Amphetamine induces excess release of striatal acetylcholine in vivo that is independent of nigrostriatal dopamine

BRAIN RESEARCH ELSEVIER

Brain Research 653 (1994) 57-65

Research report

Amphetamine induces excess release of striatal acetylcholine in vivo that is independent of nigrostriatal dopamine R.J. Mandel a,b.., G. Leanza b,d, O.G. Nilsson b, E. Rosengren c Department of Psychology, Unit'ersity of lllinois, 603 E. Daniel St., Champaign, 1L 61820. USA b Department of Medical Cell Research Unit,ersity ofLund, Biskopsgatan 5, S-223 62 Lund, Sweden c Department of Pharmacology, Unit,ersity of Lund. S-223 62 Lund, Sweden d Institute of Human Physiology, VialeA. Doria 6. 95125 ('atania, ltaly

Accepted 26 april 1994

Abstract

The effect of amphetamine on striatal acetylcholine (ACh) release was studied by an in vivo intrastriatal microdialysis technique. Although we expected systemic amphetamine to inhibit baseline striatal ACh release, the opposite was found. In addition, we found that the amphetamine-induced striatal ACh release did not depend on nigrostriatal DA since 6-hydroxydopamine (6-OHDA) lesions had no effect on amphetamine-induced ACh release. Local intrastriatal injection of amphetamine via the microdialysis probe had no effect on striatal ACh release even when the probe was located more laterally in striatum to take advantage of the medial to lateral gradient of striatal ACh and D 2 receptors. The hypothesis that amphetamine increased extracellular striatal ACh by increasing the release of biogenic amines besides dopamine was tested by pharmacological manipulations designed to specifically increase local striatal norepinephrine or serotonin levels. The serotonergic and noradrenergic manipulations had no effect on striatal ACh levels. These results indicate that amphetamine-induced release of ACh in striatum is mediated via distal brain regions that are functionally connected with the striatum. Key words: Serotonin; Norepinephrine; Apomorphine; Microdialysis; Neostriatum; Parkinson's disease

1. Introduction

One of the most fundamental concepts regarding the action of striatal D A relates to the reciprocal interaction between striatal ACh and D A [3,4,32]. Clincally, the earliest efficacious anti-Parkinsonian drugs were the alkaloids, atropine and scopolamine, which block ACh receptors [15]. Thus, in Parkinsonian patients who have low leveh~ of striatal DA, decreasing cholinergic neurotransmission improves the clinical syndrome. This clinical picture suggests that striatal D A normally inhibits striatal ACh and when D A is lost the striatal ACh is released from inhibition. In fact, the idea that D A is inhibitory to ACh release is so well established tha' in vitro ACh release is used as a pharmacological assay to study the degree of striatal

* Corresponding author. Present address: SomatixTherapy Corporation, 850 Marina Village Pkwy., Alameda, CA 94501, USA. Fax: (1) (510) 769-8533. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00524-G

D~ DA receptor stimulation [33]. In other words, in these in vitro assays, D 2 agonism is defined by the ability to inhibit ACh release in striatal slices. Since the description of a senstive assay for ACh has been recently described [17], in vivo intracerebral microdialysis for ACh has become possible. Thus, the potentia[ to study the dynamic interation between striatal D A and ACh now exists and has received much recent interest [1,5,10-12,16,27,29,35]. Contrary to the expectation of a reciprocal interaction between the control of striatal ACh by D A stimulation, in certain situations, the opposite has been found. Both systemic amphetamine and cocaine administration [10,16] has been shown to increase striatal ACh release. Systemic treatment with D 1 D A receptor specific agonists has also been shown to increase striatal ACh release [1,10,11]. However, local administration of the D 1 antagonist SCH-23390 does not reverse the D~ agonist-induced increase in striatal ACh overflow [10]. These data indicate that the D I D A receptor modulation of striatal ACh is not locally controlled within the stria-

58

R.J. Mandel et al. / Brain Research 653 (1994) 57-65

tum. Since amphetamine is known to increase the release of striatal norepinephrine and serotonin [18,20,21,26], the present study explored the possibility that amphetamine-induced increases in striatal ACh is controlled by these other biogenic amines. When we attempted to study the effect of amphetamine on in vivo striatal ACh release in normal rats, we were unable to find the reciprocal interaction between ACh and DA. To the contrary, we found that amphetamine induced greatly increased ACh release which was independent of nigrostriatal DA or any other biogenic amine in striatum.

2. Materials and methods 2.1. Subjects Young female S p r a g u e - D a w l e y rats weighing approximately 250 g at the time of the experiment were obtained from A L A B (Stockholm, Sweden), housed no more than 6 to a cage, and allowed to acclimate to the housing facility for at least 1 week before being used in any experiment. The rats were housed in a 12 h light/12 h dark cycle and were allowed ad libitum access to laboratory rat chow.

2.2. Surgery In all lesion surgeries, the rats were first anesthetized with equithesin (0.3 m l / I00 g b.wt.) and placed in a small animal stereotaxic device (David Kopf, Tujunga, CA). T h e surgical procedure to implant dialysis probes (see below) took place while the rats were anesthetized by halothane anesthesia and were allowed to awaken immediately after the surgery was completed. 6-Hydroxydopamine lesions. After a rat was m o u n t e d in the stereotaxic device, they received a two-site injection of 6 - O H D A hydrobromide (Sigma, St. Louis, MO). The first injection of 6 - O H D A ( 3 / x g / # l calculated as free base dissolved in a 0.9% s a l i n e / 2 m g / m l ascorbic acid solution) consisted of 2.5/xl infused over 2.5 min at the coordinates - 4.4 m m posterior, - 1.2 m m lateral from bregma, and - 7.8 m m ventral from the dura mater (the incisor bar was set - 2.3 m m below the interaural line). The second 6 - O H D A injection occured with the incisor bar raised to 5 m m above the interaural line at the coordinates - 4 . 0 m m posterior, - 0 . 8 m m lateral from bregma, and - 8 . 0 m m ventral from dura. O n e group of rats (n = 5) received additional injections of 25 m g / k g desipramine (DMI) i.p. in order to spare noradrenergic neurons. The other group of 6 - O H D A treated rats did not receive DMI pretreatment (n = 6). The animals were allowed 3 weeks for recovery before being used for striatal microdialysis. This two-site injection method has been shown to severely deplete dopamine from the striatum [30], mesolimbic projection to nucleus accumbens and the olfactory tubercle [14]. 5,7-Dihydroxytrypamine lesions. Thirty minutes before surgery, 6 rats received injection of 25 m g / k g DMI [6] to protect noradrenergic neurons. 5,7-Dihydroxytrypamine creatinine sulfate (5,7-DHT, 150 p.g of free base in 20 tzl of 0.9% saline and 2% ascorbic acid solution, Sigma) was injected into the right lateral ventrical at the coordinates +0.5 m m anterior, - 1 . 0 m m lateral to bregma, and - 4 . 5 ventral from dura with the incisor bar set level with the interaural plane. These rats were allowed to recover 2 - 3 weeks before being used for striatal microdialysis. This lesion method has been shown to reduce forebrain serotonin (5-HT) levels by more than 90% [6]. Dialysis probe implantation. The dialysis probes utilized in the

present experiment were of the loop type constructed as described in detail elsewhere [24,25] using saponified cellulose ester (SCE) tubing with a molecular weight cut-off of about 10 kDa. W h e n complete the probes had a diameter of 0.6 m m at their largest point. T h e probes had 4 m m of exposed SCE tubing (for a total length of 8 m m exposed to the brain). These probes have been previously demonstrated to result in 15-20% recovery of A C h [28]. The reported values have not been corrected for recovery percentages. The probes were implanted with the rats under halothane anesthesia. After the rats were sufficiently anesthetized they were placed in the stereotaxic device fitted with tubing to deliver the halothane and air mixture directly to their nose. The probes were implanted for most animals at the following coordinates: + 0.7 m m anterior, - 2 . 6 lateral to bregma, and - 6 . 0 m m ventral from dura with the incisor bar set - 2 . 3 m m below the interaural line. This placement was considered to be a medial probe placement. For the experiment comparing medial versus lateral probe placements, the lateral implantation coordinates were: + 0.7 m m anterior, + 3.5 m m lateral to bregma, and - 6 . 0 ventral from dura also with the incisor bar set -2.3 m m below the interaural line. After the correct probe placement was achieved, 3 - 4 stainless steel screws were anchored to the skull and dental acrylic was placed around the probe and the screws to hold the probe as stable as possible. The rats were then returned to cages with high walls and no tops to reduce the possibility of damaging the probes during the recovery period. The implanted rats were allowed to recover between 18 and 26 h before being used in a microdialsys experiment and most implanted rats were also used in a second dialysis experiment 24 h following the first experiment. Microdialysis procedure. Unless otherwise stated all rats undergoing microdialysis were anesthetized (continuous inhalation of 1.21.5% h a l o t h a n e / a i r mixture) and placed on a polystyrene board, with their body temperature monitored and maintained at 37°C using a heat lamp. The probe inlet cannula was connected to a microinfusion system (Carnegie Medicin AB, Stockholm, Sweden). A 40-cm piece of polyethylene tubing with an internal volume of 12 / x l / m (Carnegie Medicin AB, Stockholm, Sweden) was connected to the outlet cannula. Collection of perfusates from the probe outlet cannula occurred directly at the head of the animals. A modified Ringer solution (148.2 m M NaC1, 4 m M KC1, 1.2 m M CaC12, pH 7.4) containing 10 tzM neostigmine bromide (Sigma, St. Louis) was p u m p e d through the dialysis tubing at a flow rate of 2 / z l / m i n and a sampling time of 15 min was adopted in all experiments. After discarding the first 30-min perfusate, 30/~1 samples were collected in E p p e n d o r f microtubes. Five baseline samples were collected before performing any manipulation. The samples were immediately frozen in liquid nitrogen, and subsequently stored in a deep freeze ( - 8 5 ° C ) for between 2 days and 1 m o n t h before assay. Acetylcholine assay. ACh was assayed by an H P L C procedure first described by Potter, 1983 and later modified by Fujimoro and Y a m a m o t o [17]. Briefly, dialysis samples were injected into a column-reactor system by an automatic injector (Wisp 710B, Waters Associates). The mobile phase was 0.15 M sodium phosphate buffered (pH 8.5) containing 50 ~ M sodium octansulphonate as ion-pair reagent and 0.3 M E D T A . Acetylcholine and choline were separated on a polymeric reversed-phase column (BAS, Japan) and converted to hydrogen peroxide ( H 2 0 2) and betaine in an enzymatic post-colu m n reactor with immobilized acetycholinesterase and choline oxidase (BAS, Japan). The resultant H 2 0 2 was measured by electrochemical detection on a platinum electrode set at 500 m V compared versus an A g / A g C I reference electrode (LC4B/17A, BAS). The separation column and enzyme reactor were kept at 37°C by a column heater (LC22A/23B, BAS). Statistics. The dialysis data were analyzed using repeated measures analysis of variance (ANOVA). The null hypothesis was rejected when the probability was greater than 95% (i.e. alpha < 0.05). The d e p e n d e n t variable was the pmol of A C h per 30/xl of perfusate.

R.J. Mandel et al. /Brain Research 653 (1994) 57-65

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Fig. 1. The effect of 5.0 m g / k g amphetamine (i.p.) on striatal ACh release. The injection of amphetamine at the beginning of the sixth sampling period is indicated by an arrow• A: the pattern of ACh release measured in the lateral striatum. This striatal placement was tested because there is medial to lateral gradient of striatal D 2 receptors [22,23]. This fact, coupled with D 2 receptors" control of in vitro ACh release [33], suggested that there might be a higher probability of obtaining results more comparable to previous in vitro results• B: the pattern of ACh release from the more conventional medial striatal placement. There was no difference between the pattern of striatal ACh release measured from the different probe placements (F1,1o=0.74, P > 0.4). Ampetamine treatment lead to a highly significant approximately 2 fold average increase in extracellular striatal ACh (F~,90 = 23.7, P < 0.001, with a non-significant site X sampling period interaction). These data were collected from probes implanted 48 h before the beginning of sampling. Twenty-four h before the injection of amphetamine these animals were treated as described in Fig. 2.

animals (n = 3) with a lower dose of amphetamine (2.5 m g / k g , i.p.) with the same probe placement and the response was nearly identical to 5.0 m g / k g with slightly lower peak increases over baseline ACh values (mean percent of baseline = 200%). Since this result was unexpected, we began trying to explain this apparently anomolous result. The first variable that we decided to alter was the placement of the probe.

3. R e s u l t s

3.1. Amphetamine effect As indicated in the Introduction, the primary observation was that 5.0 m g / k g amphetamine i.p. caused increased levels of striatal ACh. These data are presented in Fig. 1A. We also tested a small number of

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Fig. 2. The effect of 10/.~M amphetamine administered locally to the striatum via the dialysis probe. A: the extracellular ACh measured through the same lateral striatal probe placement as reported in Fig. 1A. The amphetamine was administered during sample periods 6-10 as indicated by the solid black bar. There was no effect of amphetamine on the measured ACh release in the lateral striatum (demonstrated by a lack of a placement X side interaction F 1j,~ w = 0.64, P > 0.75). B: the extracellular ACh measured through the same medial striatal probe placement as reported in Fig. lB. The amphetamine was administered during sample periods 6-10 as indicated by the solid black bar. Ten ~zM amphetamine has previously been shown to cause local excess release of DA but had no effect on striatal ACh release (F~ 1,11~ = 1.1, P = 0.4).

R.J. Mandel et al. / Brain Research 653 (1994) 57-65

60

A medial to lateral gradient of striatal D 2 receptors which coincides with striatal cholinergic markers has been reported [22,23]. Therefore, we attempted to measure striatal ACh from a more lateral striatal area which is richer in both D 2 receptors and cholinergic indices. Rats were implanted with dialysis probes bilaterally, with one probe being placed at a lateral placement and the other probe located at the standard medial striatal placement. In these animals, 10 IzM amphetamine was added to the perfusion medium. This concentration of amphetamine has previously been shown to induce local release of DA [36]. The data in Fig. 2A and 2B show that the local release of DA does not affect release of striatal ACh and that probe placement did not alter this pattern of striatal ACh levels. Once again, in the lateral striatal probe placement, 5.0 mg/kg systemic amphetamine induced a large increase in extracellular ACh (Fig. 1B). These data raised the question of whether the effect of systemic amphetamine on striatal ACh was mediated by nigrostriatal DA.

3.2. Dopamine dependence In order to evaluate the role of nigrostriatal DA in this phenomenon, rats were administered unilateral 6-OHDA lesions of the nigrostriatal tract with or without the noradrenergic system protected with DMI. These rats were then implanted with bilateral microdialysis probes in the medial probe placement and administered 5.0 mg/kg amphetamine i.p. while under

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Fig. 4. The effect of 2 m g / k g apomorphine HC1 (s.c.) on striatal A C h release. A p o m o r p h i n e was injected at the beginning of the sixth 15-min sampling interval as indicated by the arrow (n = 6). There was no significant effect of apomorphine injection on striatal A C h release F9.45 = 1.3, P > 0.25.

halothane anesthesia. The data from this experiment are presented in Fig. 3A and 3B. The data in Fig. 3 clearly indicate that amphetamine-induced excess extracellular ACh is not dependent on nigrostriatal DA. There was an apparent effect of the ascending NE system on baseline ACh levels (Fig. 3B) but the amphetamine effect was completely intact as compared to the lower ACh baseline. Further indication of the independence of striatal ACh levels from DA receptor stimulation was obtained when ACh levels were measured after injection of a behaviorally active dose of the direct DA agonist apomorphine (2 mg/kg, i.p., see

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Fig. 3. The effect striatal D A denervation on systemic amphetamine-induced striatal A C h release. The injection of a m p h e t a m i n e at the beginning of the sixth sampling period is indicated by an arrow. A: the striatal A C h release measured from 5 animals with unilateral 6 - O H D A lesions of the nigrostriatal tract with a bilateral medial striatal probe placement. There was no difference between the pattern of A C h release between the intact and lesioned striatum (Fl,17 = 0.16, P = 0.7). Moreover, the pattern of A C h release in either striatum in the 6 - O H D A lesioned rats was consistent with that seen in intact rats (Fig. 1B). B: the striatal A C h release measured from 6 animals (one probe implanted in an intact striatum malfunctioned which accounts for the reported n = 5) implanted bilaterally with medial probe placements after systemic a m p h e t a m i n e treatment. In addition, these animals were unilaterally lesioned with 6 - O H D A as in (A) but the NE neurons were protected by prior DMI injection. There was an apparent affect of N E protection on the baseline levels of A C h (periods 1-5). On the other hand, the magnitude of amphetamine-induced striatal A C h release was unaffected by the protection of locus coeruleus NE neurons. Moreover, the magnitude of the a m p h e t a m i n e effect in both 6 - O H D A treatment groups is consistent with that measured in Fig. lB. ( 6 - O H D A lesion X sampling period interaction F9.153 = 0.63, P > 0.75).

R.J. Mandel et al. / B r a i n Research 653 (1994) 57-65

Fig. 4). The data from this experiment indicate that there was no effect of systemic administration of apomorphine on striatal ACh levels.

3.4. NE dependence As stated above, amphetamine also induces the release of striatal NE. We had some indication that striatal NE was not involved in the effect of systemic a m p h e t a m i n e on striatal ACh levels because animals with a lesioned ascending NE system showed the usual striatal ACh response to systemic amphetamine (Fig. 3A). However, in order to be certain that NE was not mediating the observed systemic amphetamine effect on striatal ACh, animals with the standard medial striatal probe placement were administered 5 / x M D M I in the perfusion medium over 5 sampling periods after the standard 5 baseline samples. There was no effect of adding D M I to the perfusion medium (F~,45 = 1.2, P > 0.3, data not shown). This concentration of DMI has been shown to induce excess striatal release of NE with the same probe placement and dialysis procedures [25]. These data demonstrate that excess release of striatal N E does not increase striatal ACh levels.

3.3. 5-HT dependence At this point, it is abundantly clear that the systemic amphetamine-induced increase in striatal ACh levels is not dependent on local striatal release of DA. It is well known that a m p h e t a m i n e also causes the excess releaseof other biogenic amines such as NE and 5-HT. Thus, we tested the hypothesis that the systemic amphetamine effect on striatal ACh was mediated by 5-HT release. First, a group of rats were administered 5,7-DHT lesions to remove the ascending serotonergic system and then implanted with striatal dialysis probes at the medial placement. These animals were then injected with 5.0 m g / k g amphetamine. As illustrated in Fig. 5A, 5-HT depleted rats, responded to systemic a m p h e t a m i n e in an manner identical to normal rats (cf. Fig. 1B). The second test of the 5-HT hypothesis was to sample ACh from normal striatally probe implanted rats with the drug p - c h l o r o a m o a m p h e t a m i n e (PCA, 2.0 m g / k g i.p.) which predominantly causes the excess release of 5-HT [26] in contrast to a m p h e t a m i n e (but see [31]). The data from this experiment are presented in Fig. 5B. Systemic PCA had no effect on striatal ACh levels. Thus, these two experiments appear to rule out amphetamine-induced 5-HT release as the source of increased striatal ACh after 5.0 m g / k g amphetamine.

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Fig. 5. The effect of specific manipulations of the 5-HT system on striatal A C h release. Since a m p h e t a m i n e also induces excess release of 5-HT, the two experiments reported in this figure were performed to determine if 5-HT release was responsible for the observed amphetamine-induced striatal A C h release. A: five rats were unilaterally lesioned with 5,7-DHT to remove the serotonergic innervations of the striatum. A dialysis probe was implanted in the medial striatum on the side of the lesion. The striatal serotonergic depletion had no differential effect on the amphetamine-induced striatal A C h release with a m p h e t a m i n e causing a 2-fold increase just as seen in Fig. lB. (F,, 3~ = 5.3, P < 0.001). B: the effect of systemic injection of p-chloroamphetamine (PCA). PCA has a different pharmacological profile than a m p h e t a m i n e in that 5-HT is the main transmitter released. Injection of 2.0 m g / k g which is known to cause a large increase in striatal 5-HT release had no effect on striatal A C h levels (Fg,s4 = 1.2, P > 0.3).

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R.J. Mandel et al. / Brain Research 653 (1994) 5 7 - 6 5

a m p h e t a m i n e would m i m i c the r e s p o n s e expected from these in vitro p h a r m a c o l o g i c a l experiments. T h e data i n d i c a t e (Fig. 6) that 100 /zM KC1 i n d u c e s excess release of striatal A C h b u t the effect of systemic amp h e t a m i n e is not a l t e r e d in the p r e s e n c e of KC1. Moreover, w h e n 1 0 / z M a m p h e t a m i n e is a d d e d to the KC1 in the p e r f u s i o n m e d i u m , t h e r e is n o effect of the additional a m p h e t a m i n e . T h e K + i n d u c e d d e p l o r i z a t i o n results suggest that we are actually m e a s u r i n g striatal A C h release a n d the lack of a d d i t i o n a l A C h release w h e n a m p h e t a m i n e is a d m i n i s t e r e d b e f o r e the K ÷ i n d u c e d striatal A C h increase suggests that the a m p h e t a m i n e effect is also d u e to n e u r o n a l A C h release (Fig. 6). However, t h e r e is still the possibility that a m p h e t a m i n e leads to acute cell d e a t h of striatal cholinergic i n t e r n e u r o n s a n d the i n c r e a s e d extracellular A C h release that we m e a s u r e in r e s p o n s e to a m p h e t a m i n e is d u e to l i b e r a t i o n of stored A C h d u r i n g cell death. I n o r d e r to f u r t h e r investigate w h e t h e r the amp h e t a m i n e - i n d u c e d increase in striatal A C h levels is d u e to n e u r o n a l release of A C h , we e x a m i n e d striatal A C h w h e n action p o t e n t i a l s in the vicinity of the dialysis p r o b e were blocked by i n t r a - p r o b e t e t r o d o t o x i n (T-FX, 1 /xM). T h e effect of T T X was first tested on b a s e l i n e levels of A C h a n d t h e n we a t t e m p t e d to allow b a s e l i n e A C h levels to r e t u r n to n o r m a l , a d m i n i s t e r 5.0 m g / k g a m p h e t a m i n e i.p. a n d r e p e a t the TI~X infusion.

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Fig. 7. The effects of TTX blockade of sodium channels on the levels of striatal ACh. The purpose of this experiment was to determine whether the amphetamine-induced striatal ACh release was dependent on activity-induced transmitter release. A baseline level of striatal ACh was obtained over the first 5 sampling periods and then 1 /xM TTX was added to the perfusion solution for 2 time periods as indicated by the solid bar. Three samples were taken after the cessation of TTX infusion and then no samples were taken for 1 h to attempt to allow ACh levels to return to the original baseline. At the end of the 1 h hiatus, 5 additional 'baseline' samples were obtained, after which, 5.0 mg/kg amphetamine was systemically administered (as indicated by the arrow). Forty-five rain after amphetamine administration (or 3 dialysis samples), TTX was again added to the perfusion fluid for 30 min (indicated by the second solid bar) followed by collection of the final 3 samples in the absence of TTX. TTX induced an immediate drop of striatal ACh levels (baseline vs. sampling period 6, F1.4 = 41.2, p = 0.003) which did not recover even 3 h after the cessation of the first TTX administration. However, there was a small increase in ACh coincident with the systemic injection of amphetamine (sampling periods 8-15 vs. sampling periods 16-17, F1.4 = 60.8, P = 0.001) that was abolished by the subsequent TTX infusion (sampling periods 16-17 vs. sampling periods 18-23, F1, 4 = 43.0, P = 0.003).

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5 10 15 ~ SamplingPeriod t (15min.2 Bl/min) KCL KCL

100;tM 100WV 'X Fig. 6. Effect of KCl-induced depolarization on striatal ACh release. This figure depicts the results from two sets of rats implanted with dialysis probes in the medial striatum. The first set of rats were perfused with 100 /zM KC1 through their dialysis probe during one sampling period (indicated by arrows) before and after administration of 10/zM amphetamine in the perfusion fluid (indicated by the black bar) 24 h after probe implantation. These same rats received the identical treatment 48 h after probe implantation except that they received 5.0 mg/kg i.p. before the second KC1 infusion. The second group of rats received 2 pulses of KCI infusion at the same time points as the amphetamine treated rats. The first KCl-induced ACh peak of the systemically treated rats (solid squares) is low because this was the second day of dialysis which apparently causes a general reduction in KCl-induced striatal ACh release independent of amphetamine-induced ACh release (Mandel, unpublished data).

T h e results of this e x p e r i m e n t are p r e s e n t e d in Fig. 7. T T X r e d u c e d the b a s e l i n e levels of striatal A C h to n e a r u n d e t e c t a b l e levels. T h e b a s e l i n e levels of A C h were not recovered even 3 h later w h e n a m p h e t a m i n e was a d m i n i s t e r e d . As d e m o n s t r a t e d in Fig. 7, t h e r e was a small b u t significant i m m e d i a t e increase in striatal A C h which directly after a m p h e t a m i n e a d m i n i s t r a t i o n that was completely abolished by the s u b s e q u e n t T T X infusion. T h e r e f o r e , c o n s i d e r i n g the K + data along with the T T X - i n d u c e d decrease of b a s e l i n e A C h levels the effect of 5.0 m g / k g a m p h e t a m i n e o n striatal A C h is p r o b a b l y d u e to n e u r o n a l release of ACh.

4. D i s c u s s i o n

T h e o b s e r v a t i o n that a m p h e t a m i n e elicited a large increase in extracellular striatal A C h had b e e n rep o r t e d previously [10,16]. H o w e v e r these data do not agree with in vitro, e x p e r i m e n t s in the striatal slice p r e p a r a t i o n w h e r e d o p a m i n e r g i c m a n i p u l a t i o n s of A C h overflow is the prototypical assay for D 2 r e c e p t o r func-

R.Z Mandel et aL / Brain Research 053 (1994) 57 65

tion [33] with D 2 receptor stimulation leading to a decrease in extacellular ACh [13]. In fact, the opposing interaction of ACh and D A in striatum is so fundamental that it forms the conceptual basis for the use of cholinergic blockers along with L - D O P A in treating Parkinson's disease [3]. On the other hand, several instances of DA agonists causing increased release of striatal ACh have been previously reported [1,5,10,11, 16,27] (but see [12,35]) and the lack of effect of removal of nigrostriatal DA has also been reported [2]. These previous reports of DA agonists increasing extracellular striatal ACh all come from in vivo preparations. Therefore, the present data, in agreement with these earlier reports indicate that the pharmacology of striatal ACh is quite different in vivo as compared to the in vitro striatal slice preparation. On the other hand, some previous reports [5,9,12, 35,38] using various direct DA agonist with receptor subtype specificity a n d / o r amphetamine report in vivo data which agree with the opposing A C h / D A pharmacological profile seen in vitro. The previous studies regarding decreased striatal ACh in response to amphetamine a n d / o r apomorphine [9,12] utilize the trans-striatal dialysis probe technique where the dialysis fiber runs through the striatum in the medial to lateral direction unlike dialysis procedures which utilize loop or concentric dialysis probes which have the membrane in contact with the striatum in the dorsal to ventral orientation. Therefi~re, in the present paper we tried to assess whether differences in the dopaminergic control of ACh in the medial vs. lateral striatum could account for the differences between the present data and those found with transdialysis probes especially in light of the fact that there is a medial to lateral gradient of ACh neurons in the striatum [23]. Thus, striatal ACh was measured in both the medial and lateral striatum (Fig. 1) after systemic amphetamine treatment and after intra-striatal amphetamine treatment (Fig. 2). The data from these two experiments indicate that amphetamine increases striatal DA release in both the medial and lateral striatum and that this effect is not locally controlled within striatum. The striatal probe-placement experiments do not resolve the differences between the present study and those reporting the opposite effect of amphetamine and apomorphine on striatal ACh overflow [9,12]. Another significant methodological difference between the present paper and the previous reports is that the present data were collected from anesthetized rats while the rats were awake and freely moving in the previous reports [9,12]. Therefore, it is possible that halothane anesthesia acts to remove dopaminergic control of striatal ACh. Because amphetamine is known to have noradrenergic and serotonergic releasing properties, it was possible that increased levels of these biogenic amines in

~3

striatum may have accounted for the increased striatal ACh. None of the experiments designed to evaluate the effect of increased striatal 5-HT or NE on ACh release revealed any interaction between ACh and these biogenic amines. Moreover, 6-OHDA induced degeneration of the nigrostriatal tract with or without concomitant damage to the NE-containing locus coeruleus had no effect on amphetamine-induced striatal ACh release. Thus, we are left with a situation where a drug, amphetamine, whose major neuropharmacogical effect is reported to be increased DA release, DA re-uptake blockade, and possibly inhibition of monoamine oxidase, induces excess striatal ACh release independent of local DA indices. These data also rule out the possiblity that amphetamine has direct effects on striatal ACh release. If amphetamine could directly stimulate striatal ACh release then local injection of amphetamine would be expected to increase ACh levels which was not the case (Fig. 2A,B). In addition, increased amphetamine-induced striatal ACh release is probably not specific to the high dose (5.0 m g / k g ) because a lower dose (2.5 m g / k g ) induced a similar increase in a small number of animals in this study and 2.0 m g / k g amphetamine has also been reported to induce increase striatal ACh levels [10]. Finally, a systemic dose of the direct DA agonist apomorphine which is known to cause striatally mediated stereotypic behavior [8] and reduce striatal DA release [7,37] had no effect on striatal ACh release (Fig. 4). While these apomorphine data could be interpreted to suggest that there is no dopaminergic involvement in striatal ACh release, different doses of apomorphine may provide the appropriate stimulus for striatal ACh due to different ratios of DA receptors or apomorphine-sensitivity in extra-striatal sites [9,34]. This possibility is supported by data regarding the effect of stimulation of specific DA receptor subtypes on striatal ACh release, where there is general agreement in the literature that D 1 receptor stimulation induces increased striatal ACh while D 2 receptor stimulation has the opposite effect [5,19,35]. Thus, since both DA agonists utilized in this study result in the stimulation of both D 1 and D 2 receptors, the specific dose of drugs used in the present experiment may have resulted in no net effect on dopaminergically mediated ACh release directly within the striatum. There are two possible explanations which are not directly addressed in the present set of experiments that could account for amphetamine-induced striatal ACh release. First, the HPLC assay currently in use has a detection limit of 0.2 pmol of ACh. Baseline levels of striatal ACh are lower than the detection limit so the AChE inhibitor neostigmine must be added to the perfusion fluid to artificially increase striatal ACh levels. Thus, the D A / A C h interaction may be affected

64

R.J. Mandel et aL / Brain Research 653 (1994) 57-65

by increased ACh levels present during the dialysis procedure such that the normal reciprocal interaction no longer predominates when chronic ACh levels are above the normal baseline. Although the mechanism by which striatal AChE inhibition might affect the striatal D A / A C h interaction is not known. The other possibility is that, at the high dose of amphetamine, DA release in a terminal field not affected by a nigrostriatal 6-OHDA lesion (A-8?) is mediating striatal ACh release. With regard to the later possibility, the best candidate system is frontal cortical DA release which in turn causes increased corticostriatal glutamate activity which then could induce increased levels of striatal ACh. While there are no published reports to support or refute the first possiblity, there are reports which do lend support to the second idea [10]. Moreover, the two explanations are not mutually exclusive so that with tonically increased ACh levels a frontal cortical DA interaction with the corticostriatal glutamate pathway may become more important as compared to the situation where normal striatal ACh levels are present.

Acknowledgements The authors greatfully recognize the invaluable technical assistance of Gertrude Stridsberg, AnnaKarin Old6n, Birgit Haraldsson, Alicja Flasch, and Ulla Jarl of the Department of Medical Cell Research. We also recognize the assistance of Karin Fyge of the Dept. of Pharmacology. R.J.M was supported by a Fogarty Fellowship from the NIH.

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