Phenoxybenzamine but not cyproheptadine abol- ished the enhanced activity of the flexor reflex. After chronic administration of IMI high levels of desipramine ...
Naunyn-Schmiedeberg's
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Naunyn-Schmiedeberg's Arch Pharmacol (1983) 322: 256- 260
Pharmacology
9 Springer-Verlag1983
Chronic Treatment with Imipramine: Further Functional Evidence for the Enhanced Noradrenergic Transmission in Flexor Reflex Activity J. Maj, Z. G6rka, M. Melzacka, A. Rawt6w, and A. Pilc Institute of Pharmacology, Polish Academy of Sciences, Smyrna 12, PL-31-343 Krak6w, Poland
Summary. The chronic administration of imipramine (IMI; 10 mg/kg orally, twice daily for 14 days) enhanced the flexor reflex of the hind limb in the spinal rat. This effect was maintained for at least 72 h after termination of drug administration. Phenoxybenzamine but not cyproheptadine abolished the enhanced activity of the flexor reflex. After chronic administration of IMI high levels of desipramine (DMI) were found in the spinal cord, whereas IMI was not detectable there. No correlation was found between the levels of DMI in the spinal cord and the enhancement of the flexor reflex amplitude. A single i.v. dose of DMI facilitated the flexor reflex for a short period of time. In rats treated chronically with IMI, the binding of 3H-prazosin, a Iigand of cqadrenoceptors, to spinal cord tissue was increased. The present results are a further argument for the previously advanced hypothesis that chronic administration of antidepressant drugs leads to an enhanced noradrenergic transmission, probably by increasing the number of cqadrenoceptors. Key words: Chronic imipramine - Flexor reflex Desipramine level - 3H-prazosin binding
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Introduction
Prolonged, but not acute, treatment with various typical and atypical antidepressant drugs enhances the aggressiveness induced by apomorphine or clonidine (Maj et al. 1979, 1980, 1981, 1982b, 1983). In mice chronically treated with antidepressant drugs, of various pharmacological profiles, reserpine causes an immediate increase in locomotor activity in the amine release phase which is not observed in animals treated with a single dose of antidepressant drugs (Maj et al. 1982c). All these effects might be ascribed to an ~l-adrenergic mechanism which probably consists in an increase in the number of cq-adrenoceptors. Seeking further arguments for this hypothesis we now investigated the effect of chronic imipramine (IMI) administration on: (1) the hind limb flexor reflex in the spinal rat, which is a good model for evaluating drug effects on the noradrenergic system of the spinal cord (And6n 1970) and (2) the binding of 3H-prazosin, which is as yet the most specific ligand of el-adrenoceptors (see: Discussion) in the spinal cord. Besides that, the levels of IMI and desipramine (DMI) in the spinal cord after chronic administration of IMI have been assayed. Since we found high D M I concentrations Send offprint requests to J. Maj at the above address
(and, to our surprise, no IMI) in the spinal cord of rats treated chronically with IMI, we determined the effect of a single dose of DMI on the flexor reflex as well as its concentration in the spinal cord after such administration. Methods
The experiments were performed on male Wistar rats weighing 180-250g. The animals had free access to food (Murigran) and water. The animals received IMI in a dose of 10 mg/kg (dissolved in double-distilled water) orally, twice daily (at 9 a.m. and 6 p.m.) for 14 days. Control rats were treated with the solvent. The Flexor Reflex of the Hind Limb in the Spinal Rat. The detailed description of the method was given previously (Maj et al. 1976). Rats were spinalized under light ether anaesthesia at the levels of Th 8 - 1 0 11/2 h prior to recording the reflex activity. Reflex twitches of the tibialis anterior muscle were elicited by electrical stimuli (15 V, 4Hz, lOOms) applied transcutaneously for 1 s every minute to the ipsilateral hind foot. To record twitches of the tibialis anterior muscle its distal tendon was attached to an isotonic transducer connected to a pen recorder (TZ21S, Laboratorni Pristroje, Praha, CSSR). To avoid disturbance by leg movements, the leg was fixed at the ankle and knee joints by means of specially designed holders. After 30 rain of initial recording of the reflex activity (i.e. at the 2nd h following spinalization), either the drugs under study (phenoxybenzamine, cyproheptadine, DMI) were injected or the evaluation of the reflex amplitude was begun (see below). In rats treated chronically with IMI or the solvent, the mean amplitude of ten consecutive twitches was measured 2, 24, 48 and 72h after the last dose of IMI and compared (unpaired Student's t-test) with the mean amplitude of ten twitches recorded in control rats 2 h after the last administration of the solvent. The control group consisted of 8 rats and the IMI treated groups of 5 - 9 rats. In the experiments where the effect of phenoxybenzamine or cyproheptadine on the increased flexor reflex activity of IMI rats was studied, the mean amplitude of ten twitches between 5 0 t h - 6 0 t h min after the injection of drugs or the solvent was calculated and expressed as percentage of the mean amplitude of ten twitches immediately preceding the injection. Phenoxybenzamine, cyproheptadine and the solvent were injected 2 h after the last dose of chronically administered IMI. The group injected with the solvent consisted of 3 animals, each group injected with phenoxybenzamine or cyproheptadine consisted of 6 animals.
257 In the experiments where the effect of a single dose of D M I was assessed, the amplitude of ten twitches immediately before (the control amplitude) and of five twitches 15 rain (n = 8), 1 h (n = 4) and 2 h (n = 4) after the D M I administration was calculated in each animal, The paired Student's t-test was used to estimate the statistical significance of results, Phenoxybenzamine, cyproheptadine and D M I (dissolved in double-distilled water) as well as the solvent were injected in a volume of 1 ml/kg into a tail vein.
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The Levels of l M I and D M I in the Spinal Cord. IMI and D M I were assayed spectrofluorometrically (Dingell et al. 1964) using an Aminco-Bowman apparatus at 275/410 nm. The method consisted in the extraction of the both drugs from the homogenate with 1.5 % isoamyl alcohol solution in heptane at pH = 12 and then re-extraction from the organic phase to a phosphate buffer (pH -- 5.9) - DMI, and to 0.1 N HC1 IMI. Determinations were performed in rats after (1) chronic treatment with IMI (as above); (2)the single dose of IMI (10 mg/kg, orally) ; (3) the single dose of D M I (5 mg/kg i.v.). IM! and D M I were dissolved in double-distilled water. Drugs. The drugs used were cyproheptadine hydrochloride (Merck, Sharp and Dohme, Darmstadt, FRG), desipramine hydrochloride (Ciba-Geigy, Basel, Switzerland), imipramine hydrochloride (Polfa, Starogard, Poland), phenoxybenzamine hydrochloride (Smith, Kline and French, Welwyn Garden City, Herts., UK), phentolamine (Ciba-Geigy, Basel, Switzerland), and 3H-prazosin (NEN Chemicals, Dreieich, FRG).
Results
The Flexor Reflex of the Hind Limb in the Spinal Rat After chronic treatment with IMI (10 mg/kg, orally, twice daily for 14 days) the differences between IMI and solvent treated rats were evident even on a gross inspection of the
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Binding of 3H-Prazosin. The rats treated chronically with IMI or the solvent were guillotined 48 h after the last dose. The spinal cord was excised from the vertebral column and prepared similarly as previously described for the frontal cortex (Vetulani et al. 1981). The suspension (0.7 mg protein in 450 gl of Tris-HC1 buffer, pH 7.6) was incubated for 30 rain at 30~ with 50 gl of a solution of 3H-prazosin (NEN, spec. act. 17.1 Ci/nmol: the final concentration 0.05 - 8 nM) in the presence or absence of the displacing agent phentolamine (10 gM). The incubation was terminated by vacuum filtration through Whatman GF/B filtres which, after washing (2 x 5 ml of cold Tris-HC1 buffer), were placed in scintillation vials containing Bray's fluid and counted for radioactivity (Packard B 3255 liquid scintillation counter: approx. 42 yield). The specific binding was defined as the excess of radioactivity in a sample containing no phentolamine over phentolamine containing blanks. The binding parameters were calculated by Scatchard analysis which revealed the presence of a homogenous population of 3H-prazosin binding sites (a straight Scatchard plot).
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