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Hormones and Behavior

Food competition and social experience effects on V1a receptor binding

in the forebrain of male Long–Evans hooded rats

Alicia Askewa,b,*, Fernando A. Gonzaleza,b, Jeanne M. Stahla,b, Mary C. Karoma,c,d

aCenter for Behavioral Neuroscience, Morris Brown College, Atlanta, GA 30314, USAbDepartment of Psychology, Morris Brown College, Atlanta, GA 30314, USAcDepartment of Biology, Georgia State University, Atlanta, GA 30303, USA

dDepartment of Psychology, Georgia State University, Atlanta, Georgia 30303, USA

Received 31 December 2004; revised 30 July 2005; accepted 4 August 2005

Available online 9 September 2005

Abstract

The present study investigated the effect of social status in Long–Evans hooded rats established during food competition on V1a

vasopressin receptor (V1aR) binding in the lateral septum (LS), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST),

anterior hypothalamus (AH), and central/basolateral amygdala (CeB). Serum concentration of testosterone (T) and corticosterone (CORT)

was also measured. In Experiment 1, thirty-two lever-trained weight-matched rat pairs were placed in operant chambers where a single bar

press provided access to milk reinforcement. A dominant–subordinate relationship, determined by the duration of drinking, was evident in

88% of the pairs. Sixteen rats were lever-trained but did not interact and served as no-treatment (NT) controls. In the LS, V1aR binding in the

subordinate (SUB) group was significantly higher than in the dominant (DOM) group. V1aR binding was significantly higher in the LS,

BNST, CeB, and AH in the NT group than in the other groups. The levels of CORT and T were not affected significantly by group

membership. Experiment 2 investigated whether the binding effect in the LS was related to differences in fluid consumption. The results did

not indicate a significant effect of fluid consumption. In the rat, V1aR binding in several forebrain areas seems to be affected by brief periods

of social interactions, and, in the LS, it also appears to be related to dominance status.

D 2005 Elsevier Inc. All rights reserved.

Keywords: V1a receptor; Vasopressin; Social interaction; Food competition; Lateral septum

Introduction

Researchers define dominance in terms of the results of

competitive tasks involving access to territory, mates, food,

or water. In newly formed pairs or groups of animals,

agonistic encounters generally bring about a social ranking

that reduces conflict, enabling one animal to gain priority

access to limited survival and reproductive assets. The

resident–intruder (R–I) and colony models are commonly

used for social conflict studies with rodents. These models

take advantage of rodents’ predisposition to defend their

0018-506X/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.yhbeh.2005.08.001

* Corresponding author. 503 South Broad Street, Clinton, SC 29325,

USA.

E-mail address: jaaskew@presby.edu (A. Askew).

territory, mate or litter from intrusions by conspecifics. In

both models, social conflict generally yields a winner of

agonistic encounters (dominant) and one or more defeated

(subordinate) subjects that have little control over the shared

environment (see review by Martinez et al., 1998a). Another

model involves competition for access to a limited food or

water source (Baenninger, 1970; Lanctot and Best, 2000;

Lucion and Vogel, 1994). In this food competition model,

the definition of dominance is based on the extent of control

exerted by an animal over the limited resource, with the

animal gaining priority access to the resource being

categorized as dominant.

The R– I and colony models have been used to

investigate the neurobiological substrates of agonistic

behavior and the physiological consequences/correlates of

social rank (Martinez et al., 1998a). Data from R–I studies

49 (2006) 328 – 336

A. Askew et al. / Hormones and Behavior 49 (2006) 328–336 329

indicate that many rodent forebrain areas including the

lateral septum (LS), central and basolateral nuclei of the

amygdala (CeB), medial preoptic area (MPOA), anterior

hypothalamus (AH), and bed nucleus of the stria terminalis

(BNST) play a role in social behavior (Kollack-Walker et

al., 1999; Kollack-Walker et al., 1997; Martinez et al.,

1998a,b). In rodents, many of these brain areas are either

sources of vasopressin (VP; De Vries et al., 1985) or are

targets of VP fibers (Caffe et al., 1987; De Vries et al., 1985;

Ferris and Potegal, 1988; Szot et al., 1994). Centrally

released AVP is involved in many social behaviors

including affiliation (Young et al., 1999), pair bonding

(Winslow et al., 1993), and paternal behavior (Bamshad et

al., 1994) in voles; flank-marking (Bamshad and Albers,

1996) and aggression (Ferris et al., 1997) in hamsters; and

social recognition (Englemann and Landgraf, 1994; Everts

and Koolhaas, 1997) and aggression (Koolhaas et al., 1991)

in rats. Of the three subtypes of AVP receptors, the

peripheral V1a receptor (V1aR) is pharmacologically the

most similar to central AVP receptors (Phillips et al., 1988)

and the most frequently linked to behavioral effects (Albers

et al., 1986; Ferris and Potegal, 1988; Landgraf et al., 2003;

Winslow et al., 1993).

There is an extensive literature implicating the septal area

in the regulation of various aspects of agonistic behavior in

rodents, including aggression and dominant–subordinate

relationships (Sheehan and Numan, 2000). For example,

electrolytic lesions of the septum of Long–Evans rats

reversed dominant status in a food competition setting 1

week and 3 weeks following lesion placement; no status

change was seen in septal-lesioned subordinate rats (Cos-

tanzo et al., 1977). AVP in the LS appears to be involved in

the display of aggression and/or dominance status in rats.

Compared to non-aggressive mice, aggressive mice show a

higher density of AVP neurons in the BNST (a major source

of AVP input into the LS) and of AVP innervation in the LS

(Compaan et al., 1993). Similarly, Everts et al. (1997) report

a negative correlation between LS AVP content and AVP

fiber density and intermale aggression in wild-type rats.

Social defeat and subordination in laboratory rodents

have been associated with an increase in circulating levels of

glucocorticoids (Huhman et al., 1990, 1991; Pich et al.,

1993). Defeat and subordination have also been associated

with a decrease in hypothalamic–gonadal axis (HPG)

activity that is evidenced by a decrease in leutenizing

hormone, follicle-stimulating hormone, and testosterone

(Blanchard et al., 1993; Bronson, 1973; Huhman et al.,

1991; Schuurman, 1980). In the rat, adrenal steroids may

play a role in the regulation of V1aR in several forebrain

areas (Watters et al., 1996).

In this study, we used a food competition model to

establish a dominant–subordinate relationship in pairs of

rats contending for access to a liquid reinforcer. The rat in

the pair that gained priority access as determined by the

higher duration of drinking (s) was labeled ‘‘dominant.’’

This definition of dominance was used to investigate the

relationship between social status and V1aR binding in

several areas of the rat brain (AH, BNST, MPOA, LS, and

CeB) that have been shown to regulate agonistic behaviors

in rodents. Previous literature suggests involvement of

septal AVP in the regulation of aggression and/or domi-

nance status, thus, we anticipated differences in V1aR

binding between dominant and subordinate rats in the LS.

We also explored the relationship between social status and

serum concentration of testosterone and corticosterone.

Materials and methods

Animals

The subjects were 64 male Long–Evans hooded rats

obtained from Charles Rivers Laboratories. They were

approximately 90 days old and weighed 300–400 g at the

beginning of the study. Subjects were individually housed

in hanging cages (17.8 � 24 � 17.8 cm) in the animal

facility at Morris Brown College. A 12:12 light–dark

cycle with lights off at 19:00 h was in effect in the colony

room. The rats were maintained at 95% T 3% of ad libitum

food weight-corrected for growth. Rat chow was given

daily between 15:30 and 17:00 h. Water was available ad

libitum. The Atlanta University Center Institutional Animal

Care and Use Committee approved all procedures and

protocols.

Apparatus

The food competition procedure was carried out in four

computer-controlled modular operant conditioning cham-

bers (MED Associates, Inc, ENV-007). Each chamber

(interior dimensions: 24.1 � 30.5 � 30.5 cm) was placed

inside a ventilated, sound-attenuating box (61 � 61 � 91.5

cm). A dipper was installed on the front wall of the operant

chambers to dispense 0.02 ml of the liquid reinforcement

(RNF), which was a 3:1 solution of H2O and sweetened

condensed milk. A response lever was located 2.5 cm to the

right of the dipper opening. A minimum downward force of

approximately 0.2 N operated the lever. Three small

stimulus lamps were installed in a row 3 cm above the

lever. A house lamp was located on the back wall of the

chamber, 1.5 cm below the top of the chamber. The

chambers were modified to permit rapid placement and

removal of a wire mesh partition. This partition was placed

parallel to and 17 cm from the front wall (13.5 cm from the

back wall). Separate Plexiglas doors (10 � 20 cm), hinged

at the top, were located on one side of the chamber. Rats

were inserted into and removed from the chamber through

these doors. The opening through which the partition was

slid into the chamber was located between the two doors.

Programming and data recording equipment were run by

MED/PC software (Med Associates, Inc., Version 1.20).

Logitech QuickCam Pro 3000 cameras were mounted on the

A. Askew et al. / Hormones and Behavior 49 (2006) 328–336330

back wall of the sound-attenuating boxes to produce a

digital video record of the food competition sessions.

Handling, shaping, and training

All rats were handled daily for 10 min until no visible

(urination and defecation) and audible (squealing) signs of

stress were apparent. After a minimum of 3 days of handling,

the subjects were shaped to lever press for the condensed

milk solution. Subjects were then run individually on a

program that specified that every response be immediately

followed by operation of the dipper (fixed ratio 1 [FR1]

schedule). The dipper cup remained accessible to the rat for

3.5 s. The rightmost stimulus lamp was illuminated during

milk accessibility. This signal indicated to research assistants

who scored the rats’ drinking behavior that the milk

reinforcer was available to the rat. The leftmost stimulus

lamp was briefly illuminated when the lever was depressed

to provide feedback for responding. Responses had no effect

for 1.5 s following retraction of the dipper. Since the

minimum duration of a RNF cycle was 5 s, the maximum

RNF rate was 12 RNF/min. This procedure remained in

place until average rate per session was at least 8 RNF/min

over six consecutive 30-min sessions.

Experiment 1

Food competition

After meeting the RNF rate criterion, 32 of 48 trained

rats were assigned to the food competition procedure. The

selected rats were paired by weight (N = 16 pairs). During

food competition sessions, the rats were always paired with

the same partner. A maximum of two 30-min food

competition sessions were conducted on weekdays between

9:00–12:00 h and 14:00–17:00 h. A total of 15 sessions

were run. During these sessions, the FR1 schedule

procedure described above was in effect. In an attempt to

eliminate territoriality as a motive for conflict, the rat pairs

were randomly assigned to a chamber prior to each food

competition session. One rat was placed in the chamber on

each side of the partition. Placement in the front and rear

compartments was counterbalanced across sessions for each

rat pair. After putting the rats in the chamber, the video

recording and the FR1 schedule were simultaneously

started. The partition was then removed, allowing the rats

to interact, and the door to the sound attenuation box was

closed. Immediately following each session, the chambers

were cleaned with 70% alcohol.

No-treatment controls

Prior to the experiment, the 16 trained rats not selected

for the food competition procedure were treated in the same

manner as the food competition rats. The no-treatment (NT)

control rats were not placed in the chambers, but, twice a

day for 7 days and once on the 8th day, they were placed

individually into transfer cages and moved temporarily to

the experimental room where the operant conditioning

chambers were located. NT rats were never placed in a

chamber or in a cage with another rat.

Experiment 2

Food vs. no-food

It is well known that peripheral vasopressin plays a role

in fluid homeostasis; it is secreted in response to either

increases in plasma osmolality or to decreases in plasma

volume. It follows that any measured differences in V1aR

binding among the groups (DOM, SUB) in the food

competition procedure could be occasioned by the differ-

ences in milk consumption that defined group membership

and thus confound the interpretation of results. A control

procedure (Experiment 2) was conducted to eliminate

differential fluid intake as an alternative explanation of the

results of Experiment 1.

The subjects were the 16 male Long–Evans hooded rats

not used in the food competition experiment. The apparatus

and the handling, shaping, and training procedures were

same as in the food competition procedure. Once all the rats

met the training criterion, they were divided into two

weight-matched groups. One group was randomly assigned

as the food control group (F), the other group was the no-

food control (NF). The F rats were always placed in the

front compartment of the operant chamber and, therefore,

could press the lever. The NF rats were always placed in the

rear compartment of the chamber where they did not have

access to the lever. Subjects were run individually for fifteen

30-min sessions. The partition was not removed during the

sessions. In every other respect, the procedure was identical

to the food competition procedure. It follows that the F rats

had access to the milk solution at least to the same extent as

food competition dominant rats. The NF rats were denied

access to the milk solution, and, in this respect, they were

similar to subordinate rats.

Behavioral measurements

Behavioral data were available only from the food

competition rats. Since the NT rats were never in the

chambers and the F and NF rats were alone in the chambers

during all sessions, for these groups there were no relevant

behaviors to record during the sessions.

All behavioral data were scored from the digital videos

using Event Tracker* software (Center for Behavioral

Neuroscience, 2002). The pertinent data were the frequency

and duration of overt aggressive interactions and the total

time each rat spent with its head inside the dipper opening

while the RNF signal lamp was illuminated. This last

measure was assumed to closely reflect a rat’s milk-drinking

behavior. Aggressive interactions were scored for pairs, not

for individual rats. The duration and frequency of the

following behaviors were scored for aggression: chase, bite,

‘‘on-top-of’’ (one animal pins another to the floor or sits on

A. Askew et al. / Hormones and Behavior 49 (2006) 328–336 331

top of the other), and attack. Aggressive behavior was

scored for the full duration of the initial 5 sessions.

The amount of time each rat in a pair spent drinking milk

during the food competition sessions was used to determine

dominance status. It was untenable to score the full 30-min

sessions since it would have required a minimum of 240 h

of scoring. Instead, two research assistants scored the total

duration of drinking behavior of each rat for the middle

5 min of each session. One research assistant also scored the

full 30 min for all the animals during the last two sessions.

The correlation between the 5 min and 30 min scores was

0.883. Inter-rater correlation for the 5 min scores over the 15

sessions was 0.925. Each rat’s average drinking time during

the 5-min scoring period of each of the last five sessions was

used to determine dominance status. If both rats together

spent at least 1/3 of the observation period (300 s) drinking

and one of them spent twice as much time drinking as the

other one, then that rat was categorized as dominant (DOM)

and the partner was labeled subordinate (SUB).

Sample collection

The final session occurred between the 9:00 and 14:00 h

for all groups. Pairs of rats (food competition and weight-

matched pairs of NT, F, and NF rats) were run 15–20 min

apart to allow adequate time for sample collection. One rat

from each pair was selected at random to be the first taken

out of the chamber and anesthetized. Upon removal from

the testing room, the selected rat was immediately anes-

thetized using isoflurane, and blood was collected from the

inferior vena cava. Each rat in a pair was anesthetized within

3 min after removal from the operant chamber; blood was

collected within 2 min following anesthetization. Collection

from the first animal in the pair was immediately followed

by anesthetization of the second animal. Following blood

collection, the subject was rapidly decapitated while still

under anesthesia. The brain was excised and stored at

�80-C for radioligand receptor autoradiography.

Radioimmunoassays

The collected blood was dispensed into 4 ml Gel and Clot

Activator Vacutainer* tubes (Becton Dickenson) and allowed

to clot for approximately 1 h on ice. The tubes were then spun

at 2400 rpm for a minimum of 15 min. Serum was collected

and stored at �20-C for testosterone (T) and corticosterone

(CORT) radioimmunoassays. T and CORT assays were

conducted twice. Each time, T was measured in a single

batch without extraction using antibody-coated tubes from

Diagnostic Systems Laboratories (DSL-4000). Dilution

parallelism was used to validate immunological specificity

with a recovery of 90%. Detection range of the assay was 0.09

ng/ml to 25.00 ng/ml. The intra-assay reliability was 11%.

For both CORTassays, CORTserum levels were measured in

a single batch using antibody-coated tubes from Diagnostic

Systems Laboratories (Rat Corticosterone DSL-81100).

Dilution parallelism was used to validate with a recovery of

93%. Detection range of the assay was 14.87 ng/ml to

2000.00 ng/ml. The intra-assay reliability was 9%. The inter-

assay reliability for both T and CORT was 10%.

Receptor autoradiography

The brains were cut into 20 Am sections and thaw-

mounted on superfrost slides for labeling of V1aR. Slides

were stored at �80-C until autoradiographic analysis.

Sections were processed for receptor autoradiography using125I linear Vasopressin V1aR Antagonist (NEN Life Science

Products, Inc., Boston MA, USA) as described previously

(Young et al., 2000; Francis et al., 2002). The slides were

allowed to dry and then were fixed for 2 min in 0.1%

paraformaldehyde followed by two 10-min washes in TRIS

buffer without MgCl. Slides were placed back to back in an

incubation chamber filled with tracer buffer mixed at a

concentration of 2.2 � 10�4 mol/Al and incubated for

60 min. Following incubation, slides were washed three

times in TRIS buffer with MgCl for a total of 45 min and

then dipped in distilled water. A hairdryer was used to blow-

dry the slides. Slides were taped tissue side up to a sheet of

white paper in an autoradiography cassette along with an

autoradiographic [I125] microscale (Amersham Biosciences)

and transferred to the darkroom where Kodak film Biomax

MR-2 was placed over the slides. Films were exposed to the

slides for 3 days and developed. Films were placed in a tray

of developer for 2 min followed by 30 s in tap water, 3–5

min in fixer/replenisher, and finally placed in tap water for

30 s. Optical density was quantified using Scion Image

software (http:\rsb.info.nih.gov\nih-image/) and converted

to dpm/mg tissue equivalents using 125I microscales.

Schematic representations of the quantified areas of the

LS, CeB, AH, BNST, and MPOA are depicted in Fig. 1

(Paxinos and Watson, 1998). All brain areas for specific and

nonspecific (adjacent tissue area) binding were determined

by measuring with a box that was 0.35 mm � 0.35 mm

placed on corresponding brain areas. A mean representing

dpm/mg was extrapolated from the curve created from the

microscale on the film. The specific binding mean was

subtracted from the nonspecific binding mean for each brain

section to quantify the labeling in each brain area. Signal

intensity for each area was measured unilaterally from six

sections from each animal.

Statistical analysis

The signal intensity values from the approximately 350

slides available from each area were available for analysis.

Data from individual sections representing outliers beyond

2.3 T SD were discarded. This resulted in dropping the data

from 22 (LS, 4; MPOA, 4; CeB, 0; BNST, 8; AH, 6) out of

1400 sections for the DOM, SUB, and NT groups and 11 out

of 440 sections for the F and NF groups. The values from the

remaining slides in each set for each area for each rat were

Fig. 1. Schematic representation (Paxinos and Watson, 1998) of the

quantified areas of the lateral septum (LS), amygdala (CeB), anterior

hypothalamus (AH), bed nucleus of the stria terminalis (BNST), and medial

preoptic area (MPOA).

Fig. 2. (A) Percent of pairs that engaged in aggressive bouts during the first

five sessions. (B) Mean + SEM of the total time per session spent fighting

for pairs that engaged in fighting.

A. Askew et al. / Hormones and Behavior 49 (2006) 328–336332

averaged to produce the datum for analysis. The data from

Experiment 1 and Experiment 2 were analyzed separately.

One-way between groups (DOM, SUB, NT) ANOVAs were

conducted on the V1aR binding data in the five sites [MPOA,

AH, CeB, BNST, and LS]) and on the CORT and T

measurements (a = 0.05). Since multiple tests inflate alpha,

a Bonferroni adjustment was made for the V1aR analysis.

Therefore, the chosen alpha was divided by the number of

tests so that the obtained P value was compared to an alpha of

0.05/5 = 0.01. One-wayANOVAswere also performed on the

F and NF data to determine significant differences (a = 0.05)

in V1aR binding in each of the five areas that could be

attributed to the difference in fluid intake between the two

groups. A Bonferroni adjustment of the alpha value used in

these tests was made as above so that the P value from each

ANOVA was compared to an alpha of 0.01. A planned

comparison was performed on the LS binding data from

DOM and SUB groups in Experiment 1. Post-hoc compar-

isons (Tukey’s HSD test for unequal sample sizes) were

performed for all the other V1aR binding dependent variables.

One-way ANOVAs were used to analyze the T and

CORT data from Experiment 1, and independent sample t

tests were used to analyze the hormone data from Experi-

ment 2. Because the T data for every group were severely

skewed to the right, a simple logarithmic transformation was

performed on the T data to generate approximately normal

distributions more amenable to the parametric tests. The

reported means and SEMs of the T data are the antilogs of

the means and SEMs of the distributions of the log-

transformed T data. All statistical analyses were carried

out using STATISTICA for Windows (StatSoft, Inc., Tulsa,

OK).

Results

Aggressive encounters occurred mainly during the initial

food competition sessions. As Fig. 2 shows, the percent of

rat pairs that engaged in overt aggressive behaviors dropped

drastically after the third session, and the total duration of

the aggressive bouts also decreased dramatically. The

duration the aggressive bouts varied across the rat pairs

and from encounter to encounter but the sum total of the

time spent fighting during a session was, in every case, a

very small fraction of the session duration. Aggressive

contact between the rats was rarely observed after the fifth

session.

Fig. 4. Time each rat in a pair spent drinking during the middle 5 min of

each session. Rats 77 (A) and 78 (B) spent at least twice as much time

A. Askew et al. / Hormones and Behavior 49 (2006) 328–336 333

In 14 of the 16 rat pairs, one of the rats attained dominant

status. The rats’ weights were not a factor determining

dominance status. The mean weight of the DOM rats at the

end of the experiment was 359.28 g, while the SUB rats

weighed 361.23 g. The SEM of the weight difference

between the rats in each pair was 3.44 g. Fig. 3 shows that,

during the last five sessions, most of the dominant rats

consumed milk for at least half of the scored period (300 s)

while the 14 subordinate animals drank during 10% of the

period or less. The line graphs in the panels of Fig. 4

illustrate the drinking behavior of three selected rat pairs

over the 15 food competition sessions. The drinking times

presented in panel (C) are from one of the pairs in which

dominance status was not evident. Panel (B) shows the

drinking behavior of a rat pair in which dominance status

developed in the later sessions, and panel (A) shows that in

some instances dominance emerged almost immediately. To

assess the number of sessions required to attain dominance

status, we applied the dominance criterion (see above)

beginning on the first session. When a rat in a pair attained

the criterion for five consecutive days, the session number

corresponding to the fifth day was said to represent the

number of sessions required for dominance to emerge. A

correlation between the number of sessions needed to reach

dominant status and the duration of aggressive behavior in

the initial 5 sessions was not significant.

For Experiment 1, the relationship between group

(DOM, SUB, and NT) membership and V1aR binding in

the five brain areas is depicted in the panels of Fig. 5. The

ANOVA revealed significant main effects of group member-

ship in the LS (F(2,38) = 23.4011, P < 0.00001), BNST

(F(2,41) = 26.9492, P < 0.00001), CeB (F(2,39) =

43.5634, P < 0.00001), and AH (F(2,40) = 36.2217, P <

Fig. 3. Mean drinking time during the middle 5 min of the last five sessions

for each rat pair. The darker columns represent the dominant rats. The rats

in pairs 55–56 and 54–59 failed to establish dominance status.

drinking during the last five sessions than the subordinate rats in the pairs.

Dominance status was not achieved by either of the rats in pair 54–59 (C).

0.00001). Tukey’s HSD post-hoc test indicated that V1aR

binding was significantly higher in the NT than in the DOM

and SUB groups (P < 0.00013, P < 0.01356), BNST (P <

0.00012, P < 0.00012), CeB (P < 0.00013, P < 0.00013),

and AH (P < 0.00012, P < 0.00012) and that in only in the

LS there was a significant difference between the DOM and

SUB groups (P < 0.0067). The planned comparison on the

LS data, of course, showed that the differences among the

DOM and SUB groups were significant (F(1, 38) = 9.2522,

P < 0.00425). Autoradiographic sections illustrating differ-

ences in V1aR binding that we attribute to group member-

ship in the LS are presented in Fig. 6.

In the F vs. NF comparisons, the ANOVA indicated that

the effect of group membership was not significant in any of

the brain areas. Independent samples t tests indicated that

there were no significant differences between the F and NF

groups in CORT or T serum concentrations. The mean

(TSEM) levels of CORT for the DOM, SUB, and NT were

766 T 57, 668 T 37, and 597 T 57 ng/ml respectively. The

mean levels of T for the same groups were 1.80 T 1.30,

Fig. 5. V1a receptor binding in each of the measured areas for the dominant

(DOM), subordinate (SUB), and no-treatment control (NT) groups. The

ANOVA revealed that the main effect of group membership was significant

( P < 0.00001). Post-hoc tests showed that, in the LS, BNST, CeB, and AH

receptor, binding was significantly higher in the NT than in the other two

groups ( P < 0.001) and that binding in the LS was significantly higher in

the SUB compared to the DOM group ( P < 0.006).

A. Askew et al. / Hormones and Behavior 49 (2006) 328–336334

2.73 T 1.24, and 1.18 T 1.32 ng/ml respectively. The

ANOVA indicated that the differences among these groups

in either CORT or T were not significant.

Fig. 6. Representative sections of vasopressin V1a receptor autoradiography

in the LS of SUB, DOM, and NT animals.

Discussion

In the present study, subjects were not water-deprived,

food deprivation was minimal (95% T 3%), and sessions

were conducted during the light phase of the diurnal cycle

when displays of aggression were less likely to occur. In

spite of these circumstances, we observed a clear demarca-

tion of dominance status in 88% of the rat pairs. Other

studies involving food or water competition have also been

successful in establishing dominance status (Baenninger,

1970; Peres and Leite, 2002; Price et al., 1976).

Experiment 1 investigated whether differences in V1aR

binding in the LS, MPOA, AH, BNST, and CeB are

associated with dominance status and social experience in

the rat. Only one area appeared to show differences in V1aR

binding related to dominance status. In the LS, V1aR

binding in the SUB group was significantly higher than

that observed in the DOM group, while no difference in

binding in the LS was observed for the F and NF groups.

These results suggest that the patterns of V1aR binding

observed in Experiment 1 are unrelated to fluid consump-

tion. The largest variations in V1aR binding in Experiment 1

were between the NT and the DOM and SUB groups. In the

AH, BNST, CeB, and LS, V1aR binding in the NT group

was significantly higher than in the DOM and SUB,

suggesting an effect of social experience on AVP circuitry

that is independent from dominance status. In this study,

CORT and T levels were not significantly different among

the groups.

Previous studies have reported that defeat and subordi-

nation increased glucocorticoid levels (Blanchard et al.,

1993, 1998; Huhman et al., 1990, 1991; Pich et al., 1993)

and decreased testosterone levels (Blanchard et al., 1993;

Huhman et al., 1991). It is possible that T and CORT did not

vary with social rank in our study as they have in other

studies because overt aggression was not integral to our

definition of dominance. It is also possible that, by the last

food competition session in Experiment 1, when the blood

samples were collected, the level of stress engendered by the

experimental procedure was minimal.

It should be noted that we report substantially higher

CORT levels than other studies investigating social conflict

and subordination in Long–Evans rats (Albeck et al., 1997;

Lucus et al., 2004). Differences in food access schedules

A. Askew et al. / Hormones and Behavior 49 (2006) 328–336 335

(which may shift peak CORT release; Selmaoui et al.,

2003), collection methods, housing, time of sampling, and

events prior to sampling or anesthetization (transport,

placement in anesthetic chamber) may be factors in this

variation and make these comparisons of the CORT

measurements problematic. Our T data do not appear to

contradict reports of T levels in non-stressed rats of a similar

age and obtained during a similar sampling period (Fentie et

al., 2004), but high variability in our measures, as well as

differences in collection methods, housing, and exact time

of sampling make the comparisons difficult.

It is also reasonable to consider whether the observed

differences in V1aR were due to initial differences in the

levels of stress across the groups. In Syrian hamsters,

testosterone has been shown to stimulate V1aR biosynthesis

(Johnson et al., 1995), as well as reduce V1aR binding in the

MPOA and medial preoptic nucleus (Young et al., 2000).

However, V1aR binding in the rat does not appear to be

affected by circulating levels of gonadal steroids (Tribollet

et al., 1990). Adrenal steroids, on the other hand, appear to

play an important role in the regulation of V1aR in the rat

septum. Therefore, in the rat, the differences in V1aR may

arise in response to stress-induced differences in circulating

levels of glucocorticoids (Watters et al., 1996). Though we

did not obtain differences in CORT levels between the

DOM and SUB groups when the blood samples were taken,

it is possible that stress and CORT levels may have differed

between the groups before the dominant–subordinate dyads

became stable. Future studies could address the relationship

between individual levels of aggression, neuroendocrine

responses during periods of social instability, and the level

of V1aR binding in the LS.

There is evidence that suggests that connections between

the LS and the hippocampus, MPOA, and AH regulate

social memory processes (Sheehan and Numan, 2000).

Along these lines, recent data indicate that AVP and V1aR in

the LS are important in social recognition memory in male

rats (Everts and Koolhaas, 1997; Landgraf et al., 1995,

2003). Social recognition memory may have played a role in

the development and maintenance of the stable dominant–

subordinate relationships observed in Experiment 1. The

only sensory modality that has direct connections with the

amygdala is olfaction. Information from the main and

accessory olfactory bulbs converges on the BNST and the

amygdala (Swann and Fiber, 1997), and this input may

reach the LS (see review by Sheehan and Numan, 2000).

Given these connections, it is plausible that the effects of

social experience on V1aR binding in at least the BNST, LS,

and CeB are related to the processing of social olfactory

information.

In summary, we found that, in a food competition

situation, V1aR binding in the LS is related to a rat’s status

in a dominant–subordinate dyad that evolves with minimal

displays of overt aggression under what we consider to be

relatively non-stressful conditions. We also found that

relatively short (30 min) daily periods of social interaction

between a pair of rats can produce significant differences in

V1aR binding in four of the five areas of the forebrain (LS,

BNST, CeB, and AH) considered in this study. This finding

indicates that in some areas of the rat brain V1aR can be

highly sensitive to social stimuli. Our results suggest that, at

least in rats, a social hierarchy emerges readily even when

the immediate environmental circumstances do not offer

survival or reproductive advantages associated with social

rank. These findings add to the mounting evidence that the

AVP system is an important component of the mechanisms

that underlie social behavior.

Acknowledgments

The authors would like to thank Elizabeth Ambe, Corlin

Doolittle, Linda Gardner, Rebecca Johnson, Alicia Mercer,

Elvie Montelus, Ellen Pierce, Michelle Tyson, Ashley

Weems, and the animal care technicians from Morehouse

School of Medicine for their contributions. This material is

based upon work supported by the STC Program of the

National Science Foundation under Agreement No. IBN-

9876754, NIDA-MIDARDP (5 R24 DA07256), NIGMS-

MARC U*STAR (GM T34 07666), and NIMH-COR (MH

T34 16573).

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