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Neurophysiological differences between stressed female Hyperactive and randomly bred Control rats were explored via quantifying c-fos expression in selected brain regions. The results indicated that c-fos expression was greater in Control rats in the Caudate Putamen, Dorsal Raphe, Amygdaloid nuclei, and Prefrontal Cortex. The data suggests that the amygdalostriate pathway in stressed female Hyperactive rats is activated differently than in female Control rats.
Female selectively bred hyperactive rats (Hypers) of 10-15 months demonstrate periods of alternating extreme high and low spontaneous motor activity for 12-14 weeks following exposure to a stressor. In contrast, randomly bred rats (Controls) that are over a year old respond to a stressor with symptoms of behavioral depression that typically remit within 2-6 days.
The neural basis underlying this behavior can be explored via expression of immediate early genes such as c-fos, whose transcription is initiated within minutes after cellular stimulation by neurotransmitters, growth factors, or second messengers. The neuronal activation of a brain region is indicated by elevated levels of the c-fos protein product – Fos, which is visible when stained (Dragunow & Faull, 1989; Sheng & Greenberg, 1990).
Past research shows that there are differences in Fos distribution patterns between male Hyper and Control rats. Grolley and Williams (2004) found that male Hyper and Control rats demonstrate different Fos activation patterns of the nigrostriatal pathway after exposure to a stressor. This study attempts to identify neurophysiological differences between stressed Hyper and Control female rats.
• One year old female Hyper (n=13) and Control (n=15) rats were housed two per cage in the colony and given food and water ad libitum.
• Rats were randomized into four groups based on two independent variables: rat line (Hyper or Control) and stressor [Novel Environment (NE) or Foot Shock/Novel Environment (FS/NE)].
• Rats in the NE condition were placed individually into cages in which their motor activity was recorded for a 2.5 hour period.
• Rats in the FS/NE condition first received 30 minutes of foot shock; then they were placed individually into cages in which their motor activity was recorded for a 2.0 hour period.
• After a total of 2.5 hours of treatments, all rats were perfused, their brains harvested and placed in 10% formalin solution, transferred into 25% sucrose, and cut into 50 μm slices.
• The brain slices were stained for Fos and counterstained with neutral red.
Data Analysis: Spontaneous ambulation data for the NE and FS/NE conditions were analyzed separately by a two-way analysis of variance (ANOVA) for repeated measures with time (10 minute periods) and rat lines (Hyper vs. Control) as factors. Fos was counted in the selected brain regions. The Fos counts were then analyzed by a 2X2 ANOVA (Hyper_Control x NE_FS/NE).
c-fos Expression
c-fos expression in the Caudate Putamen, Dorsal Raphe, Basolateral and Basomedial Amygdaloid Nuclei, and the Prefrontal Cortex was significantly greater in Control rats than in Hyper rats (p<.05). c-fos expression in the Locus Coeruleus (p=.054) approached being significantly greater in Control rats than in Hyper rats.
In most of the brain regions examined, c-fos expression was greater in FS/NE condition than in the NE group. Brain regions that reached significance were: Locus Coeruleus, Paraventricular Nucleus, Basolateral and Basomedial Amygdaloid Nuclei, Ventral Tegmental Area, Nucleus Accumbens Shell, Lateral and Dorsal Periaqeductal Grey. On the other hand, stressor had no influence on c-fos expression in Caudate Putamen (p=.467).
Spontaneous Motor Activity
Both Hyper and Control rats were most active when initially placed in the novel environment. The main effect of time was observed in both conditions: in NE (p<.000) and FS/NE (p<.000). In the FS/NE condition, a two-way ANOVA yielded a significant interaction of line by time (p=.019). Additionally, t-tests at individual time points yielded a significant effect of rat line at 50 min (p=.008), 90 min ( p=.025), and 110 min (p=.03) with Control rats being more active at those time points.
Decreased c-fos expression in Hyper rats in the Dorsal Raphe and Amygdaloid nuclei in comparison with Control rats suggests that Hyper rats demonstrate different neuronal activation patterns with regards to stress and emotionality-related brain regions after exposure to a stressor.
Perhaps the most interesting results are the main effects of rat line in both the Caudate Putamen and the Amygdaloid nuclei. The Caudate Putamen is connected to the Basolateral Amygdala by afferent projections that comprise the amygdalostriate pathway. Variation in neuronal activation between Control and Hyper rats in the Basolateral and Basomedial Amygdaloid nuclei could result in different activation patterns of the Caudate Putamen, which is involved in motor activity. In conclusion, this activation pattern of the amygdalostriate pathway may be involved in the behavioral differences between Hyper and Control rats.
In comparison to the study conducted by Grolley and Williams (2004), both female and male Hyper rats seemed to exhibit decreased activation of the Caudate Putamen compared to Control rats. In males, it appeared the Substantia Nigra played a role in the lower c-fos expression in the Caudate Putamen. In females, however, the Amygdaloid nuclei seemed to influence low levels of c-fos expression in the Caudate Putamen.
Future Research
A study with an increased number of male and female Hyper and Control rats could be completed, thereby providing further insight into the neurophysiological and behavioral differences between Hyper and Control rats as well as males and females.
This material is based upon work supported by the following organizations: Graduate Division of Biological and Biomedical Sciences, Emory University; LEARN program at Lafayette College; Stanley Foundation of the National Alliance for Research on Schizophrenia and Depression; and the SURE Program, Emory University.
For our project, we used Hyperactive rats that are used as a model for bipolar patients. The purpose of the experiment was to identify pathways and brain regions that become activated with a specific protein (Fos) after the subject has had exposure to a stressor. We exposed the rats to a footshock and then observed and counted the number of cells that were activated with FOS, to find any pathways that are activated after the stressor.
Perfusion, brain extraction, microtome brain slicing, brain mounting
stressor, Hyperactive rats, c-fos, FOS, Control rats
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