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Long-term effects of imipramine on striatal dopamine autoreceptor function: Involvement of both noradrenergic and serotonergic systems
Gen. Pharmac. Vol. 23, No. 3, pp. 397-401, 1992
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
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LONG-TERM EFFECTS OF IMIPRAMINE ON STRIATAL DOPAMINE AUTORECEPTOR FUNCTION: INVOLVEMENT OF BOTH N O R A D R E N E R G I C A N D SEROTONERGIC SYSTEMS CRISTOFOROSCAVONE,l MOACYRLUIZ AIZENSTEIN,1. CLEOPATRADA SILVAPLANETA2 and ROBERTODE LUCIA1 ILaborat6rio de Neuropsicofarmacologia, Departamento de Farmacologia, Instituto de Ci~ncias Biom6dicas da Universidade de S~o Paulo, Caixa Postal 4365, CEP 05508, Silo Paulo and 2Departamento de Principios Ativos Naturais e Toxicologia, UNESP, Araraquara, S~o Paulo, Brazil [Tel. 55-I 1-813-6944; Fax 55-11-813-0845] (Received 26 September 1991)
Abstract--1. The effects of apomorphine (APO) administration on DA system activity were assessed by measuring dopamine metabolite levels (HVA) in several circumstances. 2. Pretreatment with IMI reduced the effect of APO on HVA levels. 3. Pretreatments with either IDE or DMI did not reduce the effect of APO on HVA levels. 4. Reductions of either NE and 5-HT levels after DSP4 and pCPA restored the effect of APO after IMI pretreatment.
INTRODUCTION Knowledge of the mechanisms of action of antidepressant drugs is still incomplete. Several putative neurotransmitters have been implicated in the physiopathology of depression, including the dopaminergic system. Long-term administration of certain antidepressants to rats reportedly resulted in subsensitivity of dopaminergic autoreceptors in the striatum, as assessed by behavioral, biochemical and electrophysiological methods (Serra et al., 1979; Antelman and Chiodo, 1981; Maj and Wedzony, 1985; Scavone et al., 1986). Others found that locomotor responses induced by apomorphine and damphetamine were increased after chronic treatment with imipramine (IMI) and desipramine (DMI) (Spyraki and Fibiger, 1981; Maj et al., 1984), suggesting post-synaptic supersensitivity. We reported that long-term treatment with IMI may modify dopaminergic system activity by an indirect mechanism, that may involve interactions of the noradrenergic and serotonergic systems (Scavone et al., 1986). Other evidence of interactions between monoaminergic systems includes a report that lesions on the central noradrenergic neurons abolish the effect of electroconvulsive shock (ECS) inducing potentiation of the apomorphine's effect increasing motility (Green and Deakin, 1980). Other studies suggest that reduction of beta-adrenoceptor binding by antidepressants is linked to the activity of serotonergic systems, thus, antidepressants failed to reduce beta-adrenoceptor binding after inhibition of 5HT synthesis (Brunello et al., 1985; Mainier et al., 1985) or neurotoxic lesions of 5HT terminals *To whom all correspondence and reprint requests should be addressed.
(Brunello et al., 1982; Nimgaonkar et al., 1985). Since both IMI and its metabolite DMI inhibit the uptake of 5HT and the uptake of NE we performed the present experiments to investigate the influence of noradrenergic and serotonergic systems on the effect of IMI on dopaminergic activity. Moreover, we studied the influence on DA receptor sensitivity induced by long-term IMI during reduction of NE and 5HT inputs. In order to reduce serotonergic and noradrenergic activity, pCPA a tryptophan hydroxylase inhibitor and indalpine (IDE), a serotonin uptake blocker, with a toxin (DSP4) for noradrenergic neurons, were used. The results suggest that IMI acting on both serotonergic and noradrenergic systems may indirectly lead to subsensitivity of the striatal dopaminergic autoreceptors.
MATERIALS AND METHODS
Experimental animals Male Wistar rats weighing 180-250g at the beginning of the experiment were housed in groups of 8 in plastic cages 32(width) × 40(length) x 16(height) in a temperature controlled room. Animals were maintained on a 12:12hr light/dark regimen (7 :00 a.m.-7:00 p.m.) for 2 weeks prior to experimental manipulation. Food and water were given ad libitum. Drugs Imipramine hydrochloride (Ciba-Geigy, Sho Paulo), desipramine hydrochloride (Sigma Chemical Co., St Louis, Mo.), R(-) apomorphine hydrochloride (Sandoz, S~o Paulo), OL-p-chlorophenylaninemethyl ester (SigmaChemical Co.) and N 2-chloroethyl N-ethyl-2-bromobenzylamine (DSP4, Astra Lakemedel, Sweden) were freshly dissolved in distilled water. Indalpine (Rhodia, S~o Paulo) was first dissolved in hydrochloric acid 0.I N with distilled water added (1:2).
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Preliminary studies pCPA treatment schedule. 36 rats were randomly assigned to 3 groups of 12. Of the first group, 6 each received pCPA (300 mg/kg, i.p.) or saline (1.0 ml/kg, i.p.) at 8:00 a.m., 12 hr prior to physiological saline injection (1.0 ml/kg, i.p.) and they were decapitated 2 hr later (acute). The other two groups received long-term (14 days) saline (1.0 mg/kg, i.p.) treatment once daily at 8 : 0 0 p . m . On days I, 4, 8 and 12, at 8:00 a.m. half were treated with pCPA (300 mg/kg, i.p.) or saline (1.0 ml/kg, i.p.) (N = 12). Half of the animals of p C P A and saline groups were decapitated 2 hr (chronic) or 72 hr (withdraw) after the last injection. 5-HIAA and H V A levels in striatum and M H P G levels in frontal cortex were determined fluorimetrically. DSP4 treatment schedule. Animals (n = 18) received i.p. injections of IDE (10 mg/kg) at 8:00 p.m. to protect 5HT neurons and, 30 min later, a single dose of DSP-4 (50 mg/kg, i.p.) to lesion NE neurons. Their controls (N = 18) received two injections of saline (1.0ml/kg, i.p.) with a 3 0 m i n interval. At 21 days after these treatments, the animals received saline (1.0 mg/kg, i.p.) acutely or chronically (14 more days). 2 hr after the single injection (acute) and at 2 hr (chronic) and 72 hr (withdraw) after the last injection, the animals were decapitated. 5HIAA and HVA levels in striatum and M H P G levels in frontal cortex were determined fluorimetrically. Biochemical assay procedures. After decapitation the brains were rapidly taken out. The striatum and frontal cortex were dissected, immediately frozen on dry ice and stored at - 6 0 ° C until biochemical analysis. Samples were analyzed by fluorimetric assay for HVA, 5-HIAA and M H P G . Shortly, the striatum was weighed and homogenized in 0.1 M perchloric acid. After centrifugation (4000g/10min, 4°C) the supernatants were applied on Sephadex G I 0 columns; H V A and 5-HIAA from the eluate were fluorimetrically determined as described before (Westerink and Korf, 1977; Earley and Leonard, 1978). Frontal cortex was weighed and homogenized in 0.2 M zinc sulphate. After addition of a solution of 0.2 M barium hydroxide to the homogenate, it was centrifuged at 30,000g (60 min, 4°C). M H P G in the tissue extract was purified by successive chromatography on Sephadex G-10 and D E A E Sephadex A-25 (borate form). M H P G levels were determined according to Ader and Korf (1979).
Assessment of changes on striatal dopaminergic autoreceptor function Experiment l--effects of long-term IMI, IDE or DMI treatment. Animals (N = 48) received at 8:00 p.m., during 14 consecutive days, daily injections of IMI (10 mg/kg, i.p.) (N = 12), IDE (I0 mg/kg, i.p.) (N = 12), D M I (10mg/kg, i.p.) (N = 12) or saline (1.0ml/kg i.p.) (N = 12) and 72 hr after the last administration half of the animals of each group received either A P O (0.02mg/kg, s.c.) or saline
(1.0 ml/kg, s.c.). 45 min later, the rats were decapitated and striatal H V A levels were determined.
Experiment 2---effects of pCPA and IM1 associated in a long-term treatment. Rats received at 8:00p.m. IMI (10 mg/kg, i.p.) (N = 12) or saline (1.0 ml/kg, i.p.) (N = 12) daily for 14 consecutive days. On days 1, 4, 8 and 12 at 8:00 a.m. the animals (6 from IMI and 6 from saline group) were treated with p C P A (300 mg/kg, i.p.), and the remaining 6 of each group were treated with saline (1.0 mg/kg, i.p.). 7 2 h r after the last IMI or saline administration, all the animals received a single challenge dose of APO (0.02mg/kg, s.c.) or saline (1.0ml/kg, s.c.). The control group (N = 6) received only saline in the same schedule described above. 45 min later, all animals were decapitated and striatal H V A levels were determined.
Experiment 3--effects of IDE + DSP4 pretreatment on long-term 1MI administration. In this experiment rats were injected with IDE (10 mg/kg, i.p.) (N = 24) at 8:00 p.m. and 30 min later they were injected with a single dose of DSP-4 (50 mg/kg, i.p.). 21 days after this treatment, the animals received at 8:00p.m. IMI (10mg/kg) ( N = 12) or saline (1.0 mg/kg) (N = 12) i.p. daily for 14 consecutive days. 72 hr after the last daily injection, half of the animals of each group received a single challenge dose of A P O (0.02 mg/kg, s.c.) or saline (1.0 ml/kg, s.c.). The control group (N = 6) received only saline in the same schedule described above. 45 min later, the rats were decapitated and striatal HVA levels were assayed.
Data analysis The HVA, 5-HIAA and M H P G levels are expressed in pg/g net tissue ( + SEM). The significance of the differences between means in preliminary studies was computed by Student's t-test. Data from the experiments of the assessment of changes on striatal dopaminergic autoreceptors (Expts 1, 2 and 3) were submitted to a one-way analysis of variance (ANOVA) followed by the N e w m a n - K e u l s ' test for differences between pairs of means with significant F-ratios.
RESULTS
p C P A and I D E + D S P 4 treatment schedule T h e effects o f p C P A t r e a t m e n t o n H V A , 5 H I A A a n d M H P G levels a r e d e p i c t e d in T a b l e 1. T h e r e s u l t s i n d i c a t e t h a t striatal levels o f 5 - H I A A d e c r e a s e d s i g n i f i c a n t l y to a r a n g e o f 4 0 / 5 0 % o f t h e c o n t r o l v a l u e a f t e r a c u t e a n d c h r o n i c a d m i n i s t r a t i o n s , r e t u r n i n g to c o n t r o l levels o n 72 h r a f t e r t h e last i n j e c t i o n (withd r a w g r o u p ) . H V A a n d M H P G levels did n o t s h o w s i g n i f i c a n t differences in a n y t i m e s t u d i e d a f t e r p C P A treatment.
Table 1. Changes in striatal HVA and 5HIAA levels and cortical MHPG levels induced by pCPA (300 mg/kg, i.p.) or IDE (10 mg/kg, i.p.) + DSP4 (50 mg/kg, i.p.) in different intervals of treatment, pCPA was injected 12 hr prior to saline on days 1, 4, 8 and 12. IDE + DSP4 were injected 21 days before administration of saline for 1-14 days. Each value represents the mean ± SEM in #g/g tissue for 6 animals Striatum Time 1 day (acute)
Groups
HVA
Control 0.72+0.06 pCPA 0.71 _+0.04 IDE + DSP4 0.69 _+0.08 14 days Control 0.74 + 0.04 (chronic) pCPA 0.71 ± 0.07 IDE + DSP4 0.68 ± 0.05 14 days + 72 hr Control 0.69 ± 0.03 (withdraw) pCPA 0.74 ± 0.05 IDE + DSP4 0.65 ± 0.03 *P < 0.001 vs control group (Student's t-test).
5HIAA
Frontal cortex MHPG
0.29+0.08 0.15 + 0.03* 0.34 + 0.04 0.32 ± 0.06 0.13 ± 0.02* 0.35+0.08 0.29 _+0.04 0.24 + 0.09 0.27 ± 0.08
0.52_+0.11 0.49 + 0.08 0.09 ± 0.06* 0.47 _+0.08 0.44 ± 0.04 0.16_+0.09" 0.43 ± 0.06 0.48 + 0.08 0.14 ± 0.06*
Long-term effects of imipramine Table 2. Influence of long-term (14 days) IMI (10 mg/kg, i.p.), IDE (10mg/kg, i.p.) and DMI (10mg/kg, i.p.) treatments on APO effects upon HVA striatal levels. All rats received a single challenge dose of APO (0.02 mg/kg, s.c.) or saline at 72 hr after withdrawal and were decapitated 45 min later. Each value of HVA represents the mean +__SEM in #g/g tissue for 6 animals Treatments Saline + saline IMI + saline IDE + saline DMI + saline Saline + APO IMI + APO IDE ± APO DMI + APO
HVA 0.77 + 0.72 + 0.74 + 0.75 ± 0.35 + 0.69 + 0.33 ± 0.31 ±
% control
0.02 0.02 0.08 0.07 0.09* 0.05 0.07* 0.06*
100.0 93.5 96.1 97.4 45.4* 89.6 42.8* 40.2*
ANOVA: HVA: striatum F(7,40)= 32,29 (P < 0.001). *Newman-Keuls' test: saline + APO = IDE + APO = DMI + APO < saline + saline = IMI + saline = IDE + saline = DMI + saline = IMI + APO (P < 0.05).
Table 1 also summarizes the effects of IDE + DSP4 on HVA, M H P G and 5HIAA levels. M H P G was reduced in frontal cortex tissue by 80% on the first day and by 60% on the 14th day (chronic group) as well as at 72 hr after (withdraw group). No changes were observed after IDE + DSP4 administration on striatal HVA and 5HIAA levels. Assessment o f changes on striatal dopaminergic autoreceptor function
The results of Expts 1, 2 and 3 are summarized in Tables 2, 3 and 4. As shown, the effects of APO administration are dependent on the pretreatment given. Saline plus APO treatment decreased the levels of HVA compared to the saline group in all experiments. Long-term administration of IMI inhibited the reduction of HVA levels by APO, but neither IDE nor DMI long-term administration altered this effect of APO on striatal HVA levels (Table 2). Effects of pCPA pretreatment on the subsensitivity of dopaminergic autoreceptors produced by longterm IMI are depicted in Table 3. Treatment with pCPA neither changed the basal levels of HVA in the striatum nor the effect of APO in reducing the levels of this metabolite. APO reduced the levels of HVA in animals pretreated with pCPA significantly (Table 3), in contrast to the group that received only IMI in which this APO effect was blocked (Table 2). Table 3. Effects of pCPA (300 mg/kg, i.p.) and IMI (10 mg/kg, i.p.) associated in long-term treatments (14 days) on APO effects upon HVA striatal levels, pCPA was injected at 8:00 p.m. on days 1, 4, 8 and 12. All rats received a single challenge dose of APO (0.02 mg/kg, s.c.) or saline at 72 hr after withdrawal and were decapitated 45 min later. Each value of HVA represents the mean ± SEM in pg/g of tissue for 6 animals Treatments Saline + pCPA + pCPA + Saline + pCPA + pCPA +
saline + saline saline + saline IMI + saline saline + APO saline + APO IMI + APO
HVA 0.71 0.74 0.74 0.32 0.31 0.35
+ ± + + + +
0.10 0.02 0.03 0.06* 0.02* 0.02*
% control 100.0 104.2 104.2 45.0* 43.6* 49.2*
ANOVA: HVA: striatum F(5,30) = 52,52 (P < 0.001). *Newman-Keuls' test: saline + saline + APO = pCPA + saline + APO = pCPA + IMI + APO < saline + saline + saline = pCPA + saline + saline = pCPA + IMI + saline (P < 0.05).
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Table 4. Effects of IDE (10 mg/kg, i.p.)+ DSP4 (50 mg/kg, i.p.) administration before long-term (14 days) IMI (10mg/kg, i.p.) treatment upon HVA striatal levels. The IDE + DSP4 were injected twenty-one days before long-term IMI or saline treatment. All rats received a single challenge dose of APO (0.02 mg/kg, s.c.) or saline at 72 hr after withdraw and were decapitated 45 rain later. Each value of HVA represents the mean + SEM in/~g/g of tissue for 6 animals Treatments
HVA
Saline + saline + saline IDE + DSP4 + saline + saline IDE + DSP4 + IMI + saline Saline + saline + APO IDE + DSP4 -I- saline + APO IDE + DSP4 + IMI + APO
0.76 0.77 0.60 0.36 0.35 0.20
± 0.07 ± 0.09 _+0.10 ± 0.08* + 0.07* ± 0.09*
% control 100.0 101.3 78.9 47.3* 46.0* 26.3*
ANOVA: HVA: striatum F(5,30) = 25,98 (P < 0.001). *Newman-Keuls' test: saline + saline + APO = IDE + DSP4 + saline + A P O = IDE + DSP4 + IMI + APO < saline + saline+ saline = IDE + DSP4 + saline + saline = IDE + DSP4 + IMI + saline (P < 0.05).
Table 4 shows the effects of specific reduction in noradrenergic activity with IDE + DSP4 on the subsensitivity of dopaminergic autoreceptor produced by long-term IMI-treatment. Treatment with IDE + DSP4 neither changed the levels of HVA in the striatum nor the effect of APO in reducing the levels of this metabolite (Table 4). Finally, APO significantly reduced levels of HVA in animals pretreated with IDE + DSP4 (Table 4), in contrast to the group that received only IMI in which the effect of APO was blocked (Table 2). DISCUSSION
In preliminary experiments we established an effective schedule (300mg/kg at 4 days interval) for reducing 5HT activity by 30-70% of the control value, after single doses of PCPA (data not shown). Using this schedule, 5HIAA levels were remarkably decreased at the 1st or at the 14th day of IMI treatment, but not at 72 hr after IMI withdraw when APO effect was tested (see Table 1). The reduction of 5HIAA levels caused by pCPA during the chronic treatment could reflect some changes on the serotonergic neurons activity. In preliminary experiments the action of DSP4 on noradrenergic neurons was not selective since serotonergic neurons were also affected (data not shown). Thus, we gave DSP4 after IDE, a selective serotonin uptake blocker (Le Fur, 1983) which does not modify the effect of DSP4 on noradrenergic neurons. The treatment reduced M H P G without altering 5HIAA levels (Table 1), as had been found by Ross et al. (1976) with another 5HT uptake blocker, zimelidine before DSP4. In addition, since low doses of APO decrease DOPAC and HVA levels presumably by acting on the dopamine autoreceptors (Di Chiara et al., 1976), we utilized this drug (at 20pg/kg, s.c.) to evaluate changes in dopaminergic autoreceptor sensitivity after long-term IMI treatment. In agreement with our previous results (Scavone et al., 1986), rats chronically treated with IMI failed to exhibit a significant APO-induced decrease in striatal HVA levels. Other studies employing different methods (Serra et al., 1981) came to the same conclusion. However, in the present study we found that long-term treatment with either IDE or DMI, respectively norepinephrine and
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serotonin transport blockers, did not attenuate the effects of APO in reducing HVA levels, in agreement with other such studies of long-term treatment with specific serotonin or norepinephrine uptake blockers (Spyraki and Fibiger, 1981; Diggory and Buckett, 1984). The question arises of how chronic administration of IMI but not DMI or IDE, given alone or together, could reduce DA autoreceptor subsensitivity. These data are in agreement with the report of Klimek and Nielson (1987) that chronic administration of IMI resulted in a decreased total density of DA receptors. Why this was not found with DMI plus IDE (data not shown), deserves further investigation. The present data affirm complex interactions between IMI and central serotonergic and noradrenergic systems. When we reduced norepinephrine or serotonin inputs with (pCPA or IDE + DSP4) the effect of IMI on sensitivity to APO was lost (Tables 2 and 3). A possibility is that interactions of IMI at both noradrenergic and serotonergic systems may lead to a final reduction in dopaminergic receptor density. This is supported by the fact that citalopran (a 5HT uptake blocker) and not benzotropine (a DA uptake blocker) resulted in a decreased number of DA receptors (Klimek and Nielsen, 1987). Moreover various behavioral tests on animals have shown that by changing central beta-adrenergic activity, the action of apomorphine may be influenced (Mogilnicka et aL, 1984; Ortmann et al., 1985). Although further work is warranted to clarify the role of noradrenergic and serotonergic systems on dopaminergic activity, these results strongly support the suggestion that in absence of both noradrenergic and serotoninergic neural input, IMI failed to desensitize dopamine autoreceptors. In view of these findings we propose in this circumstance an indirect control of the striatal dopaminergic system caused by serotoninergic and noradrenergic systems. The occurrence of interactions among those systems have been shown before by others (for example Bunney and De Riemer, 1982; Agren, 1986). These relationships among major neurotransmitter systems suggest that any event modifying the activity of one neurotransmitter is likely to change the activity of any other linked system. For instance, single unit studies (de Montigny and Aghajanian, 1978; Menkes et al., 1980; Wang and Aghajanian, 1980) indicate that chronic antidepressant treatment enhances post-synaptic serotonergic and alpha-adrenergic responsivity in rat forebrain regions. Our results also may be relevant to the interpretation that the dopaminergic system is an important neural substrate for some forms of reinforcement or reward (e.g. Miller et al., 1990). If the pathophysiology of clinical depression includes an inability of normal environmental stimuli to mobilize this reinforcement system, the gradual facilitation of the activity of this dopaminergic system by antidepressant drugs (directly or by other indirect effects via NE and 5HT systems) would be consistent with their antidepressant properties (Spyraki and Fibiger, 1981). SUMMARY The responsiveness of rat striatal dopamine (DA) autoreceptors to apomorphine (APO) were assessed
after long-term treatment with an inhibitor of serotonin uptake indalpine (IDE), an inhibitor of NE uptake desipramine (DMI), or a mixed inhibitor of both 5HT and NE uptake imipramine (IMI). Moreover, the effects of long-term IMI treatment on DA autoreceptors were determined after reducing serotonin (5HT) or norepinephrine (NE) neuronal input, chronically with pCPA or DSP4. IDE or DMI longterm administration did not alter the reduction of homovanillic acid (HVA) levels induced by APO, but IMI treatment reduced the effect of APO on HVA levels, indicating subsensitivity of dopaminergic autoreceptors. Reducing 5HT or NE input during IMI treatment, respectively with p-chlorophenylalanine (pCPA) or IDE plus N(2-chlorethyl) N-ethyl-2-bromobenzylamine (DSP4), restored the effect of APO after long-term IMI administration. The results suggest that desensitization of DA autoreceptors induced by long-term IMI treatment is related to IMI effects upon noradrenergic and serotonergic systems. Acknowledgements--To Dr S. B. Ross (Astra Lakemedel AB-Sweden) for the gift of DSP-4. This work was supported by grants from FAPESP and CNPq.
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0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
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LONG-TERM EFFECTS OF IMIPRAMINE ON STRIATAL DOPAMINE AUTORECEPTOR FUNCTION: INVOLVEMENT OF BOTH N O R A D R E N E R G I C A N D SEROTONERGIC SYSTEMS CRISTOFOROSCAVONE,l MOACYRLUIZ AIZENSTEIN,1. CLEOPATRADA SILVAPLANETA2 and ROBERTODE LUCIA1 ILaborat6rio de Neuropsicofarmacologia, Departamento de Farmacologia, Instituto de Ci~ncias Biom6dicas da Universidade de S~o Paulo, Caixa Postal 4365, CEP 05508, Silo Paulo and 2Departamento de Principios Ativos Naturais e Toxicologia, UNESP, Araraquara, S~o Paulo, Brazil [Tel. 55-I 1-813-6944; Fax 55-11-813-0845] (Received 26 September 1991)
Abstract--1. The effects of apomorphine (APO) administration on DA system activity were assessed by measuring dopamine metabolite levels (HVA) in several circumstances. 2. Pretreatment with IMI reduced the effect of APO on HVA levels. 3. Pretreatments with either IDE or DMI did not reduce the effect of APO on HVA levels. 4. Reductions of either NE and 5-HT levels after DSP4 and pCPA restored the effect of APO after IMI pretreatment.
INTRODUCTION Knowledge of the mechanisms of action of antidepressant drugs is still incomplete. Several putative neurotransmitters have been implicated in the physiopathology of depression, including the dopaminergic system. Long-term administration of certain antidepressants to rats reportedly resulted in subsensitivity of dopaminergic autoreceptors in the striatum, as assessed by behavioral, biochemical and electrophysiological methods (Serra et al., 1979; Antelman and Chiodo, 1981; Maj and Wedzony, 1985; Scavone et al., 1986). Others found that locomotor responses induced by apomorphine and damphetamine were increased after chronic treatment with imipramine (IMI) and desipramine (DMI) (Spyraki and Fibiger, 1981; Maj et al., 1984), suggesting post-synaptic supersensitivity. We reported that long-term treatment with IMI may modify dopaminergic system activity by an indirect mechanism, that may involve interactions of the noradrenergic and serotonergic systems (Scavone et al., 1986). Other evidence of interactions between monoaminergic systems includes a report that lesions on the central noradrenergic neurons abolish the effect of electroconvulsive shock (ECS) inducing potentiation of the apomorphine's effect increasing motility (Green and Deakin, 1980). Other studies suggest that reduction of beta-adrenoceptor binding by antidepressants is linked to the activity of serotonergic systems, thus, antidepressants failed to reduce beta-adrenoceptor binding after inhibition of 5HT synthesis (Brunello et al., 1985; Mainier et al., 1985) or neurotoxic lesions of 5HT terminals *To whom all correspondence and reprint requests should be addressed.
(Brunello et al., 1982; Nimgaonkar et al., 1985). Since both IMI and its metabolite DMI inhibit the uptake of 5HT and the uptake of NE we performed the present experiments to investigate the influence of noradrenergic and serotonergic systems on the effect of IMI on dopaminergic activity. Moreover, we studied the influence on DA receptor sensitivity induced by long-term IMI during reduction of NE and 5HT inputs. In order to reduce serotonergic and noradrenergic activity, pCPA a tryptophan hydroxylase inhibitor and indalpine (IDE), a serotonin uptake blocker, with a toxin (DSP4) for noradrenergic neurons, were used. The results suggest that IMI acting on both serotonergic and noradrenergic systems may indirectly lead to subsensitivity of the striatal dopaminergic autoreceptors.
MATERIALS AND METHODS
Experimental animals Male Wistar rats weighing 180-250g at the beginning of the experiment were housed in groups of 8 in plastic cages 32(width) × 40(length) x 16(height) in a temperature controlled room. Animals were maintained on a 12:12hr light/dark regimen (7 :00 a.m.-7:00 p.m.) for 2 weeks prior to experimental manipulation. Food and water were given ad libitum. Drugs Imipramine hydrochloride (Ciba-Geigy, Sho Paulo), desipramine hydrochloride (Sigma Chemical Co., St Louis, Mo.), R(-) apomorphine hydrochloride (Sandoz, S~o Paulo), OL-p-chlorophenylaninemethyl ester (SigmaChemical Co.) and N 2-chloroethyl N-ethyl-2-bromobenzylamine (DSP4, Astra Lakemedel, Sweden) were freshly dissolved in distilled water. Indalpine (Rhodia, S~o Paulo) was first dissolved in hydrochloric acid 0.I N with distilled water added (1:2).
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Preliminary studies pCPA treatment schedule. 36 rats were randomly assigned to 3 groups of 12. Of the first group, 6 each received pCPA (300 mg/kg, i.p.) or saline (1.0 ml/kg, i.p.) at 8:00 a.m., 12 hr prior to physiological saline injection (1.0 ml/kg, i.p.) and they were decapitated 2 hr later (acute). The other two groups received long-term (14 days) saline (1.0 mg/kg, i.p.) treatment once daily at 8 : 0 0 p . m . On days I, 4, 8 and 12, at 8:00 a.m. half were treated with pCPA (300 mg/kg, i.p.) or saline (1.0 ml/kg, i.p.) (N = 12). Half of the animals of p C P A and saline groups were decapitated 2 hr (chronic) or 72 hr (withdraw) after the last injection. 5-HIAA and H V A levels in striatum and M H P G levels in frontal cortex were determined fluorimetrically. DSP4 treatment schedule. Animals (n = 18) received i.p. injections of IDE (10 mg/kg) at 8:00 p.m. to protect 5HT neurons and, 30 min later, a single dose of DSP-4 (50 mg/kg, i.p.) to lesion NE neurons. Their controls (N = 18) received two injections of saline (1.0ml/kg, i.p.) with a 3 0 m i n interval. At 21 days after these treatments, the animals received saline (1.0 mg/kg, i.p.) acutely or chronically (14 more days). 2 hr after the single injection (acute) and at 2 hr (chronic) and 72 hr (withdraw) after the last injection, the animals were decapitated. 5HIAA and HVA levels in striatum and M H P G levels in frontal cortex were determined fluorimetrically. Biochemical assay procedures. After decapitation the brains were rapidly taken out. The striatum and frontal cortex were dissected, immediately frozen on dry ice and stored at - 6 0 ° C until biochemical analysis. Samples were analyzed by fluorimetric assay for HVA, 5-HIAA and M H P G . Shortly, the striatum was weighed and homogenized in 0.1 M perchloric acid. After centrifugation (4000g/10min, 4°C) the supernatants were applied on Sephadex G I 0 columns; H V A and 5-HIAA from the eluate were fluorimetrically determined as described before (Westerink and Korf, 1977; Earley and Leonard, 1978). Frontal cortex was weighed and homogenized in 0.2 M zinc sulphate. After addition of a solution of 0.2 M barium hydroxide to the homogenate, it was centrifuged at 30,000g (60 min, 4°C). M H P G in the tissue extract was purified by successive chromatography on Sephadex G-10 and D E A E Sephadex A-25 (borate form). M H P G levels were determined according to Ader and Korf (1979).
Assessment of changes on striatal dopaminergic autoreceptor function Experiment l--effects of long-term IMI, IDE or DMI treatment. Animals (N = 48) received at 8:00 p.m., during 14 consecutive days, daily injections of IMI (10 mg/kg, i.p.) (N = 12), IDE (I0 mg/kg, i.p.) (N = 12), D M I (10mg/kg, i.p.) (N = 12) or saline (1.0ml/kg i.p.) (N = 12) and 72 hr after the last administration half of the animals of each group received either A P O (0.02mg/kg, s.c.) or saline
(1.0 ml/kg, s.c.). 45 min later, the rats were decapitated and striatal H V A levels were determined.
Experiment 2---effects of pCPA and IM1 associated in a long-term treatment. Rats received at 8:00p.m. IMI (10 mg/kg, i.p.) (N = 12) or saline (1.0 ml/kg, i.p.) (N = 12) daily for 14 consecutive days. On days 1, 4, 8 and 12 at 8:00 a.m. the animals (6 from IMI and 6 from saline group) were treated with p C P A (300 mg/kg, i.p.), and the remaining 6 of each group were treated with saline (1.0 mg/kg, i.p.). 7 2 h r after the last IMI or saline administration, all the animals received a single challenge dose of APO (0.02mg/kg, s.c.) or saline (1.0ml/kg, s.c.). The control group (N = 6) received only saline in the same schedule described above. 45 min later, all animals were decapitated and striatal H V A levels were determined.
Experiment 3--effects of IDE + DSP4 pretreatment on long-term 1MI administration. In this experiment rats were injected with IDE (10 mg/kg, i.p.) (N = 24) at 8:00 p.m. and 30 min later they were injected with a single dose of DSP-4 (50 mg/kg, i.p.). 21 days after this treatment, the animals received at 8:00p.m. IMI (10mg/kg) ( N = 12) or saline (1.0 mg/kg) (N = 12) i.p. daily for 14 consecutive days. 72 hr after the last daily injection, half of the animals of each group received a single challenge dose of A P O (0.02 mg/kg, s.c.) or saline (1.0 ml/kg, s.c.). The control group (N = 6) received only saline in the same schedule described above. 45 min later, the rats were decapitated and striatal HVA levels were assayed.
Data analysis The HVA, 5-HIAA and M H P G levels are expressed in pg/g net tissue ( + SEM). The significance of the differences between means in preliminary studies was computed by Student's t-test. Data from the experiments of the assessment of changes on striatal dopaminergic autoreceptors (Expts 1, 2 and 3) were submitted to a one-way analysis of variance (ANOVA) followed by the N e w m a n - K e u l s ' test for differences between pairs of means with significant F-ratios.
RESULTS
p C P A and I D E + D S P 4 treatment schedule T h e effects o f p C P A t r e a t m e n t o n H V A , 5 H I A A a n d M H P G levels a r e d e p i c t e d in T a b l e 1. T h e r e s u l t s i n d i c a t e t h a t striatal levels o f 5 - H I A A d e c r e a s e d s i g n i f i c a n t l y to a r a n g e o f 4 0 / 5 0 % o f t h e c o n t r o l v a l u e a f t e r a c u t e a n d c h r o n i c a d m i n i s t r a t i o n s , r e t u r n i n g to c o n t r o l levels o n 72 h r a f t e r t h e last i n j e c t i o n (withd r a w g r o u p ) . H V A a n d M H P G levels did n o t s h o w s i g n i f i c a n t differences in a n y t i m e s t u d i e d a f t e r p C P A treatment.
Table 1. Changes in striatal HVA and 5HIAA levels and cortical MHPG levels induced by pCPA (300 mg/kg, i.p.) or IDE (10 mg/kg, i.p.) + DSP4 (50 mg/kg, i.p.) in different intervals of treatment, pCPA was injected 12 hr prior to saline on days 1, 4, 8 and 12. IDE + DSP4 were injected 21 days before administration of saline for 1-14 days. Each value represents the mean ± SEM in #g/g tissue for 6 animals Striatum Time 1 day (acute)
Groups
HVA
Control 0.72+0.06 pCPA 0.71 _+0.04 IDE + DSP4 0.69 _+0.08 14 days Control 0.74 + 0.04 (chronic) pCPA 0.71 ± 0.07 IDE + DSP4 0.68 ± 0.05 14 days + 72 hr Control 0.69 ± 0.03 (withdraw) pCPA 0.74 ± 0.05 IDE + DSP4 0.65 ± 0.03 *P < 0.001 vs control group (Student's t-test).
5HIAA
Frontal cortex MHPG
0.29+0.08 0.15 + 0.03* 0.34 + 0.04 0.32 ± 0.06 0.13 ± 0.02* 0.35+0.08 0.29 _+0.04 0.24 + 0.09 0.27 ± 0.08
0.52_+0.11 0.49 + 0.08 0.09 ± 0.06* 0.47 _+0.08 0.44 ± 0.04 0.16_+0.09" 0.43 ± 0.06 0.48 + 0.08 0.14 ± 0.06*
Long-term effects of imipramine Table 2. Influence of long-term (14 days) IMI (10 mg/kg, i.p.), IDE (10mg/kg, i.p.) and DMI (10mg/kg, i.p.) treatments on APO effects upon HVA striatal levels. All rats received a single challenge dose of APO (0.02 mg/kg, s.c.) or saline at 72 hr after withdrawal and were decapitated 45 min later. Each value of HVA represents the mean +__SEM in #g/g tissue for 6 animals Treatments Saline + saline IMI + saline IDE + saline DMI + saline Saline + APO IMI + APO IDE ± APO DMI + APO
HVA 0.77 + 0.72 + 0.74 + 0.75 ± 0.35 + 0.69 + 0.33 ± 0.31 ±
% control
0.02 0.02 0.08 0.07 0.09* 0.05 0.07* 0.06*
100.0 93.5 96.1 97.4 45.4* 89.6 42.8* 40.2*
ANOVA: HVA: striatum F(7,40)= 32,29 (P < 0.001). *Newman-Keuls' test: saline + APO = IDE + APO = DMI + APO < saline + saline = IMI + saline = IDE + saline = DMI + saline = IMI + APO (P < 0.05).
Table 1 also summarizes the effects of IDE + DSP4 on HVA, M H P G and 5HIAA levels. M H P G was reduced in frontal cortex tissue by 80% on the first day and by 60% on the 14th day (chronic group) as well as at 72 hr after (withdraw group). No changes were observed after IDE + DSP4 administration on striatal HVA and 5HIAA levels. Assessment o f changes on striatal dopaminergic autoreceptor function
The results of Expts 1, 2 and 3 are summarized in Tables 2, 3 and 4. As shown, the effects of APO administration are dependent on the pretreatment given. Saline plus APO treatment decreased the levels of HVA compared to the saline group in all experiments. Long-term administration of IMI inhibited the reduction of HVA levels by APO, but neither IDE nor DMI long-term administration altered this effect of APO on striatal HVA levels (Table 2). Effects of pCPA pretreatment on the subsensitivity of dopaminergic autoreceptors produced by longterm IMI are depicted in Table 3. Treatment with pCPA neither changed the basal levels of HVA in the striatum nor the effect of APO in reducing the levels of this metabolite. APO reduced the levels of HVA in animals pretreated with pCPA significantly (Table 3), in contrast to the group that received only IMI in which this APO effect was blocked (Table 2). Table 3. Effects of pCPA (300 mg/kg, i.p.) and IMI (10 mg/kg, i.p.) associated in long-term treatments (14 days) on APO effects upon HVA striatal levels, pCPA was injected at 8:00 p.m. on days 1, 4, 8 and 12. All rats received a single challenge dose of APO (0.02 mg/kg, s.c.) or saline at 72 hr after withdrawal and were decapitated 45 min later. Each value of HVA represents the mean ± SEM in pg/g of tissue for 6 animals Treatments Saline + pCPA + pCPA + Saline + pCPA + pCPA +
saline + saline saline + saline IMI + saline saline + APO saline + APO IMI + APO
HVA 0.71 0.74 0.74 0.32 0.31 0.35
+ ± + + + +
0.10 0.02 0.03 0.06* 0.02* 0.02*
% control 100.0 104.2 104.2 45.0* 43.6* 49.2*
ANOVA: HVA: striatum F(5,30) = 52,52 (P < 0.001). *Newman-Keuls' test: saline + saline + APO = pCPA + saline + APO = pCPA + IMI + APO < saline + saline + saline = pCPA + saline + saline = pCPA + IMI + saline (P < 0.05).
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Table 4. Effects of IDE (10 mg/kg, i.p.)+ DSP4 (50 mg/kg, i.p.) administration before long-term (14 days) IMI (10mg/kg, i.p.) treatment upon HVA striatal levels. The IDE + DSP4 were injected twenty-one days before long-term IMI or saline treatment. All rats received a single challenge dose of APO (0.02 mg/kg, s.c.) or saline at 72 hr after withdraw and were decapitated 45 rain later. Each value of HVA represents the mean + SEM in/~g/g of tissue for 6 animals Treatments
HVA
Saline + saline + saline IDE + DSP4 + saline + saline IDE + DSP4 + IMI + saline Saline + saline + APO IDE + DSP4 -I- saline + APO IDE + DSP4 + IMI + APO
0.76 0.77 0.60 0.36 0.35 0.20
± 0.07 ± 0.09 _+0.10 ± 0.08* + 0.07* ± 0.09*
% control 100.0 101.3 78.9 47.3* 46.0* 26.3*
ANOVA: HVA: striatum F(5,30) = 25,98 (P < 0.001). *Newman-Keuls' test: saline + saline + APO = IDE + DSP4 + saline + A P O = IDE + DSP4 + IMI + APO < saline + saline+ saline = IDE + DSP4 + saline + saline = IDE + DSP4 + IMI + saline (P < 0.05).
Table 4 shows the effects of specific reduction in noradrenergic activity with IDE + DSP4 on the subsensitivity of dopaminergic autoreceptor produced by long-term IMI-treatment. Treatment with IDE + DSP4 neither changed the levels of HVA in the striatum nor the effect of APO in reducing the levels of this metabolite (Table 4). Finally, APO significantly reduced levels of HVA in animals pretreated with IDE + DSP4 (Table 4), in contrast to the group that received only IMI in which the effect of APO was blocked (Table 2). DISCUSSION
In preliminary experiments we established an effective schedule (300mg/kg at 4 days interval) for reducing 5HT activity by 30-70% of the control value, after single doses of PCPA (data not shown). Using this schedule, 5HIAA levels were remarkably decreased at the 1st or at the 14th day of IMI treatment, but not at 72 hr after IMI withdraw when APO effect was tested (see Table 1). The reduction of 5HIAA levels caused by pCPA during the chronic treatment could reflect some changes on the serotonergic neurons activity. In preliminary experiments the action of DSP4 on noradrenergic neurons was not selective since serotonergic neurons were also affected (data not shown). Thus, we gave DSP4 after IDE, a selective serotonin uptake blocker (Le Fur, 1983) which does not modify the effect of DSP4 on noradrenergic neurons. The treatment reduced M H P G without altering 5HIAA levels (Table 1), as had been found by Ross et al. (1976) with another 5HT uptake blocker, zimelidine before DSP4. In addition, since low doses of APO decrease DOPAC and HVA levels presumably by acting on the dopamine autoreceptors (Di Chiara et al., 1976), we utilized this drug (at 20pg/kg, s.c.) to evaluate changes in dopaminergic autoreceptor sensitivity after long-term IMI treatment. In agreement with our previous results (Scavone et al., 1986), rats chronically treated with IMI failed to exhibit a significant APO-induced decrease in striatal HVA levels. Other studies employing different methods (Serra et al., 1981) came to the same conclusion. However, in the present study we found that long-term treatment with either IDE or DMI, respectively norepinephrine and
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serotonin transport blockers, did not attenuate the effects of APO in reducing HVA levels, in agreement with other such studies of long-term treatment with specific serotonin or norepinephrine uptake blockers (Spyraki and Fibiger, 1981; Diggory and Buckett, 1984). The question arises of how chronic administration of IMI but not DMI or IDE, given alone or together, could reduce DA autoreceptor subsensitivity. These data are in agreement with the report of Klimek and Nielson (1987) that chronic administration of IMI resulted in a decreased total density of DA receptors. Why this was not found with DMI plus IDE (data not shown), deserves further investigation. The present data affirm complex interactions between IMI and central serotonergic and noradrenergic systems. When we reduced norepinephrine or serotonin inputs with (pCPA or IDE + DSP4) the effect of IMI on sensitivity to APO was lost (Tables 2 and 3). A possibility is that interactions of IMI at both noradrenergic and serotonergic systems may lead to a final reduction in dopaminergic receptor density. This is supported by the fact that citalopran (a 5HT uptake blocker) and not benzotropine (a DA uptake blocker) resulted in a decreased number of DA receptors (Klimek and Nielsen, 1987). Moreover various behavioral tests on animals have shown that by changing central beta-adrenergic activity, the action of apomorphine may be influenced (Mogilnicka et aL, 1984; Ortmann et al., 1985). Although further work is warranted to clarify the role of noradrenergic and serotonergic systems on dopaminergic activity, these results strongly support the suggestion that in absence of both noradrenergic and serotoninergic neural input, IMI failed to desensitize dopamine autoreceptors. In view of these findings we propose in this circumstance an indirect control of the striatal dopaminergic system caused by serotoninergic and noradrenergic systems. The occurrence of interactions among those systems have been shown before by others (for example Bunney and De Riemer, 1982; Agren, 1986). These relationships among major neurotransmitter systems suggest that any event modifying the activity of one neurotransmitter is likely to change the activity of any other linked system. For instance, single unit studies (de Montigny and Aghajanian, 1978; Menkes et al., 1980; Wang and Aghajanian, 1980) indicate that chronic antidepressant treatment enhances post-synaptic serotonergic and alpha-adrenergic responsivity in rat forebrain regions. Our results also may be relevant to the interpretation that the dopaminergic system is an important neural substrate for some forms of reinforcement or reward (e.g. Miller et al., 1990). If the pathophysiology of clinical depression includes an inability of normal environmental stimuli to mobilize this reinforcement system, the gradual facilitation of the activity of this dopaminergic system by antidepressant drugs (directly or by other indirect effects via NE and 5HT systems) would be consistent with their antidepressant properties (Spyraki and Fibiger, 1981). SUMMARY The responsiveness of rat striatal dopamine (DA) autoreceptors to apomorphine (APO) were assessed
after long-term treatment with an inhibitor of serotonin uptake indalpine (IDE), an inhibitor of NE uptake desipramine (DMI), or a mixed inhibitor of both 5HT and NE uptake imipramine (IMI). Moreover, the effects of long-term IMI treatment on DA autoreceptors were determined after reducing serotonin (5HT) or norepinephrine (NE) neuronal input, chronically with pCPA or DSP4. IDE or DMI longterm administration did not alter the reduction of homovanillic acid (HVA) levels induced by APO, but IMI treatment reduced the effect of APO on HVA levels, indicating subsensitivity of dopaminergic autoreceptors. Reducing 5HT or NE input during IMI treatment, respectively with p-chlorophenylalanine (pCPA) or IDE plus N(2-chlorethyl) N-ethyl-2-bromobenzylamine (DSP4), restored the effect of APO after long-term IMI administration. The results suggest that desensitization of DA autoreceptors induced by long-term IMI treatment is related to IMI effects upon noradrenergic and serotonergic systems. Acknowledgements--To Dr S. B. Ross (Astra Lakemedel AB-Sweden) for the gift of DSP-4. This work was supported by grants from FAPESP and CNPq.
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