Brain Research, 578 (1992) 317-326 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

317

BRES 17659

Amphetamine regulation of mesolimbic dopamine/cholecystokinin neurotransmission Yasmin L. Hurd a, Nils Lindefors a, Ernst Brodin a, Stefan Bren6 b, Hhkan Persson b, Urban Ungerstedt a and Tomas Hrkfelt c Karolinska Insitutet, Departments of aPharmacology, Medical Chemistry, bLaboratory of Molecular Biology and CHistology, Stockholm (Sweden) (Accepted 10 December 1991)

Key words: Nucleus accumbens; Rat; In vivo microdialysis; mRNA; Ftuoro-gold

The effects of acute and repeated amphetamine administration on mesolimbic dopamine (DA) neurons was assessed by studying DA and cholecystokinin (CCK) release in the nucleus accumbens (Acc), as well as effects on mRNA genes regulating DA and CCK synthesis in ventral tegmental area (VTA) cells in rats. Amphetamine (1.5 mg/kg) markedly increased extracellular levels of DA in the medial Acc (assessed by in vivo microdialysis) in drug-naive animals, about twice the amount released in animals repeatedly administered the drug for the previous 7 days (twice daily). CCK overflow was found to mirror the DA responses in that the very transient elevation of CCK monitored in drug-naive animals was attenuated in those with prior amphetamine use. The attenuation of both DA and CCK overflow in the medial Acc was found to be associated with a decrease in the number of CCK mRNA-positive VTA neurons (assessed by in situ hybridization histochemistry). Although the number of cells expressing CCK mRNA were decreased, the gene expression in those positive CCK and tyrosine hydroxylase mRNA cells in the VTA was significantly increased. The CCK mRNA neurons in the VTA were positively identified as those projecting to the medial Acc by the local perfusion of Fluoro-gold retrograde tracer via microdialysis probes located in the Acc. INTRODUCTION The nucleus accumbens (Acc) is a part of the striatum characterized by a prominent connectivity with limbic structures 19'35'37. A major input to the Acc arises from mesencephalic neurons of the ventral tegmental area (VTA) and medial substantia nigra compacta (SNc), and operates with the neurotransmitter dopamine (DA) 4' 10. This catecholamine has been considered to be an important neurotransmitter for the integration of motor behavior and cognitive function (see e.g. refs. 31, 33). Furthermore, there is evidence for an involvement of mesolimbic D A in reward and reinforcement based on the observation that potent psychomotor stimulants, e.g. amphetamine and cocaine, drugs with high addictive potential and with the ability to precipitate psychosis, increase D A overflow in the caudate-putamen and nucleus accumbens 7'21'22'34'54. Selective lesioning 4°, and pharmacological manipulations 51 of D A neurons in the V T A or Acc region have been shown to modify the behavioral and reinforcing properties of amphetamine and other stimulants. Furthermore, considerable evidence suggests that the primary mechanism of action of psychomotor stimulants is due to their ability to (1) stimulate D A re-

lease from presynaptic neurons (e.g. amphetamine-like drugs) or (2) inhibit the uptake of D A back into presynaptic terminal (e.g. cocaine-like drugs) 8'21'23'39. Consequently, altered mesolimbic D A neurotransmission has • been implicated as the central neurobiological mechanism underlying drug abuse and reward 15'4°, as well as mental illness such as schizophrenia 32. It has been discovered that the neuropeptide cholecystokinin (CCK) is co-localized with D A in a subpopulation of mesencephalic neurons terminating in limbic brain areas 2°'44. These mesolimbic C C K / D A fibers originating from the V T A have their most distinct projections to the medial, caudal region of the Acc 2°'52. This finding has raised the possibility that CCK participates in functions such as those associated with D A transmission, and consequently may be of importance in the psychopathology of schizophrenia and the neurochemistry of addiction. It has been demonstrated that injections of CCK produce behavioral changes similar to those induced by neuroleptic (antipsychotic) drugs 11'49"53, and that CCK can decrease the reinforcing effects of brain self-stimulation 48, or decrease amphetamine's stimulatory effect on D A neurotransmissionl. Much of the pharmacological evidence obtained re-

Correspondence: Y.L. Hurd. Present address: National Institute of Mental Health, Clinical Brain Disorder Branch, Neuroscience Center, St. Elizabeth's Hospital, Washington, D.C. 20032, USA. Fax: (1) (202) 373-6214.

318

v i v o for its ability to m a r k e d l y s t i m u l a t e m e s o l i m b i c D A transmission 41'45. A m a i n o b j e c t i v e of the i n v e s t i g a t i o n

or into 1.5 ml polyethylene tubes for determination of CCK. After the 5th consecutive sample was taken (at least 150 min following probe implantation), a subcutaneous injection of saline or amphetamine (1.5 mg/kg = 11/~mol/kg) was administered. 240 min after drug administration, the normal Krebs solution was switched (CMA 111, CMA Microdialysis AB) to a Krebs solution containing 100 mM potassium for 15 min. At the end of the experiment the animals were killed by an overdose of halothane. All samples for CCK determination were frozen (at -20°C and later stored at -80°C) immediately after collection.

was to d e t e r m i n e possible l o n g - t e r m n e u r o n a l a d a p t a -

Determination of DA and CCK in dialysate samples

tions of m e s o l i m b i c D A / C C K n e u r o n s at b o t h the n e r v e

For the determination of DA and its metabolites, dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA), 14/~1 of the total 50 pl obtained from the dialysis experiment were placed into a refrigerated autoinjector (CMA 200, Carneige Medicin AB) attached to a HPLC electrochemical detection system. A microbore (250 x 1 mm) 5 pm reverse-phase column (Brownlee Labs, Santa Barbara, USA) was used with a mobile phase containing 0.15 M phosphate buffer, 14% methanol, 0.1 mM EDTA and 0.6 mM sodium-octyl-sulfate (pH 3.8). The electrochemical detector (Bioanalytical Systems, Lafayette, Indiana) was set at an oxidation potential of 0.75 V (vs. Ag/AgCI). External standards were used to determine the concentration of DA and its metabolites with the aid of an integrator (Spectra Physics). The determination of CCK-like immunoreactivity (referred to as CCK) was carried out by radioimmunoassay similar to that reported for the analysis of other neuropeptides 5"28. Brain perfusates were assayed in the test tubes used for sample collection. Perfusate samples (100/~l), and standards (CCK-8, Peninsula, Belmont, CA, USA) dissolved in Krebs Ringer (100/xl) were first incubated for 24 h with the specific C-terminal directed gastrin/CCK antiserum 260938 and subsequently incubated with tracer (125I-labelled gastrin 17, Milab, Malmo, Sweden) for a further 72 h. After incubation, antibody-bound and free tracer were separated with the aid of anti-rabbit IgG coupled to Sepharose (Pharmacia decanting suspension, Pharmacia, Uppsala, Sweden). The detection limit of the assay was 0.1 fmol/sample.

garding C C K / D A i n t e r a c t i o n has b e e n carried o u t with e x o g e n o u s l y a p p l i e d C C K o r D A , and with in vitro tissue p r e p a r a t i o n s . F o r this r e a s o n the p r e s e n t study was d e s i g n e d to m o n i t o r in v i v o fluctuations o f D A and C C K o v e r f l o w in the m e d i a l A c c f o l l o w i n g a d m i n i s t r a t i o n of a m p h e t a m i n e , a d r u g that has b e e n well c h a r a c t e r i z e d in

t e r m i n a l and cell b o d y region. T h u s , in vivo m i c r o d i a l ysis was u s e d to m o n i t o r D A

and C C K r e l e a s e f r o m

n e r v e t e r m i n a l s in the A c c , while in situ h y b r i d i z a t i o n h i s t o c h e m i s t r y was u s e d to assess a m p h e t a m i n e - i n d u c e d changes in the e x p r e s s i o n of R N A s e n c o d i n g the ratelimiting e n z y m e in D A synthesis, tyrosine h y d r o x y l a s e ( T H ) , and p r e p r o C C K in cell b o d i e s in the V T A of animals naive to, and t h o s e p r e t r e a t e d with a m p h e t a m i n e (7 days, twice daily). To

verify

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changes in g e n e e x p r e s s i o n in the cell b o d y r e g i o n and n e u r o c h e m i c a l changes at its p r o j e c t i o n site, F l u o r o - g o l d , a fluorescent r e t r o g r a d e axonal t r a c e r 43, was i n t r o d u c e d into the m e d i a l A c c via the microdialysis p r o b e , and the l a b e l l e d cells in the m e s e n c e p h a l o n w e r e f u r t h e r characterized with in situ h y b r i d i z a t i o n histochemistry. It is evident f r o m t h e results that r e p e a t e d e x p o s u r e to a m p h e t a m i n e p r o d u c e s significant n e u r o n a l a d a p t a t i o n s o n D A and C C K n e u r o t r a n s m i s s i o n at b o t h the cell b o d y and t e r m i n a l areas. MATERIALS AND METHODS

Drug treatment and microdialysis procedure Male 250-300 g Sprague-Dawley rats, allowed free access to water and food, were subcutaneously injected twice daily during 7 days with either saline (control and acute groups) or amphetamine (1.5 mg/kg = 11/~mol/kg; amphetamine-sulfate, Sigma) (repeated amphetamine group). On day 8 (20 h after the last drug injection), the rats were anesthetized by freely breathing a 1.5% mixture of halothane and oxygen, and placed in a Kopf stereotaxic instrument, Body temperature was monitored by means of a heating pad and rectal temperature probe (CMA Microdialysis AB, Stockholm, Sweden). After the skull of the rat was exposed, two burr holes were drilled and the dura incised. Two microdialysis probes (membranes: 0.5 × 2 mm: CMA Microdialysis AB, Stockholm, Sweden) were bilaterally inserted into the Acc with coordinates relative to Bregma; A +1.7, L + 1.3 and V -7.3. One probe was continuously perfused with a Krebs Ringers solution (in mM: NaC1 138, NaHCO 3 11, KC1 5, CaC12 1, NaH2PO 4 1, glucose, pH 7.4) containing 10 mM physostigmine. The flow rate (2 #l/min) of the Krebs solution through the probe was controlled by means of a CMA/100 microinjection pump (CMA Microdialysis AB, Stockholm, Sweden). Bovine serum albumin (0.5%) and Bacitracin (0.3%) were added to the Krebs Ringer perfusion medium and perfused (5/AI min) through the second probe used for measurement of CCK levels. Perfusate samples collected during the first 30-50 min following surgery were discarded. Subsequently, perfusate samples were collected every 20 min into 0.3 ml glass vials containing 10 ~1 of 0.05 mM HCI solution to prevent breakdown of the monoamines,

Fluoro-gold perfusion and histochemistry analysis An additional group of animals receiving the same drug treatment on days 1-7 were similarly prepared for the microdialysis procedure on day 8 as described above. 1 h after implantation of the probe, the perfusion medium was switched to a Ringers solution containing 2% Fluoro-gold43. The flow rate was decreased to 0,1 pl/min and maintained at that speed for 1 h. Subsequently the flow was stopped for 5 min and then restarted with the normal Ringers solution (1 ~l/min) for an additional 15 min. The probe was slowly withdrawn and the animal's wound was sutured. 7 days following Fluoro-gold perfusion, the animals were decapitated and the brain rapidly dissected, frozen, cut on a cryostat (Leitz, Wetzlar, West Germany), thawed onto poly-L-lysine-coated slides and analyzed in a Nikon Mikrophot-FX microscope equipped for epifluorescence and proper filter combinations for Fluoro-gold43. Retrogradely labelled cells in the ventral mesencephalon were photographed (Tri-X Kodak) and the sections were then processed for in situ hybridization as described below.

In situ hybridization histochemistry procedure At the end of the microdialysis experiments (4 h after administration of amphetamine), brains were dissected out and immediately frozen on dry ice and stored at -80°C until cryostat cutting. Coronal tissue sections at the level of the VTA/ substantia nigra were cut on a cryostat (Dittes, Heidelberg, FRG) at 14/~m thickness and thawed onto poly-L-lysine-coated slides (50/~/ml). The tissues were fixed in 10% formalin in phosphate buffered saline (PBS) for 30 min, rinsed twice for 4 min in PBS, dehydrated in a graded series of ethanol solutions and delipidated in a 5 min incubation with chloroform. The sections were then air-dried. The hybridization cocktail contained 50% formamide, 4 x SSC (1 ×

319 SSC is 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0), 1 x Denhardt's solution, 1% Sarcosyl, 0.02 M Na3PO 4 (pH 7.0), 10% Dextran sulphate, 0.5 mg/ml yeast tRNA, 0.06 M DTF and 0.1 mg/ml sheared salmon sperm DNA. A 48-mer oligonucleotide complementary to rat TH mRNA encoding amino acid 445-492 of the TH polypeptide TM was used as the probe to detect TH mRNA. A 44mer oligonucleotide complementary to rat preproCCK mRNA coding for amino acid 89-103 z2 was used as the probe to detect preproCCK mRNA. PreproCCK mRNA is referred to as CCK mRNA in the text below for simplicity. The oligonucleotides was 3'-end labeled with a[35S]dATP using terminal deoxyribonucleotidyl transferase (International Biotech. Inc., New Haven, CT) to a specific activity of approximately 5 × 108 cpm/pg. The labelled probes were purified on a Nensorb column (DuPont, Wilmington, DE) prior to use. Following hybridization for 16 h at 42°C, the sections were rinsed 5 times for 15 rain each in 1 x SSC at 55°C. Finally, the sections were rinsed in autoclaved water for 2 rnin, and then dehydrated through a series of graded alcohol solutions and air-dried. The sections were exposed to X-ray film (Amersham B-max) for 7-14 days and to photographic emulsion (Kodax NTB2) for 10-21 days. After being developed, the slides were stained with Cresyl violet followed by microscopic analysis. As a control of specificity of the hybridization, an oligonucleotide probe specific for preprotachykinin A mRNA was used in the VTA.

Neuron counting and computerized image analysis The numerical density of CCK and TH mRNAmpositive neurons in VTA was analyzed using a Leitz Ortholux microscope at 313x magnification, The number of grains per neuron and the size of CCK and preprotachykinin mRNA-positive neurons were calculated at 500 × magnification using a Crystal image analysis processor (Quantel, Ltd, UK). For this purpose, the microscope was equipped with a Panasonic WV 1500 TV camera to provide a video signal for the image analysis. However, over some neurons analyzed, the high density of grains made a direct numerical count futile. Instead, the total area of grains, also including all fused grains, was divided with a standard grain area obtained from measurements of unfused grains over neurons. The area of a standard grain was arbitrarily defined as the mean area of a few small grains measured over a hybridization-positive cell.

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All data are presented as mean + S.E.M. Statistical analysis of the microdialysis data was carried out by using ANOVA and Newman-Keuls post-comparative test, while results obtained from in situ experiments was analyzed using ANOVA with Fischers postcomparitive test.

RESULTS

No pre-drug basal difference was found between the two saline-pretreated groups (n = 5; n = 6) and their basal values were combined for statistical analysis. Basal levels of neurotransmitters and metabolites analyzed in the medial Ace of saline and amphetamine pretreated rats (n = 5) were, respectively: DA, 0.02 + 0.004 and 0.025 + 0.006 pmol/8 pl; DOPAC, 6.36 + 0.42 and 5.07 + 0.52 pmol/8/d; HVA, 3.72 + 0.17 and 2.81 + 0.22 pmol/8/d; CCK, 0.822 + 0.07 and 1.069 + 0.14 fmol/ 100/A. No significant detectable difference was found between basal values of drug-naive and amphetaminetreated animals. Concentrations of CCK, and D A and its metabolites sampled from the extracellular fluid are not corrected for recovery across the microdialysis membrane as the following results are discussed in terms of relative changes.

Effects of amphetamine administration on extracellular levels of DA and CCK in the medial nucleus accumbens Acute amphetamine (11/~g/kg = 1.5 mg/kg) administration in drug naive rats (pretreated with saline) produced an immediate elevation of D A overflow (650% of basal; F2,~8 = 5.271, P < 0.01) lasting 80 min, and a

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Fig. 1. The effect of an acute subcutaneous injection of amphetamine (11/~mol/kg = 1.5 mg/kg) or saline on extracellular levels of D A in the nucleus accumbens of halothane-anaesthetized rats on day 8 of drug treatment. Animals receiving an acute challenge injection of amphetamine were pretreated with saline (©) or amphetamine ( 0 ) (11 ~mol/kg) twice daily for 7 days. Control animals ( A ) were pretreated with saline and received saline on the day of testing. The data (mean + S.E.M.) are expressed as % of basal. Statistical significance was assessed by analysis of variance and Newman-Keuls post comparison tests (*P < 0.05; **P < 0.01 compared with saline animals: ap < 0.05 compared with acute amphetamine treated animals) (n = 5-6).

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Fig. 2. The effect of an acute subcutaneous injection of amphetamine (11/~mol/kg = 1.5 mg/kg) on extracellular levels of CCKlike immunoreactivity in the nucleus accumbens of halothaneanaesthetized rats on day 8 of drug treatment. Animals receiving an acute challenge injection of amphetamine were pretreated with saline (O) or amphetamine ( 0 ) (11/~mol/kg) twice daily for 7 days, The data (mean + S.E.M.) are expressed as% of basal. Statistical significance was assessed by analysis of variance and NewmanKeuls post comparison tests (*P < 0.05 compared with basal; n = 4-5).

320 sustained reduction of D O P A C and HVA (respectively, 70 and 60% of basal; F2,16 = 23.489, P < 0.001; F2.17 = 9.057, P < 0.002) (figure not shown). However, in animals that had received amphetamine during the 7 days prior to testing, an acute challenge injection of the stimulant on day 8 produced an elevation of D A (300% of basal; P < 0.05 vs. control animals), that was significantly (P < 0.05) lower than the amphetamine-induced DA response in animals that had been pretreated with saline (Fig. 1). Four hours after the injection of amphetamine, extracellular D A levels measured during the local application of K + (100 mM) into the Acc of both treatment groups were found to be markedly (1,700%) increased from basal. The increase was nearly identical for each group, though not significantly higher than con-

trol (1,500% from basal) (data not shown). Although basal extracellular D O P A C and HVA values tended to be lower (not significantly) in animals pretreated with amphetamine, no difference was detected in the amphetamine-induced reduction of extracellular D O P A C or HVA in amphetamine-pretreated rats vs. those naive to the drug (data not shown). Complementary to D A release, CCK overflow was simultaneously assessed in the same experimental animals. Immediately following the acute amphetamine injection, a transient sharp elevation of CCK (50% over basal; P < 0.05) was evident in drug-naive animals. This-elevation returned to basal values within the next 20 min period (Fig. 2). However, similar to the D A response, the elevation of extracellular CCK that was observed with the acute injection of amphetamine in drug-naive animals was attenuated on day 8 in animals receiving prior daily (7 days) repeated injections of the stimulant (Fig. 2). When the normal Krebs Ringers flowing through the dialysis probe was switched to a Krebs solution containing 100 mM K +, CCK levels were markedly increased. The CCK release induced by the K + stimulation was significantly higher (P < 0.05) in animals previously exposed to amphetamine than those rats acutely administered amphetamine (25 + 0.25 fmol/100/~l vs. 13.5 _+ 4.5 fmol/100 ~1).

Effects of amphetamine administration on tyrosine hydroxylase mRNA and CCK mRNA levels in ventral tegmental area In situ hybridization with an oligonucleotide specific for rat TH m R N A showed an intense labelling pattern in the VTA and SNc in all drug groups (Fig. 3). Microscopic examination of the hybridized tissue sections demonstrated no significant effect of drug treatment in the number of TH mRNA-positive cells in the VTA even

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Fig. 3. Low magnification, dark-field photomicrograph of TH mRNA in situ hybridization histochemistry in mesencephalon of rats treated with either (a) saline, (b) a single dose of amphetamine (11/~mol/kg), or (c) amphetamine (11/zmol/kg) twice daily for 7 days.

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Fig. 4. Number of TH mRNA-positive neurons per mm2 (a) and the number of grains over TH mRNA-positive neurons (b) in ventral tegmental area of rats with saline (control), acute or repeated amphetamine treatment. Neurons are counted (mean + S.E.M.) over 1 mm2 in two tissue sections from five different animals. * Indicates significant difference to control group (P < 0.05).

321 though there was a trend for the number of detectable TH mRNA cells in amphetamine-pretreated animals to be reduced (Fig. 4a). However, examination of cellular expression using computerized image analysis showed a 28% increase (P < 0.05) in the number of grains over TH mRNA-positive neurons in the VTA of amphetamine-pretreated animals (Fig. 4b). In situ hybridization with an oligonucleotide specific for rat CCK mRNA revealed an intense labelling in the VTA and SNc as in the case of TH mRNA (Fig. 5). An intense signal was also seen in cerebral cortex and parts of the thalamus (Fig. 5). Microscopic examination of the hybridized tissue sections showed a 37% decrease (P < 0.05) in numerical density of CCK mRNA-positive neurons in the VTA of amphetamine-pretreated animals (Fig. 6a). However, consistent with the results of TH

mRNA, examination of cellular expression using computerized image analysis showed a 39% increase (P < 0.05) in grains over CCK mRNA-positive neurons (Fig. 6b). The decrease in numerical density of neurons expressing significant amounts of CCK mRNA was found to be of a similar magnitude as the increase in grains over the remaining CCK mRNA-positive neurons as identified by Fluoro-gold retrograde labelling (see below).

Fluoro-gold retrograde tracing Retrograde labelling by perfusion of Fluoro-gold into medial Acc displayed selective intense labelling in the VTA and scattered labelled neurons in SNc (Fig. 7A,D). Most of the retrogradely labelled neurons were CCK mRNA-positive, both in control animals (Fig. 7A-C) and in animals receiving amphetamine injection (Fig. 7D-F). DISCUSSION

Activation of presynaptic mesolimbic DA transmission is generally considered central to the mechanism of action of psychomotor stimulants such as amphetamine s' 23,sl. In the present study, in vivo microdialysis, in situ hybridization histochemistry, and Fluoro-gold retrograde labelling, were combined to assess changes in extracellular levels of neurotransmitters and changes in steadystate gene expression of mesolimbic neurons, as a result of acute and repeated administration of amphetamine. The results indicate that following repeated systemic amphetamine administration (7 days, twice daily), components of DA/CCK transmission, from the synthesis of DA and CCK to the release of these neurochemicals, are significantly altered in the mesolimbic DA/CCK pathway.

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Fig. 6. Number of CCK mRNA-positive neurons per mm 2 (a) and the number of grains over T H mRNA-positive neurons (b) in ventral tegmental area of rats with saline (control), acute or repeated amphetamine treatment. Neurons are counted (mean _+ S.E.M.) over I mm 2 in two tissue sections from five different animals. * Indicates significant difference to control group (P < 0.05).

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Fig. 7. Fluorescence micrographs (A,D), bright field (B,E) and dark field (C,F) autoradiographs of sections of the ventral mesencephalon of untreated (A-C) and amphetamine (7 days)-treated (D-F) rats. (A-C) and (D-F) show, respectively, the same sections which, after photography of retrograde Fluoro-gold labelling (A,D), were processed for CCK mRNA in situ hybridization (B,C,E,F). Arrowheads indicate CCK mRNA-positive cells; thick arrows indicate Fluoro-gold positive/hybridization negative cells; thin arrows indicate Fluoro-gold negative/ hybridization-positive cells; oblique arrows in panel E and F are used to point at a particular Fluoro-gold-positive/hybridization-negative cell shown in bright and dark field. Note that all fluorescent cells in (A) exhibit strong grain densities (cf. arrowheads in A-C). In (D) groups of cells (arrowheads) are both fluorescent and CCK mRNA-positive, whereas some cells (thick arrows) contain only Fluoro-gold, and others only CCK mRNA (thin arrows). The cell indicated by an oblique arrow (D) is Fluoro-gold-positive, but contains no CCK mRNA, although this can only be seen with dark field (cf. E and F). Bars indicates 50 mm (A = B = C; D = E = F).

Effects of amphetamine on in vivo DA and CCK release and its implications Consistent with results from other in vivo preparations, an acute injection of amphetamine greatly increased D A release in the medial Acc of drug-naive animals for 2 h, with a peak response 40 min following administration of the drug 21'23'54'55. Furthermore, the

acute injection of amphetamine to drug-naive animals caused a simultaneous rapid and very transient elevation of CCK overflow with the peak response within the first 20 min, but the magnitude of the CCK response was not as robust as that observed for D A . The amphetamineinduced overflow of both D A and CCK was, however, attenuated on day 8 in drug-experienced rats compared

323 to those receiving amphetamine for the first time. In fact, in drug pre-treated animals, a challenge injection of amphetamine was unable to significantly potentiate the release of CCK greater than basal. The in vivo differences observed here between the DA and CCK response to acute amphetamine are supported by in vitro results on striatal slice preparations in which amphetamine was also found to produce a greater, and a more long-lasting effect on DA than on CCK efflux25. It is possible that differences in the DA vs. CCK response reflect basic neurophysiological differences in amine vs. peptide neurotransmission, in addition to differences in the presynaptic mechanisms by which amphetamine may stimulate DA vs. CCK release. While it is known that amphetamine releases DA via a carriermediated non-vesicular mechanism 16'21'23'27'5°, only vesicular (depolarization-sensitive) CCK release has been demonstrated with in vitro TM and in vivo2'6"46 preparations. Based on the results of the present study it would appear that CCK release is differentially regulated by potassium and amphetamine. This is suggested because depolarization with in situ perfusion of potassium into the Acc markedly increased the release of CCK in both drug-naive and amphetamine-pretreated rats. However, the potassium-stimulated CCK release was greater in amphetamine-pretreated rats, in which a prior injection of the drug had failed to increase CCK overflow. Thus, repeated administration of amphetamine appears to alter neuronal mechanisms differentially affecting the release of CCK induced by potassium and amphetamine. It should be noted that often when monitoring the in vivo release of neuropeptides, basal levels are difficult to detect, whereas depolarization-induced release is quite evident 6'2s'3°. This may be due to factors relevant to neuropeptides such as their low extracellular concentrations, large molecular weight, low recovery from extracellular space, detected levels close to sensitivity limit, and degradation caused by extracellular peptidases. These factors might all contribute to impair the ability of in vivo microdialysis to detect discrete changes in basal neuropeptide when compared, for example, to that of DA. Nevertheless, the present study demonstrates that microdialysis is applicable for determining drug effects on in vivo CCK neurotransmission. Although an attenuation of amphetamine-induced DA release was found in this study after 7 days of repeated amphetamine treatment, other in vivo dialysis studies have reported that prior experience with stimulants produce either a greater drug-induced increase 36'4~ or no change 42 in DA overflow compared with drug-naive animals. Based on the similarities and differences between all these experiments, it would appear that significant factors for disparate dopaminergic response relate to the

doses administered and/or the period of drug treatment at which testing is carried out. While factors such as awake vs. anesthetized and/or acute vs. chronic probe implantation are important considerations, they appear to be more related to the absolute amount of DA that is recovered from the extracellular space than to the degree of change that occurs in response to challenge drug administration. This is further emphasized by recent results carried out in awake rats chronically implanted with microdialysis probes which demonstrated that DA release in the Acc of rats pretreated with cocaine, a comparable psychomotor stimulant with regard to behavioral sensitization, may be augmented, attenuated or not changed compared to first-time exposure to cocaine depending on the period of drug use, or the period of withdrawal at which testing was carried out (Guix et al., unpublished results; P. Kalivas, personal communication). The differential response of DA overflow reported with variable repeated drug treatments provide some evidence that although potentiation of DA overflow is critical to the effects of amphetamine, the absolute amount of DA that is potentiated may not directly correlate to the psychomotor stimulant properties of the drug. For example, the behavioral response to amphetamine or cocaine in animals with prior drug experience is usually more intense, has a faster onset, and a longer duration of action than those naive to the stimulant, even though DA release in these two groups does not differ42, or is only significantly different at a few time points 26'41. Thus, the absolute amount of extracellular DA cannot be expected to account for all the behavioral differences observed between drug-naive and drug-experienced animals and for the compulsive drug-seeking behavior24. Supersensitivity to the psychomotor effects induced by repeated amphetamine administration may be more closely linked to sensitized DA receptors and/or altered post-synaptic transmitter systems. If DA receptors, or receptor-effector coupling mechanisms, become supersensitive as a consequence of repeated stimulant administration 9'13'47, it would be possible for lower concentrations of synaptic DA in drug-experienced animals to produce the same or greater stimulatory response as that observed in drug-naive animals in which DA levels are higher. This hypothesis would appear to be supported by the fact that GABA overflow, monitored in the same animals from which DA and CCK overflow were assessed in this study, was only decreased in animals repeatedly administered amphetamine 29. It has been well documented that GABA release is under the inhibitory influence of DA D2 recepto rs3A7, and it appears that CCK overflow can also be inhibited via DA D2 receptor stimulation 25. Thus the reduction of amphetamine-in-

324 duced G A B A and CCK overflow in drug-experienced animals would be supportive of supersensitive D A D 2 receptors, or increased activity of secondary messengers, modulating the release of G A B A and CCK.

Effects of amphetamine on gene regulation of DA and CCK synthesis The possibility that the effects of amphetamine on D A and CCK release in the nerve terminal region were in part mediated and/or accompanied by alterations in gene expression at the cell body level was assessed by combining in situ hybridization histochemistry and Fluorogold retrograde labelling. Inclusion of Fluoro-gold tracer in the microdialysis perfusion media of probes located in the medial Acc allowed identification of CCK mRNApositive, presumably, D A neurons in the VTA which project to the medial Acc, and possibly contribute to the extracellular concentrations of D A and CCK. Only following the repeated administration of amphetamine was the number of cells with detectable CCK m R N A found to be decreased. This may in part explain the decreased CCK release in animals repeatedly administered amphetamine, assuming that the number of CCK mRNA-producing neurons correlates with total CCK release. It is possible that the decreased number of detectable CCK/DA neurons in animals pretreated with amphetamine reflect subpopulations of D A neurons with differential responses to amphetamine such that repeated exposure to amphetamine inhibits CCK m R N A expression in one population of D A neurons while the expression is upregulated in another population. Although there were no significant reductions in TH-positive m R N A cells, D A nonetheless may be similarly regulated if the subpopulation of D A cells that expresses CCK is partially reduced but the changes are masked by inclusion of other populations of TH-positive cells. It can also be speculated that the decreased number of detectable CCK/DA neurons at the cell body level is represented at the terminal level given that basal levels of D A metabolites in animals repeatedly treated with amphetamine always tended to be lower (though not significantly) than drug-naive animals, possibly due to reduced cytoplasmic DA, the substrate for monoamine oxidation to D O P A C and the substrate for carrier-mediated release induced by amphetamine. Although the number of CCK/DA neurons was reduced by repeated exposure to amphetamine, the gene REFERENCES 1 Altar, C.A., Boyar, W.C., Oei, E. and Wood, P.L., Cholecystokinin attenuates basal and drug-induced increases of limbic and striatal dopamine release, Brain Res., 460 (1988) 76-82.

expression of T H and CCK m R N A in single cells was clearly increased in the VTA of these animals. This apparent contradictory finding might reflect an upregulation of CCK and T H protein biosynthesis in populations of those active TH/CCK neurons in an attempt to maintain neurotransmission and compensate for repeated stimulation of DA/CCK neurons. The significant neuronal overlap of Fluoro-gold tracer and CCK m R N A hybridization found in those neurons expressing high levels of CCK m R N A in the VTA would also indicate that it is the neurons projecting to the medial Acc which continue to synthesize CCK after repeated amphetamine administration. A compensatory increase in CCK neurotransmission to repeated amphetamine administration can be hypothesized, based not only on the increase in CCK m R N A expression, but also from the observation that depolarizing the terminals with potassium caused a greater increase in CCK release in amphetamine-pretreated rats compared to those naive to the drug. This hypothesis might appear contradictory to the explanation of attenuated amphetamine-induced CCK and D A release associated with a significant reduction of CCK/DA-positive cells in the VTA. However, amphetamine-induced CCK and DA release might be more sensitive to loss of cell number than potassium-induced release.

Summary The present results clearly demonstrate that both at the level of release and gene expression, CCK neurotransmission mirrors the changes in D A neurotransmission in mesolimbic neurons. The results indicate that repeated amphetamine administration brings about biphasic changes in D A and CCK neurotransmission such that increased neurotransmitter release during acute exposure leads to eventual reduction in the number of active neurons, with a compensatory increase in expression of CCK and T H m R N A in mesolimbic neurons.

Acknowledgements. This work was supported by grants from the Swedish Medical Research Council (Grant nos. 8653, 6536 and 2887), from USPHS (Grant no. MH 442211; Neuroscience Center for Research in Schizophrenia), from NIMH (MH-43230), from Ake Wibergs Stiftelse and from Loo and Hans Ostermans fond. We would especially like to thank /~se Hallstr0m, Annika Olson and Lillian Sundberg for their technical assistance, Margaretha Eriksson and the staff of Animal Dept. for their dedicated care of the animals and to Monica Karlsson for her secretarial help. 2 Artaud, E, Baruch, P., Stutzmann, J.M., Saffroy, M., Godeheu, G., Barbeito, L., Herve, D., Studler, J.M., Glowinski, J. and Cheramy, A., Cholecystokinin: co-release with dopamine from nigrostriatal neurons in the cat, Neuroscience, 1 (1989) 162-171.

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cholecystokinin neurotransmission.

The effects of acute and repeated amphetamine administration on mesolimbic dopamine (DA) neurons was assessed by studying DA and cholecystokinin (CCK)...
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