35 Dp Init - Brief

8/12/2019 35 Dp Init - Brief

1/17

Brief Report

Binge

Q1

Ethanol IntoxicationHeightens Subsequent EthanolIntake in Adolescent, But NotAdult, Rats

ABSTRACT: A question still to be answered is whether ethanol initiation has agreater effect on ethanol consumption if it occurs during adolescence than inadulthood. This study assessed the effect of ethanol initiation during adolescenceor adulthood on voluntary ethanol consumption when animals were still within

the same age range. Adolescent or adult rats were given 5, 2, or 0 ethanol expo-sures. The animals were tested for ethanol consumption through two-bottle choicetests, before undergoing a 1-week deprivation. A two-bottle assessment was con-ducted after the deprivation. Adolescents, but not adults, given two ethanoladministrations during initiation exhibited significantly higher ethanol intake dur-ing the pre-deprivation period. These adolescents also exhibited a threefold in-crease in ethanol intake after 7 days of drug withdrawal, when compared withcontrols. These findings suggest that very brief experience with binge ethanolintoxication in adolescence, but not in adulthood, impacts later predisposition todrink. 2012 Wiley Periodicals, Inc. Dev Psychobiol 9999: 110, 2012.

Keywords: adolescent; rat; ethanol initiation; ethanol intake; light-dark test

INTRODUCTION

EthanolQ4 use begins most frequently during adoles-

cence, and earlier-onset ethanol initiation is associated

with a greater probability of ethanol use disorders

(DeWit, Adlaf, Offord, & Ogborne, 2000). Whether a

causal relationship exists between these variables or

whether they are manifestations of a third phenomenon

is still unclear (Schramm-Sapyta, Kingsley, Rezvani,

Swartzwelder, & Kuhn, 2009).

The prototypical pattern of ethanol self-administration

in adolescence involves ethanol binge drinking, leading

to blood ethanol concentrations 80 mg/dl. This

binge drinking appears to facilitate the escalation of

ethanol consumption (Hargreaves, Monds, Gunasekaran,

Dawson, & McGregor, 2009).

Adolescent animal models have revealed differences

between adolescents and adults in the initial response

to ethanol (Silveri & Spear, 1998, 2001). Adolescents

are more sensitive to ethanol-induced reinforcement

and social facilitation (Pautassi, Myers, Spear, Molina,

& Spear, 2008; Spear & Varlinskaya, 2005, 2010); but

less sensitive than adults to the sedative and motor

impairing, and acute hangover effects of ethanol (Dore-

mus, Brunell, Rajendran, & Spear, 2003; Ristuccia &

Spear, 2004; Silveri & Spear, 1998, 2001). This re-

sponse pattern may represent a risk factor for the devel-

opment of problematic ethanol intake.Previous studies indicated that the adolescent con-

sumption of ethanol can be further enhanced by early

exposure to ethanol during the prenatal or early postna-

tal period (Abate, Pueta, Spear, & Molina, 2008). A

scarcity of animal studies have assessed the effects of

ethanol exposure during adolescence on later adoles-

cent ethanol self-administration. An important question

to test using animal models is whether adolescent etha-

nol initiation has a differential effect on ethanol intake

than if initiation is delayed until adulthood. Hargreaves

DEV-12-077.R2(21101)

Developmental Psychobio

Mara Carolina Fabio1,2

Michael E. Nizhnikov3

Norman E. Spear3

Ricardo Marcos Pautassi1,2

1Instituto de Investigacion Medica M. y M.Ferreyra, INIMEC-CONICET, Universidad

Nacional de Cordoba, Cordoba, C.P.5000, Argentina

E-mail: [email protected]

2Facultad de Psicologa, UniversidadNacional de Cordoba, Cordoba, C.P.

5000, Argentina

3Center for Developmental Psychobiology,Binghamton University, Binghamton, New

York 13902-6000

Manuscript Received: 4 August 2012Manuscript Accepted: 12 December 2012Correspondence to: Ricardo Marcos PautassiContract grant sponsor:NIAAAQ3

Contract grant numbers: AA018164 PIP 2010-2012 SECYT-UNCPICTPRH 246

Article first published online in Wiley Online Library(wileyonlinelibrary.com).

DOI 10.1002/dev.21101 2012 Wiley Periodicals, Inc.

8/12/2019 35 Dp Init - Brief

2/17

et al. (2009) found that intermittent access to ethanol-

induced binge-like drinking in adolescent but not adult

rats. Siegmund, Vengeliene, Singer, & Spanagel (2005)

provided evidence that adolescent ethanol initiation en-

hanced stress-induced ethanol consumption. A subse-

quent study found that adult female rats that initiated

ethanol self-administration during adolescence weremore sensitive to stress-induced ethanol intake than

controls that initiated ethanol intake in adulthood (Full-

grabe, Vengeliene, & Spanagel, 2007).

Overall, these studies are consistent with epidemiolog-

ical data, in which the adolescent onset of ethanol expo-

sure promoted ethanol consumption (but see Tambour,

Brown, & Crabbe, 2008). One limitation of these long-

term access studies is that they relied on the self-admin-

istration of ethanol as the method of initiation. With this

method, level of intoxication is not controlled and the

influence of age of ethanol initiation is confounded by

baseline differences in drinking between adolescents andadults. Another limitation was the use of lengthy initia-

tion phases that did not allow the endpoint section of

intake tests to occur within the adolescent period.

One alternative is to expose animals to controlled

intragastric or intraperitoneal administrations of etha-

nol, which allow greater control over dose. Adolescent

rats repeatedly exposed to ethanol exhibited changes in

neurotransmitter systems (Pascual, Boix, Felipo, &

Guerri, 2009), that were associated with greater ethanol

intake in adulthood. This study, however, lacked a com-

parison with adult animals exposed to ethanol in a sim-

ilar fashion as adolescents.

The present study assessed the effects of brief etha-nol initiation during adolescence or adulthood on the

predisposition for voluntary ethanol consumption when

the animals were still within the same age range. Ani-

mals were given five or two intermittent exposures to

ethanol, or vehicle, and then were tested for ethanol

intake. The intake protocol involved an initial phase of

4 days in which subjects were tested with a two-bottle

procedure, followed by a 1-week ethanol deprivation

phase and a post-deprivation assessment of ethanol in-

take. The hypothesis was that passive exposure to etha-

nol augments ethanol consumption in adolescent but

not in adult rats. To examine the potential mechanismsthat underlie this putative effect, anxiety was measured

using the light/dark box test before self-administration

and on the last day of the ethanol-deprivation period.

MATERIALS AND METHODS

Experimental Design

AQ5 2 (age: adolescence or adulthood) 3 (ethanol initiation

treatment: 5-day exposure group, 2-day exposure group, and

control group treated with vehicle) factorial design was

employed, with 1013 animals in each group. Fourteen ani-

mals (7 adolescent and 7 adults) were included in an untreat-

ed (UT) condition.

Subjects

Seventy-four (39 adolescents and 35 adults) outbred male

Wistar rats were used. Adolescents and adults were 28 and70 postnatal days (PD) old, respectively, at the beginning

of procedures. Animals were born and reared in a tempera-

ture-controlled vivarium (21228C) at INIMEC-CONICET

(Cordoba, Argentina). Births were examined daily, and day

of parturition was considered PD0. Culling was conducted

on PD1 and pups remained with their respective dams until

PD 21. After weaning, animals were housed together until

PD24 when they were paired-housed according to their ex-

perimental condition. Half of the males of a given litter

were tested during adolescence, whereas the remaining half

was tested during adulthood. The procedures complied with

the National Institutes of Health Guide for Care and Use of

Laboratory Animals (National Research Council, 1996Q6)

and were approved by the Institutional Animal Care and

Use Committee.

Ethanol Administration (i.e., Initiation) Procedures

Ethanol initiation began on PD28 and PD70 for adolescents

and adults, respectively. The animals were given intragas-

tric administrations as described in Pautassi et al. (2008)

every other day and received five intubations of 2.5 g/kg

ethanol (5-day exposure group; ethanol on PD28, 30, 32,

34, and 36 [adolescents] or PD70, 72, 74, 76, and 78

[adults]), two intubations of 2.5 g/kg ethanol (2-day expo-

sure group; ethanol on PD28 and 32 and vehicle on PD30,

34, and 36 [adolescents] or ethanol on PD70 and 74 and

vehicle on PD72, 76, and 78 [adults]), or no intubations of

ethanol (control group treated with vehicle on PD28-36

[adolescent] or PD70-78 [adults]). The ethanol dose was

selected on the basis of studies from our laboratory

(Acevedo, Molina, Nizhnikov, Spear, & Pautassi, 2010) that

suggested its effectiveness in promoting ethanol intake in

adolescents. This was achieved by administering .015 ml/kg of

a 21% ethanol solution (96% proof ethanol, Porta Hnos.,

Cordoba, Argentina; vehicle: tap water).

Untreated animals experienced only standard housing dur-

ing PDs 28-36 or PDs 70-78. These animals were then tested

for sucrose intake on PDs 37 or 79, and for anxiety response

on PDs 38 or 80 (adolescent and adult subjects, respectively).

Untreated subjects were not tested for ethanol intake test. Thepurpose of including this group was to control for alterations

in sucrose preference or anxiety resulting from the manipula-

tions inherent to initiation procedures.

Homecage Ethanol Self-Administration Tests

On PD37 (adolescents) or PD79 (adults), the animals were

individually housed and exposed for 24 hr to two bottles

equipped with ball-point tubes, and filled with tap water or

1% w/v sucrose. The aim was to assess potential differences

in sucrose preference after initiation procedures.

2 Fabio et al. Developmental Psychobiology

8/12/2019 35 Dp Init - Brief

3/17

On PD39-42 or PD80-83 (self-administration phase for

adolescents and adults, respectively) the animals had continu-

ous access to two drinking bottles, one containing a 5.6% v/v

ethanol solution sweetened with 1% w/v sucrose and another

that contained water. Sweetened ethanol was chosen based on

the finding that adolescent Wistar rats did not voluntarily con-

sume unsweetened ethanol (5.6% v/v ethanol) but consumed

slightly sweetened (sucrose 1% w/v) ethanol (5.6% v/v) over-

night (Maldonado, Finkbeiner, & Kirstein, 2008). The avail-

able protocols to induce significant consumption of

unsweetened ethanol in rats are quite lengthy and, if used in

the present study, would not have allowed the post-depriva-

tion intake test to occur within the adolescent period. We pre-

viously employed unsweetened ethanol to test adolescent

self-administration (Ponce, Pautassi, Spear, & Molina, 2004,

2011), but these tests required extensive, 22-hr liquid depriva-

tion, which is a significant stressor.

The position of the water and ethanol bottles was varied

across sessions to avoid side-preference effects. On each test

day, the bottles were positioned at 11:00 AM to 12:00 PM,

and the animals were left undisturbed. On the following day(9:00 AM to 10:00 AM), the bottles were removed, weighed

to the nearest .1 g and refilled. During weighing and refilling

animals were allowed to interact with each other for 2 hr to

minimize stress-induced isolation. Ethanol intake is expressed

as grams of ethanol consumed per kilogram of body weight

(g/kg). Spillage was accounted for by placing water and etha-

nol bottles in an empty holding cage overnight. The differ-

ence in weight was subtracted from each animals intake

score to account for spillage that may have occurred over-

night. Food consumption was monitored by providing a pre-

measured amount of food and weighing the portion that

remained each day.

After the 4-day self-administration phase, the animals un-

derwent a 7-day ethanol recess phase, in which they had ad

libitum access to water and food. On PD50 or 92 (post-depri-

vation assessment for adolescents and adults, respectively)

animals were tested for 24 hr in ethanol self-administration,

through the two-bottle procedure described for the pre-depri-

vation self-administration phase. The rationale for the week

interval between the initial 4 days of self-administration and

the last ethanol intake assessment was to fit the treatment and

the test within the same adolescent period. We were also

guided by a recent study in which animals experienced a

short ethanol deprivation period between ethanol intake tests

(Acevedo et al., 2010).

Light/Dark Box Test

A light/dark box (LDB) test was conducted on PD38 and 48

(adolescents) or PD80 and 90 (adults). The aim was to detect

possible differences in anxiety-like behavior that could ex-

plain ethanol drinking patterns. The light/dark box exploits

the conflict between the animals tendency to explore a new

environment and its fear of bright light (Cancela, Bregonzio,

& Molina, 1995).

The apparatus (42 25 25 cm3) consisted of two com-

partments made of high impact acrylic, one dark

(17.5 25 25 cm3) illuminated with a 25 W red bulb, and

one white (24.5 25 25 cm3) illuminated by a 60 W

white bulb lamp. The compartments were separated by a di-

vider with a 6.5 6.5 cm2 opening at floor level. The test

lasted 5 min and began by gently placing the animal in the

center of the dark area, facing away from the white area. The

test was videotaped and then analyzed by a researcher blind

to the treatments. The latency to cross to the white compart-

ment, number of transfers from one compartment to the other

compartment, and time spent in the white compartment were

measured using Etholog software (Ottoni, 2000).

Data Analysis

The preliminary analysis indicated significant age-related dif-

ferences in the initial consumption of ethanol, with adolescent

subjects drinking almost twice as much ethanol as adults in

self-administration Session 1 [F1,65 5.17, p < .05]. Adults

subjects, on the other hand, exhibited greater overall locomo-

tion in the light/dark box test [F1,65 5.99, p < .01]. Base-

line differences between adolescents and adults have often

been observed across several variables, including ethanol in-take (Doremus, Brunell, Rajendran, & Spear, 2005), stress re-

sponsiveness (Varlinskaya & Spear, 2012), and preference for

tactile cues (Pautassi et al., 2008). We followed the statistical

approach of the latter studies and analyzed each dependent

variable using separate analyses of variance (ANOVAs) for

adolescents and adults. The loci of significant main effects or

significant interactions were analyzed using Tukey post hoc

tests. Planned comparisons were also conducted if justified by

previous hypotheses.

Self-Administration During Pre-Deprivation and Post-Depri-

vation Sessions. For each age, ethanol intake (g/kg) during

the 4-day pre-deprivation phase was analyzed using two-way

mixed-factor ANOVAs. Ethanol initiation (5-day exposure, 2-day exposure, and the control group) was the between-group

factor and session was the within-group measure. Similar

ANOVAs were used to analyze water intake (ml/100 g) and

food intake (g/kg) across the procedures. One-way ANOVAs

(comparative factor: ethanol initiation condition) were per-

formed for sucrose intake on the adaptation test day (g/kg,

the UT group was also included in this ANOVA) and for eth-

anol intake (g/kg) in the post-deprivation assessment.

Light/Dark Box Tests on PD38 and 48 (Adolescents) or PD80

and 90 (Adults). The latency to cross to the white side, num-

ber of transfers from one compartment to the other compart-

ment and time spent in the white compartment were analyzedfor adolescents and adults using two-way ANOVAs (compara-

tive factor between groups: ethanol initiation condition; with-

in-group measure: day of testing). Planned comparisons were

conducted between the UT and the vehicle-exposed group, for

each of the variables measured on the first lightdark test.

We further analyzed ethanol intake measurements using a

correlational approach. Pearsons rproductmoment correlations

were separately conducted for the adolescent and adult groups,

and for each ethanol treatment at initiation. The specific question

under analysis was to what extent initial self-administration of

ethanol predicted later ethanol self-administration during the

Developmental Psychobiology BingeQ2

Ethanol Heightens Subsequent Ethanol Intake 3

8/12/2019 35 Dp Init - Brief

4/17

initial 4-day phase and during the post-deprivation intake ses-

sion. It has been suggested that the quality of the initial expe-

rience with ethanol significantly affects later ethanol drinking

(Schramm-Sapyta et al., 2009). Moreover, it has been ob-

served that patterns of ethanol intake are established early in

adolescent rats, with subjects that drink heavily during the

first exposure to ethanol keeping this behavior on subsequent

sessions (Schramm-Sapyta et al., 2008). It is still unknown,

however, if ethanol drinking behavior follows a similar trajec-

tory in adult subjects, and if this pattern is altered in subjects

given prior ethanol initiation.

RESULTS

Body Weight, Sucrose Consumption DuringAdaptation and Food Intake

The administration of ethanol during initiation did not

significantly alter body weight during the 4-day pre-

deprivation phase or during the post-deprivation assess-ment. As expected, the ANOVAs only indicated that

body weight in the adolescents increased as a function

of days during the pre-deprivation two-bottle intake

assessment,F3,99 361.4, p < .001. Sucrose consump-

tion (g/kg) at beginning of intake tests (PD37 or PD79)

was not affected by ethanol initiation. Sucrose intake in

animals given 5-day ethanol exposure, 2-day ethanol ex-

posure, vehicle controls and in untreated controls was

.54 .08, .52 .08, .45 .06, and .34 .10; and

2.12 .47, 2.58 .47, 2.11 .39, and 1.98 .54; for

adolescents and adults, respectively.

Food intake during the 4 days in which subjects

were tested with the two-bottle procedure increased inadolescents as a function of days [F3,99 41.83,

p < .001] but remained stable in adults. These patterns

were unaffected by ethanol initiation treatment. One-

way ANOVAs indicated that food intake during the

post-deprivation session was similar across initiated

and non-initiated adolescents or adults.

Descriptive results for body weight (g) and food in-

take (g/kg) are depicted in Table 1.

Ethanol and Water Self-Administration DuringEthanol Intake Tests

Adolescent Rats. The ANOVA for the 4-day phase in

which subjects were tested with the two-bottle proce-

dure revealed a significant main effect of session,

F3,99 5.19, p < .005. The post hoc tests indicated

greater ethanol consumption during Session 1 than in

the subsequent sessions, which in turn did not differ

Table 1. Body Weight (g), Water Intake (ml/100 g of Body Weight), Food Intake (g/kg of Body Weight) and Ethanol

Intake (ml/100 g of Body Weight); in Adolescent and Adult Rats During the 4 Days in Which Subjects Were Tested With a

Two-Bottle Procedure (Pre-Deprivation Intake Sessions) and During the Post-Deprivation Intake Session

Adolescent Rats Adult Rats

5-Day Group 2-Day Group Control 5-Day Group 2-Day Group Control

Body weight (g)

Pre-deprivation intake session 1 185.92 5.91 17 6.83 4.10 182.09 5.93 443.82 13.10 433.75 10.99 456.58 9.00



Pre-deprivation intake session 4 214.54 7.08 20 7.75 5.15 214 5.56 458.27 13.56 419.42 26.16 468.92 9.39

Post-deprivation intake session 285.54 10.98 278.83 7.99 16.65 4.59 482.64 16.79 470.67 11.45 496.33 11.05

Food intake (g/kg)

Pre-deprivation intake session 1 4.24 .26 3.83 .17 4.37 .20 13.19 .65 12.59 .68 13.41 .86

Pre-deprivation intake session 2 4.72 .37 4.20 .39 4.80 .41 12.86 .84 13.63 .81 14.42 .57

Pre-deprivation intake session 3 5.24 .33 5.02 .22 5.46 .25 13.36 1.05 13.29 .78 14.15 .71

Pre-deprivation intake session 4 8.61 3.55 5.08 .24 5.68 .40 12.57 1.01 12.17 .95 13.91 .69

Post-deprivation intake session 7.86 .67 7.38 .53 8.29 .56 13.45 .70 13.74 .75 13.30 1.39

Water intake (ml/100 g)

Pre-deprivation intake session 1 9.70

1.62 8.94

1.82 12.99

1.84 11.11

1.31 9.10

1.48 7.67

1.02Pre-deprivation intake session 2 9.28 1.70 10.19 1.86 12.20 2.00 11.18 1.82 10.23 1.61 8.64 1.32

Pre-deprivation intake session 3 11.91 1.51 12.99 1.90 14.39 2.15 9.21 2.53 9.25 1.75 8.60 1.39


Post-deprivation intake session 6.76 1.36 6.68 1.38 7.98 1.58 10.63 2.66 10.28 2.24 7.63 1.66

Ethanol intake (ml/100 g)

Pre-deprivation intake session 1 7.12 1.94 10.55 2.42 5.43 1.94 2.56 1.02 5.38 1.56 3.20 .88


Pre-deprivation intake session 3 5.15 1.58 5.49 2.00 3.04 1.57 6.07 1.85 3.54 1.13 3.32 .78

Pre-deprivation intake session 4 8.69 1.57 10.07 1.94 5.78 1.63 8.80 .24 10.47 1.40 8.57 .18

Post-deprivation intake session 18.89 .62 19.40 .52 18.83 .52 7.59 1.89 6.51 1.83 7.88 1.25

Results are presented as mean standard error of the means.


8/12/2019 35 Dp Init - Brief

5/17

between each other. Guided by a priori hypothesis,

planned comparisons were conducted between the

5-day or the 2-day ethanol treatment group and the

control group, for each intake session. These analyses

revealed significantly greater ethanol intake scores

(g/kg) in adolescents given 2-day exposure than in con-

trol counterparts, during pre-deprivation intake sessions

1 and 2 (F1,33 4.61, F1,33 5.24, both ps < .05; for

Sessions 1 and 2, respectively). These results are

depicted in Figure 1.

FIGURE 1 Ethanol intake (g/kg) in adolescent (upper panels) and adult (lower panels) male

rats during the 4-day self-administration phase and during the post-deprivation assessment.

Ethanol intake during the initial 4 days of self-administration is depicted as a function of day

of assessment (Sessions 1, 2, 3, and 4) and ethanol treatment during initiation. Initiation oc-

curred on postnatal days 28-36 (PD28-36) or PD70-78 for adolescents and adults, respectively.

During initiation, the animals were given intragastric administrations every other day and re-

ceived five 2.5 g/kg ethanol intubations (5-day exposure group; ethanol on PD28, 30, 32, 34,

and 36 [adolescents] or PD70, 72, 74, 76, and 78 [adults]), two 2.5 g/kg ethanol intubations

(2-day exposure group; ethanol on PD28 and 32 and vehicle on PD30, 24, and 36 (adoles-cents) or ethanol on PD70 and 74 and vehicle on PD72, 76, and 78 [adults]), or no ethanol

intubations (control group treated with vehicle on PD28-36 [adolescents] or PD70-78 [adults]).

The initial self-administration phase (i.e., pre-deprivation assessment of ethanol intake) lasted

for 4 days, in which the animals were given continuous, 22 hr two-bottle choice between

ethanol (5.6% v/v) and water in the homecage. After the last acquisition session, the animals

had ad libitum access to only water and food for 7 days, and then a single 22-hr test of ethanol

intake was conducted (two-bottle homecage choice test between 5.6% v/v ethanol and water;

post-deprivation assessment). Adolescents but not adults given two ethanol exposures during

initiation exhibited significantly greater ethanol intake than controls during pre-deprivation

intake Sessions 1 and 2, and during the post-deprivation assessment. These significant effects

of ethanol initiation are indicated by the asterisk. The vertical bars indicate SEM.

Developmental Psychobiology Binge Ethanol Heightens Subsequent Ethanol Intake 5

8/12/2019 35 Dp Init - Brief

6/17

Ethanol initiation also appeared to heighten ethanol

intake during the post-deprivation assessment (see

Fig. 1). The one-way ANOVA revealed a significant

main effect of treatment at initiation (F2,33 3.73,

p < .005). Subsequent post hoc tests indicated signifi-

cantly greater consumption of ethanol in adolescents

given 2-day exposure than in the control group. Ani-mals that received five intubations also exhibited great-

er ethanol intake than controls, but the difference did

not achieve statistical significance.

The ANOVA for water intake during pre-deprivation

intake sessions indicated only a significant main effect

of session (F3,99 4.41, p < .005), and the post hoc

tests revealed greater water intake on Days 3 and 4 of

the pre-deprivation intake phase than on Days 1 and 2.

Treatment at initiation did not modify water intake dur-

ing pre-deprivation sessions or during the post-depriva-

tion session. The average mean (SEM) water intake

and ethanol intake (ml/100 g) in adolescents given 5-day exposure, and 2-day exposure and controls is

depicted in Table 1.

Adult Rats. Ethanol drinking in adult rats remained

stable during the 4 days in which subjects were tested

with a two-bottle procedure and was not affected by

the ethanol initiation procedures. The ANOVAs indicat-

ed a lack of significant main effects or significant inter-

actions. The planned comparisons between the 5-day or

the 2-day ethanol treatment group and the control

group did not achieved significance. Unlike adolescent

subjects, post-deprivation ethanol intake in adult rats

was insensitive to previous passive exposure to ethanol(see Fig. 1). The ANOVA revealed that ethanol-initiat-

ed and control (vehicle-treated) subjects exhibited simi-

lar levels of ethanol consumption.

The ANOVAs for water intake during the four pre-

deprivation sessions and during the post-deprivation

session indicated no significant main effects or signifi-

cant interactions. Average mean and SEM water intake

and ethanol intake (ml/100 g) across sessions is pre-

sented in Table 1.

Light/Dark Box Test

Adolescent Rats. Adolescents given 0, 2, or 5 ethanol

exposures at initiation exhibited similar latency to cross

to the white side, number of transfers from one com-

partment to the other, and similar time spent in the

bright side of the light/dark box. This pattern was fairly

similar during the first and second light/dark box tests.

The ANOVA revealed a significant main effect of day

of testing on the number of transfers, [F1,33 4.27,

p > .05], indicating a reduction of overall locomotor

activity in the second test session.

Adult Rats. Ethanol initiation did not exert significant

main effects or significantly interact with the remaining

variables in any of the measures. The number of trans-

fers tended to decrease in the second test, as indicated

by a borderline main effect of day of testing (p .056).

Planned comparisons indicated that anxiety during

the first lightdark test did not significantly differ be-tween the untreated and vehicle controls, neither in

adolescents nor in adults.

Correlation Between Measures of Ethanol Intake

Adolescent Rats. Ethanol intake was highly correlated

during the four pre-deprivation intake sessions in all of

the ethanol initiation conditions. Post-deprivation etha-

nol intake was predicted in the control groupbut not

in the 5- or 2-day exposure groupby ethanol intake

during acquisition sessions 14 (r .75, .89, .80, and

.77; respectively) (Tab. 2).

Adult Rats. In control adults ethanol intake on pre-dep-

rivation day 2 was predictive of ethanol intake on pre-

deprivation day 3 and the latter predicted ethanol intake

at pre-deprivation day 4. The animals given five ethanol

exposures exhibited a significant association between

ethanol intake on pre-deprivation day 1 and ethanol

self-administration on pre-deprivation days 2 and 4. In

this group, post-deprivation ethanol consumption was

predicted by pre-deprivation day 3. In the 2-day ethanol

initiation group ethanol intake on pre-deprivation day 1

predicted intake on pre-deprivation days 2 and 3, and

post-deprivation ethanol intake was predicted by etha-

nol intake on pre-deprivation days 1, 2, and 3 (r .81,.73, and .93, respectively).

DISCUSSION

The present study analyzed whether brief and intermit-

tent exposure to ethanol heightens later ethanol intake,

particularly after a period of ethanol deprivation, and

tested the age-specificity of this effect. Consistent with

previous studies (e.g., Doremus et al., 2005), the initial

self-administration of ethanol was greater in adoles-

cents than in adults. The most important new finding

was that adolescent rats exposed to binge ethanol intox-ication subsequently exhibited greater ethanol intake

than control subjects. Specifically, binge ethanol in-

duced a significant increase in ethanol intake in adoles-

cents during intake Sessions 1 and 2 of the initial self-

administration phase. Moreover, when tested after

7 days of drug withdrawal, adolescents given two pas-

sive ethanol intubations exhibited a significant threefold

increase in ethanol intake compared with age-matched

controls. These effects were not found in adults and no

changes in water intake were observed.


8/12/2019 35 Dp Init - Brief

7/17

Rodd-Henricks et al. (2002a, 2002b) gave adolescent

or adult ethanol-preferring rats free choice access to

ethanol or standard housing. During adulthood, ethanol

operant self-administration was greater in animals that

had been initiated to ethanol during adolescence. This

and others (e.g., Fullgrabe et al., 2007; Siegmund et al.,

2005) are similar to the present study in that they ex-

amined the predisposition for ethanol intake after ado-

lescent exposure. The previous studies, however,

employed extensive initiation phases (e.g., >7 months,

Tambour et al., 2008) that lasted past adolescence, not

allowing the intake test to occur within the adolescent

Table 2. Pearson Correlations Between Ethanol Intake (g/kg) During Acquisition Days 14 and During the

Post-Deprivation Assessment, for Adolescent and Adult Rats Given 5 [5-Day Exposure Group], 2 [2-Day Exposure Group]

or 0 [Control Group Treated With Vehicle] Administrations of 2.5 g/kg Ethanol During Ethanol Initiation Procedures

Ethanol Intake

at Acquisition

Day 1

Ethanol Intake

at Acquisition

Day 2

Ethanol Intake

at Acquisition

Day 3

Ethanol Intake

at Acquisition

Day 4

Post-Deprivation

Assessment of

Ethanol Intake

Adolescent rats

Ethanol intake at Acquisition day 1

5-Day group

2-Day group

Control


5-Day group .65

2-Day group .87

Control .82


5-Day group .69 .68

2-Day group .78 .95

Control .65 .93

Ethanol intake at Acquisition day 45-Day group .64 .76 .90

2-Day group .79 .94 .97

Control .64 .91 .99

Post-deprivation assessment of ethanol intake

5-Day group .27 .47 .27 .46

2-Day group .29 .27 .11 .21

Control .75 .89 .80 .77

Adult rats


5-Day group

2-Day group

Control


5-Day group .76

2-Day group .88

Control .12


5-Day group .53 .41

2-Day group .75 .67

Control .25 .71


5-Day group .79 .97 .44

2-Day group .06 .12 .33

Control .33 .10 .50

Post-deprivation assessment of ethanol intake

5-Day group .45 .57 .82 .57

2-Day group .81 .73 .93 .10 Control .06 .29 .08 .10

Correlations significant at p

8/12/2019 35 Dp Init - Brief

8/17

period. Moreover, in these studies the initiation phase

consisted of self-administration of ethanol, which

implies habituation of neophobia, familiarization with

the flavor of ethanol and usually results in baseline dif-

ferences in drinking across sessions. The latter factor is

particularly important when comparing adults and ado-

lescents, given the propensity of adolescents to drinksignificantly more ethanol than adults (Doremus et al.,

2005). In the present study, the choice of intragastric

intubation as the method for ethanol initiation equated

level of ethanol exposure across ages. The present

study is also significant in that it specifically investigat-

ed whether early-onset drinking in an early stage of

adolescence facilitates the escalation of ethanol con-

sumption during adolescence.

The alcohol exposure procedure employed in the

present study involved 5 or 2 binge exposures, followed

by 4 days of self-administration to a moderate concen-

tration of alcohol. Caution should be taken, therefore,when comparing the results of this study with others

that employed protracted exposure to ethanol or repeat-

ed cycles of access to and withdrawal from ethanol

(e.g., Bell et al., 2008; Spanagel, 2000). Those proce-

dures are specifically aimed to model alcoholism and

proven to induce tolerance, physical dependence, and

withdrawal symptoms (Spanagel, 2000).

Another caveat of the present study is the use of

sweetened ethanol. The enhanced predisposition for

ethanol drinking in ethanol-initiated adolescents may

have been attributable to early ethanol exposure alter-

ing the palatability of sucrose or inducing sucrose seek-

ing due to its nutritional value. There were, however,no differences in sucrose intake across ethanol initia-

tion conditions for adolescents or adults, when the

sweet tastant was tested alone against water. Likewise,

no differences were found in food intake or body

weight that could have indicated altered nutritional sta-

tus due to early ethanol exposure.

The most intriguing result of the study was the

significant increase in ethanol self-administration ob-

served in adolescents after just 2 days of ethanol ex-

posure. These animals drank more ethanol than non-

initiated counterparts during the first 2 days of self-

administration as well as during the post-deprivationassessment. What is the potential mechanism underly-

ing higher ethanol consumption in ethanol-initiated

adolescents? Ethanol exposure during passive initia-

tion or during the initial self-administration phase

could conceivably alter anxiety-like behavior. The

results of the light/dark box test, however, indicated

similar patterns of exploration in ethanol-initiated

and non-initiated animals.

Another possibility is that adolescent ethanol initia-

tion facilitates subsequent ethanol intake by altering the

normal pattern of development of specific transmitter

systems. A previous study found that chronic intermit-

tent ethanol exposure during adolescence altered basal

level of dopamine in nucleus accumbens and induced a

down-regulation of dopaminergic and glutamatergic

receptors in prefrontral cortex (PFC; Pascual et al.,

2009). Fabio, Nizhnikov, Spear, & Pautassi (2012)revealed that earlier ethanol initiation in the rat (during

the last 4 days of gestation) also altered basal neural

activity at PFC, when measured at adolescence. These

effects were associated with heightened ethanol

consumption.

Intriguingly, ethanol exposure on PD28 and PD32

increased later ethanol intake, but the 5-day exposure

treatment apparently did not. It could be that ethanol

initiation enhances subsequent ethanol intake when ini-

tiation occurs during restricted developmental time-

frames, such as during early adolescence.

Dopaminergic receptors, which modulate ethanol-in-duced appetitive learning, reach their peak at PD28,

and then decline significantly (Tarazi & Baldessarini,

2000). The present results are also in agreement with

Acevedo et al. (2010), in which ethanol exposure at

PD28 but not at PD31, enhanced subsequent ethanol

consumption. It is also possible that the 5-day exposure

treatment lacked an effect on subsequent intake due to

this treatment inducing tolerance to the appetitive

effects of ethanol.

Yet another possibility is that animals exposed twice

to ethanol on PD28 and PD32 acquired a conditioned

preference for ethanols orosensory cues due to percep-

tion of non-metabolic ethanol excretion (by urine orperspiration) in close contiguity with pharmacological

properties of the drug (Molina, Chotro, & Spear, 1989).

On the other hand, animals exposed to the 5-day expo-

sure treatment may have developed a conditioned aver-

sion, which competed with the preference acquired on

the first days of exposure. In other words, the more

protracted ethanol experience may have induced taste

aversion to ethanol chemosensory properties and this

memory affected subsequent ethanol intake.

The possibility that repeated administration of etha-

nol induced metabolic tolerance in adolescents, but not

in adults, cannot be dismissed. A recent study (VanSkike, Novier Diaz-Granados, & Matthews, 2012)

found metabolic tolerance in adolescent rats that had

been exposed to ethanol vapor 16 hr a day for 4 days.

In adolescent animals ethanol intake across the initial

4 days of self-administration was highly correlated; and

in control adolescents there was a significant associa-

tion between ethanol drinking before and after drug

deprivation. It seems that patterns of ethanol intake

were established early in adolescence and, for those

given vehicle at initiation, maintained through the post-


8/12/2019 35 Dp Init - Brief

9/17

deprivation test. Ethanol-initiated animals, on the other

hand, did not exhibit a significant correlation between

pre- and post-deprivation scores. It seems that, similar

to the results found by Schramm-Sapyta et al. (2008),

those control adolescents that consumed the most dur-

ing the first exposure to ethanol kept drinking heavily

during subsequent sessions and exhibited the highestdrinking scores at the post-deprivation assessment. Pas-

sive exposure to ethanol disrupted this normal trajecto-

ry of ethanol intake otherwise seen in adolescents.

The present study is consistent with findings sug-

gesting that onset of ethanol consumption exerts greater

impact on predisposition to drink when it occurs at ad-

olescence than when it is delayed until adulthood. The

study shows that even brief binge ethanol intoxication

during adolescence can increase drinking. Specifically,

significantly greater ethanol drinking was observed in

adolescents after just 2 days of alcohol exposure. In

agreement with recent work (Schramm-Sapyta et al.,2008), adolescents also exhibited greater susceptibly to

ethanol drinking relapse than adults.

NOTES

This work, a collaborative project between the Research

Foundation of SUNY Binghamton and Instituto Ferreyra, was

supported by NIAAA grants AA018164 to MN and grants

PIP 2010-2012, SECYT-UNC and PICTPRH 246 to RMP.

REFERENCES

Abate, P., Pueta, M., Spear, N. E., & Molina, J. C. (2008).

Fetal learning about ethanol and later ethanol responsive-

ness: evidence against safe amounts of prenatal expo-

sure. Experimental Biology and Medicine (Maywood),

233, 139154.

Acevedo, M. B., Molina, J. C., Nizhnikov, M. E., Spear,

N. E., & Pautassi, R. M. (2010). High ethanol dose during

early adolescence induces locomotor activation and

increases subsequent ethanol intake during late adoles-

cence. Developmental Psychobiology, 52, 424440.

Bell, R. L., Rodd, Z. A., Schultz, J. A., Peper, C. L., Lumeng,

L., Murphy, J. M., & McBride, W. J. (2008). Effects of

short deprivation and re-exposure intervals on the ethanoldrinking behavior of selectively bred high alcohol-

consuming rats. Alcohol, 42, 407416.

Cancela, L. M., Bregonzio, C., & Molina, V. A. (1995).

Anxiolytic-like effect induced by chronic stress is reversed

by naloxone pretreatment. Brain Research Bulletin, 36,

209213.

DeWit, D. J., Adlaf, E. M., Offord, D. R., & Ogborne, A. C.

(2000). Age at first alcohol use: a risk factor for the devel-

opment of alcohol disorders. The American Journal of

Psychiatry, 157, 745750.

Doremus, T., Brunell, S., Varlinskaya, E., & Spear, L. (2003).

Anxiogenic effects during withdrawal from acute ethanol

in adolescent and adult rats. Pharmacology, Biochemistry

and Behavior, 75, 411418.

Doremus, T. L., Brunell, S. C., Rajendran, P., & Spear, L. P.

(2005). Factors influencing elevated ethanol consumption

in adolescent relative to adult rats. Alcoholism: Clinical

and Experimental Research, 29, 17961808.

Fabio, M. C., Nizhnikov, M., Spear, N. E., & Pautassi, R. M.,

(2012). Adolescent rats exposed to ethanol during gesta-

tion exhibit altered c-fos activity in infralimbic cortex.

Poster presented at the 2012 International Society for

Biomedical Research on Alcoholism (ISBRA) World

Congress. Sapporo, Japan, September 912.

Fullgrabe, M. W., Vengeliene, V., & Spanagel, R. (2007). In-

fluence of age at drinking onset on the alcohol deprivation

effect and stress-induced drinking in female rats. Pharma-

cology Biochemistry and Behavior, 86, 320326.

Hargreaves, G. A., Monds, L., Gunasekaran, N., Dawson, B.,

& McGregor, I. S. (2009). Intermittent access to beer

promotes binge-like drinking in adolescent but not adultWistar rats. Alcohol, 43, 305314.

Maldonado, A. M., Finkbeiner, L. M., & Kirstein, C. L.

(2008). Social interaction and partner familiarity differen-

tially alter voluntary ethanol intake in adolescent male and

female rats. Alcohol, 42, 641648.

Molina, J. C., Chotro, G., & Spear, N. E. (1989). Early

(preweanling) recognition of alcohols orosensory cues

resulting from acute ethanol intoxication. Behavioral and

Neural Biology, 51, 307325.

Ottoni, E. B. (2000). EthoLog 2.2: a tool for the transcription

and timing of behavior observation sessions. Behavior

Research Methods, Instruments, & Computers, 32, 446

449.

Pascual, M., Boix, J., Felipo, V., & Guerri, C. (2009). Repeated

alcohol administration during adolescence causes changes in

the mesolimbic dopaminergic and glutamatergic systems

and promotes alcohol intake in the adult rat. Journal of Neu-

rochemistry, 108, 920931.

Pautassi, R. M., Myers, M., Spear, L. P., Molina, J. C., &

Spear, N. E. (2008). Adolescent but not adult rats exhibit

ethanol-mediated appetitive second-order conditioning.

Alcoholism: Clinical and Experimental Research, 32,

20162027.

Ponce, L. F., Pautassi, R. M., Spear, N. E., & Molina, J. C.

(2004). Nursing from an ethanol-intoxicated dam induces

short- and long-term disruptions in motor performance

and enhances later self-administration of the drug. Alco-holism: Clinical and Experimental Research, 28, 1039

1050.

Ponce, L. F., Pautassi, R. M., Spear, N. E., & Molina, J. C.

(2011). Maternal care alterations induced by repeated

ethanol leads to heightened consumption of the drug and

motor impairment during adolescence: a dose-response

analysis. Physiology and Behavior, 103, 477486.

Ristuccia, R. C., & Spear, L. P. (2004). Adolescent ethanol

sensitivity: hypothermia and acute tolerance. Annals of the

New York Academy of Sciences, 1021, 445447.

Developmental Psychobiology Binge Ethanol Heightens Subsequent Ethanol Intake 9

8/12/2019 35 Dp Init - Brief

10/17

Rodd-Henricks, Z. A., Bell, R. L., Kuc, K. A., Murphy, J. M.,

McBride, W. J., Lumeng, L., & Li, T.-K. (2002a). Effects

of ethanol exposure on subsequent acquisition and extinc-

tion of ethanol self-administration and expression of alco-

hol-seeking behavior in adult alcohol-preferring (P) rats: I.

Periadolescent exposure. Alcoholism: Clinical and Experi-

mental Research, 26, 16321641.

Rodd-Henricks, Z. A., Bell, R. L., Kuc, K. A., Murphy, J. M.,

McBride, W. J., Lumeng, L., & Li, T.-K. (2002b). Effects

of ethanol exposure on subsequent acquisition and extinc-

tion of ethanol self-administration and expression of alco-

hol-seeking behavior in adult alcohol-preferring (P) rats:

II. Adult exposure. Alcoholism: Clinical and Experimental

Research, 26, 16421652.

Schramm-Sapyta, N. L., Kingsley, M. A., Rezvani, A. H.,

Propst, K., Swartzwelder, H. S., & Kuhn, C. M. (2008).

Early ethanol consumption predicts relapse-like behavior

in adolescent male rats. Alcoholism: Clinical and Experi-

mental Research, 32, 754762.

Schramm-Sapyta, N. L., Walker, Q. D., Caster, J. M., Levin,

E. D., & Kuhn, C. M. (2009). Are adolescents morevulnerable to drug addiction than adults? Evidence from

animal models. Psychopharmacology, 206, 121.

Spanagel, R. (2000). Recent animal models of alcoholism.

Alcohol Research & Health, 24, 124131.

Siegmund, S., Vengeliene, V., Singer, M. V., & Spanagel, R.

(2005). Influence of age at drinking onset on long-term

ethanol self-administration with deprivation and stress

phases. Alcoholism: Clinical and Experimental Research,

29, 11391145.

Silveri, M., & Spear, L. (2001). Acute, rapid, and chronic

tolerance during ontogeny: Observations when equating

ethanol perturbation across age. Alcoholism: Clinical and

Experimental Research, 25, 13011308.

Silveri, M. M., & Spear, L. P. (1998). Decreased sensitivity

to the hypnotic effects of ethanol early in ontogeny.

Alcoholism: Clinical and Experimental Research, 22, 670

676.

Spear, L. P., & Varlinskaya, E. I. (2005). Adolescence. Alco-

hol sensitivity, tolerance, and intake. Recent Developments

in Alcoholism, 17, 143159.

Spear, L. P., & Varlinskaya, E. I. (2010). Sensitivity to ethanol

and other hedonic stimuli in an animal model of adoles-

cence: implications for prevention science? Developmental

Psychobiology, 52, 236243.

Tambour, S., Brown, L. L., & Crabbe, J. C. (2008). Gender

and age at drinking onset affect voluntary alcohol

consumption but neither the alcohol deprivation effect nor

the response to stress in mice. Alcoholism: Clinical and

Experimental Research, 32, 21002106.

Tarazi, F. I., & Baldessarini, R. J. (2000). Comparative post-

natal development of dopamine D(1), D(2) and D(4)

receptors in rat forebrain. International Journal of Devel-opmental Neuroscience, 18, 2937.

Van Skike, C. E., Novier, A., Diaz-Granados, J. L., &

Matthews, D. V. (2012). The effect of chronic intermittent

ethanol exposure on spatial memory in adolescent rats:

The dissociation of metabolic and cognitive tolerance.

Brain Research, 1453, 3439.

Varlinskaya, E. I., & Spear, L. P. (2012). Increases in anxiety-

like behavior induced by acute stress are reversed by

ethanol in adolescent but not adult rats. Pharmacology,

Biochemistry and Behavior, 100, 440450.


8/12/2019 35 Dp Init - Brief

11/17

AUTHOR QUERY FORM

JOURNAL: DEVELOPMENTAL PSYCHOBIOLOGYArticle: dev_21101

Dear Author,

During the copyediting of your paper, the following queries arose. Please respond to these by annotatingyour proofs with the necessary changes/additions.

& If you intend to annotate your proof electronically, please refer to the E-annotation guidelines.

& If you intend to annotate your proof by means of hard-copy mark-up, please refer to the proof mark-up symbols guidelines. If manually writing corrections on your proof and returning it as a scannedpdf via email, do not write too close to the edge of the paper. Please remember that illegible mark-ups may delay publication.

Whether you opt for hard-copy or electronic annotation of your proofs, we recommend that you provideadditional clarification of answers to queries by entering your answers on the query sheet, in addition tothe text mark-up.

Query No. Query Remark

Q1 The Journals copyeditors have taken care to format your authorship

according to journal style (First name, Middle Initial, Surname). In the

event a formatting error escaped their inspection, or there was insufficient

information to apply journal style, please take a moment to review all

author names and sequences to ensure the accuracy of the authorship in

the published article. Please note that this information will also affect

external indexes referencing this paper (e.g., PubMed).

Q2 Please reduce the short title of up to 45 characters.

Q3 Please check the grant sponsors.

Q4 As per the style, Introduction heading has been inserted, please check.

Q5 Please check the hierarchies of the headings for correctness.

Q6 National Research Council, 1996 has not been included in the Reference

List, please supply full publication details.

8/12/2019 35 Dp Init - Brief

12/17

INSTRUCTIONS FOR CHECKING PAGE PROOFS

A PDF page proof of your article is provided with these instructions. Its purpose is foryou to:

Proofread your article. Answer any queries (which, if present, are in a query list at the end of the article). Check the content and positioning of tables and figures.

It is important that you check this proof very carefully and answer all the queries. Pleasenote that only essential corrections can be made at this stage. Also note that changesyou make to your article that do not comply with the style of the Journal and those thatare grammatically incorrect will not be incorporated.

Proofreading instruct ionsRead over your article carefully, and check that:

There are no errors in the article (including data, equations and references). Author and address details are accurate.

Content and positioning of tables and figures is correct (note that some photographsin the file may appear blurry, as figures in the PDF are low resolution).

Special characters such as figure legend symbols and Greek letters have notcorrupted.

Any previously submitted amendments have been incorporated correctly.

Queries Any queries are listed on the last page of the proof, with a corresponding number in

the margin next to the relevant text. Please ensure all queries are answered in full.

Return of approval to pub lish

Add corrections or answers to any queriesusing e-Annotation.The instructions forfor using e-Annotation tools are on the following pages.

Retain a copy of your corrections for your records. Email the Production Editor your approval to publish your article (either with or

without amendment) and any corrections required. The Production Editors contactdetails are given in the covering email.

Prompt notification of your approval to publish your article is very much appreciated.

Please contact the Production Editor if you have any queries.

8/12/2019 35 Dp Init - Brief

13/17

USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

Required software to e-Annotate PDFs: Adobe Acrobat Professional or Adobe Reader (version 8.0 or

above). (Note that this document uses screenshots from Adobe Reader X)

The latest version of Acrobat Reader can be downloaded for free at:http://get.adobe.com/reader/

Once you have Acrobat Reader open on your computer, click on the Commenttab at the right of the toolbar:

1.Replace (Ins)Tool for replacing text.

Strikes a line through text and opens up a text

box where replacement text can be entered.

How to use it

Highlight a word or sentence.

Click on the Replace (Ins) icon in the Annotations

section.

Type the replacement text into the blue box that

appears.

This will open up a panel down the right side of the document. The majority of

tools you will use for annotating your proof will be in theAnnotationssection,

pictured opposite. Weve picked out some of these tools below:

2. Strikethrough (Del)Toolfor deleting text.

Strikes a red line through text that is to be

deleted.

How to use it

Highlight a word or sentence.

Click on the Strikethrough (Del) icon in the

Annotations section.

3. Add note to textTool for highlighting a section

to be changed to bold or italic.

Highlights text in yellow and opens up a text

box where comments can be entered.

How to use it

Highlight the relevant section of text.

Click on theAdd note to text icon in the


Type instruction on what should be changed

regarding the text into the yellow box that

a ears.

4. Add sticky noteTool for making notes at

specific points in the text.

Marks a point in the proof where a comment

needs to be highlighted.

How to use it

Click on theAdd sticky note icon in the


Click at the point in the proof where the comment

should be inserted.

Type the comment into the yellow box that

appears.
http://get.adobe.com/reader/http://get.adobe.com/reader/http://get.adobe.com/reader/http://get.adobe.com/reader/

8/12/2019 35 Dp Init - Brief

14/17

USING e-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

For further information on how to annotate proofs, click on the Helpmenu to reveal a list of further options:

5.Attach FileTool for inserting large amounts of

text or replacement figures.

Inserts an icon linking to the attached file in the

appropriate pace in the text.

How to use it

Click on theAttach File icon in the Annotations

section.

Click on the proof to where youd like the attached

file to be linked.

Select the file to be attached from your computer

or network.

Select the colour and type of icon that will appear

in the proof. Click OK.

6.Add stampToolfor approving a proof if no

corrections are required.

Inserts a selected stamp onto an appropriate

place in the proof.

How to use it

Click on theAdd stamp icon in the Annotations

section.

Select the stamp you want to use. (TheApproved

stamp is usually available directly in the menu that

appears).

Click on the proof where youd like the stamp to

appear. (Where a proof is to be approved as it is,

this would normally be on the first page).

7. Drawing MarkupsTools for drawing shapes, lines and freeform

annotations on proofs and commenting on these marks.Allows shapes, lines and freeform annotations to be drawn on proofs and for

comment to be made on these marks..

How to use it

Click on one of the shapes in the Drawing

Markupssection.

Click on the proof at the relevant point and

draw the selected shape with the cursor.

To add a comment to the drawn shape,

move the cursor over the shape until an

arrowhead appears.

Double click on the shape and type any

text in the red box that appears.

8/12/2019 35 Dp Init - Brief

15/17

AdditionalreprintandjournalissuepurchasesShouldyouwishtopurchaseadditionalcopiesofyourarticle,

pleaseclickonthelinkandfollowtheinstructionsprovided:

https://caesar.sheridan.com/reprints/redir.php?pub=10089&acro=DEVCorresponding

authors

are

invited

to

inform

their

co

authors

of

thereprintoptionsavailable.

Pleasenotethatregardlessoftheforminwhichtheyareacquired,

reprintsshouldnotberesold,norfurtherdisseminatedinelectronicform,nor

deployedinpartorinwholeinanymarketing,promotionaloreducational

contextswithoutauthorizationfromWiley.Permissionsrequestsshouldbe

directedtomailto:[email protected]

Forinformation

about

Pay

Per

View

and

Article

Select

click

on

the

following

link:http://wileyonlinelibrary.com/ppv
https://caesar.sheridan.com/reprints/redir.php?pub=10089&acro=DEVhttps://caesar.sheridan.com/reprints/redir.php?pub=10089&acro=DEVhttps://caesar.sheridan.com/reprints/redir.php?pub=10089&acro=DEVmailto:[email protected]:[email protected]:[email protected]://wileyonlinelibrary.com/ppvhttp://wileyonlinelibrary.com/ppvhttp://wileyonlinelibrary.com/ppvmailto:[email protected]://caesar.sheridan.com/reprints/redir.php?pub=10089&acro=DEV

8/12/2019 35 Dp Init - Brief

16/17

A. COPYRIGHT

1. The Contributor assigns to Wiley-Blackwell, during the full term of copy-right and any extensions or renewals, all copyright in and to the Contribution,

and all rights therein, including but not limited to the right to publish, repub-lish, transmit, sell, distribute and otherwise use the Contribution in whole or in

part in electronic and print editions of the Journal and in derivative worksthroughout the world, in all languages and in all media of expression now

known or later developed, and to license or permit others to do so.

2. Reproduction, posting, transmission or other distribution or use of the final

Contribution in whole or in part in any medium by the Contributor as permit-ted by this Agreement requires a citation to the Journal and an appropriate

credit to Wiley-Blackwell as Publisher, and/or the Society if applicable, suitablein form and content as follows: (Title of Article, Author, Journal Title and

Volume/Issue, Copyright [year], copyright owner as specified in the Journal).

Links to the final article on Wiley-Blackwells website are encouraged whereappropriate.

B. RETAINED RIGHTS

Notwithstanding the above, the Contributor or, if applicable, the ContributorsEmployer, retains all proprietary rights other than copyright, such as patent

rights, in any process, procedure or article of manufacture described in theContribution.

C. PERMITTED USES BY CONTRIBUTOR

1. Submitted Version. Wiley-Blackwell licenses back the following rights to

the Contributor in the version of the Contribution as originally submitted forpublication:

a. After publication of the final article, the right to self-archive on the Con-

tributors personal website or in the Contributors institutions/employers

institutional repository or archive. This right extends to both intranets andthe Internet. The Contributor may not update the submission version orreplace it with the published Contribution. The version posted must contain

a legend as follows: This is the pre-peer reviewed version of the followingarticle: FULL CITE, which has been published in final form at [Link to final

article].

b. The right to transmit, print and share copies with colleagues.

2. Accepted Version. Re-use of the accepted and peer-reviewed (but not

final) version of the Contribution shall be by separate agreement with Wiley-Blackwell. Wiley-Blackwell has agreements with certain funding agencies

governing reuse of this version. The details of those relationships, and otherofferings allowing open web use, are set forth at the following website:

http://www.wiley.com/go/funderstatement. NIH grantees should check thebox at the bottom of this document.

3. Final Published Version. Wiley-Blackwell hereby licenses back to theContributor the following rights with respect to the final published version of

the Contribution:

a. Copies for colleagues. The personal right of the Contributor only to sendor transmit individual copies of the final published version in any format to

colleagues upon their specific request provided no fee is charged, andfurther-provided that there is no systematic distribution of the Contribu-

tion, e.g. posting on a listserve, website or automated delivery.

b. Re-use in other publications. The right to re-use the final Contribution orparts thereof for any publication authored or edited by the Contributor

(excluding journal articles) where such re-used material constitutes lessthan half of the total material in such publication. In such case, any modifi-

cations should be accurately noted.

c. Teaching duties. The right to include the Contribution in teaching or

training duties at the Contributors institution/place of employment includ-ing in course packs, e-reserves, presentation at professional conferences,

in-house training, or distance learning. The Contribution may not be usedin seminars outside of normal teaching obligations (e.g. commercial semi-

nars). Electronic posting of the final published version in connection withteaching/training at the Contributors institution/place of employment is

permitted subject to the implementation of reasonable access controlmechanisms, such as user name and password. Posting the final published

version on the open Internet is not permitted.

d. Oral presentations. The right to make oral presentations based on theContribution.

4. Article Abstracts, Figures, Tables, Data Sets, Artwork and SelectedText (up to 250 words).

a. Contributors may re-use unmodified abstracts for any non-commercialpurpose. For on-line uses of the abstracts, Wiley-Blackwell encourages but

does not require linking back to the final published versions.b. Contributors may re-use figures, tables, data sets, artwork, and selectedtext up to 250 words from their Contributions, provided the following

conditions are met:

(i) Full and accurate credit must be given to the Contribution.(ii) Modifications to the figures, tables and data must be noted.

Otherwise, no changes may be made.(iii) The reuse may not be made for direct commercial purposes, or for

financial consideration to the Contributor.(iv) Nothing herein shall permit dual publication in violation of journal

ethical practices.

COPYRIGHT TRANSFER AGREEMENT

Date: Contributor name:

Contributor address:

Manuscript number (Editorial office only):

Re: Manuscript entitled

(the Contribution)

for publication in (the Journal)

published by (Wiley-Blackwell).

Dear Contributor(s):

Thank you for submitting your Contribution for publication. In order to expedite the editing and publishing process and enable Wiley-Blackwell to

disseminate your Contribution to the fullest extent, we need to have this Copyright Transfer Agreement signed and returned as directed in the Journals

instructions for authors as soon as possible. If the Contribution is not accepted for publication, or if the Contribution is subsequently rejected, this

Agreement shall be null and void. Publication cannot proceed without a signed copy of this Agreement.

CTA-A

8/12/2019 35 Dp Init - Brief

17/17

D. CONTRIBUTIONS OWNED BY EMPLOYER

1. If the Contribution was written by the Contributor in the course of theContributors employment (as a work-made-for-hire in the course of

employment), the Contribution is owned by the company/employer whichmust sign this Agreement (in addition to the Contributors signature) in the

space provided below. In such case, the company/employer hereby assigns toWiley-Blackwell, during the full term of copyright, all copyright in and to the

Contribution for the full term of copyright throughout the world as specified inparagraph A above.

2. In addition to the rights specified as retained in paragraph B above and the

rights granted back to the Contributor pursuant to paragraph C above, Wiley-

Blackwell hereby grants back, without charge, to such company/employer, itssubsidiaries and divisions, the right to make copies of and distribute the finalpublished Contribution internally in print format or electronically on the Com-

panys internal network. Copies so used may not be resold or distributed externally.However the company/employer may include information and text from the

Contribution as part of an information package included with software orother products offered for sale or license or included in patent applications.

Posting of the final published Contribution by the institution on a public accesswebsite may only be done with Wiley-Blackwells written permission, and payment

of any applicable fee(s). Also, upon payment of Wiley-Blackwells reprint fee,the institution may distribute print copies of the published Contribution externally.

E. GOVERNMENT CONTRACTS

In the case of a Contribution prepared under U.S. Government contract or

grant, the U.S. Government may reproduce, without charge, all or portions ofthe Contribution and may authorize others to do so, for official U.S. Govern-

ment purposes only, if the U.S. Government contract or grant so requires. (U.S.Government, U.K. Government, and other government employees: see notes

at end)

F. COPYRIG HT NOTICE

The Contributor and the company/employer agree that any and all copies ofthe final published version of the Contribution or any part thereof distributed

or posted by them in print or electronic format as permitted herein will includethe notice of copyright as stipulated in the Journal and a full citation to the

Journal as published by Wiley-Blackwell.

G. CONTRIBUTORS REPRESENTATIONS

The Contributor represents that the Contribution is the Contributors originalwork, all individuals identified as Contributors actually contributed to the Con-

tribution, and all individuals who contributed are included. If the Contributionwas prepared jointly, the Contributor agrees to inform the co-Contributors of

the terms of this Agreement and to obtain their signature to this Agreement ortheir written permission to sign on their behalf. The Contribution is submitted

only to this Journal and has not been published before. (If excerpts from copy-righted works owned by third parties are included, the Contributor will obtain

written permission from the copyright owners for all uses as set forth in Wiley-Blackwells permissions form or in the Journals Instructions for Contributors,

and show credit to the sources in the Contribution.) The Contributor alsowarrants that the Contribution contains no libelous or unlawful statements,

does not infringe upon the rights (including without limitation the copyright,patent or trademark rights) or the privacy of others, or contain material or

instructions that might cause harm or injury.

CHECK ONE BOX:

Contributor-owned work

Contributors signature Date

Type or print name and title

Co-contributors signature Date

Type or print name and title

Company/Institution-owned work

Company or Institution (Employer-for-Hire) Date

Authorized signature of Employer Date

U.S. Government work Note to U.S. Government EmployeesA contribution prepared by a U.S. federal government employee as part of the employees official duties, or

which is an official U.S. Government publication, is called a U.S. Government work, and is in the public

domain in the United States. In such case, the employee may cross out Paragraph A.1 but must sign (in the

Contributors signature line) and return this Agreement. If the Contribution was not prepared as part of the

employees duties or is not an official U.S. Government publication, it is not a U.S. Government work.

U.K. Government work Note to U.K. Government Employees(Crown Copyright) The rights in a Contribution prepared by an employee of a U.K. government department, agency or other

Crown body as part of his/her official duties, or which is an official government publication, belong to the

Crown. U.K. government authors should submit a signed declaration form together with this Agreement.

The form can be obtained via http://www.opsi.gov.uk/advice/crown-copyright/copyright-guidance/

publication-of-articles-written-by-ministers-and-civil-servants.htm

Other Government work Note to Non-U.S., Non-U.K. Government EmployeesIf your status as a government employee legally prevents you from signing this Agreement, please contact

the editorial office.

NIH Grantees Note to NIH GranteesPursuant to NIH mandate, Wiley-Blackwell will post the accepted version of Contributions authored by NIH

grant-holders to PubMed Central upon acceptance. This accepted version will be made publicly available

12 months after publication. For further information, see www.wiley.com/go/nihmandate.

ATTACH ADDITIONAL SIGNATURE

PAGES AS NECESSARY

(made-for-hire in thecourse of employment)

Documents

35 Dp Init - Brief