Cyp2d1 Polymorphism in Methamphetamine-Treated Rats

Neurotoxicology and Teratology, Vol. 20, No, 3, pp. 265-273, 1998 © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0892-0362/9H $19.lXl + .00

PH 50892-0362(97)00129-3

CYP2Dl Polymorphism in Methamphetamine-Treated Rats: Genetic Differences in Neonatal Mortality and Effects on Spatial Learning and Acoustic Startle CHARLES V. VORHEES,* TRACY M. REED,* MARY A. SCHILLING,*l

J. EDWARD FISHER,*2 MARY S. MORAN,* GREGG D. CAPPON* AND DANIEL W. NEBERTt

*Di vision of Developmental Biology, Children's

Hospital Research Foundation and Department of Pediatrics, University of Cincinnati, Cincinnati, 0 H; , Center fo r En vironmental Genetics, Department of En vironm ental Health, University of Cincinnati Medical Center, Cincinnati , OH Received 11 December 1996; Accept ed 10 October 1997

VO R H E ES, C. V., T. M. REED, M . A. SC H ILLI NG , J. E. FISHE R, M. S. MORA N, G . D. CA PPON AND D. W. NEBERT. C Y P2D I polym orph ism in methamph etam ine-treated rats: Genetic differences in neonatal mortality and effects on spatial learning and acoustic startle. NEU ROT O X ICO L TE R ATOL 20(3) 265-273, 199H.-d-Meth amphetamine (MA) is on e of mor e than two dozen drugs includ ed in the cytochrome P450-m edi at ed "debrisoquine oxid ati on polymorphism " panel. T he human gene (C YP2 D6) is responsible for th e " poor met ab olize r" ( PM) a nd "e xte nsive met abolizer " (E M) phenotype s for dru gs such as MA : a simila r polymorph ism (the C Y I'2D I ge ne) exists in rats . Female Black o r Da rk Agouti rats exhihit the PM phenotype , wher eas Sprague- Dawley (SD) rat s sho w th e E M tr ail. We sought to test th e possibil ity that these strains of rats might exhibit a ltered MA -induced developmental neurot oxicity. Neonatal exposure to MA o n day s 11-20 has previously heen shown to indu ce spa tia l learning deficits in Spr ague-D awley rats when tested as adults. Th e refore, in the present expe riment, on postpartum days II through 20, ACI (Black A gout i) and SD progeny were administer ed 30 mg/kg MA twice daily. MA treatment caus ed lar ger increases in mortality in A CI tha n in SD rats, suggesting th at decreased MA metabolism lead s to enhanced toxi city and letha lity. Female offspring were assessed beh aviorally as adults. No differe nces were observed in aco ustic startle or st rai ght swimmi ng cha nnel performance. In th e Morri s maze, both MA-tre at ed rat stra ins showed longer lat enci es to find the hidd en plat form du rin g acquisition, rein st atem ent , and shift trials, and spe nt less tim e in th e target quad ra nt on probe trials; no strain differ en ces in learning wer e foun d. A ltho ugh th ese data do not support our hypoth esis that MA induced development al neu rot o xicity might be e nha nced in th e AC I rat , thi s interpret a tion is tempered by th e high mort alit y ra te (05 %) o f MA-tr eat ed AC I neon at es, sugges ting a possible "survivo r e ffect" in thi s strai n. © 1998 Else vier Scienc e Inc. Cy toc hro me 1'4 50 Me thamphetamine

C YP2D I Debrisoquine polymorphism ACI inbred rat strai n Morri s maze Developm ent Black A gout i inbred rat strai n Sprague-Dawley rat strain

T HE adve nt of methamphet amin e (MA) self-administra tion by smoking has led to a sharp increase in its use (5,7) . Th is, in turn , has raised concern about the effects of in utero exposure to MA and its possible long-term effec ts on offsprin g develop ment (8,9,15,24).

In rod ents , the period of br ain developm ent equi valent to hum an embryogenesis and feto genesis extends through 3 weeks postn at ally (3), Based on this, we recentl y showed that exposur e to d-MA on postn at al days 1-10 or 11-20 results in lon g-term augmentatio n of acou stic sta rtle responses (26,28) ,

Requests for reprints should he addressed to C harles V. Vor hees . Ph.D ., D ivision of Developmental Biology, Child re n's H ospital Resear ch Foundat ion , 3333 Burnet Ave ., C incinna ti. O H 45229-3039. Tel: (5 13) 636-8622; Fa x: (5 13) 636- 39 12: E-ma il: cha rles.vor [email protected] 'Current address: Department of Biology, College of Mount SI. Joseph, C incinna ti, OH 45233. 2C urre nt address: Division of Neuroph a rm acological Drug Products, Ce nter for Drug Evaluation a nd Resear ch, U.S. Food and Drug Adm inistrat ion (HFD -120), Parklawn Bldg., Rockvill e , MD 20857.

265

266

VORHEES ET AL.

whereas onl y exposure on days 11-20 results in deficits in spatiallearning (26) . Polymorphisms of Ph ase I drug-met abo lizing enz yme s. such as the cytochromes P450 , have been discovered and characterized for more than 25 yea rs (12.14,23). One of the most thoroughly studied of these is th e pol ymorphism for debrisoquine/ sparte ine oxidation (10,14). It was found that human populations could be divided into two distinct gro ups, described as extensive (EM ) and poor metabolizers (PM). In En gland and North Am erica, about 8 % and 92% exhibit the PM and EM phenotypes, respectively. These genetic differences, although not conferring disease, ma y be important det erminants of the effects of mor e than two dozen commonly prescribed and over-the-counter drugs (12,22), including MA and similar phenylethylamines (2.19-21,29). The human gene respon sible for the debrisoquine polymorphism is CYP2D6 . Interestingly, cocaine has been sho wn to inhibit human neuronal CY P2D6 in the br ain (25) . The rat gene responsible for the debrisoqui ne polymorphi sm is CY P2Dl (1,4). Although th ere is no direct metabolic or gen et ic evidence implicating diff er ential CYP2Dl metabolism o f MA as the reason for interindividual differences in MA-induced neurotoxicit y, the re is circumsta ntial evidence to sugges t thi s poss ibilit y. For e xample, MA is known to under go P450-mediat ed met abolism (2) . Al so , adult neurotoxicity of the rel ated amp he ta mine, 3.4-m ethenedioxymethamphetamine (MD MA ), ha s re cently been show n to be diminished in the Dark Agouti (DA) inbred stra in of rats (6) . Accordingly, it was th e purpose of thi s ex pe rime nt to det ermine the develop me nta l neurotoxicity of MA in two strains o f rats known to exhibit PM and EM phenot ypes for debrisoquine/spartein e ( 1,4,16). We had anticipated two possible outcomes. First, the par ent dru g, MA. might be th e toxic ag ent so that th e EM rat s, in which MA clearance would be more rapid, would show less neurotoxicity than PM rat s. Alt ernatively, an acti ve met abolite of MA might be the ultim at e toxicant so that EM rat s would exhibit greater neurot oxic ity than PM rats . We used the ACI Black Ag outi ba sed on the report by Mat sunaga et al. (16), which sho wed that this rat exhibits the C Y P2Dl PM phenotype (16). The Sprague-Dawley (SO) rat, amo ng others, displays the EM phenotype (1,4) . H ence , we co mpa re d the ACI and SD str ain s, using two behavioral tests pr eviously found to show developmental neurotoxicity following early postnatal MA expos ure (26). These tests included the acoustic startle refl ex and spatial learning and mem ory in the Morris water maze .

METHOD

Subjects Nulliparous Sprague -Dawley CD (C ha rles River, R aleigh . NC) and ACI (Harlan Spr agu e-Dawley, Indianapolis. IN ) rat s were obtained and bred in-h ou se . ACI rats were br ed repeat edl y and delivered seve ra l litt ers prior to the litt er enrolled in our study, because thi s str ain exhibits poor reprodutive and maternal performance in pr imiparous dams. On the day aft er birth. offspring were weigh ed and randoml y cull ed to eight with preferential ret ention of females, bec au se onl y the female exhibits the PM ph en ot ype ( 16). At weaning, o nly fema les were retained.

Exp erimental Methods Litters of each strain wer e assigned to one of two tre atment groups on a maternal weight-matched bas is. On postpar tum days 11-20, MA-treated groups were injected twice daily (8 h apart ) SC with MA (30 mg/kg) and controls were injected twice dail y with distilled wat er alone . d-Methamphetamine H CI was obtained from Sigm a (St. Louis . MO). and do ses were expresse d as the free base . Inj ecti on sites were rot at ed syste ma tically, and the dosin g volume was 3 ml/kg. Offsp ring were wean ed on day 28, hou sed in sam e-sex pairs through day 42 and ind ivid ually ther eafter. ( Ra t dams do not wean their progen y in cages at da y 21 and we ha ve o bserved th at SO and ACI strains appear to survive and grow better if not prem aturel y weaned on da y 2 1: by leaving litt er s together , all dam s had wea ne d their progen y by day 28.) R at s were housed in a vivarium in compliance with Fed eral animal care guid elin es and fully accre d ited by the Association for Asse ssment and Accr editation of Laboratory An imal Ca re . The research pr o tocol was approved by the Institutional Animal Care and Use Co mmi ttee. Dams and offsp ring were weigh ed weekl y. Follow ing dosing, an imal s wer e co de d and tested by per sonnel blind with respect to treatment group assignment.

Behavioral Methods On day 50, all females from ea ch litter were tested individually for acoustic startle . Startle was assessed in a San Diego Instruments SR apparatus. R at s were placed in an acrylic cylind er and administered 51 habituation and 36 prepulse trials following a 5-min acclimation period. The st artle stimulus was

T A BL E 1 NUMBER OF LITTERS. NUMBER OF OFFSPRING TREATED. AND MORTALITY RATES AMONG MA-TREATED AND CONTROL. ACI AN D SD RATS' Treatment Group

Strain

MA Control MA Control

ACI ACI SO SO

No. Litters Treated 19 9 13 10

Total No. of Offspring Treated ( % It 70/108

( 64 .S)§~

2/52 (3.R) 18/99 (18.2)§ 0/80 (0.0)

No. of Female Offspring Dying/Tot al ( %)

38/64 (59.4)§1

2/27 (7.4) IS/80 (18.8)§ 0/69 (0.0)

No. Litters/No. OffspringTestcdt 10/26 9/25 12/65 10/69

*Treatments were: MA = d-mcthamphetamine 30 mg/kg X 2/day: control = distilled water 3 ml/kg x 2/day, on postnatal days II through 20. t Following culling on postnatal days I, these values represent the number of offspring dying/treated with either MA or water. No offspring died followingthe last dose on postnatal day 20. t Represents the number of litters and the number of offspring that survived the treatment period, which were retained following culling on PNO 1 and removal of males at weaning and that received behavioral testing. §p < 0.01 compared to their respective controls by Fisher's test. ~p < 0.01 MA-ACI vs. MA-SD by Fisher's test.

GENETIC DIFFERENCES IN METHAMPHETAMINE TOXICITY a US-dB (A scale), mixed frequency signal presented for 20 ms. The response was detected using a piezoelectric force transducer mounted on a platform beneath a horizontally mounted cylinder. Responses were recorded as voltage changes in the transducer generated by platform deflection during the first 100 ms following signal onset. Trials were of two types: those with no prepulse and those in which the startle signal was preceded by a less intense but otherwise identical signal by 70 ms for 20-ms duration (measured from prepulse signal onset to startle signal onset). Five prepulse intensities were used (70, 75, 80, 85, and 90 dB, A scale). These five, plus no-prepulse trials, constituted six trial types. Each trial type was presented six times. Test sessions began with 51 no-prepulse trials administered every 8 s. Immediately thereafter, prepulse trials began. Trial order for prepulse trials was balanced using a Latin-square design with the provision that the first trial was a no-prepulse trial. Response measures were maximum response amplitude (V max), average response amplitude (V"J, and latency to Vmax (Tm ax ) ' At 54 days of age, each female was administered four timed trials in a 15 X lS0-cm straight water channel with a wire ladder at the goal end. On each trial, the rat was placed in the channel at the opposite end from the goal (facing away from it) and allowed to discover and escape on the ladder. These trials were used to determine swimming proficiency

100

A

and motivation to escape prior to maze trials. Water temperature was 21 ± 1°C. The Morris water maze was built, as described by Morris (17,18), with several minor modifications. Our tank diameter was 178 em, and camouflage of the platform was provided by a flat black finish on the interior surfaces of the tank and a transparent acrylic platform that cannot be seen from the water's surface. The platform was 10 ern- and was submerged 2 em beneath the surface. The platform was covered with nylon screening to enhance traction. The goal was positioned in the SE quadrant and start positions were altered on every trial in a semirandom sequence. Rats received eight trials per day on 3 successive days for acquisition (2-min limit per trial, 30-s intertrial interval spent on the platform; rats reaching the 2-min limit are placed on the platform) (26). Latency to find the platform was recorded. On the fourth day, the platform was removed and four probe trials administered (1 min each). For these trials, time in the target quadrant and target site crossings were recorded. The platform was then replaced and four reinstatement trials were administered, identical to those for acquisition. On the fifth day the platform was shifted to the NW quadrant and eight shift trials were administered for the new position. Data were collected manualIy on all subjects. Near the end of the study, a video tracking system became operational and was used in addition to manual recording on the last 10litters out of 51 litters in the exper-

Preweaning Weights

r-DoSing~

SD 60

B

280

/

80

267

Postweaning Weights

260 240 220

/

/: 200 180

40 160 140

20~ .

_120

..c:

... ..c:

S100

OJ

OJ

~

SD

OJ ; : 40 ' \

0

o •

80

~

Control MA 30 mg/kg

280 }

o

160

•

Control MA 30 mg/kg

ACI 60 120

40 80

20

ACI 0-'-.-------,-----.-----.-------.

o

7

14

Age (days)

21

28

40

-'-r-----.-----.----,-----,-------, 35

42

49

56

63

70

Age (days)

FIG. 1. (A) Preweaning bndy weights (g) of female SD and ACI offspring (mcans :; SEM) prior to group assignment, during dosing (postnatal days 11-20, only the afternoon body weights are plotted), and after dosing up to weaning. (B) Postweaning body weights (g) of female SD and ACI rats (rneans z SEM) from weaning to 70 days of age. Adult behavioral testing was begun at 50 days of age. *p < 0.05, **p < 0.01, compared to their respective controls, by a posteriori Duncan comparisons. SEMs were smaller than the symbols and therefore are not visible. The pattern of differences in males was identical (not shown).

VORHEES ET AL.

268 iment. These last 10 litters were: SO: three control and two MA-treated; ACI: two control and three MA-treated. Statistical Methods

Data were analyzed using analyses of variance (ANOVA) (general linear model). For data that had repeated measure components, split-plot ANOVAs were used with day and/or trial treated as within-subject factors in the analyses. Because only females were tested, gender was not a factor. However, because multiple females per litter were assessed, litter can be an important source of variance (13). Accordingly, data obtained for all females within a litter were averaged together, such that litter remained the subject variable (an exception to this occurred for the video tracking data, where data were analyzed by subject). For split-plot analyses, sphericity tests were performed on the variance-covariance marginals for variables involving a repeated measure factor. Where matrices were nonspherical, Greenhouse-Geisser adjusted F-ratios were used. Significant interactions were further analyzed using simple-effect analyses of variance or r-tests, depending on the number of variables involved. A posteriori group comparisons were performed by the method of Duncan. Group Sizes

Our initial design was to study 10 litters in each of the four groups; however, because of markedly increased MA-induced mortality (especially in the ACI strain), additional litters were added to the MA groups. Thus, the MA-treated and control ACI groups comprised 19 litters and 9 litters, respectively, whereas the MA-treated and control SO groups consisted of 13 and 10 litters, respectively. There were 51 potential litters in the experiment, but because of high mortality among offspring in the ACI MA-treated group, there were 41 litters that completed the experiment. However, computerized video-tracking was available for only the last 10 litters in the experiment. This was caused by an unscheduled move of the maze shortly before the experiment began due to construction. However, the video-tracking system would not track under the lighting conditions available in the replacement room. Once the experi-

Preweaning growth of the female progeny was examined (Fig. 1A). Males showed a comparable pattern. Two-group X two-strain X day analyses (in which day was a repeated measures factor) were performed for progeny body weight prior to, during, and after treatment. There were no treatment or treatment X strain effects prior to MA administration. During MA treatment, there were significant treatment, strain, treatment X strain, day, day X strain, day X treatment, and day X treatment X strain effects. The three-way interaction, F( II, 407) = 10.8, p < 0.00001, was analyzed further. Simple effect t-tests, performed on each day and strain, revealed that the treatment weight differences became significant on the second day of treatment for SD rats and on the third day of treatment for ACI rats. Treatment group differences increased with time and persisted at days 21 through 28. Postweaning weights for females were examined (Fig. IB). We found significant strain, treatment, age, age X strain, and age X treatment effects. Because the main effect of strain was significant, separate ANOVAs were performed on the body weights of each strain. For SD rats, there were significant age and age X treatment effects. The age X treatment interaction, F(5, 100) = 10.7, p < 0.00001, was further analyzed for treatment differences at each time point by r-test. The MA-treated SO rats weighed significantly less than SO controls through postnatal day 42, but were not different thereafter. For ACI rats, there were significant treatment, age, and age X treatment effects. The age X treatment interaction, F(5, 85) = 4.5, P < 0.001, was further analyzed for treatment group differences as above. The MA-treated rats weighed significantly less than ACI controls through postnatal day 49, but were not different thereafter. Thus, in both strains the MA-treatcd groups showed recovery of body weights by the time behavioral testing began (50 days of age and beyond). Behavioral Effects Acoustic startle. There were no treatment, treatment X strain, treatment X block, or treatment X strain X block efTABLE 2

ment was begun, no changes in the room environment could

be made in order not to disrupt the performance of the animals. However, a new tracker and software were acquired during the course of the experiment that could track the animals under the prevailing lighting conditions and this was used on the litters remaining at the time the system was operational.

SUMMARY OF ACOUSTIC STARTLE HABITUATION AND PREPULSE INHIBITION TEST RESULTS Startle Paradigm

Habituation

RESULTS

Mortality and Growth

We found that MA treatment on postpartum days 11 through 20 caused statistically significant increases in mortality rates compared with that of the control groups (Table 1). In addition, MA-treated ACI rats were more severely affected than MA-treated SO rats, either with or without males included (p < 0.01). The causes of death were not determined. A comparison of mean age of death for MA-treated rats, comparing the two strains, showed no significant difference. The mean age of death for the MA-treated rats of both strains was identical (15.2 days). These data would suggest that diminished metabolism of MA in the ACI rat is associated with increased MA-induced morbidity and mortality, compared with MA metabolism in the SD rat. Mortality occurred in males in addition to females, whereas the CYP2DI polymorphism has only been described in females (1,4,16).

Prepulse 0 dB

90 dB

Treatment Group

Strain

Vmilx

r.:

MA Control MA Control MA Controls MA Controls MA Control MA Controls

ACI ACI SO SO ACI ACI SO SO ACT ACI SO SO

535 (65) 612 (33) 641 (82) 573 (51) 385(42) 427 (35) 506 (87) 403 (57) 150 (28) 170 (23) 146 (37) 118 (19)

30 (0.4) 32 (I) 34 (I) 36 (I) 31 (I) 32 (I) 35 (I) 35 (I) 33 (I) 33 (I) 39 (I) 39 (I)

Values represent group mean z SEM. V max is the maximum startle amplitude (mV); Tmax is the latency to V11lax (ms). Habituation refers to a test session with 51 identical trials (trial I was excluded) analyzed in five lO-trial blocks. The table is the average of all five trial blocks. Prepulse refers to a test session with 36 trials with prepulse modification analyzed in six trial blocks by prepulse intensity. Prepulses werc randomly presented as 0, 70, 75, 80, 85. or 90 dB. Only the and 90 dB prepulse trial block averages are presented.

°

GENETIC DIFFERENCES IN METHAMPHETAMINE TOXICITY

269

Because the main effect of strain was significant, simple-effect ANOYAs were performed on each strain. For both strains, the main effect of treatment was significant. This can be seen more clearly when we averaged the latencies across all acquisition trials (Fig. 3). A similar omnibus ANOYA was performed on reinstatement trials; significant effects were obtained for treatment, F( 1,37) = 6.4, P < 0.02, trial, F(3, 111) = 19.7, P < 0.00001, and trial X strain X treatment, F(3, 111) = 3.5, P < 0.05. No other main effect or interactions were significant. A posteriori group comparisons for each strain and trial and for treatment group averaged across trials showed that the MAtreated groups-independent of strain-had longer reinstatement latencies to find the hidden platform on most trials (Fig. 2) and for the average across trials (Fig. 3). A similar analysis was performed on latencies for shift trials. Significant effects obtained on shift trials were treatment, F(I,37) = 10.7, P < 0.01, and trial, F(7, 259) = P < 0.00001. We performed follow-up t-tests separately for each strain.

fects found on either the habituation or prepulse-inhibition test sessions-for either dependent variable of startle amplitude (V lllax or VavJ-by analysis of covariance with body weight as the covariate. Similarly, no treatment specific effects were found on startle latencies. Startle data are summarized in Table 2. Straight channel swimming. A two-treatment X two-strain X four-trial (within) ANOYA on straight channel swimming trials revealed no significant treatment, strain, or any interaction effects. There was a main effect of trial, F(3, 111) = 77.9, P < 0.00001, which was attributable to the fact that all treatment groups had progressively shorter latencies to escape. Morris maze. Latency to find the hidden platform in the Morris maze was studied (Fig. 2). A two-treatment X two-strain X 24-trial (within) ANOY A revealed significant effects of treatment, F( 1,37) = 7.0,p < 0.02, strain, F( 1,37) = 12.I,p < OJ)I, trial, F(23, ~51) = ~6.0, P < 0.0000 I, and trial X strain, F(23, ~51) = 3.3. p < 0.001. No other interactions were significant.

Morris Maze 100

o •

Control MA30 mg/kg

80 60 40 20

c.i ~

SD

0

rIJ

'-'

.... <:..l

....= ~

I:':

100

...:l

80 60 40 20

ACI

0 Trial: Day:

3

5 1

7

9

11

13 15

2

Acquisition

17 19 21 23

~ 3

3

5

7

3

Reinstatement

Shift

FIG. 2. Morris hidden-platform mazc latencies in female SD and ACI rats (means:+: SEM). Reinstatement trials are identical to acquistion except that they occur after the probe trials (see Fig. 3), and 'Shift' represents reversal trials in which the platform was shifted to thc opposite quadrant. *p < 0.05, **p < 0.01 compared to their respective controls by a posteriori Duncan comparisons. Note that acquisition was conducted

over 3 days. such that trial 9 and 17 mark the beginning of ncw daily sessions. Probe and reinstatement trials were conducted on the day following the last day of acquisition.and shift trials were conducted thc day after the day that probe and reinstatement trials were conducted.

270

VORHEES ET AL.

Morris Maze

60

c:::::=::J ControI _ MA30mg/kg

SD

50

40

30

c; ---
20

til

'-'

>.

C)

C

60


..J

50

ACI

40

30

20

Acquisition

Reinstatement

Shift

FIG. 3. Morris hidden platform maze latencies in female SD and ACI rats (means:+: SEM), averaged across trials for each phase of testing. Reinstatement trials are identical to acquistion except that they are given after probe trials, and 'Shift' trials are with the platform in the opposite quadrant. *p < 0.05, "» < (UJI compared with their respective controls, by a posteriori Duncan comparisons. Group comparisons showed that the MA-treated groups of both strains had longer latencies on the shift trials, averaged across trials, than their respective controls (Fig. 3). Data for probe trials were analyzed similarly to the acquisition, reinstatement, and shift phases. The time spent in the target quadrant during the probe trials showed a significant effect of treatment, F(l, 37) = 6.0,p < 0'()2, and trial, F(3, III) = 80.1, p < 0.00001. Figure 4 shows the times in the target quadrant for all four groups. As can been seen, MA-treated offspring of both strains spent less time in the target quadrant than controls. No treatment or treatment-related interactions were found for the number of platform site crossings. For the 10 litters for which video tracking was available, the data were analyzed as above, except that the analyses were by-subject, because two groups contained only two litters each and the other two groups contained only three litters each (Table 3). The results of the by-subject analyses are summarized in Table 3. In these analyses, the treatment main effect was significant for several measures of acquisition, probe, and reversal performance. For acquisition, latency and cumulative distance from target (platform) were significant, whereas a trend was present for path length. There were no significant treatment-related interactions. Strain was significant on only some measures, but for consistency with the by-litter analyses.

separate ANOV As were performed for each strain. The SO MA group had significantly longer latency, path length, and cumulative distance from platform on acquisition than SO controls. The ACI MA group showed differences in the same direction and of similar magnitude compared to ACI controls, but because of the smaller group sizes, these differences were not significant. For probe trials, there was no significant effect on number of platform site crossings. However, the percentage of time spent in the target quadrant did show a significant treatment effect and there was a trend towards a treatment effect for average distance from platform. There were no significant treatment-related interactions. Simple-effect ANOV As by strain showed that both the SO and ACI MA groups spent a lower percentage of time in the target quadrant on probe trials than their respective controls. The simple-effect ANOV As for average distance from target showed that the SO MA group was farther from the platform on probe trials than SO controls. The difference for the ACI MA group on this measure was in the same direction. but smaller and not significant. Analyses of reversal trials showed that there were treatment main effect trends in the overall ANOV A for latency and cumulative distance from target, but not for path length. Simpleeffect ANOV As by strain for latency showed that the SD MA group had longer latencies to find the platform than SO con-

271

GENETIC DIFFERENCES IN METHAMPHETAMINE TOXI CITY

Morris Maze

= _

30

~

-

-s-e

Cont rol

I\1A30mIVk g

*

*

25

" ::l

0-

.:

.

E

i=

20

15 L-_

_

'----'-_

so

AC I

FIG. 4. Morris hidden platform maze, lime (in seconds) spent in the target quadrant in female SO and ACI rats (rnea ns z SEM) avera ged across four prob e trials. *p < 0.05. compa red to their respect ive contro ls. by a poste riori Duncan comparisons.

trols, whereas the trend in the A CI MA gro up was in the same direct ion compared to ACI contro ls but was not signific ant. A simple-ef fect ANOVA by stra in performed on cumulative distan ce fro m the targ et aga in sho wed that the SD MA group was farther from th e tar get th an SO controls, whereas the A CI MA gro up showed a sma ller differen ce compar ed to A CI controls that was not significa nt. DtSCUSSION

Th e curre nt study was designed to test the hypothesis th at MA-induced development al neurot oxicit y might be altered in

ra ts exhibiting th e PM phenotype for CY P2D 1, as this en zyme is responsible for MA met ab olism . The methods that we used to assess development includ ed acoustic startle and spatial learning in th e Morris hidden platform task. Th e acou stic star tle dat a showed no effects of MA treatment in eith er stra in and therefore provided no info rmation relevant to th e hypothesis. This was unexpected because in our previous study we had found that MA tr eatm ent, on either day s 1-10 or days 11- 20. augm ents the aco ustic start le equally in females. altho ugh males receiv ing MA on da ys 1-10 were mo re affected than with days 11-20 expos ure in a prepulse inhibition star tle paradigm (26) . We have since replicat ed the finding th at day 1-10 exposur e to MA at 20 mg/kg b.i.d. result s in sta rt le augmentation (28). We cannot currently reconcile th e lack of an MA-induced startle augmentation in the present experiment with the previous findings. For the test of spatial learning in the Morris hidden platform maze in the present study, the results showed a clear MA- induced deficit in maze performance; however, the dat a comparin g the two strains do not support the hypothesis that the poor metabolizer ACI rat is mor e sensitive than the extensive met ab olizer SO rat to the learning deficit assoc iated with de velopment al MA exposure. In fact, the results among th e surviving females sugges t that th e effects in the two stra ins we re nearl y identical. Thus, although the two stra ins wer e similarly affecte d, the dat a support the original ob se rvati on s (26,27) that MA exposure on days 11-20 induces re liable Morris maze deficit s. Moreover , not onl y were both strains of MA -tr eat ed rats affect ed in initial hidd en platform learning (acq uisition), the y also sho wed probe trial deficits in time spe nt in th e target quadrant , reinstatement deficits whe n returned to the ori ginal acqu isition conditions following the unrei nforced probe tr ials, and reversa l (shift) learning deficits when the platform was relo cat ed to the opposite qu adrant. It is not eworthy that no differ enc es were found between str ains or among the gro ups for perform ance in the 150-cm stra ightcha nnel swimming test pri or to Morris maze testing, th er eby

TABLE 3 MORRIS MAZE VIDEO-TRACKING RESULTS SD

ACl

Control No. of litters No. of subjects Acquisition Latency (s) Path length * Cum. dist. fro m target* Prob e Platform crossings % Time in target quad. Av. dist. from target * Reversal Latency (s) Path length ? Cum. dist. from target *

3 22 34 (2) (22) 3 1~0 (225) 3~O

1.4 (0.2) 41 ( I ) 24 (0.5) 20 (2) 273 (2 1) 2234 (177)

MA 2 10 45 (5)t (40) 42 2~ (428) t 4~ 1

1.0 (0.2) 34 (3) t 27 ( I) t 26 ( I)t 337 (2 1) 2864 ( 179)t

Control 2 5 46 (6) 394 (41) 3370 (37~) 1.4 (0.4) 47 (3) 24 (I) 20 (3) 246(4 1) 2073 (3 1~)

MA

p-valuc

3 7 5~ (7.0) 422 (39) 4032 (424)

1.4 (0.3) (2)t 24 ( I) 3~

22 (3) 266 (28) 2389 (2~2)

0.03

0.10 0.04

0.41 0.01 (J.l 0 0.09 0.19 0.08

Values repre sent subject gro up mean (:!:SEM). Video-t racking data were only available on the last 10 litters in the study out of a tot al of 4 1 litters that were tested beca use of track ing probl ems under the existing lighting conditi ons. Toward s the end of the expe riment a new track er and software were obtained that could track under the prevailing lighting. p -value is for the Treatment Group main effect from the Tr eatm ent Gro up X Strain X Day X Trial A NO VA performed on each depend ent measure. *On e unit = 3 em. t p < 0.05 compared to contro l.

272

VORHEES ET AL.

making it unlikely that performance factors could explain the differences seen in the MA-treated rats of both strains. Furthermore, the latency data differences were confirmed by computerized video-tracking analyses of path length and cumulative distance from the platform on acquisition and reversal trials and percentage of time spent in the target quadrant and average distance from the platform on probe trials that was available on last 10 litters in the experiment. Cumulative distance from the platform on learning trials and average distance from the platform on memory trials have been suggested as improved learning indices for analysis of Morris maze data (11). These measures provide information concerning an animal's proximity to the target and, therefore, provide some assurance that animals with different latencies and/or path lengths differ because of differences in spatial navigation rather than because of other influences. One can imagine animals who have longer latencies, not because of learning difficulties, but because they swim slower, or that have longer path lengths, not because they cannot locate the target, but because of near-misses that lengthen swim distance without reflecting spatial localization deficiences. The fact that these two proximity measures were also different in MA-treated animals, especially the SDs, suggest that in fact the MA animals are having difficulty locating and remembering the target location and that these group differences do not reflect performance differences unrelated to spatial navigation. There is one important caveat to the interpretation that the spatial learning deficits of both strains are comparable that must be added. This arises from the fact that the MAtreated ACI rats show a very high mortality rate during the treatment period. Thus, the Morris maze data are based on survivors, which are a select subset of all the ACI animals born in this group. It is possible that a "survivor effect" operated in this context, resulting in the least affected ACI rats in the MA-treated group reaching adulthood for Morris maze or acoustic startle testing. A survivor effect, if present, would be likely to attenuate the differential response in the ACI strain. Evidence in support of this possibility is the observation that

the lethality of MA in the ACI strain was strikingly higher than that in the SD rats (Table 1). Furthermore, the effect of MA on ACI body weights in the preweaning period is less than that on SD body weights (Fig. lA), a finding consistent with the idea that excess mortality in the MA-treated ACI rats might lead to a decreased effect among the ACI survivors. Evidence against our hypothesis that MA-induced developmental neurotoxicity might be greater in ACI rats than in SD rats is the observation that the high mortality rate was seen in both male and female MA-treated ACI rats. The possible problem is that only ACI females are shown to exhibit the PM trait (1,4,16). If the CYP2Dl polymorphism is the critical determinant of MA-induced deaths, we would have expected that ACI males might be less severely affected than ACI females. Upon completion of this study, the Dark Agouti (DA) inbred strain of rats-developed in England as the original murine model of the CYP2D 1 polymorphism-became commercially available in the United States (Harlan Sprague-Dawley, Indianapolis, IN). Preliminary data from our laboratories suggest that MA-treated DA females exhibit Morris maze acquisition deficits, at an MA dose at which SO females are unaffected. These findings lend support to the hypothesis that the CYP2Dl polymorphism may contribute to differences in MAinduced developmental neurotoxicity, and that MA-rather than a metabolite-may be the agent primarily responsible for initiating the cascade of events that lead ultimately to neurotoxicity. These preliminary data also support our abovementioned caveat of the survivor effect. In other words, due to excessive mortality of ACI rats during the treatment period with MA, the Morris maze results performed after 50 days of age were based on a select subset of ACI survivors. ACKNOWLEDGEMENTS

This research was primarily supported as a pilot project from center grant ES06096 and partially supported by training grant ES07051 (T. M. R. M. A. S.. and 1. E. F.) and research grant DA06733 (C. V. V.).

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