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Effects of chronic antidepressant treatment on serotonin receptor activity in mice
European Journal of Pharmacology, 89 (1983) 69-76
69
Elsevier Biomedical Press
EFFECTS OF CHRONIC A N T I D E P R E S S A N T TREATMENT O N S E R O T O N I N RECEPTOR A C T I V I T Y I N MICE EITAN FRIEDMAN *, THOMAS B. COOPER and AIMEE DALLOB Departments of Psychiatry (EF, TBC) and Pharmacology (EF), New York University, School of Medicine, New York, NY 10016 and Rockland Research Institute (TBC), Orangeburg~NY, U.S.A.
Received 3 January 1983, accepted 27 January 1983
E. FRIEDMAN, T.B. COOPER and A. DALLOB, Effects of chronic antidepressant treatment on serotonin receptor activity in mice, European J. Pharmacol. 89 (1983) 69-76. The effect of acute and chronic treatments with conventional and atypical antidepressant drugs on serotonin receptor activity was assessed by the responsiveness of mice to the serotonin receptor agonist 5-methoxy-N,N-dimethyltryptamine. Acute treatment with 10 mg/kg of amitriptyline, imipramine, trazodone, mianserin or viloxazine reduced the head twitch response measured 1 h following a challenged dose of the serotonin agonist. Acute iprindole and desmethylimipramine, however, had no effect on the serotonergic response. Chronic treatment with the clinically effective antidepressants amitriptyline, imipramine, desmethylimipramine, iprindole, and trazodone produced an enhanced responsiveness to 5-MeODMT. The enhanced responsiveness was first observed 24 h after cessation of treatment with most drugs. The effect lasted for at least 48 h. Chronic treatment with the neuroleptic haloperidol did not result in altered responsivity to the serotonin agonist. Brain accumulation of imipramine and amitriptyline and their deaminated metabolites were measured. Brain drug and metabolite levels peaked 1 h following both acute and chronic treatments. Brain accumulations of amitriptyline and its metabolite were much greater than those of imipramine and its metabolite. This pharmacokinetic data is consistent with an early (1 h) antagonism of the 5-MeODMT response and the emergence of hightened responsiveness to 5-MeODMT after chronic treatment, when brain drug levels are reduced. These findings are also consistent with the greater inhibitory effect found after treatment with amitriptyline than with imipramine. It is concluded that enhanced serotonin neurotransmission which develops during chronic treatment with antidepressant drugs may be related to the clinical action of these drugs. Responsivity of serotonin receptors
Tricyclic antidepressants, in vivo
1. Introduction The clinical efficacy of the tricyclic antidepressants has been attributed to their effects on brain norepinephrine and serotonin transmitter systems (Davis, 1970). The early findings that these agents block neuronal m e m b r a n e uptake, was thought to be responsible for their therapeutic action and constituted important information which contributed to the formulations of hypotheses of affective illnesses. However, the advent of * To whom all correspondence should be addressed: Dr. Eitan Friedman, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016, U.S.A. 0014-2999/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
Brain drug levels
effective antidepressants which do not alter uptake, and the fact that there is a temporal delay in the therapeutic response to treatment with antidepressant drugs ( M a n n et al., 1980), have p r o m p t e d investigations of the chronic effects of these agents for a better understanding of their mechanism of action. The therapeutic antidepressant responses to imipramine and tranylcypromine have been shown to be dependent on serotonin synthesis, as evidenced b y reversal of their clinical effect by p-chlorophenylalanine, and not by a-methyl-p-tyrosine (Shopsin et al., 1975; 1976). There are, however, no consistent changes in serotonin turnover after chronic administration of antidepressants that
70 would account for their therapeutic actions (Sugrue, 1980; Svensson, 1978; Alpers and Himwich, 1972). Alterations in serotonin receptor activity following long term antidepressant treatment have been observed. Single-cell studies by Aghajanian (1981) have revealed postsynaptic supersensitivity to iontophoretically applied serotonin in various brain regions. Functional models of serotonin receptor activity have yielded similar conclusions (Friedman and Dallob, 1979; Jones, 1980; Mogilnicka and Klimek, 1979). In the present study we have extended our previous findings with imipramine and amitriptyline and assessed the effects of acute and chronic administration of a wide range of antidepressants and other drugs on serotonin receptor activity as measured by the response to the serotonin agonist 5-methoxy-N,N-dimethyltryptamine (5MeODMT).
2. Materials and methods
Male Swiss white mice weighing 25-40 g were housed 5 per cage with food and water ad lib. Naive animals which had been acclimated to the laboratory environment for at least 2 weeks prior to the experiment were used in the acute studies. In studies of chronic drug effects, mice were administered single daily intraperitoneal (i.p.) injections of various drugs, while control animals received repeated saline injections for 4 weeks.
control group. There were 10 animals per treatment group and most experiments were repeated on at least two separate groups of animals. Significance was determined by the two tailed Student's t-test.
2.2. Brain drug levels Whole brains were removed, weighed and stored at - 8 0 ° C until assayed for imipramine (IMI) or amitriptyline (AMI) and their respective demethylated metabolites demethylimipramine (DMI) and nortriptyline (NOR). These samples were analyzed by a gas chromatographic procedure utilizing a nitrogen phosphorous detector operated in the nitrogen mode (Cooper et al., 1975). The method originally described has been modified by incorporation of double internal standards (butriptyline and chlorimipramine in IMI determinations and protriptyline and chlorimipramine in AMI determinations), and for these assays standard curves were prepared using drug-free brain homogenates. The brain samples were homogenized in 0.1 N HC1 and aliquots were taken for analysis. To each tube an additional 200 mg wet brain homogenate from drug-free animals was added to ensure protein and lipid concentration equal to that of the standard curve and to guard against loss of drug which has been observed when inadequate amounts of carrier materials are present. The samples were then processed exactly as previously described (Cooper et al., 1975; 1976).
2.3. Drugs 2.1. Serotonergic receptor activity Serotonin-receptor activity was assessed (Come et al., 1963) by observing the head twitch response to 5-MeODMT. The head twitch response to i.p. injection of 5-MeODMT was measured at various times after acute or chronic drug treatment. The serotonin-receptor agonist was dissolved in 0.5% ascorbic acid and injected in doses of 5, 10 or 20 mg/kg. Injected animals were placed in individual cages and observed for the following 6 min. The number of head twitches was counted by an observer blind as to the treatment (Friedman and Dallob, 1979). Each drug tested was run with its
The compounds used in these studies were obtained from commercial sources or were gifts of the respective firms as indicated: 5-MeODMT (Sigma), methysergide (Sandoz), imipramine HCI (Ciba-Geigy), amitriptyline HC1 (MSD), desmethyhmipramine HCI (U.S. Vitamin), iprindole HC1 (Wyeth), trazodone HCI (Mead Johnson), viloxazine HC1 (Stuart), mianserin HC1 (Organon), haloperidol (McNeil), d, 1-propranolol HC1 (Ayerst), phentolamine HC1 (Ciba-Geigy), reserpine (Serpasil, Ciba), d,l-p-chloroamphetamine HC1 (Regis), trebenzomine (Parke-Davis). All drugs were dissolved in saline with the exception
71 o f h a l o p e r i d o l , w h i c h w a s d i s s o l v e d i n 0.1% t a r t a r i c acid. Drugs doses are given in term of their respect i v e salts.
3. R e s u l t s
3.1. Characterization of the response to 5 - M e O D M T in mice I n j e c t i o n o f t h e s e r o t o n i n r e c e p t o r a g o n i s t 5MeODMT induced head twitches in mice in a d o s e - r e l a t e d m a n n e r (fig. 1). T h i s r e s p o n s e is blocked by methysergide, a 5-HT receptor a n t a g o n i s t , b u t is n o t a l t e r e d b y t h e a d r e n e r g i c receptor antagonists phentolamine or propranolol ( t a b l e 1). T r e a t m e n t w i t h a g e n t s w h i c h i n d u c e a l o n g l a s t i n g d e p l e t i o n o f s e r o t o n i n , s u c h as res e r p i n e o r p - c h l o r o a m p h e t a m i n e p r o d u c e d a n inc r e a s e i n r e s p o n s i v e n e s s t o 5 - M e O D M T ( t a b l e 2).
3.2. Effect of antidepressants on the 5 - M e O D M T response The effect of acute doses of various antidepressant drugs on the head twitch response are
Effect of acute drug treatment on 5-MeODMT-induced head twitches. Groups of l0 mice each were injected with a single dose of the given drugs and challenged with l0 mg/kg of 5-MeODMT at the stated time. All drug doses were l0 mg/kg with the exception of 2 mg/kg of methyserglde. Each value represents the mean+S.E.M, of head twitches counted for 6 min. Treatment
Time of measurement
Twitches/6 min - -
Amitriptyline
1h 2h 1h 2h 1h 1h 1h 1h 1h 1h 0.5 h 20 rain 20 rain 1h
1.0 + 1.0 ~ 3.4 ___1.9 1.2 4- 0.7 b 3.4+0.7 3.2 + 1.0 6.0 _ 2.7 0.4+0.2 d 0.4 + 0.4 ¢ 0.4 + 0.2 c 2.8 + 1.2 c 1.0 + 1.0 a 5.0 4- 1.6 7.4 + 1.7 6.8 4- 1.8
Imipramine Iprindole Desmethylimipramine Trazadone Trebenzomine Mianserin Viloxazine Methyserglde Phentolamine Propranolol Saline
P < 0.025 drug treated vs. its saline control group, b p < 0.01, c P < 0.005, d P < 0.001.
s h o w n i n t a b l e 1. I m i p r a m i n e a n d a m i t r i p t y l i n e significantly reduced head twitches 1 h after drug administration, while no significant effect was seen 2 o r 48 h ( d a t a n o t s h o w n ) a f t e r d r u g a d m i n i s t r a tion. Trazodone, trebenzomine, viloxazine and mianserin also significantly reduced the response 1 h after acute administration. Iprindole and desmethylimipramine, however, had no significant
O3 W 115 t) H
Q rF I,I I
TABLE 1
18
b_ 5 O
TABLE 2
=ll= 5
10
DOSE
5-MeODMT
15
28
(mg/kg)
Fig. 1. Effect of chronic imipramine and amitriptyline treatment on the response to increasing doses of 5-MeODMT. Eighteen h after last IMI or AMI dose, animals were challenged with 5-MeODMT (5-20 mg/kg) and the head twitch response was quantitated. Each point represents the mean + S.E.M. of 10 animals. * P < 0.05, ** P < 0.001 treated vs. saline control. • • Saline, • IMI, • - . - • AMI.
Effects of brain serotonin depletion on serotonin receptor activity. Twitch response to an injection of 20 mg/kg of 5 MeODMT was measured on day 12 after a single injection of p-chloramphetamine (10 mg/kg) or 3 days post two daily single injections of reserpine (2.5 mg/kg). Values are the group mean 4-S.E.M. obtained from 10 animals each. Treatment
Response to 5-MeODMT
Saline p-CA Reserpine
I 1.2 + 2.6 22.8 + 2.8 a 28.3 + 3.3 a
a p < 0.05, treated compared to saline control group.
72
effects on the response to 5-MeODMT at 1 h after drug administration or at 2 and 3 h following acute drug injections (data not shown). 3.3. Effect of chronic drug treatment on the response to 5 - M e O D M T
Head twitch responses of animals repeatedly treated with amitriptyline or imipramine for 4 weeks, as a function of time after the last drug injection, are shown in fig. 2. At 1 and 18 h after the last dose of amitriptyline or imipramine the head twitch response was markedly reduced. Twenty-four and 48 h after the last drug dose, both imipramine and amitriptyline significantly potentiated the head twitch response 2-2.5 fold compared to saline treated control animals. Fig. 1 illustrates the effect of chronic imipramine and amitriptyline treatments on the head twitch response to increasing doses of 5-MeODMT (5, 10 and 20 mg/kg) 18 h after the last antidepressant drug injection. Amtriptyline significantly decreased the response at all 5-MeODMT doses employed, while imipramine was effective in reducing the twitch response at the higher (10 and 20 mg/kg) doses of agonist.
I#I W
TABLE 3 Dose response to 5-MeODMT-induced head twitches in mice 48 h after chronic IMI treatment. Mice were treated with single daily 10 m g / k g doses of IMI or with saline for 4 weeks. They received a single 5, l0 or 20 m g / k g dose of 5-MeODMT 48 h following the last IMI dose and head twitch was counted. Each value represents the mean twitches ± S.E.M. of l0 animals. Dose 5-MeODMT
Saline
Imipramine
5 mg/kg 10 m g / k g 20 m g / k g
4.0+0.5 9.0± 1.5 11.4+ 1.4
13.0± 2.7 ¢ 20.4± 3.5 b 15.5 ± 1.4 a
a P < 0.05; b P < 0.02; c P < 0.01.
Forty-eight h after the last dose in animals chronically treated with imipramine (10 mg/kg), the head twitch response was significantly potentiated at all 5-MeODMT doses tested (table 3). Animals injected repeatedly for 4 weeks with 10 mg/kg of the antidepressants, amitriptyline, imipramine, desmethylimipramine, iprindole, trazodone and 0.2 or 1 mg/kg haloperidol (1 mg/kg is more than twice the maximal dose for DOPAC elevation), were tested for their response to a challenge dose of 5-MeODMT (10 mg/kg) 48 h after the last drug injection. All antidepressants significantly potentiated the head twitch response, while haloperidol had no effect when compared to saline controls (table 4).
2o I
I--
TABLE 4
~ 15
Effect of chronic treatment on 5-MeODMT-induced head twitches.
U/ "r W
Z I-.-
x:
5
0 I
G
12
18
TIME
24
I
I
I
I
30
38
42
4B
(HOURS)
Fig. 2. Changes in the responsiveness to 5-MeODMT (5 m g / k g ) following the cessation of 4-week treatment with (10 m g / k g per day) imipramine or amitriptyline. Each point represents the mean obtained from l0 animals + S.E.M. Enhanced responsivity was obtained for both treatment groups at both 24 and 48 h (P < 0.05). • • Saline, • IMI, • - . - • AMI.
Treatment
Response to 5-MeODMT
Amitriptyline Imipramine Desmethylimipramine Iprindole Trazodone Haloperidol 0.2 m g / k g 1.0 m g / k g Saline
14.4 ± 2.2 18.9 ± 3.8 17.8 ± 2.7 19.9±2.1 14.5 ±2.4
P P P P P
10.8± 1.7 11.7 + 2.0 9.9 ± 1.1
n.s. n.s.
< < < < <
0.05 0.05 0.01 0.001 0.05
Groups of 10 mice each were treated for 4 weeks with 10 m g / k g of the listed antidepressants or with the indicated doses of haloperidol and challenged with 10 m g / k g 5-MeODMT 48 h after the last drug dose. Each value represents the mean + S.E.M. of head twitches counted over 6 min.
73 TABLE 5 Brain antidepressant drug and metabolite concentrations after acute and chronic injections in mice. Groups of mice were treated with 10 m g / k g of imipramine (IMI) or amitriptyline (AMI) for 4 weeks and sacrificed 1 or 2 h following the last dose. Acutely treated animals were injected with saline for 4 weeks prior to the administration of the drugs. Whole brains were analyzed for the parent compound and the respective demethylated metabolites desmethylimipramine (DMI) and nortriptyline (NOR). Concentrations are the m e a n + S.E.M. of 5 animals each. Imipramine
Acute IMI
1h 2h Amitriptyline
1h 2h
882+198 a 4 6 6 + 86
Chronic DMI 85+ 34 4 7 + 29
Acute
IMI 751+115 348+122
DMI 70+ 65+
7 19
Chronic
AMI
NOR
AMI
NOR
2100 + 525 513+ 95
842 + 181 292+ 68
1731 + 413 850+419
1 177 + 219 901 -t-367
a Drug and metabolite concentrations (ng/g).
3.4. Effect of acute and chronic drug administrations on brain drug and metabolite accumulations
Whole brain accumulations of IMI and AMI and their metabolites DMI and NOR are shown in table 5. Brain drug and metabolite levels peaked 1 h following both acute and chronic treatment regimens. Under both treatment schedules, AMI and NOR accumulated to a much larger extent than IMI and its demethylated product DMI. The absolute accumulation of NOR was 6-15 fold greater than that of DMI and constituted a far greater proportion (30-50%) of the accumulated drug and metabolite in brain than did DMI (815%). Furthermore, chronic treatment resulted in a greater (P < 0.05) accumulation of NOR at the 2 h point, while DMI accumulation did not change when compared to acutely treated animals.
4. Discussion
The present results demonstrate that 4 weeks of repeated treatment with antidepressant agents induced an enhancement in serotonin receptor activity. The response to the serotonin receptor agonist 5-MeODMT (Fuxe et al., 1972; Trulson and Jacobs, 1979) was used here to monitor and quantitate alterations in serotonin receptor responsiveness. The head twitch response can also
be induced by other serotonin agonists (Bedard and Pycock, 1977; Vetulani et al., 1980) and by 5-hydroxytryptophan (Corne et al., 1963). It is blocked by the serotonin receptor antagonists methysergide, cyproheptadine, brom-LSD and cinanserin (table 1; Corne et al., 1963; Sloviter et al., 1978; Peroutka et al., 1981; Friedman and Dallob, 1979) but not by the adrenergic receptor antagonists phentolamine or propranolol. An exaggerated head twitch response has also been attributed to changes in receptor sensitivity following experimental manipulations which either deplete serotonin or result in the degeneration of serotonin-containing neurons (table 2; Friedman et al., 1979; Nakamura and Fukushima, 1978). We have observed that acute treatment or shortly following the last of a series of chronic injections of the antidepressants amitriptyline, imipramine, trazodone and viloxazine, the response to 5-MeODMT was significantly reduced. This apparent serotonin receptor blockade was not noted after treatment with iprindole or desmethylimipramine, confirming that these agents have a low affinity for serotonin receptors (Peroutka and Snyder, 1980) and suggesting that this effect is not related to the mechanism by which these drugs elicit their antidepressant action. The antagonism of the 5-MeODMT response may depend on the brain concentrations of the antidepressant drug a n d / o r its metabohte at the time of testing. The
74 greater inhibition by amitriptyline observed following chronic treatment may be related to (1) the high brain concentrations achieved with this drug, (2) the higher affinity of the parent compound and its deaminated metabolite, nortriptyline at brain serotonin receptors (Peroutka and Snyder, 1980) and (3) the higher accumulation of its metabolite in brain. Enhanced responsiveness to 5-MeODMT was observed in animals treated chronically with clinically effective antidepressant drugs including the atypical agent iprindole and not after treatment with the neuroleptic, haloperidol. The emergence of enhanced serotonin receptor activity after cessation of the chronic antidepressant treatment is consistent with a direct antagonistic effect of amitriptyline and imipramine. In the clinical setting, these drugs are administered in divided doses, thus allowing the blockade to be overshadowed by the heightened responsivity to 5-HT. The temporal delay in the appearance of this response in the experimental situation seems to depend on the gradual reduction of brain drug concentrations, thus allowing the supersensitivity to be expressed. This effect lasts for at least 48 h thereafter. These results, which extend our earlier studies (Friedman and Dallob, 1979), were confirmed in a study of the behavioral signs of serotonin-induced sleep in chicks (Jones, 1980) and in the serotonergic response to 5-hydroxytryptophan and 5-MeODMT in rats (Mogilnicka and Klimek, 1979; Stolz and Marsden, 1982). In a recent study Blackshear and Sanders-Bush (1982) have not observed enhanced responsiveness to 5-MeODMT following chronic mianserin treatment. This finding may indicate that not all antidepressants elicit sensitization of the 5-HT twitch response. However, sensitization with mianserin, which is the antidepressant with the highest affinity for serotonin receptors, may require a longer washout period than the 96 h employed by the authors. The present findings are also in accord with single-cell studies which have demonstrated that chronic but not acute administration of antidepressants resulted in sensitization of the electrophysiological response to iontophoresed serotonin in hippocampus, amygdala, facial motor nucleus and ventral lateral geniculate (De Montigny and Aghajanian, 1978; McCall and
Aghajanian, 1979; Gallager and Bunney, 1979; Wang and Aghajanian, 1980). However, others have not found changes in cortical responsiveness to iontophoresed serotonin (Olpe and Schellenberg, 1981). Biochemical studies of serotonin receptors have shown that chronic treatment with antidepressant drugs resulted in no change in serotonin- 1 receptors detected with [ 3H]serotonin (Wirz-Justice et al., 1978; Bergstrom and Kellar, 1979; Peroutka and Snyder, 1980) and a decreased number of serotonin-2 receptors labeled with [3H]spiroperidol (Peroutka and Snyder, 1980). The head twitch response may be mediated via serotonin-2 receptors (Peroutka et al., 1981). However, the precise sequence of events which may follow interaction at the serotonin-2 recognition site is not well understood and does not provide a simple basis for understanding the enhanced physiological responsiveness to serotonin which is observed following chronic medication with antidepressant drugs. It is possible that chronic antidepressant drug treatment disengages the membrane recognition site and the subsequent physiologic output of the neuron. Alternatively, the adaptive changes in adrenergic receptors which develop during drug treatment may exert an influence on the activity of the serotonergic neurons. Chronic antidepressant treatment has been shown to enhance a-adrenergic and decrease fl-adrenergic receptor sensitivities (Banerjee et al., 1977; Menkes et al., 1980). Moreover, enhanced behavioral response to a serotonin agonist following ECT is abolished by lesions of the locus coeruleus (Green and Deakin, 1980). The role of altered serotonergic transmission in depressive illness was suggested (Coppen, 1967) and biochemical studies have produced evidence for deficits in serotonin or norepinephrine in subtypes of depressions (Maas et al., 1972; Asberg et al., 1975). In direct clinical tests of these hypotheses, we have observed that interruption of serotonin synthesis, but not of catecholamine synthesis will reverse the therapeutic effects of tranylcypromine or imipramine (Shopsin et al., 1975; 1976). Thus the present findings are consistent with the suggestion that enhanced serotonin neurotransmission is vital for the clinical action of antidepressant drugs.
75
Acknowledgement This work was supported by U.S.P.H.S. Grant MH 28350 and RSDA MH 00208 from the NIMH.
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Elsevier Biomedical Press
EFFECTS OF CHRONIC A N T I D E P R E S S A N T TREATMENT O N S E R O T O N I N RECEPTOR A C T I V I T Y I N MICE EITAN FRIEDMAN *, THOMAS B. COOPER and AIMEE DALLOB Departments of Psychiatry (EF, TBC) and Pharmacology (EF), New York University, School of Medicine, New York, NY 10016 and Rockland Research Institute (TBC), Orangeburg~NY, U.S.A.
Received 3 January 1983, accepted 27 January 1983
E. FRIEDMAN, T.B. COOPER and A. DALLOB, Effects of chronic antidepressant treatment on serotonin receptor activity in mice, European J. Pharmacol. 89 (1983) 69-76. The effect of acute and chronic treatments with conventional and atypical antidepressant drugs on serotonin receptor activity was assessed by the responsiveness of mice to the serotonin receptor agonist 5-methoxy-N,N-dimethyltryptamine. Acute treatment with 10 mg/kg of amitriptyline, imipramine, trazodone, mianserin or viloxazine reduced the head twitch response measured 1 h following a challenged dose of the serotonin agonist. Acute iprindole and desmethylimipramine, however, had no effect on the serotonergic response. Chronic treatment with the clinically effective antidepressants amitriptyline, imipramine, desmethylimipramine, iprindole, and trazodone produced an enhanced responsiveness to 5-MeODMT. The enhanced responsiveness was first observed 24 h after cessation of treatment with most drugs. The effect lasted for at least 48 h. Chronic treatment with the neuroleptic haloperidol did not result in altered responsivity to the serotonin agonist. Brain accumulation of imipramine and amitriptyline and their deaminated metabolites were measured. Brain drug and metabolite levels peaked 1 h following both acute and chronic treatments. Brain accumulations of amitriptyline and its metabolite were much greater than those of imipramine and its metabolite. This pharmacokinetic data is consistent with an early (1 h) antagonism of the 5-MeODMT response and the emergence of hightened responsiveness to 5-MeODMT after chronic treatment, when brain drug levels are reduced. These findings are also consistent with the greater inhibitory effect found after treatment with amitriptyline than with imipramine. It is concluded that enhanced serotonin neurotransmission which develops during chronic treatment with antidepressant drugs may be related to the clinical action of these drugs. Responsivity of serotonin receptors
Tricyclic antidepressants, in vivo
1. Introduction The clinical efficacy of the tricyclic antidepressants has been attributed to their effects on brain norepinephrine and serotonin transmitter systems (Davis, 1970). The early findings that these agents block neuronal m e m b r a n e uptake, was thought to be responsible for their therapeutic action and constituted important information which contributed to the formulations of hypotheses of affective illnesses. However, the advent of * To whom all correspondence should be addressed: Dr. Eitan Friedman, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016, U.S.A. 0014-2999/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
Brain drug levels
effective antidepressants which do not alter uptake, and the fact that there is a temporal delay in the therapeutic response to treatment with antidepressant drugs ( M a n n et al., 1980), have p r o m p t e d investigations of the chronic effects of these agents for a better understanding of their mechanism of action. The therapeutic antidepressant responses to imipramine and tranylcypromine have been shown to be dependent on serotonin synthesis, as evidenced b y reversal of their clinical effect by p-chlorophenylalanine, and not by a-methyl-p-tyrosine (Shopsin et al., 1975; 1976). There are, however, no consistent changes in serotonin turnover after chronic administration of antidepressants that
70 would account for their therapeutic actions (Sugrue, 1980; Svensson, 1978; Alpers and Himwich, 1972). Alterations in serotonin receptor activity following long term antidepressant treatment have been observed. Single-cell studies by Aghajanian (1981) have revealed postsynaptic supersensitivity to iontophoretically applied serotonin in various brain regions. Functional models of serotonin receptor activity have yielded similar conclusions (Friedman and Dallob, 1979; Jones, 1980; Mogilnicka and Klimek, 1979). In the present study we have extended our previous findings with imipramine and amitriptyline and assessed the effects of acute and chronic administration of a wide range of antidepressants and other drugs on serotonin receptor activity as measured by the response to the serotonin agonist 5-methoxy-N,N-dimethyltryptamine (5MeODMT).
2. Materials and methods
Male Swiss white mice weighing 25-40 g were housed 5 per cage with food and water ad lib. Naive animals which had been acclimated to the laboratory environment for at least 2 weeks prior to the experiment were used in the acute studies. In studies of chronic drug effects, mice were administered single daily intraperitoneal (i.p.) injections of various drugs, while control animals received repeated saline injections for 4 weeks.
control group. There were 10 animals per treatment group and most experiments were repeated on at least two separate groups of animals. Significance was determined by the two tailed Student's t-test.
2.2. Brain drug levels Whole brains were removed, weighed and stored at - 8 0 ° C until assayed for imipramine (IMI) or amitriptyline (AMI) and their respective demethylated metabolites demethylimipramine (DMI) and nortriptyline (NOR). These samples were analyzed by a gas chromatographic procedure utilizing a nitrogen phosphorous detector operated in the nitrogen mode (Cooper et al., 1975). The method originally described has been modified by incorporation of double internal standards (butriptyline and chlorimipramine in IMI determinations and protriptyline and chlorimipramine in AMI determinations), and for these assays standard curves were prepared using drug-free brain homogenates. The brain samples were homogenized in 0.1 N HC1 and aliquots were taken for analysis. To each tube an additional 200 mg wet brain homogenate from drug-free animals was added to ensure protein and lipid concentration equal to that of the standard curve and to guard against loss of drug which has been observed when inadequate amounts of carrier materials are present. The samples were then processed exactly as previously described (Cooper et al., 1975; 1976).
2.3. Drugs 2.1. Serotonergic receptor activity Serotonin-receptor activity was assessed (Come et al., 1963) by observing the head twitch response to 5-MeODMT. The head twitch response to i.p. injection of 5-MeODMT was measured at various times after acute or chronic drug treatment. The serotonin-receptor agonist was dissolved in 0.5% ascorbic acid and injected in doses of 5, 10 or 20 mg/kg. Injected animals were placed in individual cages and observed for the following 6 min. The number of head twitches was counted by an observer blind as to the treatment (Friedman and Dallob, 1979). Each drug tested was run with its
The compounds used in these studies were obtained from commercial sources or were gifts of the respective firms as indicated: 5-MeODMT (Sigma), methysergide (Sandoz), imipramine HCI (Ciba-Geigy), amitriptyline HC1 (MSD), desmethyhmipramine HCI (U.S. Vitamin), iprindole HC1 (Wyeth), trazodone HCI (Mead Johnson), viloxazine HC1 (Stuart), mianserin HC1 (Organon), haloperidol (McNeil), d, 1-propranolol HC1 (Ayerst), phentolamine HC1 (Ciba-Geigy), reserpine (Serpasil, Ciba), d,l-p-chloroamphetamine HC1 (Regis), trebenzomine (Parke-Davis). All drugs were dissolved in saline with the exception
71 o f h a l o p e r i d o l , w h i c h w a s d i s s o l v e d i n 0.1% t a r t a r i c acid. Drugs doses are given in term of their respect i v e salts.
3. R e s u l t s
3.1. Characterization of the response to 5 - M e O D M T in mice I n j e c t i o n o f t h e s e r o t o n i n r e c e p t o r a g o n i s t 5MeODMT induced head twitches in mice in a d o s e - r e l a t e d m a n n e r (fig. 1). T h i s r e s p o n s e is blocked by methysergide, a 5-HT receptor a n t a g o n i s t , b u t is n o t a l t e r e d b y t h e a d r e n e r g i c receptor antagonists phentolamine or propranolol ( t a b l e 1). T r e a t m e n t w i t h a g e n t s w h i c h i n d u c e a l o n g l a s t i n g d e p l e t i o n o f s e r o t o n i n , s u c h as res e r p i n e o r p - c h l o r o a m p h e t a m i n e p r o d u c e d a n inc r e a s e i n r e s p o n s i v e n e s s t o 5 - M e O D M T ( t a b l e 2).
3.2. Effect of antidepressants on the 5 - M e O D M T response The effect of acute doses of various antidepressant drugs on the head twitch response are
Effect of acute drug treatment on 5-MeODMT-induced head twitches. Groups of l0 mice each were injected with a single dose of the given drugs and challenged with l0 mg/kg of 5-MeODMT at the stated time. All drug doses were l0 mg/kg with the exception of 2 mg/kg of methyserglde. Each value represents the mean+S.E.M, of head twitches counted for 6 min. Treatment
Time of measurement
Twitches/6 min - -
Amitriptyline
1h 2h 1h 2h 1h 1h 1h 1h 1h 1h 0.5 h 20 rain 20 rain 1h
1.0 + 1.0 ~ 3.4 ___1.9 1.2 4- 0.7 b 3.4+0.7 3.2 + 1.0 6.0 _ 2.7 0.4+0.2 d 0.4 + 0.4 ¢ 0.4 + 0.2 c 2.8 + 1.2 c 1.0 + 1.0 a 5.0 4- 1.6 7.4 + 1.7 6.8 4- 1.8
Imipramine Iprindole Desmethylimipramine Trazadone Trebenzomine Mianserin Viloxazine Methyserglde Phentolamine Propranolol Saline
P < 0.025 drug treated vs. its saline control group, b p < 0.01, c P < 0.005, d P < 0.001.
s h o w n i n t a b l e 1. I m i p r a m i n e a n d a m i t r i p t y l i n e significantly reduced head twitches 1 h after drug administration, while no significant effect was seen 2 o r 48 h ( d a t a n o t s h o w n ) a f t e r d r u g a d m i n i s t r a tion. Trazodone, trebenzomine, viloxazine and mianserin also significantly reduced the response 1 h after acute administration. Iprindole and desmethylimipramine, however, had no significant
O3 W 115 t) H
Q rF I,I I
TABLE 1
18
b_ 5 O
TABLE 2
=ll= 5
10
DOSE
5-MeODMT
15
28
(mg/kg)
Fig. 1. Effect of chronic imipramine and amitriptyline treatment on the response to increasing doses of 5-MeODMT. Eighteen h after last IMI or AMI dose, animals were challenged with 5-MeODMT (5-20 mg/kg) and the head twitch response was quantitated. Each point represents the mean + S.E.M. of 10 animals. * P < 0.05, ** P < 0.001 treated vs. saline control. • • Saline, • IMI, • - . - • AMI.
Effects of brain serotonin depletion on serotonin receptor activity. Twitch response to an injection of 20 mg/kg of 5 MeODMT was measured on day 12 after a single injection of p-chloramphetamine (10 mg/kg) or 3 days post two daily single injections of reserpine (2.5 mg/kg). Values are the group mean 4-S.E.M. obtained from 10 animals each. Treatment
Response to 5-MeODMT
Saline p-CA Reserpine
I 1.2 + 2.6 22.8 + 2.8 a 28.3 + 3.3 a
a p < 0.05, treated compared to saline control group.
72
effects on the response to 5-MeODMT at 1 h after drug administration or at 2 and 3 h following acute drug injections (data not shown). 3.3. Effect of chronic drug treatment on the response to 5 - M e O D M T
Head twitch responses of animals repeatedly treated with amitriptyline or imipramine for 4 weeks, as a function of time after the last drug injection, are shown in fig. 2. At 1 and 18 h after the last dose of amitriptyline or imipramine the head twitch response was markedly reduced. Twenty-four and 48 h after the last drug dose, both imipramine and amitriptyline significantly potentiated the head twitch response 2-2.5 fold compared to saline treated control animals. Fig. 1 illustrates the effect of chronic imipramine and amitriptyline treatments on the head twitch response to increasing doses of 5-MeODMT (5, 10 and 20 mg/kg) 18 h after the last antidepressant drug injection. Amtriptyline significantly decreased the response at all 5-MeODMT doses employed, while imipramine was effective in reducing the twitch response at the higher (10 and 20 mg/kg) doses of agonist.
I#I W
TABLE 3 Dose response to 5-MeODMT-induced head twitches in mice 48 h after chronic IMI treatment. Mice were treated with single daily 10 m g / k g doses of IMI or with saline for 4 weeks. They received a single 5, l0 or 20 m g / k g dose of 5-MeODMT 48 h following the last IMI dose and head twitch was counted. Each value represents the mean twitches ± S.E.M. of l0 animals. Dose 5-MeODMT
Saline
Imipramine
5 mg/kg 10 m g / k g 20 m g / k g
4.0+0.5 9.0± 1.5 11.4+ 1.4
13.0± 2.7 ¢ 20.4± 3.5 b 15.5 ± 1.4 a
a P < 0.05; b P < 0.02; c P < 0.01.
Forty-eight h after the last dose in animals chronically treated with imipramine (10 mg/kg), the head twitch response was significantly potentiated at all 5-MeODMT doses tested (table 3). Animals injected repeatedly for 4 weeks with 10 mg/kg of the antidepressants, amitriptyline, imipramine, desmethylimipramine, iprindole, trazodone and 0.2 or 1 mg/kg haloperidol (1 mg/kg is more than twice the maximal dose for DOPAC elevation), were tested for their response to a challenge dose of 5-MeODMT (10 mg/kg) 48 h after the last drug injection. All antidepressants significantly potentiated the head twitch response, while haloperidol had no effect when compared to saline controls (table 4).
2o I
I--
TABLE 4
~ 15
Effect of chronic treatment on 5-MeODMT-induced head twitches.
U/ "r W
Z I-.-
x:
5
0 I
G
12
18
TIME
24
I
I
I
I
30
38
42
4B
(HOURS)
Fig. 2. Changes in the responsiveness to 5-MeODMT (5 m g / k g ) following the cessation of 4-week treatment with (10 m g / k g per day) imipramine or amitriptyline. Each point represents the mean obtained from l0 animals + S.E.M. Enhanced responsivity was obtained for both treatment groups at both 24 and 48 h (P < 0.05). • • Saline, • IMI, • - . - • AMI.
Treatment
Response to 5-MeODMT
Amitriptyline Imipramine Desmethylimipramine Iprindole Trazodone Haloperidol 0.2 m g / k g 1.0 m g / k g Saline
14.4 ± 2.2 18.9 ± 3.8 17.8 ± 2.7 19.9±2.1 14.5 ±2.4
P P P P P
10.8± 1.7 11.7 + 2.0 9.9 ± 1.1
n.s. n.s.
< < < < <
0.05 0.05 0.01 0.001 0.05
Groups of 10 mice each were treated for 4 weeks with 10 m g / k g of the listed antidepressants or with the indicated doses of haloperidol and challenged with 10 m g / k g 5-MeODMT 48 h after the last drug dose. Each value represents the mean + S.E.M. of head twitches counted over 6 min.
73 TABLE 5 Brain antidepressant drug and metabolite concentrations after acute and chronic injections in mice. Groups of mice were treated with 10 m g / k g of imipramine (IMI) or amitriptyline (AMI) for 4 weeks and sacrificed 1 or 2 h following the last dose. Acutely treated animals were injected with saline for 4 weeks prior to the administration of the drugs. Whole brains were analyzed for the parent compound and the respective demethylated metabolites desmethylimipramine (DMI) and nortriptyline (NOR). Concentrations are the m e a n + S.E.M. of 5 animals each. Imipramine
Acute IMI
1h 2h Amitriptyline
1h 2h
882+198 a 4 6 6 + 86
Chronic DMI 85+ 34 4 7 + 29
Acute
IMI 751+115 348+122
DMI 70+ 65+
7 19
Chronic
AMI
NOR
AMI
NOR
2100 + 525 513+ 95
842 + 181 292+ 68
1731 + 413 850+419
1 177 + 219 901 -t-367
a Drug and metabolite concentrations (ng/g).
3.4. Effect of acute and chronic drug administrations on brain drug and metabolite accumulations
Whole brain accumulations of IMI and AMI and their metabolites DMI and NOR are shown in table 5. Brain drug and metabolite levels peaked 1 h following both acute and chronic treatment regimens. Under both treatment schedules, AMI and NOR accumulated to a much larger extent than IMI and its demethylated product DMI. The absolute accumulation of NOR was 6-15 fold greater than that of DMI and constituted a far greater proportion (30-50%) of the accumulated drug and metabolite in brain than did DMI (815%). Furthermore, chronic treatment resulted in a greater (P < 0.05) accumulation of NOR at the 2 h point, while DMI accumulation did not change when compared to acutely treated animals.
4. Discussion
The present results demonstrate that 4 weeks of repeated treatment with antidepressant agents induced an enhancement in serotonin receptor activity. The response to the serotonin receptor agonist 5-MeODMT (Fuxe et al., 1972; Trulson and Jacobs, 1979) was used here to monitor and quantitate alterations in serotonin receptor responsiveness. The head twitch response can also
be induced by other serotonin agonists (Bedard and Pycock, 1977; Vetulani et al., 1980) and by 5-hydroxytryptophan (Corne et al., 1963). It is blocked by the serotonin receptor antagonists methysergide, cyproheptadine, brom-LSD and cinanserin (table 1; Corne et al., 1963; Sloviter et al., 1978; Peroutka et al., 1981; Friedman and Dallob, 1979) but not by the adrenergic receptor antagonists phentolamine or propranolol. An exaggerated head twitch response has also been attributed to changes in receptor sensitivity following experimental manipulations which either deplete serotonin or result in the degeneration of serotonin-containing neurons (table 2; Friedman et al., 1979; Nakamura and Fukushima, 1978). We have observed that acute treatment or shortly following the last of a series of chronic injections of the antidepressants amitriptyline, imipramine, trazodone and viloxazine, the response to 5-MeODMT was significantly reduced. This apparent serotonin receptor blockade was not noted after treatment with iprindole or desmethylimipramine, confirming that these agents have a low affinity for serotonin receptors (Peroutka and Snyder, 1980) and suggesting that this effect is not related to the mechanism by which these drugs elicit their antidepressant action. The antagonism of the 5-MeODMT response may depend on the brain concentrations of the antidepressant drug a n d / o r its metabohte at the time of testing. The
74 greater inhibition by amitriptyline observed following chronic treatment may be related to (1) the high brain concentrations achieved with this drug, (2) the higher affinity of the parent compound and its deaminated metabolite, nortriptyline at brain serotonin receptors (Peroutka and Snyder, 1980) and (3) the higher accumulation of its metabolite in brain. Enhanced responsiveness to 5-MeODMT was observed in animals treated chronically with clinically effective antidepressant drugs including the atypical agent iprindole and not after treatment with the neuroleptic, haloperidol. The emergence of enhanced serotonin receptor activity after cessation of the chronic antidepressant treatment is consistent with a direct antagonistic effect of amitriptyline and imipramine. In the clinical setting, these drugs are administered in divided doses, thus allowing the blockade to be overshadowed by the heightened responsivity to 5-HT. The temporal delay in the appearance of this response in the experimental situation seems to depend on the gradual reduction of brain drug concentrations, thus allowing the supersensitivity to be expressed. This effect lasts for at least 48 h thereafter. These results, which extend our earlier studies (Friedman and Dallob, 1979), were confirmed in a study of the behavioral signs of serotonin-induced sleep in chicks (Jones, 1980) and in the serotonergic response to 5-hydroxytryptophan and 5-MeODMT in rats (Mogilnicka and Klimek, 1979; Stolz and Marsden, 1982). In a recent study Blackshear and Sanders-Bush (1982) have not observed enhanced responsiveness to 5-MeODMT following chronic mianserin treatment. This finding may indicate that not all antidepressants elicit sensitization of the 5-HT twitch response. However, sensitization with mianserin, which is the antidepressant with the highest affinity for serotonin receptors, may require a longer washout period than the 96 h employed by the authors. The present findings are also in accord with single-cell studies which have demonstrated that chronic but not acute administration of antidepressants resulted in sensitization of the electrophysiological response to iontophoresed serotonin in hippocampus, amygdala, facial motor nucleus and ventral lateral geniculate (De Montigny and Aghajanian, 1978; McCall and
Aghajanian, 1979; Gallager and Bunney, 1979; Wang and Aghajanian, 1980). However, others have not found changes in cortical responsiveness to iontophoresed serotonin (Olpe and Schellenberg, 1981). Biochemical studies of serotonin receptors have shown that chronic treatment with antidepressant drugs resulted in no change in serotonin- 1 receptors detected with [ 3H]serotonin (Wirz-Justice et al., 1978; Bergstrom and Kellar, 1979; Peroutka and Snyder, 1980) and a decreased number of serotonin-2 receptors labeled with [3H]spiroperidol (Peroutka and Snyder, 1980). The head twitch response may be mediated via serotonin-2 receptors (Peroutka et al., 1981). However, the precise sequence of events which may follow interaction at the serotonin-2 recognition site is not well understood and does not provide a simple basis for understanding the enhanced physiological responsiveness to serotonin which is observed following chronic medication with antidepressant drugs. It is possible that chronic antidepressant drug treatment disengages the membrane recognition site and the subsequent physiologic output of the neuron. Alternatively, the adaptive changes in adrenergic receptors which develop during drug treatment may exert an influence on the activity of the serotonergic neurons. Chronic antidepressant treatment has been shown to enhance a-adrenergic and decrease fl-adrenergic receptor sensitivities (Banerjee et al., 1977; Menkes et al., 1980). Moreover, enhanced behavioral response to a serotonin agonist following ECT is abolished by lesions of the locus coeruleus (Green and Deakin, 1980). The role of altered serotonergic transmission in depressive illness was suggested (Coppen, 1967) and biochemical studies have produced evidence for deficits in serotonin or norepinephrine in subtypes of depressions (Maas et al., 1972; Asberg et al., 1975). In direct clinical tests of these hypotheses, we have observed that interruption of serotonin synthesis, but not of catecholamine synthesis will reverse the therapeutic effects of tranylcypromine or imipramine (Shopsin et al., 1975; 1976). Thus the present findings are consistent with the suggestion that enhanced serotonin neurotransmission is vital for the clinical action of antidepressant drugs.
75
Acknowledgement This work was supported by U.S.P.H.S. Grant MH 28350 and RSDA MH 00208 from the NIMH.
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