The Ca2 Channel Blockade Changes the Behavioral and Biochemical
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The Ca21 Channel Blockade Changes the Behavioral and Biochemical Effects of Immobilization Stress Jacek Mamczarz, Ph.D., Bogusława Budziszewska, Ph.D., Lucyna Antkiewicz-Michaluk, Ph.D., and Jerzy Vetulani, Ph.D. We investigated how the effects of chronic immobilization D1 receptors in the cerebral cortex nor D2 dopamine stress in rats are modified by Ca21 channel blockade receptors in the striatum. A single restraint session caused preceding restraint sessions. The application of nifedipine (5 an elevation of blood corticosterone level that remained mg/kg) shortly before each of seven daily 2 h restraint unaffected by nifedipine pretreatment, but the reduction of sessions prevented the development of sensitized response to this response during the eighth session was significantly amphetamine as well as the stress-induced elevation of the less expressed in nifedipine-treated rats. We conclude that densities of L-type Ca21 channels in the hippocampus and L-type calcium channel blockade prevents development of significantly reduced the elevation of the densities of several stress-induced adaptive responses. 3 [ H]nitrendipine binding sites in the cortex and D1 [Neuropsychopharmacology 20:248–254, 1998] dopamine receptors in the limbic forebrain. Neither stress, © 1999 American College of Neuropsychopharmacology. a nor nifedipine affected the density of 1-adrenoceptors and Published by Elsevier Science Inc. 1 KEY WORDS: Immobilization stress; Ca2 channel; ences between the action of stress and psychostimulants, Dopamine receptors; Corticosterone; Amphetamine particularly regarding the involvement of N-methyl-D- sensitization; Nifedipine aspartate (NMDA) receptors (Tolliver et al. 1996), the Repeated stress has several consequences for the re- relationship between stress and addictive stimulants is sponse of an organism to environmental stimuli and to of interest, because the stress seems to potentiate and psychotropic drugs. One of those effects is sensitization accelerate the development of addiction and reinstates of animals to the action of psychostimulants (Robinson the drug-seeking behavior after a prolonged with- et al. 1985). It was believed that stress and psychostimu- drawal period (Ahmed and Koob 1997; Shaham et al. lants act similarly on the mesocortical dopamine system 1996). Repetitive unavoidable stress causes several neu- and that the mechanisms of development of sensitiza- rochemical changes that may be regarded as adaptive tion by those treatments are similar (Antelman et al. changes, but on the other hand, may be causally related 1980). Although recent studies have demonstrated differ- to the induction of sensitization to psychostimulants and potentiation of addictive properties of drugs. We previously postulated that L-type Ca21 channels play an important role in the development of adaptive From the Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland. changes and that their blockade may prevent adaptive Address correspondence to: Dr. Jerzy Vetulani, Institute of Phar- processes at the neurochemical and behavioral levels macology, Polish Academy of Sciences, Smetna 12, 31-343 Krakow, (Antkiewicz-Michaluk et al. 1994a,b; 1995). This mecha- Poland. 21 Received April 6, 1998; revised June 29, 1998; accepted July 21, nism may explain prevention by Ca channel blocking 1998. agents of morphine dependence, dopaminergic symp- NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 3 © 1999 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/99/$– see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(98)00071-2 NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 3 Nifedipine Counteracts Stress Effects 249 toms after withdrawal from chronic neuroleptic treat- Preparation of Tissue for Receptor Studies and ment, or neurochemical and behavioral effects of LSD General Procedure (Antkiewicz-Michaluk et al. 1997). In the present study, 1 The rats were guillotined 2 h after the end of the last im- we investigated whether L-type Ca2 channel blockade mobilization, the brains were rapidly removed, and with nifedipine applied before every restraint session placed on an ice-cold glass plate, and the cerebral cor- would affect the potentiating effect of immobilization tex, hippocampus, striatum, and limbic forebrain (con- stress on amphetamine hypermotility and antagonize sisting of limbic cortex, olfactory bulb, preoptic area, the neurochemical consequences of repetitive immobili- nucleus accumbens septi, and the amygdala) were dis- zation stress. sected, and the tissues were stored at 2708C. On the day of assay, the tissues were homogenized (Polytron) at 08C in 20 vol of 50 mm Tris-HCl buffer pH 7.6, if not MATERIALS AND METHODS stated otherwise. The homogenate was centrifuged at Animals 1,000 g and 08C for 10 min, and the supernatant was re- centrifuged at 25,000 g for 30 min. The pellet was reho- The experiment was carried out on male Wistar rats, mogenized in the original volume of the buffer. The 250 to 280 g. The animals were kept in groups of four or membrane preparation (fraction P of Whittaker and six (in 55 3 34 3 19 cm cages). The cages, made of 2 Barker 1972) was adjusted with the Tris-HCl buffer to opaque plastic and having sawdust bedding, were kept contain the appropriate concentration of protein (as- in a room at 218C, under 12/12 h light/dark cycle. The sayed according to Lowry et al. 1951). The radioligands animals had free access to food and water. The immobi- were prepared in six concentrations. The incubation lization procedure and activity measurements were car- mixture contained 450 ml of homogenate, 50 ml of radio- ried out in another room. ligand solution, and 50 ml of buffer or displacer, if not stated otherwise, and was incubated in a shaking water Drugs bath for 30 min. All incubations were carried out in du- plicates. The incubation was terminated by rapid filtra- The repetitive treatment was applied once daily for 7 tion through Whatman GF/C filters that were afterward consecutive days. Nifedipine (Polfa) was prepared as rinsed twice with 5 ml of ice-cold buffer and placed in 3 suspensions in 1% Tween 80 immediately before injec- ml of Aquascint (Biocare) solution and counted for ra- tion and was given in a dose of 5 mg/kg IP 15–20 min dioactivity in a Beckman LS 3801 counter. The specific before immobilization of rats. Amphetamine sulphate binding was defined as the difference between the total (Sigma), 0.75 mg/kg SC, was dissolved in 0.9% NaCl and and unspecific binding and expressed in fmole/mg of given after the period of adaptation of a rat in the acto- protein. The B and K values were calculated by meter. The injections were made in a volume of 2 ml/kg. max D Scatchard analysis; the following procedures differed, depending upon the kind of receptor investigated. Immobilization Stress The animals were transferred to a room of lower tem- The Assay of L-type Ca21 Channels 8 perature (18 C) and placed for 2 h in restraint cages of (Dihydropyridine Binding Sites) in the Cortex 3 3 maximum dimensions 17.0 5.5 5.5 cm. The bottom and Hippocampus and movable front and back were made of translucent plastic, the sides and top with metal rods whose posi- [3H]Nitrendipine (NEN, s.a. 78.3 Ci/mmol) was used in tion was adjustable, to assure complete restraint of the final concentrations of 0.05 to 3 nmol/l, 10 mmol/l nife- animals. dipine served to assess the unspecific binding. Solu- tions of [3H]nitrendipine were prepared in darkness. Cortices from a single animal or hippocampi pooled Locomotor Activity and from two animals were used for an assay. The final con- Amphetamine Hypermotility centration of membrane preparation was approx. 1.2 The motor activity was measured in Columbus, Ohio, mg/ml of protein. The samples were incubated at 258C. infrared actometers (Autotrack) and expressed as the length of the path covered in centimeters during the re- The Assay of D Dopamine Receptors ([3H]SCH- cording period. The rats were placed in the actometers 1 23390 Binding Sites) in the Limbic Forebrain without injection for a 60 min adaptation period, and they were injected with saline. Sixty minutes later, am- [3H]SCH-23390 (NEN, s.a. 85.5 Ci/mmol) was prepared phetamine was administered, and the recording was in concentrations of 0.06 to 2.0 nmol/l, 5 mmol/l SCH- continued for another 120 min. The tests were carried 23390 served to assess the unspecific binding and 10 out 24 h after the last immobilization session. mm serotonin to block serotonin receptors. The tissue 250 J. Mamczarz et al. NEUROPSYCHOPHARMACOLOGY 1999–VOL. 20, NO. 3 was homogenized in 40 vol of 50 mm Tris-HCl buffer (20,000 dmp/sample), and 100 ml of solution of anticor- pH 7.4, the final homogenate of P2 contained 0.3 to 0.4 ticosterone antibody A906RIT Ab, Bioproducts in ge- mg/ml of protein. The incubation was carried out at 308C. latine-containing buffer in the amount resulting in ap- proximately 50% [3H]corticosterone binding in the sample without the standard. The samples were incu- 3 The Assay of D2 Dopamine Receptors [ H]Spiperone bated overnight (12–16 h) at 4 to 68C. Free and bound Binding Sites) in the Striatum corticosterone were separated using dextran-coated [3H]Spiperone (Amersham, s.a. 16.4 Ci/mmol) was pre- charcoal: 0.0625% dextran (Dextran T 70, Pharmacia) pared in concentrations of 0.06 to 2 nmol/l, and 10 and 0.625% charcoal (activated, Sigma) in gelatine-con- mmol/l spiperone was used as the displacer. The striata taining buffer. The samples were incubated for 10 min m were homogenized in 20 vol of Tris-HCl buffer pH 7.4, and centrifuged at 1,000 g for 20 min. Samples of 200 l of supernatant were taken to scintillator for radioactiv- the final P2 pellet was suspended in 50 mm Tris-HCl buffer pH 7.1 containing 120 mm NaCl, 5 mm KCl, 1 ity assay.