Prepubertal Fischer 344 RatsDisplay Stronger Morphine-Induced Taste Avoidance ThanPrepubertal Lewis Rats

ABSTRACT: The present report asked if the previously reported differences inmorphine-induced conditioned taste avoidance between adult F344 and LEWrats (F344> LEW) are also evident in prepubescence (early adolescence). Toassess this possibility, adult (Experiment 1) and prepubertal (Experiment 2)F344 and LEW rats were assessed for their ability to acquire morphine-inducedtaste avoidance (0, 3.2, 10, or 18mg/kg) in a modified taste avoidanceprocedure. In each experiment, rats of both strains were given repeated pairingsof saccharin and morphine followed by a final two-bottle avoidance test. Adultand prepubertal F344 subjects displayed a more rapid acquisition of theavoidance response as well as stronger suppression of consumption than theirLEW counterparts. These data suggest the strains differ in their sensitivity to theaversive effects of morphine and that this differential sensitivity is evident earlyin development and is developmentally stable. The basis for these straindifferences in morphine-induced avoidance was discussed. � 2013 WileyPeriodicals, Inc. Dev Psychobiol 56: 979–988, 2014.

Keywords: strain differences; prepubertal; early adolescence; adult; F344;LEW; morphine-induced taste avoidance; development

INTRODUCTION

Recent work on conditioned taste avoidance (CTA)

learning between the inbred F344 and LEW rat strains

has demonstrated strain differences in drug-induced

taste avoidance that vary depending on the drug of

abuse tested (for cocaine, see Davis & Riley, 2007;

Glowa, Shaw, & Riley, 1994; for nicotine, see Pesca-

tore, Glowa, & Riley, 2005; for ethanol, see Roma,

Flint, Higley, & Riley, 2006). One compound for which

such strain differences have been well characterized is

morphine. Specifically, Lancellotti, Bayer, Glowa,

Houghtling, and Riley (2001) reported that the F344

strain displayed dose-dependent morphine-induced taste

avoidance, while the LEW strain did not acquire

avoidance at any dose tested, even after repeated

conditioning trials (Davis, Cobuzzi, & Riley, 2012; see

also Gomez-Serrano, Kearns, & Riley, 2009; for a

review, see Riley, 2011). That the F344 animals show

stronger avoidance is interesting in that they also self-

administer less morphine (Ambrosio, Goldberg, &

Elmer, 1995; Garcıa-Lecumberri et al., 2011; Martın

et al., 1999, 2003) and form weaker place preferences

(Davis, Roma, Dominguez, & Riley, 2007; Grakalic,

Schindler, Baumann, Rice, & Riley, 2006) than the

LEW strain, suggesting that morphine intake in these

strains may be impacted by the relative balance of

these affective properties.

Although the differences between the F344 and

LEW strains with respect to morphine-induced taste

avoidance are consistently observed in adults (see

above), it remains unknown if such effects are evident

early in life and are developmentally stable or if they

Manuscript Received: 2 July 2013Manuscript Accepted: 30 September 2013The authors declared that they have no conflicts of interest.Correspondence to: Zachary E. HurwitzContract grant sponsor: Mellon FoundationContract grant sponsor: American University Artists and Scholars

FellowshipArticle first published online in Wiley Online Library

(wileyonlinelibrary.com): 28 October 2013DOI 10.1002/dev.21176 � � 2013 Wiley Periodicals, Inc.

Developmental Psychobiology

Zachary E. Hurwitz

Jennifer L. Cobuzzi

Andrew P. Merluzzi

Bradley Wetzell

Anthony L. Riley

Psychopharmacology LaboratoryDepartment of Psychology

American University4400 Mass. Ave., NW, Washington, DC

20016E-mail: zacharyehurwitz@gmail.com

are a function of specific developmental histories that

result in differential behavior in adulthood. Interesting-

ly, strain differences have been reported to vary over

development for a number of physiological and behav-

ioral endpoints in a variety of rodent strains. For

example, Wilking et al. (2012) reported that nicotine

preference varied as a function of age and dose in the

C3H/Ibg and C57bl/6j mouse strains. Specifically, there

were no differences between the strains across various

doses tested in late adolescence and adulthood. Howev-

er, in early adolescence C3H/Ibg subjects were more

sensitive than the C57bl/6j strain at 10mg concentration

while in middle adolescence C3H/Ibg subjects emitted

less behavior than C57bl/6j mice at 20 and 30mgconcentrations (for other examples of developmental

differences in strain comparisons, see Allam, 2012;

Fairless et al., 2012; Farid, Martinez, Geyer, &

Swerdlow, 2000; Moore, Forrest, & Boehm, 2013;

Moore, Linsenbardt, Melon, & Boehm, 2011; Paylor,

Baskall-Baldini, Yuva, & Wehner, 1996; Satinder,

1981; Sinaiko & Mirkin, 1974; Tonkiss, Shultz, &

Galler, 1992; Vogl, Atchley, & Xu, 1994).

Although age differences across a variety of strains

have been well characterized, little has been examined

with the F344 and LEW rat lines, especially studies

examining the ontogenesis of strain differences in drug

reactivity (see Gomez-Serrano, Sternberg, & Riley,

2002; Gomez-Serrano, Tonelli, Listwak, Sternberg, &

Riley, 2001). In the only developmental assessment of

behavioral differences between the two strains, Siviy,

Love, DeCicco, Giordano, and Seifert (2003) reported

that strain differences in play behavior (LEW>F344)

were present and comparable at all developmental

periods assessed. Although suggestive that these pheno-

typic differences are highly heritable and stable, no

developmental assessments have been made with the

F344 and LEW strains in relation to their relative

sensitivities to drugs of abuse yet alone their ability to

acquire drug-induced taste avoidance.

It is important to note in this context that prepubertal

(early adolescent) and adult outbred rats differ dramati-

cally in taste avoidance learning (Anderson, Agoglia,

Morales, Varlinskaya, & Spear, 2012; Anderson, Varlin-

skaya, & Spear, 2010; Cobuzzi et al., 2013; Hurwitz,

Merluzzi, & Riley, 2013; Infurna & Spear, 1979;

Schramm-Sapyta et al., 2007; Schramm-Sapyta, Morris,

& Kuhn, 2006; Shram, Funk, Li, & Le, 2006; Vetter-

O’Hagen, Varlinskaya, & Spear, 2009; Wilmouth &

Spear, 2004) with prepubertal rats exhibiting significant-

ly weaker taste avoidance relative to their adult counter-

parts. Such results suggest that prepubescent rats are less

sensitive to the aversive effects of such drugs than adults,

a differential sensitivity that may confer an increased

susceptibility to the subsequent use and abuse of these

compounds (see Doremus-Fitzwater, Varlinskaya, &

Spear, 2010; Misanin, Anderson, & Hinderliter, 2009;

Schramm-Sapyta et al., 2010; Spear, 2013).

Given that differences with other strains have been

shown to vary over development and that outbred

prepubertal and adult rats differ significantly in drug-

induced avoidance across a range of drugs of abuse, it

might be predicted that the patterns evident in F344 and

LEW may differ across development. If prepubertal

F344 and LEW rats do not differ in their avoidance

patterns (and as such differ from the pattern displayed by

adults), one might argue that some unique developmental

history interacting with the unique genetic backgrounds

of the two strains may mediate the results typically seen

in adults. Conversely, if the strain differences observed

in adults are also present in prepubescence, it would

suggest that these differences in avoidance learning are

highly heritable, substantiating the use of these strains as

genetic models for drug sensitivity, use and abuse

(Beitner-Johnson, Guitart, & Nestler, 1991; Guitart et al.,

1993; Kosten et al., 1997). To address this issue, pre-

pubescent rats of the F344 and LEW stains were given

access to a novel saccharin solution and injected with

varying doses of morphine (Experiment 2). The specific

procedure employed (see below) was modified from the

typical taste avoidance procedure to accommodate their

developmental window and allow for assessment of

avoidance learning under conditions that minimize the

effects of fluid deprivation (and concomitant weight loss;

see Hurwitz et al., 2013). This modification employs

deprivation procedures not generally used in work with

adults. Consequently, prior to this assessment, adult

F344 and LEW subjects were tested under similar

conditions to assure that the often-reported differences

between the two strains in adult animals were evident

under the modified procedure (Experiment 1).

METHOD

Subjects

One hundred thirty-nine experimentally naıve male

F344 and LEW rats (Harlan Laboratories, Indianapolis,

IN) arrived at the facility on PND 21, weighing

approximately 40 g. Experiments 1 and 2 were each run

in two replicates with subjects in all dose groups and

both strains represented in each replicate. Food and

water were available ad libitum unless stated otherwise.

Procedures recommended by the National Research

Council (1996) and the Committee on Guidelines for

the Care and Use of Animals in Neuroscience and

Behavioral Research (2003) were followed at all times.

The research was reviewed and approved by the

American University IACUC.

980 Hurwitz et al. Developmental Psychobiology

Apparatus

Upon arrival to the animal colony, subjects in each

strain were initially handled and then group housed (2–

4 rats per bin) in polycarbonate bins (23 cm� 44 cm

� 21 cm). All subjects were maintained on a 12:12

light–dark cycle (lights on at 0800 hr) and at an

ambient temperature of 23˚C. All conditioning and

testing occurred during the light phase of the light–dark

cycle at 0800 hr and in the same room in which the

animals were housed. During adaptation and condition-

ing, animals were transferred to individual hanging

wire-mesh test cages (24.3 cm� 19 cm� 18 cm) but

were returned to their group-housed bins after condi-

tioning trials (see details below).

Drugs

Morphine sulfate was generously supplied by the

National Institute on Drug Abuse. For behavioral

testing, morphine sulfate was dissolved in sterile

isotonic saline (.9%) at a concentration of 5mg/ml and

was subsequently filtered through a .2mm filter to

remove any contaminants before being administered

subcutaneously (SC) at a dose of 3.2, 10, or 18mg/kg.

Morphine administered at these doses and by this route

has been reported to induce dose-dependent avoidance

in outbred rats (Hurwitz et al., 2013) and in the F344

and LEW strains (Davis et al., 2012; Gomez-Serrano

et al., 2009; Lancellotti et al., 2001). Sterile isotonic

saline was also filtered before being administered to

vehicle controls and was given equivolume to the

highest dose of morphine administered (18mg/kg).

Volume of the injection was manipulated in favor of

concentration given the influence concentration has on

drug absorption and distribution.

Morphine-Induced Taste Avoidance

Experiment 1: Adults. Procedures were adapted from

Hurwitz et al. (2013). Specifically, F344 (n¼ 35) and

LEW subjects (n¼ 36) were brought into the laboratory

on PND 21 and maintained on ad libitum food and

water until PND 77 to permit the control of their

developmental environment (housing conditions, han-

dling, and light/dark cycle). Subjects were weighed and

handled and had their water consumption measured

from PND 77–83 at which point water adaptation began.

On PND 84, subjects in each strain underwent full fluid

deprivation at 0800 hr and on the following day (PND

85) they were removed from their group-housed bin at

0800 hr, weighed and placed into individual test cages

where they were given 45-min access to tap water in

graduated 50-ml Nalgene tubes beginning at 0830 hr.

This specific procedure was used to minimize the

potential stress associated with more severe deprivation

schedules, for example, extended 20-min daily access,

in adolescent subjects (see Anderson et al., 2010; Hur-

witz et al., 2013). Under this modified deprivation

procedure, fluid consumption is generally lower, neces-

sitating longer access to assure sufficient consumption.

After 45min, bottles were removed, consumption was

recorded and subjects remained in the hanging cages for

an additional 20min before being returned to their

group-housed bin and given ad libitum water until

0800 hr the following day (PND 86). This procedure

was repeated one additional time to adapt animals to

consuming fluid in the test cages prior to being given

access to saccharin during conditioning. On the day

prior to the first saccharin conditioning day, water access

was again fully restricted before subjects underwent

conditioning in the test cages. On this first conditioning

trial (PND 89), subjects were weighed and handled (as

previously described) and given 45-min access to a

novel saccharin solution (1 g/L) in the test cages after

which they remained for an additional 20min. At this

point, subjects in each strain (independent of their

group-housed bin) were assigned to one of four groups

such that saccharin intake was comparable among

groups. Based on these group assignments, subjects in

each strain were injected with either morphine (3.2, 10,

or 18mg/kg, SC) or vehicle and then returned to their

group-housed bins and given ad libitum water until

0800 hr the following day. This procedure yielded

Groups F0 (n¼ 9), L0 (n¼ 9), F3.2 (n¼ 8), L3.2

(n¼ 8), F10 (n¼ 8), L10 (n¼ 9), F18 (n¼ 10), and L18

(n¼ 10) where the letter refers to the strain of the

subject and the number indicates the dose of morphine

administered. On the next day, subjects in each bin had

their fluid consumption restricted before the next

saccharin conditioning trial. This 2-day procedure

(saccharin-water recovery-full deprivation) was repeated

four times. On the day after the fourth cycle (PND 97),

subjects were transferred to test cages where two 50-ml

Nalgene tubes (one containing tap water; the other

containing the .1% saccharin solution) were affixed to

the cage for 45min and consumption of both solutions

was recorded. Placement of the bottles was counter-

balanced across all subjects to prevent positioning

effects. After the 45min, the bottles were removed,

consumption was recorded and subjects were returned to

their home cages where water was available ad libitum.

Experiment 2: Prepubescents. The procedures de-

scribed above were identical for the prepubescent rats

with the following exceptions. F344 and LEW rats

(n¼ 34 per strain) were brought into the facility at

PND 21 and were weighed and handled and had their

water consumption measured until PND 29. Following

Developmental Psychobiology Prepubertal Strain Differences in Morphine CTA 981

water adaptation (PND 29–32; see above), conditioning

began. During conditioning (PND 33–40), subjects in

each strain were given four saccharin-morphine (or

vehicle) pairings until the two-bottle test, which was

administered on PND 41. The group designations were

similar to those in Experiment 1: Groups F0 (n¼ 9), L0

(n¼ 9), F3.2 (n¼ 8), L3.2 (n¼ 8), F10 (n¼ 8), L10

(n¼ 8), F18 (n¼ 9), and L18 (n¼ 9).

Statistical Analysis

Acquisition. A 2 (Replicate)� 2 (Strain)� 4 (Dose)� 4

(Trial) mixed model ANOVA on saccharin consumption

(ml) on the four conditioning trials was run for each

experiment to determine differences between the strains as

a function of dose and trial. One-way ANOVAs and

Tukey’s HSD posthost tests were employed where merited

by significant interactions to determine differences

between the strains and doses across trials. To determine if

there were differences in saccharin consumption between

Conditioning Trials 1 and 4, Bonferroni-corrected t-tests

were employed (a¼ .0125) for each group.

Two-Bottle Test. A 2 (Replicate)� 2 (Strain)� 4

(Dose) univariate ANOVA was run for each experiment

on the percent saccharin consumed on the two-bottle

test. One-way ANOVAs and Tukey’s HSD post hoc

tests were used where merited to evaluate differences

in the percent saccharin consumed between replicates

and strains at the different dose groups.

RESULTS

Experiment 1: Adults

Acquisition. Over conditioning, F344 subjects drank

significantly less saccharin than LEW subjects, indica-

tive of greater morphine-induced taste avoidance. The 2

(Replicate)� 2 (Strain)� 4 (Dose)� 4 (Trial) mixed-

model ANOVA yielded significant effects of Trial [F

(3, 156)¼ 11.611], Strain [F (1, 55)¼ 159.148], Dose

[F (3, 55)¼ 37.403] as well as significant Trial�Repli-

cate [F (3, 156)¼ 6.950], Trial�Strain [F (3,

156)¼ 23.220], Trial�Dose [F (9, 156)¼ 22.869],

Strain�Dose [F (3, 55)¼ 1.389], Replicate�Strain [F

(1, 55)¼ 7.536] and Trial� Strain�Dose [F (9,

156)¼ 6.098] interactions.

Although on Trial 1 F344 subjects did not differ in

saccharin consumption, on Trials 2–4 all drug-treated

F344 subjects drank less saccharin than vehicle-treated

controls with Group 18 drinking less saccharin than

Group 3.2. On Trial 1, LEW subjects also did not differ

in saccharin consumption. On Trials 2–4, Group 18

drank less than Group 0. On all trials, F344 subjects

consumed significantly less saccharin than LEW sub-

jects. Tukey’s HSD revealed that on Trial 1 Groups F0,

F3.2, and F18 drank significantly less saccharin than

Groups L0, L3.2, and L18, respectively, with no differ-

ences between Groups F10 and L10. On Trials 2–4,

Groups F0 and L0 did not differ but Groups F3.2, F10,

and F18 drank significantly less saccharin than Groups

L3.2, L10, and L18, respectively (see Fig. 1).

Over conditioning, Group F0 displayed a significant

increase in saccharin intake from Trials 1 to 4 [t

(8)¼ 5.812]. Group F3.2 [t (7)¼ 3.036] exhibited no

significant changes in saccharin consumption, while

Group F10 [t (7)¼ 10.583] and Group F18 [t

(7)¼ 11.239] significantly decreased saccharin intake

over trials. Group L0 [t (8)¼ 3.440] exhibited a

significant increase in saccharin consumption over trials,

while Groups L3.2 [t (7)¼ 1.843], L10 [t (8)¼ .000],

and L18 [t (9)¼ 1.467] did not display any significant

changes in saccharin intake from Trials 1 to 4.

Two-Bottle Test. On the two-bottle test, the percent

saccharin consumed by morphine-injected adult F344

A B

FIGURE 1 Mean (�SEM) saccharin consumption (ml) by F344 (A) and LEW (B) adults

during acquisition. #Significant differences between Group 3.2 and Group 18. �Significantdifferences between Group 0 and Groups 3.2, 10 and 18. $Significant differences between Group

0 and Group 18.

982 Hurwitz et al. Developmental Psychobiology

subjects was less than that of adult LEW subjects. The

2 (Replicate)� 2 (Strain)� 4 (Dose) univariate

ANOVA on the percent saccharin consumed on the

two-bottle test revealed significant effects of Dose [F

(3, 55)¼ 25.403] and Strain [F (1, 55)¼ 9.508] as well

as a significant Dose� Strain interaction [F (3,

55)¼ 9.674]. All drug-treated F344 subjects drank a

significantly lower percentage of saccharin relative to

vehicle-treated F344 subjects. Within-strain analyses of

the LEW strain indicated that Group L18 drank a

significantly lower percentage of saccharin relative to

Group L0 with no other differences. Subsequent use of

Tukey’s HSD revealed that Groups F3.2 and F10 drank

a significantly lower percentage of saccharin than

Groups L3.2 and L10, with no strain differences

between Groups F0 and L0 and F18 and L18 (see

Fig. 2).

Experiment 2: Prepubescents

Acquisition. Over conditioning, F344 subjects drank

significantly less saccharin than LEW subjects, indica-

tive of greater morphine-induced taste avoidance. The 2

(Replicate)� 2 (Strain)� 4 (Dose)� 4 (Trial) mixed-

model ANOVA yielded significant effects of Trial [F

(3, 156)¼ 7.651], Strain [F (1, 52)¼ 198.754], Dose [F

(3, 52)¼ 21.722], and Replicate [F (1, 52)¼ 17.960] as

well as significant Trial�Replicate [F (3,

156)¼ 12.594], Trial� Strain [F (3, 156)¼ 37.621],

Trial�Dose [F (9, 156)¼ 14.885], Strain�Dose [F (3,

52)¼ 6.818], and Trial� Strain�Dose [F (9,

156)¼ 6.098] interactions.

Although F344 subjects did not differ in saccharin

consumption on the initial exposure to saccharin, on

Trial 2 Groups 10 and 18 drank significantly less than

Group 0. On Trials 3 and 4, all drug-treated F344

subjects differed from vehicle-treated subjects. On Trial

4, Group 10 drank less than Group 3.2. LEW subjects

did not differ in saccharin consumption at any point

over conditioning. On all trials, F344 subjects con-

sumed significantly less saccharin than LEW subjects.

Tukey’s HSD revealed that on Trial 1, F0 and F3.2

drank significantly less saccharin than L0 and L3.2,

respectively, with Groups F10 and F18 drinking compa-

rable levels of saccharin relative to Groups L10 and

L18. On Trial 2, Groups F0 and F3.2 did not differ

from Groups L0 and L3.2, while Groups F10 and F18

drank significantly less saccharin than Groups L10 and

L18. On Trials 3 and 4, Groups F0 and L0 did not

differ, while all drug-treated F344 subjects consumed

significantly less saccharin than their respective drug-

treated LEW subjects (see Fig. 3).

Over conditioning, Group F0 displayed a significant

increase in saccharin intake from Trials 1 to 4 [t

(8)¼ 5.812]. Group F3.2 [t (7)¼ 3.036] exhibited no

significant changes in saccharin consumption, while

Group F10 [t (7)¼ 10.583] and Group F18 [t

(7)¼ 11.239] significantly decreased saccharin intake

over trials. Group L0 [t (8)¼ 3.440] exhibited a

significant increase in saccharin consumption over trials,

while Groups L3.2 [t (7)¼ 1.843], L10 [t (8)¼ .000],

and L18 [t (9)¼ 1.467] did not display any significant

changes in saccharin intake from Trials 1 to 4.

Two-Bottle Test. On the two-bottle test, the percent

saccharin consumed by morphine-injected F344 sub-

jects was less than that of LEW subjects. The 2

(Replicate)� 2 (Strain)� 4 (Dose) univariate ANOVA

on the percent saccharin consumed revealed a signifi-

cant 3-way interaction [F (3, 52)¼ 3.349]. Subsequent

2 (Strain)� 4 (Dose) univariate ANOVAs on each

replicate revealed significant effects of Dose and Strain

as well as a significant Dose� Strain interaction

(Replicate One: [F (3, 22)¼ 19.348]; [F (1,

22)¼ 121.513]; [F (3, 22)¼ 5.120]; Replicate Two: [F

(3, 30)¼ 9.485]; [F (1, 30)¼ 14.727]; [F (3,

30)¼ 13.578]; respectively). Subsequent one-way

A B

FIGURE 2 Mean (�SEM) percent saccharin consumed by F344 (A) and LEW (B) adults.�Significant differences between Group 0 and Groups 3.2, 10 and 18. ��Significant differencesbetween Group 0 and Group 18.

Developmental Psychobiology Prepubertal Strain Differences in Morphine CTA 983

ANOVAs on the percent saccharin consumed in Repli-

cate One indicated significant differences between

groups [F (7, 29)¼ 31.796]. Tukey’s HSD indicated

that Groups F0 and L0 did not differ while all drug-

treated F344 subjects drank a significantly lower

percentage of saccharin than drug-treated LEW sub-

jects. The one-way ANOVA on Replicate Two also

indicated significant differences between groups [F (7,

37)¼ 12.181]. Subsequent use of Tukey’s HSD on

Replicate Two indicated Groups F0 and L0, F3.2 and

L3.2, and F10 and L10 did not differ while F18

drank a significantly lower percentage of saccharin than

Group L18.

Given that for each replicate, the general effect was

similar (i.e., F344>LEW), data from each replicate

were pooled for presentation (see Fig. 4).

DISCUSSION

Given that strain differences have been reported to vary

over development for a number of rodent strains and

that robust differences have been observed in taste

avoidance learning induced by a variety of drugs of

abuse in outbred prepubertal and adult rats (see above),

Experiment 2 assessed if the often-reported differences

in morphine-induced avoidance learning in adult F344

and LEW rats are also evident in prepubescence. As

described, prepubescent F344 rats acquired the avoid-

ance at a faster rate and to a stronger degree than

animals in the LEW strain, a pattern that paralleled the

results of Experiment 1 with adults under the modified

deprivation procedure and those previously reported

with adults in these strains (Davis et al., 2012; Gomez-

Serrano et al., 2009; Lancellotti et al., 2001). The

present results demonstrate that differences in mor-

phine-induced taste avoidance are evident as early as

prepubescence and are developmentally stable, suggest-

ing that these differences are highly heritable (for other

developmental strain assessments, see Allam, 2012;

Fairless et al., 2012; Farid et al., 2000; Moore

et al., 2011, 2013; Paylor et al., 1996; Satinder, 1981;

Sinaiko & Mirkin, 1974; Tonkiss et al., 1992; Vogl

et al., 1994; Wilking et al., 2012).

A B

FIGURE 4 Mean (�SEM) percent saccharin consumed by F344 (A) and LEW (B)

prepubescents. �Significant differences between Group 0 and Groups 3.2, 10, and 18.

A B

FIGURE 3 Mean (�SEM) saccharin consumption (ml) by F344 (A) and LEW (B)

prepubescents during acquisition. #Significant differences between Group 0 and Groups 10 and

18. �Significant differences between Group 0 and Groups 3.2, 10 and 18. ^Significant differences

between Group 3.2 and Group 10.

984 Hurwitz et al. Developmental Psychobiology

Although strain differences are clearly evident in

morphine-induced taste avoidance as early as prepubes-

cence, the basis for these differences are unknown. One

obvious possibility is that the two strains may differ in

blood and/or brain levels of morphine. Interestingly, mor-

phine plasma levels do not differ between adult F344 and

LEW rats (Davis & Riley, 2007; Gosnell & Krahn, 1993;

Lancellotti et al., 2001), although one report has demon-

strated that F344 animals have higher brain morphine

levels relative to LEW animals 30min following mor-

phine injection (Gosnell & Krahn, 1993). It remains

unknown whether adolescents show a similar pattern of

peripheral and/or central distribution and how that may

relate to strain differences in avoidance learning.

Given that the F344 strain displays greater stress

reactivity then the LEW strain (Dhabhar, McEwen, &

Spencer, 1993; Dhabhar, Miller, McEwen, & Spencer,

1995; Sternberg et al., 1992; for a review see Kosten &

Ambrosio, 2002), the reported strain differences in

morphine-induced taste avoidance could be due to

potential stress associated with the specific procedures

utilized in conditioning taste avoidance in the present

experiments, for example, deprivation, handling, injec-

tion. Despite the differential stress reactivity in these

strains (F344>LEW), work assessing the effects of

stress on the acquisition and expression of taste avoid-

ance in outbred subjects is mixed, typically with the

results indicating no direct relationship between stress

and the degree of taste avoidance conditioned (Ander-

son, Hinderliter, & Misanin, 2006; Bourne, Calton,

Gustavson, & Schachtman, 1992; Bowers, Amit, &

Gringras, 1996; Misanin, Kaufhold, Paul, Hinderliter, &

Anderson, 2006; Revusky & Reilly, 1989). It is impor-

tant to note that the vast majority of this work has been

done in adults, precluding any conclusions as to how

stress might differentially impact prepubescent F344

and LEW subjects. Interestingly, in the one direct

assessment of the effects of different stressors (isolation

housing, restraint stress) on the acquisition of ethanol-

induced taste avoidance in outbred prepubertal and adult

rats stress had no significant effect on ethanol-induced

taste avoidance in either age group (Anderson

et al., 2010).

It is possible that differences in taste, learning and

memory processing mediate the differences between

the F344 and LEW strains. These are unlikely, howev-

er, given that the reported differences between the two

strains are highly dependent upon the specific drug

examined. For example, although F344 rats display

greater morphine-induced taste avoidance than LEW

rats, the difference is reversed for cocaine (i.e., LEW>F344; Davis & Riley, 2007; Glowa et al., 1994). For

other compounds such as LiCl (Foynes & Riley, 2004),

there are no strain differences. The most parsimonious

explanation for the present results is that morphine is

differentially aversive in these strains. What accounts

for this differential sensitivity, however, remains un-

known (for a discussion of the nature of avoidance

learning, see Verendeev and Riley, 2012), although

when c-fos activity in the brainstem is examined in the

two strains, cellular activity in avoidance-related areas

(Grabus, Glowa, & Riley, 2004) are differentially

activated following morphine and in ways that parallel

the ability of morphine to induce aversions in the two

strains, that is, morphine which induces greater avoid-

ance in the F344 rat induces significantly greater

activity in these areas than that seen in the LEW strain.

Such assessments are limited to a few drugs and in

adults, so it is again unclear if these results generalize

to other ages.

The present data are suggestive of highly heritable

behavioral differences between the two strains; howev-

er, they do not argue that such differences cannot be

impacted by a host of environmental challenges. In

fact, a number of behavioral differences between the

strains are impacted by stress (Grakalic et al., 2006;

Siviy et al., 2003), diurnal cycle (Gomez-Serrano et al.,

2009), and maternal rearing (Gomez-Serrano et al.,

2001, 2002; Gomez-Serrano & Riley, 2006; Riley,

2011; Roma, Davis, & Riley, 2007; Siviy et al., 2003).

Further, it is unknown to what degree prenatal experi-

ence impacts the behavioral differences reported here.

What is clear is that the differences in morphine-

induced taste avoidance learning in these two strains

are evident early in life and are developmentally stable.

Further assessments are needed to determine the point

at which these behavioral differences become evident

and the basis for this stability.

NOTES

This work was supported in part by grants from the Mellon

Foundation to ALR and ZEH and the American University

Artists and Scholars Fellowship to APM.

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