five drugs 24 h before the experiment. NE and 7-aminobutyric acid (GABA) were administered microiontophoretically during periods of 60 sec with various ejection currents. In chronically treated rats a statistically significant reduction in the sensitivity of cortical cells to NE was observed with all four antidepressants. However, in rats treated chronically with carbamazepine, no desensitisation was observed. Desensitization to NE following chronic treatment with desipramine developed within the first 10 days of treatment. Following a ten-day treatment, there was a 33 percent and by the end of the fourth week a 43 percent reduction in sensitivity to NE. The sensitivity of these cingulate neurons to GABA was unchanged after chronic treatment with the antidepressant drugs. In conclusion, the present study demonstrates that chronic treatment with antidepressant drugs leads to a postsynaptic subsensitivity of cortical neurons to NE. Antidepressant drugs

Cingulate cortex

Noradrenaline

I. Introduction Antidepressant drugs which have a beneficial action in the treatment of depressed patients (e.g. tricyclic antidepressants, monoamine oxidase inhibitors) are thought to exert their effect b y increasing the concentration o f biogenic amines at their receptors. However, there is a striking discrepancy b e t w e e n the time course of the biochemical and pharmacological effects o f antidepressant drugs which are produced within hours or less and their therapeutic effects which take several days or weeks to develop. Biochemical data published recently seem to offer an explanation for that discrepancy. The chronic administration o f antidepressant

GABA

Rat

drugs causes a decrease in the ability of norepinephrine (NE) to stimulate adenosine cyclic 3',5'-monophosphate (cAMP) (Schultz, 1976; Vetulani et al.,1976). This effect was observed with tricyclic antidepressants and the novel drug Iprindol (Vetulani et al., 1976). in addition, three laboratoriesreported that this loss of responsiveness to catecholamines was accompanied by a decreased density of ~-adrenergic receptors (Banerjee et al., 1977; Wolfe et al., 1978; ClementsJewery, 1978). These findings suggest that the therapeutic action of antidepressants can be related to postsynaptic adaptive alterationsin the sensitivityto noradrenaline rather than to acute presynaptic acute effects (Vetulani et al., 1976). ~

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We have recently found that stimulation of the locus coeruleus elicited inhibitory effects on many cells in the cingulate cortex of the rat by activating noradrenergic fibers innervating this area (Dillier et al., 1978). Our data indicated that these effects are mediated through the activation of ~-receptors. Since the number of ~-receptors is reduced in rats treated chronically with antidepressant drugs, we were expecting to find a reduced sensitivity of cortical cells to NE. Using electrophysiological techniques we tested this hypothesis in the present study by comparing the sensitivity of cingulate cortical cells t o locally, i.e. microiontophoretically applied NE in rats treated either acutely or chronically with various antidepressant drugs. We report here that the responsiveness of cingulate neurons to NE was indeed reduced after chronic administration of various antidepressant drugs.

H.-R. OLPE, A. SCHELLENBERG

daily for 4 weeks. Eight control rats were treated identically with the vehicle. Groups of 16 rats were used in the time course study. Half of the animals were treated once daffy with 10 mg/kg i.p. of desipramine during either 3, 6, or 10 days. The other half of the rats received one daffy injection of saline during the same time periods. Twenty-four h before the experiment started, these control rats received one injection of desipramine. The following drugs were used: desipramine hydrochloride (10 mg/kg: Pertofran ®, CibaGeigy), maprotiline hydrochloride (25 mg/kg; Ludiomil ®, Ciba-Geigy), clomipramine hydrochloride (10 mg/kg; Anafranil®, Ciba-Geigy), tranylcypromine sulfate (1 mg/kg); Parnate ®, Smith, Kline and French), and carbamazepine (30 mg/kg; Tegretol®, Ciba-Geigy). All antidepressant drugs were dissolved in physiolog, ical saline solution. The experiments were performed 24 h after the last drug injection. 2.2. Microiontophoretic experiments

2. Materials and methods 2.1. Drug treatment

The experiments were performed on 144 male rats (SIV 50) initially weighing 200-220 g which were kept in groups of 6 to a cage. We compared the chronically (4 weeks) to the acutely treated animals in five series of experiments. Thirty-two rats (8 rats per dose and drug) received one daily intraperitoneal (i.p.) injection of one of the four antidepressant drugs at 8 am each for 4 weeks. Thirtytwo rats (8 rats per dose and drug) received one daffy injection of the vehicle over the same period followed by a single dose of the corresponding antidepressant drug one day before the microiontophoretic experiment (acutely treated rats). I n another series of experiments (16 rats) w e compared the sensitivity of neurons to NE in rats which had received either one injection of desipramine or one injection of the vehicle 24 h before the experiment. Eight rats received carbamazepine dissolved in methylcellulose orally once

The rats were anaesthetized with chloral hydrate (400 mg/kg i.p.) and fixed in a stereotaxic apparatus. The conventional microiontophoretic technique was used as previously reported (Dillier et al., 1978). The following compounds were tested: NE (0.5 M, pH 3.5) and 7-aminobutyric acid (GABA 0.5 M, pH 3.5). N E and GABA were administered to each rat near 4 spontaneously active, nonidentified cells of the cingulate cortex. Each electrode was used in one chronically and one acutely treated animal. NE was applied during periods of 1 min at intervals of 4 min using currents of 10, 30 and 100 nA. The current was increased progressively before each successive drug application. Retaining currents of 10 n A were used routinely for both drugs. GABA was administered during periods of 1 min at intervals of 2 rain. The first two applications of GABA were performed by reducing the retaining current from 10 to either 5 or 0 nA. Ejection currents of 10 and 40 nA were used for subsequent applications. The depressant effects of NE and GABA were

DESENSITISATION BY ANTIDEPRESSANTS d e t e r m i n e d f o r each dose b y m e a s u r i n g t h e m a x i m u m discharge r e d u c t i o n . This e f f e c t was expressed as p e r c e n t a g e o f t h e baseline activity. D a t a are p r e s e n t e d as m e a n s -+ S.E.M. E a c h value is t h e m e a n o f 32 values o b t a i n e d in a g r o u p o f 8 rats, C h a n g e s in sensitivity were c a l c u l a t e d b y c o m p a r i n g t h e m e a n firing depression p r o d u c e d w i t h ejection c u r r e n t s o f 1 0 0 nA. I n t h e t i m e c o u r s e s t u d y , desensitisat i o n was expressed f o r each g r o u p o f rats b y calculating t h e m e a n r e d u c t i o n in sensitivity observed with e j e c t i o n c u r r e n t s o f 1 0 0 n A . T h e K o l o m o g o r o f - S m i r n o f f test was used t o assess t h e degree o f statistical significance. O n l y t h e value o b t a i n e d f r o m t h e first d r u g a p p l i c a t i o n t o each cell were s u b j e c t e d t o statistical analysis since t h e values f o r subseq u e n t d r u g a d m i n i s t r a t i o n s were n o t i n d e p e n d e n t o f t h e first one.

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9 TABLE 1 The inhibitory effects of iontophoretically administered NE on cingulate cortical cells of rats treated either with 10 mg/kg i.p. of desipramine or with saline (24 h before the experiment) are shown. Each group consisted of 8 animals. NE was applied iontophoretically for 1 rain in each animal near to 4 spontaneously active neurons using ejection currents of 10, 30 and 100 nA. The maximal depression of firing rate observed during each application of NE was expressed as percentage of the spontaneous discharge rate. Data are presented as means -+ S.E.M.

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Fig. 1. The cell depressant action of iontophoretically applied NE on a cingulate cortical cell from an acutely (A) and a chronically (B) treated rat (desip~mine 10 mg/kg i.p.; 4 weeks) is shown. Note difference in sensitivity.

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H.-R. OLPE, A. SCHELLENBERG

3. Results Microiontophoretically applied NE produced a dose
desipramine or treated with the vehicle 24 h before the experiment (table 1). Following the chronic administration of all o f the four antidepressant drugs, we observed a statistically significant reduction in the sensitivity of cortical cells as compared to those recorded in acutely treated animals. This reduction of the mean firing depression was observed with all three ejection currents

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Fig. 2. Dose-response curves of the cell depressant action of NE applied microiontophoretically to cortical cells of rats treated either chronically (solid line) or acutely (dashed line) during 4 weeks with various antidepressant drugs. Each value is the mean of 32 values obtained in a group of 8 rats. * P < 0.01; ** P < 0.001. A, Tranylcypromine; B, clomipramine; C, maprotiline; D, desipramine.

reduction of sensitivity

TABLE 2

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Fig. 3. The t i m e course o f development o f desensitisation is shown as a function o f the days of treatment with desipramine. Groups of rats were treated with either 10 mg/kg i.p. o f desipramine or the vehicle. Differences in sensitivity of cingulate cortical cells were calculated by comparing the mean firing depression produced by NE (ejection current: 100 nA, 60 sec) in treated and untreated animals. ±S.D.

The inhibitory action of microiontophoretically applied NE on cingulate cortical cells of rats treated for 4 weeks with one daily injection of either carbamazepine (30 mg/kg p.o.) or methylcellulose is shown. Each group consisted of 8 animals. NE was applied iontophoretically for 1 rain in each animal near to 4 spontaneously active neurons using ejection currents of 10, 30 and 100 nA. The maximal depression of the firing rate observed during each applicaLion of NE was expressed as percentage of the spontaneous discharge rate. Data are presented as means -+ S.E.M.

Carbamazepinetreated Saline-treated

10 nA

30 nA

100 nA

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used for the local administration of NE (figs. 1 and 2). In acutely treated animals the maximal firing depression produced by NE with ejection currents of 100 nA ranged between 52 and 69%. In the chronically treated rats, however, the maximal mean firing depression ranged between 31 and 52% only. This reduction of the responsiveness of neurons from chronically treated animals as compared to acutely treated animals was calculated by comparing the maximal mean firing depression evoked with 100 nA currents. The reduction in responsiveness was 24% for tranylcypromine, 35% for clomipramine, 38% for maprotiline and 43% for desipramine. In contrast to chronic treatment with antidepressant drugs, the chronic treatment of rats with carbamazepine did not produce a statistically significant reduction in sensitivity of cingulate cortical cells to NE (table 2). Fig. 3 shows the time course of desensitisation towards NE during prolonged treatment with desipramine. Following 3 or 6 days' treatment, there was only a 1-3 percent reduction in sensitivity. At the tenth day of treatment, the sensitivity of the neurons of the treated animals was 33 percent less than that recorded in control animals. By the end of the fourth week of treatment, the reduction of sensitivity amounted to 43 percent. GABA elicited a dose-dependent reduction of the firing rate of all neurons tested. The chronic treatment with all of the four antidepressant drugs did n o t markedly change the sensitivity of cingulate neurons toward the cell depressant action of GABA (Fig. 4). Almost identical dose-response curves were obtained in acutely and chronically treated animals indicating that postsynaptic GABA receptors were not affected by the chronic administration of these antidepressant drugs.

4. Discussion The neurons of rats treated once with desipramine did not show an increased sensitivity

H.-R. OLPE, A. SCHELLENBERG

to NE as compared to neurons of rats treated once with saline. This absence of a potentiating effect was probably due to the low brain levels of the antidepressant drug. Twentyfour h following a single injection of 10 rag/ kg i.p. of this compound, its level was reported to be about 100 times lower than 1 h after injection (Vetulani et al., 1976). The main finding of this study was the marked reduction in the sensitivity of cingulate neurons to NE following the chronic administration of various antidepressant drugs as compared to the effect of their acute administration. This phenomenon was observed with one monoamine oxidaseinhibiting as well as with three NE uptakeinhibiting compounds. In contrast, carbamazepine, which is structurally related to the tricyclic antidepressants, did not induce desensitisation. This potent antiepileptic drug is devoid of any NE uptake-inhibiting properties up to a dose of 100 mg/kg (P. Baumann, personal communication). With desipramine there was a close correlation between the electrophysiologically determined change in the sensitivity to NE, the reduction of the fi-receptor binding sites and the diminished responsiveness of the adenylate cyclase system to NE. Chronic treatment with this drug (10 mg/kg i.p., 21 days) Was reported to result in a 35--45% decrease in the accumulation of adenosine cyclic 3',5'-monophosphate in response to stimulation with a maximally effective concentration of isoproterenol (Wolfe et al., 1978). This diminution in responsiveness was accompanied by a 35--45% decrease in the density of fi-adrenergic receptors (Wolfe et al., 1978) and by a 43% reduction in the sensitivity of cortical cells to the cell depressant action of NE reported in the present study. The present study confirms that there is a close relationship between the time course of development of reduced NE sensitivity of cortical cens in rodents and the clinical onset of action of antidepressants. The clinical effects of antidepressants are generally observed after 1--3 weeks of treatment.

DESENSITISATION BY ANTIDEPRESSANTS

Desensitisation of cingulate cortical cells developed within 6--10 days of treatment with desipramine. De Montigny and Aghajanian (1978) found no evidence of a change in the sensitivity to noradrenaline of neurons located in the ventral nucleus of the lateral geniculate body and in the dorsal hippocampus after chronic treatment with various antidepressant drugs. Instead these authors reported an increased responsiveness of these neurons to serotonin. This discrepancy with regard to the results obtained with noradrenaline is most likely due to the lower dosage and shorter treatment used in that study as well as to possible regional differences. Gabaergic neurons are not thought to be centrally involved in the beneficial action of antidepressant drugs. GABA was included in this study in order to demonstrate that these drugs do not induce a general desensitisation of central receptor sites. Our results obtained with GABA prove that there is no unspecific, general change in excitability of cingulate cortical cells following chronic treatment with antidepressants. In agreement with our results, De Montigny and Aghajanian (1978) reported no change in the responsiveness of neurons to GABA. However, the chronic administration of these drugs may exert presynaptic effects on gabaergic neurons which cannot be detected with this method. A functional interaction between the serotonergic and noradrenergic systems has been postulated on the basis of biochemical, pharmacological and anatomical evidence (Samanin and Garattini, 1976). It is thus conceivable that the postsynaptic hypersensitivity to serotonin and hyposensitivity to noradrenaline are functionally linked phenomena and that the beneficial effects of antidepressant drugs are brought about by a

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shift in the balance between the two cell systems. In conclusion the present study shows that the chronic treatment of rats with antidepressant drugs which increase the noradrenaline concentration in the synaptic cleft, by inhibiting either NE uptake or monoamine oxidase activity, induces subsensitivity of the cortical cells to the depressant action of NE.

References Banerjee, S.P., L.S. Kung, S.J. Riggi and S.K. Chanda, 1977, Development of /~-adrenergic receptor subsensitivity by antidepressants, Nature 268, 455. Clements-Jewery, S., 1978, The development of cortical ~-adrenoceptor subsensitivity in the rat by chronic treatment with trazodone, doxepin and mianserine, Neuropharmacology 17,779. De Montigny, C. and G.K. Aghajanian, 1978, Tricylic antidepressants: Long-term treatment increases responsivity of rat forebraln neurons to serotonin, Science 202, 1303. Dillier, N., J. Laszlo, B. Miiller,W.P. Koella and H.-R. Olpe, 1978, Activation of an inhibitory noradrenergic pathway projecting from the locus coeruleus to the cingulate cortex of the rat, Brain Res. 154, 61. Samanin, R. and S. Garattini, 1976, The serotonergic system in the brain and its possible functional connections with other arninergic systems. Life Sci. 17, 1201. Schultz, J., 1976, Psychoactive drug effects on a system which generates cyclic A M P in brain, Nature 261, 417. Vetulani, J., R.J. Stawarz, J.V. Dingell and F. Sulser, 1976. A possible c o m m o n mechanism of action of antidepressant treatments, Naunyn-Schmiedeb. Arch. Pharmacol. 293, 109 Wolfe, B.B., T.K. Harden, J.R. Sporn and P.B. Molinoff, 1978, Presynaptic modulation of betaadrenergic receptors in rat cerebral cortex after treatment with antidepressants, J. Pharmacol. Exp. Ther. 207, 446.