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Cholinergic-dopaminergic interactions and the mechanisms of action of antidepressants
European Journal of Pharmacology, 94 (1983) 193-201
193
Elsevier
C H O L I N E R G I C - D O P A M I N E R G I C I N T E R A C T I O N S AND T H E M E C H A N I S M S OF A C T I O N O F ANTIDEPRESSANTS MATHEW T. MARTIN-IVERSON,JEAN-FRANCOIS LECLERE and HANS C. FIBICJER * Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C. V6T 1 W5, Canada
Received 1 March 1983, revised MS received 24 June 1983, accepted 25 July 1983
M.T. MARTIN-IVERSON, J.-F. LECLERE and H.C. FIBIGER, Cholinergic-doparninergic mechanisms of action of antidepressants, European J. Pharmacol. 94 (1983) 193-201.
interactions and the
A series of experiments was performed to evaluate the mechanism(s) by which chronic administration of desipramine (DMI) facilitates the locomotor stimulant action of d-amphetamine, a response thought to be dependent on the mesolimbic dopaminergic system. Prior lesions of central noradrenergic or serotonergic neurons, induced by neonatal treatment with 6-hydroxydopamine (6-OHDA) or intraventricular injections of 5,7-dihydroxytryptamine (5,7-DHT) respectively, failed to block DMI-induced facilitation of amphetamine hypermotility. Chronic administration of DMI did not significantly influence specific [3H]spiperone binding in the striatum or the nucleus accumbens. In other experiments it was found that chronic administration of some (amitryptyline, imipramine, mianserin, iprindole) but not all (zimelidine, nomifensine, fluoxetine) antidepressants enhanced the locomotor response to d-amphetamine. The weak anticholinergic effects of the latter compounds suggest that the positive results obtained with the former drugs may be related to their anticholinergic properties. This hypothesis is consistent with the observation that chronic administration of scopolamine also increased the locomotor response to d-amphetamine. The results suggest that the facilitation by chronic DMI of amphetamine-induced locomotor activity is not mediated by primary actions of this tricylic antidepressant on central noradrenergic or serotonergic systems. In addition, the results argue against an effect of DMI on dopamine receptors as measured by [3H]spiperone binding. Instead, the facilitation of the amphetamine response by some of these antidepressant compounds may be related to their anticholinergic effects. The results are viewed as being consistent with a dopaminergic-cholinergic hypothesis of affective illness and suggest that the anticholinergic properties of some antidepressant compounds may contribute to their therapeutic effects. 5,7-Dihydroxytryp tamine Noradrenaline Anticholinergic
Dopamine receptors Locomotor activity d-Amphetamine
Antidepressants 6-Hydroxydopamine
1. I n t r o d u c t i o n
A variety of procedures that are known to be useful in the treatment of depression have been shown to enhance the function of central dopamine (DA)-containing systems. This includes chronic administration of tricyclic antidepressants (Serra et al., 1979; Chiodo and Antelman, 1980; Spyraki and Fibiger, 1981), electroconvulsive shock
* To whom all correspondence should be addressed. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
Tricyclics Acetylcholine
Dopamine Serotonin
(Green and Deakin, 1980; Serra et al., 1981a; BaUdin et al., 1982) and rapid eye movement sleep deprivation (Serra et al., 1981b). In the case of electroconvulsive shock (ECS), Green and Deakin (1980) have reported that the ECS-induced potentiation of apomorphine-induced motility is blocked by prior lesions of central noradrenergic (NA) neurons. It appears, therefore, that the effects of ECS on D A systems m a y be indirect and secondary to a primary action on noradrenergic neurons. Given the marked similarities in the effects of ECS and chronic tricyclic antidepressants
194 (TCAs) on DA agonist-induced behaviours (see, for example, Spyraki and Fibiger, 1981; Wielosz, 1981), one purpose of the present study was to investigate the role of NA and serotonin (5-HT) systems in the TCA-induced potentiation of a DA mediated behavior, amphetamine-induced locomotor activity (Roberts et al., 1975; Kelly and Iversen, 1976). Spyraki and Fibiger (1981) reported that the locomotor stimulant effects of d-amphetamine were enhanced in animals that had been treated chronically with desipramine. A second purpose of these experiments was to investigate the extent to which other antidepressant compounds share this property. Finally, we sought to determine if chronic administration of desipramine, in a regimen that is k n o w n to enhance d - a m p h e t a m i n e - i n d u c e d locomotor activity, changes the characteristics of DA receptor binding in the striatum and nucleus accumbens.
2. Materials and methods
2.1. Drug treatments 2.1.1. 6-Hydroxydopamine Pregnant Wistar rats (Woodlyn Laboratories, Ontario) were housed in a light (12 h cycle) and temperature (23°C) controlled room with free access to food and water. On the day of birth, females were culled from the litters. The remaining male neonates (6-11 per litter) received 6-hydroxydopamine HBr (100 mg base and 1.0 mg ascorbate dissolved in 1.0 ml of 0.9% saline) or ascorbatesaline, injected (1 /~l/g body weight, i.p.) every second day from Day 1 to Day 11 after birth. This procedure was chosen because similar regimens selectively lesion NA-containing axons in the forebrain that originate in the locus coeruleus (Taylor et al., 1972). At 5 weeks the mothers were removed from the cages. At 8 weeks of age the rats were housed with males from other litters (5 per cage). Each cage contained both 6-OHDA- and vehicletreated rats. The animals were grouped housed in this manner for the duration of the experiment. At 9 weeks of age, half of the 6-OHDA and vehicle groups were injected twice daily (08 : 00 and 17 : 00)
for 15 days with desipramine HC1 (DMI, 5 m g / k g i.p.) and half were injected with vehicle. Amphetamine-stimulated locomotor activity was recorded 3 days after withdrawal from DMI. There were 4 groups in this experiment (6-OHDA + Vehicle, 6-OHDA + DMI, Vehicle + Vehicle and Vehicle + DMI, n = 14 per group).
2.1.2. 5, 7-Dihydroxytryptamine Other groups of male Wistar rats (Woodlyn), weighing 200-250 g, were housed (6 per cage) under the same light and temperature conditions as above. Beginning one week after arrival, they were injected with DMI (25 m g / k g i.p.) to protect NA-containing neurons. Thirty minutes later, they received bilateral intraventricular injections of 75 /Lg (base) of 5,7-dihydroxytryptamine creatine sulphate (5,7-DHT, dissolved in 0.9% saline (10 btl) containing 0.1 m g / m l ascorbate) or vehicle into each lateral ventricle. After the intraventricular injections, the animals were singly housed for the duration of the experiment. Beginning ten days after surgery, these rats were given twice daily injections of DMI (5 m g / k g i.p.) or vehicle for 15 days, as in the previous experiment. Amphetamine-induced locomotor activity was recorded on the third day after withdrawal from chronic DMI. There were 4 groups in this experiment (5,7-DHT + Vehicle, 5,7-DHT + DMI, Vehicle + Vehicle, and Vehicle + DMI, n = 8 per group).
2.1.3. Antidepressants Male Wistar rats (Woodlyn Laboratories) weighing 200-250 g were housed 6 per cage with free access to food and water. Environmental conditions were as described above. Groups of animals (n = 7 to 12) were injected twice daily (08 : 00 and 19:00) for 15 days with one of the following compounds: desipramine HCI (5 mg/kg), nomifensine 2HC1 (5 mg/kg), amitriptyline HC1 (5 mg/kg), mianserin HC1 (10 mg/kg), zimelidine (10 mg/kg), iprindole (10 mg/kg), fluoxetine (8 mg/kg), and scopolamine hydrobromide (1 mg/kg). Animals receiving amitriptyline were given an additional injection at 13:00 because of the short half-life of this compound when injected intraperitoneally in rats (D. Coscina, personal communication). All drugs were dissolved in 0.9%
195
saline and the solutions were prepared daily. On the third day after withdrawal from the chronic drug regimen, d-amphetamine-induced locomotor activity was recorded after 1 h habituation as described below.
2.2. Locomotor activity Locomotor activity was measured in circular (61 cm) activity cages (BRS/LVE), transected by 6 red photocell beams. Photobeam interruptions were recorded on electromechanical counters, and automatically printed out every 10 min. After 1 h of habituation, which began at either 09:00 or 13 : 00 h, rats were injected with d-amphetamine sulphate (1.5 m g / k g i.p.). Locomotor activity was recorded for 2 h after the injections. Animals from each treatment group were tested at the same times to ensure a balanced design.
2.3. Amine assays Two days after behavioral testing, eight 6OHDA- and 5-vehicle-treated rats were killed by cervical dislocation, after which their brains were dissected on ice. The NA content of cerebral cortex, hippocampus and hypothalamus, and the DA content of the striatum were determined. Eight 5,7D H T - and 8 vehicle-treated rats were killed by cervical dislocation, and their brains were dissected on ice, 2-3 weeks after behavioral testing. As in the first experiment, NA content of cerebral cortex, hippocampus and hypothalamus, and DA levels in the striatum were determined. The concentration of 5-HT in these brain regions was also determined for rats treated with 5,7-DHT or vehicle. Dissections were made as previously described (Roberts et al., 1975), and amine determinations were conducted according to modifications of McGeer and McGeer (1962).
2.4. [ 3H]spiperone binding Male Wistar rats (Woodlyn) weighing 275-300 g at the beginning of the experiment received twice daily injections of desipramine HCI (5 m g / k g i.p. at 08 : 00 and 18 : 00, n = 30) or 0.9% saline vehicle (n = 30) for 14 days. On the third day of
withdrawal from this regimen the rats were killed by cervical fracture. The brains were removed from the skull and placed on a freezing microtome which was used to obtain frozen coronal sections about 0.5 mm thick. With the aid of a dissecting microscope the nucleus accumbens and the striatum were dissected from these sections which were kept cold on ice. Tissue samples were weighed and then immediately frozen at - 80°C until they were assayed for [3H]spiperone binding sites. Tissue from 6 to 7 animals was pooled and homogenized using an Ultra Turrax in 40 v / w ice-cold Tris-HC1 buffer 50 mM, p H 7.7. The homogenate was centrifuged for 15 min at 40000 × g at 4°C. The pellet was washed by resuspension and recentrifugation two times. The final pellet was suspended in Tris-HC1 50 mM p H 7.6 containing 120 mM NaC1, 5 mM KCI, 2 mM CaC12, 1 mM MgCI 2 0.1% ascorbic acid, 10 # M pargyline; 400 volumes per original wet weight of nucleus accumbens tissue and 800 volumes per original wet weight of striatal tissue. Incubation mixtures were composed of 3 ml tissue suspension; 0.1 ml [3H]spiperone (concentration 0.03 to 0.7 nM) dissolved in 10% ethanol containing R 43 448 in 100-fold excess over the [3H]spiperone concentration; and 0.1 ml 10% ethanol for total binding or domperidone solution in 1000-fold excess over the [3H]spiperone concentration to assess non-specific binding. The [3H]spiperone concentrations assayed in duplicate were 0.03, 0.05, 0.06, 0.1, 0.2, 0.3, 0.55 and 0.7 nM. 100-Fold excess R 43 448 was added to all assays to prevent binding of [3H]spiperone to 5-HT S2-receptor binding sites (Leysen et al., 1978). Incubations were run for 10 min at 37°C and then rapidly filtered through G F / B glass fibre filters and rinsed 3 times with 5 ml ice-cold buffer. Filters were transferred to counting vials, 10 ml of Instagel was added and vials were vigorously shaken for 10 min. After having kept the vials at least 6 h in the dark, the radioactivity was estimated in a liquid scintillator counter equipped with external standard and built-in computer for DPM calculation. Five different groups of control animals and DMI-treated animals were assayed independently.
196
2.5. Drugs and chemicals These compounds were generously supplied by the following companies: desipramine HC1 (CibaGeigy), iprindole (Wyeth), fluoxetine (Eli Lilly), d - a m p h e t a m i n e sulphate (Smith, Kline and French), zimelidine (Astra), amitriptyline HC1 (Merck Sharp and Dohme), mianserin HC1 (Beecham). Scopolamine HBr, 6-hydroxydopamine HBr and 5,7-dihydroxytryptamine creatine sulphate were purchased from Sigma. The drugs were dissolved in 0.9% saline. [3H]Spiperone (specific radioactivity 25.7 C i / n m o l ) was from New England Nuclear, Boston, USA. R43448, 1-(4-fluorop h e n y l m e t h y l ) - N - ( - [ 2 - ( 2 - p y r i d i n y l / e t h y l 1]-4piperidinyl)-lH-benzinadazol-2-amine, was from Janssen Pharmaceutica.
2.6. Statistics Locomotor activity was analyzed by two factor analysis of variance. Orthogonal comparisons were made where appropriate. Student's t-tests were employed for the biochemical results.
*
~¢,:x-
jo x
4
>> --
i 3
T
L) < O
2
O O O © ..J
1 I®,,,
V VEH.
E
H. DMI
-OHDA VEH.
DMI
Fig. 1. d-Amphetamine-induced(1.5 mg/kg) locomotor activity expressed as percent of habituation (see text). Rats were injected as neonates with 6-OHDA or its vehicle (VEH), and about 2 months later received repeated injections of DMI or its vehicle (VEH). Three days after cessation of the DMI or vehicle injections each animal was 161acedin an activity cage for a habituation period (1 h) after which it received d-amphetamine and locomotor activity was recorded for another 2 h. * Different from VEH+VEH, P < 0.05. ** Different from 6OHDA + VEH, P < 0.01.
3. Results
3.1. Effect of neonatal treatment with 6-OHDA on facilitation of d-amphetamine-induced locomotor activity by chronic D M I Neither neonatal pretreatment with 6 - O H D A nor chronic treatment with D M I had a significant effect on locomotor activity during habituation (F(1,52) = 1.79, P > 0.05; F(1,52) = 0.12, P > 0.05, respectively). Post-amphetamine locomotor activity was analyzed as percent of each animal's habituation activity, since the locomotor activity of control animals after amphetamine correlates significantly with activity during habituation (r = 0.482, df = 12, P < 0.05). The long-term D M I treatment significantly (F(1,52) = 12.96, P < 0.001) potentiated the m o t o r stimulant effects of d-amphetamine (fig. 1). Pretreatment with 6O H D A did not have a significant effect on amphetamine-induced locomotor activity (F(1,52) = 1.38, P > 0.05). In addition, there was no sig-
nificant interaction between 6 - O H D A treatment and D M I treatment (F(1,52)= 1.86, P > 0.05). Thus, pretreatment with 6 - O H D A did not influence facilitation of amphetamine-mediated locomotor activity induced by chronic treatment with DMI.
3.2. Effect of 6-OHDA on brain amine levels Neonatal pretreatment with 6 - O H D A resulted in depletions of N A below measurable levels in the hippocampus (all values are means _+S.E.M. in n g / g wet weight of tissue: 340 _+ 20 (control), 0 _+ 0 (6-OHDA)) and substantial depletion in the cerebral cortex (260 _+ 30 (control), 40 + 30 (6OHDA), P < 0.005), while having no effects on hypothalamic levels. DA levels in the striatum were not affected by the 6 - O H D A pretreatment.
197
These results compare favourably with prior reports (Taylor et al., 1972; Versteeg et al., 1975), and confirm that 6-OHDA administered by the present regimen selectively depletes NA terminals in the distal projection fields of the locus coeruleus.
3.3. Effect of intraventricular injections of 5, 7-DHT on facilitation of d-amphetamine-induced locomotor activity by chronic DMI Neither 5,7-DHT nor DMI treatment produced significant effects on locomotor activity during habituation (F(1,28)= 2.36 and 2.44, respectively, P > 0.05 in both cases). Nor was the interaction between 5,7-DHT and DMI during habituation significant (F interaction = 1.07, P > 0.05). Postamphetamine locomotor activity was expressed as percent of each animal's habituation activity, due to the significant correlation between the two behaviors in control animals (r = 0.82, df = 6, P < 0.01). Both 5,7-DHT and DMI produced significant main effects on the motor stimulant effects of
9
7
<
amphetamine (F(1,28) = 5.02, P < 0.05, and 23.11, P < 0.001, respectively). As can be observed in fig. 2, rats treated with 5,7-DHT exhibited higher levels of activity after amphetamine than did controls, and DMI treatment increased the stimulant effects of amphetamine in rats both with and without prior intraventricular injections of 5,7-DHT. There was no significant interaction between 5,7-DHT and DMI (F(1,28)= 1.24, P > 0.05). Thus, although central serotonin depletion enhanced the effect of amphetamine on locomotor activity, it did not influence the action of long-term treatment with DMI on this behavior.
3.4. Effect of 5, 7-DHT on brain amine levels Intraventricular injections of 5,7-DHT reduced 5-HT levels (means + S.E.M. in n g / g wet weight of tissue) in hippocampus (270 + 20 (control), 150 ___20 (5,7-DHT), P < 0.005), hypothalamus (550 ___ 60 (control), 350 + 30 (5,7-DHT), P < 0.005), cerebral cortex (280 + 30 (control), 110 + 10 (5,7DHT), P < 0.005) and striatum (290 + 20 (control), 180 + 20 (5,7-DHT), P < 0.05). Levels of NA were slightly reduced in the hippocampus (400 + 20 (control), 330 + 20 (5,7-DHT)) but were unaffected in other regions. Striatal DA levels were not altered by 5,7-DHT.
3.5. Effect of chronic administration of various antidepressants on d-amphetamine-induced locomotor activity
5
o=3 .J 1
V VEH.
E
H. DMI
5,7 DHT VEH.
DMI
Fig. 2. d-Amphetamine-induced (1.5 mg/kg) locomotor activity expressed as percent of habituation (see text). Rats were given bilateral intraventricular injections of 5,7-DHT or its vehicle (VEH), and 10 days later received repeated injections of DMI or its vehicle (VEH). Three days after cessation of the DMI or vehicle injections, each animal was placed in an activity cage for a habituation period (1 h) after which it received damphetamine and locomotor activity was recorded for another 2 h. * Different from V E H + V E H , P < 0.05. ** Different from 5,7-DHT.+ VEH, P < 0.01.
As can be observed in fig. 3, three days after withdrawal from chronic administration, some but not all of these antidepressants facilitated the locomotor response (2 h) to d-amphetamine. Thus, both of the tricyclic compounds (desipramine and amitriptyline) enhanced this response. In addition, two of the atypical antidepressants, iprindole and mianserin, resulted in an effect that was significantly greater than controls. On the other hand, chronic administration of zimelidine, fluoxetine, or nomifensine did not alter the locomotor stimulant response to d-amphetamine. Finally, chronic administration of the antimuscarinic compound, scopolamine, resulted in an enhanced response to d-amphetamine.
198 TABLE 1
lOO-.i.iii11
......
iiill
[3 H]Spiperone binding in the nucleus accumbens and striatum after withdrawal from chronic DMI. Animals received injections of desipramine HC1 (5 mg/kg, twice daily) for 15 days. Brain regions were assayed on the third day of DMI withdrawal as described in Methods. Bma~ and K D values were derived from Scatchard plots. Mean values + S.E.M. obtained from 5 independently performed experiments are presented. Scatchard plots, constructed with 8 points from assays in the concentration range of 0.03 to 0.7 nM [3H]spiperone were linear in all cases; regression lines were calculated according to the method of least squares. The mean protein content in the pooled tissues of control animals was 39.2+ 1.3 m g / g and of DMI treated animals was 38.5 + 1.8 mg/g. There are no statistically significant differences between the values from DMI treated groups and their respective control groups. Accumbens
FLU
ZlM
NOM
('lO)
(7)
Oo)
IPR
DMI
MI
AMI
(lO) (lO) (10) (10)
SCOP (12)
Fig. 3. The effect of chronic administration of various antidepressants or scopolamine on d-amphetamine-induced (1.5 mg/kg) locomotor activity. Animals were habituated to the photocell cages for 1 h prior to the injection of amphetamine. For each group total activity (2 h) after amphetamine was expressed as a percent of the control group activity. The eight treated groups are designated FLU (fluoxetine), ZIM (zimelidine), N O M (nomifensine), IPR (iprindole), DMI (desipramine), MI (mianserin), AMI (amitriptyline) and SCOP (scopolamine). The number of rats in each group is given in parentheses. Different from control group * P < 0.05, ** P < 0.025, Ordinate: percent of control activity.
3.6. Effect of chronic administration of desipramine on [3H]spiperone binding in the nucleus accumbens and striatum The results of this experiment are given in table 1. Chronic administration of DMI for 15 days did not change [3H]spiperone binding in either the nucleus accumbens or the striatum. Thus, neither the number (Bronx) nor the affinity (KD) of the dopamine receptors in these regions appeared to be influenced by the chronic drug treatment.
Controls DMI
Striatum
Bm.~ fmol/mg protein
KD nM
Bm.x fmol/mg protein
KD nM
431 +23 460+30
0.09 + 0.02 0.09+0.02
867 + 58 980+53
0.066_+0.006 0.067+0.007
4. Discussion
The present results confirm the recent finding that long-term administration of DMI can increase some of the behavioral effects of dopamine agonists (Spyraki and Fibiger, 1981). Specifically, DMI increased amphetamine-stimulated locomotor activity, a behavior thought to be mediated by the mesolimbic DA system (Kelly and Iversen, 1976; Koob et al., 1981). Depletions of NA in the projection fields of the locus coeruleus did not alter the locomotor response to d-amphetamine after withdrawal from chronic DMI. This contrasts with a report by Green and Deakin (1980) who found that lesions of NA neurons blocked the enhanced apomorphine-induced motility that occurs in ECS-treated rats. This discrepancy may reflect differences in mechanisms of action of tricyclic antidepressants and ECS, in DA agonists (d-amphetamine as compared to apomorphine), in the measures of locomotor activity, or in the brain regions depleted of NA. Facilitation of amphetamine-induced motility after disruption of central 5-HT functions has
199
been well documented (e.g. Carter and Pycock, 1978, 1979). Similar effects were obtained in the present experiments inasmuch as compared to controls the 5,7-DHT-treated rats exhibited enhanced locomotor activity in response to d-amphetamine (fig. 2). Despite this increase in baseline, 5,7-DHT-treated animals still showed the DMI-induced potentiation of the amphetamine response. It is apparent, therefore, that intact central 5-HT systems are not necessary for DMI to produce its effects on amphetamine-induced locomotor activity. The present results indicate that not all clinically effective antidepressants enhance amphetamine-induced locomotor activity after withdrawal from chronic administration. Of the compounds that were examined, both tricyclics (desipramine and amitriptyline) were effective. Spyraki and Fibiger (1981) previously demonstrated that chronic administration of another tricyclic, imipramine, also produces this effect. Thus, all of the tricyclic antidepressant compounds that have been investigated to date have been found to potentiate the locomotor stimulant effects of d-amphetamine. These results indicate that it would be worthwhile to examine if this is a property that is common to all tricyclic antidepressants. Of the so-called 'atypical' antidepressants that were examined in this model of mesolimbic DA function, both mianserin and iprindole produced significant effects. On the other hand, nomifensine, zimelidine and fluoxetine were not effective. The latter two compounds are potent and highly selective inhibitors of 5-HT uptake (Wong et al., 1976; Ogren et al., 1981). These results suggest that chronic inhibition of 5-HT uptake is not the mechanism by which some of these compounds produce their effect on the amphetamine locomotor response, a conclusion that is consistent with the results of the 5,7-DHT experiment discussed above. Inasmuch as direct effects on either noradrenergic or serotonergic systems do not appear to be the basis of the enhanced response to amphetamine observed after chronic administration of some antidepressant compounds, the question arises as to whether other common properties of these drugs can be identified. Some of these agents have weak effects on DA uptake (Friedman et al., 1977;
Randrup and Braestrup, 1977), but it is unlikely that chronic blockade of DA uptake is an important factor because nomifensine, which is a potent inhibitor of DA uptake (Hoffmann, 1977), failed to significantly enhance the amphetamine response. In addition, the absence of any changes in specific [3H]spiperone binding in the nucleus accumbens and striatum argues against a direct effect of chronic DMI on dopaminergic receptors in these regions. Previous reports have also failed to observe changes in binding measures of DA receptors after chronic antidepressants (Peroutka and Snyder, 1980; Tang et al., 1981). However, there are reports of decreased [3H]spiperone binding in the striatum (Koide and Matsushita, 1981) and a decreased binding of [3H]DA (Lee and Tang, 1982). These effects were not observed until 21 days or longer of treatment with twice the dosage of DMI used in the present experiments. The decrease in [3H]DA binding was also observed after chronic treatment with nomifensine. Thus, a relationship between alterations in DA receptors and enhancement of amphetamine-induced locomotor activity by chronic antidepressants appears unlikely. One of the most common side effects of many antidepressant compounds concerns their anticholinergic properties (Snyder and Yamamura, 1977). Hall and Ogren (1981) have compared the antimuscarinic potency of a number of antidepressants using a receptor binding technique and it is interesting that those compounds having some anticholinergic effects (amitriptyline, imipramine, desipramine, iprindole, mianserin) are those that potentiate the amphetamine response, while the least potent antimuscarinics failed to influence this response (nomifensine, zimelidine). At the time when the amphetamine response was increased in animals withdrawn from chronic DMI, Spyraki and Fibiger (1981) did not find any evidence of residual anticholinergic action. Therefore, to the extent that central anticholinergic properties of these drugs are responsible for the enhanced amphetamine response, this is most likely due to some lasting, presently unspecified consequence of chronic blockade of muscarinic receptors rather than to a continuation of the antimuscarinic action itself. In order to explore further the hypothesis
200
that chronic blockade of muscarinic receptors by some of these antidepressant compounds may contribute to the facilitation of the amphetamine response, a group of animals received repeated injections of scopolamine. On the third day of withdrawal from the chronic drug regimen these rats also showed an enhanced response to damphetamine (fig. 3), thereby supporting this hypothesis. The mesolimbic DA projection has been associated with reward or hedonic mechanisms (Koob et al., 1978; Phillips and Fibiger, 1978), as well as with amphetamine-induced motor activity. Furthermore, long-term DMI treatment may increase the rewarding properties of intracranial self-stimulation obtained from electrodes in the A10 region of the ventromedial tegmentum (Fibiger and Phillips, 1981). As 'anhedonia' is an important feature of depression, the possibility that the anticholinergic properties of these antidepressant compounds is the mechanism by which they enhance the function of the mesolimbic DA projection, raises the question as to whether the anticholinergic effects of these drugs may contribute to their therapeutic efficacy in the treatment of depression. Janowsky and collaborators (Janowsky et al., 1972; Risch et al., 1980) have proposed a cholinergic-adrenergic hypothesis of depression and mania in which depression is associated with hyperactive cholinergic function. There is evidence for increased central cholinergic tone in depressed patients (Sitaram et al., 1980; Risch, 1982). In addition, intravenous administration of the cholinesterase inhibitor, physostigmine, causes depressive effects on mood (Risch et al., 1980), leads to slowed thoughts, and decreases speech and spontaneous behavior (Davis et al., 1976). Given these findings, it seems plausible that the anticholinergic properties of antidepressant drugs may contribute to their therapeutic effects. It is noteworthy that there is considerable support for a functionally significant balance between central dopaminergic and cholinergic systems (cf. Butcher, 1977; De Souza and Palermonto, 1982) and the present results suggest that this theoretical framework may also be of value when applied to the neurobiology of depression. In this regard, it is interesting that there are anecdotal reports that
anticholinergic drugs have antidepressant effects (see Janowksy et al., 1972). Clearly, it would be worthwhile to examine this in a controlled, systematic manner. On the other hand, compounds that lack significant anticholinergic effects (e.g. nomifensine, zimelidine) must produce their clinically beneficial actions through other mechanisms. It would be naive, however, to assume that the neurobiological substrates of affect would have only one pharmacological point at which their function could be influenced, and that antidepressants must all act on the same neuronal systems.
Acknowledgements Supported by the Medical Research Council. M.T.M.-I. is supported by an MRC Studentship. J.-F. was supported by a postdoctoral Fellowship from Delegation Generale a la Recherche Scientifique et Technique (France). Excellent technical assistance was provided by Elaine Chan and Stella Atmadja. We thank Dr. J. Leysen, Janssen Pharmaceutica, for conducting the dopamine receptor binding assays and for her valuable comments on an earlier draft of this manuscript.
References Balldin, J., A.K. Granerus, G. Lindstedt, K. Modish and J. Walinder, 1982, Neuroendocrine evidence for increased responsiveness of dopamine receptors in humans following electroconvulsive therapy, Psychopharmacology 76, 371. Butcher, L.L., 1977, Nature and mechanisms of cholinergicmonoaminergic interactions in the brain, Life Sci. 21, 1207. Carter, C.J. and C.J. Pycock, 1978, Differential effects of central serotonin manipulation on hyperactive and stereotyped behaviour, Life Sci. 23, 953. Carter, C.J. and C.J. Pycock, 1979, The effects of 5,7-dihydroxytryptamine lesions of extrapyramidal and mesolimbic sites on spontaneous motor behavior, and amphetamine-induced stereotypy, Naunyn-Schmiedeb. Arch. Pharmac. 308, 51. Chiodo, L.A. and S.M. Antelman, 1980, Tricyclic antidepressants induce subsensitivity of presynaptic dopamine autoreceptors, European J. Pharmacol. 64, 203. Davis, K.L., L.E. Hollister, J. Overall, A. Johnson and K. Train, 1976, Physostigmine effects on cognitive and affect in normal subjects, Psychopharmacologia 51, 23. De Souza, H. and J. Palermonto, 1982, A quantitative study of cholinergic-dopaminergic interactions in the central nervous system, Pharmacology 24, 222. Fibiger, H.C. and A.G. Phillips, 1981, Increased intracranial
201 self-stimulation in rats after long-term administration of desipramine, Science 214, 683. Friedman, E., F. Fung and S. Gershon, 1977, Antidepressant drugs and dopamine uptake in different brain regions, European J. Pharmacol. 42, 47. Green, A.R. and J.F.W. Deakin, 1980, Brain noradrenaline depletion prevents ECS-induced enhancement of serotoninand dopamine-mediated behavior, Nature 285,232. Hall, H. and S.-O. Ogren, 1981, Effects of antidepressant drugs on different receptors in the brain, European J. Pharmacol. 70, 393. Hoffman, I., 1977, A comparative review of the pharmacology of nomifensine, Br. J. Clin. Pharmacol. 4, 69S. Janowsky, D.S., M.K. E1-Yousef, J.M. Davis and H.J. Sekerke, 1972, A cholinergic-adrenergic hypothesis of mania and depression, Lancet 2, 632. Kelly, P.H. and S.D. Iversen, 1976, Selective 6-OHDA induced destruction of mesolimbic dopamine neurons: abolition of psychostimulant induced locomotor activity in rats, European J. Pharmacol. 40, 45. Koide, T. and H. Matsushita, 1981, An enhanced sensitivity of muscarinic cholinergic receptor associated with dopaminergic receptor supersensitivity after chronic antidepressant treatment, Life Sci. 28, 1139. Koob, G.F., P.J. Fray and S.D. Iversen, 1978, Self-stimulation at the lateral hypothalamus and locus coeruleus after specific unilateral lesions of the dopamine system, Brain Res. 146, 123. Koob, G.F., L. Stinus and M. LeMoal, 1981, Hyperactivity and hypoactivity produced by lesions to the mesolimbic dopamine system, Behav. Brain Res. 3, 341. Lee, T. and S.W. Tang, 1982, Reduced presynaptic dopamine receptor density after chronic antidepressant treatment in rats, Psychiat. Res. 7, 111. Leysen, J.E., W. Gommeren and P.M. Leduron, 1978, Distinction between dopaminergic and serotonergic components of neuroleptic binding sites in limbic brain areas, Biochem. Pharmacol. 28, 447. McGeer, E.G. and P.L. McGeer, 1962, Catecholamine content in the spinal cord, Can. J. Biochem. 40, 1141. Ogren, S.-O., S.B. Ross, H. Hall, A.C. Holm and A.L. Renyi, 1981, The pharmacology of zimelidine: a 5-HT selective reuptake inhibitor, Acta Psychiat. Scand. 63, 127. Peroutka, S.J. and S.H. Snyder, 1980, Long-term antidepressant treatment decreases spiperidol labelled serotonin receptor binding, Science 215, 88. Phillips, A.G. and H.C. Fibiger, 1978, The role of dopamine in maintaining intracranial self-stimulation in the ventral tegmentum, nucleus accumbens, and medial prefrontal cortex, Can. J. Psychol. 32, 58. Randrup, A. and C. Braestrup, 1977, Uptake inhibition of biogenic amines by newer antidepressant drugs: relevance
to the dopamine hypothesis of depression, Psychopharmacology 53, 309. Risch, S.C., 1982, fl-Endorphin hypersecretion in depression: possible cholinergic mechanisms, Biol. Psychiat. 17, 1071. Risch, S.C., R.M. Cohen, D.S. Janowsky, N.H. Kalin and D.L. Murphy, 1980, Mood and behavioral effects of physostigmine on humans are accompanied by elevation in plasma fl-endorphin and cortisol, Science 209, 1545. Roberts, D.C.S., A.P. Zis and H.C. Fibiger, 1975, Ascending catecholamine pathways and amphetamine-induced locomotor activity: importance of dopamine and apparent non-involvement of norepinephrine, Brain Res. 93, 441. Serra, G., A. Argiolas, F. Fadda, M.R. Melis and G.L. Gessa, 1981a, Repeated electroconvulsive shock prevents the sedative effect of small doses of apomorphine, Psychopharmacology 73, 194. Serra, G., A. Argiolas, V. Klimek, F. Fadda and G.L. Gessa, 1979, Chronic treatment with antidepressants prevent the inhibitory effect of small doses of apomorphine on dopamine synthesis and motor activity, Life Sci. 25, 415. Serra, G., M.R. Melis, A. Argiolas, F. Fadda and G.L. Gessa, 1981b, REM sleep deprivation induces subsensitivity of dopamine receptors mediating sedation in rats, European J. Pharmacol. 72, 131. Sitaram, N., J.I. Nurnberger, E.S. Gershon and J.C. Gillin, 1980, Faster cholinergic REM sleep induction in euthymic patients with primary affective illness, Science 208, 200. Snyder, S.H. and H.I. Yamamura, 1977, Antidepressants and the muscarinic acetylcholine receptor, Arch. Gen. Psychiat. 34, 236. Spyraki, C. and H.C. Fibiger, 1981, Behavioral evidence for supersensitivity of postsynaptic dopamine receptors in the mesolimbic system after chronic administration of desipramine, European J. Pharmacol. 74, 195. Tang, S.W., P. Seeman and S. Kwan, 1981, Differential effect of chronic desipramine and amitriptyline treatment on rat brain adrenergic and serotonergic receptors, Psychiat. Res. 4, 129. Taylor, K.M., D.W.J. Clark, R. Laverty and E.L. Phelan, 1972, Specific noradrenergic neurones destroyed by 6-hydroxydopamine injection into newborn rats, Nature New Biol. 239, 247. Versteeg, D.H.G., J.M. Van Ree and A.P. Provoost, 1975, Neonatal 6-hydroxydopamine treatment: noradrenaline levels and in vitro 3H-catecholamine synthesis in discrete brain regions of adult rats, Life Sci. 15, 2127. Wielosz, M., 1981, Increased sensitivity to dopaminergic agonists after repeated electroconvulsive shock (ECS) in rats, Neuropharmacol. 20, 941. Wong, D.T., J.S. Horny, F.P. Bymaster, K.L. Houser and B.B. Molay, 1976, A selective inhibitor of serotonin uptake Lilly 110140 3-p-trifluoromethyl phenoxy-n-methyl-3-phenylpropylamine, Life Sci. 15, 471.
193
Elsevier
C H O L I N E R G I C - D O P A M I N E R G I C I N T E R A C T I O N S AND T H E M E C H A N I S M S OF A C T I O N O F ANTIDEPRESSANTS MATHEW T. MARTIN-IVERSON,JEAN-FRANCOIS LECLERE and HANS C. FIBICJER * Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C. V6T 1 W5, Canada
Received 1 March 1983, revised MS received 24 June 1983, accepted 25 July 1983
M.T. MARTIN-IVERSON, J.-F. LECLERE and H.C. FIBIGER, Cholinergic-doparninergic mechanisms of action of antidepressants, European J. Pharmacol. 94 (1983) 193-201.
interactions and the
A series of experiments was performed to evaluate the mechanism(s) by which chronic administration of desipramine (DMI) facilitates the locomotor stimulant action of d-amphetamine, a response thought to be dependent on the mesolimbic dopaminergic system. Prior lesions of central noradrenergic or serotonergic neurons, induced by neonatal treatment with 6-hydroxydopamine (6-OHDA) or intraventricular injections of 5,7-dihydroxytryptamine (5,7-DHT) respectively, failed to block DMI-induced facilitation of amphetamine hypermotility. Chronic administration of DMI did not significantly influence specific [3H]spiperone binding in the striatum or the nucleus accumbens. In other experiments it was found that chronic administration of some (amitryptyline, imipramine, mianserin, iprindole) but not all (zimelidine, nomifensine, fluoxetine) antidepressants enhanced the locomotor response to d-amphetamine. The weak anticholinergic effects of the latter compounds suggest that the positive results obtained with the former drugs may be related to their anticholinergic properties. This hypothesis is consistent with the observation that chronic administration of scopolamine also increased the locomotor response to d-amphetamine. The results suggest that the facilitation by chronic DMI of amphetamine-induced locomotor activity is not mediated by primary actions of this tricylic antidepressant on central noradrenergic or serotonergic systems. In addition, the results argue against an effect of DMI on dopamine receptors as measured by [3H]spiperone binding. Instead, the facilitation of the amphetamine response by some of these antidepressant compounds may be related to their anticholinergic effects. The results are viewed as being consistent with a dopaminergic-cholinergic hypothesis of affective illness and suggest that the anticholinergic properties of some antidepressant compounds may contribute to their therapeutic effects. 5,7-Dihydroxytryp tamine Noradrenaline Anticholinergic
Dopamine receptors Locomotor activity d-Amphetamine
Antidepressants 6-Hydroxydopamine
1. I n t r o d u c t i o n
A variety of procedures that are known to be useful in the treatment of depression have been shown to enhance the function of central dopamine (DA)-containing systems. This includes chronic administration of tricyclic antidepressants (Serra et al., 1979; Chiodo and Antelman, 1980; Spyraki and Fibiger, 1981), electroconvulsive shock
* To whom all correspondence should be addressed. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
Tricyclics Acetylcholine
Dopamine Serotonin
(Green and Deakin, 1980; Serra et al., 1981a; BaUdin et al., 1982) and rapid eye movement sleep deprivation (Serra et al., 1981b). In the case of electroconvulsive shock (ECS), Green and Deakin (1980) have reported that the ECS-induced potentiation of apomorphine-induced motility is blocked by prior lesions of central noradrenergic (NA) neurons. It appears, therefore, that the effects of ECS on D A systems m a y be indirect and secondary to a primary action on noradrenergic neurons. Given the marked similarities in the effects of ECS and chronic tricyclic antidepressants
194 (TCAs) on DA agonist-induced behaviours (see, for example, Spyraki and Fibiger, 1981; Wielosz, 1981), one purpose of the present study was to investigate the role of NA and serotonin (5-HT) systems in the TCA-induced potentiation of a DA mediated behavior, amphetamine-induced locomotor activity (Roberts et al., 1975; Kelly and Iversen, 1976). Spyraki and Fibiger (1981) reported that the locomotor stimulant effects of d-amphetamine were enhanced in animals that had been treated chronically with desipramine. A second purpose of these experiments was to investigate the extent to which other antidepressant compounds share this property. Finally, we sought to determine if chronic administration of desipramine, in a regimen that is k n o w n to enhance d - a m p h e t a m i n e - i n d u c e d locomotor activity, changes the characteristics of DA receptor binding in the striatum and nucleus accumbens.
2. Materials and methods
2.1. Drug treatments 2.1.1. 6-Hydroxydopamine Pregnant Wistar rats (Woodlyn Laboratories, Ontario) were housed in a light (12 h cycle) and temperature (23°C) controlled room with free access to food and water. On the day of birth, females were culled from the litters. The remaining male neonates (6-11 per litter) received 6-hydroxydopamine HBr (100 mg base and 1.0 mg ascorbate dissolved in 1.0 ml of 0.9% saline) or ascorbatesaline, injected (1 /~l/g body weight, i.p.) every second day from Day 1 to Day 11 after birth. This procedure was chosen because similar regimens selectively lesion NA-containing axons in the forebrain that originate in the locus coeruleus (Taylor et al., 1972). At 5 weeks the mothers were removed from the cages. At 8 weeks of age the rats were housed with males from other litters (5 per cage). Each cage contained both 6-OHDA- and vehicletreated rats. The animals were grouped housed in this manner for the duration of the experiment. At 9 weeks of age, half of the 6-OHDA and vehicle groups were injected twice daily (08 : 00 and 17 : 00)
for 15 days with desipramine HC1 (DMI, 5 m g / k g i.p.) and half were injected with vehicle. Amphetamine-stimulated locomotor activity was recorded 3 days after withdrawal from DMI. There were 4 groups in this experiment (6-OHDA + Vehicle, 6-OHDA + DMI, Vehicle + Vehicle and Vehicle + DMI, n = 14 per group).
2.1.2. 5, 7-Dihydroxytryptamine Other groups of male Wistar rats (Woodlyn), weighing 200-250 g, were housed (6 per cage) under the same light and temperature conditions as above. Beginning one week after arrival, they were injected with DMI (25 m g / k g i.p.) to protect NA-containing neurons. Thirty minutes later, they received bilateral intraventricular injections of 75 /Lg (base) of 5,7-dihydroxytryptamine creatine sulphate (5,7-DHT, dissolved in 0.9% saline (10 btl) containing 0.1 m g / m l ascorbate) or vehicle into each lateral ventricle. After the intraventricular injections, the animals were singly housed for the duration of the experiment. Beginning ten days after surgery, these rats were given twice daily injections of DMI (5 m g / k g i.p.) or vehicle for 15 days, as in the previous experiment. Amphetamine-induced locomotor activity was recorded on the third day after withdrawal from chronic DMI. There were 4 groups in this experiment (5,7-DHT + Vehicle, 5,7-DHT + DMI, Vehicle + Vehicle, and Vehicle + DMI, n = 8 per group).
2.1.3. Antidepressants Male Wistar rats (Woodlyn Laboratories) weighing 200-250 g were housed 6 per cage with free access to food and water. Environmental conditions were as described above. Groups of animals (n = 7 to 12) were injected twice daily (08 : 00 and 19:00) for 15 days with one of the following compounds: desipramine HCI (5 mg/kg), nomifensine 2HC1 (5 mg/kg), amitriptyline HC1 (5 mg/kg), mianserin HC1 (10 mg/kg), zimelidine (10 mg/kg), iprindole (10 mg/kg), fluoxetine (8 mg/kg), and scopolamine hydrobromide (1 mg/kg). Animals receiving amitriptyline were given an additional injection at 13:00 because of the short half-life of this compound when injected intraperitoneally in rats (D. Coscina, personal communication). All drugs were dissolved in 0.9%
195
saline and the solutions were prepared daily. On the third day after withdrawal from the chronic drug regimen, d-amphetamine-induced locomotor activity was recorded after 1 h habituation as described below.
2.2. Locomotor activity Locomotor activity was measured in circular (61 cm) activity cages (BRS/LVE), transected by 6 red photocell beams. Photobeam interruptions were recorded on electromechanical counters, and automatically printed out every 10 min. After 1 h of habituation, which began at either 09:00 or 13 : 00 h, rats were injected with d-amphetamine sulphate (1.5 m g / k g i.p.). Locomotor activity was recorded for 2 h after the injections. Animals from each treatment group were tested at the same times to ensure a balanced design.
2.3. Amine assays Two days after behavioral testing, eight 6OHDA- and 5-vehicle-treated rats were killed by cervical dislocation, after which their brains were dissected on ice. The NA content of cerebral cortex, hippocampus and hypothalamus, and the DA content of the striatum were determined. Eight 5,7D H T - and 8 vehicle-treated rats were killed by cervical dislocation, and their brains were dissected on ice, 2-3 weeks after behavioral testing. As in the first experiment, NA content of cerebral cortex, hippocampus and hypothalamus, and DA levels in the striatum were determined. The concentration of 5-HT in these brain regions was also determined for rats treated with 5,7-DHT or vehicle. Dissections were made as previously described (Roberts et al., 1975), and amine determinations were conducted according to modifications of McGeer and McGeer (1962).
2.4. [ 3H]spiperone binding Male Wistar rats (Woodlyn) weighing 275-300 g at the beginning of the experiment received twice daily injections of desipramine HCI (5 m g / k g i.p. at 08 : 00 and 18 : 00, n = 30) or 0.9% saline vehicle (n = 30) for 14 days. On the third day of
withdrawal from this regimen the rats were killed by cervical fracture. The brains were removed from the skull and placed on a freezing microtome which was used to obtain frozen coronal sections about 0.5 mm thick. With the aid of a dissecting microscope the nucleus accumbens and the striatum were dissected from these sections which were kept cold on ice. Tissue samples were weighed and then immediately frozen at - 80°C until they were assayed for [3H]spiperone binding sites. Tissue from 6 to 7 animals was pooled and homogenized using an Ultra Turrax in 40 v / w ice-cold Tris-HC1 buffer 50 mM, p H 7.7. The homogenate was centrifuged for 15 min at 40000 × g at 4°C. The pellet was washed by resuspension and recentrifugation two times. The final pellet was suspended in Tris-HC1 50 mM p H 7.6 containing 120 mM NaC1, 5 mM KCI, 2 mM CaC12, 1 mM MgCI 2 0.1% ascorbic acid, 10 # M pargyline; 400 volumes per original wet weight of nucleus accumbens tissue and 800 volumes per original wet weight of striatal tissue. Incubation mixtures were composed of 3 ml tissue suspension; 0.1 ml [3H]spiperone (concentration 0.03 to 0.7 nM) dissolved in 10% ethanol containing R 43 448 in 100-fold excess over the [3H]spiperone concentration; and 0.1 ml 10% ethanol for total binding or domperidone solution in 1000-fold excess over the [3H]spiperone concentration to assess non-specific binding. The [3H]spiperone concentrations assayed in duplicate were 0.03, 0.05, 0.06, 0.1, 0.2, 0.3, 0.55 and 0.7 nM. 100-Fold excess R 43 448 was added to all assays to prevent binding of [3H]spiperone to 5-HT S2-receptor binding sites (Leysen et al., 1978). Incubations were run for 10 min at 37°C and then rapidly filtered through G F / B glass fibre filters and rinsed 3 times with 5 ml ice-cold buffer. Filters were transferred to counting vials, 10 ml of Instagel was added and vials were vigorously shaken for 10 min. After having kept the vials at least 6 h in the dark, the radioactivity was estimated in a liquid scintillator counter equipped with external standard and built-in computer for DPM calculation. Five different groups of control animals and DMI-treated animals were assayed independently.
196
2.5. Drugs and chemicals These compounds were generously supplied by the following companies: desipramine HC1 (CibaGeigy), iprindole (Wyeth), fluoxetine (Eli Lilly), d - a m p h e t a m i n e sulphate (Smith, Kline and French), zimelidine (Astra), amitriptyline HC1 (Merck Sharp and Dohme), mianserin HC1 (Beecham). Scopolamine HBr, 6-hydroxydopamine HBr and 5,7-dihydroxytryptamine creatine sulphate were purchased from Sigma. The drugs were dissolved in 0.9% saline. [3H]Spiperone (specific radioactivity 25.7 C i / n m o l ) was from New England Nuclear, Boston, USA. R43448, 1-(4-fluorop h e n y l m e t h y l ) - N - ( - [ 2 - ( 2 - p y r i d i n y l / e t h y l 1]-4piperidinyl)-lH-benzinadazol-2-amine, was from Janssen Pharmaceutica.
2.6. Statistics Locomotor activity was analyzed by two factor analysis of variance. Orthogonal comparisons were made where appropriate. Student's t-tests were employed for the biochemical results.
*
~¢,:x-
jo x
4
>> --
i 3
T
L) < O
2
O O O © ..J
1 I®,,,
V VEH.
E
H. DMI
-OHDA VEH.
DMI
Fig. 1. d-Amphetamine-induced(1.5 mg/kg) locomotor activity expressed as percent of habituation (see text). Rats were injected as neonates with 6-OHDA or its vehicle (VEH), and about 2 months later received repeated injections of DMI or its vehicle (VEH). Three days after cessation of the DMI or vehicle injections each animal was 161acedin an activity cage for a habituation period (1 h) after which it received d-amphetamine and locomotor activity was recorded for another 2 h. * Different from VEH+VEH, P < 0.05. ** Different from 6OHDA + VEH, P < 0.01.
3. Results
3.1. Effect of neonatal treatment with 6-OHDA on facilitation of d-amphetamine-induced locomotor activity by chronic D M I Neither neonatal pretreatment with 6 - O H D A nor chronic treatment with D M I had a significant effect on locomotor activity during habituation (F(1,52) = 1.79, P > 0.05; F(1,52) = 0.12, P > 0.05, respectively). Post-amphetamine locomotor activity was analyzed as percent of each animal's habituation activity, since the locomotor activity of control animals after amphetamine correlates significantly with activity during habituation (r = 0.482, df = 12, P < 0.05). The long-term D M I treatment significantly (F(1,52) = 12.96, P < 0.001) potentiated the m o t o r stimulant effects of d-amphetamine (fig. 1). Pretreatment with 6O H D A did not have a significant effect on amphetamine-induced locomotor activity (F(1,52) = 1.38, P > 0.05). In addition, there was no sig-
nificant interaction between 6 - O H D A treatment and D M I treatment (F(1,52)= 1.86, P > 0.05). Thus, pretreatment with 6 - O H D A did not influence facilitation of amphetamine-mediated locomotor activity induced by chronic treatment with DMI.
3.2. Effect of 6-OHDA on brain amine levels Neonatal pretreatment with 6 - O H D A resulted in depletions of N A below measurable levels in the hippocampus (all values are means _+S.E.M. in n g / g wet weight of tissue: 340 _+ 20 (control), 0 _+ 0 (6-OHDA)) and substantial depletion in the cerebral cortex (260 _+ 30 (control), 40 + 30 (6OHDA), P < 0.005), while having no effects on hypothalamic levels. DA levels in the striatum were not affected by the 6 - O H D A pretreatment.
197
These results compare favourably with prior reports (Taylor et al., 1972; Versteeg et al., 1975), and confirm that 6-OHDA administered by the present regimen selectively depletes NA terminals in the distal projection fields of the locus coeruleus.
3.3. Effect of intraventricular injections of 5, 7-DHT on facilitation of d-amphetamine-induced locomotor activity by chronic DMI Neither 5,7-DHT nor DMI treatment produced significant effects on locomotor activity during habituation (F(1,28)= 2.36 and 2.44, respectively, P > 0.05 in both cases). Nor was the interaction between 5,7-DHT and DMI during habituation significant (F interaction = 1.07, P > 0.05). Postamphetamine locomotor activity was expressed as percent of each animal's habituation activity, due to the significant correlation between the two behaviors in control animals (r = 0.82, df = 6, P < 0.01). Both 5,7-DHT and DMI produced significant main effects on the motor stimulant effects of
9
7
<
amphetamine (F(1,28) = 5.02, P < 0.05, and 23.11, P < 0.001, respectively). As can be observed in fig. 2, rats treated with 5,7-DHT exhibited higher levels of activity after amphetamine than did controls, and DMI treatment increased the stimulant effects of amphetamine in rats both with and without prior intraventricular injections of 5,7-DHT. There was no significant interaction between 5,7-DHT and DMI (F(1,28)= 1.24, P > 0.05). Thus, although central serotonin depletion enhanced the effect of amphetamine on locomotor activity, it did not influence the action of long-term treatment with DMI on this behavior.
3.4. Effect of 5, 7-DHT on brain amine levels Intraventricular injections of 5,7-DHT reduced 5-HT levels (means + S.E.M. in n g / g wet weight of tissue) in hippocampus (270 + 20 (control), 150 ___20 (5,7-DHT), P < 0.005), hypothalamus (550 ___ 60 (control), 350 + 30 (5,7-DHT), P < 0.005), cerebral cortex (280 + 30 (control), 110 + 10 (5,7DHT), P < 0.005) and striatum (290 + 20 (control), 180 + 20 (5,7-DHT), P < 0.05). Levels of NA were slightly reduced in the hippocampus (400 + 20 (control), 330 + 20 (5,7-DHT)) but were unaffected in other regions. Striatal DA levels were not altered by 5,7-DHT.
3.5. Effect of chronic administration of various antidepressants on d-amphetamine-induced locomotor activity
5
o=3 .J 1
V VEH.
E
H. DMI
5,7 DHT VEH.
DMI
Fig. 2. d-Amphetamine-induced (1.5 mg/kg) locomotor activity expressed as percent of habituation (see text). Rats were given bilateral intraventricular injections of 5,7-DHT or its vehicle (VEH), and 10 days later received repeated injections of DMI or its vehicle (VEH). Three days after cessation of the DMI or vehicle injections, each animal was placed in an activity cage for a habituation period (1 h) after which it received damphetamine and locomotor activity was recorded for another 2 h. * Different from V E H + V E H , P < 0.05. ** Different from 5,7-DHT.+ VEH, P < 0.01.
As can be observed in fig. 3, three days after withdrawal from chronic administration, some but not all of these antidepressants facilitated the locomotor response (2 h) to d-amphetamine. Thus, both of the tricyclic compounds (desipramine and amitriptyline) enhanced this response. In addition, two of the atypical antidepressants, iprindole and mianserin, resulted in an effect that was significantly greater than controls. On the other hand, chronic administration of zimelidine, fluoxetine, or nomifensine did not alter the locomotor stimulant response to d-amphetamine. Finally, chronic administration of the antimuscarinic compound, scopolamine, resulted in an enhanced response to d-amphetamine.
198 TABLE 1
lOO-.i.iii11
......
iiill
[3 H]Spiperone binding in the nucleus accumbens and striatum after withdrawal from chronic DMI. Animals received injections of desipramine HC1 (5 mg/kg, twice daily) for 15 days. Brain regions were assayed on the third day of DMI withdrawal as described in Methods. Bma~ and K D values were derived from Scatchard plots. Mean values + S.E.M. obtained from 5 independently performed experiments are presented. Scatchard plots, constructed with 8 points from assays in the concentration range of 0.03 to 0.7 nM [3H]spiperone were linear in all cases; regression lines were calculated according to the method of least squares. The mean protein content in the pooled tissues of control animals was 39.2+ 1.3 m g / g and of DMI treated animals was 38.5 + 1.8 mg/g. There are no statistically significant differences between the values from DMI treated groups and their respective control groups. Accumbens
FLU
ZlM
NOM
('lO)
(7)
Oo)
IPR
DMI
MI
AMI
(lO) (lO) (10) (10)
SCOP (12)
Fig. 3. The effect of chronic administration of various antidepressants or scopolamine on d-amphetamine-induced (1.5 mg/kg) locomotor activity. Animals were habituated to the photocell cages for 1 h prior to the injection of amphetamine. For each group total activity (2 h) after amphetamine was expressed as a percent of the control group activity. The eight treated groups are designated FLU (fluoxetine), ZIM (zimelidine), N O M (nomifensine), IPR (iprindole), DMI (desipramine), MI (mianserin), AMI (amitriptyline) and SCOP (scopolamine). The number of rats in each group is given in parentheses. Different from control group * P < 0.05, ** P < 0.025, Ordinate: percent of control activity.
3.6. Effect of chronic administration of desipramine on [3H]spiperone binding in the nucleus accumbens and striatum The results of this experiment are given in table 1. Chronic administration of DMI for 15 days did not change [3H]spiperone binding in either the nucleus accumbens or the striatum. Thus, neither the number (Bronx) nor the affinity (KD) of the dopamine receptors in these regions appeared to be influenced by the chronic drug treatment.
Controls DMI
Striatum
Bm.~ fmol/mg protein
KD nM
Bm.x fmol/mg protein
KD nM
431 +23 460+30
0.09 + 0.02 0.09+0.02
867 + 58 980+53
0.066_+0.006 0.067+0.007
4. Discussion
The present results confirm the recent finding that long-term administration of DMI can increase some of the behavioral effects of dopamine agonists (Spyraki and Fibiger, 1981). Specifically, DMI increased amphetamine-stimulated locomotor activity, a behavior thought to be mediated by the mesolimbic DA system (Kelly and Iversen, 1976; Koob et al., 1981). Depletions of NA in the projection fields of the locus coeruleus did not alter the locomotor response to d-amphetamine after withdrawal from chronic DMI. This contrasts with a report by Green and Deakin (1980) who found that lesions of NA neurons blocked the enhanced apomorphine-induced motility that occurs in ECS-treated rats. This discrepancy may reflect differences in mechanisms of action of tricyclic antidepressants and ECS, in DA agonists (d-amphetamine as compared to apomorphine), in the measures of locomotor activity, or in the brain regions depleted of NA. Facilitation of amphetamine-induced motility after disruption of central 5-HT functions has
199
been well documented (e.g. Carter and Pycock, 1978, 1979). Similar effects were obtained in the present experiments inasmuch as compared to controls the 5,7-DHT-treated rats exhibited enhanced locomotor activity in response to d-amphetamine (fig. 2). Despite this increase in baseline, 5,7-DHT-treated animals still showed the DMI-induced potentiation of the amphetamine response. It is apparent, therefore, that intact central 5-HT systems are not necessary for DMI to produce its effects on amphetamine-induced locomotor activity. The present results indicate that not all clinically effective antidepressants enhance amphetamine-induced locomotor activity after withdrawal from chronic administration. Of the compounds that were examined, both tricyclics (desipramine and amitriptyline) were effective. Spyraki and Fibiger (1981) previously demonstrated that chronic administration of another tricyclic, imipramine, also produces this effect. Thus, all of the tricyclic antidepressant compounds that have been investigated to date have been found to potentiate the locomotor stimulant effects of d-amphetamine. These results indicate that it would be worthwhile to examine if this is a property that is common to all tricyclic antidepressants. Of the so-called 'atypical' antidepressants that were examined in this model of mesolimbic DA function, both mianserin and iprindole produced significant effects. On the other hand, nomifensine, zimelidine and fluoxetine were not effective. The latter two compounds are potent and highly selective inhibitors of 5-HT uptake (Wong et al., 1976; Ogren et al., 1981). These results suggest that chronic inhibition of 5-HT uptake is not the mechanism by which some of these compounds produce their effect on the amphetamine locomotor response, a conclusion that is consistent with the results of the 5,7-DHT experiment discussed above. Inasmuch as direct effects on either noradrenergic or serotonergic systems do not appear to be the basis of the enhanced response to amphetamine observed after chronic administration of some antidepressant compounds, the question arises as to whether other common properties of these drugs can be identified. Some of these agents have weak effects on DA uptake (Friedman et al., 1977;
Randrup and Braestrup, 1977), but it is unlikely that chronic blockade of DA uptake is an important factor because nomifensine, which is a potent inhibitor of DA uptake (Hoffmann, 1977), failed to significantly enhance the amphetamine response. In addition, the absence of any changes in specific [3H]spiperone binding in the nucleus accumbens and striatum argues against a direct effect of chronic DMI on dopaminergic receptors in these regions. Previous reports have also failed to observe changes in binding measures of DA receptors after chronic antidepressants (Peroutka and Snyder, 1980; Tang et al., 1981). However, there are reports of decreased [3H]spiperone binding in the striatum (Koide and Matsushita, 1981) and a decreased binding of [3H]DA (Lee and Tang, 1982). These effects were not observed until 21 days or longer of treatment with twice the dosage of DMI used in the present experiments. The decrease in [3H]DA binding was also observed after chronic treatment with nomifensine. Thus, a relationship between alterations in DA receptors and enhancement of amphetamine-induced locomotor activity by chronic antidepressants appears unlikely. One of the most common side effects of many antidepressant compounds concerns their anticholinergic properties (Snyder and Yamamura, 1977). Hall and Ogren (1981) have compared the antimuscarinic potency of a number of antidepressants using a receptor binding technique and it is interesting that those compounds having some anticholinergic effects (amitriptyline, imipramine, desipramine, iprindole, mianserin) are those that potentiate the amphetamine response, while the least potent antimuscarinics failed to influence this response (nomifensine, zimelidine). At the time when the amphetamine response was increased in animals withdrawn from chronic DMI, Spyraki and Fibiger (1981) did not find any evidence of residual anticholinergic action. Therefore, to the extent that central anticholinergic properties of these drugs are responsible for the enhanced amphetamine response, this is most likely due to some lasting, presently unspecified consequence of chronic blockade of muscarinic receptors rather than to a continuation of the antimuscarinic action itself. In order to explore further the hypothesis
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that chronic blockade of muscarinic receptors by some of these antidepressant compounds may contribute to the facilitation of the amphetamine response, a group of animals received repeated injections of scopolamine. On the third day of withdrawal from the chronic drug regimen these rats also showed an enhanced response to damphetamine (fig. 3), thereby supporting this hypothesis. The mesolimbic DA projection has been associated with reward or hedonic mechanisms (Koob et al., 1978; Phillips and Fibiger, 1978), as well as with amphetamine-induced motor activity. Furthermore, long-term DMI treatment may increase the rewarding properties of intracranial self-stimulation obtained from electrodes in the A10 region of the ventromedial tegmentum (Fibiger and Phillips, 1981). As 'anhedonia' is an important feature of depression, the possibility that the anticholinergic properties of these antidepressant compounds is the mechanism by which they enhance the function of the mesolimbic DA projection, raises the question as to whether the anticholinergic effects of these drugs may contribute to their therapeutic efficacy in the treatment of depression. Janowsky and collaborators (Janowsky et al., 1972; Risch et al., 1980) have proposed a cholinergic-adrenergic hypothesis of depression and mania in which depression is associated with hyperactive cholinergic function. There is evidence for increased central cholinergic tone in depressed patients (Sitaram et al., 1980; Risch, 1982). In addition, intravenous administration of the cholinesterase inhibitor, physostigmine, causes depressive effects on mood (Risch et al., 1980), leads to slowed thoughts, and decreases speech and spontaneous behavior (Davis et al., 1976). Given these findings, it seems plausible that the anticholinergic properties of antidepressant drugs may contribute to their therapeutic effects. It is noteworthy that there is considerable support for a functionally significant balance between central dopaminergic and cholinergic systems (cf. Butcher, 1977; De Souza and Palermonto, 1982) and the present results suggest that this theoretical framework may also be of value when applied to the neurobiology of depression. In this regard, it is interesting that there are anecdotal reports that
anticholinergic drugs have antidepressant effects (see Janowksy et al., 1972). Clearly, it would be worthwhile to examine this in a controlled, systematic manner. On the other hand, compounds that lack significant anticholinergic effects (e.g. nomifensine, zimelidine) must produce their clinically beneficial actions through other mechanisms. It would be naive, however, to assume that the neurobiological substrates of affect would have only one pharmacological point at which their function could be influenced, and that antidepressants must all act on the same neuronal systems.
Acknowledgements Supported by the Medical Research Council. M.T.M.-I. is supported by an MRC Studentship. J.-F. was supported by a postdoctoral Fellowship from Delegation Generale a la Recherche Scientifique et Technique (France). Excellent technical assistance was provided by Elaine Chan and Stella Atmadja. We thank Dr. J. Leysen, Janssen Pharmaceutica, for conducting the dopamine receptor binding assays and for her valuable comments on an earlier draft of this manuscript.
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