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Reserpine enhances amphetamine stereotypies without increasing amphetamine-induced changes in striatal dialysate dopamine
Brain Research, 505 (1989) 83-90 Elsevier
83
BRES 15056
Reserpine enhances amphetamine stereotypies without increasing amphetamine-induced changes in striatal dialysate dopamine Clifton W. Callaway, Ronald Kuczenski and David S. Segal Department of Psychiatry, University of California, San Diego, La Jolla, CA (U.S.A.) (Accepted 23 May 1989) Key words: Amphetamine; Dopamine; Dialysis; Reserpine; Stereotypy; Striatum
Indirect evidence suggests that amphetamine (AMPH) releases dopamine (DA) from an extravesicular, cytoplasmic pool. Disruption of vesicular DA storage by reserpine has been hypothesized to increase the concentration of extravesicular DA available for release by AMPH, which is consistent with the observation that reserpine does not prevent but augments the behavioral response to AMPH. In order to more directly test this hypothesis, the in vivo microdialysis technique was used to concurrently examine the behavioral and striatal dopaminergic response to AMPH (1.25 or 2.5 mg/kg) 24 h following reserpine pretrcatmcnt (2.5 mg/kg). Reserpine decreased tissue levels of DA by approximately 90% and reduced baseline dialysate DA concentrations by approximately 80%. Reserpine augmented the behavioral effects of AMPH, particularly increasing the occurrence and intensity of stereotypies. In contrast, reserpine did not alter the amount or duration of AMPH-induced DA release. This observation confirms that DA release by AMPH does not depend on vesicular stores but is inconsistent with the hypothesis that augmentation of behavior by reserpine results from increased striatal DA eelease.
INTRODUCTION Behavioral effects of amphetamine (AMPH) have been attributed to central release of dopamine (DA). A M P H putatively releases D A from a newly synthesized, cytoplasmic pool IT, while only very high doses of AMPH appear to recruit vesicular stores of D A 7. Consistent with this alleged mechanism of action, disruption of D A vesicular stores by reserpine does not attenuate and may intensify AMPH-induced behaviors 11'24'26'27'32'34. This potentiation has been proposed to result from an increased concentration of cytoplasmic D A available for release by A M P H n. However, some indirect evidence suggests that reserpine pretreatment may not alter D A release by A M P H 3'2°'22. Recently, direct measurement of extracellular transmitter concentrations in freely moving animals has become possible with the in vivo microdialysis technique t2'37'4°. This technique has confirmed that the behavioral response to AMPH is associated with an increase in D A release ]3'ts'30'37'4°. However, some dissociations between the quantitative features of D A release and the behavioral effects of A M P H have also been noted ~s'3° and therefore the relationship between D A and AMPl-l-induced behavior requires further clarification. In the present study, the microdialysis technique was
used to correlate behavioral changes with extracellular D A coqcentrations during reserpine-induced depletion of vesicular D A and subsequent challenge with AMPH. Because the most prominent effect of reserpine on the A M P H response is to intensify stereotypies 27, and evidence indicates that the nigrostriatal D A pathway is involved in the expression of these behaviors 4't5'1~, DA release in striatum was examined. The present results suggest that A M P H releases D A from a reserpine insensitive (i.e. extravesicular) pool, but that the reserpine potentiation of the behavioral response to AMPH is not associated with a corresponding increase in striatal DA release.
MATERIALS AND METHODS Animals Male Sprague-Dawley rats (Simonsen Labs) (300-325 g) were housed 4 per cage with food and water available ad lihitum in an animal colony which was maintained at constant temperature and humidity, on a reverse white light-red light cycle (white lights on 18.00-06.00 h). Following at least one week of habituation to the animal colony, a guide cannula was surgically implanted in each animal according to procedures previously described in detailIs. Briefly, each animal was anesthetized with 2.0% halothane in air and placed in a stereotaxic device (Kopf). A unilateral stainless steel guide cannula extending 2.6 mm below the skull surface was implanted through a burr hole, and the assembly was fixed in place with dental acrylic anchored by 4 skull screws. A stainless steel stylet extending 0,1 mm below the tip of the cannula was introduced to
Correspondence: Ronaid Kuczenski, Department of Psychiatry (M-003), University of California, San Diego, La Jolla, CA 92093, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
84 maintain patency. Following surgery, animals were housed individually and a!lowed at least one week to recover before receiving any treatment. Microdialysis probes were constructed of Spectra/Por hollow fiber (MW cut-aft 6000, o.d. 250 l~m) extending 2.25 mm beyond the end of a 25.gauge stainless steel tube. Probes were aimed at the anterior striatum (1.0 mm anterior and +2.8 mm lateral to bregma, 6.2 mm below dura) 2"~.The dialysis membrane was attached to the stainless steel tube and sealed at its free end with an epoxy plug. Glass capillar~ tubing (fused silica, 1501to.d. x75/~ i.d. x 50 cm) was passed inside the stainless steel tube and dialysis tubing within 0.25 mm of the sealed end of the probe. Probes were perfused by artificial cerebrospinal fluid ([in mM:] NaCI 147, CaCI2 2.3, MgCI2 0.9, KCI 4.0) delivered by a microinfusion pump (3-3.5 id/min) via 50 cm of Micro-line ethyl vinyl acetate tubing connected to a fluid swivel. Dialysate was collected through the glass capillary tubing in vials containing 10 ld of 1 N HCI. The tubing was supported by the fluid swivel with a counterweight, thereby allowing unrestricted movement by the rat, and samples were collected outside the experimental chamber thus minimizing disturbance of the animal. Individual probe recoveries, which ranged from 1.5% to 3.5%, were estimated by sampling a standard DA/DOPAC solution in vitro.
Biochemistry Dialysate samples (60-70/4) were collected every 20 min, and DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were assayed in 50-/d aliquots by HPLC with electrochemical detection (HPLC-EC). The HPLC-EC consisted of a 150 mmx4.6 mm ODS-CIS 5 #m column (Waters) maintained at 35 °C. Mobile phase (0.08 M citric acid, 7% MeOH, 0.1 mM Na2EDTA and 0.8 mM octane sulfonate adjusted to pH 3.7-4.2) was delivered at 1.2 ml/min by a Waters mtMel 510 pump. Amines were detected with a Waters 460 detector with glassy carbon electrode maintained at +0.65 V relative to a Ag/AgCI reference electrode. Concentrations of extracellular substances, corrected for individual probe recoveries, were estimated from peak areas using a Waters Maxima 820 data station. Amine content of whole caudates was also determined by HPLC.EC. Tissue was sonicated in 1.0 ml of mobile phase and centrifuged. Filtered supernatant (50 ld) was directly injected into the HPLC-EC. A separate column (250 mmx4.6 mm, 10 ~m, ODS.CI8), mobile phase t~ lind detector (glassy carbon electrode set at +0.7 V) were used for tissue analyses.
Behavior Experiments were conducted in 12x12x15 inch sound-attenuated, temperature, and humidity.controlled chambers, previously described in detail :~, Cage crossings were detected through contacts in :he floor grid, and rearings were registered when the animal contacted a wall touch#ate 5 inches above the floor. Contacts with water in a sipper tube or with a metal food container provided measures of drinking and eating. Automated behavioral data were collected continuously by computer. Animals were also videotaped to allow direct observational rating of behavior as described previouslyTM, After AMPH administration, the percentage of time during which the animal engaged in focussed sniffing, repetitive head movements or oral stereotypies was recorded.
Drugs S(+)-AMPH (NIDA) was dissolved in saline and injected subcutaneously in a volume of 1 ml/kg body weight. Doses refer to weight of free base. Reserpine was used directly from the commercially available viais (2.5 mg/ml, Serpasil, CIBA) and was injected intraperitoneally.
P;ocedures Each rat was placed in a testing chamber eL~, the day prior to treatment (15.00-16.00 h) to allow for ac61imation to the test environment and for adequate equilibration of the dialysis probe. All drugs were administered at approximately 10.00 h. Animals
received either reserpine (2.5 mg/kg) or an equal volume of saline, followed 24 h later by AMPH (1.25 or 2.5 mg/kg). In order to determine the time course of biochemical and behavioral changes following reserpine, microdialysis samples were collected from several animals every 20 min from 80 min before through 4 h after reserpine administration, and again at 6 and 12 h after reserpine. Reserpine-induced behavioral changes were also monitored with both automated and observational procedures. At the end of the experiment, each animal was sacrificed, the brain removed, and the cannulated side fixed in 3% formalin for histological verification of probe placement. The whole caudate was quickly hand-dissected from the non-cannulated side of the brain and stored at -80 °C for subsequent assay of amine content. RESULTS
Response to reserpine The temporal pattern of effects of reserpine on dialysate neurotransmitter and metabolite concentrations for two animals are presented in Fig. 1. Following reserpine, D A concentrations declined over 3 h and remained depressed for the remainder o f the 24 h preceding A M P H administration. Concomitantly, D O P A C and H V A concentrations increased and returned to near baseline concentrations by 6 h. The peak concentrations of dialysate D O P A C and H V A following reserpine occurred at 4 0 - 8 0 min and 80-120 min respectively. In contrast, concentrations of 5 - H I A A increased following reserpine and remained elevated for the remainder of the 24 h prior to A M P H administration. Consistent with previous findings TM, no change in dialysate D A , D O P A C , H V A or 5 - H I A A cottcentrati~ns were observed following saline administration (data not shown). Animals pretreated with reserpine exhibited a characteristic syndrome which included ptosis, hunch-back posture, piloerection and diarrhea. During the hour prior to A M P H administration, crossovers and rearings were significantly reduced in reserpinized animals compared to saline-treated control animals (Fig. 2). Consistent with the time course of the reserpine response presented in Fig. 1, baseline dialysate concentrations of D A on the day of A M P H administration were significantly lower in reserpinized animals. Furthermore, baseline 5 - H I A A concentrations were about two-fold greater in reserpinepretreated animals, but D O P A C and H V A concentrations were not significantly changed (see legend to Fig. 1). The decrease in extraceUular D A concentration in reserpinized animals corresponded to about a 90% depletion of tissue D A levels, (Fig. 2). However, in 3 reserpine-pretreated animals there was significantly less depletion of tissue D A . The behavior of these less-depleted animals was not significantly different from saline controls and they actually exhibited an increase in baseline dialysate concentrations o f D A (Fig. 2).
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Houri Fig. 1. Temporal pattern of striatal neurochemical responsc in dialysates following reserpine (2.5 mg/kg, i.p.) administration. Data from two individual animals are presented. Reserpine was adminisered at time 0. For all animals, absolute dialysate concentrations of neurochemicals (nM_S.E.M.) 24 h after saline or 2.5 mg/kg reserpine, respectively were: DA, 35.8 +_ 3.0, 6.6 _. 1.4 (P < 0.001); DOPAC, 5207 +_703, 6789 -+ 579 (n.s.); HVA, 4326 +_ 487, 5876 _+ 578 (n.s.); 5-HIAA, 1352 +_ 108, 2835 +_ 254 (P < 0.00l).
Response to A M P l f A M P H induced characteristic dose-dependent changes in the behavior of all animals tested. In control animals, the low dose (1.25 mg/kg) o f A M P H produced a continuous period of increased locomotor activity without focussed stereotypies (Fig. 3). In contrast, reserpi-
nized animals exhibited a multiphasic response to this dose of A M P H . Immediately following drug administration (0-10 rain), locomotor activity was significantly increased followed by a subsequent decrease (20-40 min), which corresponded to the appearance primarily of focussed sniffing. Following a post-stereotypy period of
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Fig. 2. Tissue DA content, striatal dialysate DA concentration, and locomotor activity foRowing saline (n = 12) or 2.5 mg/kg reserpine (n = 13). Locomotor activity, measured as combined crossovers and rearings, and dialysate DA concentration were obtained 24 h later daring the l-h interval preceding AMPH administration. Reserpine reduced tissue DA 28 h after injection by about 90% in 10 animals (closed bars), from 120.1 + 8.5 to 12.0 _+ 1.2 pmol/mg, but by less than 80%, to 56.8 + 15.1, in 3 animals (hatched bars). Data are presented as percent of saline, mean +- S.E.M. (**P < 0.01; ***P < 0.0{)1).
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Fig. 3. Behavioral response to AMPH (1.25 mg/kg, s.c.) 24 h followingpretreatment with saline (n : 5, open symbols)or reserpine (2.5 mg/kg) (n = 4, closed symbols). Stereotypyis presented as percentage of total time engaged in focussed sniffing(%FSN). Data are presented as mean + S.E.M. Histograms represent cumulated activity during the indicated interval. (**P < 0.0l; ***P < 0.001). locomotor activity (40-120 min), ptosis and hunch-back posture reappeaced in these animals. A multiphasic response to the higher dose of AMPH (2.5 mg/kg) was observed in both saline-treated and reserpinized animals (Fig. 4). However, whereas control animals exhibited primarily repetitive head movements during the stereotypy phase, reserpinized animals displayed oral stereotypies, typical of a higher AMPH dose. Furthermore, as with the lower dose of AMPH, the
poststereotypy locomotor Fhase ( i 2 0 ~ 4 0 min) was significantly attenuated in reserpinized animals. The reserpine-pretreated animals with less depletion of D A did not differ from control animals in their response to 1.25 mg/kg (n = 2) or 2.5 mg/kg (n = 1) A M P H (data not shown). The low dose of AMPH (1.25 mg/kg) induced a 10-fold increase of dialysate DA concentrations (Fig. 5A), and the high dose increased DA concentrations about 20-fold
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Fig. 4. Behavioral response to AMPH (2.5 mg/kg, s.c.) 24 h followingpretreatment with s~line (n = 7, open symbols) or reserpine (2.5 mg/kg) (n = 6, closed symbols). Stereotypy is presented as percentage of total time engaged in repetitive head movements (%RHM) or oral stereotypies (%ORL). Data are presented as mean _+ S.E.M. Histograms represent cumulated activity during the indicated interval. (**P < 0.01; ***P < 0.001).
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Fig. 5. Temporal pattern of AMPH-induced changes in striatal dialysate DA concentrations 24 h following pretreatment with saline (open symbols),or reserpine (2.5 mg/kg, i.p.) (dosed symbols).A: DA concentrations following1.25 mg/kgAMPH. B: DA concentrations following 2.5 mg/kgAMPH. Insets: BaselineDA concentrationsduring the 20-min interval precedingAMPH administration. (**P < 0.01; ***P< 0.001). (Hg. 5B). However, although baseline concentrations of DA were significantly lower in reserpine-treated animals, reserpine did not alter the AMPH-induced increase of DA. Likewise, DOPAC and HVA concentrations were reduced to a similar extent in reserpinized and control animals following AMPH administration. Neither dose of AMPH altered concentrations of 5-HIAA (data not
shown). DISCUSSION The present study used the microdialysis technique to assess the role of vesicular DA in AMPH-induced DA release, and to examine the mechanism~ by which reserpine potentiates the behavioral response to AMPH. Concurrent measures of behavioral and neurochemicai changes provide a more accurate indication of the role of specific neurotransmitter systems in the various components of the AMPH response than has been accomplished using in vitro and post mortem techniques or with microdialysis in anesthetized animals.
Effects of reserpine Consistent with its alleged depletion of vesicular pools, reserpine produced a marked reduction of tissue DA, and a delayed but prolonged decrease in striatal extracellular DA concentration. The decrease in baseline dialysate DA concentration in animals with tissue DA levels depleted by approximately 90% supports the view that vesicular pools are involved in the impulse-mediated
neurotransmitter release process. However, we also found that reserpine did not reduce but actually increased striatal DA release in 3 animals (Fig. 2) whose tissue levels of DA were depleted by less than 80% of controls. This observation is consistent with the view that striatal DA stores exceed normal requirements, and, in fact, recent work with 6-hydroxydopamine indicates that a substantial reduction of tissue DA can occur without a corresponding decrease in extracellular DA concentratioJ~! (Scgal and Kuczenski, unpublished observations) 25' 39. It has been shown that reserpine increases nigrostriatal dopaminergic neuronal activitys, perhaps as a consequence of DA depletion in nigral dendrites, which appear to be more sensitive to disrup~ion by reserpine 14' as and which regulate impulse traffic in these neurons t°. The disruption of 90% of striatal vesicular DA stores may preclude an enhanced DA release accompanying increased neuronal activity in most reserpine-pretreated animals. In contrast, the residual vesicular DA pool in the less-depleted group may be sufficiently large to respond to the increased nigrostriatal impulse flow. The transient increase of dialysate DOPAC concentrations immediately following reserpine parallels a similar increase in tissue levels6'33'3s, and presumably reflects the release of vesicular DA to the cytoplasm where it is rapidly degraded by MAO. The temporal delay between the increase in DOPAC and HVA is consistent with the role of HVA as a metabolite of DOPAC. Twenty-four h after reserpine administration, when extracellular DA concentrations were substantially reduced, DOPAC and
88
HVA had returned to pre-reserpine baseline levels (Fig. 1). If DOPAC and HVA were derived from released DA, dialysate concentrations should decline correspondingly. The absence of a decrease in steady-state extraceUular DOPAC and HVA, supports previous results LS"ls'36 which indicate that these metabolites originate primarily from degradation of a cytoplasmic pool of DA to which released DA does not substantially contribute. Behavioral activity during the hour prior to AMPH administration (i.e. 23-24 h after reserpine pretreatment) was reduced only in those reserpine-treated animals which exhibited reduced DA release (Fig. 2). This observation is consistent with previous reports that >90% DA depletion is required to produce gross behavioral changes 25'39, thus further indicating that available DA is in excess of normal demands. The increased levels of dialysate DA in the less-depleted group did not correspond to any obvious change in motor behavior, although more subtle behavioral alterations may have been missed. Further studies will be required to more thoroughly characterize the behavioral profiles associated with different levels of reserpine-induced DA depletion. In accordance with previous observationsa~, reserpine also significantly reduced tissue levels of 5-hydroxytryptamine (5-HT), although not to the same extent as DA. Since dialysate 5-HT was not measured, it is not known whether reserpine produced a corresponding decrease in extracellular 5-HT or whether, as with DA, a threshold level of depletion is necessary to alter 5-HT release, Both extracellular and tissue concentrations of 5-rliAA, the primary metabolite of 5-HT, were found to exhibit a persistent increase following reserpine, in contrast to the relatively transient increase in DA metabolites. The reserpine-induced impairment of vesicular storage in 5-HT terminals may occur more slowly than in DA terminals, thus resulting in a more gradual exposure of 5-HT to cytoplasmic MAO, and a more prolonged increase in metabolite.
Effect of AMPH As previously reported 11.24.26.27.32.34, reserpine augmented the behavioral response to AMPH. This effect was most apparent in the form of an intensification of the stereotypy produced by each of the two doses of AMPH. It has previously been hypothesized n that this potentiation is mediated by a reserpine-induced increase in the AMPH releasable (cytoplasmic) DA pool. Because striatal dopaminergic mechanisms have been implicated in AMPH-induced stereotypies, striatal DA release in response to AMPI-! would be expected to be facilitated by reserpine pretreatment. However. while the present results confirm that AMPH release of DA does not require vesicular neurotransmitter stores, striatal DA
release induced by AMPH was unaffected by reserpine (Fig. 5). Likewise, reserpine failed to enhance AMPHinduced DA release in vitro 2°'22 or in the dialysates of anesthetized rats 2. These results are consistent with our previous studies TM indicating no simple quantitative relationship between AMPH-induced DA release and the appearance of specific components of the stereotypy response, and suggest that the intensification of AMPHinduced behaviors by reserpine does not involve increased striatal DA release. In contrast to the potentiation of AMPH-induced stereotypies by reserpine pretreatment, reserpine actually reduced the duration of the AMPH behavioral response. The reemergence of a reserpinized state in these animals might reflect a more rapid depletion of AMPH-releasable DA. However, at a time when behavioral activity was reduced in the reserpinized group (120-240 rain) (Fig. 4), extracelluiar DA concentrations were not significantly lower (Fig. 5B). Thus there appears to be a dissociation between the temporal pattern of the behavioral and dopaminergic response profiles produced by acute AMPH Is which further indicates that factors besides striatal DA release maintain the behavioral response to AMPH. Other striatal DA mechanisms may underly the augmentation of the AMPH response by reserpine. For example, the low baseline levels of DA release in the reserpinized animals may have resulted in an up-regulation of postsynaptic DA receptors. Consistent with this hypothesis, pretreatment with reserpine has been reported to increase behaviors induced by direct DA receptor agonists 2~.33. However, whereas up-regulation of DA receptors in the presence of continued AMPHinduced DA release should prolong the AMPH behaw ioral response, reserpine-pretreated animals actually exhibited a truncated AMPH behavioral profile (Fig. 4). Likewise, the enhanced baseline DA release in the less depleted animals might be expected to result :,a downregulation of DA receptors, leading to an attenuated AMPH behavioral response. In contrast, however, the behavioral'~esponse to AMPH in these animals w a s n o t decreased in intensity, although, as with the other reserpine-treated animals, the duration of the AMPH behavioral response appeared to be truncated (data not shown). It is also conceivable that interactions between DA and other neurotransmitter systems may be responsible for the augmented AMPH response. Inhibition of 5-HT or norepinephrine activity has been reported to enhance various components of the AMPH response 9,2s, and bo:h of these monoamines are depleted by reserpine. In this regard, it is interesting to note that, whereas tissue 5-HT levels were significantly reduced in the reserpinized
89 group, the less DA-depleted animals did not exhibit a significant 5 - H T depletion. In summary, it appears that D A c,+,mcentrations are far in excess o f normal physiological requirements and that depletion o f about 90% is necessary before impulsemediated D A release is reduced. Moreover, the results o f this study support the view that AMPH-induced D A release is not dependent o n the vesicular D A pool. Although reserpine pretreatment both potentiated and abbreviated the A M P H behavioral response, neither the
magnitude nor the duration of A M P H - i n d u c e d striatal D A release was altered. Together with previous observations of dissociations between striatal D A release and behavior, these data suggest that the A M P H response involves additional non-dopaminergic neurotransmitter systems.
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1 Braestrup, C., Biochemical differentiation of amphetamine vs methylphenidate and nomifensine in rats, J. Pharm. Pharmacol., 29 (1977) 463-472. 2 Butcher, S.P., Fairbrother, I.S., Kelly, J.S. and Arbuthnott, G.W., Amphetamine-induced dopamine release in the rat striatum: an in vivo microdialysis study, J. Neurochem., 50 (1988) 346-355. 3 Chiueh, C.C. and Moore, K.E., d-Amphetamine-induced release of "newly synthesized" and "stored" dopamine from the caudate nucleu~ in vivo, J. PharmacoL Exp. Ther., 192(3) (1985) 642-653. 4 Creese, I. and iversen, S.D., The role of forebrain dopamine systems in amphetamine-induced stereotypy in the adult rat following neonatal treatment with 6-hydroxydopamine, Psychopharmacology, 39 (1974) 345-357. 5 Di Giulio, A.M., Groppetti, A., Cattabeni, E, Galli, C.L., Maggi, A., Algeri, S. and Ponzio, F., Significance of dopamine metabolites in the evaluation of drugs acting on dopaminergic neurons, Eur. J. Pharmaeol., 52 (1978) 201-207. 6 Fadda, E, Argiolas, A., Stefanini, E. and Gessa, G.L., Differential effect of psychotropie drugs on dihydroxy phenylacetic acid (DOPAC) in the rat substantia nigra and caudate nucleus, Life Sci., 21 (1977) 411-418. 7 Fischer, J.F. anti Chn, A.K., Chemical release of dopamine from striatal homogenates: evidence for an exchange diffusion model, 1. PharmacoL Exp. Ther., 192 (1979) 642-653. 8 German, D.C., McMillen, B.A., Sanghera, M,K., Saffer, S.I, and Shore, P.A., Effect of severe dopamine depletion on dopamine neuronal impulse flow and on tyrosine hydroxylase regulation, Brain Res. Bull., 6 (1981) 131-134. 9 Geyer, M.A., Masten, G. and Segal, D.S., Behavioral effects of xylamine-induced depletions of brain norepinephrine: interactions with amphetamine, Behav. Brain Res., 21 (1986) 55-64. 10 Groves, P.M., Wilson, C.J., Young, S.J. and Rebec, G.V., Self-inhibition by dopaminergic neurons, Science, 190 (1975) 522-529. 11 Hong, M., Jenner, P. and Marsden, C.D., Comparison of the acute actions of amine-depleting drugs and dopamine receptor antagonists on dopamine function in the brain in rats, Neuropharmacology, 26 (1987) 237-245. 12 Imperato, A. and Di Chiara, G., Dopamine release and metabolism in awake rats after systemic neuroleptics as studied by trans-striatal dialysis, I. Neurosci., 5(2) (1985) 297-306. 13 lmperato, A., Tanda, G., Frau, R. and Di Chiara, G., Pharmacological profile of dopamine receptor agonists as studied by brain dialysis in behaving rats, J. PharmacoL Exp. Ther., 245 (1988) 257-264. 14 Kebabian, J.W., Saavedra, J.M. and Axelrod, J., A sensitivt~ enzymatic-radioisotopic assay for 3,4-dihydroxyphenylacetic acid, J. Neurochem., 28 (1977) 795-801. 15 Kelly, P., Seviour, P. and Iversen, S.D., Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum, Broin Re,
Acknowledgements. This research was supported by U.S. Public Health Service Grants DA-04157 and DA-01568 and by a Research Scientist Award MH-70183 to D.S.S.C.W.C. was supported by h'~edieal Student Training Grant M-07198.
90 (Eds.), Handbook of Psychopharmacology, Plenum, New York, 1978, pp. 197-219. 32 Smith, C.B., Enhancement by reserpine and a-methyl DOPA of the effects of d-amphetamine upon the locomotor activity of mice, J. Pharmacol. Exp. Ther., 447 (1963) 335-340. 33 Starr, B.S., Start, M.S. and Kilpatrick, I.C., Behavioural role of dopamine D1 receptors in the reserpine-treated mouse, Neuroscience, 22 (1987) 179-188. 34 Stolk, J.M. and Rech, R.H., Enhanced stimulant effects of d-amphetamine on the spontaneous locomotor activity of rats treated with reserpine, J. PharmacoL Exp. Ther.. 158 (1967) 140-149. 35 Tonon, G., Saiani, L., Spano, EE and Trabucchi, M., Differential effect of reserpine on dopaminergic receptor function in rat substantia nigra and caudate nucleus, Brain Research, 160 (1979) 553-558. 36 Westerink, B.H. and Spaan, S.J., On the significance of endogenous 3-methoxytyramine for the effects of centrally acting
drugs on dopamine release in the rat brain, J. Neurochem., 38 (1982) 680-688. 37 Westerink, B.H. and Tuinte, M.H., Chronic use of intracerebral dialysis for the in vivo measurement of 3,4-dihydroxyphenylethylamine and its metabolite 3,4-dihydroxyphenylacetic acid, J. Neurochem., 46 (1986) 181-185. 38 Westerink, B.H.B., Effects of drugs on the formation of 3-methoxy-tyramine, a dopamine metabolite, in the substantia nigra, striatum, nucleus accumbens and tuberculum olfactnrium of the rat, J. Pharm. Pharmacol., 31 (1979) 94-99. 39 Zetterstrom, T., Herrera-Marschitz, M. and Ungerstedt, U., Simultaneous measurement of dopamine release and rotational behaviour in 6-hydroxydopamine denervated rats using intracerebral dk~¢vsis, Brain Research, 376 (1986) 1-7. 40 Zetterstrom, T., Sharp, T., Marsden, C.A. and Ungerstedt, U., In vivo measurement of dopamine and its metabolites by intracerebral dialysis: changes after d-amphetamine, I. Neurochem., 41 (1983) 1769-1773.
83
BRES 15056
Reserpine enhances amphetamine stereotypies without increasing amphetamine-induced changes in striatal dialysate dopamine Clifton W. Callaway, Ronald Kuczenski and David S. Segal Department of Psychiatry, University of California, San Diego, La Jolla, CA (U.S.A.) (Accepted 23 May 1989) Key words: Amphetamine; Dopamine; Dialysis; Reserpine; Stereotypy; Striatum
Indirect evidence suggests that amphetamine (AMPH) releases dopamine (DA) from an extravesicular, cytoplasmic pool. Disruption of vesicular DA storage by reserpine has been hypothesized to increase the concentration of extravesicular DA available for release by AMPH, which is consistent with the observation that reserpine does not prevent but augments the behavioral response to AMPH. In order to more directly test this hypothesis, the in vivo microdialysis technique was used to concurrently examine the behavioral and striatal dopaminergic response to AMPH (1.25 or 2.5 mg/kg) 24 h following reserpine pretrcatmcnt (2.5 mg/kg). Reserpine decreased tissue levels of DA by approximately 90% and reduced baseline dialysate DA concentrations by approximately 80%. Reserpine augmented the behavioral effects of AMPH, particularly increasing the occurrence and intensity of stereotypies. In contrast, reserpine did not alter the amount or duration of AMPH-induced DA release. This observation confirms that DA release by AMPH does not depend on vesicular stores but is inconsistent with the hypothesis that augmentation of behavior by reserpine results from increased striatal DA eelease.
INTRODUCTION Behavioral effects of amphetamine (AMPH) have been attributed to central release of dopamine (DA). A M P H putatively releases D A from a newly synthesized, cytoplasmic pool IT, while only very high doses of AMPH appear to recruit vesicular stores of D A 7. Consistent with this alleged mechanism of action, disruption of D A vesicular stores by reserpine does not attenuate and may intensify AMPH-induced behaviors 11'24'26'27'32'34. This potentiation has been proposed to result from an increased concentration of cytoplasmic D A available for release by A M P H n. However, some indirect evidence suggests that reserpine pretreatment may not alter D A release by A M P H 3'2°'22. Recently, direct measurement of extracellular transmitter concentrations in freely moving animals has become possible with the in vivo microdialysis technique t2'37'4°. This technique has confirmed that the behavioral response to AMPH is associated with an increase in D A release ]3'ts'30'37'4°. However, some dissociations between the quantitative features of D A release and the behavioral effects of A M P H have also been noted ~s'3° and therefore the relationship between D A and AMPl-l-induced behavior requires further clarification. In the present study, the microdialysis technique was
used to correlate behavioral changes with extracellular D A coqcentrations during reserpine-induced depletion of vesicular D A and subsequent challenge with AMPH. Because the most prominent effect of reserpine on the A M P H response is to intensify stereotypies 27, and evidence indicates that the nigrostriatal D A pathway is involved in the expression of these behaviors 4't5'1~, DA release in striatum was examined. The present results suggest that A M P H releases D A from a reserpine insensitive (i.e. extravesicular) pool, but that the reserpine potentiation of the behavioral response to AMPH is not associated with a corresponding increase in striatal DA release.
MATERIALS AND METHODS Animals Male Sprague-Dawley rats (Simonsen Labs) (300-325 g) were housed 4 per cage with food and water available ad lihitum in an animal colony which was maintained at constant temperature and humidity, on a reverse white light-red light cycle (white lights on 18.00-06.00 h). Following at least one week of habituation to the animal colony, a guide cannula was surgically implanted in each animal according to procedures previously described in detailIs. Briefly, each animal was anesthetized with 2.0% halothane in air and placed in a stereotaxic device (Kopf). A unilateral stainless steel guide cannula extending 2.6 mm below the skull surface was implanted through a burr hole, and the assembly was fixed in place with dental acrylic anchored by 4 skull screws. A stainless steel stylet extending 0,1 mm below the tip of the cannula was introduced to
Correspondence: Ronaid Kuczenski, Department of Psychiatry (M-003), University of California, San Diego, La Jolla, CA 92093, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
84 maintain patency. Following surgery, animals were housed individually and a!lowed at least one week to recover before receiving any treatment. Microdialysis probes were constructed of Spectra/Por hollow fiber (MW cut-aft 6000, o.d. 250 l~m) extending 2.25 mm beyond the end of a 25.gauge stainless steel tube. Probes were aimed at the anterior striatum (1.0 mm anterior and +2.8 mm lateral to bregma, 6.2 mm below dura) 2"~.The dialysis membrane was attached to the stainless steel tube and sealed at its free end with an epoxy plug. Glass capillar~ tubing (fused silica, 1501to.d. x75/~ i.d. x 50 cm) was passed inside the stainless steel tube and dialysis tubing within 0.25 mm of the sealed end of the probe. Probes were perfused by artificial cerebrospinal fluid ([in mM:] NaCI 147, CaCI2 2.3, MgCI2 0.9, KCI 4.0) delivered by a microinfusion pump (3-3.5 id/min) via 50 cm of Micro-line ethyl vinyl acetate tubing connected to a fluid swivel. Dialysate was collected through the glass capillary tubing in vials containing 10 ld of 1 N HCI. The tubing was supported by the fluid swivel with a counterweight, thereby allowing unrestricted movement by the rat, and samples were collected outside the experimental chamber thus minimizing disturbance of the animal. Individual probe recoveries, which ranged from 1.5% to 3.5%, were estimated by sampling a standard DA/DOPAC solution in vitro.
Biochemistry Dialysate samples (60-70/4) were collected every 20 min, and DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were assayed in 50-/d aliquots by HPLC with electrochemical detection (HPLC-EC). The HPLC-EC consisted of a 150 mmx4.6 mm ODS-CIS 5 #m column (Waters) maintained at 35 °C. Mobile phase (0.08 M citric acid, 7% MeOH, 0.1 mM Na2EDTA and 0.8 mM octane sulfonate adjusted to pH 3.7-4.2) was delivered at 1.2 ml/min by a Waters mtMel 510 pump. Amines were detected with a Waters 460 detector with glassy carbon electrode maintained at +0.65 V relative to a Ag/AgCI reference electrode. Concentrations of extracellular substances, corrected for individual probe recoveries, were estimated from peak areas using a Waters Maxima 820 data station. Amine content of whole caudates was also determined by HPLC.EC. Tissue was sonicated in 1.0 ml of mobile phase and centrifuged. Filtered supernatant (50 ld) was directly injected into the HPLC-EC. A separate column (250 mmx4.6 mm, 10 ~m, ODS.CI8), mobile phase t~ lind detector (glassy carbon electrode set at +0.7 V) were used for tissue analyses.
Behavior Experiments were conducted in 12x12x15 inch sound-attenuated, temperature, and humidity.controlled chambers, previously described in detail :~, Cage crossings were detected through contacts in :he floor grid, and rearings were registered when the animal contacted a wall touch#ate 5 inches above the floor. Contacts with water in a sipper tube or with a metal food container provided measures of drinking and eating. Automated behavioral data were collected continuously by computer. Animals were also videotaped to allow direct observational rating of behavior as described previouslyTM, After AMPH administration, the percentage of time during which the animal engaged in focussed sniffing, repetitive head movements or oral stereotypies was recorded.
Drugs S(+)-AMPH (NIDA) was dissolved in saline and injected subcutaneously in a volume of 1 ml/kg body weight. Doses refer to weight of free base. Reserpine was used directly from the commercially available viais (2.5 mg/ml, Serpasil, CIBA) and was injected intraperitoneally.
P;ocedures Each rat was placed in a testing chamber eL~, the day prior to treatment (15.00-16.00 h) to allow for ac61imation to the test environment and for adequate equilibration of the dialysis probe. All drugs were administered at approximately 10.00 h. Animals
received either reserpine (2.5 mg/kg) or an equal volume of saline, followed 24 h later by AMPH (1.25 or 2.5 mg/kg). In order to determine the time course of biochemical and behavioral changes following reserpine, microdialysis samples were collected from several animals every 20 min from 80 min before through 4 h after reserpine administration, and again at 6 and 12 h after reserpine. Reserpine-induced behavioral changes were also monitored with both automated and observational procedures. At the end of the experiment, each animal was sacrificed, the brain removed, and the cannulated side fixed in 3% formalin for histological verification of probe placement. The whole caudate was quickly hand-dissected from the non-cannulated side of the brain and stored at -80 °C for subsequent assay of amine content. RESULTS
Response to reserpine The temporal pattern of effects of reserpine on dialysate neurotransmitter and metabolite concentrations for two animals are presented in Fig. 1. Following reserpine, D A concentrations declined over 3 h and remained depressed for the remainder o f the 24 h preceding A M P H administration. Concomitantly, D O P A C and H V A concentrations increased and returned to near baseline concentrations by 6 h. The peak concentrations of dialysate D O P A C and H V A following reserpine occurred at 4 0 - 8 0 min and 80-120 min respectively. In contrast, concentrations of 5 - H I A A increased following reserpine and remained elevated for the remainder of the 24 h prior to A M P H administration. Consistent with previous findings TM, no change in dialysate D A , D O P A C , H V A or 5 - H I A A cottcentrati~ns were observed following saline administration (data not shown). Animals pretreated with reserpine exhibited a characteristic syndrome which included ptosis, hunch-back posture, piloerection and diarrhea. During the hour prior to A M P H administration, crossovers and rearings were significantly reduced in reserpinized animals compared to saline-treated control animals (Fig. 2). Consistent with the time course of the reserpine response presented in Fig. 1, baseline dialysate concentrations of D A on the day of A M P H administration were significantly lower in reserpinized animals. Furthermore, baseline 5 - H I A A concentrations were about two-fold greater in reserpinepretreated animals, but D O P A C and H V A concentrations were not significantly changed (see legend to Fig. 1). The decrease in extraceUular D A concentration in reserpinized animals corresponded to about a 90% depletion of tissue D A levels, (Fig. 2). However, in 3 reserpine-pretreated animals there was significantly less depletion of tissue D A . The behavior of these less-depleted animals was not significantly different from saline controls and they actually exhibited an increase in baseline dialysate concentrations o f D A (Fig. 2).
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Houri Fig. 1. Temporal pattern of striatal neurochemical responsc in dialysates following reserpine (2.5 mg/kg, i.p.) administration. Data from two individual animals are presented. Reserpine was adminisered at time 0. For all animals, absolute dialysate concentrations of neurochemicals (nM_S.E.M.) 24 h after saline or 2.5 mg/kg reserpine, respectively were: DA, 35.8 +_ 3.0, 6.6 _. 1.4 (P < 0.001); DOPAC, 5207 +_703, 6789 -+ 579 (n.s.); HVA, 4326 +_ 487, 5876 _+ 578 (n.s.); 5-HIAA, 1352 +_ 108, 2835 +_ 254 (P < 0.00l).
Response to A M P l f A M P H induced characteristic dose-dependent changes in the behavior of all animals tested. In control animals, the low dose (1.25 mg/kg) o f A M P H produced a continuous period of increased locomotor activity without focussed stereotypies (Fig. 3). In contrast, reserpi-
nized animals exhibited a multiphasic response to this dose of A M P H . Immediately following drug administration (0-10 rain), locomotor activity was significantly increased followed by a subsequent decrease (20-40 min), which corresponded to the appearance primarily of focussed sniffing. Following a post-stereotypy period of
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Fig. 2. Tissue DA content, striatal dialysate DA concentration, and locomotor activity foRowing saline (n = 12) or 2.5 mg/kg reserpine (n = 13). Locomotor activity, measured as combined crossovers and rearings, and dialysate DA concentration were obtained 24 h later daring the l-h interval preceding AMPH administration. Reserpine reduced tissue DA 28 h after injection by about 90% in 10 animals (closed bars), from 120.1 + 8.5 to 12.0 _+ 1.2 pmol/mg, but by less than 80%, to 56.8 + 15.1, in 3 animals (hatched bars). Data are presented as percent of saline, mean +- S.E.M. (**P < 0.01; ***P < 0.0{)1).
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Fig. 3. Behavioral response to AMPH (1.25 mg/kg, s.c.) 24 h followingpretreatment with saline (n : 5, open symbols)or reserpine (2.5 mg/kg) (n = 4, closed symbols). Stereotypyis presented as percentage of total time engaged in focussed sniffing(%FSN). Data are presented as mean + S.E.M. Histograms represent cumulated activity during the indicated interval. (**P < 0.0l; ***P < 0.001). locomotor activity (40-120 min), ptosis and hunch-back posture reappeaced in these animals. A multiphasic response to the higher dose of AMPH (2.5 mg/kg) was observed in both saline-treated and reserpinized animals (Fig. 4). However, whereas control animals exhibited primarily repetitive head movements during the stereotypy phase, reserpinized animals displayed oral stereotypies, typical of a higher AMPH dose. Furthermore, as with the lower dose of AMPH, the
poststereotypy locomotor Fhase ( i 2 0 ~ 4 0 min) was significantly attenuated in reserpinized animals. The reserpine-pretreated animals with less depletion of D A did not differ from control animals in their response to 1.25 mg/kg (n = 2) or 2.5 mg/kg (n = 1) A M P H (data not shown). The low dose of AMPH (1.25 mg/kg) induced a 10-fold increase of dialysate DA concentrations (Fig. 5A), and the high dose increased DA concentrations about 20-fold
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Fig. 5. Temporal pattern of AMPH-induced changes in striatal dialysate DA concentrations 24 h following pretreatment with saline (open symbols),or reserpine (2.5 mg/kg, i.p.) (dosed symbols).A: DA concentrations following1.25 mg/kgAMPH. B: DA concentrations following 2.5 mg/kgAMPH. Insets: BaselineDA concentrationsduring the 20-min interval precedingAMPH administration. (**P < 0.01; ***P< 0.001). (Hg. 5B). However, although baseline concentrations of DA were significantly lower in reserpine-treated animals, reserpine did not alter the AMPH-induced increase of DA. Likewise, DOPAC and HVA concentrations were reduced to a similar extent in reserpinized and control animals following AMPH administration. Neither dose of AMPH altered concentrations of 5-HIAA (data not
shown). DISCUSSION The present study used the microdialysis technique to assess the role of vesicular DA in AMPH-induced DA release, and to examine the mechanism~ by which reserpine potentiates the behavioral response to AMPH. Concurrent measures of behavioral and neurochemicai changes provide a more accurate indication of the role of specific neurotransmitter systems in the various components of the AMPH response than has been accomplished using in vitro and post mortem techniques or with microdialysis in anesthetized animals.
Effects of reserpine Consistent with its alleged depletion of vesicular pools, reserpine produced a marked reduction of tissue DA, and a delayed but prolonged decrease in striatal extracellular DA concentration. The decrease in baseline dialysate DA concentration in animals with tissue DA levels depleted by approximately 90% supports the view that vesicular pools are involved in the impulse-mediated
neurotransmitter release process. However, we also found that reserpine did not reduce but actually increased striatal DA release in 3 animals (Fig. 2) whose tissue levels of DA were depleted by less than 80% of controls. This observation is consistent with the view that striatal DA stores exceed normal requirements, and, in fact, recent work with 6-hydroxydopamine indicates that a substantial reduction of tissue DA can occur without a corresponding decrease in extracellular DA concentratioJ~! (Scgal and Kuczenski, unpublished observations) 25' 39. It has been shown that reserpine increases nigrostriatal dopaminergic neuronal activitys, perhaps as a consequence of DA depletion in nigral dendrites, which appear to be more sensitive to disrup~ion by reserpine 14' as and which regulate impulse traffic in these neurons t°. The disruption of 90% of striatal vesicular DA stores may preclude an enhanced DA release accompanying increased neuronal activity in most reserpine-pretreated animals. In contrast, the residual vesicular DA pool in the less-depleted group may be sufficiently large to respond to the increased nigrostriatal impulse flow. The transient increase of dialysate DOPAC concentrations immediately following reserpine parallels a similar increase in tissue levels6'33'3s, and presumably reflects the release of vesicular DA to the cytoplasm where it is rapidly degraded by MAO. The temporal delay between the increase in DOPAC and HVA is consistent with the role of HVA as a metabolite of DOPAC. Twenty-four h after reserpine administration, when extracellular DA concentrations were substantially reduced, DOPAC and
88
HVA had returned to pre-reserpine baseline levels (Fig. 1). If DOPAC and HVA were derived from released DA, dialysate concentrations should decline correspondingly. The absence of a decrease in steady-state extraceUular DOPAC and HVA, supports previous results LS"ls'36 which indicate that these metabolites originate primarily from degradation of a cytoplasmic pool of DA to which released DA does not substantially contribute. Behavioral activity during the hour prior to AMPH administration (i.e. 23-24 h after reserpine pretreatment) was reduced only in those reserpine-treated animals which exhibited reduced DA release (Fig. 2). This observation is consistent with previous reports that >90% DA depletion is required to produce gross behavioral changes 25'39, thus further indicating that available DA is in excess of normal demands. The increased levels of dialysate DA in the less-depleted group did not correspond to any obvious change in motor behavior, although more subtle behavioral alterations may have been missed. Further studies will be required to more thoroughly characterize the behavioral profiles associated with different levels of reserpine-induced DA depletion. In accordance with previous observationsa~, reserpine also significantly reduced tissue levels of 5-hydroxytryptamine (5-HT), although not to the same extent as DA. Since dialysate 5-HT was not measured, it is not known whether reserpine produced a corresponding decrease in extracellular 5-HT or whether, as with DA, a threshold level of depletion is necessary to alter 5-HT release, Both extracellular and tissue concentrations of 5-rliAA, the primary metabolite of 5-HT, were found to exhibit a persistent increase following reserpine, in contrast to the relatively transient increase in DA metabolites. The reserpine-induced impairment of vesicular storage in 5-HT terminals may occur more slowly than in DA terminals, thus resulting in a more gradual exposure of 5-HT to cytoplasmic MAO, and a more prolonged increase in metabolite.
Effect of AMPH As previously reported 11.24.26.27.32.34, reserpine augmented the behavioral response to AMPH. This effect was most apparent in the form of an intensification of the stereotypy produced by each of the two doses of AMPH. It has previously been hypothesized n that this potentiation is mediated by a reserpine-induced increase in the AMPH releasable (cytoplasmic) DA pool. Because striatal dopaminergic mechanisms have been implicated in AMPH-induced stereotypies, striatal DA release in response to AMPI-! would be expected to be facilitated by reserpine pretreatment. However. while the present results confirm that AMPH release of DA does not require vesicular neurotransmitter stores, striatal DA
release induced by AMPH was unaffected by reserpine (Fig. 5). Likewise, reserpine failed to enhance AMPHinduced DA release in vitro 2°'22 or in the dialysates of anesthetized rats 2. These results are consistent with our previous studies TM indicating no simple quantitative relationship between AMPH-induced DA release and the appearance of specific components of the stereotypy response, and suggest that the intensification of AMPHinduced behaviors by reserpine does not involve increased striatal DA release. In contrast to the potentiation of AMPH-induced stereotypies by reserpine pretreatment, reserpine actually reduced the duration of the AMPH behavioral response. The reemergence of a reserpinized state in these animals might reflect a more rapid depletion of AMPH-releasable DA. However, at a time when behavioral activity was reduced in the reserpinized group (120-240 rain) (Fig. 4), extracelluiar DA concentrations were not significantly lower (Fig. 5B). Thus there appears to be a dissociation between the temporal pattern of the behavioral and dopaminergic response profiles produced by acute AMPH Is which further indicates that factors besides striatal DA release maintain the behavioral response to AMPH. Other striatal DA mechanisms may underly the augmentation of the AMPH response by reserpine. For example, the low baseline levels of DA release in the reserpinized animals may have resulted in an up-regulation of postsynaptic DA receptors. Consistent with this hypothesis, pretreatment with reserpine has been reported to increase behaviors induced by direct DA receptor agonists 2~.33. However, whereas up-regulation of DA receptors in the presence of continued AMPHinduced DA release should prolong the AMPH behaw ioral response, reserpine-pretreated animals actually exhibited a truncated AMPH behavioral profile (Fig. 4). Likewise, the enhanced baseline DA release in the less depleted animals might be expected to result :,a downregulation of DA receptors, leading to an attenuated AMPH behavioral response. In contrast, however, the behavioral'~esponse to AMPH in these animals w a s n o t decreased in intensity, although, as with the other reserpine-treated animals, the duration of the AMPH behavioral response appeared to be truncated (data not shown). It is also conceivable that interactions between DA and other neurotransmitter systems may be responsible for the augmented AMPH response. Inhibition of 5-HT or norepinephrine activity has been reported to enhance various components of the AMPH response 9,2s, and bo:h of these monoamines are depleted by reserpine. In this regard, it is interesting to note that, whereas tissue 5-HT levels were significantly reduced in the reserpinized
89 group, the less DA-depleted animals did not exhibit a significant 5 - H T depletion. In summary, it appears that D A c,+,mcentrations are far in excess o f normal physiological requirements and that depletion o f about 90% is necessary before impulsemediated D A release is reduced. Moreover, the results o f this study support the view that AMPH-induced D A release is not dependent o n the vesicular D A pool. Although reserpine pretreatment both potentiated and abbreviated the A M P H behavioral response, neither the
magnitude nor the duration of A M P H - i n d u c e d striatal D A release was altered. Together with previous observations of dissociations between striatal D A release and behavior, these data suggest that the A M P H response involves additional non-dopaminergic neurotransmitter systems.
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Acknowledgements. This research was supported by U.S. Public Health Service Grants DA-04157 and DA-01568 and by a Research Scientist Award MH-70183 to D.S.S.C.W.C. was supported by h'~edieal Student Training Grant M-07198.
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