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Hyperactive rats (Hypers) exhibit several unique
behavioral patterns in comparison to normal rats that have not been
selectively bred (Non-Select rats [NS]). Such patterns include increased
baseline motor activity, extreme hyperactivity following exposure
to mild stressors, and prolonged behavioral depression in response
to strong, uncontrollable stressors. This study attempted to identify
regions of the brain that are differentially activated in Hyper
and NS rats. The activation of such regions was identified by quantifying
the expression of the gene c-fos which is induced by exposure to
stressful conditions. Hyper and NS rats were exposed to one of two
stressor conditions: Novel Environment (NE) or Foot Shock (FS).
Rats in the NE group were housed individually for 2.5 hours, whereas
those in the FS group first received 0.5 hours of FS followed by
2 hours of individual housing. Spontaneous motor activity was recorded
for all animals during the individual housing period. Following
individual housing, rats were immediately sacrificed and perfused
with 10% formalin. Their brains were harvested, sectioned and stained
for Fos, the protein product of c-fos. Levels of Fos were recorded
in various brain regions chosen based on their roles in stress,
emotionality, escape response, motor activity and dopaminergic regions
of the mesocorticolimbic system. Hyper and NS rats differed in regards
to both spontaneous motor activity and c-fos expression. During
the first 20 minutes of the individual housing period, the activity
of Hypers in both the NE and FS groups was greater than that of
the NS rats. However, during the remainder of the individual housing
session, the activity of the Hypers was less than that of the NS
rats. In most of the brain regions examined, NS rats expressed more
c-fos than Hypers. This difference was statistically significant
in the Caudate Putamen and Prefrontal Cortex. However, in the Substantia
Nigra, Hypers expressed more c-fos than did NS rats. The differences
in c-fos expression in the Caudate Putamen and Substantia Nigra
indicate that the nigrostriatal dopamine pathway may be responsible
for differences in motor activity between Hyper and NS rats.
This study compares c-fos expression in Hyperactive
(Hyper) rats and Non-Select (NS, not selectively bred) rats in response
to a stressor. Hyper rats have been selectively bred to exhibit
increased baseline motor activity and large motor responses to a
stressor (Weiss et al., 1995). Two-five days after exposure to a
mild stressor, Hyper rats that are 3-4 months old respond with extreme
hyperactivity (lasting 2-3 days) followed by a return to baseline
activity levels. Additionally, Hyper rats that are over one year
of age show prolonged behavioral depression (lasting 30-40 days)
in response to a strong uncontrollable stressor. In contrast, a)
NS rats that are 3-4 months old very rarely respond to a mild stressor
with hyperactivity and b) NS rats that are over one year of age
respond to a strong uncontrollable stressor with symptoms of behavioral
depression, but these typically remit within 4-6 days. Expression
of the gene c-fos immediately and transiently follows neuronal activation,
and is readily induced by exposure to a stressor (Dragunow &
Faull, 1989; Sheng & Greenberg, 1990). Its protein product,
Fos, is visible when stained and can be used as a marker for activated
neurons. By comparing relative concentrations of Fos in various
brain regions, the present experiment attempted to identify brain
regions that show different activation patterns in Hyper and NS
rats in response to a stressor.
One year-old male Hyper (n=14) and NS (n=14) rats
were used. Rats were housed in the colony 2 per cage, maintained
at a 12:12 light/dark cycle and given food and water ad libitum.
Rats were randomized into 4 groups based on two independent variables:
rat type - Hyper or NS - and stressor - Novel Environment (NE) or
Foot Shock (FS). Rats in NE condition were placed individually into
cages in which their motor ambulatory activity was measured for
a 2.5 hour period. Rats in the FS condition received 30 minutes
of foot shock in a shock chamber, and then were placed individually
into cages in which their motor ambulatory activity was measured
for a 2.0 hour period. At the conclusion of the 2.5 hour treatments,
all rats were perfused with 10% formalin and their brains removed
and placed into 10% formalin. 48 hours later, each brain was mounted
on a freezing microtome and cut into 50 m slices. Brain slices were
stained for Fos, mounted on slides, and counter-stained with neutral
red. Data analysis: motor activity (ambulation counts) was analyzed
by a 2-way analysis of variance with main factors of time (20 minute
periods) and rat line (Hyper vs. NS), using repeated measures over
the time factor. Data analysis: cells expressing c-fos were counted
in several brain regions. While counting, researchers were kept
blind to the experimental conditions received by each rat. These
counts were then analyzed using 2x2 (Hyper/NS x NE/FS) analyses
of variance.
-In most of the brain regions examined, c-fos expression was greater
in NS rats than in Hyper rats. c-fos expression in the Caudate Putamen
and the Prefrontal Cortex was significantly greater in NS rats than
in Hyper rats. c-fos expression in the Medial Postereoventral Amygdaloid
Nucleus approached being significantly greater in NS rats than in
Hypers (p=.053).
-In contrast to these effects, c-fos expression in the Substantia
Nigra approached being significantly greater in Hyper rats than
in NS rats (p=.107).
-In almost all of the brain regions examined, c-fos expression was
greater in the FS condition than in the NE condition. c-fos expression
was significantly greater in the FS condition than in the NE condition
for the following brain regions: Locus Coeruleus, Dorsal Raphe,
Paraventricular Nucleus, Arcuate Nucleus, Medial Postereoventral
Amygdaloid Nucleus, Substantia Nigra, Ventral Tegmentum, Nuclues
Accumbens Shell, Prefrontal Cortex, Claustrum and Lateral Septal
Nucleus, Ventral.
-When placed in individual housing, Hyper rats were more active than NS
rats during the first 20 minutes, but less active during the remaining time.
Most of the brain regions examined showed higher c-fos expression
in NS rats than in Hyper rats. Since neural activation in many of
the brain regions examined reflects stress and/or emotionality,
these results therefore indicate that brain regions associated with
stress and/or emotionality were more activated in NS rats than in
Hyper rats by the experimental conditions used. Perhaps the most
dramatic results seen were in brain regions that comprise the Nigrostriatal
pathway (i.e., Substantia Nigra [cell body region] and Caudate Putamen
[projection field]). In the Substantia Nigra, c-fos expression was
somewhat greater in Hyper rats than in NS rats, whereas in the Caudate
Putamen, c-fos expression was significantly greater in NS rats.
Since dopamine release triggers c-fos expression in post-synaptic
neurons, it appears that the decreased expression of c-fos in the
Caudate Putamen of Hyper rats is the result of highly activated
Substantia Nigra neurons releasing low amounts of dopamine. This
activation pattern of the nigrostriatal pathway may be involved
in the post-stressor behavioral pattern that is characteristic of
Hyper rats.
This material is based upon work supported by the following organizations:
Howard Hughes Medical Institute under Grant No. 52003727
LEARN program at Lafayette College
Stanley Foundation of the National Alliance for Research on Schizophrenia
and Depression
In this study, we compared post-stressor brain activation in two
distinct rat lines, Hyperactive and Non-Select. Hyperactive rats
have been selectively bred and serve as an animal model of depression.
Non-Select rats served as the control rats. Rats were removed from
their home cages, stressed and their activity levels were monitored.
At the end of the stressor session, their brains were removed, sectioned
and stained for a protein, Fos, whose presence is indicative of
neuronal activation. This protein was counted in brain regions relating
to stress, emotionality and motor activity. We found significantly
more Fos in the Prefrontal Cortex and Caudate Putamen of Non-Select
rats than Hyperactive rats. Interestingly, in the Substantia Nigra,
the brain region that projects to the Caudate Putamen, there was
more Fos in the Hyperactive rats. It appears that the interaction
of the Substantia Nigra and Caudate Putamen is different in Hyperactive
and Non-Select rats and may be involved in their post-stressor behavioral
differences.
tissue preparation, immunological assay perfusion, microscopy,
animal handling
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