Prior methamphetamine self-administration attenuates serotonergic deficits induced by subsequent high-dose methamphetamine administrations

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Drug and Alcohol Dependence 126 (2012) 87– 94

Contents lists available at SciVerse ScienceDirect

Drug and Alcohol Dependence

jo u rn al hom epage: www.elsev ier .com/ locate /drugalcdep

rior methamphetamine self-administration attenuates serotonergic deficitsnduced by subsequent high-dose methamphetamine administrations

isa M. McFadden, Madison M. Hunt, Paula L. Vieira-Brock, Janice Muehle, Shannon M. Nielsen,cott C. Allen, Glen R. Hanson, Annette E. Fleckenstein ∗

epartment of Pharmacology and Toxicology University of Utah, Salt Lake City, UT 84112, United States

r t i c l e i n f o

rticle history:eceived 3 January 2012eceived in revised form 19 April 2012ccepted 21 April 2012vailable online 28 May 2012

eywords:ethamphetamine

elf-administrationerotoninippocampus

a b s t r a c t

Background: Pre-clinical studies indicate that high-dose, non-contingent methamphetamine (METH)administration both rapidly and persistently decreases serotonergic neuronal function. Despite researchindicating the hippocampus plays an important role in METH abuse and is affected by METH use, effectsof METH self-administration on hippocampal serotonergic neurons are not well understood, and werethus an important focus of the current study. Because humans often administer METH in a binge-likepattern, effects of prior METH self-administration on a subsequent “binge-like” METH treatment werealso examined.Methods: Rats were treated as described above, and sacrificed 1 or 8 d after self-administration or 1 h or7 d after the final binge METH or saline exposure. Hippocampal serotonin (5-hydroxytryptamine; 5HT)content and transporter (SERT) function were assessed.Results: METH self-administration per se had no persistent effect on hippocampal 5HT content or SERTfunction. However, this treatment attenuated the persistent, but not acute, hippocampal serotonergicdeficits caused by a subsequent repeated, high-dose, non-continent METH treatment administered 1 dthe last self-administration session. No attenuation in persistent deficits were seen when the high-dose

administration of METH occurred 15 d after the last self-administration session.Conclusions: The present findings demonstrate that METH self-administration alters serotonergic neu-rons so as to engender “tolerance” to the persistent serotonergic deficits caused by a subsequent METHexposure. However, this “tolerance” does not persist. These data provide a foundation to investigatecomplex questions including how the response of serotonergic neurons to METH may contribute tocontingent-related disorders such as dependence and relapse.

. Introduction

Methamphetamine (METH) is a highly addictive substance thats abused worldwide. Abusers often display structural and neuro-hemical changes within the brain and higher rates of psychiatricisorders and cognitive deficits (for review, see Barr et al., 2006;hang et al., 2007; but see also Hart et al., 2012). For example,ositron emission tomography has indicated decreased serotonin5-hydroxytryptamine; 5HT) transporter (SERT) densities in sev-ral brain regions in abstinent METH users (Sekine et al., 2006).dditionally, increased depression scores and impaired memoryerformance on a word recall test have been correlated with

educed hippocampal volumes in METH abusers (Thompson et al.,004). Reductions in gray matter volumes of both the amygdala andippocampus have been reported in subjects with METH-induced

∗ Corresponding author. Tel.: +1 801 585 7474; fax: +1 801 585 5111.E-mail address: [email protected] (A.E. Fleckenstein).

376-8716/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.drugalcdep.2012.04.020

© 2012 Elsevier Ireland Ltd. All rights reserved.

psychosis (Orikabe et al., 2011). Further, functional magnetic res-onance imaging has indicated decreased hippocampal activationin METH abusers during an empathy task (Kim et al., 2010). Theselatter findings emphasize the need for study of the neurochemicalchanges in the hippocampus following METH exposure.

Preclinical data indicate that hippocampal serotonergic neuronsare vulnerable to non-contingent high-dose METH administra-tion. For example, METH, given in a “binge-like” pattern (e.g., 3–6injections, 7.5–50 mg/kg/injection, 2–8-h intervals), decrease rathippocampal SERT function, [125I]RTI55 binding to SERT and 5HTcontent as assessed 72 h to 3 months after the METH injections(Farfel and Seiden, 1995; Haughey et al., 2000; Johnson-Davis et al.,2003; Richards et al., 1993; Schröder et al., 2003). Further, trypto-phan hydroxylase (TPH) activity is decreased 30 d (Hotchkiss andGibb, 1980), and 5HT content is decreased 35 d, following a binge

METH exposure (Herring et al., 2010). These changes are not uniqueto the hippocampus, as persistent decreases in 5HT content, SERTfunction and TPH activity have also been reported in the stria-tum (Bowyer et al., 2008; Danaceau et al., 2007; Haughey et al.,

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000; Herring et al., 2010; Hotchkiss and Gibb, 1980; Stephansnd Yamamoto, 1996). Additionally, serotonergic axonal degener-tion has been reported using immunocytochemistry in animalshat receive large METH doses (Axt and Molliver, 1991).

Of relevance, pretreatment with escalating-dose administra-ions of METH attenuated the decreased hippocampal 5HT contentaused by a binge METH treatment, although this effect appearsependent upon the length of time between the binge exposure andonoamine assessment. For example, such pretreatment atten-

ates hippocampal 5HT deficits as assessed 7 d (Johnson-Davist al., 2003), but not 1 d (Cadet et al., 2009; Graham et al., 2008),fter a binge METH treatment. Further, escalating-dose pretreat-ent attenuated the decreases in hippocampal [125I]RTI-55 binding

aused by a binge METH treatment as assessed 7 d later (Belchert al., 2008). Finally, pretreatment with escalating doses of METHffords protection against subsequent binge METH-induced sero-onergic deficits when the binge is administered within 1 week, butot 14 or 31 d, after the final escalating-dose exposure (Danaceaut al., 2007).

The hippocampus is important for METH reinforcement. Forxample, when METH is administered directly into the dorsal hip-ocampus, rats self-administer the drug and develop conditionedlace preference for the location where it was administered (Ricoynd Martinez, 2009). Further, METH self-administration disruptsbject-in-place recognition, which is thought to rely upon perirhi-al cortex-prefrontal cortex-hippocampal circuitry (Reichel et al.,012). Despite research indicating that the hippocampus plays an

mportant role in METH abuse and is affected by METH use, effectsf METH self-administration on hippocampal serotonergic neuronsre not well understood.

Human METH addiction is characterized by escalating drugse and the loss of the ability to control drug intake sometimesesulting in high-dose exposure to the drug of abuse (Koob andolkow, 2010). Previous research utilizing this self-administrationodel has demonstrated that 7 d of METH self-administration

0.12 mg/infusion; McFadden et al., 2012) lead to: (1) an escalationn daily drug intake; (2) brain METH concentrations 1 h after the lastelf-administration session that are comparable to those found inuman postmortem studies (Kalasinsky et al., 2001); (3) persistentecreases in dopamine transporter immunoreactivity within thetriatum which were similar in magnitude to those observed usingET imaging of human METH abusers (Chang et al., 2007); and (4)n attenuation of the striatal dopaminergic deficits induced by aubsequent binge METH treatment.

Because of the importance of serotonergic neurons and the hip-ocampus, the present study evaluated SERT function and 5HTontent after METH self-administration. It was hypothesized thatETH self-administration would lead to resistance to serotonergic

eficits. To test this, a high-dose METH regimen was adminis-ered after METH self-administration. Results revealed that prior

ETH self-administration did not cause persistent (herein defineds 7–8 d following last METH exposure) serotonergic deficits per se.owever, such treatment attenuated the persistent, but not acute,

erotonergic deficits induced by a subsequent binge METH treat-ent given 1 d after the start of the last self-administration session.

he attenuation of persistent deficits was not seen when the bingexposure occurred 15 d after the last self-administration session.hese data may provide important insight into the neurochemicaleficits reported in human METH abusers.

. Methods

.1. Animals

Male Sprague-Dawley rats (275–300 g; Charles River Laboratories, Portage, MI)ere housed four rats/cage. Following surgery, each rat was individually housed in

transparent plastic cage. Water was available in their home cage ad libitum. During

l Dependence 126 (2012) 87– 94

food training, rats were food restricted such that no rat dropped below 90% of theirbody weight at the time the experiment was initiated. Rats were maintained underthe same 14:10 h light/dark cycle in the animal facility and in the operant cham-bers. Animals were sacrificed by decapitation. All experiments were approved bythe University of Utah Institutional Animal Care and Use Committee, in accordancewith the National Institutes of Health Guide for the Care and Use of Laboratory Ani-mals. Lever pressing behavior, core body temperatures and striatal dopaminergicassessments from rats utilized to generate Figs. 1 and 2 were published previously(McFadden et al., 2012).

2.2. Drugs

Racemic-METH hydrochloride (Research Triangle Institute; Research TrianglePark, NC) was dissolved in 0.9% sterile saline. Doses are expressed as free base.Rats were anesthetized using ketamine (90 mg/kg; Hospira Inc., Lake Forest, IL,USA) and xylazine (7 mg/kg; Sigma–Aldrich, St. Louis, MO, USA). The antibiotic cefa-zolin (10 mg/ml; Schein Pharmaceutical, Florham Park, NJ, USA) was dissolved inheparinized saline (63.33 U/ml; Sigma, St. Louis, MO, USA). Flunixin meglumine(1.1 mg/kg; MWI Veterinary Supply, Meridian, ID, USA) was used for post-surgeryanalgesia.

2.3. Food training and surgery

Food training and self-administration occurred in an operant chamber(30.5 cm × 25.5 cm × 30.5 cm; Coulbourn Instruments, Whitehall, PA USA) asdescribed in McFadden et al., 2011. Prior to surgery, each rat was trained to press fora 45-mg food pellet during four overnight 14-h sessions. Following food training,rats were anesthetized and an indwelling catheter was implanted. The catheter wasconstructed as described previously (Frankel et al., 2008, 2011). The outlet of thecatheter was implanted subcutaneously in the back and the free end of the Silastictubing was inserted 25 mm into the right jugular vein and secured to the surround-ing tissue with sutures. Each rat received flunixin meglumine on the day of thesurgery and the day following the surgery. Immediately following surgery and dailythereafter, each rat was infused with 0.1 ml of cefazolin followed by 0.05 ml of hep-arinized saline and heparinized glycerol through the catheter. Catheter patency wasconfirmed by infusing 0.03 ml (20 mg/ml) of xylazine.

2.4. Self-administration and METH challenge

Rats underwent 7 d of self-administration (8 h/session; FR1; 0.12 mg/infusionMETH or saline) during the light cycle in a room maintained at 29 ± 1 ◦C to pro-mote lever pressing (Cornish et al., 2008). For each active lever press, an infusionpump connected to a liquid swivel (Coulbourn Instruments) suspended outsidethe operant chamber delivered 10 �l of METH or saline per infusion over a 5-sduration through a polyethylene tube located within a spring leash (CoulbournInstruments) tethered to the rat. During this period, both levers were retracted. Fol-lowing the infusion, the levers remained retracted for an additional 20 s. The activelever was counterbalanced within each group. Pressing the inactive lever resultedin no programmed consequences although it was recorded. Rectal temperatureswere measured using a digital thermometer (Physiotemp Instruments, Clifton, NJ)approximately 30 min after the end of each session. Animals were sacrificed 1 or 8 dafter the start of the last self-administration session or received a binge of METH orsaline as described below.

Twenty-four hours after the start of the last self-administration session, ratswere challenged with 4 injections of METH (7.5 mg/kg/injection; 2-h interval) orsaline (1 ml/kg/injection). This time point was chosen based on research utiliz-ing escalating doses of METH (see Danaceau et al., 2007; O’Neil et al., 2006). Inthe initial binge experiment, all animals received drug in an ambient temperatureof 22–23 ◦C. In subsequent studies, METH self-administering rats challenged withMETH (METH/METH) were maintained in a warm environment (25 ◦C) to promotehyperthermia. Previous research in our laboratory has shown that this ambient tem-perature permits binge METH-induced that would have otherwise been attenuatedby prior exposure to METH during development (see McFadden et al., 2011). Animalswere sacrificed 1 h or 7 d after the binge exposure. Another group of rats received abinge of METH or saline 15 d following the final self-administration session and weresacrificed 7 d later. Rectal temperatures were measured using a digital thermometerapproximately 30 and 90 min after each injection.

2.5. Synaptosomal [3H]5HT uptake

[3H]5HT uptake was determined using a rat hippocampal synaptosomal prepa-ration as previously described (Hadlock et al., 2011). In brief, synaptosomes wereprepared by homogenizing freshly dissected hippocampal tissue in ice-cold 0.32 Msucrose pH 7.4, and centrifuged (800 × g, 12 min; 4 ◦C). A small section of the leftanterior hippocampus was quickly frozen on dry ice and retained for determining

5HT content. The supernatants were centrifuged (22,000 × g, 15 min; 4 ◦C) and theresulting pellets were resuspended in ice-cold assay buffer (in mM: 126 NaCl, 4.8KCl, 1.3 CaCl2, 16 sodium phosphate, 1.4 MgSO4, 11 glucose and 1 ascorbic acid;pH 7.4) and 1 �M pargyline. Samples were incubated for 10 min at 37 ◦C and theassays initiated by the addition of [3H]5HT (5 nM final concentration). Following

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Fig. 1. METH self-administration attenuates the persistent effects of a subsequent binge METH treatment when administered in at a similar ambient temperature (22–23 ◦C).Rats self-administered METH (0.12 mg/infusion) or saline (10 �l/infusion) for 7 d (8 h/d). Twenty-four hours after the beginning of the final self-administration session, ratsr on, s.c5 om all

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eceived METH (4 × 7.5 mg/kg/injection, s.c., 2-h intervals) or saline (1 ml/kg/injectiHT tissue content (Panel B) were assessed in the hippocampus. **p < 0.05 differs fr

ncubation for 3 min, samples were placed on ice to stop the reaction. Samples werehen filtered through GF/B filters (Whatman, Florham Park, NJ) soaked previously in.05% polyethylenimine. Filters were rapidly washed three times with 3 ml of ice-old 0.32 M sucrose buffer using a filtering manifold (Brandel, Gaithersburg, MD).

ig. 2. METH self-administration attenuates the persistent effects of a subsequent bingeelf-administration in a warmer environment (25 ◦C). Rats self-administered METH (0.12eginning of the final self-administration session, rats received METH (4 × 7.5 mg/kg/injere sacrificed 7 d later, and SERT function (Panel A) and 5HT tissue content (Panel B) wiffers from SAL/SAL group; #p < 0.05 METH/METH differs from SAL/METH group.

., 2-h intervals; Panel C), were sacrificed 7 d later, and SERT function (Panel A) and other groups; #p < 0.05 METH/METH differs from SAL/METH group.

Nonspecific values were determined in the presence of 10 �M fluoxetine. Radioac-tivity trapped in filters was counted using a liquid scintillation counter. Proteinconcentrations were determined using the Bio-Rad Protein Assay (Bio-Rad Labora-tories Inc., Hercules, CA).

METH treatment when administered in animals with a previous history of METH mg/infusion) or saline (10 �l/infusion) for 7 d (8 h/d). Twenty-four hours after theection, s.c., 2-h intervals) or saline (1 ml/kg/injection, s.c., 2-h intervals; Panel C),ere assessed in the hippocampus. **p < 0.05 differs from all other groups; *p < 0.05

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.6. 5HT and 5-hydroxyindoleacetic acid (5HIAA) content

The anterior portion of the left hippocampus or striatum was sonicated for–5 s in 1 ml tissue buffer (0.1 M phosphate/citrate buffer, pH 2.5, containing0% methanol) according to Haughey et al. (2000). Fifty �l were injected onto aartisphere C-18 reverse-phase analytical column (5-�m spheres; 250 × 4.6 mm;hatman, Clifton, NJ, USA). Mobile phase consisting of 0.05 M sodium phosphate,

.03 M citrate buffer, 0.1 M EDTA, 0.030–0.035% sodium octylsulfate, and 20–25%ethanol (pH 2.85, flow rate = 0.75 ml/min) was used. Serotonin was detected using

n ampherometric electrochemical detector with the working electrode potentialet at +0.70 V relative to an Ag+/AgCl reference electrode.

.7. Statistical analysis

Statistical analysis was conducted in SAS 9.2 (Cary, NC, USA). Statistical anal-ses among groups were conducted using a t-test, analysis of variance (ANOVA)r repeated measures ANOVA followed by Newman–Keuls posthoc analyses. Viola-ions of the sphericity assumption resulted in the use of a Huynh–Feldt correction.he data represent means ± standard error of the mean (S.E.M.) of 5–14 rats/group.

. Results

The pattern of METH self-administration was consistent withhat described in McFadden et al. (2012). Only animals that methe criteria largely based on Brennen et al. (2010) were included.pecifically, rats were included if they: (1) pressed an averagef more than 10 active lever presses per d; and (2) the ratio ofctive/inactive lever presses was ≥2:1. Discrimination between thective and inactive lever exceeded 5:1 in METH self-administeringnimals by the last day of self-administration. Further, an escala-ion in daily METH intake occurred in all METH self-administrationxperiments similar to that described in McFadden et al. (2012;ee Figs. 4A and 6A therein). Of note, in the studies describedelow, some saline self-administering/METH challenged rats suc-umbed to METH treatment. However, there were no fatalitiesmong METH self-administering/METH challenged rats in any ofhe studies described below.

METH self-administration did not cause persistent serotoner-ic deficits as assessed 8 d after the last session. Specifically,his treatment (described in Section 2) did not alter hip-ocampal [3H]5HT uptake (Saline: 1.53 ± 0.10 fmol/�g protein;ETH: 1.54 ± 0.07 fmol/�g protein; t(12) = 0.73, ns), 5HT content

Saline: 4.99 ± 0.49 pg/�g protein; METH: 5.67 ± 0.38 pg/�g pro-ein; t(12) = 1.10, ns) or 5HIAA content (Saline: 8.51 ± 0.67 pg/�grotein; METH: 8.60 ± 0.54 pg/�g protein; t(12) = 0.11). Core bodyemperatures were 38.0 ± 0.1 ◦C in saline self-administering and8.5 ± 0.1 ◦C in METH self-administering rats (t(12) = 4.32, p < 0.05).n this study, rats self-administered a total of 26.33 ± 1.49 mg of

ETH over the course of the 7 d of self-administration.A separate group of animals were allowed to self-administer

or 7 days and were sacrificed 1 d after the start of the last self-dministration session. No differences were found in hippocampal3H]5HT uptake (t(12) = 0.62, ns; Saline: 1.26 ± 0.04 fmol/�g pro-ein; METH: 1.31 ± 0.08 fmol/�g protein), 5HT hippocampal tissueontent (t(13) = 0.56, ns; Saline: 5.06 ± 0.21 pg/�g protein; METH:.33 ± 0.47 pg/�g protein), or 5HIAA content (t(13) = 0.43, ns;aline: 7.05 ± 0.35 pg/�g protein; METH: 6.75 ± 0.61 pg/�g pro-ein). These METH self-administering animals consumed a total of2.33 ± 1.82 mg METH over the course of self-administration.

Results presented in Fig. 1 indicate that 7 d of METH self-dministration attenuated the persistent hippocampal serotoner-ic deficits caused by a subsequent binge METH treatment admin-stered 1 d after the start of the last self-administration session,s assessed 7 d later. METH self-administration attenuated deficitsn [3H]5HT uptake (F(2,29) = 3.59, p < 0.05; Fig. 1A), 5HT content

F(2,29) = 15.88, p < 0.05; Fig. 1B), and 5HIAA content (F(2,29) = 9.14,

< 0.05; Saline/Saline: 5.27 ± 0.15 pg/�g protein; METH/METH:.43 ± 0.32 pg/�g protein; Saline/METH: 3.03 ± 0.29 pg/�g pro-ein) relative to Saline/METH controls. No attempt was made to

l Dependence 126 (2012) 87– 94

maintain equal hyperthermia during the binge, which resulted inthe METH/METH group maintaining a cooler body temperaturesduring the first 2.5 h following METH administration compared tothe Saline/METH group (F(16,224) = 7.70, p < 0.05; reprinted withpermission from McFadden et al., 2012). Animals self-administered20.99 ± 1.33 mg METH over the course of self-administration.

In subsequent experiments, an attempt to maintain equalhyperthermia in the METH/METH and Saline/METH groups wasdone as described in the Section 2. Results presented in Fig. 2 indi-cate that, as in Fig. 1, 7 d of METH self-administration attenuatedthe persistent hippocampal SERT deficits caused by a subse-quent binge METH treatment, as assessed 7 d later. In particular,METH self-administration attenuated deficits in [3H]5HT uptake(F(2,16) = 14.00, p < 0.05; Fig. 2A) and 5HIAA content (F(2,14) = 9.73,p < 0.05; Saline/Saline: 9.23 ± 1.02 pg/�g protein; METH/METH:5.57 ± 0.71 pg/�g protein; Saline/METH: 3.66 ± 0.95 pg/�g pro-tein) relative to Saline/METH controls. Resembling effects inFig. 1B, prior METH self-administration trended toward attenuat-ing binge-induced deficits in 5HT content (although the attenuationwas not statistically significant; F(2,14) = 4.13, p < 0.05; Fig. 2B).Results presented in Fig. 2C indicate that despite the warmerambient environment, prior METH self-administration attenu-ated the increase in core body temperatures caused by thesubsequent binge METH treatment as assessed during the first2.5 h after treatment (F(2,16) = 103.3, p < 0.05; Fig. 2C; reprintedwith permission from McFadden et al., 2012). In this study,rats self-administered 21.68 ± 1.86 mg METH over the course ofself-administration.

To investigate if alterations in the acute binge-induced effectsof METH are associated with the attenuation of persistent deficitsseen in Fig. 2, animals were sacrificed 1 h following the lastinjection of the binge-exposure. Results presented in Fig. 3indicate that prior METH self-administration did not attenuatethe rapid decreases in [3H]5HT uptake (F(2,22) = 17.31, p < 0.05;Fig. 3A), 5HT content (F(2,22) = 68.94, p < 0.05; Fig. 3B), or 5HIAAcontent (Saline/Saline: 12.71 ± 1.34 pg/�g protein; METH/METH:4.55 ± 0.32 pg/�g protein; Saline/METH: 3.84 ± 0.18 pg/�g protein;F(2,22) = 38.32, p < 0.05) caused by a subsequent binge METH treat-ment, as assessed 1 h after the binge exposure. Similarly and despiteefforts to maintain hyperthermia, the METH/METH group hadattenuated the hyperthermia (F(14,154) = 12.09, p < 0.05; Fig. 3C),as assessed 30 min following the first and third injection of METH.Rats self-administered a total of 21.61 ± 1.23 mg METH during the7 d of self-administration.

Results presented in Fig. 4 indicate that prior self-administrationdid not afford protection against persistent deficits when the bingeof METH was given 15 d after the last self-administration session.Binge METH-induced decreases in [3 H]5HT uptake (F(2,19) = 11.39,p < 0.05, Fig. 4A), hippocampal 5HT content (F(2,20) = 16.47,p < 0.05; Fig. 4B) and hippocampal 5HIAA content (F(2,20) = 9.88,p < 0.05; Saline/Saline: 5.29 ± 0.23 pg/�g protein; METH/METH:3.19 ± 0.36 pg/�g protein; Saline/METH: 2.89 ± 0.47 pg/�g pro-tein) were similar between METH/METH and Saline/METHgroups. Further, similar hyperthermia was achieved between theMETH/METH and Saline/METH groups during the binge of METH(F(14,140) = 6.02, p < 0.05; Fig. 4C). Animals self-administered20.57 ± 0.83 mg METH.

Noteworthy, the impact of METH self-administration on 5HTsystems was not unique to the hippocampus. Prior METH self-administration also attenuated the persistent (F(2,15) = 50.76,p < 0.05; Fig. 5A), but not the acute deficits (F(2,21) = 110.70,p < 0.05; Fig. 5B) in striatal 5HT content caused by a subsequent

binge treatment when administered 1 d after the start of the lastself-administration session and were treated as in Figs. 2 and 3.No attenuation was seen in depletions of striatal 5HT contentwhen the binge was given 15 d after the last self-administration

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Fig. 3. METH self-administration does not alter the acute effects of a subsequent binge METH treatment. Rats self-administered METH (0.12 mg/infusion) or saline( lf-admo last ini ffers f

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10 �l/infusion) for 7 d (8 h/d). Twenty-four hours after the beginning of the final ser saline (1 ml/kg/injection, s.c., 2-h intervals; Panel C), were sacrificed 1 h after the

n the hippocampus. *p < 0.05 differs from SAL/SAL group; #p < 0.05 METH/METH di

ession (F(2,20) = 13.72, p < 0.05; Fig. 5C). Of note, 5HT contentithin the striatum did not significantly differ in METH and

aline self-administering rats 1 d after the start of the last self-dministration session (t(13) = 1.30, ns; Saline: 7.17 ± 0.37 pg/�grotein; METH: 6.52 ± 0.32 pg/�g protein).

ig. 4. METH self-administration does not alter the persistent effects of a subsequeats self-administered METH (0.12 mg/infusion) or saline (10 �l/infusion) for 7 d (8 h4 × 7.5 mg/kg/injection, s.c., 2-h intervals) or saline (1 ml/kg/injection, s.c., 2-h intervalontent (Panel B) were assessed in the hippocampus. *p < 0.05 differs from SAL/SAL group

inistration session, rats received METH (4 × 7.5 mg/kg/injection, s.c., 2-h intervals)jection, and SERT function (Panel A) and 5HT tissue content (Panel B) were assessedrom SAL/METH group.

4. Discussion

These findings are among the first to characterize the effectsMETH self-administration on SERT function in the hippocam-pus. Consistent with human postmortem studies measuring SERT

nt binge of METH when given 15 d after the last self-administration session./d). Fifteen days after the final self-administration session, rats received METHs; Panel C), were sacrificed 7 d later, and SERT function (Panel A) and 5HT tissue.

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Fig. 5. For panels A, B and C rats were treated the same as in Figures 2, 3, or 4, respectively. Striatal 5HT tissue content was assessed 7 d (Panel A) or 1 h (Panel B) after theb ationM from

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inge treatment of METH administered 1 d after the start of the last self-administrETH administered 15 d after the last self-administration session. **p < 0.05 differs

mmunoreactivity (Kish et al., 2009), the present study demon-trates that METH self-administration per se did not lead toersistent changes in SERT function in the hippocampus 1 or 8 dfter the last self-administration session. In contrast, others haveound that METH self-administration causes slight, but significant,ecreases in SERT immunoreactivity in the hippocampus (Reichelt al., 2012). Differences in the dosing paradigms (e.g., length ofETH access) may contribute to the discrepancies.Results also reveal that although METH self-administration

aradigm per se did not cause apparent serotonergic deficits, it didlter the impact of a subsequent METH binge exposure 1 d afterhe last self-administration session in 22–23 ◦C and 25 ◦C rooms.n particular, prior METH self-administration attenuated the per-istent SERT deficits resulting from an experimenter-administeredETH as assessed 7-d later in the hippocampus and 5HT content

n the striatum. This effect of self-administration is reminiscent offfects of escalating-dose regimens of experimenter-administeredETH. For example, escalating-dose administration of METH prior

o a binge treatment attenuated the METH-induced decreases inERT as assessed by [125I]RTI-55 binding and decreases in 5HTissue content in both the hippocampus and striatum 7 d afterhe binge METH treatment (Belcher et al., 2008; Johnson-Davist al., 2003). This protection is also similar to the effect of priorETH self-administration on binge treatment-induced alterations

n dopaminergic neuronal function in that the former attenuateshe persistent dopaminergic deficits caused by the binge treatmentMcFadden et al., 2012).

In contrast to the ability of METH self-administration to atten-ate persistent serotonergic deficits, the prior experience withETH did not attenuate the acute effects of METH as assessed 1-

after binge METH treatment in the hippocampus and striatum.hese findings are similar to reports by Cadet et al. (2009) that

scalating-dose METH treatment did not attenuate the decreasen hippocampal and striatal 5HT content 1 d after the binge of

ETH. The findings of the present study suggest that the attenua-ion of persistent binge METH-induced deficits afforded prior METH

session. Striatal tissue 5HT content (Panel C) was 7 d after the binge treatment ofall other groups; *p < 0.05 differs from SAL/SAL group.

self-administration are not a consequence of attenuating the acuteeffects of METH on SERT function.

The attenuations of persistent deficits afforded by prior METHself-administration were only significant when the binge of METHoccurred 1 d after the start of the last self-administration session.When the binge of METH was administered 15 d after the last self-administration session, prior METH self-administration no longerafforded protection. These findings are consistent with escalating-dose METH pretreatment in that protection by METH pretreatmentwas only seen when the binge of METH was administered within7 d of the end of pretreatment but not 14 or 31 d. These findingssuggest that the serotonergic system may be vulnerable to deficitswhen relapse occurs after prolonged abstinence. Future studies willexamine if reinstatement of METH self-administration after 15 d ofabstinence leads to serotonergic deficits.

Of interest, alterations in hyperthermia may contribute tothe attenuations in persistent serotonergic deficits. Reductions inMETH-induced hyperthermia and SERT deficits in animals witha history of prior METH self-administration occurred when thebinge was administered 1 d after the last session in both the ani-mals maintained in 22–23 ◦C and 25 ◦C rooms. Similar to otherstudies, these finding suggest that the attenuation of persistentserotonergic deficits may be due to reduction in hyperthermia(Kiyatkin and Sharma, 2009; Sharma et al., 2007). This sugges-tion is based, at least in part, on findings by Bowyer et al. (2008)that METH-induced 5HT depletions within the striatum occurredonly in hyperthermic mice maintained in a warm environment(Bowyer et al., 2008). METH-induced hyperthermia promotes theformation of oxygen radicals and reduces tryptophan hydroxy-lase activity (Fleckenstein et al., 1997; see also Stone et al., 1989),which may contribute to oxidative damage (Tata and Yamamoto,2007; Yamamoto et al., 2010). Additionally, increased tempera-

ture has been shown to increase human SERT uptake of [3 H]DAin HEK-293 cells, which may further promote oxidative damage(Saldana and Barker, 2004). Thus, attenuation of binge-inducedhyperthermia may have reduced oxygen radical formation thereby

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iminishing persistent serotonergic deficits when administeredithin 1 d after the last self-administration session. When the binge

f METH was administered 15 d after the last self-administrationession, similar hyperthermia was achieved and similar serotoner-ic deficits occurred. Although alterations in hyperthermia mayontribute to these findings, further research is needed to inves-igate the other mechanisms that may underlie these changes,uch as alterations in glutamate regulation, corticosteroids, neu-otrophic factors, and microglia activation (for reviews see Cadetnd Krasnova, 2009; Fleckenstein et al., 2007; Tata and Yamamoto,007; Yamamoto et al., 2010).

In summary, the results of the current study are the firsto demonstrate that the self-administration of METH prior toxperimenter-administered binge of METH administered 1 d afterhe last self-administration session attenuated the METH-inducederotonergic deficits 7 d following the METH binge challenge. Thebility of prior self-administration to attenuate these deficits isikely attributable in part to its ability to attenuate binge-inducedyperthermia. In contrast, prior self-administration did not alterhe acute effects of the binge METH treatment, suggesting that dif-erences exist in mechanisms underlying the acute and persistentffects of the binge treatment. Further, no protection was affordedy prior METH self-administration when the binge of METH wasiven 15 d after the last self-administration session. These find-ngs are of importance, since human addicts often escalate METHsage until they lose of control of their ability to limit drug intakend resulting in binge-like exposure (Koob and Volkow, 2010).lthough these data suggest that recent drug use may temporarilyfford protection against METH-induced serotonergic deficits, ourndings suggest that this protection is no longer effective if relapseo METH use occurs after prolonged abstinence. Lastly, these datarovide a foundation to investigate complex questions includingow the response of serotonergic neurons to METH may contributeo contingent-related disorders such as dependence and relapse.

ole of funding source

Funding for this study was provided by NIH grants: DA19447,3367, 11389, 04222, 00378, 00869, 033097; the NIH had no furtherole in study design; in the collection, analysis and interpretationf data; in the writing of the report; or in the decision to submit theaper for publication.

ontributors

Authors McFadden, Fleckenstein, and Hanson designed thetudy. Authors McFadden, Hunt, Allen, Muehle, Vieira-Brock, andielsen conducted the experiments and undertook the statis-

ical analysis. Authors McFadden and Fleckenstein wrote theanuscript. All authors contributed to and have approved the finalanuscript.

onflict of interest

All authors declare that they have no conflicts of interest.

eferences

xt, K.J., Molliver, M.E., 1991. Immunocytochemical evidence formethamphetamine-induced serotonergic axon loss in the rat brain. Synapse 9,302–313.

arr, A.M., Panenka, W.J., MacEwan, G.W., Thornton, A.E., Lang, D.J., Honer, W.G.,

Lecomte, T., 2006. The need for speed: an update on methamphetamine addic-tion. J. Psychiatry Neurosci. 31, 301–313.

elcher, A.M., Feinstein, E.M., O’Dell, S.J., Marshall, J.F., 2008. Methamphetamineinfluences on recognition memory: comparison of escalating and single-daydosing regimens. Neuropsychopharmacology 33, 1453–1463.

l Dependence 126 (2012) 87– 94 93

Bowyer, J.F., Robinson, B., Ali, S., Schmued, L.C., 2008. Neurotoxic-related changesin tyrosine hydroxylase, microglia, myelin, and the blood-brain barrier inthe caudate-putamen from acute methamphetamine exposure. Synapse 62,193–204.

Brennan, K.A., Colussi-Mas, J., Carati, C., Lea, R.A., Fitzmaurice, P.S., Schenk, S.,2010. Methamphetamine self-administration and the effect of contingency onmonoamine and metabolite tissue levels in the rat. Brain Res 131, 137–146.

Cadet, J.L., Krasnova, I.N., 2009. Molecular bases of methamphetamine-induced neu-rodegeneration. Int. Rev. Neurobiol. 88, 101–119.

Cadet, J.L., Krasnova, I.N., Ladenheim, B., Cai, N.S., McCoy, M.T., Atianjoh, F.E.,2009. Methamphetamine preconditioning: differential protective effects onmonoaminergic systems in the rat brain. Neurotox. Res. 15, 252–259.

Chang, L., Alicata, D., Ernst, T., Volkow, N., 2007. Structural and metabolic brainchanges in the striatum associated with methamphetamine abuse. Addiction102 (Suppl. 1), 16–32.

Cornish, J.L., Clemens, K.J., Thompson, M.R., Callaghan, P.D., Dawson, B., McGregor,I.S., 2008. High ambient temperature increases intravenous methamphetamineself-administration on fixed and progressive ratio schedules in rats. J. Psy-chopharmacol. 22, 100–110.

Danaceau, J.P., Deering, C.E., Day, J.E., Smeal, S.J., Johnson-Davis, K.L., Fleckenstein,A.E., Wilkins, D.G., 2007. Persistence of tolerance to methamphetamine-inducedmonoamine deficits. Eur. J. Pharmacol. 559, 46–54.

Farfel, G.M., Seiden, L.S., 1995. Role of hypothermia in the mechanism of pro-tection against serotonergic toxicity. II. Experiments with methamphetamine,p-chloroamphetamine, fenfluramine, dizocilpine and dextromethorphan. J.Pharmacol. Exp. Ther. 272, 868–875.

Fleckenstein, A.E., Wilkins, D.G., Gibb, J.W., Hanson, G.R., 1997. Interaction betweenhyperthermia and oxygen radical formation in the 5-hydroxytryptaminergicresponse to a single methamphetamine administration. J. Pharmacol. Exp. Ther.283, 281–285.

Fleckenstein, A.E., Volz, T.J., Riddle, E.L., Gibb, J.W., Hanson, G.R., 2007. New insightsinto the mechanism of action of amphetamines. Annu. Rev. Pharmacol. Toxicol.47, 681–698.

Frankel, P.S., Hoonakker, A.J., Hanson, G.R., 2008. Differential response of neu-rotensin to methamphetamine self-administration. Ann. N. Y. Acad. Sci. 1139,112–117.

Frankel, P.S., Hoonakker, A.J., Alburges, M.E., McDougall, J.W., McFadden, L.M.,Fleckenstein, A.E., Hanson, G.R., 2011. Effect of methamphetamine self-administration on neurotensin systems of the basal ganglia. J. Pharmacol. Exp.Ther. 336, 809–815.

Graham, D.L., Noailles, P.A., Cadet, J.L., 2008. Differential neurochemicalconsequences of an escalating dose-binge regimen followed by single-day multiple-dose methamphetamine challenges. J. Neurochem. 105,1873–1885.

Hadlock, G.C., Webb, K.M., McFadden, L.M., Chu, P.W., Ellis, J.D., Allen, S.C.,Andrenyak, D.M., Vieira-Brock, P.L., German, C.L., Conrad, K.M., Hoonakker,A.J., Gibb, J.W., Wilkins, D.G., Hanson, G.R., Fleckenstein, A.E., 2011. 4-Methylmethcathinone (mephedrone): neuropharmacological effects of adesigner stimulant of abuse. J Pharmacol Exp Ther. 339, 530–536.

Hart, C.L., Marvin, C.B., Silver, R., Smith, E.E., 2012. Is cognitive functioning impairedin methamphetamine users? A critical review. Neuropsychopharmacology 37,586–608.

Haughey, H.M., Fleckenstein, A.E., Metzger, R.R., Hanson, G.R., 2000. The effectsof methamphetamine on serotonin transporter activity: role of dopamine andhyperthermia. J. Neurochem. 75, 1608–1617.

Herring, N.R., Gudelsky, G.A., Vorhees, C.V., Williams, M.T., 2010. (+)-Methamphetamine-induced monoamine reductions and impaired egocentriclearning in adrenalectomized rats is independent of hyperthermia. Synapse 64,773–785.

Hotchkiss, A.J., Gibb, J.W., 1980. Long-term effects of multiple doses of metham-phetamine on tryptophan hydroxylase and tyrosine hydroxylase activity in ratbrain. J. Pharmacol. Exp. Ther. 214, 257–262.

Johnson-Davis, K.L., Fleckenstein, A.E., Wilkins, D.G., 2003. The role of hyperthermiaand metabolism as mechanisms of tolerance to methamphetamine neurotoxic-ity. Eur. J. Pharmacol. 482, 151–154.

Kalasinsky, K.S., Bosy, T.Z., Schmunk, G.A., Reiber, G., Anthony, R.M., Furukawa, Y.,Guttman, M., Kish, S.J., 2001. Regional distribution of methamphetamine inautopsied brain of chronic human methamphetamine users. Forensic Sci. Int.116, 163–169.

Kim, Y.T., Lee, J.J., Song, H.J., Kim, J.H., Kwon, D.H., Kim, M.N., Yoo, D.S., Lee, H.J., Kim,H.J., Chang, Y., 2010. Alterations in cortical activity of male methamphetamineabusers performing an empathy task: fMRI study. Hum. Psychopharmacol. 25,63–70.

Kish, S.J., Fitzmaurice, P.S., Boileau, I., Schmunk, G.A., Ang, L.C., Furukawa, Y., Chang,L.J., Wickham, D.J., Sherwin, A., Tong, J., 2009. Brain serotonin transporter inhuman methamphetamine users. Psychopharmacology (Berl.) 202, 649–661.

Kiyatkin, E.A., Sharma, H.S., 2009. Acute methamphetamine intoxication brainhyperthermia, blood-brain barrier, brain edema, and morphological cell abnor-malities. Int. Rev. Neurobiol. 88, 65–100.

Koob, G.F., Volkow, N.D., 2010. Neurocircuitry of addiction. Neuropsychopharma-cology. 35, 217–238.

McFadden, L.M., Hoonakker, A.J., Vieira-Brock, P.L., Stout, K.A., Sawada, N.M., Ellis,J.D., Allen, S.C., Walters, E.T., Nielsen, S.M., Gibb, J.W., Alburges, M.E., Wilkins,D.G., Hanson, G.R., Fleckenstein, A.E., 2011. Methamphetamine treatment duringdevelopment attenuates the dopaminergic deficits caused by subsequent high-dose methamphetamine administration. Synapse 65, 771–777.

Page 8

9 lcoho

M

O

O

R

R

R

S

4 L.M. McFadden et al. / Drug and A

cFadden, L.M., Hadlock, G.C., Allen, S.C., Vieira-Brock, P.L., Stout, K.A., Ellis, J.D.,Hoonakker, A.J., Anderyak, D.M., Neilson, S.M., Wilkins, D.G., Hanson, G.R., Fleck-enstein, A.E., 2012. Methamphetamine self-administration causes persistentstriatal dopaminergic alterations and mitigates the deficits caused by a sub-sequent methamphetamine exposure. J. Pharmacol. Exp. Ther. 340, 295–303.

’Neil, M.L., Kuczenski, R., Segal, D.S., Cho, A.K., Lacan, G., Melega, W.P., 2006. Esca-lating dose pretreatment induces pharmacodynamic and not pharmacokinetictolerance to a subsequent high-dose methamphetamine binge. Synapse 60,465–473.

rikabe, L., Yamasue, H., Inoue, H., Takayanagi, Y., Mozue, Y., Sudo, Y., Ishii, T.,Itokawa, M., Suzuki, M., Kurachi, M., Okazaki, Y., Kasai, K., 2011. Reduced amyg-dala and hippocampal volumes in patients with methamphetamine psychosis.Schizophr. Res. 132, 183–189.

eichel, C.M., Ramsey, L.A., Schwendt, M., McGinty, J.F., See, R.E., 2012.Methamphetamine-induced changes in the object recognition memory circuit.Neuropharmacology 62, 1119–1126.

ichards, J.B., Baggott, M.J., Sabol, K.E., Seiden, L.S., 1993. A high-dose metham-phetamine regimen results in long-lasting deficits on performance of areaction-time task. Brain Res. 627, 254–260.

icoy, U.M., Martinez Jr., J.L., 2009. Local hippocampal methamphetamine-inducedreinforcement. Front. Behav. Neurosci. 3, 47.

aldana, S.N., Barker, E.L., 2004. Temperature and 3,4-methylenedioxy-methamphetamine alter human serotonin transporter-mediated dopamineuptake. Neurosci. Lett. 354, 209–212.

l Dependence 126 (2012) 87– 94

Schröder, N., O’Dell, S.J., Marshall, J.F., 2003. Neurotoxic methamphetamine regimenseverely impairs recognition memory in rats. Synapse 49, 89–96.

Sekine, Y., Ouchi, Y., Takei, N., Yoshikawa, E., Nakamura, K., Futatsubashi, M., Okada,H., Minabe, Y., Suzuki, K., Iwata, Y., Tsuchiya, K.J., Tsukada, H., Iyo, M., Mori, N.,2006. Brain serotonin transporter density and aggression in abstinent metham-phetamine abusers. Arch. Gen. Psychiatry 63, 90–100.

Sharma, H.S., Sjoquist, P.O., Ali, S.F., 2007. Drugs of abuse-induced hyperthermia,blood-brain barrier dysfunction and neurotoxicity: neuroprotective effects of anew antioxidate compound H-290/51. Curr. Pharm. Des. 13, 1903–1923.

Stephans, S., Yamamoto, B., 1996. Methamphetamines pretreatment and the vul-nerability of the striatum to methamphetamine neurotoxicity. Neuroscience 72,593–600.

Stone, D.M., Johnson, M., Hanson, G.R., Gibb, J.W., 1989. Acute inactivation of trypto-phan hydroxylase by amphetamine analogs involves the oxidation of sulfhydrylsites. Eur. J. Pharmacol. 172, 93–97.

Tata, D.A., Yamamoto, B.K., 2007. Interactions between methamphetamine andenvironmental stress: role of oxidative stress, glutamate and mitochondrialdysfunction. Addiction 102 (Suppl. 1), 49–60.

Thompson, P.M., Hayashi, K.M., Simon, S.L., Geaga, J.A., Hong, M.S., Sui, Y., Lee,

J.Y., Toga, A.W., Ling, W., London, E.D., 2004. Structural abnormalities inthe brains of human subjects who use methamphetamine. J. Neurosci. 24,6028–6036.

Yamamoto, B.K., Moszczynska, A., Gudelsky, G.A., 2010. Amphetamine toxicities:classical and emerging mechanisms. Ann. N. Y. Acad. Sci. 1187, 101–121.