Repeated, intermittent treatment with amphetamine induces neurite outgrowth in rat pheochromocytoma cells (PC12 cells)

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1 Brain Research 951 (2002) locate/ bres Research report Repeated, intermittent treatment with amphetamine induces neurite outgrowth in rat pheochromocytoma cells (PC12 cells) a a a,b a, Yang Hae Park, Lana Kantor, Kevin K.W. Wang, Margaret E. Gnegy * a Department of Pharmacology, 2220 MSRB III, University of Michigan School of Medicine, 1150 W. Medical Center Dr, Ann Arbor, MI , USA b CNS Molecular Sciences, Pfizer Global R&D, Ann Arbor, MI 48105, USA Accepted 20 June 2002 Abstract Repeated, intermittent treatment with amphetamine (AMPH) leads to long-term neurobiological adaptations in rat brain including an increased number and branching of dendritic spines. This effect depends upon several different cell types in the intact brain. Here we demonstrate that repeated, intermittent AMPH treatment induces neurite outgrowth in cultured PC12 cells without the requirement for integrated synaptic pathways. PC12 cells were treated with 1 mm AMPH for 5 min a day, for 5 days. After 10 days of withdrawal, there was an increase in the percentage of cells with neurites ( 30%) and the length of neurites as well as an increase in the level of GAP-43 and neurofilament-m. Neurite outgrowth was enhanced as withdrawal time was increased. Neurite outgrowth was much greater following repeated, intermittent treatment with AMPH compared to continuous or single treatment with AMPH. Pretreatment with cocaine, a monoamine transporter blocker, inhibited the AMPH-mediated increase in neurite outgrowth. Neither NGF antibody nor DA receptor antagonists blocked AMPH-induced neurite outgrowth, demonstrating that AMPH-induced neurite outgrowth is not dependent on endogenous NGF release or DA receptors. Thus we have demonstrated that repeated, intermittent treatment with AMPH has a neurotrophic effect in PC12 cells. The effect requires the action of AMPH on the norepinephrine transporter, and shares characteristics in its development with other forms of sensitization but does not require an intact neuroanatomy Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Drugs of abuse: amphetamine and other stimulants Keywords: Amphetamine; Cocaine; Norepinephrine transporter; Neurite outgrowth; Nerve growth factor; PC12 cell; Cell differentiation 1. Introduction AMPH challenge in animals [32]. The sensitization develops and strengthens with time after the discontinuation Amphetamine (AMPH) is a synthetic psychomotor of AMPH [28,30]. AMPH must be given intermittently for stimulant that acts as a substrate for monoamine transpor- the development of behavioral sensitization since the ters such as the dopamine transporter and norepinephrine continuous infusion of AMPH via a subcutaneous pellet transporter. AMPH exerts its physiological effects by does not result in behavioral sensitization [29,32]. In enhancing the transport of dopamine (DA), norepinephrine addition, a single treatment with AMPH can induce long- (NE) and serotonin from nerve terminals into the synapse term behavioral and neurochemical sensitization in rats through a reversal of their respective plasmalemmal trans- [39]. porters [36]. Repeated, intermittent treatment with AMPH Repeated, intermittent psychostimulant treatment also results in a number of long-term neurobiological adapta- tions in vivo [31,41,42]. Neuroadaptations developing after repeated AMPH include DA autoreceptor subsensitivity [41], enhanced basal levels of extracellular DA in the results in a behavioral sensitization, which is characterized by an increased locomotor response to a subsequent *Corresponding author. Tel.: ; fax: address: (M.E. Gnegy) /02/$ see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S (02) ventral tegmental area [17], enhanced stimulus-induced DA release [18,20,33] and enhanced phosphorylation of

2 44 Y.H. Park et al. / Brain Research 951 (2002) synaptic proteins [14]. The neuroadaptations exhibit differ- inactivated fetal bovine serum, 5% heat-inactivated horse ent time courses following discontinuation of AMPH. For serum, 2 mm L-glutamine, 100 mg/ ml of streptomycin and instance, the enhanced AMPH-induced DA release takes at 100 U/ ml of penicillin (Gibco) and were incubated at 10% 4 least 10 days to develop but is extremely persistent. There CO 2. To induce neurite outgrowth, 5310 cells/ ml were 2 is morphological evidence that AMPH can remodel plated in 75 cm tissue culture flask at 10% CO2 in the synapses in rat brain by increasing the density of synapses same growth medium. PC12 cells were treated with 1 mm and number of dendritic spines in rat medial prefrontal amphetamine (Sigma, St Louis, MO, USA) for 5 min/ day, cortex, nucleus accumbens and prefrontal cortex [34,35]. for 5 days. After 10 days without drugs (withdrawal), The sustained, glutamate-dependent increases in basic pictures of neurite outgrowth in PC12 cells were taken fibroblast growth factor (b-fgf) immunoreactivity elicited with a digital camera (SPOT RT) (Diagnostic Instruments) by repeated, intermittent AMPH in both the ventral at 2003 phase contrast using a Leica DMI RB inverted tegmentum and substantia nigra pars compacta [7] may microscope. For the continuous treatment with AMPH, contribute to this effect. Axonal growth and dendritic PC12 cells were treated with 1 mm AMPH for 5 days. extension are key morphological features characterizing AMPH was kept in the media, which was changed every neuronal development. Regulation of neurite outgrowth is other day during treatment days to retain AMPH activity. also an important aspect of neuronal regeneration from In experiments designed to block potential endogenous injuries or neuropathological conditions. Repeated AMPH NGF release, NGF antibody (0.5 mg/ ml) was kept in the treatment enhanced neural sprouting and synaptogenesis in media all the time until the experiment was done. Fresh the neocortex after neocortical infarction in rats [37]. This NGF antibody was added to the media every other day to suggests that AMPH may have neuroprotective effects, maintain the neutralizing activity. To block NET or DA which can be beneficial for neurodegenerative diseases receptors, 10 mm cocaine or 1 mm SCH23390/ 1 mm such as Parkinson s disease. sulpiride, respectively, were given 30 min before each We examined whether repeated, intermittent AMPH AMPH treatment. could elicit this neuronal adaptation in a cultured cell line that can undergo neurite extension and is responsive to 2.3. Quantification of neurite outgrowth AMPH. Therefore, rat pheochromocytoma cells (PC12 cells) were used for this study. PC12 cells contain an For each culture condition, randomly chosen individual AMPH-sensitive NE transporter [2], produce neurites [10] areas (containing $80 cells) were scored in pictures taken and express growth-associated proteins on exposure to under phase-contrast optics. A neurite was counted when a nerve growth factor (NGF) [16,25,47]. We examine the cellular process was longer than the diameter of the cell ability of AMPH to increase neurite outgrowth in PC12 body. The percentage of neurite outgrowth was calculated cells, the timing of AMPH treatment and withdrawal as the number of cells with neurites divided by total cell requirements, and whether the NE transporter, NGF and numbers [9,26]. The length of neurite was calculated as the dopamine receptors are required for these phenomena. average length of 10 selected cells with neurites. Each experiment was conducted in triplicate. 2. Materials and methods 2.4. Immunoblotting 2.1. Chemicals PC12 cells were lysed for 15 min in 1 mm Tris, ph 7.4 with protease inhibitor cocktail tablets, Complete Mini (1 D-Amphetamine sulfate and sulpiride were purchased tablet/ 10 ml of buffer) (Roche, Mannheim, Germany) and from Sigma (St Louis, MO, USA). Cocaine was purchased homogenized in same buffer. Nuclei and undisrupted cells from University of Michigan hospital pharmacy (Ann were removed by spinning at 10003g. Supernatants were Arbor, MI, USA). Nerve growth factor 2.5S (NGF) was collected and centrifuged for 30 min at g and purchased from Harlan Bioproducts for Science (In- 4 8C. The pellet was resuspended in 50 mm Tris HCl, 10 dianapolis, IN, USA). Anti-gb-NGF antibody was pur- mm MgCl2 and 1 mm EGTA, ph 7.6. Protein conchased from R&D systems (Minneapolis, MN, USA). centration was determined by a modified Lowry method SCH23390 was purchased from Schering (Bloomfield, NJ, (Bio-Rad D-C protein assay kit). Each lane contained 15 USA). mg protein. Resuspended pellets were resolved by electrophoresis on 8% SDS PAGE gels and transferred to 2.2. Cell culture polyvinylidene difluoride membranes (0.2 mm). Membranes were blocked with 5% milk in Tris-buffered saline 2 PC12 cells were maintained in a 75 cm tissue culture containing 0.1% Tween 20 (TBST) and incubated in 2.5% flask at 10% CO2 in growth medium composed of Dulbec- milk in TBST with mouse monoclonal anti GAP-43 co s modified Eagle s medium from BioWhittaker (Walk- (Zymed, San Francisco, CA, USA) (1:500 dilution, 1 h) ersville, MD, USA) supplemented with 10% (v/ v) heat- and mouse monoclonal anti-neurofilament-m (Zymed)

3 Y.H. Park et al. / Brain Research 951 (2002) (1:1000 dilution, 1 h). After three washes with TBST, ( 1.8 fold) and neurofilament-m ( two fold) concurrent membranes were incubated with a biotinylated secondary with AMPH-induced neurite outgrowth. The increase in antibody and avidin-conjugated alkaline phosphatase the level of GAP-43 and neurofilament-m has been (Amersham Pharmacia Biotech). The immunoblots were reported after the differentiation of PC12 cells [23,25]. developed with nitroblue tetrazolium and 5-bromo-4-chlo- These results suggest AMPH has a neurotrophic effect in ro-3-indolyl phosphate. To quantify the result, the density PC12 cells. of each band was measured using densitometry and the NIH images program (Scion) Repeated, intermittent treatment and withdrawal time are required for AMPH-induced neurite outgrowth 2.5. Data analysis in PC12 cells Statistical significance between two groups was de- To determine whether there is a requirement for retermined using a two-tailed Student s t-test. Analyses peated AMPH treatments, PC12 cells were treated with 1 among three or more groups were conducted using one- mm AMPH for 5 min, for only 1 day and given 10 days way analysis of variance (ANOVA) with post-test Tukey withdrawal (Fig. 3). A single treatment with AMPH (13 Kramer multiple comparison analysis. AMPH) induced neurite outgrowth with respect to vehicle treatment (13 veh). However, repeated treatment with AMPH (5 min/day for 5 days; 53 AMPH) showed more 3. Results robust neurite outgrowth than the single treatment with AMPH (13 AMPH). Although there was an effect with 3.1. Repeated, intermittent amphetamine induces neurite only one AMPH treatment, the percentage of cells with outgrowth in PC12 cells neurites was still very low. We then investigated whether the intermittent treatment was required or whether continu- PC12 cells were treated with 1 mm AMPH for 5 ous exposure to AMPH would also increase neurite min/ day for 5 days. After 10 days of withdrawal from outgrowth. Continuous treatment with 1 mm AMPH was AMPH, the PC12 cells were examined for neurite out- investigated with respect to repeated, intermittent treatment growth. Repeated, intermittent AMPH treatment resulted in with AMPH. PC12 cells were treated with 1 mm AMPH enhanced neurite outgrowth compared to vehicle treatment for 5 days. AMPH was kept in the media, which was in PC12 cells (Fig. 1). As quantified in Fig. 2A, repeated, changed every other day during treatment days to retain intermittent AMPH treatment significantly increased in the AMPH activity. After 10 days of withdrawal, neurite percentage of cells with neurites ( 30%) and the length of outgrowth was quantified and compared to that resulting neurites ( 21.4 mm) with respect to vehicle treatment. Fig. repeated, intermittent treatment with AMPH (53 AMPH). 2B shows a significant increase in the level of GAP-43 The result showed that, in contrast to repeated, intermittent Fig. 1. Repeated, intermittent treatment with AMPH induced neurite outgrowth in PC12 cells. PC12 cells were treated with vehicle (A) or 1 mm AMPH (B) for 5 min per day for 5 days. After 10 days of withdrawal from AMPH, pictures of PC12 cells were taken by digital camera SPOT RT (Diagnostic Instruments) at 2003 phase contrast. Scale bar represents 100 mm.

4 46 Y.H. Park et al. / Brain Research 951 (2002) Fig. 2. (A) Quantification analysis of neurite outgrowth after repeated, intermittent treatment of AMPH (5 min per day for 5 days, 10 days of withdrawal) and vehicle (veh) in PC12 cells. AMPH-pretreated cells show a significant increase in the percentage of cells with neurites (%) and the length of neurites (mm). Values represent means6s.e.m. n$3. *P,0.05, **P,0.01 compared to veh-treated control by two-tailed, unpaired t-test. (B) The increase in the level of GAP-43 and neurofilament-m after repeated, intermittent treatment with 1 mm AMPH. Western blot analysis was carried out as described in Material and methods. Representative blots for GAP-43 and neurofilament-m are shown. The density of each band was quantified and compared using densitometry. Values represent means6s.e.m. n55. *P,0.05 compared to veh-treated control by two-tailed, unpaired t-test. AMPH, continuous treatment with AMPH did not result in veloped and strengthened gradually as withdrawal time enhanced neurite outgrowth. This result demonstrated that increased (Fig. 4). intermittent treatment is required for AMPH-induced neurite outgrowth. To examine the length of time following 3.3. AMPH-induced neurite outgrowth depends on AMPH for significant neurite outgrowth to become appar- norepinephrine transporter (NET) in PC12 cells ent, the percentage of cells with neurites was determined during various withdrawal times. Although some enhance- AMPH has to bind to norepinephrine transporter (NET) ment was apparent by 3 days after the last AMPH and be transported to the cell or nerve terminal in order to treatment, significant AMPH mediated neurite outgrowth release catecholamines [36]. In a recent paper, we showed was evident by 8 days following discontinuation of that our PC12 cells contain a norepinephrine transporter AMPH. Therefore, AMPH-induced neurite outgrowth de- but not the dopamine transporter [21]. However, AMPH

5 Y.H. Park et al. / Brain Research 951 (2002) can also easily diffuse through cellular membranes. To investigate whether AMPH-induced neurite outgrowth is dependent on NET, PC12 cells were pretreated with 10 mm cocaine, a NET blocker, 15 min before AMPH treatment. Unlike AMPH, cocaine blocks the uptake and release of dopamine without acting as a substrate. As shown in Fig. 5, cocaine pretreatment significantly blocked AMPH-induced neurite outgrowth. Cocaine alone did not affect vehicle-induced neurite outgrowth. It suggests AM- PH-induced neurite outgrowth is dependent on NET in PC12 cells The relationship between AMPH and NGF in terms of neurite outgrowth Since AMPH can elicit release of a growth factor in vivo [8] and PC12 cells are highly responsive to NGF, PC12 cells were treated with AMPH and NGF to see if AMPH would potentiate the effect of NGF (Fig. 6A). After 5 days treatment with 1 mm AMPH, PC12 cells were Fig. 3. Repeated, intermittent treatment was required for AMPH-induced treated with 5 ng/ml of NGF to induce neurite outgrowth neurite outgrowth. PC12 cells were treated with a single dose (13 Veh or during off AMPH time. The low dose of 5 ng/ml NGF 13AMPH) or repeated, intermittent doses (53 veh or 53 AMPH). PC12 was used to permit visualization of any potentiation effect cells were also treated continuously with vehicle or 1 mm AMPH (C-veh of AMPH. NGF was added to the media on days 2 and 6 or C-AMPH) as described in Material and methods. Values represent means6s.e.m. n53. ANOVA, P, In post-hoc Tukey analysis, after stopping AMPH. On withdrawal day 10, the neurite [ P,0.05 versus 13 veh; *P,0.05, ***P,0.001 versus 53 Veh. outgrowth was quantified. There was no significant difference between NGF only, AMPH only or AMPH plus NGF treatment in the percentage of cells with neurites. AMPH was neither potentiative nor additive to NGF in terms of neurite outgrowth in PC12 cells. To ensure that the effects of AMPH were not mediated by NGF, PC12 cells were also treated with NGF antibody to block potential endogenous NGF release. Before the experiment, the neutralization effect of NGF antibody was investigated and 0.5 mg/ ml NGF antibody was found to neutralize external NGF growth effects (Fig. 6D,E). NGF antibody was kept in the media during the treatment and withdrawal time. Fresh NGF antibody was added to the media every other day to maintain the neutralization activity. As shown in Fig. 6A C, NGF antibody did not block AMPH-induced neurite outgrowth. This result shows repeated, intermittent AMPH-induced neurite outgrowth is not dependent on endogenous NGF release AMPH-induced neurite outgrowth does not depend on DA receptors in PC12 cells The principal effect of AMPH is to elicit the release of DA and norepinephrine [36]. There is some evidence that Fig. 4. AMPH-induced neurite outgrowth is dependent on withdrawal PC12 cells have D2 receptors [13,46]. To examine whether time. PC12 cells were treated with 1 mm AMPH for 5 min/ day for 5 days DA receptors play a role in AMPH-induced neurite and neurite outgrowth was measured at various times following the final outgrowth, DA receptor antagonists were used. PC12 cells AMPH treatment in PC12 cells. AMPH-induced neurite outgrowth was were pretreated with 1 mm SCH23390, a D1 receptor strengthened as withdrawal time increased. Values represent means6s.e.m. n$3. ANOVA, P,0.0001, In post-hoc Tukey analysis, antagonist or 1 mm sulpiride, a D2 receptor antagonist, 30 AMPH significantly differed from vehicle after withdrawal day 8 and 10. min before AMPH treatment. As shown in Fig. 7, neither *P,0.01, **P, the D1 nor the D2 antagonist blocked AMPH-induced

6 48 Y.H. Park et al. / Brain Research 951 (2002) Fig. 5. Cocaine pretreatment blocked AMPH-induced neurite outgrowth. PC12 cells were pretreated with 10 mm cocaine 15 min before 1 mm AMPH treatment (5 min per day for 5 days). After 10 days of withdrawal from drug treatment, the percentage of cells with neurites was calculated as described in Material and methods. Cocaine pretreatment blocked AMPH-induced neurite outgrowth (1cocaine). Cocaine alone did not show a significant difference from vehicle (veh). Values represent means6s.e.m. n$3. ANOVA, P, In post-hoc Tukey analysis, AMPH significantly differed from veh, cocaine and AMPH1cocaine at *P, neurite outgrowth, suggesting DA receptors are not re- nucleus accumbens and prefrontal cortex [34,35,38]. Our quired for AMPH-induced neurite outgrowth in PC12 results, however, demonstrate that AMPH can directly cells. elicit this effect without the need of intact synaptic pathways or even DA receptors. The in vivo effects of AMPH in inducing neuronal 4. Discussion sprouting could be mediated by enhanced production of a growth factor. Repeated, intermittent AMPH treatment In this study, we have made the novel observation that elicits an increase in the level of astrocytic b-fgf in repeated, intermittent AMPH induces neurite outgrowth in dopamine cell body areas [8]. Although AMPH initiates PC12 cells. Repeated, intermittent treatment with AMPH the effect, the increase in astrocytic b-fgf production increased the percentage of cells with neurites and the appears to be more directly mediated by glutamate. PC12 length of the neurites in these cells. The AMPH-induced cells are highly responsive to the growth-enhancing effects neurite outgrowth was characterized by an increase in of NGF and can produce NGF in response to stress stimuli, GAP-43 and neurofilament-m, which also occurs during such as serum deprivation [9]. Mena et al. [26] demon- NGF-initiated neurite outgrowth and differentiation in strated that L-dopa could potentiate NGF-induced neurite PC12 cells [16,23,25]. Our observations are in agreement outgrowth in PC12 cells. The action of AMPH in increaswith an in vivo study of Stroemer et al. [36] in which ing neurite outgrowth in PC12 cells, however, is not repeated, intermittent AMPH resulted in an enhanced mediated by an AMPH-mediated increase in secretion of neuronal sprouting and synaptogenesis following unilateral NGF since the AMPH treatment was not blocked by neocortical ischemia in the rat. Significant increases in antibody to NGF. Neither did AMPH potentiate the action GAP-43 were found ipsilateral to the lesion from 3 to 14 of NGF. AMPH is unlikely to be stimulating the formation days following initiation of widely-spaced injections of of b-fgf in our experiments since RT-PCR failed to detect AMPH. In vivo effects of repeated intermittent AMPH on m-rna for b-fgf in PC12 cells [45]. enhancement of neuronal growth in an intact brain have Several requirements for development of the AMPHalso been demonstrated. Repeated, intermittent AMPH induced neurite outgrowth in PC12 cells are similar to leads to an increase in dendritic branching and the density requirements for development of other neuroadaptations of dendritic spines on medium spiny neurons in the and behavioral expressions following repeated AMPH. The

7 Y.H. Park et al. / Brain Research 951 (2002) Fig. 6. NGF does not mediate AMPH-induced neurite outgrowth. (A) PC12 cells were treated with 1 mm AMPH (5 min per day, 5 days treatment and 10 days of withdrawal); 5 ng/ml of NGF was added at post-amph day 2 and 6. AMPH was not additive to NGF in terms of neurite outgrowth in PC12 cells. The NGF antibody (0.5 mg/ ml) was kept in the media during the treatment and withdrawal time. NGF antibody did not block AMPH-induced neurite outgrowth. Values represent means6s.e.m. n53. ANOVA, P, In post-hoc Tukey analysis, *P,0.05, **P,0.01, ***P,0.001 versus vehicle. (B E) NGF antibody did not block AMPH-induced neurite outgrowth. (B and C) PC12 cells were treated with AMPH and NGF antibody (0.5 mg/ml) to neutralize potential endogenous NGF release. PC12 cells were treated with 1 mm AMPH 5 min/day, for 5 days without (B) or with (C) 0.5 mg/ml of NGF antibody. Pictures were taken as described in Material and methods. Scale bar represents 100 mm. (D and E) Neutralizing effect of NGF antibody on external NGF effect on PC12 cells. PC12 cells were plated on six-well plates and treated with 10 ng/ml NGF without (D) or with (E) 0.5 mg/ml of NGF antibody. After 48 h, pictures were taken.

8 50 Y.H. Park et al. / Brain Research 951 (2002) compared to the continuous administration of L-dopa [4]. On the other hand, continuous infusion but not repeated administration of apomorphine reduced protein and mrna content of tyrosine hydroxylase in rats [15], suggesting that continuous exposure could lead to a down-regulation of some activities. Continuous treatment of PC12 cells with L-dopa for h elicited a variable neurite formation, but the neurites were delicate in appearance and did not last beyond 3 days [26]. Although some neurotoxicity was apparent after high doses of L-dopa (100 mm) [26], in our experiments no apparent reduction in cell number was noted following continuous administration with the comparatively low dose of 1 mm AMPH. Therefore the pulsatile nature of the drug administration is crucial for the development of enhanced responsiveness. A third characteristic in the development of AMPHinduced neurite outgrowth in the PC12 cells that is shared with other neuroadaptations in response to AMPH is that Fig. 7. DA receptor antagonist pretreatment did not block AMPH-induced of progressive enhancement and persistence following neurite outgrowth. PC12 cells were pretreated with 1 mm withdrawal from AMPH. Some effects, such as behavioral SCH23390 or 1 mm sulpiride 30 min before vehicle or 1 mm AMPH sensitization, begin within days after stopping repeated treatment (5 min per day for 5 days). After 10 days of withdrawal from AMPH, while others, such as enhanced stimulus-induced drug treatment, the percentage of cells with neurites was calculated as described in Material and methods. DA receptor antagonist alone did not DA release take a week or more to develop [31]. AMPH- show a significant difference from vehicle. Values represent induced neurite outgrowth was not evident until at least 3 means6s.e.m. n$3. ANOVA, P, In post-hoc Tukey analysis, days after discontinuing AMPH but was progressively [ **P,0.01 versus veh; P,0.01 versus SCH23390; *P,0.05 versus enhanced with time. The effect persisted through at least sulpiride. 10 days following withdrawal from AMPH. The reason for this slow neurite outgrowth is unclear. Although NGFfirst is our finding that the AMPH-induced neurite out- induced neurite outgrowth is evident within days, slower growth developed after one AMPH dose but was far more rates of induction of neurite outgrowth in PC12 cells have pronounced following repeated AMPH. An enhancement been reported in response to growth factors such as in both locomotor behavior and dopamine release to a epidermal growth factor and to proteasome inhibitors challenge of AMPH has been reported following a single [12,27]. dose of AMPH [33,39,40]. These effects, as is our It was important to examine whether active transport observation of neurite outgrowth, are progressively en- into the cell through NET was required [36]. AMPH could hanced following repeated AMPH treatments. The increase elicit this effect by simply diffusing across the membranes in values for vehicle treatment between 1 and 5 days is or binding to the newly-reported trace amine receptor likely due to the mild stress to the cells of changing the [3,36]. We found that blockade of the transporter with media daily. However, the action of AMPH was always cocaine inhibited the effect of AMPH. Therefore, the greater than vehicle. neurotrophic effects of AMPH, in addition to the be- A second requirement shared with development of other havioral effects and catecholamine-releasing effects, reneuroadaptations following repeated AMPH is the fact that quired uptake of AMPH through the transporter into the AMPH must be administered intermittently rather than cell. Cocaine alone did not change the level of vehiclecontinuously for the neurite outgrowth to occur. Continu- induced neurite outgrowth. Continuous cocaine (10 mg/ ous administration of AMPH in an animal, in which blood ml) has been reported to inhibit nerve growth factorlevels are maintained at high levels for days, leads to a induced neurite outgrowth in PC12 cells [43] but cocaine behavioral tolerance and neurotoxicity rather than the alone was not toxic. The NGF-inhibiting role of cocaine behavioral sensitization seen after intermittent AMPH appeared to be mediated through dopamine D1 receptors [24,29,32]. For instance, acoustic startle in mice in re- [44]. In addition, D1 receptors are involved in AMPHsponse to a test dose of AMPH was enhanced following mediated sensitization in VTA [1]. Since the main effect of repeated, intermittent but not continuous AMPH [22]. A AMPH is to elicit DA release, it is possible that AMPHsimilar dichotomy in responses was reported following induced neurite outgrowth is dependent on dopamine continuous versus intermittent treatment with other dopa- receptors. However, we found dopamine receptor antagominergic agents. Intermittent treatment of 6-hydroxy- nists do not block AMPH-induced neurite outgrowth, dopamine-treated rats with L-dopa led to a robust rotation- suggesting that dopamine receptors are not involved in the al response to the dopamine D2 agonist, quinpirole, neurotrophic effects of AMPH. Similarly, dopamine re-

9 Y.H. Park et al. / Brain Research 951 (2002) ceptors did not appear to be involved in the NGF-potentiat- and D-2 dopamine agonists, Eur. J. Pharmacol. 168 (1989) 291 ing effects of L-dopa on PC12 cells [26] [5] A.E. Fleckenstein, R.R. Metzger, M.L. Beyeler, J.W. Gibb, G.R. At this time, we do not know the mechanism for the Hanson, Oxygen radicals diminish dopamine transporter function in effect of AMPH on neurite outgrowth. Possibilities include rat striatum, Eur. J. Pharmacol. 334 (1997) a role for protein kinases, including protein kinase C, and [6] A.E. Fleckenstein, R.R. Metzger, D.G. Wilkins, J.W. Gibb, G.R. oxidative processes. We have demonstrated that inhibitors Hanson, Rapid and reversible effects of methamphetamine on of protein kinase C block the ability of AMPH to release dopamine transporters, J. Pharmacol. Exp. Ther. 282 (1997) dopamine through the plasmalemmal dopamine transporter [7] C. Flores, D. Rodaros, J. Stewart, Long-lasting induction of as- [19]. AMPH could be acting through either oxidant or trocytic basic fibroblast growth factor by repeated injections of antioxidant pathways. Methamphetamine causes oxygen amphetamine: blockade by concurrent treatment with a glutamate radical formation and decreases the activity of dopamine antagonist, J. Neurosci. 18 (1998) transporter in rat striatum [5,6]. Mild pro-oxidant treatbasic [8] C. Flores, A.N. Samaha, J. Stewart, Requirement of endogenous fibroblast growth factor for sensitization to amphetamine, J. ments have been suggested to protect cells from death by Neurosci. 20 (2000) RC55. up-regulating GSH [11]. On the other hand, L-dopa and [9] J.S. Gill, A.E. Schenone, J.L. Podratz, A.J. Windebank, Autocrine apomorphine are proposed to potentiate the neurotrophic regulation of neurite outgrowth from PC12 cells by nerve growth effects of NGF through synergistic induction of antioxidant factor, Brain Res. Mol. Brain Res. 57 (1998) pathways [26]. We are currently investigating the mechaline [10] L.A. Greene, A.S. Tischler, Establishment of a noradrenergic clonal of rat adrenal pheochromocytoma cells which respond to nerve nism of AMPH-induced nerve outgrowth. growth factor, Proc. Natl. Acad. Sci. USA 73 (1976) In summary, repeated, intermittent treatment with [11] S.K. Han, C. Mytilineou, G. Cohen, L-DOPA up-regulates gluta- AMPH shows neurotrophic effect in PC12 cells. AMPH thione and protects mesencephalic cultures against oxidative stress, has to bind to a monoamine transporter to elicit neurite J. Neurochem. 66 (1996) outgrowth and this effect is not mediated by NGF or [12] K. Hashimoto, G. Guroff, Y. Katagiri, Delayed and sustained dopamine receptors. Understanding the regulation of the activation of p42/ p44 mitogen-activated protein kinase induced by proteasome inhibitors through p21(ras) in PC12 cells, J. Neurochem. neurite outgrowth in PC12 cells after repeated, intermittent 74 (2000) treatment with AMPH will give greater insights into the [13] K. Inoue, K. Nakazawa, T. Watano, M. Ohara-Imaizumi, K. neuronal adaptations following withdrawal from repeated Fujimori, A. Takanaka, Dopamine receptor agonists and antagonists AMPH. They could also contribute to the therapeutics of enhance ATP-activated currents, Eur. J. Pharmacol. 215 (1992) neurodegenerative disease such as Parkinson s disease [14] S. Iwata, G.H.K. Hewlett, M.E. Gnegy, Amphetamine increases the phosphorylation of neuromodulin and synapsin I in rat striatal synaptosomes, Synapse 26 (1997) Acknowledgements [15] S. Iwata, M. Nomoto, S. Kaseda, S. Tanoue, M. Shimosaka, T. Fukuda, TH protein and mrna in nigrostriatal dopaminergic The authors would like to thank Dr Ronald Holz for his neurons are down-regulated by continuous but not intermittent apomorphine, Brain Res. Mol. Brain Res. 82 (2000) generous gift of PC12 cells. 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