8 L
rats were treated with benzodiazepines or vehicle and 1900 h. Animals were anesthetized by co-administration of
tested on a repeated trial of the elevated plus-maze ketamine and penthothal 110 and 10 mg k i.p., respec-
Experiment 4. tively and positioned in a stereotaxic frame David-Kopf
Instruments. The skull was exposed and the incisor bar adjusted such that bregma and lambda were at the same
2. Materials and methods height. To lesion the MPFC a knife blade was positioned at
2.5 mm anterior to bregma. The sharp pointed tip of a 2.1. Animals and surgery
3-mm wide scalp blade was lowered 7.0 mm below dura such that each side of the blade extended to a maximum of
Male Wistar rats weighing 250–300 g were single 1.5 mm lateral to the midsagital suture see Fig. 1.
housed with food and water ad libitum, and the room Sham-operated had a craniotomy and the longitudinal sinus
temperature was kept at 228C. Lights were on from 0700 to was cut. In both type of surgical procedure, bleeding from
Fig. 1. Diagrammatic representation of A rat brain sagital section showing the lines of transection and bregma T and B, respectively and B coronal sections 2.5 mm anterior to Bregma through the rat brain showing the target area shaded of the MPFC. Animals with track knife outside of this target
area were excluded from statistical analysis. C Photomicrograph of a typical horizontal brain section stained for hematoxylin-eosin showing the extent of mechanical damage and gliosis 10 days after surgery. Level: frontal pole 3-mm depth from dura. Bar 0.5 mm.
L .E. Gonzalez et al. Brain Research 887 2000 7 –15
9
Fig. 1. continued
sinus was stopped by pressing over the area for about 1 line crossing and rears using a computational timer coun-
min. Animals were allowed to recover for 10 days prior to ter apparatus.
behavioral testing. 2.3.2. The plus-maze
The plus-maze was made of wood and consisted of two 2.2. Drugs
opposite open arms 50310 cm, and two opposite arms enclosed by 40 cm high walls. The arms were connected
Diazepam and
D
-amphetamine sulphate Sigma, St. by a 10310 cm central square, and thus the maze formed a
Louis, MO, USA were prepared in 0.9 sodium chloride ‘plus’ shape. The maze was elevated 50 cm above the
solution. A drop of Tween 80 was added to disolve floor. A closed circuit TV camera was vertically mounted
diazepam. over the maze and the behavior was scored from a monitor
in an adjacent room. All scores were directly entered into 2.3. Apparatus
an IBM computer. Changes in the of time spent on the open arms indicate changes in anxiety [34] whereas the
2.3.1. The social interaction test arena number of closed arm entries is the best measure of
The social interaction test arena was a wooden box general activity in the maze [4,11]. Testing took place in a
60360 cm, with 35 cm high walls and was lit by low light room lit by dim light ,50 lux from a light source
50 lux. A camera was vertically mounted above the arena directly above the plus-maze.
and the rats were observed on a monitor in an adjacent room. Test sessions were also video recorded for sub-
2.4. Behavioral testing sequent analysis. The arena was divided into nine squares
2
of 20 cm each by placing a grid over the video screen. An Testing took place randomly for condition or treatment
observer blind to the drug treatment scored the number of between 08:00 and 12:00 h.
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
