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Responding for Brain Stimulation Reward

in the Bed Nucleus of the Stria Terminalisin Alcohol-Preferring Rats Following Alcohol and Amphetamine Pretreatments

WILLIAM J.A. EILER, II,1 LATHEN HARDY, III,1 JOSUHA GOERGEN,1 REGAT SEYOUM,1

BOIKAI MENSAH-ZOE,1 AND HARRY L. JUNE2,3*1 Psychobiology of Addictions Program, Department of Psychology, Indiana University-Purdue University,

Indianapolis, Indiana 462022 Division of Alcohol and Drug Abuse, Department of Psychiatry, University of Maryland School of Medicine,

Baltimore, Maryland 212013 Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine,

Baltimore, Maryland 21201

KEY WORDS brain stimulation reward (BSR); alcohol preferring (P) and non-pre-ferring (NP) rats; alcohol; amphetamine; SCH 23390

ABSTRACT The bed nucleus of the stria terminalis (BNST) has been reported to

release increased levels of extracellular dopamine (DA) following the systemic admin-

istration of abused drugs in outbred rats. This study examined the BNST as a novel

locus for supporting operant responding for brain stimulation reward (BSR) in rats

bred for alcohol preference while determining any potentiating effects of ethanol

(EtOH) (0.125–1.25 g/kg, i.p.) and amphetamine (0.25–1.60 mg/kg, i.p.) on BSR within

the BNST. Also examined was the capability of D1 receptor blockade to attenuate any

observed potentiation. Following surgical implantation, alcohol-preferring (P) and non-

preferring (NP) rats responded to a range of descending frequencies (300–20 Hz) as

evaluated by a rate-frequency paradigm. The results revealed that the BNST was

capable of supporting BSR in P but not NP rats. Also, amphetamine pretreatment pro-

duced a significant leftward shift in the rate-frequency function in P rats with signifi-cant reductions observed in three other measures of reward threshold, while EtOH

only lowered the minimum frequency needed to produce responding. The effects of sys-

temic amphetamine were successfully attenuated by the unilateral infusion of the D1

receptor antagonist SCH 23390 (5.0 mg) into the contralateral nucleus accumbens. The

results suggest the BNST is capable of supporting BSR performance in P, but not NP

rats, possibly due to increased sensitivity to the electrical stimulation-induced DA

release of BSR in the innately DA ‘‘deficient’’ limbic system of P rats. Synapse

61:912–924, 2007. VVC 2007 Wiley-Liss, Inc.

INTRODUCTION

It is suggested that individuals that self-administer

drugs of abuse may be sensitive not only to a singledrug or reinforcer, but to multiple types of rein-

forcers. Thus, it is believed that individuals that

habitually use drugs of abuse posses an increased

abuse liability, or the increased likelihood to abuse

any drug type. One method used to evaluate the rein-

forcing potential of various drugs is brain stimulation

reward (BSR) (Lewis, 1993). BSR is often referred to,

and now accepted as a model of drug-induced eupho-

ria (Kornetsky and Bain, 1990; Lewis, 1993). While

other neurotransmitter systems are likely involved,

many studies have established that the rewarding

effects of BSR may depend on the ability of the brain

stimulation to activate the mesocorticolimbic dopa-

mine (DA) system (Lewis, 1993; Wise, 1996). BSR canbe used to discreetly activate brain substrates within

Contract grant sponsor: National Institute of Alcohol Abuse and Alcoholism(NIAAA); Contract grant numbers: AA10406, AA11555; Contract grant spon-sor: National Institute of General Medical Science (NIGMS); Contract grantnumbers: AA10422, GM47818; Contract grant sponsor: National Heart, Lung,and Blood Institute, NIH; Contract grant number: T35M.

*Correspondence to: Harry L. June, Division of Alcohol and Drug Abuse,Department of Psychiatry, University of Maryland School of Medicine, 701West Pratt Street, Rm 597, Baltimore, MD 21201, USA.E-mail: [email protected]

Received 17 August 2003; Accepted 29 March 2007

DOI 10.1002/syn.20437

Published online in Wiley InterScience (www.interscience.wiley.com).

VVC 2007 WILEY-LISS, INC.

SYNAPSE 61:912–924 (2007)



the reward circuitry as opposed to a more systemic

activation by other paradigms used to measure the

reinforcing properties of drugs (e.g., conditioned place

preference, drug discrimination, i.v. drug infusion)

(Phillips and Fibiger, 1989; Stellar and Stellar, 1985).

Another positive feature of the BSR paradigm is thatdose-response functions similar to those seen in tradi-

tional pharmacological studies can be obtained by the

use of the curve-shift paradigm in which the stimula-

tion frequency or current intensity can be systemati-

cally varied (Kling-Petersen and Svensson, 1993;

Liebman 1983; Wise and Rompre, 1989; Wise, 1996).

While a number of drugs have been evaluated

using the BSR paradigm, amphetamine has been

reported to be the most reliable drug to facilitate BSR

performance. Amphetamine consistently produces

leftward shifts in the rate frequency function and also

lowers BSR threshold while enhancing lever-press

responding (for review see Schaefer and Michael,

1987; Wise, 1996). Less consistent, however, are the

reports investigating alcohol’s effects on BSR perform-

ance (Bain and Kornetsky, 1989; Lewis and June,

1990; Moolten and Kornetsky, 1990; Schaefer and

Michael, 1987, 1992). While a clear explanation for

the varied effects of alcohol is yet to emerge, Lewis

(1993) has suggested route of administration, time of

testing following administration, and the various

threshold determination methods as likely contribut-

ing factors in these discrepancies. The choice of which

substrate within the mesolimbic DA system to place

the stimulating electrode may also contribute to these

inconsistencies. For this study, we evaluated the bed

nucleus of the stria terminalis (BNST) which repre-sents a novel locus for the BSR paradigm, but has

been shown to function as a neurobiological substrate

for the reinforcing actions of alcohol as well as psy-

chostimulants (Day et al., 2001; Eiler et al., 2003;

Koob, 1999; Leshner and Koob, 1999).

The BNST, a component of the mesolimbic reward

system, receives a number of afferent and efferent

connections from putative drug reward areas as

depicted in Figure 1 (Alheid and Heimer, 1988; Alheid

et al., 1995; Eiler et al., 2003). Of particular interest

is that DA neurons from the ventral tegmental area

(VTA) project directly to the BNST, in a manner anal-

ogous to the nucleus accumbens (NAC) and ventralpallidum both of which have been shown to support

BSR (Mogenson et al., 1979; Panagis et al., 1997).

Figure 1 also illustrates the number of GABAergic

afferents and efferents between the BNST and

numerous limbic structures. The GABA and DA sys-

tems within the BNST have emerged as pivotal neu-

ronal systems in regulating the reinforcing properties

of alcohol and psychostimulants (Carboni et al., 2000;

Eiler et al., 2003; Epping-Jordan et al., 1998; Hyytia

and Koob, 1995) with evidence emerging that GABA

may indirectly regulate DA by modulating the rein-

forcing/activational effects of psychostimulants

(Austin and Kalivas, 1990; Gong et al., 1997) and

alcohol (Harvey et al., 2002; Hodge et al., 1995; June

et al., 2001).

To further examine the role of abuse liability, we

chose to use subjects selectively bred for alcohol pref-

erence. While several rat lines have been developed

through selective breeding to produce high and low

alcohol drinking variants with the alcohol perferring/

nonperferring (P/NP) lines most widely used andstudied (Cloninger, 1987; McBride and Li, 1998; Mur-

phy et al., 2002). The P rat is particularly useful as

an animal model as it fulfills all criteria (for review

see Cicero, 1979; Lester and Freed, 1973) to accept-

ably model human alcoholism to the satisfaction of

the alcohol research community (McBride and Li,

1998; Murphy et al., 2002). While its utility in study-

ing alcoholism is clear, the P rat may also be useful

in making inferences to the reinforcing potential (i.e.,

abuse liability) of drugs other than alcohol, specifi-

cally the psychostimulant amphetamine.

The purpose of the present study was to determine

if the BNST could be used in a manner analogous toother putative reward loci, as a novel locus to support

lever-press responding contingent on direct electrical

stimulation in rats selectively bred to consume alco-

hol (Phillips and Fibiger, 1989; Stellar and Stellar,

1985) To accomplish this, we employed a BSR para-

digm utilizing the curve-shift model coupled with an

array of parameters to assess BSR performance. Sec-

ondly we attempted to determine if reward potentiat-

ing effects of alcohol and amphetamine could be

observed on BSR within the BNST of P and NP rats.

The final purpose was to determine if the D1 DA

Fig. 1. Schematic illustration of the interrelationship of dopami-nergic and GABAergic inputs and outputs and their connectivitybetween the VTA via the medial forebrain bundle (MFB)/lateral

hypothalamus (LH) to the bed nucleus of the stria terminalis(BNST) and other extended amygdala loci.

913BSR IN BNST OF P RATS FOLLOWING EtOH AND A mp

Synapse DOI 10.1002/syn



receptor played a role in regulating the BSR potenti-

ating effects of amphetamine.

MATERIALS AND METHODS

Subjects

Male P rats (n ¼ 10) and NP rats (n ¼ 9) of the S50generation (Indiana University, Indianapolis, IN)

weighing between 410 and 546 g at the time of test-

ing were used as subjects. The animals were individu-

ally housed in shoebox cages in a temperature and

humidity controlled room with food and water avail-

able ad libitum. Behavioral training took place

between 9 a.m. and 3 p.m. All experimental proce-

dures were conducted in strict compliance with the

NIH Guide for the Care and Use of Laboratory Ani-

mals.

Stereotaxic surgery

Rats were anesthetized with sodium pentobarbital

(45 mg/kg, i.p.) and placed in a stereotaxic apparatus.

The animals were then implanted with platinum elec-

trodes with a diameter of 0.3 mm (Plastics One, Roa-

noke, VA) into the bed nucleus of the stria terminalis

(BNST) of the left hemisphere. The Teflon insulated

electrode was fixed to the skull with stainless steel

screws and dental acrylic. Animals were also concur-

rently implanted with a contralateral 22-gauge guide

cannula in the NAC within the right hemisphere. In

relation to bregma, the coordinates for the BNST

were as follows: AP À0.5, ML 62.5, DV À7.6, with an

8.78 lateral angle from the skull. The coordinates for

the NAC were as follows: AP +1.4; ML 61.0; DV –6.0

from the skull. The cannulas were aimed 1 mm above

the intended brain loci. A stylet which protruded

1 mm beyond the tip of the guide cannulae was

inserted when the injector was not in place. All coor-

dinates were made using the rat brain atlas of Paxi-

nos and Watson (1998).

Histology

After the completion of all experimental procedures,

the rats were sacrificed via CO2 inhalation. The loca-

tion of the electrode was immediately lesioned by an

isolated physiological stimulator [Model A13-65](Coulbourn Instruments, Allentown, PA) using three-

3 s, 4 milliamphere bursts with a 1 s separation

between bursts. Next, to identify the guide cannulae

location within the NAC, cresyl violet acetate (0.50

ml) was injected into the infusion site, and the brains

were removed and frozen. Subsequently, histological

evaluations were performed to determine the place-

ment of the electrodes. Once the brains were sec-

tioned on a microtone at 50 mm and placed on slides

they were stained with cresyl violet acetate. The sec-

tions were examined under a light microscope with

lesions and cannula placements indicated on draw-

ings adapted from the rat brain atlas of Paxinos and

Watson (1998).

Behavioral apparatus

Behavioral training and testing were conducted in

10 standard operant chambers (Coulbourn Instru-

ments, Allentown, PA) equipped with a removable

lever enclosed in a sound attenuating cubicle. Each

lever press triggered an electrical pulse of a given

current intensity supplied by an isolated physiological

stimulator [Model A13-65] (Coulbourn Instruments,

Allentown, PA). The stimulation was a 0.2 s train of

100 Hz biphasic rectangular pulses of 1.0 ms duration

similar to our previous research (Lewis and June,

1990). Responses and reinforcements were controlled

and recorded during a 20 min operant session by PC

computers using the Jay-Shake-Li ICSS operant soft-

ware program (Shake-Li and Huston, 2002).

Behavioral training

After surgical implantation, the BSR group was

trained to respond to electrical stimulation under an

FR1 schedule of reinforcement. This was accom-

plished through shaping via successive approxima-

tion. During this training phase the current of the

electrical stimulus was varied to determine the cur-

rent for each rat that evoked optimal responding. The

rats continued to respond for electrical stimulation at

their optimal current, or the current level that main-

tained the highest level of responding (50–350 m A)until their responding stabilized. For this study, stabi-

lization is defined as daily responses within 620% of

the average responses for 5 consecutive days.

BSR parameters

The BSR model and calculation of BSR parameters

used in the present study are based on the earlier

work by Kling-Petersen and Svensson (1993) and

emulates the experimental paradigm employed by

Shake-Li and Huston (2002). Four dependent varia-

bles were collected for the present study: (1) the

frequency that corresponds with 50% of responding,often referred to as EF50 or half maximal frequency/

responding, (2) the minimum/lowest frequency capa-

ble of maintaining BSR, (3) the maximum frequency

producing the highest rate of lever pressing through-

out the BSR session, and (4) total number of lever

presses (i.e., responses) produced during the 20 min

session. The first three variables are used to deter-

mine the BSR threshold (Kling-Petersen and Svens-

son 1993; Shake-Li and Huston, 2002). It is these

variables that produce the curve shift that is indica-

tive of a change in threshold (Lewis, 1993; Stellar

914 W.J.A. EILER ET AL.




and Stellar 1985; Wise, 1989). The final two variables

are important in the formation of the asymptote of

the rate/frequency curve. The asymptote is an essen-

tial component of BSR analysis, as it indicates

whether or not an experimental manipulation pro-

duces a motoric effect (Lewis 1993; Miliaressis et al.1986; Stellar and Stellar, 1985). A motor enhance-

ment due to an experimental manipulation is mani-

fested by an increase in the asymptote relative to con-

trol levels, whereas a decrease in the asymptote indi-

cate a reduction in motor capacity (Stellar and

Stellar, 1985; Wise, 1989) All data were collected

using the ICSS computer program developed by

Shake-Li and Huston (2002).

Experiment 1: examination of basal sensitivity

to the reinforcing effect of BSR in P and

NP rats following EtOH or

amphetamine pretreatment

The animals ( P ¼ 10, NP ¼ 9) were initially trained

to bar press under an FR1 schedule of reinforcement

at a current level of 100 l A employing a frequency of

100 Hz. which produced sustained lever pressing at

100 l A. This current level was determined to be the

optimal current for BSR performance. After the ani-

mals had established a consistent level of responding,

the current level was adjusted for each animal to

reach a maximal level of lever press responding. This

current was operationally defined as the optimal cur-

rent (100%) level. For the P rats, this current level

ranged from 250 to 150 l A, while in the NP rats it

ranged from 300 to 200 l A. After animals were stabi-lized on the optimal current under an FR1 schedule,

the current was set to 50% of the animals optimal

current level, since it has been suggested that the

current level that produces the maximal level of

responses may not be the preferred (i.e., efficacious)

for the rats, or most sensitive to the drug treatments

(Stellar and Stellar, 1985; Lewis, 1993). Further, as

noted earlier, submaximal current levels produce sub-

maximal response rates and may be more indicative

of the rewarding efficacy of the stimulation (Liebman,

1983). The rate/frequency function was then gener-

ated by presenting a series of frequencies in a de-

scending manner from 300 to 20 Hz: (i.e., 300, 260,220, 180, 140, 100, 80, 60, 40, and 20 Hz). The rate/

frequency function was generated over session time of

20 min and 30 s. Three, 1 s priming pulses were

presented prior to each tested frequency.

After responding was stabilized animals were given

1 of 4 doses (0.125, 0.35, 0.75, 1.25 g/kg i.p.) of etha-

nol (EtOH) (10% w/v), or 1 of 4 doses (0.25, 0.75, 1.25,

1.60 mg/kg i.p.) of amphetamine 5 min before the

BSR session. A minimum of 3 and a maximum of 4

days of no testing separated each EtOH and ampheta-

mine drug dose administration. Twenty-four hours

post drug administration, we examined the effects of

all drug treatments to determine residual drug

effects. All rats received the same dose of EtOH or

amphetamine on the same day and the doses were

administered in random order using a random num-

ber generator.

Experiment 2: effect of SCH 23390 on the basal

sensitivity to the reinforcing effect of BSR in P

rats following amphetamine pretreatment

SCH 23390 was mixed prior to each infusion. The

drugs were mixed into 0.25 ml of sterile saline. The

SCH 23390 was then unilaterally infused into

the NAC at a rate of 0.1 ml/min for 2.5 min using a

Harvard infusion pump. The injector tip was left in

the cannula for an additional minute to facilitate the

diffusion of all injected drug from the injector tip into

the brain loci. The animals were then either placed

into the operant chamber or given an i.p. injection of 0.75 mg/kg of amphetamine and then placed in the

operant chamber 5 min following the injection. After

the rats received the initial infusion, subsequent infu-

sions were not performed until responding returned

to baseline.

Drug preparation

EtOH (95%) (AAPER Alcohol and Chemical, Shel-

byville, KY) was diluted with saline to produce 10%

(w/v) solution and injected at volumes sufficient to

produce 0.25–1.25 g/kg doses. d-Amphetamine was

dissolved in saline and injected in a volume of 1 ml/

kg. The SCH 23390 was mixed readily with sterile sa-line immediately before the experiments and was

injected unilaterally using a Harvard Pump. The d-

Amphetamine and SCH 23390 were purchased from

Sigma Drug Company (St. Louis, MO).

Data analysis

Separate repeated measures ANOVAs were used on

P and NP responding data, maximum frequency data,

minimum frequency data, and EF50 data separately

to further delineate within subject effects. A t-test

post hoc analysis was conducted on all significant

ANOVA’s.

RESULTS

Figures 2A and 2B show a reconstruction of serial

coronal sections of the rat brain illustrating the loca-

tion of the unilateral electrodes tip in the BNST of

the (A) P and (B) NP rats. The electrode tips were

well localized within the mediolateral to dorsolateral

BNST of both rat lines. Figure 2C illustrates a recon-

struction of serial coronal sections of the rat brain

depicting the location of the unilateral cannula im-

plantation in the NAC; the cannulae were located in





both the shell and core. Figure 3 depicts photomicro-

graphs of the actual unilateral placements in the

mediolateral and dorsolateral BNST for 3 P (A–C)

and 2 NP (D–E) rats following electrode lesioning.

Figures 3F and 3G depict the actual placements for 2

P rats in separate photomicrographs illustrating the

extent of the lesion sustained as a result of the uni-

lateral guide cannula in the NAC.

Experiment 1: examination of basal sensitivity

to the reinforcing effect of BSR in P and

NP rats following EtOH or

amphetamine pretreatment

Effects of EtOH on BSR performance

P rats. Figure 4A illustrates the rate of lever

pressing as a function of stimulation frequency in P

rats following EtOH administration (n ¼ 7). The rate-

frequency curves show that relative to placebo, the

amount of stimulation that sustained responding was

not changed at the lower frequencies with any of the

EtOH doses, but beginning at 150 Hz, a leftward shift

in the rate frequency function began to emerge forthe 0.75 and 1.25 g/kg doses. Figure 4B shows that

EtOH produced a dose-dependent reduction in mini-

mum frequency resulting in a significant effect of

dose [ F (4,24) ¼ 4.82, P < 0.01]. Minimum frequencies

ranged from 49 6 11 to 130 6 20 Hz. Post hoc analy-

ses determined that both the 0.75 ( P < 0.01) and 1.25

g/kg ( P < 0.02) doses significantly lowered minimum

frequency compared with placebo. Figure 4C shows

that none of the EtOH treatments altered the EF50

parameter relative to placebo resulting in a non-

significant effect of dose [ F (4,24) ¼ 0.88, P < 0.49].

Fig. 3. Representative histological photomicrographs of theactual unilateral placements in the mediolateral and dosolateralBNST for 3 alcohol-preferring (P) ( A–C) and 2 alcohol-nonpreferring(NP) (D–E) rats following electrode lesioning. Figures (F and G)depict the actual placements for 2 P rats in separate photomicro-graphs illustrating the extent of the lesion sustained as a result of the unilateral guide cannula in the NAC.

Fig. 2. Reconstruction of serial coronal sections of the rat brainsillustrating the location of the unilateral electrode’s tip in the bed

nucleus of the stria terminalis (BNST) of the ( A ) P (n ¼ 7) and (B)NP rats (n ¼ 6). C: Illustrates a reconstruction of serial coronal sec-tions of the P rat brains (n ¼ 7) depicting the location of the unilat-eral cannula implantation in the nucleus accumbens (NAC) (contra-lateral hemisphere). Each rat is represented by one solid black circlein all figures. Coronal sections are adapted from the rat brain atlasof Paxinos and Watson (1998).





Figures 4D and 4E also show that EtOH did not sig-

nificantly alter total responding or maximal frequency

[F(4,24) ¼ 1.67, P < 0.19], [ F (4,24) ¼ 1.43, P < 0.25],

respectively.

NP rats. The rate-frequency curve for NP rats (n

¼ 6) following EtOH administration is illustrated in

Figure 5A. The plot illustrates the erratic perform-

ance of the NP line, clearly demonstrating the inabil-

ity of BSR to act as a sufficient stimulus to elicit sta-

ble behavior in this rat line. Panel B of Figure 5

depicts the failure of EtOH (0.35 or 1.25 g/kg) to al-

ter the minimum frequency needed to produce

responding [ F (2,10) ¼ 2.18, P > 0.05]. EtOH alsofailed to produce any effect on the maximum fre-

quency parameter [ F (2,10) ¼ 2.94, P > 0.05] as illus-

trated in Figure 5C. Figure 5D shows the effect of

the two tested doses of EtOH on the total lever-press

responding in NP rats. Of importance in this figure

is the low level of basal responding for BSR produced

by the NP rat line, a mere 7% of the number of

responses produced in the P line. As with the mini-

mum and maximum frequencies, EtOH failed to al-

ter the total number of responses [ F (2,10) ¼ 0.57, P

> 0.05]. It was impossible to calculate the EF50 in

these animals due to both the low levels of respond-

ing and the inconsistent response to the varying fre-

quencies.

Effects of amphetamine on BSR performance

P rats. Figure 6A illustrates the rate of lever

pressing as a function of stimulation frequency follow-

ing amphetamine treatments. The rate-frequency

curves show that relative to placebo, the amount of

stimulation that sustained responding was reduced

with all doses of amphetamine as indicated by left-

ward shifts in the rate frequency function. Figure 6Bshows that amphetamine produced a reduction in

minimum frequency resulting in a significant effect of

dose, with no further reductions being observed with

the 1.6 mg/kg dose relative to the 1.25 mg/kg dose

[ F (4,24) ¼ 2.85, P < 0.05]. Post hoc analyses con-

firmed that the 0.75–1.60 mg/kg doses significantly

lowered minimum frequency compared with placebo

( P 0.05). Figure 6C shows that amphetamine pro-

duced a reduction on the EF50 parameter relative to

placebo [ F (4,24) ¼ 5.45, P < 0.01]. However, similar

to the minimum frequency, no further reductions

Fig. 4. Rats were tested on a 300–20 Hzdescending frequency schedule, and at asubmaximal current intensity (50% opti-mal current) following i.p. administrationof EtOH (10% v/v) (0.0–1.25 g/kg) across a20 min operant session in P rats ( n ¼ 7).EtOH was administered 5 min prior tothe BSR session. A : BSR rate-frequencycurves. For clarity the standard error of measurements (SEM) are omitted on thisand subsequent rate frequency graphs.B: Minimum frequency. C: Effectivefrequency (EF50) [threshold parameter].D: Total responding. E: Maximal fre-quency. Bars represent the standard errorof measurements (SEM) in this and subse-quent figures. ** P < 0.01, * P < 0.05 fromplacebo control.





were observed with the 1.6 mg/kg dose relative to the

1.25 mg/kg dose; however, the 0.25–1.60 mg/kg doses

all significantly lowered the EF50 parameter com-

pared with placebo ( P 0.05). Figure 6D shows mean

total responding following the 4 doses of ampheta-

mine. Mean total responding ranged from 277 6 82 to

652 6 170. Amphetamine elevated responding with

all the tested doses relative to placebo; however, the

overall ANOVA failed to reach statistical significance[ F (4,24) ¼ 1.69, P ! 0.05]. While an analysis of var-

iance did not demonstrate significance, it is clear that

all tested doses of amphetamine trended toward a sig-

nificant increase in responding for BSR. Figure 6E

shows that amphetamine produced reductions on the

maximum frequency measure with all tested doses,

resulting in a significant effect of doses [ F (4,24) ¼

2.89, P < 0.05]. Post hoc analyses confirmed that the

0.25–1.60 mg/kg doses significantly lowered the maxi-

mum frequency compared with placebo condition ( P <

0.05).

NP rats. Figure 7A illustrates the rate of lever

pressing as a function of stimulation frequency follow-ing amphetamine treatments in NP rat line (n ¼ 6)

and as with the EtOH condition this plot also demon-

strates the inability of BSR to act as a sufficient

stimulus to elicit stable behavior in this rat line even

following the administration of a potent psychostimu-

lant. Figure 7B shows that the 0.25 mg/kg dose of

amphetamine was sufficient ( P < 0.05) to alter the

minimum frequency needed to produce responding

[ F (2,10) ¼ 4.81, P < 0.05]. The 0.25 mg/kg dose of am-

phetamine also produced a significant ( P < 0.05)

effect on the maximum frequency parameter [ F (2,10)

¼ 4.54, P < 0.05] as illustrated in Figure 7C. Figure

7D shows the effect of the two tested doses of amphet-

amine on the total lever-press responding in NP rats.

As with EtOH, amphetamine failed to alter the total

number of responses [ F (2,10) ¼ 3.48, P > 0.05].

Again, the low levels of responding and the inconsis-

tent response to the varying frequencies made it

impossible to calculate the EF50 parameter in these

animals.

Experiment 2: effect of SCH 23390 on the basal

sensitivity to the reinforcing effect of BSR in P

rats following amphetamine pretreatment

Effects of SCH 23390 on BSR performance in

P rats with and without amphetamine

Figure 8A illustrates the rate of lever pressing as a

function of stimulation frequency following the con-

tralateral microinjection of SCH 23390 into the NAC.

None of the drug treatments reached statistical sig-

nificance on any of the BSR parameters (data notshown). Figure 8B illustrates the rate of lever press-

ing as a function of stimulation frequency with am-

phetamine and SCH 23390 alone, and the combina-

tion of SCH 23390 (5.0 mg) and amphetamine (0.75

mg/kg, i.p.). The SCH 23390 and the amphetamine

data are redrawn from Figures 6 and 8. The rate-

frequency curves show that when rats were microin-

jected with a 5.0 mg dose of SCH 23390 in the NAC it

attenuated the reinforcing efficacy of amphetamine,

as indicated by a rightward, albeit downward shift in

the rate/frequency function. Figure 8C shows that

Fig. 5. Rats were tested on a 300–20 Hz descending frequency schedule,and at a submaximal current intensity(50% optimal current) following i.p.administration of EtOH (10% v/v)(0.0–1.25 g/kg) across a 20 min oper-ant session in NP rats (n ¼ 6). EtOHwas administered 5 min prior to theBSR session. A : BSR rate-frequencycurves. B: Minimum frequency.C: Effective frequency (EF50) [thresh-

old parameter]. D: Total responding.E: Maximal frequency. ** P < 0.01, * P< 0.05 from placebo control.





when the 5.0 mg dose of SCH 23390 was given imme-

diately prior to the 0.75 mg/kg amphetamine dose, it

significantly ( P < 0.05) attenuated the reduction on

the minimum frequency parameter. Given alone, con-

tralateral administration of the D1 antagonist was

without effect. These data profiles resulted in a signif-

icant ANOVA [ F (3,18) ¼ 3.22, P < 0.04]. Figure 8D

shows that when the 5.0 lg dose of SCH 23390 was

given immediately prior to amphetamine, it did

attenuate amphetamine’s reduction of the EF50

parameter, however; this effect was not significant

( P > 0.05). These data profiles resulted in a signifi-cant ANOVA [ F (3,18) ¼ 4.87, P 0.01]. Figure 8E

shows that when the 5.0 lg dose of SCH 23390 was

given immediately prior to the amphetamine it com-

pletely ( P < 0.01) reversed the marked amphetamine-

induced enhancement on lever-press responding.

Given alone, the D1 antagonist produced a reduction

on responding. These data profiles yielded a signifi-

cant main effect of drug treatment [ F (3,18) ¼ 11.77, P

0.01]. No significant effects were observed on the

maximum frequency measurement (Fig. 8F) with the

combination treatment.

DISCUSSION

The primary purpose of this study was to determine

the ability of the BNST to support BSR responding in

animals with a genetic predisposition to alcohol. To

accomplish this we tested an animal model of human

alcoholism, the P rat, and its contrasting counterpart,

the NP, utilizing a rate-frequency BSR paradigm to

evaluate the effects of EtOH and amphetamine on le-

ver-press responding contingent on direct electrical

stimulation of the BNST. We further investigated the

capacity of the D1 receptor antagonist SCH 23390

centrally administered into the contralateral NAC inreversing the potentiating effects of systemically

delivered amphetamine on BSR.

The initial finding of this study was that while the

P rat consistently lever-pressed for direct electrical

stimulation of the BNST, the NP rat line failed to

dependably respond to such stimulation producing

only one response per minute on average in contrast

to the $16 responses made by the P rats under the

same conditions. It is not surprising that the BNST

can support BSR responding as a number of neuro-

anatomical studies have demonstrated that reciprocal

Fig. 6. Rats were tested on a 300–20 Hzdescending frequency schedule, and at asubmaximal current intensity (50% opti-mal current) following i.p. administrationof amphetamine (0.0–1.60 mg/kg) across a20 min operant session in P rats ( n ¼ 7).

Amphetamine was administered 5 minprior to the BSR session. A : BSR rate-fre-quency curves. B: Minimum frequency. C:Effective frequency (EF50). D: Totalresponding. E: Maximal frequency. ** P <

0.01, * P < 0.05 from placebo control.





projections exist from the BNST to both the lateral

hypothalamus and the VTA (Eiler et al., 2003; Mac-

Donald, 1991; Sun and Cassell, 1993), two established

DA reward loci that have been demonstrated to regu-

late BSR (Phillips and Fibiger, 1989; Stellar and Stel-

lar, 1985). It is also important to note that the BNST

neurons have been shown to be antidromatically acti-

vated via stimulation of their efferent fibers in thehypothalamus (Dalsass and Siegel, 1987). Such evi-

dence makes the inability of the BNST to support

responding for electrical stimulation in the NP rat

line somewhat difficult to explain. One possible expla-

nation is the inherently deficient DA tone seen in the

P rat line (Murphy et al., 2002). It is conceivable that

this deficiency renders the P rat more sensitive to

increases in DA release in the terminal fields of the

mesolimbic pathway creating a more ‘‘rewarding’’

effect of such a DA release. Therefore, the NP rat line

may not be alone in its ability to support BNST BSR.

Such a hypothesis could be addressed by replicating

this study using outbred Wistar rats, the parentstrain of the P and NP lines, to determine if the

BNST can only support BSR responding in a DA defi-

cient limbic system as seen in the P rat line.

The second objective of this study was to evaluate

what effect, if any, EtOH and amphetamine may have

on the reward threshold of BSR in the BNST of the P

and NP rat lines. To determine the effect of EtOH on

lever-press responding for electrical stimulation of the

BNST, both rat lines were given EtOH systemically

just prior to BSR testing. Not surprisingly, EtOH pre-

treatment had no effect on BSR in NP animals.

EtOH, however, was able to alter one parameter of

reward threshold in the P rat line. Both the 0.75 and

1.25 g/kg doses of EtOH were sufficient to reduce the

minimum frequency needed to elicit responding with

no effects seen in the other threshold parameters. As

discussed in the introduction, many conflicting

reports exist in the literature in relation to facilita-

tion of BSR performance following alcohol administra-tion (Bain and Kornetsky, 1989; Lewis and June,

1990; Moolten and Kornetsky, 1990; Schaefer and Mi-

chael, 1987, 1992). Lewis and June (1990, 1994)

showed that reward threshold was consistently low-

ered with i.p. administration of alcohol in doses of

0.25–0.75 g/kg with stimulating lateral hypothalamic

(LH) electrodes, but not mesencephalic ventral norad-

renergic bundle (VNB) sites. Kornetsky and his

colleagues (Bain and Kornetsky, 1989; Moolten and

Kornetsky, 1990) reported that oral alcohol self-

administration (i.e., subject administered) lowered

reward threshold when the lateral hypothalamic

region of the MFB was stimulated; however, i.p. (ex-perimenter administered) alcohol was ineffective. In

agreement with Kornetsky and his colleagues, Schae-

fer and Michael (1987, 1992) have consistently failed

to observe a threshold lowering effect with i.p. or

intragastrically administered alcohol. The findings of

this study suggest that even within the BNST, alcohol

is a less potent reinforcer when compared with other

drugs of abuse. A recent microdialysis study by Car-

boni et al. (2000) appears to support this hypothesis

by demonstrating that, within the BNST, the

enhancement of extracellular DA release by morphine

Fig. 7. Rats were tested on a300–20 Hz descending frequencyschedule, and at a submaximal cur-rent intensity (50% optimal cur-rent) following i.p. administrationof amphetamine (0.0–1.60 mg/kg)across a 20 min operant session inNP rats (n ¼ 6). Amphetamine wasadministered 5 min prior to theBSR session. A : BSR rate-frequency

curves. B: Minimum frequency.C: Effective frequency (EF50). D: Totalresponding. E: Maximal frequency.** P < 0.01, * P < 0.05 from placebocontrol.





and cocaine was far greater than seen with alcohol.

Therefore, it is possible that alcohol’s diminished rein-

forcing profile may be related to its reduced capacityin enhancing DA in the mesolimbic reward circuitry.

Amphetamine was also evaluated in both lines for

its potential effect on the reward threshold of BNST

mediated BSR. Unlike with EtOH, the NP animals

did exhibit a potentiation of BSR on the reward

threshold as seen by a decrease in both the minimum

and maximum frequencies needed to support respond-

ing following a 0.25 mg/kg dose of amphetamine.

However, it must be stated that these data are highly

suspect and probably contribute little to scientific

understanding due to the low levels and erratic

responding seen in the NP line for BSR. Response

profiles like these make interpreting these data diffi-cult; however, they do serve to clearly demonstrate

the NP line’s reduced sensitivity to BNST mediated

BSR reward when compared to the P rat line. The P

rats, with their robust, consistent rates of responding

also show a potentiation of the BSR reward threshold

with significant reductions in the EF50, as well as,

the minimum and maximum frequency parameters in

nearly all tested doses (except the 0.25 mg/kg dose on

minimum frequency). Of the tested doses the 1.25

mg/kg dose appears to be the most effectual. These

effects are consistent with a well established litera-

ture on outbred rats with electrodes in the MFB or

VTA (Esposito et al., 1980; Schafer and Michael,

1992; Wise, 1996). Amphetamine is reputed to be themost optimal drug to study leftward shifts in the rate

frequency function in BSR studies (Gallistel and Kar-

ras, 1984; Kling-Petersen and Svensson, 1993; Wise

and Munn, 1993) since it produces an impulse-inde-

pendent release of DA from nerve terminals (Carboni

et al., 1989; Hurd and Ungerstedt, 1989), and blocks

DA inactivation by blocking its reuptake (Heikkila

et al., 1975; Wise, 1996). The data of the present

study are also in agreement with recent findings from

this laboratory using amphetamine in P and NP rats

with electrodes in the MFB (Eiler et al., 2005). These

data demonstrated that amphetamine facilitated BSR

threshold measures to a greater degree in P rat whencompared with NP rats. Unlike the present study,

however, NP rats readily initiated BSR responding

following electrode inplantation in the MFB. Together,

these data suggest that alcohol preferring rats are

more sensitive to the rewarding effects of ampheta-

mine relative to their nonpreferring (NP) counter-

parts.

While it is possible that amphetamine, a potent

psychostimulant, may merely increase the perform-

ance capacity, locomotor activity, of the P rats to a

greater level than seen in NP rats, explaining the dif-

Fig. 8. Rats were tested on a 300–20Hz descending frequency schedule, and at asubmaximal current intensity (50% optimalcurrent) following central infusion of SCH23390 (5.0 mg) into the NAC and/or i.p.administration of amphetamine (0.75 mg/ kg) across a 20 min operant session in Prats (n ¼ 7). A : BSR rate-frequency curves

following contralateral microinjections of SCH 23390 (2.5 and 5.0 lg) in the NAC. B:Illustrates the rate of lever pressing as afunction of stimulation frequency with am-phetamine (0.75 mg/kg, i.p.) (redrawn fromFig. 6A) and SCH 23390 (5.0 lg) alone, andthe combination of the two (5.0 lg SCH23390 + 0.75 mg/kg amphetamine, i.p.). C:Minimum frequencies (D) Effective fre-quency (EF50) (E) Total responding (F)Maximal frequency. ** P < 0.01 from pla-cebo control, * P < 0.05 from placebo con-trol, { P < 0.05 from amphetamine.





ferential responding, we do not believe this is the

case. While the asymptotic function of the rate-

response curve was elevated relative to placebo by

the higher doses of amphetamine tested, other

research groups have suggested that increases in

response rates may occur independently of changes inBSR threshold (Markou and Koob, 1991) and strong

evidence supports the notion that performance altera-

tions do not significantly change reward threshold

values (Edmonds and Gallistel, 1974; Miliaressis and

Rompre, 1987). Wise (1996) further contends that

drugs of abuse generally increase simple response

rate when optimal stimulation parameters are cho-

sen. More importantly, simple increases in activity

would be thought to increase responding throughout

the 20 min testing session; however, reductions in the

EF50 /half-maximal responding parameter, one of the

most highly accepted measures of BSR reward thresh-

old (Miliaressis et al., 1986; Stellar and Stellar, 1985;

Wise and Rompre 1989), provide evidence that the

increased responding levels are sensitive to frequency

and not simply increased locomotor activity.

Lastly, this study attempted to determine the capa-

bility of the D1 DA receptor antagonist SCH 23390

centrally administered into the NAC to attenuate the

potentiation of the BSR reward threshold produced

by the moderate 0.75 mg/kg dose of amphetamine.

Because of the lack of consistent data in the NP line,

this portion of the study was conducted only on in P

rats. It was found that SCH 23390 alone (2.5 and 5.0

mg) was unable to affect any of the tested BSR pa-

rameters; however, the 5.0 mg dose was sufficient to

significantly reverse the reductions observed on totalresponding and minimum frequency with a 0.75 mg/

kg dose of amphetamine. Also, while not statistically

significant, the 5.0 mg dose of SCH 23390 did mildly

reverse the reduction in EF50 seen with amphetamine

alone. Recent work from our laboratory shows similar

effect of SCH 23390 on amphetamine potentiated

BSR reward in the MFB (Eiler et al., 2006). These

data are consistent with a recent study single-unit

recording study by Cheer et al. (2005) that demon-

strates that SCH 23390 attenuates neural activity in

the NAC during intracranial self-stimulation in the

MFB. They suggest that NAC neurons are preferen-

tially inhibited by GABA A receptors following MFBstimulation. This data suggests that D1 blockade by

SCH 23390 in this study may attenuate the effects of

amphetamine by the inhibition of GABA interneurons

projecting directly to the BNST and other limbic

structures and/or longer relays back to the VTA.

While the mechanism is still unclear, the potentiation

of the BSR reward threshold produced by ampheta-

mine appears to be at least partially regulated by the

DA D1 receptor system within the NAC.

It is important to note that this study did not inves-

tigate the potential effects other neurotransmitter

systems may have on BSR and its regulation in the

BNST. Substantial neuroanatomical and neurochemi-

cal studies have shown that the BNST is uniquely

positioned such that GABA, norepinephrine, and

opioid neurotransmission could independently or

through interactions with the DA systems, regulatereward related behaviors of the BNST. For example,

the BNST receives one of the densest NE fiber inputs

in the CNS, receiving axonal projections from the

ventral [A1, A2] and dorsal [A6] noradrenergic cell

body groupings (Phelix et al., 1992; Swanson and

Hartman, 1975). These noradrenergic fibers travel

through the MFB in close conjunction with the DA

fibers projecting from the VTA (Swanson and Hart-

man, 1975; Ungerstedt, 1971). The BNST also has a

fair density of opioid receptors (Mansour et al., 1987,

1995; Uhl et al., 1978) and receives direct enkepha-

lin-containing fibers originating from the central nu-

cleus of the amygdala via the stria terminalis (Uhl

et al., 1978). Finally, as depicted in Figure 1,

GABAergic afferents originating from several limbic

loci project to the BNST. The BNST also sends

GABAergic fibers to the VTA. This nexus of converg-

ing neurotransmitter systems renders the BNST a

focal point where DA, NE, GABA, and opioids, may

converge to regulate the reinforcing properties of

drugs of abuse and subsequently BSR (cf. Koob, 1999;

Koob and LeMoal, 2001).

In conclusion, the present study demonstrated that

the BNST is a novel BSR reward locus capable of sup-

porting BSR performance in P, but not NP rats. We

hypothesize that the ‘‘innately’’ deficient DA system

within P rats may increase their sensitivity to ele-vated concentrations of DA in the terminal fields of

the BNST and other reward areas such as the NAC.

Amphetamine was also found to markedly lower BSR

threshold measures and enhanced response rates

while alcohol was without effect on all but the mini-

mum frequency parameter. Thus, the reinforcing

potency of alcohol on BSR performance was markedly

less compared with amphetamine. This undoubtedly

reflects the relative amount of extracelluar DA

released within terminals of the BNST, NAC and

other DA sensitive loci. It was also found that unlia-

teral infusion of the D1 antagonist SCH 23390 was

capable of attenuating the effects of amphetamine onBSR. In short, the BNST appears to be a viable neu-

ral substrate capable of assessing the reinforcing, and

hence, abuse liablity of drugs of abuse, particularly in

rats with a predisposition to alcoholism.

ACKNOWLEDGMENTS

The authors thank Drs. T.-K. Li, currently at

NIAAA, and Larry Lumeng and the Alcohol Research

Center, Indiana University School of Medicine for pro-

viding the P and NP rats.





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