MOLECULAR

RESEARCH EL S E VI E R Molecular Brain Research 21 (1994) 359-362

Short Communication

Acute amphetamine or methamphetamine alters opioid peptide mRNA expression in rat striatum

April J.W. Smith, Jacqueline F. McGinty * Department of Anatomy and Cell Biology, East Carolina University School of Medicine, Greenville, NC 27858, USA

(Accepted 5 November 1993)

Abstract

The effects of a single dose of amphetamine or methamphetamine on opioid peptide gene expression in the dorsal and ventral striatum of rats were investigated by quantitative in situ hybridization 3, 6, or 18 h after injection. Although amphetamine-treated rats exhibited significantly different behaviors than those treated with methamphetamine, both drugs caused a patchy increase in preprodynorphin, but not preproenkephalin, mRNA in the caudate at all 3 time points. No changes were detected in the nucleus accumbens. These data indicate that prolonged elevation of preprodynorphin expression may alter the responsiveness of striatonigral neurons to subsequent amphetamine exposure.

Key words: Preprodynorphin; Preproenkephalin; In situ hybridization; Caudate; Nucleus accumbens

A consensus has emerged that repeated administra- tion of indirect or direct dopamine agonists results in increased synthesis of the opioid peptide, preprodynor- phin (PPD), but not preproenkephalin (PPE), in stria- tonigral neurons [1,3,5,8,10,11,14]. In contrast, it has been demonstrated that a single dose of apomorphine or amphetamine (AMPH) causes a decrease in striatal dynorphin immunoreactivity within 1 hour [11,14], sug- gesting that an abrupt decrease in synthesis a n d / o r an increase in local release of dynorphin occurs as a response to dopaminergic agonists. Therefore, it was somewhat surprising when Hanson and colleagues demonstrated that a single dose of methamphetamine (METH) causes a robust and prolonged (6-48 h) in- crease in dynorphin immunoreactivity in the caudate- putamen [5]. More recently, Hurd and Herkenham [7] demonstrated an increase in PPD mRNA in the dorso- lateral caudate-putamen 2 h after a single dose of cocaine but not AMPH. The relatively delayed and prolonged elevation of dynorphin immunoreactivity af- ter METH [5] and the possibility of a different re- sponse to AMPH over time, prompted this study of the effects of acute AMPH or METH on PPD and PPE

* Corresponding author. Fax: (1) (919) 816-2850. Internet: mcginty@merlin.med.ecu.edu

0169-328x/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0169-328X(93)E0210-Q

gene expression in rat striatum 3, 6, and 18 h after injection.

These experiments were conducted in accordance with the NIH Guide for the Care and Use of Labora- tory Animals. Adult, male Wistar rats (200-300 g; Charles River, Raleigh, NC) were housed individually in clear plastic cages and kept on a 12 h l ight /dark schedule with food and water ad libitum. After accli- mating to their surroundings, the rats were weighed and injected once with either d-amphetamine sulfate (5 mg /kg , i.p., calculated as the salt, diluted in sterile saline, n--12) , methamphetamine HCI (15 mg/kg , i.p., calculated as the salt, diluted in sterile saline, n = 12), or an equal volume of sterile saline alone (i.p., n = 12). The behavior of the rats was rated at 5 min intervals for 1 hour, using a 10-point scale modified from Ellinwood and Balster [2]: (1) asleep, inactive; (2) alert, normal in place activities; (3) increased activity; (4) hyperactivity with jerky movements; (5) slow pat- terned (repetitive exploration); (6) fast patterned (rep- etitive exploration with hyperactivity); (7) repetitive sniffing a n d / o r rearing in one location; (8) continuous sniffing, gnawing, or licking to the exclusion of other activity; (9) motionless; and (10) dyskinesia, seizures.

At 3, 6, or 18 h after the injection, the rats ( n - - 4 per group) were anesthetized with Equithesin (5 ml /kg ) and decapitated. The brains were removed from the

3 6 0 A..I It ' . Smith, .1. t" Mc(iint~ ' 3,.lolecular Brain Research 21 ¢ 1994) 359-362

skull and immediately frozen in isopentanc at 40°C and stored at - 7 0 ° C until ready to section for in situ hybridization histochemistry. Twelve /xm sections were collected throughout the striatum on twice ge la t in / chromalum-subbed slides. The slides were postfixed for 10 min with 4% paraformaldehyde in 10 mM phos- phate buffered saline, pretreated for 10 min with 0.25% acetic anhydride/0.1 M triethanolamine in 0.9% NaCI, defatted in chloroform and alcohol and dried. Oligonu- cleotide probes, 48 bases in length, for preprodynor- phin (PPD; bases 862-909) [15] and preproenkephal in (PPE; bases 388-435) [15], were synthesized and puri- fied by the ECU DNA Synthesizer Core Laboratory. The probes were labeled at the 3' end with e-[3sS]de- oxyadenosine tr iphosphate (1075 C i / m m o l ; New Eng- land Nuclear), using terminal deoxynucleotidyl trans- ferase (Boehringer Mannheim Biochemicals) in a pro- tocol modified from Young and colleagues [15]. Pre- treated slides were incubated with 1 × 10 ~ cpm/25 /xl hybridization buf fe r / sec t ion overnight (16-20 h) at 37°C in a humid environment. The slides were washed

A

9

B E H A V I O R A L R A T I N G S C A L E

8 $*AUC = 445.21

7 *AUC = 361.25

5 ~ C O N T I

- -O- - - A M P H

' I 3

2 AUC = 105.63

0 10 20 30 40 50 60 70

TIME (IN MINUTES)

B

P R E P R O D Y N O R P H I N

2000 - e, "-

z

12oo ~

! - - o - - CONT ] i ~ A M P H

soo ! - - , , - - M ~ r n

400 ~ ~ T z

[ L 1 . . . . . . i

0 3 6 9 12 15 18

TIME (IN HOURS)

Fig. 1. A: behavioral rating of rats every 5 min for 60 min after drug injection. AUC = area under the curve. * Statistical significance (P <0.05) over controls. $ Statistical significance (P <0.05) from AMPH-treated rats. B: quantitative image analysis of PPD hybridiza- tion signal in the dorsomedial caudate-putamen in integrated density units at 3, 6 and 18 h after AMPH, METH, or saline injection. • Significant difference from control (P < 0.0167).

stringently in 1 × SSC (sterile sodium chloride, sodium citrate buffer), then in 2 × SSC/50% fl~rmamide a~ 40°C, followed by two rinses in 1 × SSC. The slides were dehydrated in an ascending alcohol series and exposed, along with 14C standards (American Radiola- beled Chemicals), to Kodak X-OMAT film for 1 week (PPE) or 2 weeks (PPD). After film development, the slides were dipped in emulsion (Kodak NTB-3, IBI: diluted 1:1 with 0.1% Dreft), exposed for 3 (PPE) oi 4 (PPD) weeks, then developed with Dektol (1:1 with water) and fixed with Kodak fixer.

Quantification of the opioid hybridization signals on X-ray films was performed using Image software (W. Rasband, NIMH) and a Macintosh Ilci. The 14C stand- ards were measured, plotted against known d p m / m g , and converted to 35S equivalences to generate a cali- bration curve. With transmittance values calibrated in d p m / m g , the dorsomedial caudate was measured us- ing a circle 150 × 150 pixels and the dorsomedial shell of the nucleus accumbens was measured using an oval 25 × 45 pixels, at AP 0.7 mm rostral to Bregma [12], in three adjacent sections per animal. Quantitative changes were recorded as (1) the number of labeled pixels per area (AREA), (2) mean density in d p m / m g of tissue and (3) integrated density which is the prod- uct of A R E A and mean density.

Statistical analysis of the hybridization data was performed using a nested two-way A N O V A followed by a least square means comparison of groups• Behav- ioral data were analyzed by calculating area under the curve (AUC) for the rating values plotted against time. A N O V A followed by a Tukey's comparison test was performed on the AUC values•

Animals treated with A M P H initially exhibited in- creased locomotor activity with some late onset (30 min) of stereotypies (rearing, sniffing). METH-t rea ted animals showed maximal stereotypies (sniffing, chew- ing, swimming motion) within 10 min of the injection. The AUC values of the AMPH- and METH-t rea ted groups were significantly increased over those of the control group (Fig. 1A). In addition, the AUC values of the METH-t rea ted animals were significantly greater than those of the AMPH-t rea ted group (Fig. 1A).

The expression of PPD m R N A in the striatum of AMPH- and METH-t rea ted animals is illustrated in Fig. 2. PPD expression in the caudate-putamen, but not in the nucleus accumbens, increased in a patchy motif at 3, 6 and 18 h after a single injection of either drug. Quantification indicated that the integrated den- sity was significantly increased, due to significant in- creases in A R E A and mean density, at 3, 6 and 18 h following a single M E T H injection as compared to those of the control group (Fig. 1B). Over the time course, integrated density was increased 163%, 3110% and 289% above control values. AMPH-t rea ted rats also demonstrated a significant increase in the inte-

A.J. IV. Smith, J.F. McGinty / Molecular Brain Research 21 (1994) 359-362 361

grated density for striatal PPD mRNA at 3, 6 and 18 h; however, at 18 h, the AMPH-induced increase was declining (Fig. 1B). The integrated density in the AMPH-treated group was increased 164%, 270% and 187% above control levels at 3, 6 and 18 h, respec- tively.

PPE mRNA levels were not significantly changed at 3, 6, or 18 h after AMPH and METH injections as compared to those in controls. However at 3 h, the PPE hybridization measurements tended to be higher in AMPH- and lower in METH-treated rats than in controls. This trend resulted in the AREA, mean den- sity and integrated density of AMPH-induced PPE mRNA being significantly greater than METH-induced PPE mRNA measurements 3 h after injection (data not shown).

This study demonstrated that, although acute ad- ministration of AMPH elicits behaviors that differ sig- nificantly from METH, both drugs produce a signifi-

cant and prolonged increase in PPD mRNA expression in the mediodorsal caudate nucleus. These data extend the observations of Hanson and colleagues [5] who reported that a single injection of METH increased striatal dynorphin immunoreactivity from 6-48 h after injection. Because dynorphin immunoreactivity is de- creased in the striatum 1 h after a single injection of apomorphine or AMPH [11,14], presumably it is de- creased in response to METH as well. The present study indicates that this decrease is followed by a compensatory increase in PPD synthesis which be- comes evident 3 h after a single dose of either AMPH or METH.

Multiple doses of AMPH [11,14], METH [4,5], 3,4- methylenedioxy-methamphetamine [13] or apomor- phine [9,10,11] in several different injection paradigms, increases dynorphin immunoreactivity 6-24 h following the last injection. The D1 receptor antagonist, SCH 23390 [4], or the NMDA receptor antagonist, MK-801

Fig. 2. Photomicrographs of coronal sections (AP 0.7 nun rostral to Bregma) from emulsion-dipped slides. The sections were hybridized with 3ss-labeled preprodynorphin oligonucleotide after (A) saline, (B-D) AMPH, or (E-G) METH at 3 h (B, E), 6 h (C, F), or 18 h (D, G). Large circle in A depicts area in caudate quantified; parentheses surround area of nucleus accumbens shell quantified; small arrowheads indicate difference in the number of patchy areas expressing preprodynorphin mRNA in A and B. Bar = 1 ram.

3~2 .4../H.'. 3mith, .1 F :SIc'(;tt~It, /.,~vIlolectdar Brain Reseapz'h 21 t 1994) 350-302

[13], prevents this stimulant-induced increase in dynor- phin immunoreactivity, but sulpiride, a D 2 receptor antagonist, does not [5,9]. These data suggest that dopamine and glutamate play an important role in regulating dynorphin immunoreactivity and mRNA ex- pression in the dorsal striatum.

This study and others [5,7] demonstrate that in- creases in striatal PPD synthesis occur after single high doses of stimulants that induce stereotypical behaviors and precede the expression of behavioral sensitization. Recently, Heidbreder and colleagues [6] have provided a clue to the possible functional significance of stimu- lant-induced alterations in the striatonigral dynorphin system. They demonstrated that the kappa opioid ago- nist, U69593, blocks the locomotor stimulating and behavioral sensitizing effects of cocaine. Thus, it is possible that dynorphin is released from striatonigral neurons in response to a single dose of cocaine or amphetamines to hinder the induction of behavioral sensitization.

We thank James B. Daunais and W. Todd Bohler for technical support and advise and Dr. James Esinhart for statistical help. This research was supported by DA03982.

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