- Email: [email protected]
Central dopaminergic transmission is selectively increased in the limbic system of rats chronically exposed to antidepressants
European Journal of Pharmacology, 180 (1990) 31-35
31
Elsevier EJP 51288
Central dopaminergic transmission is selectively increased in the limbic system of rats chronically exposed to antidepressants G r a z i e l l a M. D e M o n t i s , P a o l a D e v o t o , G i a n Luigi G e s s a t, D o m e n i c o M e l o N , A n n a Porcella, Pierluigi S a b a , G i n o S e r r a a a n d A l e s s a n d r o T a g l i a m o n t e Institute of Pharmacology and Biochemical Pathology, and 1 Department of Neuroscience, University of Cagliari, Via Porcell 4, 09100 Cagliari, Italy
Received 30 January 1990, accepted 13 February 1990
Repeated electroconvulsive shock (ECS) exposure produced a decrease of [3H]SCH 23390 binding sites and a reduced response of adenylate cyclase activity to dopamine D-I receptor stimulation in the rat limbic area analogous to that previously observed in rats chronically treated with imipramine. These effects were completely prevented by the repeated administration of a small dose of a-methyl-p-tyrosine (a-MPT), associated with the tricyclic compound. Increased dopaminergic transmission seems to be involved in the mechanism of antidepressant action. Rats chronically treated with imipramine showed a decrease of dihydroxyphenylacetic acid (DOPAC) concentration restricted to the limbic area. Finally, both imipramine and desipramine blocked the uptake of [3H]dopamine in the limbic system with a 100-fold greater potency than that observed in the ba~sal ganglia. Antidepressants; Dopamine D 1 receptors; DOPAC (dihydroxyphenylacetic acid); Dopamine uptake
1. Introduction
Behavioral evidence indicates that chronic, but not acute, treatment with antidepressants results in the potentiation of those responses to dopamine agonists (Serra et al., 1988) that are considered to be mediated by the mesolimbic dopamine system (Spyraki and Fibiger, 1981; Wedzony and Maj, 1983). On the other hand, the behavioural responses that are attributed to the stimulation of dopamine receptors in the striatum are not modified (Spyraki and Fibiger, 1981). Behavioural changes are associated with a decrease in the number of D-1 receptors in limbic areas, whereas the decreases in the striatum are much less pro-
Correspondence to: G.M. De Montis, Institute of Pharmacology and Biochemical Pathology, University of Cagliari, Via Porcell 4, 09100 Cagliari, Italy.
nounced (Klimek and Nielsen, 1987). More recently we have shown that the imipramine-induced reduction in D - I receptors is associated with a decreased sensitivity of adenylate cyclase to dopamine, an effect which is restricted to the limbic areas (De Montis et al., 1989). Chronic antidepressant treatments are known to produce down-regulation of a-adrenoceptors, /3-adrenoceptors and 5-HT receptors (Sugrue, 1983), a change which is considered to be secondary to the enhanced stimulation of the above receptors by an increased synaptic concentration of the specific neurotransmitters (Sulser et al., 1978). These considerations prompted us to investigate if the down-regulation of dopamine D-1 receptors might also depend on their enhanced stimulation by dopamine. In agreement with such a hypothesis, the present results show that the down-regulation of D-1 receptors induced by chronic imipramine is pre-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
32 vented by a-methyl-p-tyrosine (a-MPT), and that both imipramine and desipramine potently block ([3H]dopamine uptake in the limbic areas, being almost ineffective in the caudate nucleus. Finally, our results show that chronic imipramine reduces the content of dihydroxyphenylacetic acid (DOPAC), the deaminated dopamine metabolite, in the limbic system but not in the striatum.
increase obtained with the stimulation (Wmax) were calculated by linear regression analysis of EadieHofstee plots obtained from concentration-response curves. Dopamine stimulation was always tested in the presence of I ~M sulpiride. The protein content was determined by the method of Lowry et al. (1951).
2.3. [3H]SCH 23390 binding 2. Materials and methods
2.1. Animals Experiments were carried out on male SpragueDawley rats (Charles River, Como, Italy, 175-200 g) kept with a controlled environmental temperature of 22°C, and a 12-h light-dark cycle with water and standard laboratory food available ad libitum. One group of rats was treated twice daily for 2-3 weeks with imipramine, 10 m g / k g i.p.; a second group received the combination of imipramine and a-MPT, the latter was given at the dose of 50 m g / k g i.p. once a day; the controls received saline 1 ml/kg. Electroconvulsive shock (ECS, 150 V) was delivered through ear-clip electrodes to unanesthetized rats. Only animals responding with tonic-clonic convulsions were used. The animals received one single shock daily (at 10:00 a.m.) for 8 days. Control animals were handled in the same manner, but no current was passed. The rats were decapitated 24 h after the last treatment, the striata and the limbic system (containing the olfactory tubercle, the nucleus accumbens and the septum) were dissected out and used immediately for adenylate cyclase assay or were frozen at - 8 0 °C for [3H]SCH 23390 binding or dopamine and DOPAC evaluation.
2.2, Adenylate cyclaz'e Adenylate cyclase activity was assayed in membranes prepared from striata and limbic system according to Olianas et al. (1983), The results are expressed as pmol of cAMP formed per mg of protein per min. The concentration of dopamine required to induce half-maximal activation of adenylate cyclase activity (K,n) and the maximal
The tissues were homogenized with a Polytron FT 10 for 20 s in 100 volumes (w : v) of ice-cold 50 mM Tris-HCl buffer, p H 7.4 and centrifuged for 10 min at 48000 × g. The pellet was washed once by resuspension and recentrifugation in 100 volumes of the same buffer. The final pellet was resuspended in 200 volumes of 50 mM Tris-HCl buffer, pH 7.4. The buffer contained (mM) 120 NaC1, 5 KCI, 2 CaC12 and 1 MgC12. Binding was assayed according to Billard et al. (1984), with minor modifications. Briefly, 400/~1 of membrane suspension were incubated with different concentrations of [3H]SCH 23390 (specific activity 85 Ci/mmol, Amersham) ranging from 0.098 to 3 nM, in a final volume of 0.5 ml. Non-specific binding was determined in the presence of 10/~M cisflupentixol. The reaction was stopped after 20 rain incubation at 37 o C by adding 4 ml of ice-cold Tris-HC1 buffer, pH 7.4, followed by filtration under vacuum through Whatman G F / B filters.
2.4. Determination of dopamine and DOPA C Dopamine and DOPAC were measured by HPLC (Waters) with electrochemical detection (Waters 460) according to Giorgi et al. (1987). The concentrations of dopamine and DOPAC were calculated by linear regression analysis by means of a Waters 745 Data Module Integrator.
2.5. [3H]Dopamine uptake Striata and limbic system were homogenized in ice-cold 0.32 M sucrose containing 10 mM glucose and 10 m M Tris-HC1 pH 7.4. The crude mitochondrial-synaptosomal pellet (P2) was resuspended in the same buffer and assayed according
33 TABLE 1
TABLE 2
[3H]SCH 23390 binding and dopamine (DA)-stimulated adenylate cycla.se (A.C.) in the limbic system of chronically treated rats. The data are the means+S.E, of at least six experiments performed in triplicate. Bm~ is expressed as fmol/mg protein, K a as nM, Vm~ as pmol/mg protein, K m as /tM. The basal values of A.C. activity for the different treatment groups were not significantly different from that of controls: 60.38 + 9.30 pmol/mgP per min. The values for the control group shown in the table are the means of the values obtained from controls treated with saline (for imipramine (IMI) experiments) and controls for ECS experiments treated as described in Methods, the values for the two control groups not being significantly different. Statistical analysis was done with a two-tailed Student's t-test.
[3H]Dopamine uptake inhibition in untreated brain synaptosomes. The data are the means + S.E. of at least four separate experiments. Eight drug concentrations were tested in duplicate in each experiment.
Treat-
[3H]SCH23390 binding
DA-stimulated A.C.
ment
Bmax
Vmax
None ECS IMI tx-MPT a-MPT +IMI
220.4+18.8 0.45+0.04 33.3+4.6 6.78+0.91 166.1+15.4 a 0.42+0.02 22.1+5.3 a 5.88+0.85 160.9+20.3 ~ 0.40+0.03 20.9+3.8 a 6.19+0.71 260.3+22.5 0.45+0.03 35.6+5.2 7.61+0.65 251.0+21.9
Kd
Concentration causing 50% inhibition of uptake (/tM) Drug
Limbic
Striatum
Imipramine Desipramine Chlorimipramine Mianserine Amitriptyline Mazindole Cocaine Benztropine
3.1 +0.42 0.5 + 0.04 1.1 +0.12 540 + 6.2 1.3 + 0.09 0.0053 + 0.0003 23 +0.3 0.22 +0.02
250 + 3.2 25 + 0.2 4.0 ___ 0.5 1000 + 12.4 4.9 + 0.6 0.008 + 0.0005 34 + 0.6 0.07 + 0.008
Km
0.43+0.04 37.8+__4.1 6.49+0.58
P < 0.01 with respect to control values. to S c h o e m a k e r an d N i c k o l s o n (1983). T h e [3H]d o p a m i n e c o n c e n t r a t i o n was 100 n M .
3. Results As expected f r o m previous results ( K l i m e k and Nielsen, 1987), c h r o n i c t r e a t m e n t with i m i p r a m i n e or with repeated E C S p r o d u c e d a r e d u c t i o n in [3H ] S C H 23390 b i n d i n g sites (table 1). M o r e o v e r we found that b o t h c h r o n ic i m i p r a m i n e a n d rep e a t e d E C S caused a decrease in the response of l i m b i c a d e n y l a t e cyclase to d o p a m i n e . In agreem e n t with previous results ( D e M o n t i s et al., 1989), the i m i p r a m i n e - a n d E C S - i n d u c e d reduc-
tion of D-1 r ecep t o r s in the s t r i a t u m was m u c h smaller t h a n that o b s e r v e d in the limbic system, whereas the ch an g es in a d e n y l a t e cyclase sensitivity were restricted to the l i m b i c structures. As table I shows, the changes in [ 3 H ] SCH 23390 b i n d i n g and in a d e n y l a t e cyclase i n d u c e d by i m i p r a m i n e were p r e v e n t e d by the daily treatm e n t with ct-MPT. T h e latter, given alone, at the daily dose of 50 m g / k g , showed a t e n d e n c y to increase D-1 r e c e p t o r n u m b e r . T h e increase failed to reach significance a n d bad no effect on cyclase sensitivity to d o p a m i n e . T r e a t m e n t with a - M P T r e d u c e d the d o p a m i n e , D O P A C an d n o r a d r e n a line c o n t e n t in the same area, by 16, 24 and 17%, respectively. Since these results suggested that an e n h a n c e d s y n a p t i c c o n c e n t r a t i o n of d o p a m i n e m i g h t be responsible for the d o w n - r e g u l a t i o n of the D-1 r e c e p t o r - a d e n y l a t e cyclase complex, we c o m p a r e d the i n f l u e n c e of i m i p r a m i n e on [3H]d o p a m i n e u p t a k e in the s t r i a t u m an d in the l i m b i c system.
"FABLE 3 Effect of chronic imipramine on dopamine metabolism. Dopamine and DOPAC are expressed as ng/mg protein. The values are the means + S.E. of at least three experiments performed in quadruplicate. Statistical analysis was done with a two-tailed Student's t-test. Limbic system Dopamine DOPAC
Striatum
Saline
Imipramine
Saline
Imipramine
27.07 + 7.01 5.53 + 1.04
25.25 + 5.4 3.62 + 1.80 a
104.44 + 14.19 15.07 + 2.76
101.18 + 21.57 14.84 +_4.02
a p < 0.05 with respect to control values.
34 Table 2 shows the effect of different drugs on the synaptosomal uptake of dopamine. Imipramine inhibited [3H]dopamine uptake in synaptosomes of limbic areas and striatum. However, the ICs0 for the uptake was about two orders of magnitude lower for limbic than for striatal synaptosomes. A similar potency ratio was observed with desipramine. Table 3 shows that chronic treatment with imipramine caused a reduction in D O P A C concentration in the limbic structures while no changes were produced in the striatum.
4. Discussion Long-term treatment of rats with imipramine or repeated ECS administration produced a decrease in D-1 receptor number and a reduced potency of dopamine to stimulate adenylate cyclase activity. These changes were localized almost selectively within the limbic structures and were prevented by the coadministration of a-MPT. Alone, the latter compound had no significant effect on the number of D-1 receptors or on cyclase sensitivity to dopamine. Our findings suggest that an active synthesis of catecholamines is needed for the down-regulation of the D-1 receptor-adenylate cyclase complex induced by imipramine or ECS, and that these changes are the consequence of an increased concentration of dopamine in the synaptic cleft. This conclusion is supported by the finding that imipramine, and even more so its major metabolite, desipramine, inhibited the uptake of [3H]dopamine in synaptosomal preparations from limbic areas but not from striatum. The ICs0 of the tricyclic antidepressants was within the concentration range that can be reached in brain following the systemic administration of the drugs at the dosage schedule used in the present study (Vetulani et al., 1976). The fact that chronic imipramine decreased the concentration of DOPAC, the deaminated metabolite of dopamine, which is considered to reflect the amount of dopamine recaptured by nerve terminals (Roth et al., 1976), suggests that imipramine also reduces dopamine uptake in vivo. This effect was restricted to the limbic areas. Since the limbic
areas, unlike the striatum, are rich in both noradrenergic and dopaminergic nerve terminals, the question arises as to whether dopamine can be captured by noradrenergic terminals as well by dopaminergic ones. Such a possibility might explain the potency of imipramine and desipramine to block dopamine uptake in synaptosomal preparations from limbic areas and the relative ineffectiveness in striatal synaptosomes, Accordingly, the finding that imipramine induced a reduction in the concentration of D O P A C would imply that dopamine is normally captured not only by dopaminergic but by noradrenergic nerve terminals as well. The question therefore arises as to whether dopamine in noradrenergic neurons might play the role not only of the precursor of noradrenaline but also that of cotransmitter (Fadda et al., 1984). In general, chronic imipramine administration or repeated ECS seems to produce similar changes in dopaminergic, noradrenergic and serotoninergic transmission. These changes seem to be secondary to the inhibition of monoarnine uptake and to the increased concentration of the monoamines in the synaptic cleft.
References Billard, W., V. Ruperto, G. Crosby, L.C. lorio and A. Barnett, 1984, Characterization of the binding of [3H]SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum, Life Sci. 35, 1885. De Montis, G.M., P. Devoto, G.L. Gessa, D. Meloni, A. Porcella, P. Saba, G. Serra and A. Tagliamonte, 1989, Chronic imipramine reduces [3H]SCH 23390 binding and DA-sensitive adenylate cyclase in limbic system, European J. Pharmacol. 167, 299. Fadda, F., M. Marcou, Z.L. Rossetti, E. Mosca and G.L. Gessa, 1984, Evidence for terminal autoreceptors, in: Dopaminergic Neurons of the Mesocortic',d system. Catecholamines: Neuropharmacologyand Central Nervous System Theoretical Aspects, 13-18, eds. E. Usdin, A. Carlsson, A. Dahlstrom, J. Engel (Alan R. Liss, Inc.). Giorgi, O., G. De Montis, M.L. Porceddu, S. Mele, G. Calderini, G. Toffano and G. Biggio, 1987, Developmental and age-related changes in D-1 dopamine receptors and dopamine content in the rat striatum, Dev. Brain Res. 35, 283. Klimek, V. and M. Nielsen, 1987, Chronic treatment with antidepressants decreases the number of [3H]SCH 23390
35 binding sites in rat striatum and the limbic system, European J. Pharmacol. 139, 163. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Olianas, M.C., P. Onali, N.H. Neff and E. Costa, 1983, Adenylate cyclase activity of synaptic membranes from rat striatam, Mol. Pharmacol. 23, 393. Roth, R.H., L.C. Muffin and J.R. Waiters, 1976, Central dopaminergic neurons: effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid, European J. Pharmacol. 36, 163. Schoemaker, H. and V.J. Nickolson, 1983, Dopamine uptake by rat striatal synaptosomes: time- and temperature-dependent decay and protection by dithiothreitol and dopamine, J. Neurochem. 40, 922. Serra, G., M. Collu, P. D'Aquila, L. Pani and G.L. Gessa, 1988, Behaviourai supersensitivity of D-2 dopaminergic receptors induced by chronic treatment with imipramine, Neurosci. Lett. (Supplement) 33, $180 (Abstract).
Spyraki, C. and H.C. Fibiger, 1981, Behavioural evidence for supersensitivity of postsynaptic dopamine receptors in the mesolimbic system after chronic administration of desipramine, European J. Pharmacol. 74, 195. Sugrue, M.F., 1983, Chronic antidepressant therapy and assodated changes in central monoaminergic receptor functioning, Pharmacol. Ther. 21, 1. Sulser, F., J. Vetulani and P.L. Mobley, 1978, Mode of action of antidepressant drugs, Biochem. Pharmacol. 27, 257. Vetulani, J., R.J. Stawarz, J.V. Dingell and F. Sulser, 1976, A possible common mechanism of action of antidepressant treatments, Naunyn-Schmiedeb. Arch. Pharmacol. 293, 109. Wedzony, K. and J. May, 1983, The effect of repeated treatment with imipramine on the locomotor hyperactivity induced in rats by amphetamine administered into the nucleus accumbens, 8th Congress of the Polish Pharmacological Society, Warsaw (Abstracts) p. 181.
31
Elsevier EJP 51288
Central dopaminergic transmission is selectively increased in the limbic system of rats chronically exposed to antidepressants G r a z i e l l a M. D e M o n t i s , P a o l a D e v o t o , G i a n Luigi G e s s a t, D o m e n i c o M e l o N , A n n a Porcella, Pierluigi S a b a , G i n o S e r r a a a n d A l e s s a n d r o T a g l i a m o n t e Institute of Pharmacology and Biochemical Pathology, and 1 Department of Neuroscience, University of Cagliari, Via Porcell 4, 09100 Cagliari, Italy
Received 30 January 1990, accepted 13 February 1990
Repeated electroconvulsive shock (ECS) exposure produced a decrease of [3H]SCH 23390 binding sites and a reduced response of adenylate cyclase activity to dopamine D-I receptor stimulation in the rat limbic area analogous to that previously observed in rats chronically treated with imipramine. These effects were completely prevented by the repeated administration of a small dose of a-methyl-p-tyrosine (a-MPT), associated with the tricyclic compound. Increased dopaminergic transmission seems to be involved in the mechanism of antidepressant action. Rats chronically treated with imipramine showed a decrease of dihydroxyphenylacetic acid (DOPAC) concentration restricted to the limbic area. Finally, both imipramine and desipramine blocked the uptake of [3H]dopamine in the limbic system with a 100-fold greater potency than that observed in the ba~sal ganglia. Antidepressants; Dopamine D 1 receptors; DOPAC (dihydroxyphenylacetic acid); Dopamine uptake
1. Introduction
Behavioral evidence indicates that chronic, but not acute, treatment with antidepressants results in the potentiation of those responses to dopamine agonists (Serra et al., 1988) that are considered to be mediated by the mesolimbic dopamine system (Spyraki and Fibiger, 1981; Wedzony and Maj, 1983). On the other hand, the behavioural responses that are attributed to the stimulation of dopamine receptors in the striatum are not modified (Spyraki and Fibiger, 1981). Behavioural changes are associated with a decrease in the number of D-1 receptors in limbic areas, whereas the decreases in the striatum are much less pro-
Correspondence to: G.M. De Montis, Institute of Pharmacology and Biochemical Pathology, University of Cagliari, Via Porcell 4, 09100 Cagliari, Italy.
nounced (Klimek and Nielsen, 1987). More recently we have shown that the imipramine-induced reduction in D - I receptors is associated with a decreased sensitivity of adenylate cyclase to dopamine, an effect which is restricted to the limbic areas (De Montis et al., 1989). Chronic antidepressant treatments are known to produce down-regulation of a-adrenoceptors, /3-adrenoceptors and 5-HT receptors (Sugrue, 1983), a change which is considered to be secondary to the enhanced stimulation of the above receptors by an increased synaptic concentration of the specific neurotransmitters (Sulser et al., 1978). These considerations prompted us to investigate if the down-regulation of dopamine D-1 receptors might also depend on their enhanced stimulation by dopamine. In agreement with such a hypothesis, the present results show that the down-regulation of D-1 receptors induced by chronic imipramine is pre-
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
32 vented by a-methyl-p-tyrosine (a-MPT), and that both imipramine and desipramine potently block ([3H]dopamine uptake in the limbic areas, being almost ineffective in the caudate nucleus. Finally, our results show that chronic imipramine reduces the content of dihydroxyphenylacetic acid (DOPAC), the deaminated dopamine metabolite, in the limbic system but not in the striatum.
increase obtained with the stimulation (Wmax) were calculated by linear regression analysis of EadieHofstee plots obtained from concentration-response curves. Dopamine stimulation was always tested in the presence of I ~M sulpiride. The protein content was determined by the method of Lowry et al. (1951).
2.3. [3H]SCH 23390 binding 2. Materials and methods
2.1. Animals Experiments were carried out on male SpragueDawley rats (Charles River, Como, Italy, 175-200 g) kept with a controlled environmental temperature of 22°C, and a 12-h light-dark cycle with water and standard laboratory food available ad libitum. One group of rats was treated twice daily for 2-3 weeks with imipramine, 10 m g / k g i.p.; a second group received the combination of imipramine and a-MPT, the latter was given at the dose of 50 m g / k g i.p. once a day; the controls received saline 1 ml/kg. Electroconvulsive shock (ECS, 150 V) was delivered through ear-clip electrodes to unanesthetized rats. Only animals responding with tonic-clonic convulsions were used. The animals received one single shock daily (at 10:00 a.m.) for 8 days. Control animals were handled in the same manner, but no current was passed. The rats were decapitated 24 h after the last treatment, the striata and the limbic system (containing the olfactory tubercle, the nucleus accumbens and the septum) were dissected out and used immediately for adenylate cyclase assay or were frozen at - 8 0 °C for [3H]SCH 23390 binding or dopamine and DOPAC evaluation.
2.2, Adenylate cyclaz'e Adenylate cyclase activity was assayed in membranes prepared from striata and limbic system according to Olianas et al. (1983), The results are expressed as pmol of cAMP formed per mg of protein per min. The concentration of dopamine required to induce half-maximal activation of adenylate cyclase activity (K,n) and the maximal
The tissues were homogenized with a Polytron FT 10 for 20 s in 100 volumes (w : v) of ice-cold 50 mM Tris-HCl buffer, p H 7.4 and centrifuged for 10 min at 48000 × g. The pellet was washed once by resuspension and recentrifugation in 100 volumes of the same buffer. The final pellet was resuspended in 200 volumes of 50 mM Tris-HCl buffer, pH 7.4. The buffer contained (mM) 120 NaC1, 5 KCI, 2 CaC12 and 1 MgC12. Binding was assayed according to Billard et al. (1984), with minor modifications. Briefly, 400/~1 of membrane suspension were incubated with different concentrations of [3H]SCH 23390 (specific activity 85 Ci/mmol, Amersham) ranging from 0.098 to 3 nM, in a final volume of 0.5 ml. Non-specific binding was determined in the presence of 10/~M cisflupentixol. The reaction was stopped after 20 rain incubation at 37 o C by adding 4 ml of ice-cold Tris-HC1 buffer, pH 7.4, followed by filtration under vacuum through Whatman G F / B filters.
2.4. Determination of dopamine and DOPA C Dopamine and DOPAC were measured by HPLC (Waters) with electrochemical detection (Waters 460) according to Giorgi et al. (1987). The concentrations of dopamine and DOPAC were calculated by linear regression analysis by means of a Waters 745 Data Module Integrator.
2.5. [3H]Dopamine uptake Striata and limbic system were homogenized in ice-cold 0.32 M sucrose containing 10 mM glucose and 10 m M Tris-HC1 pH 7.4. The crude mitochondrial-synaptosomal pellet (P2) was resuspended in the same buffer and assayed according
33 TABLE 1
TABLE 2
[3H]SCH 23390 binding and dopamine (DA)-stimulated adenylate cycla.se (A.C.) in the limbic system of chronically treated rats. The data are the means+S.E, of at least six experiments performed in triplicate. Bm~ is expressed as fmol/mg protein, K a as nM, Vm~ as pmol/mg protein, K m as /tM. The basal values of A.C. activity for the different treatment groups were not significantly different from that of controls: 60.38 + 9.30 pmol/mgP per min. The values for the control group shown in the table are the means of the values obtained from controls treated with saline (for imipramine (IMI) experiments) and controls for ECS experiments treated as described in Methods, the values for the two control groups not being significantly different. Statistical analysis was done with a two-tailed Student's t-test.
[3H]Dopamine uptake inhibition in untreated brain synaptosomes. The data are the means + S.E. of at least four separate experiments. Eight drug concentrations were tested in duplicate in each experiment.
Treat-
[3H]SCH23390 binding
DA-stimulated A.C.
ment
Bmax
Vmax
None ECS IMI tx-MPT a-MPT +IMI
220.4+18.8 0.45+0.04 33.3+4.6 6.78+0.91 166.1+15.4 a 0.42+0.02 22.1+5.3 a 5.88+0.85 160.9+20.3 ~ 0.40+0.03 20.9+3.8 a 6.19+0.71 260.3+22.5 0.45+0.03 35.6+5.2 7.61+0.65 251.0+21.9
Kd
Concentration causing 50% inhibition of uptake (/tM) Drug
Limbic
Striatum
Imipramine Desipramine Chlorimipramine Mianserine Amitriptyline Mazindole Cocaine Benztropine
3.1 +0.42 0.5 + 0.04 1.1 +0.12 540 + 6.2 1.3 + 0.09 0.0053 + 0.0003 23 +0.3 0.22 +0.02
250 + 3.2 25 + 0.2 4.0 ___ 0.5 1000 + 12.4 4.9 + 0.6 0.008 + 0.0005 34 + 0.6 0.07 + 0.008
Km
0.43+0.04 37.8+__4.1 6.49+0.58
P < 0.01 with respect to control values. to S c h o e m a k e r an d N i c k o l s o n (1983). T h e [3H]d o p a m i n e c o n c e n t r a t i o n was 100 n M .
3. Results As expected f r o m previous results ( K l i m e k and Nielsen, 1987), c h r o n i c t r e a t m e n t with i m i p r a m i n e or with repeated E C S p r o d u c e d a r e d u c t i o n in [3H ] S C H 23390 b i n d i n g sites (table 1). M o r e o v e r we found that b o t h c h r o n ic i m i p r a m i n e a n d rep e a t e d E C S caused a decrease in the response of l i m b i c a d e n y l a t e cyclase to d o p a m i n e . In agreem e n t with previous results ( D e M o n t i s et al., 1989), the i m i p r a m i n e - a n d E C S - i n d u c e d reduc-
tion of D-1 r ecep t o r s in the s t r i a t u m was m u c h smaller t h a n that o b s e r v e d in the limbic system, whereas the ch an g es in a d e n y l a t e cyclase sensitivity were restricted to the l i m b i c structures. As table I shows, the changes in [ 3 H ] SCH 23390 b i n d i n g and in a d e n y l a t e cyclase i n d u c e d by i m i p r a m i n e were p r e v e n t e d by the daily treatm e n t with ct-MPT. T h e latter, given alone, at the daily dose of 50 m g / k g , showed a t e n d e n c y to increase D-1 r e c e p t o r n u m b e r . T h e increase failed to reach significance a n d bad no effect on cyclase sensitivity to d o p a m i n e . T r e a t m e n t with a - M P T r e d u c e d the d o p a m i n e , D O P A C an d n o r a d r e n a line c o n t e n t in the same area, by 16, 24 and 17%, respectively. Since these results suggested that an e n h a n c e d s y n a p t i c c o n c e n t r a t i o n of d o p a m i n e m i g h t be responsible for the d o w n - r e g u l a t i o n of the D-1 r e c e p t o r - a d e n y l a t e cyclase complex, we c o m p a r e d the i n f l u e n c e of i m i p r a m i n e on [3H]d o p a m i n e u p t a k e in the s t r i a t u m an d in the l i m b i c system.
"FABLE 3 Effect of chronic imipramine on dopamine metabolism. Dopamine and DOPAC are expressed as ng/mg protein. The values are the means + S.E. of at least three experiments performed in quadruplicate. Statistical analysis was done with a two-tailed Student's t-test. Limbic system Dopamine DOPAC
Striatum
Saline
Imipramine
Saline
Imipramine
27.07 + 7.01 5.53 + 1.04
25.25 + 5.4 3.62 + 1.80 a
104.44 + 14.19 15.07 + 2.76
101.18 + 21.57 14.84 +_4.02
a p < 0.05 with respect to control values.
34 Table 2 shows the effect of different drugs on the synaptosomal uptake of dopamine. Imipramine inhibited [3H]dopamine uptake in synaptosomes of limbic areas and striatum. However, the ICs0 for the uptake was about two orders of magnitude lower for limbic than for striatal synaptosomes. A similar potency ratio was observed with desipramine. Table 3 shows that chronic treatment with imipramine caused a reduction in D O P A C concentration in the limbic structures while no changes were produced in the striatum.
4. Discussion Long-term treatment of rats with imipramine or repeated ECS administration produced a decrease in D-1 receptor number and a reduced potency of dopamine to stimulate adenylate cyclase activity. These changes were localized almost selectively within the limbic structures and were prevented by the coadministration of a-MPT. Alone, the latter compound had no significant effect on the number of D-1 receptors or on cyclase sensitivity to dopamine. Our findings suggest that an active synthesis of catecholamines is needed for the down-regulation of the D-1 receptor-adenylate cyclase complex induced by imipramine or ECS, and that these changes are the consequence of an increased concentration of dopamine in the synaptic cleft. This conclusion is supported by the finding that imipramine, and even more so its major metabolite, desipramine, inhibited the uptake of [3H]dopamine in synaptosomal preparations from limbic areas but not from striatum. The ICs0 of the tricyclic antidepressants was within the concentration range that can be reached in brain following the systemic administration of the drugs at the dosage schedule used in the present study (Vetulani et al., 1976). The fact that chronic imipramine decreased the concentration of DOPAC, the deaminated metabolite of dopamine, which is considered to reflect the amount of dopamine recaptured by nerve terminals (Roth et al., 1976), suggests that imipramine also reduces dopamine uptake in vivo. This effect was restricted to the limbic areas. Since the limbic
areas, unlike the striatum, are rich in both noradrenergic and dopaminergic nerve terminals, the question arises as to whether dopamine can be captured by noradrenergic terminals as well by dopaminergic ones. Such a possibility might explain the potency of imipramine and desipramine to block dopamine uptake in synaptosomal preparations from limbic areas and the relative ineffectiveness in striatal synaptosomes, Accordingly, the finding that imipramine induced a reduction in the concentration of D O P A C would imply that dopamine is normally captured not only by dopaminergic but by noradrenergic nerve terminals as well. The question therefore arises as to whether dopamine in noradrenergic neurons might play the role not only of the precursor of noradrenaline but also that of cotransmitter (Fadda et al., 1984). In general, chronic imipramine administration or repeated ECS seems to produce similar changes in dopaminergic, noradrenergic and serotoninergic transmission. These changes seem to be secondary to the inhibition of monoarnine uptake and to the increased concentration of the monoamines in the synaptic cleft.
References Billard, W., V. Ruperto, G. Crosby, L.C. lorio and A. Barnett, 1984, Characterization of the binding of [3H]SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum, Life Sci. 35, 1885. De Montis, G.M., P. Devoto, G.L. Gessa, D. Meloni, A. Porcella, P. Saba, G. Serra and A. Tagliamonte, 1989, Chronic imipramine reduces [3H]SCH 23390 binding and DA-sensitive adenylate cyclase in limbic system, European J. Pharmacol. 167, 299. Fadda, F., M. Marcou, Z.L. Rossetti, E. Mosca and G.L. Gessa, 1984, Evidence for terminal autoreceptors, in: Dopaminergic Neurons of the Mesocortic',d system. Catecholamines: Neuropharmacologyand Central Nervous System Theoretical Aspects, 13-18, eds. E. Usdin, A. Carlsson, A. Dahlstrom, J. Engel (Alan R. Liss, Inc.). Giorgi, O., G. De Montis, M.L. Porceddu, S. Mele, G. Calderini, G. Toffano and G. Biggio, 1987, Developmental and age-related changes in D-1 dopamine receptors and dopamine content in the rat striatum, Dev. Brain Res. 35, 283. Klimek, V. and M. Nielsen, 1987, Chronic treatment with antidepressants decreases the number of [3H]SCH 23390
35 binding sites in rat striatum and the limbic system, European J. Pharmacol. 139, 163. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Olianas, M.C., P. Onali, N.H. Neff and E. Costa, 1983, Adenylate cyclase activity of synaptic membranes from rat striatam, Mol. Pharmacol. 23, 393. Roth, R.H., L.C. Muffin and J.R. Waiters, 1976, Central dopaminergic neurons: effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid, European J. Pharmacol. 36, 163. Schoemaker, H. and V.J. Nickolson, 1983, Dopamine uptake by rat striatal synaptosomes: time- and temperature-dependent decay and protection by dithiothreitol and dopamine, J. Neurochem. 40, 922. Serra, G., M. Collu, P. D'Aquila, L. Pani and G.L. Gessa, 1988, Behaviourai supersensitivity of D-2 dopaminergic receptors induced by chronic treatment with imipramine, Neurosci. Lett. (Supplement) 33, $180 (Abstract).
Spyraki, C. and H.C. Fibiger, 1981, Behavioural evidence for supersensitivity of postsynaptic dopamine receptors in the mesolimbic system after chronic administration of desipramine, European J. Pharmacol. 74, 195. Sugrue, M.F., 1983, Chronic antidepressant therapy and assodated changes in central monoaminergic receptor functioning, Pharmacol. Ther. 21, 1. Sulser, F., J. Vetulani and P.L. Mobley, 1978, Mode of action of antidepressant drugs, Biochem. Pharmacol. 27, 257. Vetulani, J., R.J. Stawarz, J.V. Dingell and F. Sulser, 1976, A possible common mechanism of action of antidepressant treatments, Naunyn-Schmiedeb. Arch. Pharmacol. 293, 109. Wedzony, K. and J. May, 1983, The effect of repeated treatment with imipramine on the locomotor hyperactivity induced in rats by amphetamine administered into the nucleus accumbens, 8th Congress of the Polish Pharmacological Society, Warsaw (Abstracts) p. 181.