Psychopharmacology (2005) 181: 4859DOI 10.1007/s00213-005-2219-1

ORIGINAL INVESTIGATION

Douglas S. Ramsay . Karl J. Kaiyala .Brian G. Leroux . Stephen C. Woods

Individual differences in initial sensitivity and acutetolerance predict patterns of chronic drug toleranceto nitrous-oxide-induced hypothermia in rats

Received: 4 August 2004 / Accepted: 20 January 2005 / Published online: 19 March 2005# Springer-Verlag 2005

Abstract Rationale: A preventive strategy for drug addic-tion would benefit from being able to identify vulnerableindividuals. Understanding how an individual responds dur-ing an initial drug exposure may be useful for predictinghow that individual will respond to repeated drug admin-istrations. Objectives: This study investigated whetherindividual differences in initial drug sensitivity and acutetolerance can predict how chronic tolerance develops.Methods: During an initial 3-h administration of 60% ni-trous oxide (N2O), male LongEvans rats were screenedfor N2Os hypothermic effect into subsets based on beinginitially insensitive (II), sensitive with acute tolerance (AT),or sensitive with no intrasessional recovery (NR). Animalsin each individual difference category were randomly as-signed to receive six 90-min exposures of either 60% N2Oor placebo gas. Core temperature was measured telemet-rically. Results: Rats that exhibited a comparable degree

of hypothermia during an initial N2O exposure, but dif-fered in acute tolerance development, developed differentpatterns of chronic tolerance. Specifically, the NR groupdid not become fully tolerant over repeated N2O expo-sures while the AT group developed an initial hyperther-mia followed by a return of core temperature to controllevels indicative of full tolerance development. By the sec-ond N2O exposure, the II group breathing N2O becamehyperthermic relative to the placebo control group andthis hyperthermia persisted throughout the multiple N2Oexposures. Conclusions: Individual differences in initialdrug sensitivity and acute tolerance development predictdifferent patterns of chronic tolerance. The hypothesis issuggested that individual differences in opponent-adaptiveresponses may mediate this relationship.

Keywords Inhalant . Addictive vulnerability . Addiction .Drug dependence . Intrasessional tolerance . Allostasis .Homeostasis . Regulation

Introduction

Drug abuse is a major public health and economic problemworldwide (McGinnis and Foege 1999; Single et al. 1999;Fenoglio et al. 2003). While advances are being made tounderstand the neurobiology of addictions for devisingnovel pharmacological therapies (OBrien 1997), an alter-native approach is to identify especially vulnerable indi-viduals to utilize preventive strategies (Piazza and LeMoal1996). Individual differences in key characteristics couldthen be used to identify at-risk individuals before they be-come addicted to abusable drugs (Crabbe et al. 1999;Schuckit et al. 2001). The adaptations an individual makesduring an initial drug exposure may indicate how that in-dividual will respond to future drug exposures.Adaptation models of drug addiction are popular be-

cause they account for central features of the drug addictionprocess, including tolerance, dependence, and withdrawal(Jaffe 1985; Peper 2004). These phenomena are thought tobe manifestations of a common underlying adaptive re-

D. S. Ramsay (*) . K. J. Kaiyala . B. G. LerouxDepartment of Dental Public Health Sciences,University of Washington,P.O. Box 357475 Seattle, WA, 98195-7475, USAe-mail: ramsay@u.washington.eduTel.: +1-206-5432034Fax: +1-206-6854258

D. S. RamsayDepartment of Orthodontics,University of Washington,Seattle, WA, USA

D. S. RamsayDepartment of Pediatric Dentistry,University of Washington,Seattle, WA, USA

B. G. LerouxDepartment of Biostatistics,University of Washington,Seattle, WA, USA

S. C. WoodsDepartment of Psychiatry,University of Cincinnati Medical Center,Cincinnati, OH, USA

sponse that develops with repeated drug use. In brief, thedrug initially perturbs a biologically regulated system,eliciting adaptive changes that reduce the perturbation (i.e.,tolerance develops). Over repeated drug administrations,the individual comes to function in a drug-adapted or-dependent state wherein drug-induced perturbations areeffectively countered by acquired neutralizing or compen-satory adaptations. If the drug effect is removed, the com-pensatory effects caused by these adaptations becomeunopposed and result in withdrawal. A premise of adap-tation models is that addiction results from adaptive re-sponses made when confronted with repeated drug-inducedregulatory disturbances. Presumably, individuals who donot become addicted despite repeated drug exposures arenot making those same adaptations. This suggests the hy-pothesis that individuals differ quantitatively or qualitativelyin terms of the adaptive responses elicited by comparabledrug exposures. Individuals could differ in the magnitudeof the disturbance required to initiate adaptive responses,or vulnerable individuals may react to a drug-induced per-turbation with rigorous adaptive responses while individ-uals with sluggish or weak adaptive responses would beless susceptible to becoming addicted. Investigating thishypothesis requires identifying how individuals differ inthe adaptive responses that are elicited during the initialadministration of a drug.Identifying the response(s) an individual makes during

an initial drug administration is not obvious. When a de-pendent measure is assessed before and after a drug ad-ministration, the resulting change in the measure is casuallyreferred to as either the drug effect or the individualsresponse to the drug. Yet, neither description is accurate.What is typically being measured represents an integral ofmany ongoing processes that include both the direct phar-macological effect of the drug as well as the adaptive re-sponses that are elicited by the drug-induced regulatorydisturbance (Dworkin 1993; Ramsay and Woods 1997;Kaiyala and Ramsay 2005). The challenge is to distinguishthe individuals response from the direct effect of the drugand from the effects of other cooccurring processes (e.g.,handling, circadian rhythms) that influence that same mea-sure (Brick et al. 1984; Peris and Cunningham 1986). Pro-posed methods for evaluating the response an individualmakes during an initial drug exposure include assessinginitial drug sensitivity, acute tolerance, and precipitatedwithdrawal (Ramsay and Woods 1997). Human and animaldata suggest that these processes have predictive value forunderstanding how an individual will react to chronic drugexposure. Humans who appear initially insensitive to al-cohol are at greater risk of becoming addicted than aremore drug-sensitive individuals (Schuckit and Smith 1996;Heath et al. 1999). Schuckit (1994) has described initialinsensitivity to alcohol as ... a potent predictor of futurealcohol abuse or dependence (p. 341). McBride and Li(1998) reported that initial sensitivity and acute (within-session) tolerance are by far the most generalizable and ro-bust responses to ethanol found in association with ethanolpreference in rodents (p. 346). Kest and colleagues (2002)quantified naloxone-precipitated withdrawal during an ini-

tial administration of morphine in 11 inbred mouse strainsand found that .... the differential genetic liability to mor-phine physical dependence begins with, and is predictedby, the first morphine exposure (p. 463).The goal of the present study was to investigate the

relationship between individual differences in acute toler-ance and sensitivity during an initial drug administrationand the development of chronic tolerance. Core body tem-perature was selected as the dependent variable because itcan be measured accurately, continuously, and noninva-sively and because it reflects the operation of a well-stud-ied, centrally regulated control system. This is importantbecause regulatory principles are at the foundation of nu-merous influential theories relevant to drug addiction(e.g., Solomon 1980; Poulos and Cappell 1991; Koob andLeMoal 1997; Siegel et al. 2000). Our drug choice wasinfluenced by the ease, speed, and accuracy with whichsteady-state drug concentrations could be achieved andmaintained. The pharmacologically active gas, nitrous oxide(N2O), is well suited for this purpose. N2O reaches steady-state concentrations quickly (Eger 1985) and its concen-tration can be measured accurately and continuously usinginfrared spectroscopy. N2O causes a pronounced hypo-thermia at typical ambient temperatures (Quock et al. 1987;Pertwee et al. 1990). Outbred LongEvans rats exhibit widebetween-subject variation in initial sensitivity and acutetolerance to N2O-induced hypothermia (Ramsay et al. 1999)and these differences are reliable characteristics of an in-dividual (Kaiyala et al. 2001). Chronic tolerance also de-velops to N2O hypothermia (Ramsay et al. 1999).This research addresses a long-standing question con-

cerning the relationship between acute and chronic toler-ance (Kalant et al. 1971; Tabakoff and Rothstein 1983; Wuet al. 2001). Based on the premise that adaptive responsesplay a role in tolerance development, two primary hypoth-eses were evaluated. The first was that sensitive indi-viduals who have significant acute tolerance developmentare likely to develop chronic tolerance more quickly and toa greater degree than sensitive individuals who developlittle acute tolerance. The second was that initially insen-sitive individuals will quickly become fully tolerant to asmall initial hypothermic effect and then remain insensitive/tolerant over repeated drug administrations. This hypoth-esis is consistent with contemporary explanations for ini-tial insensitivity. If individuals are insensitive because thedrug has little or no pharmacological effect on their tis-sues, it seems unlikely that repeated drug exposure wouldcause a drug effect to develop. Alternatively, if some indi-viduals are insensitive because of an effective or powerfuladaptive response that effectively counters the drugs hy-pothermic effect within an initial exposure, effective reg-ulation of core temperature should continue over repeateddrug exposures.

Methods

Subjects Male LongEvans rats (ten squads of 40 ratseach, Simonsen Laboratory, Gilroy, CA) weighing 200

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250 g were housed in groups of two to three per tub withfree access to pelleted chow and water. The housing roomhad a 12 h:12 h light/dark cycle (07001900 h) and anambient temperature of 221C. The animal proceduresused in this research were approved by the University ofWashingtons IACUC and were conducted in accordancewith the Guidelines for the Care and Use of Mammals inNeuroscience and Behavioral Research (National Re-search Council 2003).

Surgery for core temperature measures At least 7 daysafter arriving in the lab, each rat was anesthetized (0.5 ml/kg of a solution containing 90.9mg/ml ketamine and 1.8 mg/ml xylazine, i.p.) and a telemetric temperature sensor (E-mitters from Mini-Mitter Co., Bend, OR) was implantedinto the peritoneal cavity. Rats recovered for 7 days be-fore the experiment.

N2O delivery/temperature data acquisition system Acustom-built, automated system controlled gas deliveryand data acquisition. Medical grade oxygen (O2), nitrogen(N2), and N2O were blended to produce an outflow of 30%O2, 10% N2, and 60% N2O (referred to as 60% N2O). N2and O2 were blended to produce an outflow consisting of30% O2 and 70% N2 (referred to as placebo gas). Twolines affixed to outlets in each chambers lid provided alow-resistance path by which exhaust gas flowed to a fumehood. Effluent gas sampling [N2O, O2, and CO2 by aninfrared gas analyzer (Normocapoxy, Datex InstrumentsCorp., Helsinki, Finland)] and data acquisition were con-trolled by a program written in LabView 5.0 (NationalInstruments, Austin, TX) installed on a Macintosh IIcicomputer. This system is described fully elsewhere (Kaiyalaet al. 2001).Each exposure chamber (Columbus Instruments, Colum-

bus, OH) was made of clear Lexan and had a removable lidsecured with four latches and sealed with a closed-cell sil-icone foam rubber gasket. The floor of the chamber wascovered with a layer of wood shavings. The chambers in-ternal dimensions were 181831 cm, yielding a volume of10 l. With a gas inflow (and outflow) rate of 3.0 l/min, thetheoretical half-time by which the 10-l chamber achievesthe target gas composition is 2.3 min (Lewis et al. 1987).The chambers gas environment is calculated to reach 97%of the administered gas concentration in 11.6 min. Infraredgas analyses confirmed this figure and indicated that 60%N2O steady-state gas concentrations were achieved in thisstudy. Each test chamber was placed on a platform thatenergized the implanted temperature sensor and receivedits signal. Temperature data were acquired using VitalViewsoftware (Mini-Mitter Co.). During all sessions, a blackvinyl drape visually isolated each chamber. All gas admin-istrations occurred in the light part of the cycle and com-menced at least 2 h after lights on and ended at least 2 h priorto lights off.

Experimental design and procedures During the initialscreening session, each rat received a 2-h administration ofplacebo gas followed immediately by a 3-h administration

of 60% N2O. A squad of 40 rats was screened over a periodof 11 days after which ten rats (25%) were retained thatexhibited: (1) minimal hypothermia while receiving N2O(designated as initially insensitive, II); (2) a large hypo-thermic effect that persisted throughout the N2O adminis-tration (designated sensitive with no recovery, NR); or (3) alarge hypothermic effect followed by a robust return oftemperature toward baseline (designated as sensitive withacute tolerance, AT). These categorizations were based onZ-scores of the regression residuals (Kaiyala et al. 2001)for: (1) the size of the drug effect (degree of sensitivity)adjusted for baseline temperature, and (2) the degree ofrecovery from the drug effect (acute tolerance) adjusted forbaseline temperature and drug effect.Each of the ten retained rats was randomly assigned to

receive either placebo gas or 60% N2O for six additionalexposures on an every-other-day schedule beginning 4 or5 days after screening. Rats assigned to the 60% N2Ogroup received 2 h of placebo gas followed by 1.5 h of60% N2O at each gas exposure session while rats assignedto the placebo group received 3.5 h of placebo gas.

Individual difference group assignment Categorization ofrats into the three individual difference groups was doneusing methods described previously (Kaiyala et al. 2001).Temperature values were recorded at 30-s intervals over the5-h screening session. These were smoothed using naturalsmoothing splines with 10 df (Hastie 1992) to minimizethe influence of occasional aberrant temperature values.The smoothed temperature values were used to calculatethe following three parameters (see Fig. 1 for illustration):(1) baseline core temperature, equal to the average of thesmoothed temperature values for the 3-min period prior todrug administration; (2) drug effect, equal to the differencebetween baseline temperature and the minimum smoothedtemperature value after drug onset; and (3) acute tolerance

Fig. 1 Core temperature data (dashed line) from a single rat aresmoothed (solid line) and calculations of baseline temperature,minimum temperature after drug onset, and the final temperature aremade using the smoothed data. The drug effect equals 1.07C (i.e.,37.24 minus 36.17), the drug effect regression residual equals0.01637, and the drug effect Z-score equals 0.0308. Acute toleranceequals 0.73C (i.e., 36.90 minus 36.17), the acute tolerance re-gression residual equals 0.01099, and the acute tolerance Z-scoreequals 0.0399. This rat is depicted in the scatterplot of Fig. 2a asthe most average rat

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(i.e., intrasessional recovery), equal to the difference be-tween the mean of the smoothed values over the final 15 minof drug administration minus the minimum smoothed tem-perature. After a squad of rats completed screening, regres-sion residuals were calculated for each rats drug effect andacute tolerance scores (Nagoshi et al. 1986; Kaiyala et al.2001). The drug effect regression residual is an adjusted drugeffect score that is not confounded by baseline core tem-perature; this adjustment is important because on aver-age, rats with higher baseline temperatures can exhibitsignificantly greater N2O-induced hypothermia (Kaiyalaet al. 2001). The drug effect regression residual was calcu-lated by subtracting from the drug effect score the regression-predicted mean drug effect score given the rats observedbaseline core temperature (T):

Drug Effect RegressionResidual

Drug Effect Score 1 T

Based on the 387 rats that completed the screeningsession, the values of the beta coefficients for the drug effectregression residual calculation were: (intercept)=1.698and 1 (coefficient for baseline temperature)=0.07391. Ap-plying this formula to the data in Fig. 1 yields a drug effectregression residual of 0.01637=1.071(1.698+0.0739137.243).The acute tolerance regression residual is an adjusted

acute tolerance score that is not confounded by baselinecore temperature or the drug effect score; this adjustment isimportant because mean acute tolerance magnitude variesdirectly with the drug effect which is influenced by baselinetemperature. The acute tolerance regression residual wascalculated by subtracting from the acute tolerance score theregression-predicted mean acute tolerance score given therats observed baseline core temperature (T) and drug effect:

Acute Tolerance Regression Residual Acute Tolerance Score 1 T 2 Drug Effect Score

Based on the 387 rats that completed the screening ses-sion, the values of the beta coefficients for the acute tol-erance regression residual calculation were: (intercept)=1.790, 1 (coefficient for baseline temperature)=0.03887and 2 (coefficient for drug effect)=0.03750. Applyingthis formula to the data provided in Fig. 1 yields an acutetolerance regression residual of 0.01099=0.733[1.79+(0.03887)37.243+0.37501.071].To operationally define which rats were exemplars of

high/low initial sensitivity and which rats exemplifiedinitial sensitivity with high/low acute tolerance develop-ment, Z-scores were used to identify where each rat waslocated within the distribution of drug effect regressionresiduals (i.e., initial sensitivity) and the distribution ofacute tolerance regression residuals (i.e., acute tolerancedevelopment). Selection criteria for group assignment werebased on critical values for independent normal distribu-

tions for drug effect and acute tolerance Z-scores and weredesigned to assign 25% of the screened population into oneof the three groups. Z-scores were calculated by dividingeach rats drug effect and acute tolerance regression re-siduals by the corresponding standard deviation for thoseregression residuals. Based on the 387 rats that completedthe screening session, the standard deviation of the drugeffect regression residuals was 0.5316 and the standarddeviation of the acute tolerance regression residuals was0.2755. The individual rats data provided in Fig. 1 can beconverted to Z-scores as follows: the drug effect Z-scoreequals 0.0308 (i.e., 0.01637/0.5316) and the acute toler-ance Z-score equals 0.0399 (i.e., 0.01099/0.2755). Be-cause both Z-score values are close to zero, this rat was quiteaverage in both the magnitude of the hypothermic drugeffect and the amount of acute tolerance that developed.Thus, this rat would not be a good exemplar of high or lowinitial sensitivity, nor of high or low acute tolerance de-velopment (and it was not considered further).

Data analysis Temperatures for each of the six test ses-sions were summarized by the baseline temperature (meanof temperature values over the 15-min interval prior todrug administration) and the change from baseline to themean temperature over three intervals: (1) 030 min afterdrug onset, (2) 3060 min after drug onset, and (3) 6090min after drug onset. Baseline temperature values andchanges from baseline temperature were analyzed by fit-ting linear models using Generalized Estimating Equations(GEEs; Liang and Zeger 1986) to account for the cor-relation between repeated measures on the same animal.The individual difference group (II, AT, or NR) and thedrug condition (N2O or placebo) were factors and session(27) was used as a continuous independent variable. Alltwo-way interactions and the three-way interaction werealso included in the models. Statistical significance was de-termined using empiricalWald tests (Liang and Zeger 1986).One GEE analysis was performed for baseline tempera-tures and three separate GEE analyses were performed forchanges from baseline over each of the three time intervals(030, 3060, and 6090 min). The analysis of temper-ature changes included the baseline temperature as a pre-caution to protect against spurious associations due todifferences between groups in baseline temperature.

Results

Of the 400 rats (ten squads) initially screened, 13 had un-usable data resulting from E-mitter failure. Of the 387remaining rats, 96 were categorized into the three indi-vidual difference groupings as follows: II=34, AT=37,NR=25. These 96 rats had extreme values for the combi-nation of drug effect and acute tolerance Z-scores (Fig. 2a).The mean temperature curves during the initial screeningsession with 60% N2O for each of the three groups dem-onstrate that the groups fit the intended profiles (Fig. 2b).The average Z-scores for each of the selected groups of ratswere: 1.48 (drug effect) and 0.18 (acute tolerance) for the

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II group, 0.96 (drug effect) and 1.36 (acute tolerance) forthe AT group, and 1.13 (drug effect) and 1.70 (acutetolerance) for the NR group. These mean Z-scores reflecthighly selected subgroups of animals; the probability ofrandomly selecting three groups with these characteristicsis vanishingly small. The mean (SD) baseline core temper-ature for each individual difference group was: 37.23C(0.45) for the II group, 37.11C (0.48) for the AT group, and37.27C (0.31) for the NR group. After the onset of N2O,the mean (SD) maximal hypothermia (i.e., lowest coretemperature) for each individual difference group was:37.00C (0.48) for the II group, 35.58C (0.44) for the ATgroup, and 35.61C (0.60) for the NR group. Since rats ineach individual difference group were assigned randomlyto receive placebo gas or 60% N2O during the subsequentsix test sessions, there was a similar distribution of the drugeffect and acute tolerance regression residual Z-scores forthe placebo and N2O conditions, within each individualdifference grouping (Fig. 2a).Figure 3 depicts how core temperature changed over six

additional N2O exposures as a function of an individualsinitial (in)sensitivity and acute tolerance development toN2O. GEE analysis provided no evidence for differencesin baseline temperature by individual difference group (p=0.68), by drug condition (p=0.39), or by session (p= 0.44)(see Table 1). The two-way interactions (GroupSession,GroupDrug, DrugSession) and the three-way interaction(GroupDrugSession) were also nonsignificant (p=0.32,0.17, 0.55, and 0.45, respectively). Results from GEE anal-ysis of temperature change from baseline over repeatedN2O or placebo exposures demonstrated distinct patternsfor the three individual difference groups. Results are pre-sented separately below for each of the three 30-min post-baseline time intervals.

For the 0- to 30-min time interval (Fig. 4), there weresignificant differences among individual difference groupsin the effect of N2O at Session 2 (p

the effect in the 0- to 30-min interval, namely, a hyper-thermia of 0.18C (compare with 0.22C for 030 min),and there was no evidence for a change in core tempera-ture across sessions (p=0.44). For the AT group, there wasa significant (p

with 0.22 and 0.18C for the other two time intervals), andthere was no evidence for a change in core temperatureacross sessions (p=0.99). For the AT group, there wasa significant (p

tial N2O administration, the AT group exhibited significanthyperthermia during the initial 30 min of N2O inhalationfor each of the final three sessions (Fig. 4, 030 min). Thedirect observation of hyperthermia argues that a warmingadaptive response is recruited during the development ofchronic tolerance. This adaptive response occurs shortlyafter N2O onset in anticipation of the upcoming drug ef-fect. Furthermore, the hyperthermia is of sufficient magni-tude to counter N2Os hypothermic drug effect and bringscore temperature to control levels during the final hourof the last three sessions. Presumably, the NR rats alsoactivate a warming adaptive response during chronic tol-erance development. However, the less robust/effectiveadaptive responses made by the NR group are not suffi-cient to create a statistically significant hyperthermia andare thus insufficient to fully counter the hypothermic ef-fects of N2O. Based on adaptation models of addiction,individuals with a weaker adaptive response may be ex-pected to be less vulnerable to developing addiction.The second hypothesis tested was whether rats that are

initially insensitive to N2O hypothermia (II) would devel-op altered core temperature during repeated N2O admin-istrations. We expected the II group to rejoin its controlgroup rapidly and to remain that way over the remainderof the exposure sessions. However, by the second N2Oadministration, the II group surpassed the temperature ofthe placebo control group (i.e., moved in the hyperthermicdirection) and this prolonged hyperthermic effect persistedacross the remainder of the sessions (Figs. 3, 4). Based onthese differences between the N2O group and placebocontrol group, it is inaccurate to view N2O as being rela-tively inert (i.e., having little impact on temperature regu-latory mechanisms) in rats that appear initially insensitiveto N2Os hypothermic effects.A provocative implication of these findings has to do

with the nature of regulatory control systems in initiallyinsensitive individuals. During the initial N2O adminis-tration, II rats may rapidly activate adaptive regulatorymechanisms that minimize the hypothermic drug perturba-tion such that core temperature is defended (i.e., little drugeffect is observed). However, during subsequent drug ad-ministrations, these warming responses are so robust thatthey cause core temperature to overshoot control valuesand the rat becomes hyperthermic. Indeed, the II rat ex-periences a withdrawal-like effect in the presence of thedrug. This type of dysregulation could contribute to a rapidescalation of drug taking. As can occur for drug-dependentindividuals who take a drug to ameliorate withdrawal ef-fects (Baker et al. 2004), II rats could attempt to restorenormothermia by countering the withdrawal-like hyperther-mia caused by their own dysregulated warming response byincreasing the hypothermic drug effect through increaseddrug consumption. It may be this adaptive over-responsive-ness of the regulatory system in initially insensitive individ-uals that puts them at increased vulnerability for addiction.Future research will need to assess whether the hyperther-mia observed in II rats during repeated N2O administrationsreflects a perturbation away from the defended core tem-perature or, alternatively, represents a drug-induced allo-

static (Koob and LeMoal 2001) increase in the defendedcore temperature. It is important to distinguish betweendrug-induced changes in body temperature that result frombeing forced away from the set point versus those that resultfrom a change in set point temperature because these dif-ferent scenarios activate quite different regulatory responses(Gordon 2001).Direct observation of hyperthermia in the II group during

subsequent N2O exposures (Sessions 27) suggests that anopponent regulatory response is present during those ses-sions. The II rats may also make an adaptive opponent re-sponse within minutes of the initial N2O exposure that issufficiently robust to prevent significant hypothermia fromdeveloping and thereby cause the appearance of initial in-sensitivity. However, without direct evidence of hyperther-mia during the initial exposure, this hypothesis dependsupon an unobserved underlying process. To investigatepossible underlying processes, our lab has begun to mea-sure metabolic heat production and heat loss, the two prox-imal determinants of core temperature. We simultaneouslyquantify heat production using indirect calorimetry, heatloss using direct calorimetry, and core temperature viatelemetry. On average, rats become hypothermic to asteady-state administration of 60% N2O because of arapid reduction in heat production and an increase in heatloss (Kaiyala and Ramsay 2005). Unpublished individualdifference data from our lab indicate that it is possible fora rat to appear initially insensitive to N2Os hypothermiceffects despite exhibiting an N2O-induced increase in heatloss that is comparable to what is observed in rats thatexhibit a significant hypothermia to N2O. In this case, therat appears initially insensitive at the level of core tem-perature because the typical increase in heat loss is largelyoffset by an increase in heat production. This observationsupports the hypothesis that although a drug can exert itspharmacological effect in an initially insensitive individ-ual, little or no change is evident in the dependent mea-sure because the individuals adaptive response effectivelycounters the drug-induced regulatory disturbance (Dworkin1993; Ramsay and Woods 1997). If effective opponentadaptive responses are occurring in the II group duringtheir initial N2O exposure, it is unclear why these re-sponses become excessive (i.e., dysregulated) and resultin a persistent hyperthermia with repeated exposure. Re-search that identifies the specific unconditioned responsesthat are triggered by a drugs initial effect is likely toprovide insight into the nature of the conditioned re-sponses that develop with repeated drug exposures andmodify the measured variable (Dworkin 1993; Ramsayand Woods 1997; Siegel et al. 2000). The developmentof hyperthermia in the II group is consistent with thepossibility that increased heat production is the uncon-ditioned response and that the ... conditioned reflexresembles the unconditioned reflex, and as it develops, itaugments the effect of the unconditioned reflex (Dworkin1993, p. 38).The findings of this study prompt a discussion about

the interpretation and measurement of chronic tolerance.Chronic tolerance is a reduction in the magnitude of drug-

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induced change in a dependent measure that develops overrepeated drug exposures. This can be seen as a shift to theright in the doseeffect curve indicating that a larger drugdose is required to elicit the same magnitude of effect in atolerant individual as is observed in a drug-nave controlsubject (L et al. 1992). Chronic tolerance can also be mea-sured as the degree of reduction in a drug-induced changein a dependent measure that develops when a constantdrug dose is administered repeatedly. The present studyused the latter method by repeatedly administering a 60%N2O concentration to evaluate chronic tolerance develop-ment. In the case of chronic tolerance development, thelargest drug-induced deviation from baseline (or a controlgroup defined baseline) typically occurs during an initialdrug administration and then the magnitude of this dis-crepancy diminishes over repeated administrations as tol-erance develops. Conceptually, there would be no tolerancedevelopment if the initial degree of drug-induced devia-tion did not change over repeated drug administrationswhile full/complete tolerance development would occurif the amount of drug-induced change in the dependentvariable diminishes to the extent that it is indistinguishablefrom baseline or control values.Common methods to quantify chronic tolerance devel-

opment include measuring the change over sessions ineither the maximum deviation from baseline (i.e., sensi-tivity) or the area under the curve (i.e., the total or inte-grated effect). The different patterns of chronic tolerancedata collected for the three individual difference groupsillustrate shortcomings of this analytical approach (Fig. 3).During the first 30 min of the final three drug adminis-tration sessions, the NR group is considered the most tol-erant of the three groups because its core temperature ismost similar to that of its control group (Fig. 4). Yet, theinability of the NR group to mount a more powerful hy-perthermic response during this initial 30-min period mayexplain why these rats are the least tolerant during the finalhour of the drug administration (Fig. 4). During the finalthree drug administration sessions, the AT group is the mostchronically tolerant of the groups when considering thefinal hour of the drug exposure (Fig. 4). The AT groupshyperthermic deviation away from control values duringthe first 30 min of the final drug exposure sessions maybe responsible for the full chronic tolerance that resultsduring the following hour. How then should the II groupbe described in terms of tolerance development consider-ing that they began with a high degree of initial insensi-tivity or innate tolerance (Kalant et al. 1971)? Relativeto their initial exposure to N2O, the II group moved fur-ther away from their control values during subsequent ex-posures (i.e., Sessions 27) that could be described asdeveloping more than 100% tolerance (Fig. 4). Should theII group be described as being hypertolerant or overlytolerant? The point is that if there are different patterns ofchronic tolerance development (see Fig. 3), then simplesummary measures such as area under the curve or maxi-mum drug effect serve to mask these patterns. Further, theexistence of these distinct patterns of change over re-

peated drug administrations illustrate the limitations ofour current use of the term tolerance.

Perspectives There is a wealth of human and animal liter-ature, primarily from research on ethanol, that investigateswhether characteristics measured during an initial drugadministration predict chronic tolerance or the magnitudeof subsequent drug consumption. For example, early workindicated that young men with a family history of alco-holism report being less sensitive to ethanols effects thando control subjects (Schuckit 1980). Schuckit and col-leagues (2001) estimate that 40% of children of alco-holics are relatively insensitive to ethanol while only 10%of children of nonalcoholics have this characteristic. Ani-mal research has investigated whether these initial intra-sessional factors are associated with subsequent chronictolerance development or drug consumption by using ro-dents from outbred and inbred strains, as well as knock-out mice and selectively bred lines for drug consumption,drug sensitivity, and acute tolerance [e.g., outbred strains(Tabakoff and Culp 1984; San-Marina et al. 1989; Khannaet al. 1990b, 1991); inbred strains (Crabbe et al. 1982,1994; Tabakoff and Culp 1984; Khanna et al. 1990a;Gehle and Erwin 2000); knock-out mice (Naassila et al.2002); lines selectively bred for consumption (Tampierand Mardones 1999; Tampier et al. 2000; Bell et al.2001); lines selectively bred for sensitivity (Khanna et al.1985; Crabbe et al. 1989; Limm and Crabbe 1992; Crabbe1994; Browman et al. 2000; Deitrich et al. 2000; Draskiet al. 2001; Palmer et al. 2002); lines selectively bred foracute tolerance (Deitrich et al. 2000; Rustay et al. 2001;Wu et al. 2001)]. While the scientific literature is in generalagreement that initial sensitivity and acute tolerance arerelevant to the subsequent development of chronic toler-ance and drug consumption, a coherent description of thisrelationship has not emerged. Newlin and Thomson (1990)describe the human literature on whether offspring of al-coholics are more or less sensitive to alcohol as beingremarkably inconsistent (p. 385). Khanna and colleagues(1990b) describe the animal literature on the relationshipbetween sensitivity and tolerance to alcohol consumptionas being inconclusive, because of contradictory results(p. 429). More recently, Ponomarev and Crabbe (2002)state that It is not clear how initial sensitivity and acutetolerance individually contribute to alcohol abuse (p. 257)and they suggest a novel method to improve animal re-search on the topic. Much of this literature discusses thefactors that contribute to this lack of clarity. Describingsome of these factors places our current study in propercontext.Too few intermittent measurements of a dependent var-

iable can make it difficult to assess initial sensitivity (i.e.,observe the maximal drug effect) and to measure acute tol-erance (Khanna et al. 1985; San-Marina et al. 1989). Like-wise, too few measurements of the drug concentration alsocomplicate this assessment especially when drug concen-trations are rapidly changing (L et al. 1992; Deitrich et al.2000; Gehle and Erwin 2000; Rustay et al. 2001). Fur-

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thermore, venous blood measurements of the drug con-centration often do not reflect the drug concentration in thebrain (Wu et al. 2001; L et al. 1992). To circumvent thesemethodological problems, a compelling rationale has beenmade for measuring acute tolerance at steady-state drug con-centrations for ethanol (Ramchandani et al. 1999; Froehlichet al. 2001) and for other drugs as well (Kissin et al. 1996;Kaiyala et al. 2001). Measuring a dependent variable con-tinuously at steady-state drug concentrations facilitates themeasurement of initial sensitivity and acute tolerance.Many dependent variables have been used to measure

drug effects (L et al. 1992). Estimates of drug sensitivityand tolerance often do not generalize across dependentmeasures but instead are task-specific (San-Marina et al.1989; Deitrich et al. 2000). Interpreting the relationship be-tween initial sensitivity, acute tolerance, and chronic toler-ance is facilitated when they are investigated in the samestudy with a consistent dependent measure. Some depen-dent variables are affected by the measurement procedureitself and associated handling. A good example is coretemperature measurement in rodents where the act of tak-ing a rectal temperature changes core temperature and canalter the apparent magnitude of the drug effect (Peris andCunningham 1986). Dependent measures assessing drugeffects can also be influenced by intoxicated practice (Wengeret al. 1981; L and Kalant 1992; L et al. 1992; Zack andVogel-Sprott 1995). These are but examples of the factorsthat make it difficult to draw general conclusions from stud-ies that use different methods and dependent measures.There is often both a conceptual and an analytical con-

founding of initial sensitivity and acute and chronic tolerance.Numerous investigators have recognized that measures ofinitial sensitivity often represent a mixture of sensitivityand acute tolerance development (San-Marina et al. 1989;Khanna et al. 1990a, b, 1991; Ramsay and Woods 1997;Deitrich et al. 2000; Gehle and Erwin 2000; Ponomarev andCrabbe 2002). Research investigating how initial drugsensitivity, acute tolerance, and chronic tolerance are re-lated is susceptible to finding spurious associations be-cause a baseline value can influence the size of the drugeffect (sensitivity) that in turn can influence the amount ofacute and chronic tolerance that can develop. For example,the absence of a large initial drug effect (i.e., as seen in aninitially insensitive individual) prevents a large amount ofacute or chronic tolerance from developing because there islittle deviation to recover from when a drug effect is small(Khanna et al. 1985; San-Marina et al. 1989). The recom-mended strategy for testing these relationships is to useresidual scores from regression analyses to quantify indi-vidual differences in initial sensitivity and tolerance ratherthan difference or ratio scores (Nagoshi et al. 1986; Grantet al. 1989). Unfortunately, the improper use of differencescores to relate initial sensitivity and tolerance can com-plicate interpretation of the literature (e.g., see Fig.1 ofSan-Marina et al. 1989).The experimental paradigm selected for our current

study has numerous strengths. N2O is a hypothermic drugthat equilibrates quickly throughout the body and is wellsuited for steady-state administrations (Eger 1985). N2O is

inhaled and thus there are no surgical procedures neededto enable drug administration. This made it feasible to in-vestigate individual differences using a large number ofsubjects because of the ease with which prolonged steady-state drug concentrations could be achieved over multiplesessions. Pharmacokinetic explanations for changes in thedependent variable during a steady-state N2O administra-tion are eliminated because there are no significant meta-bolic pathways for N2O (Trudell 1985). Core temperaturedata are collected continuously and noninvasively at aknown steady-state N2O concentration. This facilitates themeasurement of initial sensitivity and acute and chronictolerance. Using core temperature as the primary depen-dent variable also builds on an extensive body of drugtolerance research. The experimental design incorporatesmeasures of initial sensitivity, acute tolerance, and chronictolerance in the same study. The data analytical proceduresemploy regression residual scores to avoid spurious find-ings that could result from using simple difference scores.Outbred rats were used to study individual differences sothat the predictive utility of any associations could be rel-evant to a heterogeneous population (San-Marina et al.1989). The ability to screen large numbers of rats effi-ciently permitted the use of a selective screening process tocreate distinct individual difference groups that enhancedour ability to visualize the effect that these differences hadon chronic tolerance development. Thus, this experimen-tal paradigm was able to address and circumvent many ofthe problematic factors common to this type of research.Of course, the present study also has its limitations. It is

not known whether the relationships found in the presentstudy apply to other drugs, species, or dependent variables.It is possible to evaluate whether these findings generalizeto ethanol-induced hypothermia in rats by using the steady-state ethanol infusion method described by Froehlich andcolleagues (2001). It is also important to determine whetherthe relationships observed in this study reflect a processthat is common to other regulated physiological systems orif the findings are specific to the temperature regulatorysystem. If unusually adaptive control systems exist in someindividuals for psychophysiologically regulated variablesthat motivate drug taking (e.g., hedonic or affective state),then being initially insensitive in regard to such variablescould be the result of a relatively prompt and excessiveactivation of adaptive responses causing withdrawal-likesymptoms that could motivate an escalation of drug use.Our observation of a withdrawal-like shift at the level ofcore temperature in the II rats (Fig. 3) suggests a novelhypothesis to explain the documented association betweeninitial drug (alcohol) insensitivity and addictive vulnera-bility. However, extending our present research paradigmto investigate the effect of individual differences in sen-sitivity and tolerance on drug consumption is hampered bythe minimal work on N2O self-administration methods.N2O is a drug of abuse (Layzer 1985) and although self-administration methods exist for monkeys (Grubman andWoods 1982; Nemeth and Woods 1982; Wood et al. 1977),there is only a single published study investigating N2Oself-administration methods in the rat (Ramsay et al. 2003).

57

Finally, chronic tolerance development was evaluated overseven N2O exposure sessions. It is possible that the ob-served individual differences in chronic tolerance devel-opment could change with additional exposure sessions.

Conclusion The present data provide new insight into thephenomenon of chronic drug tolerance. As chronic tolerancedevelops over repeated drug administrations, the prevailingbelief has been that the perturbed variable is returning to-ward a baseline or control value. The among-subject var-iability in the speed and magnitude of chronic tolerancedevelopment typically has been viewed as error variabilitythat obscures a single common underlying trajectory. Inthe present study, stringent criteria were used to select in-dividual outbred rats for further study based on reliable in-dividual difference characteristics in sensitivity and acutetolerance (i.e., NR, AT, and II) that were assessed during aninitial drug exposure. Importantly, these individual differ-ences predict qualitatively different patterns of chronictolerance development. We believe the effect of these in-dividual differences on chronic tolerance has gone unno-ticed because research often emphasizes group means thatcan obscure meaningful individual differences. The causesof these individual differences in chronic tolerance de-velopment may be relevant to understanding individualdifferences in addictive vulnerability and warrant furtherinvestigation.

Acknowledgements This investigation was supported by theNational Institute on Drug Abuse (DA010611 and DA016047). Wealso acknowledge Chris Prall and Chae Watson for their technicalcontributions to this study.

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Individual differences in initial sensitivity and acute tolerance predict patterns of chronic drug tolerance to nitrous-oxide-induced hypothermia in ratsAbstractAbstractAbstractAbstractAbstractAbstractIntroductionMethodsSubjectsSurgery for core temperature measuresN2O delivery/temperature data acquisition systemExperimental design and proceduresIndividual difference group assignmentData analysis

ResultsDiscussionPerspectivesConclusion

References

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