Effects of acute and chronic administration of diazepam ondelay discounting in Lewis and Fischer 344 ratsSally L. Huskinson and Karen G. Anderson

Impulsive choice is often examined using a delay-

discounting procedure, where there is a choice between

two reinforcers of different magnitudes presented at

varying delays. Individual discounting rates can be

influenced by many factors including strain differences and

drug effects. Lewis (LEW) and Fischer 344 (F344) rats have

behavioral and neurochemical differences relevant to delay

discounting and were used to examine effects of acute

and chronic administration of diazepam on impulsive

choice. Consistent with the previous literature,

larger-reinforcer choice decreased as a function of

increasing delays for all rats, and steeper discounting

functions were obtained for LEW relative to F344 rats.

Acute and chronic administration of diazepam resulted

in differential effects between rat strains and sometimes

between subjects within the same rat strain. Overall,

larger-reinforcer choice remained unchanged across

multiple phases of the experiment for LEW rats. For F344

rats, larger-reinforcer choice increased following the acute

administration of smaller doses of diazepam and

decreased following the acute administration of the

largest dose tested. Decreases in larger-reinforcer

choice occurred for F344 rats during chronic and

postchronic administration and persisted throughout

a nondrug return-to-baseline phase. These results

suggest potential directions for future investigation of

environmental, genetic, and neurochemical variables

involved in delay discounting. Behavioural Pharmacology

23:315–330 �c 2012 Wolters Kluwer Health | Lippincott

Williams & Wilkins.

Behavioural Pharmacology 2012, 23:315–330

Keywords: choice, delay discounting, diazepam, Fischer 344, impulsive,Lewis, rat, self-control, temporal discounting

Department of Psychology, West Virginia University, Morgantown, West Virginia,USA

Correspondence to Karen G. Anderson, PhD, Department of Psychology,West Virginia University, PO Box 6040, Morgantown, WV 26506, USAE-mail: karen.anderson@mail.wvu.edu

Received 8 June 2011 Accepted as revised 15 April 2012

IntroductionImpulsive behavior is associated with problem behaviors

and disorders including attention-deficit/hyperactivity dis-

order, gambling, substance abuse, and obesity (Kollins,

2003; Reynolds, 2006; Winstanley et al., 2006; Perry and

Carroll, 2008; Weller et al., 2008; Rasmussen et al., 2010).

A greater understanding of the neurological, genetic, and

behavioral mechanisms that underlie impulsive behavior

may enhance the understanding and treatment of problem

disorders. Impulsive choice in humans and animals is often

assessed in the laboratory using a delay-discounting

procedure (e.g., Mazur, 1987; Bickel et al., 1999).

In delay-discounting procedures, impulsive choice is

examined by providing subjects with a choice between

two reinforcers of different magnitudes presented at

varying delays (Ainslie, 1975; Mazur, 1987). Impulsive

choice is operationally defined as choosing a smaller, more

immediate reinforcer over a larger, more delayed reinfor-

cer and self-controlled choice as the reverse. The

effectiveness of the larger, delayed reinforcer in control-

ling choice generally decreases as a function of increasing

delay, and its subjective value may become less than the

smaller, immediate reinforcer. In other words, choice

becomes more impulsive as the delay to the larger

reinforcer increases. The rate of discounting may be

quantified using various measures including indifference

points (e.g., Anderson and Woolverton, 2005), mean

adjusted delay (e.g. Perry et al., 2005), area under the

curve (AUC; Myerson et al., 2001), and k value (Mazur,

1987). Steeper rates of discounting are indicated by

shorter indifference points and mean adjusted delays,

smaller AUC, and larger k values.

Substance users discount delayed reinforcers more steeply

than nonusers (see Reynolds, 2006 for a review). For

example, alcohol (Petry, 2001), nicotine (Bickel et al.,1999; Mitchell, 1999; Reynolds et al., 2004b), opioid,

(Madden et al., 1997, 1999), cocaine (Coffey et al., 2003),

and methamphetamine abusers (Hoffman et al., 2006;

Monterosso et al., 2007) discount delayed reinforcers more

steeply than nonusers or ex-users. Drug-dependent parti-

cipants also discount hypothetical cigarettes (Bickel et al.,1999), heroin (Madden et al., 1997, 1999), and cocaine

(Coffey et al., 2003) more steeply than hypothetical money.

Laboratory research with nonhuman animals also indi-

cates a relation between drug self-administration and

delay discounting. Rats categorized as high on impulsive

choice subsequently self-administered more freely avail-

able, orally delivered ethanol (Poulos et al., 1995), more

quickly acquired intravenous cocaine self-administration,

and, over the last five sessions of acquisition, self-

administered more cocaine, compared with rats categor-

ized as low impulsive (Perry et al., 2005, 2008a). Rats

categorized as high impulsive also lever pressed more for

Original article 315

0955-8810 �c 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/FBP.0b013e3283564da4

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

nicotine infusions at fixed-ratio (FR) values of 20 and 25

compared with those categorized as low impulsive

(Diergaarde et al., 2008). These results suggest that

higher rates of impulsive choice may precede substance

abuse. Other laboratory research has shown that exposure

to a drug, through experimenter administration or self-

administration, may affect the performance on subse-

quent delay-discounting tasks (Simon et al., 2007; Mendez

et al., 2010). Simon et al. (2007) exposed two groups of

rats to either a high dose of cocaine (30 mg/kg) or saline

for 14 days. After 3 weeks with no dosing, they were

tested on a delay-discounting task. The group of rats

exposed to cocaine chose the larger reinforcer less often

than the group exposed to saline. This suggests that

exposure to a commonly abused drug such as cocaine may

somehow increase impulsive choice.

Examination of different rat strains has shed light on the

roles of behavioral and neurochemical factors in impulsive

choice and their relation to substance use. Comparisons

between Lewis (LEW) and Fischer 344 (F344) rats

provide evidence of behavioral differences that may be

relevant to delay discounting and substance abuse. In

general, LEW rats more readily self-administer opioids

(Suzuki et al., 1988b; Garcia-Lecumberri et al., 2010),

cocaine (Kosten et al., 1997; Freeman et al., 2009), and

ethanol (Suzuki et al., 1988a) compared with F344 rats.

LEW rats also make more impulsive choices on some

delay-discounting tasks compared with F344 rats (Ander-

son and Woolverton, 2005; Madden et al., 2008; Anderson

and Diller, 2010; Garcia-Lecumberri et al., 2010; Huskin-

son et al., 2012). The procedures used, however, may not

be equally sensitive to strain differences as Wilhelm and

Mitchell (2009) did not report differences between LEW

and F344 rats using an adjusting-amount procedure.

LEW and F344 rats also have neurochemical differences that

may be relevant to delay discounting. LEW rats have fewer

dopamine (DA) receptors (specifically D2 and D3) and DA

transporters in some brain regions than F344 rats (Flores

et al., 1998), and LEW rats have lower levels of serotonin

(5-HT) and fewer 5-HT1A receptors in some brain regions

compared with F344 rats (Burnet et al., 1992; Selim and

Bradberry, 1996). In general, previous research suggests that

lower levels of DA and 5-HT are related to greater rates of

impulsive choice (Wogar et al., 1993; Mobini et al., 2000;

see Cardinal et al., 2003 for a review). In addition, the

administration of DA and 5-HT agonists generally increases

self-controlled choice, whereas antagonists generally increase

impulsive choice (Poulos et al., 1996; Cardinal et al.,2000; Wade et al., 2000; Winstanley et al., 2003; Perry et al.,2008b; Huskinson et al., 2012). There are mixed results,

however, as sometimes agonists increase impulsive choice

(Evenden and Ryan, 1996, 1999; Cardinal et al., 2000; Perry

et al., 2008b; Slezak and Anderson, 2009).

Compared with DA and 5-HT agonists and antagonists,

there is much less research on the effects on impulsive

choice of the administration of central nervous system

depressants such as benzodiazepines. Benzodiazepines

have facilitative actions on the central nervous system

inhibitory neurotransmitter system g-aminobutyric acid

(GABA), with a specific action at the GABAA receptor site

(e.g., Gielen et al., 2012), in addition to actions on DA and

5-HT systems (Stein et al., 1975; Roth et al., 1988; Finlay

et al., 1995). Benzodiazepines may also differentially

affect LEW and F344 rats. LEW rats developed less

physical dependence and withdrawal compared with

F344 rats following repeated exposure to food mixed

with diazepam (a benzodiazepine agonist; Suzuki et al.,1992). LEW and F344 rats have basal differences in gene

expression in GABA-related neurons that project from the

nucleus accumbens to the ventral pallidum (Sharp et al.,2011), and LEW rats have more benzodiazepine receptors

in the hypothalamus compared with F344 rats (Smith

et al., 1992). It is unclear, however, whether or how

different basal levels of gene expression and benzodiaze-

pine receptor density are related to delay discounting.

Effects of benzodiazepine administration on delay dis-

counting with humans and nonhuman animals are mixed.

Two studies have examined effects of acute administration

of benzodiazepines on delay discounting in humans. The

administration of therapeutically effective doses of diaze-

pam (5 and 10 mg) and larger doses of diazepam (20 mg)

did not affect the rates of discounting in human

participants (Reynolds et al., 2004a; Acheson et al., 2006).

Effects of acute benzodiazepine administration on impul-

sive choice in rats are mixed. Using a T-maze procedure,

bretazenil (a partial benzodiazepine agonist) decreased

larger-reinforcer choice at a 15-s delay, whereas alprazolam

(a full benzodiazepine agonist) exerted no effect at a 15-s

delay and increased larger-reinforcer choice at a 25-s delay

(Bizot et al., 1999). Diazepam and muscimol (a GABA

agonist) decreased larger-reinforcer choice at a 15-s delay

and exerted no effect at a 25-s delay (Bizot et al., 1999).

Others using a T-maze procedure found that diazepam

decreased larger-reinforcer choice at 15- and 30-s delays,

and other benzodiazepines (nitrazepam, chlordiazepoxide,

and clobazam) exerted the same effect at a 15-s delay

(Thiebot et al., 1985).

Using a different delay-discounting procedure, where rats

chose between a lever that delivered a smaller reinforcer

immediately versus a lever that delivered a larger reinforcer

after varying delays, chlordiazepoxide decreased larger-

reinforcer choice depending on the dose and the presence

of a cue signaling the delay (Cardinal et al., 2000), whereas

diazepam increased larger-reinforcer choice (Evenden and

Ryan, 1996) or exerted no effect (Charrier and Thiebot,

1996). It is possible that the various procedures used led to

the mixed findings across studies.

To date, effects of administration of benzodiazepine on

delay discounting with LEW and F344 rats have not been

examined. Using two rat strains that differ in the baseline

316 Behavioural Pharmacology 2012, Vol 23 No 4

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

rates of discounting and their physiological response to

benzodiazepines may provide an insight into the beha-

vioral and biological determinants of impulsive choice.

The present experiment evaluated effects of exposure to

acute, chronic, and postchronic administration of diaze-

pam on delay discounting in LEW and F344 rat strains.

This study was designed to assess whether LEW and

F344 rats differ in larger-reinforcer choices at baseline,

and whether the administration of diazepam differentially

affects larger-reinforcer choice.

MethodsSubjects

Eight experimentally naive male LEW and eight experi-

mentally naive male F344 rats (Harlan Sprague–Dawley

Inc., Indianapolis, Indiana, USA) served as subjects and

were about 3 months old at the start of experimentation.

Consistent with known strain differences, LEW rats

weighed more than F344 rats (e.g., Harlan Sprague–

Dawley Inc.; Christensen et al., 2009) at the start of

experimentation, and this strain difference was observed

throughout the experiment. The mean weights for LEW

rats ranged from 298 g (SEM = 4.0) at the start to 409 g

(SEM = 7.2) at the end, and the mean weights for F344

rats ranged from 283 g (SEM = 3.6) at the start to 400 g

(SEM = 5.0) at the end. The differences obtained were

statistically significant during 7 of the 12 months of

experimentation (P values < 0.05). Rats were housed

individually with free access to water in their home cages.

Temperature and humidity were maintained at constant

levels, and a reverse 12-h light–dark cycle was in place.

All sessions were conducted during the dark phase of the

light–dark cycle at approximately the same time each day.

The subjects were fed approximately 10–12 g of food one

half hour following each experimental session. This

schedule resulted in about 21 h of food restriction before

the start of each session. All procedures were carried out

in accordance with West Virginia University’s Animal Care

and Use Committee.

Apparatus

Experimental sessions were conducted in eight standard

operant-conditioning chambers for rats, each enclosed in a

melamine sound-attenuating cubicle (Med Associates, St

Albans, Vermont, USA). Each chamber contained a working

area of 30.5� 24.5� 21.0 cm, a grid floor, and a 45-mg

pellet dispenser with a pellet receptacle that was centered

between two retractable response levers. The levers were

11.5 cm apart from each other and required a force of at

least 0.25 N for a response to be recorded. The levers were

4.8 cm wide, protruded 1.9 cm into the chamber, and were

elevated 8 cm from the grid floor. Two 28 V stimulus lights,

2.5 cm in diameter, were 7 cm above each lever. Each

chamber contained a 28 V house light on the wall opposite

to the wall containing the operandum. A ventilation fan

circulated air and served to mask extraneous noise.

Equipment was interfaced to a computer, and experimental

sessions and data collection were programmed and

conducted using MedPC-IV software (Med Associates).

Procedure

Initial training

During the initial lever-press training, food was delivered

according to a conjoint variable time (VT) 1 min, FR 1

schedule. During these initial training sessions, both levers

were extended into the chamber, and one 45-mg food

pellet was delivered every minute on average and following

a press on either lever. Sessions lasted until 60 food pellets

were delivered. If at least 40 of the 60 total food pellet

deliveries were not earned according to the FR 1

contingency after four sessions under the conjoint VT

1 min, FR 1 schedule, lever pressing was shaped using

reinforcement of successive approximations. Following the

hand-shaping procedure, lever pressing was reinforced

according to a FR 1 schedule with both levers extended,

and a press on either lever resulted in the delivery of a food

pellet. Once lever pressing was established by either

method (free-operant acquisition or hand shaping), it was

reinforced according to a FR 1 schedule, where only one

lever had a programmed consequence of food delivery. After

five food pellets were earned for presses on a single lever,

an extinction schedule for that lever commenced, and the

FR 1 contingency was alternated to the second lever. This

alternating procedure occurred within each session until

40 food pellets were earned. Daily sessions continued until

the subjects reliably earned food for presses on both levers.

General delay-discounting procedure

After training sessions were complete, delay-discounting

sessions began. Sessions began with a 10-min blackout

period, during which the chamber was completely dark.

Following the blackout period, trials grouped into five

blocks of eight began. Blocks consisted of forced-choice and

free-choice trials that started every 100 s. The first two

trials in each block were forced-choice trials with one

randomly determined lever extended into the chamber, and

the cue light directly above it illuminated. Following a lever

press (FR 1), the lever was retracted into the chamber, the

cue light darkened, and either a single food pellet was

delivered immediately or three food pellets were delivered

after a delay, depending on which lever was pressed. At the

start of the second forced-choice trial, the other lever was

extended into the chamber, the cue light directly above it

was illuminated, and the other outcome was available

dependent on a single lever press (FR 1). The lever (left or

right) associated with the larger-reinforcer was counter-

balanced across subjects and remained constant within and

across sessions throughout the study.

After exposure to both outcomes during forced-choice trials,

the last six trials in each block were free-choice trials. During

these trials, both cue lights were illuminated, both levers

were extended into the chamber, and both alternatives were

available. Following a press on either lever (FR 1), both levers

Diazepam and delay discounting Huskinson and Anderson 317

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

retracted, both cue lights darkened, and one immediate or

three delayed food pellets were delivered, depending on

which lever was pressed. The house light always remained on

during delay periods, flashed each time a food pellet was

delivered, whether immediately or after a delay, and was

turned off after food delivery until the start of the next trial.

If a lever press did not occur within 30 s of trial onset (for

forced-choice and free-choice trials), the lever(s) retracted,

the cue light(s) and house light darkened, no food was

delivered, and the trial was recorded as an omission.

In the first block of eight trials, the delay to the larger

reinforcer was always 0 s. Delay-discounting sessions

began with no delay across all five blocks of trials. When

larger-reinforcer choice was at least 80% in each block,

and for no more than three sessions, the delays were

increased to a series of 0, 1, 2, 4, and 6 s across blocks.

The 6-s delay series remained in effect until the total

number of larger-reinforcer choices during the free-choice

trials in the 0-s block was at or above 80%. The delays

were then increased to 0, 2, 4, 8, and 16 s across blocks.

After the 16-s delay series, the delay series was increased or

decreased as necessary for individual subjects to obtain

discounting functions with no ceiling or floor effects (i.e.,

near exclusive choice for the larger or smaller reinforcer).

Only if ceiling effects were obtained during the 16-s delay

series were the delays increased to 0, 5, 10, 20, and 40 s across

blocks, and if necessary, to 0, 10, 20, 40, and 60 s across

blocks. Sessions ended after 40 total (10 forced-choice and 30

free-choice) trials and were initially conducted 5 days/week.

Six months into the experiment, sessions were conducted

7 days/week. No systematic differences were observed

between those subjects that received five compared with

seven sessions per week during the baseline phase.

Baseline

Once choice was maintained at a single delay series, a

baseline was established. The baseline delay series was in

effect for a minimum of 20 sessions and until responding

was stable. Stability was defined as 80% or greater choice

for the larger reinforcer in the 0-s delay block, less than 20%

variation in the total larger-reinforcer choice during each of

the last five sessions from the grand mean, and no

increasing or decreasing trends between the total number

of larger-reinforcer choices across the last five sessions. To

ensure sensitivity to the variations in reinforcer magnitude,

all delays during a single session were set equal to zero once

per week (0-s probe sessions) (Evenden and Ryan, 1996,

1999; Cardinal et al., 2000; Diller et al., 2008; Huskinson

et al., 2012). These sessions continued until larger-

reinforcer choice was at least 80% across all blocks of trials.

Acute diazepam administration

After establishing a stable delay-discounting baseline,

acute effects of diazepam were assessed. Either vehicle or

diazepam (1.0, 3.0, and 10.0 mg/kg) was administered by

an intraperitoneal injection twice per week, separated by

at least two sessions, as long as two criteria were fulfilled.

First, at least 80% of lever presses occurred on the lever

associated with the larger reinforcer in each block during

sessions when all delays were set equal to zero. Second,

during the control session immediately preceding vehicle

or drug sessions, at least 80% of lever presses occurred on

the lever associated with the larger reinforcer in the first

block when both delays were 0 s. Before the first

diazepam administration, vehicle was administered at

least twice. Initially, half of LEW and half of F344 rats

received smaller doses of diazepam (0.3, 0.56, and

1.0 mg/kg) in descending order, and the other half received

them in ascending order. Because these doses did not result

in any observed behavioral effects, it became necessary to

increase the doses, and hence, all analyses include only 1.0,

3.0, and 10.0 mg/kg of diazepam. Each of the latter doses of

diazepam was determined at least twice for each subject,

and intermediate doses (1.7 and 5.6 mg/kg) were adminis-

tered when warranted. A third or a fourth determination of

a particular dose occurred when there was considerable

variability in larger-reinforcer choice between the first and

the second determinations.

Chronic diazepam administration

Once acute drug effects were determined, a dose of

diazepam that resulted in the largest change in percent

larger-reinforcer choice and maintained responding at 80%

or greater for the larger reinforcer in the first block was

administered daily (chronic dose). If no change in percent

larger-reinforcer choice occurred during acute administra-

tion, the largest dose of diazepam that did not decrease

larger-reinforcer choice below 80% in the first block of trials

was used. To determine effects of chronic exposure to

diazepam, experimental sessions were conducted 7 days/

week, and the chronic dose was administered daily before

the start of each session. Chronic administration occurred

for a minimum of 30 sessions and until choice was stable or

a maximum of 50 sessions, to ensure approximately the

same amount of chronic drug exposure across subjects. In

this phase, stability was defined as no more than 20%

variation in the total number of larger-reinforcer choices

during each of the last five sessions from the grand mean

and no increasing or decreasing trends in the total number

of larger-reinforcer choices across the last five sessions.

During this phase, 0-s probe sessions were not conducted.

Postchronic diazepam administration

Following chronic administration of the (single) chronic

dose of diazepam, the dose–response function was

redetermined to allow for comparisons of larger-reinforcer

choice during acute and postchronic diazepam exposure.

During this phase, daily diazepam administration (se-

lected chronic dose) continued. The chronic dose was

administered daily, except that two to three times per

week, the chronic dose was substituted with a previously

318 Behavioural Pharmacology 2012, Vol 23 No 4

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

administered dose of diazepam (1.0, 3.0, and 10.0 mg/kg)

or vehicle. Each dose and vehicle administration was

determined at least twice for each subject and dose–

response determinations were separated by at least two

sessions with the chronic dose. A third or a fourth

determination of a particular dose occurred if there was

considerable variability in larger-reinforcer choice be-

tween the first and the second determinations.

Baseline replication

After the determination of the second (postchronic) dose–

response function, diazepam administration ceased, and

subjects that completed the chronic phase were exposed to

0-s probe sessions where all delays were set equal to 0 s

across all five blocks of trials. These sessions were

conducted until larger-reinforcer choice was at least 80%

across all blocks for each subject. Once this criterion was

fulfilled, the conditions were the same as those during the

terminal baseline phase. Sessions were conducted 7 days/

week, with the baseline delay series in effect, except once

per week when 0-s probe sessions were conducted until

larger-reinforcer choice was at least 80% across all blocks of

trials. This phase was in effect for a minimum of 20

sessions, except for subjects LEW-2 and LEW-8 that

received 19 and 15 sessions, respectively, and until choice

was stable. The stability criteria were the same as those

used during the initial baseline phase. LEW-2 and LEW-8

did not fulfill the minimum session requirement as a result

of equipment malfunction; however, the choice data from

the last five sessions did fulfill the stability criteria.

Drugs

Diazepam was obtained from Sigma-Aldrich (St Louis,

Missouri, USA) and was dissolved in 0.9% sodium chloride

plus varying concentrations of Tween 80 (2, 3, 5, and 10%).

Diazepam was injected in a volume of 1.0 ml/kg. Vehicle

(0.9% sodium chloride plus varying concentrations of Tween

80) or diazepam (1.0, 3.0 and 10.0 mg/kg) was administered

through intraperitoneal injections immediately before the

experimental sessions. Three drops of ethanol were added to

every 5.0 ml of solution only for the largest dose.

Data analysis

Percent choice for the larger reinforcer was the main

dependent measure used. From this measure, indiffer-

ence points and AUC were calculated for each subject in

each phase. Delay-discounting functions for each phase

were plotted as percent choice for the larger reinforcer

across increasing delays in each block. Using nonlinear

regression, a function was fit to the data, and indifference

points were interpolated on the basis of these functions.

AUC was derived by calculating the area of trapezoids

that were formed by drawing vertical lines from the

normalized x-axis to each obtained percent choice for the

larger reinforcer at each delay. The areas of these

trapezoids were added together and divided by the entire

possible area of the graph (Myerson et al., 2001). It is

important to note that because the x-axis was normalized

when calculating AUC, this measure does not take into

account different delay series used in the present study.

Using this measure, all delay series were transformed into

a scale that ranged from 0 to 1.

To assess differences in strains following baseline, acute,

postchronic, and baseline-replication phases, one-way

analysis of variance was carried out. To assess effects of

drug within strain, repeated-measures analyses of var-

iance were used. Rat strain was a between-subjects

variable and indifference points and AUC for acute,

chronic, and postchronic doses of diazepam were within-

subjects variables. Planned comparisons were carried out

between each dose of diazepam and vehicle and between

each acute dose compared with each postchronic dose.

Independent-samples t-tests were carried out to examine

strain differences in the number of 0-s probe sessions and

number of sessions in each phase.

ResultsBaseline

The number of sessions conducted during the baseline

phase and the number of 0-s probe sessions conducted

during baseline for individual subjects are presented

in Table 1. There was no significant difference in the

mean number of sessions for LEW and F344 rats to reach

stability during the baseline phase. LEW rats did, however,

require more 0-s probe sessions during the baseline phase

compared with F344 rats [t(14) = 3.10, P < 0.01].

Figure 1 shows the mean percent larger-reinforcer choice as a

function of delay to the larger reinforcer (shown as blocks of

trials) for LEW (closed symbols) and F344 (open symbols)

rats during the last five sessions of baseline (circles) and for

all 0-s probe sessions (squares) conducted during baseline.

During 0-s probe sessions, the mean larger-reinforcer choice

remained above 80% across all blocks of trials for both

strains. During sessions when the delay to the larger

reinforcer increased, the choice for that alternative decreased

for all subjects, and this occurred to a greater extent for LEW

compared with F344 rats. The finding that the choice for the

larger reinforcer decreased at shorter delays for LEW

compared with F344 rats is also indicated in Fig. 2, which

shows the mean indifference points (Fig. 2a) and the mean

AUC (Fig. 2b) for each strain during baseline (see Appendix

for data from individual subjects). LEW rats had shorter

mean indifference points (M = 6.6 s, SEM = 0.8 s) and

smaller mean AUCs (M = 0.40, SEM = 0.04) compared

with F344 rats (M = 17.3 s, SEM = 4.4 s; M = 0.61, SEM =

0.04, respectively) [F(1,15) = 5.88, P < 0.05; F(1,15) =

16.34, P < 0.01, respectively].

Acute diazepam administration

Subject F344-8 died before acute drug administration;

acute analyses are therefore based on eight LEW and

Diazepam and delay discounting Huskinson and Anderson 319

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

seven F344 rats. Table 1 shows the number of sessions

conducted during the acute phase and the number of 0-s

probe sessions conducted during this phase for individual

subjects. There were no significant differences between

the mean number of sessions for LEW and F344 rats to

complete the acute drug phase or the number of 0-s

probe sessions conducted during this phase.

Figure 3 shows the mean indifference points (Fig. 3a) and

the mean AUC (Fig. 3b) for LEW (closed symbols) and

F344 (open symbols) rats as a function of acute doses of

Table 1 Sessions conducted with each subject

Subjects Delay series BL 0-s probes Baseline 0-s probes acute Acute Chronic Postchronic 0-s probes Baseline replication

LEW-1 16 15 20 24 82 32 39 1 22LEW-2 40 16 53 14 57 43 20 1 19LEW-3 16 33 35 30 61 32 22 2 22LEW-4 16 4 22 5 30 31 25 1 21LEW-5 16 27 34 29 68 32 39 3 20LEW-6 16 16 29 36 71 36 46 2 29LEW-7 40 15 38 16 57 – – – –LEW-8 16 13 44 16 40 34 40 6 15M (SEM) 17.4 (3.1)* 34.4 (3.9) 21.3 (3.6) 58.3 (5.9) 34.3 (1.6) 33.0 (3.5) 2.3 (0.7) 21.1 (4.2)F344-1 16 9 35 15 54 30 37 1 20F344-2 16 9 29 16 46 33 41 3 23F344-3 40 7 35 21 68 40 22 3 24F344-4 40 5 23 21 63 35 29 2 24F344-5 16 7 27 17 63 36 44 1 20F344-6 16 6 37 6 73 – – – –F344-7 40 10 21 15 51 30 31 2 20F344-8 16 7 27 – – – – – –M (SEM) 7.5 (0.6)* 29.3 (2.1) 15.9 (1.9) 59.7 (3.7) 34.0 (1.6) 34.0 (3.3) 2.0 (0.4) 21.8 (0.8)

For each subject, the delay series, followed by the numbers of 0-s probes conducted during baseline (BL 0-s probes), sessions conducted during baseline, 0-s probesduring acute, sessions conducted during acute, chronic, and postchronic phases, 0-s probe sessions after the completion of drug phases (0-s probes), and baselinereplication sessions. Means and SEM are shown for each strain separately. Single asterisks represent strain differences at P < 0.05.BL, baseline; F344, Fischer 344; LEW, Lewis.

Fig. 1

Delay to larger reinforcer(s)Bloc

k 5

Block 4

Block 3

Block 2

Block 1

Per

cent

cho

ice

for l

arge

r rei

nfor

cer

0

20

40

60

80

100

LEWLEW 0-s probesF344F344 0-s probes

Mean percent larger-reinforcer choice as a function of delay blocks forthe last five baseline sessions (circles) and for 0-s probe sessions(squares) conducted during the terminal (baseline) delay series forLEW (closed symbols) and F344 (open symbols) rats. Error barsrepresent the SEM. The horizontal axis is composed of blocks ratherthan delay values as two LEW and three F344 rats responded in a 0-,5-, 10-, 20-, and 40-s delay series, and six LEW and five F344 ratsresponded in a 0-, 2-, 4-, 8-, and 16-s delay series. During 0-s probesessions, the delay to the larger reinforcer was 0 s across all five blocksof trials. F344, Fischer 344; LEW, Lewis.

Fig. 2

Baseline

∗∗

∗25

(a)

(b)

20

15

Indi

ffere

nce

poin

t(s)

10

5

0

1.0

LEW

F344

0.8

0.6

0.4

0.2

0.0

Are

a un

der t

he c

urve

Mean indifference points (a) and area under the curve (b) from the lastfive sessions of baseline for LEW (filled bars) and F344 (open bars)rats. Error bars represent the SEM. Single and double asterisksrepresent strain differences at P < 0.05 and P < 0.01, respectively.F344, Fischer 344; LEW, Lewis.

320 Behavioural Pharmacology 2012, Vol 23 No 4

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

diazepam (see Appendix for data from individual

subjects). It is important to note that baseline strain

differences in impulsive choice persisted throughout the

acute phase, as the mean indifference points were shorter

and the mean AUCs were smaller for LEW compared with

F344 rats during control sessions (nondrug sessions)

[F(1,14) = 7.88, P < 0.05; F(1,14) = 6.75, P < 0.05, re-

spectively]. LEW rats also had shorter mean indifference

points following vehicle administration relative to F344

rats [F(1,14) = 4.68, P = 0.05] (differences between

strains indicated by asterisks). In addition, the mean

indifference points were shorter and the mean AUCs

were smaller for LEW compared with F344 rats following

the administration of 1.0 mg/kg of diazepam [F(1,14) =

6.71, P < 0.05; F(1,14) = 10.31, P < 00.01, respectively].

The opposite (i.e., greater impulsive choice for F344 rats)

occurred at the largest dose of diazepam. The mean AUC

at this dose decreased to a smaller value for F344 relative

to LEW rats [F(1,14) = 8.54, P < 0.05].

Considering each strain separately, the acute administration

of diazepam resulted in different effects. For LEW rats

(Fig. 3, closed symbols), there was no significant effect of

dose on the mean indifference points or AUCs relative to

vehicle administration. Following the administration of

10.0 mg/kg of diazepam, however, there appeared to be an

increase in the mean indifference points relative to vehicle

administration. The mean indifference points at this dose

were largely affected by two outliers: large increases in

indifference points relative to vehicle were observed for

LEW-2 and LEW-3 at this dose. Further examination of

individual-subject data (see Appendix) for LEW rats

indicated that acute diazepam did affect larger-reinforcer

choice; however, the effect occurred at different doses. For

all subjects (eight of eight), at least one of the three doses

administered increased AUC relative to vehicle. For LEW-

5, LEW-6, and LEW-7, the observed increase was small;

however, statistical analysis comparing AUC obtained

following acute vehicle administration to the largest AUC

obtained for each subject across the three doses adminis-

tered showed a significant increase in AUC [F(1,7) = 6.91,

P < 0.05]. The analysis included AUC at the 1.0 mg/kg

dose for LEW-6 and LEW-7, at 3.0 mg/kg for LEW-1, LEW-

5, and LEW-8, and at 10.0 mg/kg for LEW-2, LEW-3, and

LEW-4 relative to vehicle administration. AUCs obtained

following the administration of the doses included in the

analysis are highlighted in bold in the Appendix.

For F344 rats (Fig. 3, open symbols), diazepam exerted dose-

dependent effects on the mean indifference points and

AUCs [F(4,24) = 5.89, P < 0.01; F(4,24) = 12.25, P < 0.01,

respectively]. Although not statistically significant, compared

with vehicle administration, there was a slight increase in

indifference points and AUC in six of seven and five of seven

F344 rats following the acute administration of 1.0 and

3.0 mg/kg of diazepam, respectively (see Appendix). In-

difference points and AUCs did not increase following at

least one of these doses for only one subject (F344-6). When

this subject’s data were not included in the analyses, the

mean AUCs following 1.0 mg/kg were significantly greater

than mean AUC following vehicle administration [F(1,5) =

7.66, P < 0.05], and the mean indifference points following

3.0 mg/kg were significantly longer than the mean indiffer-

ence points following vehicle administration [F(1,5) = 6.84,

P < 0.05]. There was also a significant decrease in the mean

indifference points and AUC following the administration of

the largest dose of diazepam (10.0 mg/kg) relative to vehicle

administration [F(1,6) = 9.45, P < 0.05; F(1,6) = 15.81,

P < 0.01, respectively] (indicated by single and double

crosses). The observed decrease in larger-reinforcer choice

at the 10.0 mg/kg dose of diazepam relative to vehicle

occurred for six of the seven F344 rats.

Fig. 3

Are

a un

der t

he c

urve

0.0

0.2

0.4

0.6

0.8

∗ ∗∗∗

++

C V 1.0 3.0 10.0

C V 1.0 3.0 10.0

Indi

ffere

nce

poin

t(s)

0

5

10

15

20

25

Diazepam (mg/kg)

∗ ∗

∗

+

LEW acute (n=8)F344 acute (n=7)

(a)

(b)

Mean indifference points (a) and area under the curve (b) as a functionof the doses of diazepam [control (C), vehicle (V), 1.0, 3.0, and10.0 mg/kg] during the acute phase for LEW (closed symbols) andF344 (open symbols) rats. Error bars represent the SEM. Single anddouble asterisks represent the significance levels of P < 0.05 andP < 0.01, respectively, where differences were found between ratstrains at a particular dose. Single and double crosses represent thesignificance levels of P < 0.05 and P < 0.01, respectively, wheredifferences were found between vehicle and a particular dose ofdiazepam. F344, Fischer 344; LEW, Lewis.

Diazepam and delay discounting Huskinson and Anderson 321

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Figure 4 shows effects of acute administration of

diazepam on indifference points (Fig. 4a and c) and

AUCs (Fig. 4b and d) for individual LEW (Fig. 4a and b)

and F344 (Fig. 4c and d) rats, expressed as the proportion

of scores obtained under vehicle administration. Data

points that fall above the horizontal dashed line indicate

an increase in indifference points and AUCs following the

administration of diazepam relative to vehicle adminis-

tration, and data points that fall below the horizontal

dashed line indicate a decrease in indifference points and

AUCs following the administration of diazepam relative

to vehicle administration. These data are similar to those

described above for Fig. 3 and show the between-subject

variability that occurred for LEW and F344 rats following

the administration of each dose of diazepam. For LEW

rats, between-subject variability increased as a function of

increasing doses of diazepam. As noted above, however,

comparisons between AUCs obtained following vehicle

administration and the largest AUC obtained following

diazepam administration for individual LEW rats indi-

cated a significant increase in AUC relative to vehicle

(see the bold values in Appendix B for the values and

doses included in the analysis).

For F344 rats (Fig. 4, open symbols), between-subject

variability in indifference points and AUC was observed

across all doses of diazepam administered. As described

above for Fig. 3, increases in indifference points and

AUCs were obtained for most F344 rats following the

administration of smaller doses of diazepam and de-

creases in indifference points and AUCs were obtained

for most F344 rats following the administration of the

largest dose of diazepam.

Chronic diazepam administration

Subjects LEW-7 and F344-6 did not receive chronic drug

administration because a much longer period of time was

required to determine the baseline delay series compared

with other subjects. Considering the lifespan of these rat

strains, completion of the chronic phase would likely not

have been possible or healthy for these subjects. Analyses

throughout the rest of the experiment are thus based on

seven LEW and six F344 rats. The chronic dose of

diazepam administered for each subject is listed in the

Appendix, and the number of sessions conducted during

the chronic phase for individual subjects is shown

in Table 1. There was no significant difference between

the mean number of sessions required for LEW and F344

rats to complete the chronic phase.

Figure 5 shows the mean percent larger-reinforcer choice

for acute vehicle administration and the first and last five

sessions of chronic diazepam administration (30–50

sessions) as a function of delay to the larger reinforcer

(shown as blocks of trials) for LEW (Fig. 5a) and F344

(Fig. 5b) rats (see Appendix for data from individual

Fig. 4

LEW acute (n=8) F344 acute (n =7)

Diazepam (mg/kg)1.0

0.0

1.0

0.5

1.5

2.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

3.0 10.0 1.0 3.0 10.00.0

0.5

1.0

1.5

2.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Pro

port

ion

of v

ehic

le A

UC

P

ropo

rtio

n of

veh

icle

indi

ffere

nce

poin

t(s)

(a) (c)

(b) (d)

Indifference points (a, c) and AUC (b, d) for individual subjects as a function of doses of diazepam (1.0, 3.0, and 10.0 mg/kg) expressed asproportion of vehicle during the acute phase for LEW (a, b) and F344 (c, d) rats. Data points above the dashed line indicate increases and datapoints below the dashed line indicate decreases in indifference points and AUC for individual subjects. AUC, area under the curve; F344, Fischer344; LEW, Lewis.

322 Behavioural Pharmacology 2012, Vol 23 No 4

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

subjects). Data from acute vehicle administration were

included here to compare chronic drug effects to a

nondrug phase. There was a differential effect of chronic

diazepam administration between rat strains. For LEW

rats, the mean larger-reinforcer choice during chronic

diazepam administration (first five sessions and last five

sessions) was similar to the levels obtained during acute

vehicle administration. For individual LEW rats, however,

indifference points and AUCs often did change during

the first five and the last five sessions of chronic

administration; however, the effects were not systematic.

For F344 rats, the mean larger-reinforcer choice during

chronic diazepam administration decreased relative to

acute vehicle administration (Fig. 5b). Within the first

five sessions of chronic diazepam administration, there

was an overall decrease in larger-reinforcer choice for four

of six F344 rats. For individual subjects, the observed

decrease in larger-reinforcer choice only occurred for

F344 rats that received the largest doses (5.6 and

10.0 mg/kg) of diazepam, and a slight increase across

the first five sessions of chronic diazepam occurred for

F344 rats that received the smallest dose (1.0 mg/kg) of

diazepam chronically. The mean AUC during the last five

sessions of chronic diazepam administration for F344 rats

tended to decrease relative to acute vehicle administra-

tion [F(1,5) = 6.49, P = .051].

Postchronic diazepam administration

Table 1 shows the number of sessions conducted during

the postchronic phase for individual subjects. There was

no difference between the mean number of sessions

required for LEW and F344 rats to complete this

phase. Figure 6 shows the mean indifference points

(Fig. 6a and c) and the mean AUCs (Fig. 6b and d) for

LEW (left panels) and F344 (right panels) rats as a

function of acute and postchronic doses of diazepam (see

Appendix for data from individual subjects). Again, for

LEW rats, there was no significant effect of dose on the

mean indifference points or AUC during the postchronic

phase, and the mean indifference points and AUC during

postchronic diazepam administration did not differ

significantly from those obtained during acute diazepam

administration (Fig. 6a and b). The large increases that

had occurred for LEW-2 and LEW-3 during the acute

administration of the 10.0 mg/kg dose of diazepam were

no longer observed when it was administered during the

postchronic phase.

For F344 rats, there was an overall decrease in larger-

reinforcer choice from the acute to the postchronic

diazepam phases, as indicated by an overall decrease in

the mean indifference points and AUC across all doses of

diazepam administered (Fig. 6c and d). When postchronic

vehicle was administered (nondrug sessions), the mean

AUC for F344 rats decreased significantly relative to acute

vehicle administration [F(1,5) = 16.71, P < 0.01]. The

mean indifference points and AUCs for F344 rats also

decreased significantly from acute to postchronic adminis-

tration of 1.0 [F(1,5) = 8.31, P < 0.05; F(1,5) = 68.65,

P < 0.01, respectively] and 3.0 mg/kg [F(1,5) = 6.59,

P = 0.05; F(1,5) = 8.99, P < 0.05, respectively] of diaze-

pam. The mean indifference points and AUCs at the

largest dose administered (10.0 mg/kg) remained low across

the acute and the postchronic phases. Considering these

results in combination with the decreases in larger-

reinforcer choice within the first five sessions of chronic

diazepam administration, a baseline shift occurred for F344

rats, which remained present throughout the chronic and

the postchronic phases of diazepam administration.

Fig. 5

Delay to larger reinforcer(s)

0

20

40

60

80

100

Acute vehicleFirst five chronicLast five chronic

Block 4

Block 5

Block 3

Block 2

Block 1

Block 4

Block 5

Block 3

Block 2

Block 1

0

20

40

60

80

100

LEWn = 7

F344n = 6

Per

cent

cho

ice

for l

arge

r rei

nfor

cer

(a) (b)

Mean percent larger-reinforcer choice as a function of delay during acute vehicle administration (closed squares) and the first (open squares) and thelast (open circles) five sessions of chronic diazepam administration for LEW (a) and F344 (b) rats. Error bars represent the SEM. The horizontal axisis composed of blocks as one LEW and three F344 rats responded in a 0-, 5-, 10-, 20-, and 40-s delay series, and six LEW and three F344 ratsresponded in a 0-, 2-, 4-, 8-, and 16-s delay series. F344, Fischer 344; LEW, Lewis.

Diazepam and delay discounting Huskinson and Anderson 323

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Baseline replication

Table 1 shows the number of 0-s probe sessions

conducted after the completion of all drug phases and

the number of sessions conducted during the baseline-

replication phase. There was no significant difference

between the mean number of 0-s probe sessions and the

mean number of sessions during baseline replication for

LEW and F344 rats. Figure 7 shows the mean percent

larger-reinforcer choice as a function of delay to the larger

reinforcer (shown as blocks of trials) for only those LEW

(Fig. 7a) and F344 (Fig. 7b) rats that completed all drug

phases. Choice data are from the last five sessions of the

baseline phase conducted at the beginning of experi-

mentation (before drug administration) and the last five

sessions of the baseline-replication phase during which

drug administration had ceased. During the initial base-

line phase, LEW rats chose the larger-reinforcer less than

F344 rats. This was indicated by shorter mean indiffer-

ence points and smaller mean AUC for LEW rats.

Differences in larger-reinforcer choice were no longer

present during baseline replication (see Appendix for

data from individual subjects) as similar rates of

discounting occurred for LEW and F344 rats.

DiscussionConsistent with previous research (e.g., Anderson and

Woolverton, 2005; Madden et al., 2008), delay discounting

was observed for both LEW and F344 rat strains in that,

as delay to the larger reinforcer increased, the choice for

that alternative decreased. Delay series for individual

subjects were functionally determined and, despite

obtaining steeper discounting functions and smaller

AUC for LEW rats, intermediate functions were obtained

for both strains. LEW rats had shorter indifference points

compared with F344 rats, and this finding is consistent

with previous research using the same discounting

procedure, where delays increased systematically within

sessions (Anderson and Woolverton, 2005; Anderson and

Diller, 2010; Garcia-Lecumberri et al., 2010; Huskinson

et al., 2012), and using a different procedure where delays

increased systematically between sessions (Madden et al.,2008). Despite the significant differences in the mean

indifference points at baseline, there was some overlap

between strains. For example, relatively long indifference

points were observed for two LEW rats that were similar

to the four shortest indifference points observed for F344

rats.

Fig. 6

Indi

ffere

nce

poin

t(s)

0

5

10

15

20

25

0

5

10

15

20

25Acute DZPPostchronic DZP

Are

a un

der t

he c

urve

0.0

0.2

0.4

0.6

0.8

VV 1.0 3.0 10.0 1.0 3.0 10.0

VV 1.0 3.0 10.0 1.0 3.0 10.00.0

0.2

0.4

0.6

0.8

Diazepam (mg/kg)

F344LEW n = 7

LEWn = 7

n = 6

F344n = 6

∗∗

∗∗∗

∗∗

(a) (c)

(b) (d)

Mean indifference points (a, c) and area under the curve (b, d) as a function of doses of diazepam [vehicle (V), 1.0, 3.0, and 10.0 mg/kg] during acute(closed symbols) and postchronic (open symbols) drug administration for LEW (a, b) and F344 (c, d) rats. Error bars represent the SEM. Single anddouble asterisks represent significance levels of P < 0.05 and P < 0.01, respectively, where differences were found between acute and postchronicadministration at a particular dose. DZP, diazepam; F344, Fischer 344; LEW, Lewis.

324 Behavioural Pharmacology 2012, Vol 23 No 4

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

In the current study, LEW rats, on average, required more

0-s probe sessions during the baseline phase compared

with F344 rats. It is possible that LEW rats were less

sensitive to differences in reinforcer amount, and this

could have contributed toward differences in the baseline

rates of discounting. Differences in the number of

sessions required to pass 0-s probe sessions were no

longer present during the acute phase; yet, rates of

discounting were steeper for LEW compared with F344

rats during nondrug (vehicle and control) sessions. It is

also possible that sensitivity to reinforcer delay is

different for LEW compared with F344 rats. The

procedure used, however, cannot separate the effects of

reinforcer amount and delay, and strain differences in

discounting may result from differential sensitivity to

reinforcer amount, delay, or both.

Neurochemical differences could have also contributed

toward differences in the baseline rates of discounting

between these rat strains. In general, LEW rats have lower

levels of DA and 5-HT in various brain regions relative to

F344 rats (Burnet et al., 1992; Selim and Bradberry,

1996; Flores et al., 1998), and the depletion of these

neurotransmitters is related to steeper rates of discount-

ing (Wogar et al., 1993; Mobini et al., 2000; Kheramin et al.,2004). Neurochemical measures were not used in the

present experiment; however, future research correlating

discounting rates and neurotransmitter levels could

provide empirical support for this relation.

It is important to note that, in addition to differences in

DA and 5-HT, LEW and F344 rats have other physiolo-

gical differences relevant to GABA and benzodiazepines.

For example, LEW and F344 rats differ in hypothalamic–

pituitary–adrenal (HPA) axis function, with LEW rats

showing hyporesponsive HPA axis function following

stress exposure (see Kosten and Ambrosio, 2002 for a

review). LEW rats also have lower total plasma concen-

trations of corticosterone compared with F344 rats

(Smith et al., 1992). Although HPA axis function is

related to drug abuse (see Kosten and Ambrosio, 2002 for

a review), it is not clear how or whether these systems are

related to delay discounting. The differences obtained in

delay discounting at baseline between LEW and F344 rats

could have resulted from differences specific to one or a

combination of the various neurochemical differences

between these rat strains. Future research is necessary to

determine whether or how other neurotransmitter

systems are related to delay discounting.

During the acute drug phase, steeper rates of discounting

continued to occur for LEW relative to F344 rats during

control and vehicle sessions; thus, the baseline differences

persisted throughout acute diazepam administration. For

F344 rats, the mean indifference points and AUCs did

decrease slightly during the control and the vehicle

sessions, relative to those obtained at baseline, but this

effect did not occur for LEW rats. It is possible that larger-

reinforcer choice gradually declines with extended expo-

sure to the task, particularly for subjects with initially

shallower discounting functions. In the current study, the

two F344 rats with the longest indifference points and the

largest AUCs (F344-3 and F344-7) showed the greatest

decrease from baseline to control sessions. However, if

extended exposure to the discounting task was responsible

for the observed decreases in larger-reinforcer choice for

F344 rats, it would be likely that this effect would have also

occurred for LEW rats.

Fig. 7

Delay to larger reinforcer(s)Bloc

k 1

Block 2

Block 3

Block 4

Block 5

Block 1

Block 2

Block 3

Block 4

Block 5

Per

cent

cho

ice

for l

arge

r rei

nfor

cer

0

20

40

60

80

100BaselineBaseline replication

0

20

40

60

80

100Baseline Baseline replication

LEWn = 7

F344n = 6

(a) (b)

Mean percent larger-reinforcer choice as a function of delay blocks for the last five sessions conducted during the baseline phase (circles) and thelast five sessions from the baseline-replication phase (squares) for only those LEW (a) and F344 (b) rats that completed all phases of the experiment.Error bars represent the SEM. The horizontal axis is composed of blocks rather than delay values as one LEW and three F344 rats responded in a 0-,5-, 10-, 20-, and 40-s delay series, and six LEW and three F344 rats responded in a 0-, 2-, 4-, 8-, and 16-s delay series. F344, Fischer 344; LEW,Lewis.

Diazepam and delay discounting Huskinson and Anderson 325

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

The finding that acute administration of diazepam did

not alter reinforcer choice in LEW rats is consistent with

other research examining the effects of acute diazepam

administration on different behavioral measures in LEW rats.

Takahashi et al., (2001) examined the effects of different

doses of diazepam on an elevated plus-maze task in LEW

rats relative to spontaneously hypertensive rats. Doses that

affected behavior in spontaneously hypertensive rats had no

effect on behavior in LEW rats. LEW rats were also less

sensitive to the behavioral effects of chlordiazepoxide,

another benzodiazepine, on a successive negative-contrast

procedure compared with F344 rats (Freet et al., 2006).

Within the same procedure, chlordiazepoxide increased the

overall consumption of a sucrose solution for F344, but not

LEW rats (Freet et al., 2006).

Perhaps delay discounting is another measure that, in LEW

rats, is not sensitive to effects of benzodiazepines.

Examination of individual-subject data, however, suggested

otherwise. Acute diazepam did not affect the mean larger-

reinforcer choice in LEW rats when the effects of each dose

were compared separately, but different doses of diazepam

did have effects for individual subjects. For example, at

least one of the doses administered increased AUC in LEW

rats relative to vehicle, but which dose had this effect was

variable across subjects and was obscured by presenting

only the means for each dose. Increased larger-reinforcer

choice in LEW rats is consistent with Evenden and Ryan’s

(1996) findings that diazepam increased larger-reinforcer

choice in Sprague–Dawley rats. In studies that report no

effect (Takahashi et al., 2001; Freet et al., 2006), it is

possible that individual LEW rats were insensitive to

benzodiazepine administration or that effects were ob-

served for individual LEW rats but were obscured by

reporting only the group means. This highlights the

importance of reporting individual-subject data, particularly

in experiments that use small-n designs.

At least one of the two smallest doses (1.0 and 3.0 mg/kg)

of acute diazepam increased indifference points and AUCs

for all except one F344 rat, and this is also consistent with

the results of Evenden and Ryan (1996) with diazepam in

Sprague–Dawley rats. Conversely, the largest dose of

diazepam decreased larger-reinforcer choice in F344 rats,

and this is consistent with the finding of Cardinal et al.(2000) that relatively large doses of chlordiazepoxide

decrease larger-reinforcer choice in Lister hooded rats. It

should be noted that neither Evenden and Ryan (1996)

nor Cardinal et al. (2000) reported individual-subject data.

These studies are used here as a comparison, however, as

both studies used a delay-discounting procedure that was

similar to the one used in the present study. Most of the

research examining effects of benzodiazepines on delay

discounting has used a T-maze procedure. Comparisons

between the current study and those using a T-maze

procedure are more difficult because there are many

procedural differences.

It is also possible that discrepant findings with benzo-

diazepines and delay discounting result from the admin-

istration of an insufficient range of doses. Evenden and

Ryan (1996) found increases in larger-reinforcer choice

following smaller diazepam doses (0.3 and 1.0 mg/kg).

Other doses of diazepam were not tested. Cardinal et al.(2000) found decreases in larger-reinforcer choice follow-

ing the administration of the largest chlordiazepoxide

dose tested (10.0 mg/kg). These researchers used multi-

ple doses, but did not examine effects of doses smaller

than 1.0 mg/kg. The present experiment found increases

and decreases in larger-reinforcer choice depending on

the strain and the dose tested.

Decreases in larger-reinforcer choice observed for F344

rats following acute diazepam administration may be

related to effects of benzodiazepine administration on

DA and 5-HT. For example, benzodiazepines decreased

the extracellular levels of DA and 5-HT neuronal activity

in specific brain regions (Stein et al., 1975; Finlay et al.,1995). Administration of the largest dose may have

resulted in lower levels of DA and 5-HT that contributed

to steeper rates of discounting in F344 rats. Larger-

reinforcer choice did not decrease for LEW rats following

acute diazepam administration. LEW rats already have

lower levels of DA and 5-HT, and it is possible that a floor

effect occurred with these rats. Future research could

correlate DA and 5-HT levels with rates of discounting

following benzodiazepine administration.

Decreases in larger-reinforcer choice observed for F344

rats following acute diazepam administration may also be

related to HPA axis function, corticosterone concentra-

tions, and benzodiazepine receptor density (Smith et al.,1992; Kosten and Ambrosio, 2002). Although benzodia-

zepines have actions on DA and 5-HT systems (Stein

et al., 1975; Roth et al., 1988; Finlay et al., 1995), they are

primarily known for their actions on GABA (e.g., Gielen

et al., 2012). The involvement of these systems in delay

discounting has received much less attention relative to

DA and 5-HT. As indicated above, more research is

required to determine the role of systems other than DA

and 5-HT in delay discounting.

Overall, changes in larger-reinforcer choice were not

systemic for LEW rats during chronic administration. For

F344 rats that received the largest doses of diazepam as

the chronic dose (four of six), larger-reinforcer choice

decreased during the first five sessions of chronic

administration. With these subjects, larger-reinforcer

choice decreased rapidly and remained low across the

chronic phase. By the end of the chronic phase, larger-

reinforcer choice had decreased relative to acute vehicle

for all six F344 rats. That more systematic effects

occurred for F344 relative to LEW rats is consistent with

other studies examining chronic diazepam exposure in

LEW and F344 rats. LEW rats had fewer signs of physical

dependence and withdrawal compared with F344 rats

326 Behavioural Pharmacology 2012, Vol 23 No 4

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

following chronic exposure to and removal of food mixed

with diazepam, despite the two strains having similar

blood-diazepam levels (Suzuki et al., 1992).

Compared with acute administration, postchronic diaze-

pam administration had no effect on the mean percent

larger-reinforcer choice for LEW rats. Indifference points

and AUCs across all doses were similar to those obtained

during both acute and postchronic vehicle administration.

The variability in indifference points and AUCs across

subjects decreased for LEW rats following postchronic

diazepam administration. For F344 rats, the overall

decrease in larger-reinforcer choice that occurred during

the first 5 days of chronic diazepam administration

remained during the postchronic phase. Across all doses

administered, including vehicle, indifference points and

AUCs were similar to those obtained following the acute

administration of the largest dose of diazepam. Carryover

effects may have contributed to the finding that similar

indifference points and AUCs were obtained across all

doses of diazepam administered during the postchronic

phase. Before this phase, the chronic dose of diazepam

had been administered for 30–50 days. During this phase,

the chronic dose continued to be administered daily,

except two to three times per week, when it was

substituted with vehicle and other doses of diazepam.

During the baseline-replication phase, when all injections

ceased, the rate of discounting for LEW rats was similar to

the rate of discounting observed for these rats during the

initial baseline phase. Thus, discounting in LEW rats

remained, at least on average, unchanged across multiple

phases of the experiment. For F344 rats, the overall

decrease in larger-reinforcer choice observed early in

chronic diazepam exposure persisted into the baseline-

replication phase. It seems that the baseline shift in F344

rats could have occurred for at least three reasons. First,

chronic diazepam could have resulted in a relatively

permanent baseline shift in larger-reinforcer choice. Rats

that completed chronic and postchronic phases were

exposed to daily diazepam administration for 2–3 months.

If the initial baseline shift that occurred within the first

five sessions of chronic administration was not relatively

permanent, one might expect larger-reinforcer choice to

return-to-baseline levels as individual subjects became

tolerant to the effects of diazepam. This did not occur

throughout chronic administration nor did rates of

discounting return to the initial baseline levels following

a minimum of 20 sessions on the delay-discounting task

when diazepam was no longer administered.

The second and third potential reasons for an overall

decrease in larger-reinforcer choice for F344 rats could have

been repeated exposure to the delay-discounting task and

subject maturation, respectively. Exposure to diazepam

across the multiple phases, exposure to the discounting

task, and subject maturation were confounded throughout

experimentation. It is possible that as F344 rats aged or

experienced continued exposure to the task, larger-

reinforcer choice would have decreased anyway. However,

the finding that aged F344 rats have shallower discounting

functions compared with young F344 rats, using a

procedure similar to the one used in the current study,

would predict the opposite (Simon et al., 2010). The

findings of Simon et al. (2010) do not rule out extended

exposure to the discounting task as a potential contributor

to decreases in larger-reinforcer choice, as both aged and

young F344 rats were exposed to the task for a short period

of time. That the same overall decrease in larger-reinforcer

choice did not occur for LEW rats, however, makes the

effects of both age and exposure to the task less plausible.

The present study is the first to report acute, chronic, and

postchronic effects of diazepam on delay discounting in

LEW and F344 rats. LEW and F344 rats have behavioral

and neurochemical differences that are correlated with

their different baseline rates of delay discounting. Acute

administration of diazepam had differential effects on

larger-reinforcer choice in LEW and F344 rats and for

individual subjects within each strain. Chronic administra-

tion of diazepam did not systematically affect larger-

reinforcer choice in LEW rats and resulted in an overall

decrease in larger-reinforcer choice for F344 rats that

persisted throughout a baseline-replication phase.

Using two rat strains that differ in baseline rates of delay

discounting and their physiological response to benzo-

diazepines has shed light on the relative influence of

behavioral and biological determinants of impulsive

choice. For example, differences in delay discounting

were found with genetically different rat strains under

baseline conditions and following acute, chronic, and

postchronic administration of diazepam despite similar

environmental histories for both strains. These findings

suggest a genetic component in determining impulsive

choice and subsequent drug effects. However, there was

substantial within-strain variability in each of the

conditions throughout experimentation, suggesting an

environmental component in determining impulsive

choice and subsequent drug effects. The results dis-

cussed have also raised many questions about, and

potential directions for future investigation of, more

specific environmental, genetic, and neurochemical vari-

ables involved in delay discounting and the effects of

benzodiazepines on delay discounting.

AcknowledgementsThe authors thank Amber Barse, Meagan Follett, and

Christopher Krebs for their help in conducting portions of

the study. This study was supported by DA019842

awarded to Karen G. Anderson from the National

Institute on Drug Abuse.

Conflicts of interest

There are no conflicts of interest.

Diazepam and delay discounting Huskinson and Anderson 327

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Appendix A. Indifference points for each subject during baseline, acute diazepam (control, VEH, 1.0, 3.0, and 10.0 mg/kg), first five (chronic) and last

five (chronic) sessions of chronic diazepam administration, postchronic diazepam (VEH, 1.0, 3.0, and 10.0 mg/kg), and baseline replication at the end

of experimentation (baseline 2)

Acute dose (mg/kg)

Subjects Delay series Baseline Control VEH 1.0 3.0 10.0

LEW-1 16 7.67 9.78 7.89 9.04 10.78 9.57LEW-2 40 10.25 10.17 10.92 9.73 12.85 63.20LEW-3 16 8.09 6.84 5.89 8.34 14.52 20.66LEW-4 16 4.27 5.05 5.30 5.25 5.25 6.76LEW-5 16 3.52 4.01 4.92 4.90 5.40 3.19LEW-6 16 7.38 5.33 6.18 6.70 3.11 2.65LEW-7 40 6.33 8.89 9.35 8.68 7.97 9.46LEW-8 16 4.91 6.07 3.73 4.75 7.41 5.00M (SEM) 6.55 (0.8) 7.02 (0.8) 6.77 (0.9) 7.17 (0.7) 8.41 (1.4) 15.06 (7.2)F344-1 16 8.88 7.34 4.96 7.59 5.26 5.26F344-2 16 9.67 9.70 5.79 14.64 12.09 2.25F344-3 40 42.88 12.98 15.00 17.05 18.61 5.35F344-4 40 16.96 14.23 12.60 13.03 17.36 5.08F344-5 16 8.26 7.45 6.51 8.61 5.56 3.00F344-6 16 15.32 14.91 21.28 14.37 3.80 3.00F344-7 40 27.95 21.91 21.04 35.20 28.90 1.53F344-8 16 8.38 – – – – –M (SEM) 17.29 (4.4) 12.65 (1.9) 12.45 (2.6) 15.78 (3.5) 13.08 (3.5) 3.64 (0.6)

Postchronic Dose (mg/kg)

Subjects Delay series Chronic dose (mg/kg) First 5 chronic Last 5 chronic VEH 1.0 3.0 10.0 Baseline 2LEW-1 16 1.7 18.32 11.95 7.79 13.39 14.86 17.45 7.22LEW-2 40 10.0 45.89 92.16 16.02 8.76 10.00 17.22 7.59LEW-3 16 10.0 9.98 2.48 1.50 3.30 2.89 3.96 5.60LEW-4 16 10.0 3.89 7.26 4.22 3.85 4.00 5.43 6.02LEW-5 16 10.0 3.46 5.02 3.22 3.89 5.69 3.13 5.12LEW-6 16 5.6 2.79 3.72 5.78 3.29 5.84 4.89 5.31LEW-7 40 – – – – – – – –LEW-8 16 3.0 5.22 6.04 4.50 5.60 6.51 4.93 4.96M (SEM) 12.79 (5.9) 18.38 (12.4) 6.15 (1.8) 6.01 (1.4) 7.11 (1.5) 8.14 (2.4) 5.97 (0.4)F344-1 16 1.0 5.58 3.92 5.05 5.11 5.61 1.30 10.34F344-2 16 1.0 9.89 5.44 3.24 4.54 5.28 2.62 4.14F344-3 40 10.0 3.06 3.70 4.87 4.62 4.62 3.88 4.44F344-4 40 10.0 0.29 2.27 6.80 5.35 2.99 1.39 7.13F344-5 16 10.0 2.00 6.43 3.72 3.52 4.63 5.14 4.07F344-6 16 – – – – – – – –F344-7 40 5.6 5.37 4.00 4.82 6.12 3.43 3.43 5.43F344-8 16 – – – – – – – –M (SEM) 4.37 (1.8) 4.29 (0.6) 4.75 (0.5) 4.88 (0.4) 4.43 (0.4) 2.96 (0.6) 5.93 (1.0)

F344, Fischer 344; LEW, Lewis; VEH, vehicle.

Diazepam and delay discounting Huskinson and Anderson 329

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Appendix B. Area under the curve for each subject during baseline, acute diazepam (control, VEH, 1.0, 3.0, and 10.0 mg/kg), first five (chronic) and

last five (chronic) sessions of chronic diazepam administration, postchronic diazepam (VEH, 1.0, 3.0, and 10.0 mg/kg), and baseline replication at the

end of experimentation (baseline 2)

Acute dose (mg/kg)

Subjects Delay series Baseline Control VEH 1.0 3.0 10.0

LEW-1 16 0.519 0.576 0.519 0.542 0.618 0.583LEW-2 40 0.360 0.358 0.392 0.351 0.399 0.653LEW-3 16 0.548 0.485 0.412 0.516 0.664 0.815LEW-4 16 0.369 0.361 0.398 0.380 0.359 0.477LEW-5 16 0.271 0.341 0.353 0.346 0.379 0.253LEW-6 16 0.488 0.394 0.441 0.443 0.234 0.198LEW-7 40 0.279 0.333 0.342 0.344 0.339 0.315LEW-8 16 0.400 0.437 0.319 0.347 0.508 0.385M (SEM) 0.404 (0.04) 0.411 (0.03) 0.397 (0.02) 0.409 (0.03) 0.438 (0.05) 0.460 (0.08)F344-1 16 0.546 0.533 0.387 0.544 0.372 0.401F344-2 16 0.602 0.608 0.422 0.736 0.635 0.151F344-3 40 0.790 0.415 0.434 0.462 0.490 0.142F344-4 40 0.488 0.426 0.396 0.417 0.462 0.188F344-5 16 0.558 0.556 0.452 0.539 0.417 0.206F344-6 16 0.742 0.779 0.817 0.633 0.346 0.198F344-7 40 0.625 0.532 0.530 0.656 0.589 0.162F344-8 16 0.563 – – – – –M (SEM) 0.614 (0.04) 0.550 (0.05) 0.491 (0.06) 0.570 (0.04) 0.473 (0.04) 0.207 (0.03)

Acute dose (mg/kg)

Subjects Delay series First 5 chronic Last 5 chronic VEH 1.0 3.0 10.0 Baseline 2LEW-1 16 0.779 0.638 0.503 0.663 0.792 0.781 0.513LEW-2 40 0.629 0.658 0.432 0.337 0.385 0.451 0.317LEW-3 16 0.577 0.217 0.094 0.219 0.250 0.342 0.383LEW-4 16 0.275 0.510 0.365 0.287 0.344 0.393 0.400LEW-5 16 0.227 0.360 0.319 0.302 0.392 0.264 0.375LEW-6 16 0.202 0.265 0.423 0.237 0.417 0.354 0.371LEW-7 40 – – – – – – –LEW-8 16 0.410 0.425 0.358 0.396 0.464 0.409 0.348M (SEM) 0.443 (0.08) 0.439 (0.07) 0.356 (0.05) 0.349 (0.06) 0.435 (0.06) 0.428 (0.06) 0.387 (0.02)F344-1 16 0.396 0.327 0.358 0.370 0.403 0.180 0.648F344-2 16 0.598 0.400 0.275 0.367 0.424 0.224 0.294F344-3 40 0.090 0.088 0.118 0.104 0.104 0.085 0.106F344-4 40 0.060 0.071 0.182 0.142 0.115 0.053 0.210F344-5 16 0.196 0.450 0.273 0.250 0.349 0.380 0.325F344-6 16 – – – – – – –F344-7 40 0.258 0.098 0.139 0.208 0.089 0.089 0.183F344-8 16 – – – – – – –M (SEM) 0.266 (0.08) 0.239 (0.07) 0.224 (0.04) 0.240 (0.05) 0.247 (0.07) 0.168 (0.05) 0.295 (0.08)

Bold values indicate largest obtained area under the curve following acute diazepam administration.F344, Fischer 344; LEW, Lewis; VEH, vehicle.

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