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Enhanced stress-induced dopamine release in the prefrontal cortex of amphetamine-sensitized rats
European Journal of Pharmacology, 237 (1993) 65-71
65
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53119
Enhanced stress-induced dopamine release in the prefrontal cortex of amphetamine-sensitized rats Takashi Hamamura
a n d H a n s C. F i b i g e r
Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
Received 3 December 1992, revised MS received 15 March 1993, accepted 23 March 1993
This study examined the extent to which chronic d-amphetamine administration sensitizes animals to some behavioral and neurochemical effects of foot shock stress. Rats received daily injections of saline for 14 days or d-amphetamine (2 mg/kg 7 days and 4 mg/kg 7 days). After a 7 day drug abstinent period, extracellular dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid concentrations were measured in the medial prefrontal cortex using in vivo microdialysis in freely moving rats. The behavioral responses to mild foot shock stress were enhanced in the d-amphetamine-pretreated subjects. Concomitant with this behavioral sensitization, d-amphetamine-pretreated subjects showed greater stress-induced increases in extracellular dopamine in the medial prefrontal cortex than in controls, d-Amphetamine (2 mg/kg)-induced stereotyped behavior was also enhanced in the amphetamine-pretreated animals compared to controls; however, d-amphetamine-induced increases in extracellular dopamine in the medial prefrontal cortex were not enhanced in the amphetamine-pretreated group. These results suggest that the mesocortical dopaminergic system is involved in cross-sensitization between d-amphetamine and stress, but not in d-amphetamine-induced behavioral sensitization.
Amphetamine; Dopamine; Prefrontal cortex; Stress; Sensitization
1. Introduction
Acutely, a m p h e t a m i n e produces psychomotor stimulation and euphoria in humans; however, repeated exposure to a m p h e t a m i n e generates a very different pattern of effects, including a spectrum of psychotic symptoms (Bell, 1973; Ellinwood et al., 1973; Angrist et al., 1974; Fibiger, 1991). In humans with a history of stimulant drug abuse, a paranoid state can readily be re-induced by low doses of m e t h a m p h e t a m i n e (Sato et al., 1983, 1992). In addition, a paranoid psychotic state can be re-induced in former m e t h a m p h e t a m i n e abusers by psychological stressors (Utena, 1966; Sato et al., 1992). Psychological stress also plays an important role in relapse in schizophrenic patients (Dohrenwend and Egri, 1981). In animal studies, repeated administration of amphetamine causes a progressive augmentation of loco-
Correspondence to: H.C. Fibiger, Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3. Tel. (1) (604) 822-7039, fax (1) (604) 822-7981.
motion and stereotyped behavior; this amphetamineinduced behavioral sensitization has been proposed to be an animal model of paranoid psychosis (Segal and Mandell, 1974; Robinson and Becker, 1986). Stress-induced behaviors are also enhanced in animals with a history of exposure to chronic amphetamine, a phenomenon referred to as cross-sensitization (Antelman et al., 1980; Antelman and Chiodo, 1983). There is considerable evidence that stress increases dopamine metabolism in the medial prefrontal cortex of rats ( H e r m a n et al., 1982; Deutch et al., 1985; Kaneyuki et al., 1991). T h e r e is also evidence that the effects of stress on regional dopamine metabolism are enhanced in amphetamine-sensitized rats (Robinson et al., 1987). A m p h e t a m i n e also increases dopamine release in the medial prefrontal cortex of rats (Moghaddam and Bunney, 1989; Maisonneuve et al., 1990; Kashihara et al., 1991). The present study sought to confirm that there is behavioral cross-sensitization to stress in animals given repeated daily injections of amphetamine. In addition, the extent to which chronic a m p h e t a m i n e may sensitize dopaminergic mechanisms in the medial prefrontal cortex to stress was also determined.
66 2. Materials and methods
2.1. Materials and experimental protocol Male Wistar rats weighing 220-240 g at the start of treatment for chronic experiments, and 350-390 g for acute studies were used. The difference in weights between the chronic and acute groups was imposed to ensure that the animals had similar body weights at the time of surgery. The rats were housed with free access to food and water under a 12 h light-12 h dark cycle (lights on at 7:30 h). Two or three rats were housed in each cage.
detector system for the m e a s u r e m e n t of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid. These compounds were separated by ion-pair reversephase chromatography using a column (150 x 4 . 6 mm, Nucleosil 5 / z m ODS-C18) and a mobile phase consisting of 0.1 M acetic acid buffer (pH 4.1) containing 0.7-0.8 m M octanesulfonic acid, 0.01 mM N a 2 E D T A and 13% methanol. The mobile phase was delivered by a dual-piston p u m p ( B I O - R A D , 1350) at a rate of 1.1 / z l / m i n . T h e e l e c t r o c h e m i c a l detection system (Coulochem II, ESA) was set as follows: guard cell, + 400 mV; oxidation electrode, + 400 mV and reduction electrode, - 3 0 0 inV.
2.4. Histology 2.2. Surgery and microdialysis procedure Rats were anaesthetized with sodium pentobarbital (60 m g / k g i.p.) and placed in a stereotaxic frame. The skull was exposed and a hole was drilled for the implantation of a dialysis probe into the medial prefrontal cortex (coordinates: rostral +4.8 m m from bregma, lateral + 0.7 m m from the midline, and ventral - 5 . 5 m m from the dura) according to the atlas of Pellegrino et al. (1979). The microdialysis probe, a variant of concentric type ( D a m s m a et al., 1992), was implanted and secured with dental cement and skull screws. The active dialysis portion was 4.0 m m in length and was made of copolymer of polyacrylonitrile and sodium methallyl sulfonate (40 000 molecular cutoff; i.d. 0.24 mm; Hospal Inc.). The inlet and outlet were m a d e from fused silica capillary tubing (i.d. 7.5 /xm; o.d. 150 /zm; Polymicro Technologies, Phoenix, A Z ) and were enclosed in 22 gauge stainless-steel tubing. After surgery rats were housed individually for 2 days in Plexiglas cages for recovery. Dialysis experiments began 48 h after surgery. Rats were placed in a shock box (Plexiglas 35 x 25 x 35 cm with a grid floor) for experiment 2 and in a Plexiglas cage with a solid floor for the other experiments. The inlet of the probe was connected to a microinfusion p u m p (Harvard) and the probe outlet to an autoinjector (Valco Inst. Inc.) using polyethylene tubing (PE-10, Clay Adams). The probe was perfused with artificial CSF containing (mM) 147 NaC1, 3.0 KC1, 1.0 MgCI2, 1.3 CaCl 2 and 1.0 sodium phosphate buffer (pH 7.4) at a rate of 5 /zl/min. The perfusate was automatically injected into the analytical system every 20 min. All experiments were started after a 3 h habituation period.
2.3. Biochemical assay The perfusates were injected directly into a reverse-phase ion-pair H P L C with an electrochemical
After completion of the experiments, rats were killed by an overdose of pentobarbital. The brains were removed and fixed in 4% paraformaldehyde for at least 4 days. Using a cryostat, 50 /zm coronal sections were obtained, stained with cresyl violet, and the position of each probe was determined with the aid of a microscope.
2.5. Statistical analysis The means of three samples immediately before treatment was regarded as the basal value and the change in levels of extracellular dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid are expressed as a percentage of the basal value. Statistical analysis of data was carried out using the Mann-Whitney U-test for behavioral scores, one-way A N O V A for the basal levels of dopamine and its metabolites and two-way A N O V A for changes in dopamine and its metabolites. Individual comparisons were performed using Scheffd's F-test.
2.6. Experiment 1 To determine if dopamine present in the dialysate samples that were obtained from the medial prefrontal cortex reflected neuronal release, the effects of perfusate containing nomifensine (10 -6 M; Hoechst) or tetrodotoxin (10 -6 M; Sigma), and of a Ca2+-free perfusion solution were evaluated (n = 4). Artificial CSF with CaC12 removed contained 2.3 mM MgCI 2.
2. 7. Experiment 2 A m p h e t a m i n e - p r e t r e a t e d group: rats (n = 8) were injected i.p. once daily with d-amphetamine (2 m g / k g per ml) for 7 consecutive days, and amphetamine (4 m g / k g per ml) for the next 7 consecutive days. Am-
67
phetamine was dissolved in 0.9% saline. Control group: rats (n = 8) were injected with saline (1 m l / m g ) for 14 consecutive days. Five days after the last injection, probes were implanted in the medial prefrontal cortex of both groups. Two days after probe implantation, each rat was placed in a shock box. After a 3 h habituation period, foot shock (0.4 mA, 10 s duration, 50 s interval for 20 min) was applied. During foot shock each animal was observed for the presence or absence of several behaviors. The presence or absence of each behavior was recorded in 20 10 s periods; thus, the maximal score for each behavior was 20. The scored behaviors were as follows: walking with exploratory behavior, running in the cage, rearing, crouching, flinch and jump. One hundred minutes after the last foot shock, each animal was injected with d-amphetamine (2 m g / k g i.p.). After the amphetamine injection, behavioral assessment was conducted for 40 min, and the intensity of stereotypy was estimated according to the rating scale of Akiyama et al. (1982): 0, asleep or still; i, locomotion with normal exploration and a normal pattern of sniffing; 2, hyper-locomotion with repetitive exploratory behavior, rearing or increased rate of sniffing; 3, discontinuous sniffing with periodic locomotor activity; 4, continuous sniffing without locomotion. Throughout the experiment dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid from the medial prefrontal cortex were measured and behavior was recorded by video tape for subsequent behavioral rating.
2.8. Experiment 3 To determine if 2 m g / k g of d-amphetamine maximally increased extracellular dopamine concentrations in the medial prefrontal cortex, naive rats were injected i.p. with d-amphetamine at either 2 m g / k g (n = 8) or 4 m g / k g (n = 6).
3. Results
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significant decrease in extracellular levels to 9_+ 5% (F(3,9) = 63.8, P < 0.01).
3.2. Experiment 2 Foot shock elicited a number of behaviors (fig. 2) that were significantly enhanced in the amphetaminesensitized animals: running (P < 0.01); flinch (P < 0.01); jump (P < 0.02) and vocalization (P < 0.01). For the saline control group basal values for dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid were 1.00 _ 0.14, 65.6 _+ 9.4 and 114.5 _+ 11.5 fmol/min. Basal values from the d-amphetamine-pretreated subjects were: dopamine, 1.14 _+ 0.10; 3,4-dihydroxyphenylacetic acid, 91.1 _+ 15.4 and homovanillic acid, 162.8 + 21.1 fmol/min. There were no significant differences in these basal values between the amphetamine- and saline-pretreated groups.
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3.1. Experiment 1 In drug-naive subjects, perfusion with nomifensine for 20 min significantly increased extracellufar dopamine concentrations (F(7,21)= 50.5, P < 0.01) with the maximal change being 368 _+ 40% (mean _+ S.E.M.) (fig. 1). After dopamine returned to basal levels, perfusion with a CaZ+-free solution caused a significant decrease in dopamine levels to 11 + 2% of basal values (F(3,9) = 45.9, P < 0.01). Reperfusion with normal artificial CSF restored extracellular dopamine to 63 + 7% of the basal values. Subsequent perfusion with tetrodotoxin ( 1 0 - 6 M ) for 60 min caused a further (10 -6 M)
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Fig. 2. Behavioral ratings of amphetamine-pretreated and control rats during foot shock. Behavior was scored as present or absent over 20 periods of 10 s each; thus, the maximal score for each behavior component is 20. Values represent m e a n s ± S . E . M . (n = 8). Open bars: control group. Shaded bars: amphetamine-pretreated group. * P < 0.05 amphetamine-pretreated group vs. control group.
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TIME(min) Fig. 3. Dialysate concentrations of dopamine obtained from medial prefrontal cortex in response to foot shock and amphetamine (2 m g / k g i.p.) in rats that had previously received repeated injections of saline ( o ) o r amphetamine (e). Values represent means _+S.E.M. (n = 8). * P < 0.05 amphetamine-pretreated vs. control group.
During foot shock, extracellular dopamine concentrations increased and the maximal change was observed in perfusates collected during the 0-20 min sampling period (fig. 3). The maximal increases were to 363 _+ 37% of the baseline values in the amphetaminepretreated group and 225 _+ 22% in the saline-pretreated animals. The increase in extracellular dopamine was significantly greater in the amphetamine-sensitized group than in the saline-pretreated group (group effect, F(1,14)= 8.064, P < 0.05; time effect, F(6,84)= 63.565, P < 0.01; group x time interaction, F(6,84) = 6.539, P < 0.01). Extracellular 3,4-dihydroxyphenylacetic acid was also increased by footshock with the maximal change being observed at 20-40 min (group effect, F(1,14)= 2.852 n.s.; time effect, F(6,84)= 36.748, P <0.01; g r o u p × time interaction, F(6,84) = 2.471, P < 0.05) (fig. 4). The maximal increases were to 186 _+ 19% of the baseline value in the amphetamine-pretreated group and 152 +_ 8% in saline-pretreated group. Extracellular ho-
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TIME(min) Fig. 5. Dialysate concentrations of homovanillic acid obtained from medial prefrontal cortex in response to foot shock and amphetamine (2 m g / k g i.p.) in rats that had previously received repeated injections of saline ( o ) or amphetamine (e). Values represent means_+ S.E.M. (n = 8). * P < 0.05 amphetamine-pretreated vs. control group.
movanillic acid also increased after footshock stress and the maximal change was observed in perfusates collected at 20-40 min (fig. 5). In the amphetaminepretreated group the increase in extracellular homovanillic acid was significantly greater than in the saline-pretreated group (group effect F(1,14)= 5.366, P < 0.05; time effect, F(6,84) = 27.742, P < 0.01; group × time interaction, F(6,84) = 2.929, P < 0.05). The maximal increases were to 156 _+ 12% of baseline values in the amphetamine-pretreated group and 130_+ 6% in saline-pretreated group. Amphetamine produced marked increases in extracellular dopamine in the medial prefrontal cortex of both groups. However, no significant differences were observed between the amphetamine-pretreated and saline-pretreated groups (fig. 3). While extracellular concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid decreased after amphetamine challenge, there were no significant differences between the two groups (figs. 4, 5). After the amphetamine (2
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TIME(min) Fig. 6. B e h a v i o r a l s t e r e o t y p y r a t i n g s a f t e r a m p h e t a m i n e (2 m g / k g ) in r a t s t h a t h a d p r e v i o u s l y r e c e i v e d r e p e a t e d i n j e c t i o n s o f saline ( o ) o r a m p h e t a m i n e (e). * p < 0.05 a m p h e t a m i n e - p r e t r e a t e d vs. c o n t r o l group.
69
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TIME(min) Fig. 7. Dialysate concentrations of dopamine obtained from the medial prefrontal cortex following a m p h e t a m i n e 2 m g / k g ( o , n = 8) and 4 m g / k g (e, n = 6). Values represent means_+ S.E.M. * P < 0.05 2 m g / k g a m p h e t a m i n e vs. 4 m g / k g amphetamine.
m g / k g ) challenge, the amphetamine-pretreated group displayed significantly augmented stereotyped behavior compared to the saline-pretreated group (fig. 6).
3.3. Experiment 3 Acute injection of d-amphetamine (4 m g / k g ) markedly increased extracellular dopamine concentrations (fig. 7), the maximum increases being to 1010 _+ 142% of the baseline values. Compared to the effects of acute injections of 2 m g / k g , the higher dose increased dopamine to a significantly greater extent (dose effect, F(1,12) = 18.598, P < 0.01; time effect, F(9,108) = 34.016, P < 0.01; dose x time interaction, F(9,108) = 4.83, P < 0.01).
4. Discussion
Experiment 1 demonstrated that extracellular dopamine measured in dialysates from the medial prefrontal cortex primarily reflects neural activity, inasmuch as perfusion with tetrodotoxin or a CaZ+-free solution greatly decreased dialysate concentrations of this neurotransmitter. Experiment 2 demonstrated that the amphetamine-pretreated group showed significantly enhanced behavioral responses to foot shock. A number of stressors, including foot shock, tail pinch, food deprivation and restraint have been reported to produce behavioral cross-sensitization with amphetamine (Antelman and Eichler, 1978; Antelman et al., 1980; Antelman and Chiodo, 1983; H e r m a n et al., 1984; Robinson et al., 1985a,b). Concomitant with this behavioral cross-sensitization, the amphetamine-pretreated group also showed augmented increases in dopamine release in the medial prefrontal cortex. These results are consistent with some previous reports. For example, Robinson et al. (1987) have reported that
amphetamine pretreatment produces a more rapid onset in foot shock-induced increases in 3,4-dihydroxyphenylacetic a c i d / d o p a m i n e ratios in the medial prefrontal cortex. In earlier work, however, Robinson and coworkers (1985a) reported that previous exposure to d-amphetamine produced enduring enhancement in mesocortical dopamine utilization in the absence of foot shock stress, such that the addition of foot shock stress did not further enhance dopamine utilization in the frontal cortex of amphetamine-pretreated animals. In the present study amphetamine pretreatment did not change basal concentrations of extracellular dopamine and its metabolites in the medial prefrontal cortex; however, foot shock stress produced an augmented increase of extracellular dopamine and its metabolites in the amphetamine-pretreated subjects. The interpretation of the augmented dopaminergic response to footshock in the amphetamine-pretreated group is uncertain because the functional significance of dopamine release in the medial prefrontal cortex during stress is unclear. D'Angio et al. (1988) have obtained evidence that the increase in cortical dopamine metabolism induced by stressors is not a direct neurochemical correlate of emotional responses caused by aversive stimuli but may rather underlie heightened attention or activation of cognitive processes in attempts to cope with stressors. Specifically, these authors found that a number of stressful stimuli increased extracellular 3,4-dihydroxyphenylacetic acid concentrations in the medial prefrontal cortex of low but not high emotion rats. In the present study, damphetamine-pretreated rats showed more intense behavioral responses to foot shock. Dopamine release in medial prefrontal cortex was also augmented. While the most parsimonious explanation for these observations is that the increase in dopamine release is directly related to the increased emotional responsiveness of the amphetamine-pretreated animals, the data from D'Angio et al. (1988) suggest that this may not be the case. In this regard, Imperato et al. (1991) have shown with microdialysis that dopamine release in the medial prefrontal cortex is transiently enhanced first during restraint stress and again when the animal is released from restraint. Therefore, there does not appear to be an exclusive relationship between dopamine release in the medial prefrontal cortex and exposure to stressful stimuli, despite the fact that a variety of stressors, as well as the anxiogenic /3-carboline FG7142, can enhance dopamine release in this part of the brain (Bradberry et al., 1991). A unifying hypothesis about the functional significance of dopamine release in the medial prefrontal cortex does not appear to be possible at the present time and must await the results of additional experiments. Sorg and Kalivas (1993) have recently reported that chronic cocaine pretreatment reduces the increase of
70
extracellular dopamine in the medial prefrontal cortex produced by foot shock stress, an effect that is opposite to the results reported here. Both cocaine and amphetamine can induce psychosis and behavioral sensitization; moreover, cocaine and amphetamine show cross-sensitization, both behaviorally and neurochemically (Shuster et al., 1977; Kazahaya et al., 1989; Akimoto et al., 1990). It seems unlikely, therefore, that these disparate results are due to pharmacological differences between amphetamine and cocaine. Earlier, Kalivas and Duffy (1989) reported that a cocaine sensitization regimen enhanced increases in tissue concentrations of 3,4-dihydroxyphenylacetic acid in the medial prefrontal cortex produced by foot shock stress and concluded that cocaine pretreatment enhanced stressinduced increases in dopamine metabolism in medial prefrontal cortex. These latter findings are more consistent with the results reported here. They are also consistent with findings by Robinson et al. (1987) showing that footshock-induced increases in dopamine utilization in the medial prefrontal cortex are enhanced in amphetamine-sensitized rats. At present, there does not appear to be a satisfactory explanation for the discrepancy between the present results and those of Sorg and Kalivas (1993). Experiment 2 showed that there was behavioral sensitization to amphetamine in the amphetamine-pretreated group. However, and somewhat surprisingly, d-amphetamine-induced increases in extracellular dopamine in the medial prefrontal cortex of the amphetamine-pretreated group did not differ from those in the saline-pretreated group. Experiment 3 demonstrated that this result was not due to a ceiling effect by showing that dopamine output in the medial prefrontal cortex can be increased to a greater extent than is produced by 2 m g / k g of d-amphetamine (fig. 6). Robinson et al. (1988) have reported that amphetamine produces an augmented increase in extracellular dopamine in the nucleus accumbens of amphetamine-sensitized animals. Similar effects of repeated amphetamine injections on amphetamine-induced dopamine release have also been observed in striatum (Kazahaya et al., 1989; Hamamura et al., 1991). In contrast to these observations, Segal and Kuczenski (1992) have reported that repeated amphetamine administration attenuates subsequent effects of amphetamine on dopamine release in nucleus accumbens and striatum. The reasons for this discrepancy are again not immediately apparent but may be related to the fact that Segal and Kuczenski (1992) used somewhat shorter drug-abstinent periods than other investigators, and that the d-amphetamine challenge (2.5 m g / k g ) induced unusually large increases (about 5000%) in extracellular dopamine concentrations, values several times higher than in other reports (Robinson et al., 1988; Westerink et al., 1989; Hama-
mura et al., 1991). Kalivas and Duffy (1993) have obtained evidence that the effects of a cocaine challenge can vary significantly as a function of the abstinent period after repeated cocaine administration, with these neurochemical indices of sensitization becoming apparent only at longer abstinent periods. In conclusion, the present study has confirmed that amphetamine-pretreated rats show behavioral crosssensitization to foot shock stress and enhanced behavioral responses to amphetamine. In the medial prefrontal cortex of the amphetamine-pretreated group, stress-induced increases in extracellular dopamine were augmented while amphetamine-induced increases were not affected. This suggests that while dopaminergic mechanisms in the medial prefrontal cortex may be involved in behavioral hetero-sensitization between amphetamine and footshock stress, they do not mediate amphetamine homo-sensitization (i.e. amphetamine sensitization to itself).
Acknowledgements We gratefully acknowledge C.H.M. Beck for his valuable comments. Thanks also to Catriona Wilson for outstanding technical assistance. This work is supported by grants from the Medical Research Council of Canada (PG23) and Bristol-Myers/Squibb. T. Hamamura is supported by the Uehara Memorial Foundation, Japan.
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Maisonneuve, I.M., R.W. Keller and S.D. Glick, 1990, Similar effects of d-amphetamine and cocaine on extracellular dopamine levels in medial prefrontal cortex of rats, Brain Res. 535, 221. Moghaddam, B. and B.S. Bunney, 1989, Differential effect of cocaine on extracellular dopamine levels in rat medial prefrontal cortex and nucleus accumbens comparison to amphetamine, Synapse 4, 156. Pellegrino, L.J., A.S. Pellegrino and A.J. Cushman, 1979, A Stereotaxic Atlas of the Rat Brain (Plenum, New York). Robinson, T.E. and J.B. Becker, 1986, Enduring changes in the brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis, Brain Res. Rev. 11, 157. Robinson, T.E., J.B. Becker, C.J. Moore, E. Castaneda and G. Mittleman, 1985a, Enduring enhancement in frontal cortex dopamine utilization in an animal model of amphetamine psychosis, Brain Res. 343, 374. Robinson, T.E., A.L. Angus and J.B. Becker, 1985b, Sensitization to stress: the enduring effects of prior stress on amphetamine-induced rotational behavior, Life Sci. 37, 1039. Robinson, T.E., J.B. Becker, E.A. Young, H. Akil and E. Castaneda, 1987, The effects of footshock stress on regional brain dopamine metabolism and pituitary /3-endorphin release in rats previously sensitized to amphetamine, Neuropharmacology 26, 679. Robinson, T.E., P.A, Jurson, J.A. Bennett and K.M. Bentgen, 1988, Persistent sensitization of dopamine neurotransmission in ventral striatum (nucleus accumbens) produced by prior experience with (+)-amphetamine: a microdialysis study in freely moving rats, Brain Res. 462, 211. Sato, M., C.C. Chen, K. Akiyama and S. Otsuki, 1983, Acute exacerbation of paranoid psychotic state after long-term abstinence in patient with previous methamphetamine psychosis, Biol. Psychiat. 18, 429. Sato, M., Y. Numach and T. Hamamura, 1992, Relapse of paranoid psychotic state in methamphetamine model of schizophrenia, Schizophr. Bull. 18, 115. Segal, D.S. and R. Kuczenski, 1992, In vivo microdialysis reveals a diminished amphetamine-induced DA response corresponding to behavioral sensitization produced by repeated amphetamine pretreatment, Brain Res. 571,330. Segal, D.S. and A.J. Mandell, 1974, Long-term administration of d-amphetamine: progressive augmentation of motor activity and stereotypy, Pharmacol. Biochem. Behav. 2, 249. Shuster, L., G. Yu and A. Bates, 1977, Sensitization to cocaine stimulation in mice, Psychopharmacology 52, 185. Sorg, B.A. and P.W. Kalivas, Effects of cocaine and footshock stress on extracellular dopamine levels in the medial prefrontal cortex, Neuroscience (in press). Utena, H., 1966, Behavioral aberration in methamphetamine-intoxicated animals and chemical correlates in the brain, Progress in Brain Research: Correlative Neuroscience-Clinical Studies, Vol. 21B, eds. T. Tokizane and J.P. Schad6 (Elsevier, Amsterdam) p. 192. Westerink, B.H.C., R.M. Hofsteede, J. Tuntler and J.B. De Vries, 1989, Use of calcium antagonism for the characterization of drug-evoked dopamine release from the brain of conscious rats determined by microdialysis, J. Neurochem. 52, 722.
65
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53119
Enhanced stress-induced dopamine release in the prefrontal cortex of amphetamine-sensitized rats Takashi Hamamura
a n d H a n s C. F i b i g e r
Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
Received 3 December 1992, revised MS received 15 March 1993, accepted 23 March 1993
This study examined the extent to which chronic d-amphetamine administration sensitizes animals to some behavioral and neurochemical effects of foot shock stress. Rats received daily injections of saline for 14 days or d-amphetamine (2 mg/kg 7 days and 4 mg/kg 7 days). After a 7 day drug abstinent period, extracellular dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid concentrations were measured in the medial prefrontal cortex using in vivo microdialysis in freely moving rats. The behavioral responses to mild foot shock stress were enhanced in the d-amphetamine-pretreated subjects. Concomitant with this behavioral sensitization, d-amphetamine-pretreated subjects showed greater stress-induced increases in extracellular dopamine in the medial prefrontal cortex than in controls, d-Amphetamine (2 mg/kg)-induced stereotyped behavior was also enhanced in the amphetamine-pretreated animals compared to controls; however, d-amphetamine-induced increases in extracellular dopamine in the medial prefrontal cortex were not enhanced in the amphetamine-pretreated group. These results suggest that the mesocortical dopaminergic system is involved in cross-sensitization between d-amphetamine and stress, but not in d-amphetamine-induced behavioral sensitization.
Amphetamine; Dopamine; Prefrontal cortex; Stress; Sensitization
1. Introduction
Acutely, a m p h e t a m i n e produces psychomotor stimulation and euphoria in humans; however, repeated exposure to a m p h e t a m i n e generates a very different pattern of effects, including a spectrum of psychotic symptoms (Bell, 1973; Ellinwood et al., 1973; Angrist et al., 1974; Fibiger, 1991). In humans with a history of stimulant drug abuse, a paranoid state can readily be re-induced by low doses of m e t h a m p h e t a m i n e (Sato et al., 1983, 1992). In addition, a paranoid psychotic state can be re-induced in former m e t h a m p h e t a m i n e abusers by psychological stressors (Utena, 1966; Sato et al., 1992). Psychological stress also plays an important role in relapse in schizophrenic patients (Dohrenwend and Egri, 1981). In animal studies, repeated administration of amphetamine causes a progressive augmentation of loco-
Correspondence to: H.C. Fibiger, Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3. Tel. (1) (604) 822-7039, fax (1) (604) 822-7981.
motion and stereotyped behavior; this amphetamineinduced behavioral sensitization has been proposed to be an animal model of paranoid psychosis (Segal and Mandell, 1974; Robinson and Becker, 1986). Stress-induced behaviors are also enhanced in animals with a history of exposure to chronic amphetamine, a phenomenon referred to as cross-sensitization (Antelman et al., 1980; Antelman and Chiodo, 1983). There is considerable evidence that stress increases dopamine metabolism in the medial prefrontal cortex of rats ( H e r m a n et al., 1982; Deutch et al., 1985; Kaneyuki et al., 1991). T h e r e is also evidence that the effects of stress on regional dopamine metabolism are enhanced in amphetamine-sensitized rats (Robinson et al., 1987). A m p h e t a m i n e also increases dopamine release in the medial prefrontal cortex of rats (Moghaddam and Bunney, 1989; Maisonneuve et al., 1990; Kashihara et al., 1991). The present study sought to confirm that there is behavioral cross-sensitization to stress in animals given repeated daily injections of amphetamine. In addition, the extent to which chronic a m p h e t a m i n e may sensitize dopaminergic mechanisms in the medial prefrontal cortex to stress was also determined.
66 2. Materials and methods
2.1. Materials and experimental protocol Male Wistar rats weighing 220-240 g at the start of treatment for chronic experiments, and 350-390 g for acute studies were used. The difference in weights between the chronic and acute groups was imposed to ensure that the animals had similar body weights at the time of surgery. The rats were housed with free access to food and water under a 12 h light-12 h dark cycle (lights on at 7:30 h). Two or three rats were housed in each cage.
detector system for the m e a s u r e m e n t of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid. These compounds were separated by ion-pair reversephase chromatography using a column (150 x 4 . 6 mm, Nucleosil 5 / z m ODS-C18) and a mobile phase consisting of 0.1 M acetic acid buffer (pH 4.1) containing 0.7-0.8 m M octanesulfonic acid, 0.01 mM N a 2 E D T A and 13% methanol. The mobile phase was delivered by a dual-piston p u m p ( B I O - R A D , 1350) at a rate of 1.1 / z l / m i n . T h e e l e c t r o c h e m i c a l detection system (Coulochem II, ESA) was set as follows: guard cell, + 400 mV; oxidation electrode, + 400 mV and reduction electrode, - 3 0 0 inV.
2.4. Histology 2.2. Surgery and microdialysis procedure Rats were anaesthetized with sodium pentobarbital (60 m g / k g i.p.) and placed in a stereotaxic frame. The skull was exposed and a hole was drilled for the implantation of a dialysis probe into the medial prefrontal cortex (coordinates: rostral +4.8 m m from bregma, lateral + 0.7 m m from the midline, and ventral - 5 . 5 m m from the dura) according to the atlas of Pellegrino et al. (1979). The microdialysis probe, a variant of concentric type ( D a m s m a et al., 1992), was implanted and secured with dental cement and skull screws. The active dialysis portion was 4.0 m m in length and was made of copolymer of polyacrylonitrile and sodium methallyl sulfonate (40 000 molecular cutoff; i.d. 0.24 mm; Hospal Inc.). The inlet and outlet were m a d e from fused silica capillary tubing (i.d. 7.5 /xm; o.d. 150 /zm; Polymicro Technologies, Phoenix, A Z ) and were enclosed in 22 gauge stainless-steel tubing. After surgery rats were housed individually for 2 days in Plexiglas cages for recovery. Dialysis experiments began 48 h after surgery. Rats were placed in a shock box (Plexiglas 35 x 25 x 35 cm with a grid floor) for experiment 2 and in a Plexiglas cage with a solid floor for the other experiments. The inlet of the probe was connected to a microinfusion p u m p (Harvard) and the probe outlet to an autoinjector (Valco Inst. Inc.) using polyethylene tubing (PE-10, Clay Adams). The probe was perfused with artificial CSF containing (mM) 147 NaC1, 3.0 KC1, 1.0 MgCI2, 1.3 CaCl 2 and 1.0 sodium phosphate buffer (pH 7.4) at a rate of 5 /zl/min. The perfusate was automatically injected into the analytical system every 20 min. All experiments were started after a 3 h habituation period.
2.3. Biochemical assay The perfusates were injected directly into a reverse-phase ion-pair H P L C with an electrochemical
After completion of the experiments, rats were killed by an overdose of pentobarbital. The brains were removed and fixed in 4% paraformaldehyde for at least 4 days. Using a cryostat, 50 /zm coronal sections were obtained, stained with cresyl violet, and the position of each probe was determined with the aid of a microscope.
2.5. Statistical analysis The means of three samples immediately before treatment was regarded as the basal value and the change in levels of extracellular dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid are expressed as a percentage of the basal value. Statistical analysis of data was carried out using the Mann-Whitney U-test for behavioral scores, one-way A N O V A for the basal levels of dopamine and its metabolites and two-way A N O V A for changes in dopamine and its metabolites. Individual comparisons were performed using Scheffd's F-test.
2.6. Experiment 1 To determine if dopamine present in the dialysate samples that were obtained from the medial prefrontal cortex reflected neuronal release, the effects of perfusate containing nomifensine (10 -6 M; Hoechst) or tetrodotoxin (10 -6 M; Sigma), and of a Ca2+-free perfusion solution were evaluated (n = 4). Artificial CSF with CaC12 removed contained 2.3 mM MgCI 2.
2. 7. Experiment 2 A m p h e t a m i n e - p r e t r e a t e d group: rats (n = 8) were injected i.p. once daily with d-amphetamine (2 m g / k g per ml) for 7 consecutive days, and amphetamine (4 m g / k g per ml) for the next 7 consecutive days. Am-
67
phetamine was dissolved in 0.9% saline. Control group: rats (n = 8) were injected with saline (1 m l / m g ) for 14 consecutive days. Five days after the last injection, probes were implanted in the medial prefrontal cortex of both groups. Two days after probe implantation, each rat was placed in a shock box. After a 3 h habituation period, foot shock (0.4 mA, 10 s duration, 50 s interval for 20 min) was applied. During foot shock each animal was observed for the presence or absence of several behaviors. The presence or absence of each behavior was recorded in 20 10 s periods; thus, the maximal score for each behavior was 20. The scored behaviors were as follows: walking with exploratory behavior, running in the cage, rearing, crouching, flinch and jump. One hundred minutes after the last foot shock, each animal was injected with d-amphetamine (2 m g / k g i.p.). After the amphetamine injection, behavioral assessment was conducted for 40 min, and the intensity of stereotypy was estimated according to the rating scale of Akiyama et al. (1982): 0, asleep or still; i, locomotion with normal exploration and a normal pattern of sniffing; 2, hyper-locomotion with repetitive exploratory behavior, rearing or increased rate of sniffing; 3, discontinuous sniffing with periodic locomotor activity; 4, continuous sniffing without locomotion. Throughout the experiment dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid from the medial prefrontal cortex were measured and behavior was recorded by video tape for subsequent behavioral rating.
2.8. Experiment 3 To determine if 2 m g / k g of d-amphetamine maximally increased extracellular dopamine concentrations in the medial prefrontal cortex, naive rats were injected i.p. with d-amphetamine at either 2 m g / k g (n = 8) or 4 m g / k g (n = 6).
3. Results
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significant decrease in extracellular levels to 9_+ 5% (F(3,9) = 63.8, P < 0.01).
3.2. Experiment 2 Foot shock elicited a number of behaviors (fig. 2) that were significantly enhanced in the amphetaminesensitized animals: running (P < 0.01); flinch (P < 0.01); jump (P < 0.02) and vocalization (P < 0.01). For the saline control group basal values for dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid were 1.00 _ 0.14, 65.6 _+ 9.4 and 114.5 _+ 11.5 fmol/min. Basal values from the d-amphetamine-pretreated subjects were: dopamine, 1.14 _+ 0.10; 3,4-dihydroxyphenylacetic acid, 91.1 _+ 15.4 and homovanillic acid, 162.8 + 21.1 fmol/min. There were no significant differences in these basal values between the amphetamine- and saline-pretreated groups.
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3.1. Experiment 1 In drug-naive subjects, perfusion with nomifensine for 20 min significantly increased extracellufar dopamine concentrations (F(7,21)= 50.5, P < 0.01) with the maximal change being 368 _+ 40% (mean _+ S.E.M.) (fig. 1). After dopamine returned to basal levels, perfusion with a CaZ+-free solution caused a significant decrease in dopamine levels to 11 + 2% of basal values (F(3,9) = 45.9, P < 0.01). Reperfusion with normal artificial CSF restored extracellular dopamine to 63 + 7% of the basal values. Subsequent perfusion with tetrodotoxin ( 1 0 - 6 M ) for 60 min caused a further (10 -6 M)
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Fig. 2. Behavioral ratings of amphetamine-pretreated and control rats during foot shock. Behavior was scored as present or absent over 20 periods of 10 s each; thus, the maximal score for each behavior component is 20. Values represent m e a n s ± S . E . M . (n = 8). Open bars: control group. Shaded bars: amphetamine-pretreated group. * P < 0.05 amphetamine-pretreated group vs. control group.
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TIME(min) Fig. 3. Dialysate concentrations of dopamine obtained from medial prefrontal cortex in response to foot shock and amphetamine (2 m g / k g i.p.) in rats that had previously received repeated injections of saline ( o ) o r amphetamine (e). Values represent means _+S.E.M. (n = 8). * P < 0.05 amphetamine-pretreated vs. control group.
During foot shock, extracellular dopamine concentrations increased and the maximal change was observed in perfusates collected during the 0-20 min sampling period (fig. 3). The maximal increases were to 363 _+ 37% of the baseline values in the amphetaminepretreated group and 225 _+ 22% in the saline-pretreated animals. The increase in extracellular dopamine was significantly greater in the amphetamine-sensitized group than in the saline-pretreated group (group effect, F(1,14)= 8.064, P < 0.05; time effect, F(6,84)= 63.565, P < 0.01; group x time interaction, F(6,84) = 6.539, P < 0.01). Extracellular 3,4-dihydroxyphenylacetic acid was also increased by footshock with the maximal change being observed at 20-40 min (group effect, F(1,14)= 2.852 n.s.; time effect, F(6,84)= 36.748, P <0.01; g r o u p × time interaction, F(6,84) = 2.471, P < 0.05) (fig. 4). The maximal increases were to 186 _+ 19% of the baseline value in the amphetamine-pretreated group and 152 +_ 8% in saline-pretreated group. Extracellular ho-
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TIME(min) Fig. 5. Dialysate concentrations of homovanillic acid obtained from medial prefrontal cortex in response to foot shock and amphetamine (2 m g / k g i.p.) in rats that had previously received repeated injections of saline ( o ) or amphetamine (e). Values represent means_+ S.E.M. (n = 8). * P < 0.05 amphetamine-pretreated vs. control group.
movanillic acid also increased after footshock stress and the maximal change was observed in perfusates collected at 20-40 min (fig. 5). In the amphetaminepretreated group the increase in extracellular homovanillic acid was significantly greater than in the saline-pretreated group (group effect F(1,14)= 5.366, P < 0.05; time effect, F(6,84) = 27.742, P < 0.01; group × time interaction, F(6,84) = 2.929, P < 0.05). The maximal increases were to 156 _+ 12% of baseline values in the amphetamine-pretreated group and 130_+ 6% in saline-pretreated group. Amphetamine produced marked increases in extracellular dopamine in the medial prefrontal cortex of both groups. However, no significant differences were observed between the amphetamine-pretreated and saline-pretreated groups (fig. 3). While extracellular concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid decreased after amphetamine challenge, there were no significant differences between the two groups (figs. 4, 5). After the amphetamine (2
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TIME(min) Fig. 6. B e h a v i o r a l s t e r e o t y p y r a t i n g s a f t e r a m p h e t a m i n e (2 m g / k g ) in r a t s t h a t h a d p r e v i o u s l y r e c e i v e d r e p e a t e d i n j e c t i o n s o f saline ( o ) o r a m p h e t a m i n e (e). * p < 0.05 a m p h e t a m i n e - p r e t r e a t e d vs. c o n t r o l group.
69
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TIME(min) Fig. 7. Dialysate concentrations of dopamine obtained from the medial prefrontal cortex following a m p h e t a m i n e 2 m g / k g ( o , n = 8) and 4 m g / k g (e, n = 6). Values represent means_+ S.E.M. * P < 0.05 2 m g / k g a m p h e t a m i n e vs. 4 m g / k g amphetamine.
m g / k g ) challenge, the amphetamine-pretreated group displayed significantly augmented stereotyped behavior compared to the saline-pretreated group (fig. 6).
3.3. Experiment 3 Acute injection of d-amphetamine (4 m g / k g ) markedly increased extracellular dopamine concentrations (fig. 7), the maximum increases being to 1010 _+ 142% of the baseline values. Compared to the effects of acute injections of 2 m g / k g , the higher dose increased dopamine to a significantly greater extent (dose effect, F(1,12) = 18.598, P < 0.01; time effect, F(9,108) = 34.016, P < 0.01; dose x time interaction, F(9,108) = 4.83, P < 0.01).
4. Discussion
Experiment 1 demonstrated that extracellular dopamine measured in dialysates from the medial prefrontal cortex primarily reflects neural activity, inasmuch as perfusion with tetrodotoxin or a CaZ+-free solution greatly decreased dialysate concentrations of this neurotransmitter. Experiment 2 demonstrated that the amphetamine-pretreated group showed significantly enhanced behavioral responses to foot shock. A number of stressors, including foot shock, tail pinch, food deprivation and restraint have been reported to produce behavioral cross-sensitization with amphetamine (Antelman and Eichler, 1978; Antelman et al., 1980; Antelman and Chiodo, 1983; H e r m a n et al., 1984; Robinson et al., 1985a,b). Concomitant with this behavioral cross-sensitization, the amphetamine-pretreated group also showed augmented increases in dopamine release in the medial prefrontal cortex. These results are consistent with some previous reports. For example, Robinson et al. (1987) have reported that
amphetamine pretreatment produces a more rapid onset in foot shock-induced increases in 3,4-dihydroxyphenylacetic a c i d / d o p a m i n e ratios in the medial prefrontal cortex. In earlier work, however, Robinson and coworkers (1985a) reported that previous exposure to d-amphetamine produced enduring enhancement in mesocortical dopamine utilization in the absence of foot shock stress, such that the addition of foot shock stress did not further enhance dopamine utilization in the frontal cortex of amphetamine-pretreated animals. In the present study amphetamine pretreatment did not change basal concentrations of extracellular dopamine and its metabolites in the medial prefrontal cortex; however, foot shock stress produced an augmented increase of extracellular dopamine and its metabolites in the amphetamine-pretreated subjects. The interpretation of the augmented dopaminergic response to footshock in the amphetamine-pretreated group is uncertain because the functional significance of dopamine release in the medial prefrontal cortex during stress is unclear. D'Angio et al. (1988) have obtained evidence that the increase in cortical dopamine metabolism induced by stressors is not a direct neurochemical correlate of emotional responses caused by aversive stimuli but may rather underlie heightened attention or activation of cognitive processes in attempts to cope with stressors. Specifically, these authors found that a number of stressful stimuli increased extracellular 3,4-dihydroxyphenylacetic acid concentrations in the medial prefrontal cortex of low but not high emotion rats. In the present study, damphetamine-pretreated rats showed more intense behavioral responses to foot shock. Dopamine release in medial prefrontal cortex was also augmented. While the most parsimonious explanation for these observations is that the increase in dopamine release is directly related to the increased emotional responsiveness of the amphetamine-pretreated animals, the data from D'Angio et al. (1988) suggest that this may not be the case. In this regard, Imperato et al. (1991) have shown with microdialysis that dopamine release in the medial prefrontal cortex is transiently enhanced first during restraint stress and again when the animal is released from restraint. Therefore, there does not appear to be an exclusive relationship between dopamine release in the medial prefrontal cortex and exposure to stressful stimuli, despite the fact that a variety of stressors, as well as the anxiogenic /3-carboline FG7142, can enhance dopamine release in this part of the brain (Bradberry et al., 1991). A unifying hypothesis about the functional significance of dopamine release in the medial prefrontal cortex does not appear to be possible at the present time and must await the results of additional experiments. Sorg and Kalivas (1993) have recently reported that chronic cocaine pretreatment reduces the increase of
70
extracellular dopamine in the medial prefrontal cortex produced by foot shock stress, an effect that is opposite to the results reported here. Both cocaine and amphetamine can induce psychosis and behavioral sensitization; moreover, cocaine and amphetamine show cross-sensitization, both behaviorally and neurochemically (Shuster et al., 1977; Kazahaya et al., 1989; Akimoto et al., 1990). It seems unlikely, therefore, that these disparate results are due to pharmacological differences between amphetamine and cocaine. Earlier, Kalivas and Duffy (1989) reported that a cocaine sensitization regimen enhanced increases in tissue concentrations of 3,4-dihydroxyphenylacetic acid in the medial prefrontal cortex produced by foot shock stress and concluded that cocaine pretreatment enhanced stressinduced increases in dopamine metabolism in medial prefrontal cortex. These latter findings are more consistent with the results reported here. They are also consistent with findings by Robinson et al. (1987) showing that footshock-induced increases in dopamine utilization in the medial prefrontal cortex are enhanced in amphetamine-sensitized rats. At present, there does not appear to be a satisfactory explanation for the discrepancy between the present results and those of Sorg and Kalivas (1993). Experiment 2 showed that there was behavioral sensitization to amphetamine in the amphetamine-pretreated group. However, and somewhat surprisingly, d-amphetamine-induced increases in extracellular dopamine in the medial prefrontal cortex of the amphetamine-pretreated group did not differ from those in the saline-pretreated group. Experiment 3 demonstrated that this result was not due to a ceiling effect by showing that dopamine output in the medial prefrontal cortex can be increased to a greater extent than is produced by 2 m g / k g of d-amphetamine (fig. 6). Robinson et al. (1988) have reported that amphetamine produces an augmented increase in extracellular dopamine in the nucleus accumbens of amphetamine-sensitized animals. Similar effects of repeated amphetamine injections on amphetamine-induced dopamine release have also been observed in striatum (Kazahaya et al., 1989; Hamamura et al., 1991). In contrast to these observations, Segal and Kuczenski (1992) have reported that repeated amphetamine administration attenuates subsequent effects of amphetamine on dopamine release in nucleus accumbens and striatum. The reasons for this discrepancy are again not immediately apparent but may be related to the fact that Segal and Kuczenski (1992) used somewhat shorter drug-abstinent periods than other investigators, and that the d-amphetamine challenge (2.5 m g / k g ) induced unusually large increases (about 5000%) in extracellular dopamine concentrations, values several times higher than in other reports (Robinson et al., 1988; Westerink et al., 1989; Hama-
mura et al., 1991). Kalivas and Duffy (1993) have obtained evidence that the effects of a cocaine challenge can vary significantly as a function of the abstinent period after repeated cocaine administration, with these neurochemical indices of sensitization becoming apparent only at longer abstinent periods. In conclusion, the present study has confirmed that amphetamine-pretreated rats show behavioral crosssensitization to foot shock stress and enhanced behavioral responses to amphetamine. In the medial prefrontal cortex of the amphetamine-pretreated group, stress-induced increases in extracellular dopamine were augmented while amphetamine-induced increases were not affected. This suggests that while dopaminergic mechanisms in the medial prefrontal cortex may be involved in behavioral hetero-sensitization between amphetamine and footshock stress, they do not mediate amphetamine homo-sensitization (i.e. amphetamine sensitization to itself).
Acknowledgements We gratefully acknowledge C.H.M. Beck for his valuable comments. Thanks also to Catriona Wilson for outstanding technical assistance. This work is supported by grants from the Medical Research Council of Canada (PG23) and Bristol-Myers/Squibb. T. Hamamura is supported by the Uehara Memorial Foundation, Japan.
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