10 L
2.4.1. The social interaction test 2.6. Histology
Rats were tested under low light, unfamiliar test con- ditions for anxiogenic or anxiolytic effects [9]. A day prior
At the end of the behavioural testing all animals were to testing, operated animals were weighed and allocated to
overdosed with chloroform and the brains perfused in- pairs; the partners were non-operated, untreated rats of
tracardially with 0.9 saline followed by 4 formalde- similar weight, which had been singly housed for the same
hyde solution. Brains were removed from the skull and length of time as the operated animals. Both animals of a
fixed with paraffin. Coronal 25 mm microtome sections, pair were placed in low light 50 lux in the social
stained by hematoxylin-eosin, were performed to evaluate interaction arena for a 5-min period. An observer blind to
the position and extensions of the lesion [33]. To further rat condition scored the time spent in social interaction.
visualize the extension of the mechanical damage and Behaviors were only scored when initiated by the operated
gliosis, preliminary studies included horizontal brain sec- animal and consisted of sniffing, following, allogrooming,
tions Fig. 1C. wrestling, biting and kicking. A surgical scar on the head
allowed the scorer to distinguish the operated animals from 2.7. Statistics
their partner. Behavioral scores from experiments 1 and 3 were
2.4.2. Elevated plus-maze analyzed by Student t-tests. Data from experiments 2 and 4
Each rat was placed in the central square of the plus- were subjected to analyses of variance with drug and
maze facing a closed arm and its behavior observed for 5 lesion as independent factors Two-way ANOVA. The
min by an observer blind to the treatment. The number of significance obtained by Newman-Keuls’ post-hoc test is
entries onto open and closed arms and the times spent in shown in the figures.
open and closed arms and in the central square were scored. The arena was thoroughly wiped with damp tissue
after each trial.
3. Results
2.5. Allocation to experimental groups 3.1. Histology
Except for experiments 3 and 4, different sets of rats were allocated to each experiment. Numbers in brackets
The stereotaxic knife cut produced lesions of uniform correspond to animals with correct lesion location.
size and comparable volumes. Data from animals with predominant damage on the left n54: two from experi-
2.5.1. Experiment 1 ment 1 and two from experiment 2 and right n52, from
Effect of the MPFC lesion on the behavior in the social experiments 3 and 4 sides were excluded from the study.
interaction test. Rats were allocated to sham n59 and The corpus callosum and nucleus accumbens were spared
MPFC lesion n59 groups. in all the animals included in the study. Fig. 1A shows a
diagram of the lesion target area. 2.5.2. Experiment 2
Effect of amphetamine 2 mg kg on the behavior in the
3.2. Experiment 1 social interaction test
. Rats were allocated to the following groups: sham1vehicle n58; sham1amphetamine n58;
Rats with MPFC lesions significantly spent more time in MPFC lesion1vehicle n57; MPFC lesion1amphetamine
social interaction than controls P,0.002, Fig. 2. No n57.
significant difference in locomotor activity was observed, see Fig. 2. MPFC damaged rats increased social interaction
2.5.3. Experiment 3 by displaying extensive social investigation sniffing,
Effect of the MPFC lesion on the behavior in the allogrooming, and following. Aggressive behavioral fea-
plus-maze . Rats were allocated to sham n510 and MPFC
tures wrestling, biting and kicking were not observed. lesion n510 groups.
2.5.4. Experiment 4 3.3. Experiment 2
Effect of diazepam 1 mg kg on the behavior in a
plus-maze second trial . Rats tested on experiment 3 were
The effect of amphetamine on time spent in social retested in the plus-maze apparatus for 5 min. Animals
interaction varied depending on whether the animal brain were allocated to the following groups: sham1vehicle
was intact or transected [drug3lesion, F1,2654.85, P, n55; sham1diazepam 1 mg kg n55; MPFC lesion1
0.01; lesion, F1,26516.67, P,0.0001; drug, F1,265 vehicle n55; MPFC lesion1diazepam 1 mg kg n55.
3.47, P,0.074]. Thus, systemic administration of amphet-
L .E. Gonzalez et al. Brain Research 887 2000 7 –15
11
Fig. 2. Mean 6S.E.M. time s spent in social interaction by rats with medial prefrontal cortex lesions and sham in low light unfamiliar test
condition. Unpaired t-test: T1653.7, P,0.002. There was no differ- ence in locomotor activity [T1650.3, P50.1].
amine decreased the time spent in social interaction in sham but not in MPFC damaged Fig. 3A. As in experi-
ment 1, MPFC damaged rats increased social interaction exclusively at the expense of social investigation.
Amphetamine increased locomotor activity [F1,265 16.3, P,0.0001] see Fig. 3B. However, this effect was
greater in lesioned than in sham animals and there was a significant drug3lesion interaction [F1,2653.34, P,
0.05]. However, the lesion itself had no effect on locomotor activity [F1,2651.6, NS].
There were effects of the lesions [F1,26513.59, P, 0.001], and drug treatment [F1,2655.81, P,0.02] on
number of rearing and there was significant drug3lesion
Fig. 3. Behavioral measures in the social interaction test by MPFC and
interaction [F1,2654.7, P,0.05]. However, post-hoc
sham rats after IP injections of saline or d-amphetamine 2 mg kg in low
analysis showed that rearing only increased in sham but
light, unfamiliar test conditions. A Mean 6S.E.M. time s spent in
not in lesioned rats following amphetamine administration.
social interaction. Sham-amphetamine and lesion-vehicle groups differ significantly from sham-vehicle P,0.05 and 0.01, respectively. B
In contrast, only the drug factor was significant for time
Mean 6S.E.M. number of line crossing. P,0.05 sham-amphetamine
spent in
self-grooming [F1,26516.97,
P,0.0001],
vs. sham-vehicle. P,0.001 lesioned-amphetamine vs. sham-vehicle
whereas the lesion and drug3lesion factors were not
and lesioned-vehicle. C Mean 6S.E.M. number of rearing. P,0.01
significant [F1,2650.01, NS and F1,2650.1, NS,
sham-amphetamine vs. sham vehicle. D Mean 6S.E.M. time spent
respectively]. Post-hoc analysis showed that amphetamine
grooming. P,0.05 both groups sham and lesioned amphetamines groups vs. their controls.
abolished self-grooming in both lesioned and sham animals to the same extent.
It is worth noting that number of rears and time spent in self-grooming from lesioned and sham animals were
3.5. Experiment 4 similar Figs. 3C and D.
The effect of diazepam on percentage of time spent on the open arms varied depending on whether the animal
3.4. Experiment 3 brain was intact or transected [drug3lesion, F1,1653.39,
P,0.04; lesion, F1,1656.23, P,0.02; drug, F1,165 Rats with MPFC lesions significantly spent more per-
4.03, P,0.061, see Fig. 4]. Further analysis showed that centage of time on the open arms P,0.001 and made
MPFC lesioned rats did not differ from the sham group in more entries to the open arms P,0.01 than sham rats
this measure. However, only MPFC lesioned rats increased Fig. 4A. No significant difference in closed arm entries
the percentage of time spent on open arms as compared to was observed Table 1.
sham-vehicle group following administration of diazepam.
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was confirmed that the lesion produced a primary change in anxiety measures, which does not depend on a particular
motor activity pattern. Lesioned animals reduced open arms exploration as did sham ones in the second trial of
the elevated plus-maze. This implies that MPFC transected rats conserved their ability for spatial discrimination and
for changing exploratory strategy.
Increased social behavior in rats following MPFC lesions have been reported elsewhere [6,23]. However, a
behavioral analysis on anxiety related measures in the social interaction test was lacking. The increase in social
interaction reported here was at the expense of social investigation and not related to augmented aggression.
The results in the plus-maze trial 1 confirm the be- havioral pattern observed in the social interaction test.
MPFC transected animals explore the open arms more than controls without altering the entries to closed arms, which
is considered the best measure of locomotion on this model [4,11]. During a plus-maze trial rodents progressively
reduce open arm exploration due to their natural aversion to open spaces [12,13,17,38,46]. Therefore, the animal
identifies and avoids an anxiogenic area. In contrast, social interaction increases as a function of familiarity with the
Fig. 4. Mean 6S.E.M. time s spent on, and number of entries onto,
arena that becomes less aversive over the time [9,35].
open arms of the elevated plus-maze by MPFC and sham rats. A TRIAL 1, there was a significant difference between groups [ time open: T18
The fact that MPFC transections selectively decrease
3.2, P,0.005; number open: T1852.7, P,0.01]; B TRIAL 2,
anxiety as measured by tests whose situational arrange-
after IP injections of saline or diazepam 1 mg kg. P,0.01 lesioned-
ments serve distinct cognitive processes suggests that the
diazepam vs. sham-vehicle group.
MPFC transections primarily altered the general emotional response. Further support for this view comes from the
No effects were observed in other plus-maze measures see plus-maze second trial experiment. In this test situation
Fig. 4B and Table 1. both sham and MPFC transected animals learned to avoid
the open arms. However, MPFC transected animals sig- nificantly differed from sham-vehicle group when treated
4. Discussion
