The effects of methamphetamine on core body temperature in the rat Part 2: an escalating regimen

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1 Psychophrmcology (2008) 198: DOI /s ORIGINAL INVESTIGATION The effects of methmphetmine on core ody temperture in the rt Prt 2: n esclting regimen Benit J. Myles & Kren E. Sol Received: 31 Mrch 2007 / Accepted: 20 Decemer 2007 / Pulished online: 26 April 2008 # Springer-Verlg 2008 Astrct Rtionle Methmphetmine (METH) induces hyperthermi, which is diminished with chronic tretment in dosedependent mnner. Our ojective ws to determine whether the temperture responses produced y chronic, esclting-dose METH regimen nd chronic, 5.0 mg/kg dose regimen. Methods Rts received pretretment injections of sline, 5.0 mg/kg METH, 10.0 mg/kg METH (second comprison group), or n esclting-meth regimen (2 9 mg/kg) for 12 dys. On dy 13, ll four groups were chllenged with 10.0 mg/kg METH. Temperture mesurements were mde telemetriclly t 24 C mient temperture. Results Esclting pretretment produced hyperthermi; with successive eposures, the hyperthermic pek shifted to the right. The 5.0-mg/kg-pretretment group initilly showed no chnge in temperture t 60 min post-tretment ut developed hypothermi t 60 min with chronic tretment; t 3 h post-tretment, significnt hyperthermi ws present nd did not diminish with chronic tretment. After the 10.0-mg/kg-METH chllenge, the sline-pretretment group ws hyperthermic, nd the 10.0-mg/kg-pretretment group ws hypothermic; the 5.0 mg/kg nd esclting pretretment groups were intermedite nd were not different from ech other. At 3 h post-chllenge, no group differences were pprent. Dopmine (DA) nd serotonin (5-HT) were not depleted when mesured 2 weeks fter tretment ended. K. E. Sol (*) Deprtment of Psychology, University of Mississippi, Oford, MS 38677, USA e-mil: ksol@olemiss.edu B. J. Myles : K. E. Sol Deprtment of Phrmcology, University of Mississippi, Oford, MS 38677, USA Conclusions (1) Esclting nd 5.0-mg/kg regimens produced different temperture profiles during the 12-dy pretretment period ut similr diminished response to the 10.0-mg/kg-METH chllenge on dy 13. (2) The diminished temperture responses with chronic tretment occurred in the sence of long-term DA nd 5-HT depletions. Keywords Methmphetmine. Drug use. Rt. Temperture. Tolernce Humns who use the mphetmines often egin y tking smll doses; however, with repeted use, tolernce occurs, cusing users to consume lrger mounts. This develops into secondry high-dose pttern of multiple dosing throughout the dy. Users typiclly self-dminister the drug chroniclly throughout the dy (consuming up to 1 g until lter in the dy, then sleeping t night) or in pttern of multiple dosing over severl dys (consuming up to 2 4 g over n pproimte 3-dy intervl of periodic dministrtion) (Cho nd Meleg 2002). Studies with lortory nimls indicte tht tolernce develops to methmphetmine (METH)-induced hyperthermi. Becuse humn drug users esclte their doses, we were interested in evluting tolernce to hyperthermi in reltion to n esclting-dose METH regimen in the lortory rt. In niml studies of temperture nd neurotoicity, mny reserchers choose to mimic humn-dosing ptterns of estlished users. Thus, reserchers hve used multiple high-dose ptterns dministering totl of four doses t 2-hour intervls to study drug effects (Alers nd Sonsll 1995; Bowyer et l. 1992; Gygi et l. 1996; Johnson-Dvis et l. 2002, 2003; Riddle et l. 2002; Sol et l. 2000, 2001; Schmidt et l. 1985). In ddition to mimicking the ptterns nd dosing intervls of the estlished user, reserch hs lso focused

2 314 Psychophrmcology (2008) 198: on simulting the initil esclting ptterns of use, which reflect the erly phse of drug use. Accordingly, ptterns of escltion hve een used, which rnge from once-dily tretments (Roinson et l. 1988) to multiple-dosing regimens (Gygi et l. 1996; Johnson-Dvis et l. 2003; Segl nd Kuczenski 1997). Regimens of once-dily tretments my yield greter clrity in dt (Cho et l. 2001; Miller nd O'Cllghn 2003). Becuse temperture chnges occur up to 6 h post-injection, chnges occurring during rpid multiple-injection prdigm would e difficult to interpret. We emined the core temperture effects of n esclting-meth pretretment regimen, which incremented once per dy cross 12 dys. Previously, we showed tht chronic tretment with 10 mg/kg/dy METH ut not 1.0 mg/kg/dy resulted in diminished hyperthermi over 12-dy period t 24 C mient temperture (Myles et l. 2008). The effect with 5.0 mg/kg ws intermedite. Furthermore, when using n intermittent, esclting pttern of METH pretretment, the development of tolernce to hyperthermi ws evident (Johnson-Dvis et l. 2003). The purpose of this study ws to determine whether (1) n esclting-dose regimen (incresing from 2.0 to 9.0 mg/kg METH) nd (2) constnt dily dose regimen of 5.0 mg/kg would result in similr chnges in core temperture fter 12-dy period of chronic eposure. Both regimens hd equivlent totl doses. We lso wnted to determine if the ltered temperture response with chronic tretment ws independent of the neurotoic effects of METH. Mterils nd methods Sujects Twenty-four mle Sprgue Dwley rts, g upon rrivl (Hrln, Indinpolis, IN, USA), were used. The rts were individully housed in hnging stinless steel wire cges, mintined on 12-h light/drk cycle (lights on 7:00 AM). They hd free ccess to food nd wter ecept during the 7-h test session. All procedures were crried out ccording to the Guide for the Animl Cre nd Use of Lortory Animls (NRC 1996) nd were pproved y the Institutionl Animl Cre nd Use Committee t the University of Mississippi. Surgery For the mesurement of core temperture, rdio trnsmitters (Mini-mitter, model #VM-FH disc) were implnted into the domen of ll rts. Animls were nesthetized with ketmine (80 mg/kg), ylzine (10 mg/kg), nd tropine (5 mg/kg). Supplementl nesthesi ws with ketmine s needed. Ketoprofen (5 mg/kg) ws given s post-surgicl nlgesi. Drugs (+)-Methmphetmine hydrochloride (SIGMA, St. Louis, MO, USA) ws dissolved in sline vehicle (0.09% NCl solution). A constnt injection volume of 1 ml/kg ws used. METH doses were clculted s the METH HCl slt. Ech rt (n=6/tretment) received intrperitonel (i.p.) injections of vehicle, 5.0 mg/kg, 10.0 mg/kg, or n esclting regimen of METH (2.0, 2.5, 3.0, nd 3.5 mg/kg on dys 1, 2, 3, nd 4, respectively; 4.0, 4.5, 5.0, nd 5.5 mg/kg on dys 5, 6, 7, nd 8, respectively; 6.0, 7.0, 8.0, nd 9.0 mg/kg on dys 9, 10, 11, nd 12, respectively) for 12 consecutive dys, once/ dy, followed y chllenge dose of 10 mg/kg METH on dy 13. Procedure After 1-week qurntine, surgery ws performed. Two weeks fter surgery, the rts egn tretments. Ech rt received once-dily sline injections for 4 dys efore ech regimen (HAB dy-lock 1 4). This ws followed y one of the following four regimens: dily injections of sline, 5.0 mg/kg, 10.0 mg/kg, or n esclting regimen of METH for 12 pretretment dys. On dy 13, rts from ll pretretment groups were chllenged with 10.0 mg/kg METH. This resulted in totl of 17 test-dys/rt. Note tht the sline-pretretment group received 16 dily injections of sline, followed y 10.0 mg/kg METH on the chllenge dy. Body weight ws mesured t the strt of ech test dy. The rts were housed in computer-controlled environmentl chmers mintined t 24 C for 7 h/dy during the 17-dy test period. Tretment occurred 2 h fter the rts were plced into the chmers. Telemetric temperture signls were utomticlly recorded once per minute. Two procedures descried ove were different from those used in our compnion pper (Myles et l. 2008): the ddition of the HAB1 4 period llowed us to investigte whether repeted sline or METH resulted in crry-over effects t the eginning of successive sessions; recording seline temperture for 2 h insted of 1 h ech dy, llowed core temperture to level off nd stilize t the time of dily METH or sline tretment. In prior reserch (Myles et l. 2008), we noticed the presence of slivtion fter tretment with METH. Becuse slivtion is mechnism for het loss, we were interested in systemticlly studying its role in METH s effects on core temperture in this report. Animls were given score rnging from 0 to 3 depending on the degree of slivtion oserved t 15-min intervls during the temperture

3 Psychophrmcology (2008) 198: mesurement periods: 0 = no slivtion; 1 = sliv noted on floor of test chmer, with smll mount on the ody; 2 = wet forelims nd hind lims; 3 = wet ventrl ody surfce. The rter ws not lind to tretment. The highest vlue recorded over ech 4-dy lock ws used for dt nlysis. Two weeks fter the completion of testing, the nimls were euthnized y decpittion, hving their rins rpidly dissected on ice for high-performnce liquid chromtogrphy (HPLC) neurochemicl nlysis. Neurochemicl nlysis Dopmine (DA) nd 5-HT concentrtions in the stritum, hippocmpus, nd hypothlmus were nlyzed postmortem using HPLC. Internl stndrd (DHBA) nd 0.01 N HClO 4 were dded to individul rin regions. Smples were homogenized with n ultrsonic tissue disrupter nd centrifuged twice t 14,000 rpm for 20 min. The superntnt ws drwn off nd stored on ice for HPLC nlysis. Instrumenttion included ESA HPLC pump (model #580); Rheodyne injection vlve (model #7010); Adsorosphere Ctecholmine mm column (C 18, 3-μm prticle size); ESA Coulochem II coulometric detector (model #5200), with pplied potentils of 100 (E1) nd +330 mv (E2); nd EZ-Chrome, Chromtogrphy Dt System (version 6.7). Moile phse consisted of 20.7 g/2 L N 2 H 2 PO 4, g/2 L OSA, nd 500 μl of 100 mm EDTA/2 L. The voltile component of the moile phse ws 7.5% cetonitrile. The solution ws titrted to ph of 3.1 using phosphoric cid. Dt nlysis Temperture Dt for 4 dys were comined for nlysis; this resulted in four, 4-dy locks, nmely one hitution sline dy-lock (HAB dy-lock 1 4), followed y three METH (M1 4, M5 8, M9 12), or sline (S1 4, S5 8, S9 12) dy-locks. For ech pretretment regimen, two-wy within-sujects nlysis of vrince (ANOVA) ws used to nlyze the effect of dy-lock nd session time ( 105 nd 60 nd 0 min pre-injection, 30, 60, 120, 180, 240, nd 300 min post-injection). To evlute chnges in seline performnce for the sline-pretretment group, we followed up significnt min effects of session time (collpsed cross dy-locks) with the modified Bonferroni post hoc test (p=(df)(.05)/numer of comprisons (Keppel 1991); p<.05) compring time 0 (immeditely efore injection) with 105, 60, +30, nd +60 min. To evlute crry-over effects in the METH pretretment groups, we followed up significnt min effects of dylock or significnt interctions with one-wy ANOVAs t specific time points efore injection (times 105, 60, nd 0 min). Significnt one-wy ANOVAs t ech time point were followed y three post hoc pir-wise comprisons: HAB dy-lock 1 4 ws compred to METH dy-locks 1 4, 5 8, nd 9 12 (modified Bonferroni correction, p< 0.05). By compring ech dy-lock to HAB1 4, we demonstrted whether previous drug eposure influenced core temperture t the strt of ech session. To determine the effects of initil METH eposure (dylock 1 4) nd chronic METH eposure (dy-locks 1 4, 5 8, nd 9 12), we followed up significnt min effects of dy-lock or interctions with one-wy ANOVAs t specific time points post-injection. Significnt one-wy ANOVAs were followed y four post hoc tests. HAB dy-lock 1 4 ws compred to METH dy-lock 1 4 to determine initil drug-induced chnges in temperture; METH dy-lock 1 4, METH dy-lock 5 8, nd METH dy-lock 9 12 were compred to ech other to determine the effect of chronic eposure (modified Bonferroni correction, p<0.0375). These tests were crried out t different session times post-tretment depending on the dose of METH. To ssess the effect of the chllenge injection on the 13th METH tretment dy, two-wy ANOVAs were conducted. One of the two-wy ANOVAs nlyzed the sline group: dy-lock (sline dy-lock 9 12 vs. dy 13) session time ( 105, 60, 0, 30, 60, 120, 180, 240, nd 300 min postinjection). This ws followed y one-wy within-sujects ANOVA compring sline dy-lock 9 12 to METH chllenge dy 13 t ech time point (modified Bonferroni correction of p<0.044). This nlysis llowed us to demonstrte the hyperthermic chllenge effect in nimls without history of METH eposure. The other two-wy ANOVA nlyzed the METH chllenge dy (pretretment session time) to determine the effects of different pretretment regimens upon chllenge. This ws followed y one-wy etween-sujects ANOVA t ech time point. Before injection, sline ws compred to ech METH pretretment group (modified Bonferroni correction of 0.05); fter injection, ll four groups were compred to ech other (modified Bonferroni correction of 0.025). Slivtion Slivtion dt were nlyzed using the non-prmetric test of Kruskl Wllis for ech dy-lock, followed y the Mnn Whitney U (modified Bonferroni, p<.05). Body weight A two-wy ANOVA (pretretment dy-lock) ws used to nlyze the ody weights of treted nimls. Follow-up

4 316 Psychophrmcology (2008) 198: one-wy ANOVAs were conducted, nd pir-wise comprisons were mde etween the different pretretment groups t ech of the three pretretment dy-locks (modified Bonferroni correction, p<0.05). Neurochemistry Using one-wy ANOVAs for ech rin region (stritum, hippocmpus, nd hypothlmus), we nlyzed the postmortem tissue concentrtions of DA nd 5-HT for the following four groups: sline-pretretment/meth chllenge, esclting-meth-pretretment/meth chllenge, 5.0-mg/kg-METH pretretment/meth chllenge, nd 10.0-mg/kg-METH pretretment/meth chllenge. Results Temperture METH pretretment Rts received chronic tretment with one of the following four regimens: sline, 5.0 mg/kg METH, esclting-meth doses, or 10.0 mg/kg METH. For ech pretretment regimen, we nlyzed the effects of chronic eposure on core temperture using one-wy ANOVAs (dy-lock session time). For sline pretretment, the min effect of session time ws significnt [F(8,32) =87.8, p=0.0000]. The min effect of dy-lock nd the session time dy-lock interction were not significnt. For 5.0-mg/kg-METH pretretment, the min effect of session time [F(8,32) =19.3, p=0.000] nd the interction etween session time nd dy-lock [F (24,96) =11.8, p=0.0000] were significnt, ut the min effect of dy-lock ws orderline [F(3,12) =3.2, p=0.061]. For esclting pretretment, there were significnt min effects of dy-lock [F(3,15) =19.8, p=0.0000] nd session time [F(8,40) =9.0, p=0.000]; the interction ws lso significnt [F(24,120) =19.2, p=0.0000]. For 10.0-mg/kg- METH pretretment, there were significnt min effects of dy-lock [F(3,15) =6.8, p=0.004] nd session time [F (8,40) =17.9, p=0.0000]; the interction ws lso significnt [F(24,120) =10.2, p=0.0000]. Anlysis of temperture efore nd fter injection for sline pretretment We eplored the significnt min effect of session time in the sline-pretretment group y compring seline time 0 to 105, 60 (pre-injection), +30, nd +60 min (postinjection), collpsed cross dy-locks. Only the 0 vs. +60 min post-injection comprison filed to rech significnce. At 105 nd 60 min (pre-injection), core temperture decresed s the session progressed. Temperture incresed t 30 min post-tretment, reflecting the effects of stress (hndling) or rousl from sleep on core temperture. Note tht this effect reversed itself t 60 min post-injection, t which point there ws no difference from seline time 0 (dt not shown). Tests for crry-over effects from preceding tretment dys As stted ove, for the sline-pretretment group, the min effect of dy-lock nd the dy-lock session time interction were not significnt. For 5.0-mg/kg-METH pretretment, dy-locks were significntly different t 105 [F(3,12) =26.2, p=0.0000] nd 60 min [F(3,12) = 8.3, p=0.003]. At 105 min, HAB dy-lock 1 4 ws greter thn M1 4, M5 8, nd M9 12. At 60 min, HAB dy-lock 1 4 ws greter thn M5 8 nd M9 12 (Fig. 1). For esclting pretretment, dy-locks were significntly different t 105 [F(3,15) =33.3, p=0.0000] nd 60 min [F(3,15) =3.5, p=0.041]. At 105 min, HAB dy-lock 1 4 ws greter thn M1 4, M5 8, nd M9 12. At 60 min, HAB dy-lock 1 4 ws greter thn M1 4 nd M9 12 (Fig. 2). For 10.0-mg/kg-METH pretretment, dy-locks were significntly different t 105 [F(3,15) =30.0, p= ] nd 60 min [F(3,15)=9.7, p=0.0008]. At 105 min, HAB dy-lock 1 4 ws greter thn M1 4, M5 8, nd M9 12. At 60 min, HAB dy-lock 1 4 ws greter thn M1 4, M5 8, nd M9 12 (Fig. 3). These dt demonstrte tht for ll pretretments ecept sline, crryover effects were seen t ll 3 dy-locks: M1 4, M5 8, Degrees C y z y z Core Temperture: 5.0 mg/kg c N=5 HAB 1-4 N=5 METH 1-4 N=5 METH 5-8 N=5 METH Minutes Fig. 1 Core temperture (men±sem). Rts received 4 dys of sline injection (HAB 1 4) followed y 12 dys of 5.0 mg/kg METH (METH 1 4, 5 8, nd 9 12). HAB 1 4 ws significntly different from METH 1 4, y HAB 1 4 ws significntly different from METH 5 8, z HAB1 4 ws significntly different from METH 9 12, METH 1 4 ws significntly different from METH 5 8, METH 1 4 ws significntly different from METH 9 12, c METH 5 8 ws significntly different from METH HAB 1 4 Hitution dylock 1 4, METH 1 4 METH dy-lock 1 4, METH 5 8 METH dylock 5 8, METH 9 12 METH dy-lock 9 12

5 Psychophrmcology (2008) 198: Degrees C y z Core Temperture: Esclting Dose z N=6 HAB 1-4 N=6 METH 1-4 N=6 METH 5-8 N=6 METH Minutes Fig. 2 Core temperture (men±sem). Rts received 4 dys of sline injection followed y 12 dys of n esclting-meth regimen. HAB 1 4 ws significntly different from METH 1 4, y HAB 1 4 ws significntly different from METH 5 8, z HAB1 4 ws significntly different from METH 9 12, METH 1 4 ws significntly different from METH 5 8, METH 1 4 ws significntly different from METH 9 12, c METH 5 8 ws significntly different from METH HAB 1 4 Hitution dy-lock 1 4, METH 1 4 METH dy-lock 1 4, METH 5 8 METH dy-lock 5 8, METH 9 12 METH dy-lock 9 12 nd M9 12. The significnce of this finding is tht some spect of chronic drug eposure resulted in diminished core temperture t the strt of the net test session, efore new drug eposure. c c c the initil dy-lock t 60, 120, nd 180 min post-tretment (M1 4 ws greter thn HAB dy-lock 1 4). There were lso significnt effects of chronic drug eposure t severl time points. At 60 min, M5 8 ndm9 12 were less thn M1 4. At 120 min, M5 8 ndm9 12 were less thn M1 4; M9 12wslessthnM5 8. At 180 min, M1 4 ws less thn M5 8. At 240 min, M1 4 ws less thn M5 8 ndm9 12; M5 8 ws less thn M9 12. At 300 min, M1 4 ndm5 8 were less thn M9 12. As cn e seen in Fig. 2, the esclting regimen resulted in right-shift of the pek temperture response over dy-locks. For the 10.0-mg/kg-pretretment regimen, follow-up one-wy ANOVAs showed significnt effects of dy-lock t 30 min [F(3,15) =4.3, p=0.0215], 60 min [F(3,15) =5.2, p=0.0115], 90 min [F(3,15) =7.5, p=0.0027], 120 min [F(3,15)=4.6, p=0.0173], 180 min [F(3,15)=6.8, p= ], 240 min [F(3,15) =44.0, p=0.0000], nd 300 min [F(3,15) =49.2, p=0.0000]. Pirwise comprisons indicted tht 10.0-mg/kg-METH pretretment resulted in hyperthermi during the initil dy-lock t 120, 180, 240, nd 300 min (M1 4 ws greter thn HAB dy-lock 1 4). There were lso significnt effects of chronic drug eposure t the following time points: 30 min, M9 12 ws less thn M1 4; 60 min, M5 8 nd M9 12 were less thn M1 4; 90 min, M5 8 nd M9 12 were less thn M1 4; 120 min, M5 8 nd M9 12 were less thn M1 4; 180 min, M9 12 ws less thn M1 4 (Fig. 3). Tests for initil hyperthermi (dy-lock 1 4) nd effects due to chronic tretment As stted ove, for the sline-pretretment group, the min effect of dy-lock nd the dy-lock session time interction were not significnt. For the 5.0-mg/kg-pretretment regimen, follow-up onewy ANOVAs showed significnt effects of dy-lock t 60 min [F(3,12) =5.6, p=0.0125], 120 min [F(3,12) =4.3, p=.0276], 180 min [F(3,12) =45.0, p=0.0000], 240 min [F(3,12)=11.5, p=0.0008], nd 300 [F(3,12)=6.8, p= ]. Pirwise comprisons showed tht 5.0-mg/kg- METH pretretment resulted in hyperthermi during the initil dy-lock t 120, 180, nd 240 min post-tretment (M1 4 ws greter thn HAB dy-lock 1 4). Chronic effects were seen t 60 min where M9 12 ws lower thn M1 4 nd M5 8 nd t 300 min where M9 12 ws greter thn M1 4 (Fig. 1). For the esclting-dose-pretretment regimen, follow-up one-wy ANOVAs showed significnt effects of dy-lock t 60 min [F(3,15)=8.5, p=0.0016], 120 min [F(3,15)= 25.8, p=0.0000], 180 min [F(3,15)=19.0, p=0.0000], 240 min [F(3,15)=58.3, p=0.0000], nd 300 min [F(3,15)= 32.7, p=0.0000]. Pirwise comprisons indicted tht esclting-meth pretretment resulted in hyperthermi during Degrees C Core Temperture: 10.0 mg/kg 37.0 y 36.5 z y z 36.0 N=6 HAB 1-4 N=6 METH N=6 METH 5-8 N=6 METH Minutes Fig. 3 Core temperture (men±sem). Rts received 4 dys of sline injection followed y 12 dys of 10.0 mg/kg METH. HAB 1 4 ws significntly different from METH 1 4, y HAB 1 4 ws significntly different from METH 5 8, z HAB1 4 ws significntly different from METH 9 12, METH 1 4 ws significntly different from METH 5 8, METH 1 4 ws significntly different from METH 9 12, c METH 5 8 ws significntly different from METH HAB 1 4 Hitution dy-lock 1 4, METH 1 4 METH dy-lock 1 4, METH 5 8 METH dy-lock 5 8, METH 9 12 METH dy-lock 9 12

6 318 Psychophrmcology (2008) 198: The 10.0-mg/kg chllenge on dy 13 To determine the effects of 10.0 mg/kg METH in rts without prior history of METH, two-wy ANOVA ws performed on the dt from the sline-pretretment group: dy-lock (dy-lock 9 12 vs. dy 13) nd session time. There were significnt effects of dy-lock [F(1,4) =103, p=0.0005] nd session time [F(8,32) =9.0, p=0.0000], nd there ws significnt interction [F(8,32)=11.7, p= ]. Post hoc nlysis showed no significnt effects of dy-lock t 105 min, 60 min, nd injection time 0 min. However, t 30, 60, 120, 180, 240, nd 300 min, significnt drug-induced hyperthermi ws evident fter the 10.0-mg/kg chllenge (Fig. 4; note tht sline dy-lock 9 12 is not included in this figure). A second two-wy ANOVA ws conducted to compre the effects of the four different pretretment conditions (sline, 5.0 mg/kg, esclting, 10.0 mg/kg METH) on the temperture response fter 10.0-mg/kg-METH-chllenge injection. There were significnt effects of pretretment [F(3,18) =7.9, p=0.0014] nd session time [F(8,144) =29.5, p=0.0000], nd there ws significnt interction [F(24,144) =4.6, p=0.0000]. Follow-up one-wy ANOVAs indicted significnt crry-over effects t 105 min [F(3,18) =6.0, p=0.0049]; significnt post-injection effects were found t 30 min [F(3,18)=4.5, p=0.0159], 60 min [F (3,18) =19.2, p=0.0000], nd 120 min [F(3,18) =4.5, p= ]. Before injection, t 105 min (crry-over effects), the sline-pretretment group ws greter thn ll METH pretretment groups. At 30 min post-injection (chllenge effects), the 10.0-mg/kg-METH-pretretment group ws less thn the sline-pretretment group, with the sline Degrees C Core Temperture: 10.0 mg/kg METH Chllenge Dy * 36.5 * * * N=5 Sline 36.0 * N=6 Esclte * N=5 5mg/kg 35.5 N=6 10mg/kg * Minutes Fig. 4 Core temperture (men±sem). All rts received 10.0-mg/ kg-meth chllenge on dy 13 fter 12 dys of pretretment with sline, 5.0 mg/kg METH, n esclting-dose-meth regimen, or 10.0 mg/kg METH. * Significntly different from the chronic slinepretretment significntly different from the chronic mg/kg-meth-pretretment group Tle 1 Oserved slivtion during drug dministrtion Pretretment Degree of slivtion group ehiiting hyperthermi nd the 10.0 mg/kg group ehiiting hypothermi. At 60 min, the sline group ws significntly greter thn the 5.0 mg/kg, the esclting regimen, nd the 10.0-mg/kg-METH-pretretment groups; the 5.0 mg/kg nd the esclting-meth groups were greter thn the 10.0-mg/kg-METH group; however, there ws no difference etween the 5.0 mg/kg nd esclting- METH groups. At 120 min, the sline group ws greter thn the esclting regimen nd 10.0 mg/kg groups (Fig. 4). Oservtions: slivtion Five of si rts in ech group were oserved for slivtion. There were significnt min effects of tretment t dy-lock 1 4 (p=.0018), dy-lock 5 8 (p=.0095), dy-lock 9 12 (p=.0149), ut not on chllenge dy 13. Post hoc nlysis showed tht slivtion ws incresed in the 10.0-mg/kgpretretment group compred to the sline-pretretment group t ll 3 dy-locks (Tle 1). Weight mesurements Dy of erliest ppernce Numer of rts Sline 2 Dy 13 chllenge 1 of mg/kg 1 3 8th METH dy 1 of 5 Esclting 1 3 9th nd 11th METH dy 2 of mg/kg 1 3 1st, 2nd, 3rd, nd 8th METH dy 5of5 0 No slivtion, niml does not pper wet, 1 slivtion onto the floor, smll mount on the ody, 2 wet forelims nd hind lims, 3 ventrl ody surfce ppers wet A two-wy ANOVA (pretretment dy-lock) ws performed. There ws significnt effect of pretretment [F(3,18)=3.4, p= ], nd the interction ws significnt [F(6,36)=13.2, p=0.0000]. Post hoc nlysis indicted tht for dy-lock 5 8, sline ws only different from mg/kg-meth pretretment. At dy-lock 9 12, sline ws different from the three METH pretretment groups (Tle 2). Tle 2 Averge dily weight (grms, men±sem) during the 12- dy pretretment period Pretretment Dy-lock 1 4 Dy-lock 5 8 Dy-lock 9 12 Sline 377± ± ± mg/kg 370± ± ±1.8 Esclting 375± ± ± mg/kg 355± ± ±9.7 Different from sline, sme dy-lock

7 Psychophrmcology (2008) 198: Tle 3 Post-mortem DA nd 5-HT tissue concentrtions (nnogrm per milligrm tissue, men±sem) in the hypothlmus, hippocmpus, nd stritum Sline pretretment 5.0-mg/kg pretretment Esclting pretretment 10.0-mg/kg pretretment Hypothlmus DA 0.311± ± ± ± HT 0.523± ± ± ±0.01 Hippocmpus DA 5-HT 0.311± ± ± ±0.01 Stritum DA ± ± ± ± HT 0.302± ± ± ±0.01 There were no significnt differences etween pretretment groups. Neurochemistry There were no significnt depletions of DA or 5-HT in stritum, hippocmpus, or hypothlmus for ny of the METH pretretment groups (Tle 3). Discussion The effect of chronic, esclting-meth regimen on core temperture ws investigted in rts. METH tretment egn fter ll groups received 4 dys of sline eposure (HAB dy-lock 1 4) to hitute the nimls to the testing/injection procedures. Both initil (METH or sline dy-locks 1 4) nd chronic (METH or sline dy-locks 5 8 nd 9 12) pretretment regimens were then evluted. A finl chllenge dose of 10.0 mg/kg METH ws dministered to ll pretretment groups on the 13th tretment dy, for totl of 17 dys of temperture testing. Consistent with prior reserch, initil dministrtion resulted in hyperthermi (Lewnder 1971; Myles et l. 2008; Thornhill et l. 1977), chronic dministrtion resulted in susequent diminished core temperture response (Lewnder 1971; Myles et l. 2008), nd the mimum temperture increse fter the 5.0 mg/kg dose (24 C) occurred t 3 h post-tretment (Myles et l. 2008). Esclting-dose regimen The primry focus of this report ws to determine () whether n esclting-meth regimen would result in diminishing temperture response over chronic tretment period nd () how the degree of temperture chnge would relte to tht found with chronic, non-esclting regimen with totl dose tht ws equivlent to the esclting regimen. The esclting regimen involved one injection per dy, incresing the dose ech dy; the totl-dose comprison group involved the sme dose (5.0 mg/kg) ech dy. The individul ptterns demonstrted y these two groups differed cross the 12-dy pretretment period. With successive dys, the 5.0-mg/kg-pretretment group demonstrted diminishing core temperture t 1 h post-injection nd consistent hyperthermi t 3 h (Fig. 1). The esclting regimen resulted in right-shift in the core temperture profile over the 12-dy pretretment period (Fig. 2). We recently showed tht single injections (0.5 to 5.0 mg/kg METH) resulted in longer pek delys with incresing dose (Phelps et l. 2005). This finding suggested tht, in ddition to chronic tretment, the shift seen here with the esclting regimen ws ecuse of the incresing dose. We lso demonstrted tht even though the 5.0 mg/kg nd the esclting regimen groups (equivlent totl-dose groups) showed different temperture profiles during the 12-dy regimen, they demonstrted similr ptterns when chllenged with 10.0 mg/kg METH (tretment dy 13, Fig. 4). This is prticulrly evident t the 60 min posttretment time point, when the chronic 5.0 mg/kg nd esclting pretretment groups were different from the sline nd 10.0-mg/kg-pretretment groups, ut not from ech other. These findings suggest tht grdully incresing regimen nd constnt-mount dily regimen hve similr long-term effects on core temperture, when the totl dose nd durtion of eposure (once dy over 12-dy period) re held constnt. Whether this conclusion cn e generlized to higher doses will need further testing. Two-phse temporl response to METH dministrtion Also importnt in this study ws the time-dependent response of core temperture for the different pretretment groups. Chronic METH (5.0 nd 10.0 mg/kg) resulted in diminishing core temperture response t 1 h post-tretment. By 2 nd 4 h post-tretment (5.0 nd 10.0 mg/kg METH, respectively), METH dy-lock 1 4 showed hyperthermi, yet none of the METH dy-locks differed from ech other (Figs. 1 nd 3). This finding indictes tht chronic tretment did not diminish the temperture response 2 4 h into the session. In ddition, on the 10.0-mg/kg chllenge dy, t 1 h post-meth, the sline-pretretment group demonstrted hyperthermi, the 10-mg/kg-pretretment group showed hypothermi, nd the 5.0-mg/kg nd esclting regimens showed n intermedite core temperture response. How-

8 320 Psychophrmcology (2008) 198: ever, y 3 h post-injection, no group differences eisted (Fig. 4). This pttern of results supports the suggestion of our prior work (Myles et l. 2008) tht METH induces twophse temperture response: mllele erly phse nd less fleile lter phse. Long-term neurochemicl, neurontomicl, nd cognitive chnges hve een identified in humns with history of METH use (Chng et l. 2007; Chng et l. 2005; Chng et l. 2002; Simon et l. 2002; Thompson et l. 2004; Volkow et l. 2001; Wilson et l. 1996). Neurochemicl chnges re ltered y differences in core temperture (Bowyer et l. 1994). Intermittent pretretment with METH results in tolernce to neurotoicity induced y susequent high-dose tretment; this tolernce to neurotoic consequences, in prt, my e due to tolernce to hyperthermi (Dnceu et l. 2007; Riddle et l. 2002). After pretretment with n esclting intermittent regimen, Johnson- Dvis et l. (2003) reported significnt tolernce to METH-induced hyperthermi t 90 min post-tretment ut not lter during high-dose regimen; the METH preeposure lso protected ginst dmge induced y the high-dose regimen. Bsed on the results of Johnson-Dvis et l. (2003) nd our findings tht the phse 1 temperture response my e more fleile thn the phse 2 response, we suggest tht phse 1 temperture responses might e responsile for enhnced neurotoicity induced y METH, s well s the protection fforded y cooling procedures; erly intervention my wield greter influence on the longterm consequences of METH. Mechnisms of METH-induced chronic temperture chnges Reductions in core temperture, fter chronic tretment with stimulnts, re often discussed s tolernce to n cute hyperthermic response. In our compnion report (Myles et l. 2008), we found tht the chnge in temperture fter chronic METH tretment ws sometimes n induction of hypothermi reltive to previously neutrl seline insted of diminished hyperthermi. This finding ws supported in the present report s well. At 60 min posttretment, the nimls receiving chronic 5.0 mg/kg METH filed to show n initil incresed core temperture (dylock 1 4) ut demonstrted decresed temperture response with repeted tretment (Fig. 1). See Myles et l. (2008) for further discussion. Slivtion When testing t the 10.0 mg/kg dose, two different temperture ptterns emerged t 60 min posttretment. The initil tretment period (METH dy-lock 1 4, Fig. 3) filed to show n incresed temperture until 120 min post-tretment. This finding represents divergence from our previous work (Myles et l. 2008), in which tretment (dy-lock 1 4) resulted in significnt increse t 60 min, nd from this report where the sline-pretretment group showed significnt increse 60 min fter the 10.0-mg/kg-METH chllenge. The reson for this discrepncy is not cler ut my e relted to fctors not controlled for cross the eperiments. For emple, environmentl vriles such s humidity or suject fctors such s slivtion my hve plyed role. In the rt, spreding sliv on the fur contriutes to het loss during therml stress, nd continuous eposure to het cn cuse hypertrophy of the slivry glnds (Gordon 1990, 1993). In our compnion report, we noted the presence of slivtion ut did not systemticlly mesure it. We did so in this report. Rts receiving chronic 10.0 mg/kg (ut not chronic 5.0 mg/kg or the esclting regimen) slivted significntly more thn sline-treted rts during ll 3 dy-locks of the METH pretretment period. In this study, slivtion my hve ttenuted METH-induced hyperthermi for the 10.0 mg/kg pretretment group (dy-lock 1 4) nd contriuted to susequent temperture decreses throughout the METH tretment period. The etent to which slivtion plys role in cooling rts chroniclly treted with METH will require further study. Anorei It hs een shown tht clorie-restricted rts show lower ody tempertures thn rts fed d liitum (Duffy et l. 1989; Duffy et l. 1997). Rts in our chronic regimen groups eperienced significnt weight loss during the pretretment period (Tle 2). During the 5 8 dy-lock, only the 10.0-mg/kg group differed significntly from sline, ut t the 9 12 dy-lock, ll METH pretretment groups were different from sline. Anorei, therefore, my e contriuting fctor to the development of hypothermi during the phse 1 response. Neurochemistry METH induces trnsient DA depletions, which, in prt, my e ecuse of diminished synthesis (Bowyer et l. 1992). METH-induced 5-HT nd DA depletions re lso ssocited with neurotoicity (Bowyer et l. 1994; O'Cllghn nd Miller 1994; Sol et l. 2001; Wgner et l. 1980). In this report, DA nd 5-HT postmortem tissue concentrtions were not depleted when mesured 2 weeks fter completion of tretment (Tle 3). We hve not ddressed the role of trnsient decreses in monomine tissue levels post-meth; however, our findings tht depletions were not present 2 weeks fter the completion of tretment suggest tht the effects of chronic METH tretment on core temperture re not ecuse of the onset of longer-term degenertion of DA or 5-HT on terminls. See Myles et l. (2008). Contet Environmentl contet hs een identified s n importnt determinnt for tolernce to certin drug effects

9 Psychophrmcology (2008) 198: (Poulos nd Cppell 1991). In prticulr, Hinson et l. (1991) found tht tolernce developed to mphetmineinduced hyperthermi in one environment, ut hyperthermi ws reinstted when injected with mphetmine in n environment previously ssocited with sline injections. One spect of our dt suggests tht environmentl contet my hve een fctor in the temperture reductions we oserved with chronic eposure. In our prior report (Myles et l. 2008), we found tht oth sline nd METH decresed seline temperture during chronic tretment regimen (seline is defined s temperture mesurements efore dily injections). In the present report, we modified our design (4 dys of sline lone for ll groups (HAB dylock 1 4) nd longer seline periods within ech dily session) nd found tht chronic tretment with METH, ut not sline, decresed seline temperture. This pttern is demonstrted in Figs. 1 3, where the hitution dys (HAB dy-lock 1 4) showed significntly higher core temperture compred to the METH tretment dy-locks (2 h efore tretment). The sme finding is lso evident in Fig. 4 on chllenge dy 13: the sline-pretretment nimls hd significntly higher core ody temperture t 2 h efore injection compred to ll other pretretment groups. Contet (temperture test chmers) my hve triggered this preprtory response. Alterntively, this decresed core temperture during seline periods my hve een triggered y internl responses to repeted METH tretment rther thn the environment. In sum, while the sence of long-lsting DA/5-HT depletions suggests n sence of neurotoicity, other fctors including short-term monomine depletions, slivtion, norei, nd contet my hve contriuted to the ltered temperture response fter chronic METH. Summry Initil eposure to METH (dy-lock 1 4) incresed core temperture t 24 C mience. The timing of this increse ws dose dependent. Susequent decreses in temperture were evident with chronic eposure. A constnt dily regimen (5.0 mg/kg/dy) nd n esclting regimen of the sme totl dosge produced different temporl ptterns of core temperture response; however, these regimens demonstrted similr decrese in temperture (reltive to the sline-pretretment group) when given chllenge dose of METH. The differentil response to chronic METH t 1 vs. 3 h post-tretment suggests two seprte mechnisms of thermoregultion. The ttenuted temperture response fter chronic METH tretment is unrelted to neurotoicity s indeed y centrl DA or 5-HT tissue concentrtions mesured 2 weeks fter the end of tretment. Other potentil contriutors to the chronic temperture response include short-term monomine depletion, slivtion, nd norei. Chronic eposure to METH lso produced decrese in seline temperture, possily relted to Pvlovin conditioning. 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