Bachelor project Psychobiology 2020
Voluntary wheel running in rats increases ∆FosB
expression in a hippocampus subregion-dependent
manner
Maaike Kesting
11602368
Supervisor: Dr. Joram Mul
Second supervisor: Gideon Meerhof
Examiner: Joost van Kordelaar
Abstract
High levels of stress afects the lifestyles of almost every human being. It can lead to negative efects, such as depression and learning and memory impairments in the hippocampus. Therefore, it is very important to find a therapy to reduce the negative efects of high levels/chronic stress. It is known for several decades, that exercise can reduce the maladaptive efects of stress in the brain. However, it is not yet clear what the mechanism of exercise is on these maladaptive efects in the brain. What is known, is that physical activity induces the transcription factor ∆FosB in the nucleus accumbens (NAc), and this promoted resilience to chronic stress in mice. Because of the minimal amount of research about the functional consequences of exercise-induction of ∆FosB in the hippocampus, this was investigated in this study using voluntary wheel running (VWR) as a model of exercise. Because of the close functional relation of a limbic corticostriatal network between the NAc and the
hippocampus, it is hypothesized that the VWR group has significant more ∆FosB than the control group. The experiment consisted of one group of 12 rats that had four weeks of physical activity by means of a running wheel and the other group (12 rats) was housed sedentary. After the four weeks of running, the brains were removed and immunohistochemistry was used to visualize ∆FosB-positive neurons. The ∆FosB-positive neurons were quantified. There was no significant diference found in the suprapyramidal dentate gyrus (DGsp), cornu ammonis 2 (CA2) and cornu ammonis 3 (CA3) of the hippocampus between the two groups. However, VWR rats had a higher average number of ∆FosB-positive neurons in the infrapyramidal dentate gyrus (DGip) and cornu ammonis 1 (CA1) than SED Rats. DG is important for information processing that leads to the production of episodic memories and the CA1 is important for spatial learning. This could mean that exercise could help by processing events, which could possibly lead to a reduction of the negative efects of high levels of stress. Furthermore, physical activity could have an efect on the CA1, because of the spatial activity which is increased during wheel running. Future research could focus on these subregions of the
hippocampus for improving exercise-based interventions against the negative efects of chronic stress.
Introduction
Stress, and even high levels of stress, afect almost every human being during their lifetime. High levels of stress and chronic stress can lead to negative efects in the brain, such as depression (Katz, Roth & Carroll, 1981) and learning and memory impairments in the hippocampus (Song et al., 2006). Exercise reduces the maladaptive efects of stress in the brain (Keller & Seraganian, 1984; Roth & Holmes, 1987; McCann & Holmes, 1984). However, it is not yet clear what the mechanism of exercise is on these maladaptive efects of stress in the brain. When this mechanism is fully understood, exercise-based interventions could be improved in reducing the negative efects of high levels of stress.
Stress induces the very stable transcription factor ∆FosB in the brain (Perrotti et al., 2004; Chen et al 1997). Therefore, ∆FosB can accumulate in a neuron upon repeated stimulation of that neuron (Chen et al., 1997). ∆FosB belongs to the Fos family and is a splice variant of the FosB protein and due to its functional changes, it gets very stable and has a half-life around eight days (Carle, T. L.,2007). In comparison, the other proteins of the Fos family have a half-life of several hours (Carle, T. L.,2006). Normal expression of ∆FosB reduces stress and depression (as in that it reduces social avoidance and anhedonia) (Nestler, 2015; Vialou et al., 2010). Physical activity induces the transcription factor ∆FosB in the nucleus accumbens (NAc), this resulted in resilience to stress (Mul et al., 2018). Additionally,
physical activity induces ∆FosB in the ventral striatum and in the medial DS 9a region for reward and movement (Kravitz & Kreitzer, 2012). Because the NAc and hippocampus are functional components of a limbic corticostriatal network (Ito, Robbins, Pennartz & Everitt, 2008), it is likely that physical activity also induces ∆FosB in the hippocampus. This makes it interesting to investigate the influence of physical activity on ∆FosB in the hippocampus. A model for exercise in rodents is voluntary wheel running (VWR).
Previous research already found that long-term physical activity through voluntary wheel running significantly increased ∆FosB in all the subregions of the hippocampus in mice (Nishijima, Kawakami & Kita, 2013). However, research still needs to be done because Nishijima, Kawakami & Kita (2013) used mice for their research, while rats have more cognitive similarities with humans (Ellenbroek & Youn, 2016). This leads to the research question: what is the efect of voluntary wheel running on ∆FosB expression in the hippocampus of rats?
Furthermore, viral-mediated blockade of ∆FosB, and as well, surprisingly, overexpression of ∆FosB in the hippocampus impairs learning and memory and increases anxiety (Eagle et al., 2015).
Furthermore, Eagle et al. (2015) found that overexpression of ∆FosB increases immature dendritic spines on pyramidal cells of the CA1, whereas viral-mediated blockade of ∆FosB reduces the number of immature and mature spine types. These data suggest that ∆FosB plays an important yet complex role in hippocampus-dependent learning and memory.
General note:
The behavioral experiments were performed before this research, just as cutting the brain slices and obtaining the images of the slices by cohort 1 and 2.
To assess how voluntary wheel running modulates expression of ∆FosB in the hippocampus, we used rats that were either housed sedentary (SED) or with a voluntary running wheel (i.e. VWR). The voluntary wheel running (VWR) rats had a running wheel for a month in their cages which was always available. The sedentary (SED) rats had a cage without a running wheel. After this month the brains were removed and were cut in slices using the Paxinos Rat brain Atlas (2007, 6th edition) for the exact
location. An immunohistochemistry was performed so that the ∆FosB-positive neurons could be localized. In this study, the number of ∆FosB-positive neurons in the SED and VWR rat brain slices were collected using ImageJ Fiji and the average number of ∆FosB-positive neurons between VWR and SED rats were compared for several regions of the hippocampus. The five subregions of the hippocampus (dentate gyrus infrapyramidal (DGip), dentate gyrus suprapyramidal (DGsp), cornu ammonis 1, 2 and 3 (CA1, CA2 and CA3)) and the hippocampus as a whole were investigated for the cohorts separately. Furthermore, the hippocampus and its subregions of cohort 1 and cohort 2 were analyzed together to increase power. Additionally, the hippocampus was analyzed in a subregion-dependent manner in bregma -4.44 till -5.64. Based on previous research that VWR induces ∆FosB in the NAc (Mul et al., 2018) and in the hippocampus (Nishijima, Kawakami & Kita, 2013) of mice, we hypothesized that VWR increased the average number of ∆FosB-positive neurons in the hippocampus of VWR rats compared to SED controls.
Materials & methods
General noteThe behavioral experiments were performed before this research, just as cutting the brain slices, performing immunohistochemistry and obtaining the images of the slices by cohort 1 and 2. The current study is from collecting the data of both cohorts to analyzing and interpreting the data.
Animals and VWR
Twenty-four male WU Wistar rats, weighing between 220 and 260 grams at arrival, were used in this study. Cohort one had twelve rats and cohort two also had twelve rats for the experiments. The rats were group-housed (4 rats per cage) for seven days to habituate to the Netherlands Institute of Neuroscience (NIN). Water was freely available and the diet was controlled (CD; Teklad Global Diet 2918: 18.6% protein, 44.2% carbohydrate and 6.2% fat; 3.1 kcal/gram). The lights were on from 7:00 till 19:00 per day and the rats had a wooden gnawing stick and red rat retreat as cage enrichment. After the habituation period of one week, the rats were individually housed.
All experiments were approved according to the Dutch legal ethical guidelines by the animal care committee of the Royal Dutch Academy of Science.
The rats were divided in two groups: housed without a running wheel (sedentary; SED) or with a running wheel (voluntary wheel running; VWR). The VWR rats were individually placed in a cage containing a running wheel (43 cm Ø; 1.068 m/revolution; Techniplast) and the SED rats were
individually placed in a standard type 3 cage. The groups were normalized based on body weight. The cage register program (Dept. Biomedical Engineering, UMC Utrecht, the Netherlands) registered the wheel revolutions.
After 28 days of VWR, the wheels were blocked for 24 hours to ensure that only ∆FosB, and not the other Fos family member proteins, was expressed in the hippocampus (∆FosB is stable enough to stay present after the 24 hours) (Mul et al., 2018; Perrotti et al., 2004).
Preparation of the brains for the immunohistochemistry
After the 24 hours of wheel blockade, the rats were sacrificed during the first halve of the light phase. This was done by anaesthetizing using pentobarbital and briefly perfused with 0.1M PBS. Hereafter, the rats were perfused with 4% paraformaldehyde in 0.01M PBS and then the brains were isolated and stored overnight in 4% paraformaldehyde at four degrees. For cryoprotection, the brains were transferred to 30% sucrose/0.01M TBS solution and were then stored at -80 degrees.
The brains were sliced on a cryostat (Leica CM1950 for cohort 1 and Cryostar NX50 for cohort 2) in coronal sections of 35 µm, ranging from approximately bregma 5.16mm to 0.84mm (Set 1) and -1.08 to -6.72 (Set 2). The Paxinos Rat Brain Atlas (2007, 6th edition) was used for determining the position
in the brain during cutting. They used six containers with a cryoprotectant solution (30% glycerol, 30% ethylene glycol and 40% 0,1 M PBS) to divide the serial brain slices. This resulted in 210µm (6*35µm) between every brain-slice and was stored at -20 degrees.
Immunohistochemistry
For the immunohistochemical staining, identical numerical containers were chosen from each brain and each series. The immunohistochemistry was used to visualize the ∆FosB-positive neurons. Slices were washed in TBS (0.15M NaCl, 0.05M Tris, pH set at 7.6) for three times five minutes. After each incubation step this washing procedure was also performed. Then, slices were pretreated with 10% v/v methanol, 3% v/v H202 (Merck) in TBS to block the endogenous peroxidase activity for ten minutes. Subsequently, the slices were incubated for 60 minutes at room temperature with a FosB specific primary antibody (art.#2251, Lot#3, Cell Signalling) which was diluted 1:3000 in supermix (0.25% w/v gelatin, 0.5% v/v Triton X-100 in TBS, pH set at 7.6). This incubated overnight at four degrees. The day after, the slices were incubated for 60 minutes at room temperature with a secondary antibody diluted 1:400 (biotinylated anti-rabbit IgG BA-1000, vector laboratories).
Hereafter, supermix was added which contained avidin-biotin complex (1:800). At last, a DAB solution (0.05% w/v 3.3-Diaminobenzidine, 0.23% w/v nickelammoniumsulphate (Merck) and 0.01% v/v H2O2 (Merck)) was used to stain all slices. After four minutes of staining, the staining reaction was stopped by using purified water. Slices were then rinsed in TBS and mounted onto Superfrost ++ glass slides (Thermo Scientific) and at last were coverslipped with Entallan.
Imaging and quantification of ∆FosB-positive neurons
For cohort 1, the images of the brain slices of twelve rats were taken using a Zeiss Axioskop
microscope. For cohort two, the brain slices of the other twelve rats were scanned using an Intellisite ultra-fast scanner (Philips Eindhoven). In the current study, an unblinded analysis was done, because we decided to have the same threshold per rat to reduce variety in number of ∆FosB-positive neurons per rat. The threshold was made as optimal as possible, by ensuring that there is a minimal amount of noise and a maximal amount of neurons. Moreover, it was checked if the backgrounds of the images had a similar amount of gray color and if there were any blurry images that needed to be excluded. The images were converted to 8-bit grayscale images, at a resolution of 7680x4320 pixels (8K). One VWR rat of cohort 1 was excluded due to images that were out of focus.
In this study, ImageJ Fiji was used to manually draw the regions of interest (ROIs) and to quantify the ∆FosB-positive neurons. The rat brain atlas (Watson and Paxinos, 2007, 6th edition) was used to
identify the regions of interest (ROI). The following procedure was performed to collect the number of ∆FosB-positive neurons (see diagram 1 for a flowchart of this procedure):
First, make certain that every image is an 8-bit image. Second, when an image is opened, set the right threshold (by image→ adjust→ threshold). Then a ROI can be manually drawn and it is necessarily to save it correct (by pressing ‘T’). Open the ROI manager before drawing if there are already ROIs present, otherwise it will only save the previous saved ROIs. When all the ROIs are drawn in the image, convert to mask (by process → binary → convert to mask) and then watershed to divide the particles which are too close to each other (by process→ binary → watershed). To collect the number of ∆FosB-positive neurons, select the ROI and analyze the neurons (by analyze → analyze particles (size= "3-infinity", select "pixel units", show: "nothing", select "display results", select "clear results". Select "summarize"). In this way, only the neurons larger than three pixels are included to reduce the noise. A summary will pop up which include the “counts” (number of ∆FosB-positive neurons).
Diagram 1: Flowchart of the procedure for collecting ∆FosB-positive neurons.
Per cortical slice, the number of ∆FosB-positive neurons were averaged between the bilateral sides for every subregion. Finally, an excel sheet was made per rat with the average number of ∆FosB-positive neuronsper subregion per bregma. Furthermore, the mean of the average number of ∆FosB-positive neurons for every subregion per rat was calculated between bregma -3.12 till -5.64 (Table 1). This bregma range was chosen, because there was a minimal number of 6 slices per rat in this range (see appendix A for the atlas pictures of the hippocampus subregions per used bregma).
Table 1: Excel sheet per rat. An example of an excel sheet for one rat. It shows the average number of ∆FosB-positive neuronsper subregion per bregma ranging from bregma -3.12 till -5.64. NP; No particles, NIB; Not in bregma.
Statistical Analysis
The subregions of the hippocampus (DGip, DGsp, CA1, CA2 & CA3) and the hippocampus with all the subregions together were analyzed. An unpaired two-tailed t-test was used for comparing the VWR rats and the SED rats. A p-value of <0.05 is considered significant.
First, for cohort 1 and separate for cohort 2, the average number of ∆FosB-positive neurons of the VWR rats and the SED rats were compared for the hippocampus and also for its subregions. Second, the data of cohort 1 and 2 were combined to increase the power of this study. Now, the average number of ∆FosB-positive neurons of VWR rats of both cohorts and the SED rats of both cohorts were compared for the hippocampus and also for its subregions.
And last, the dorsal side of the subregions DGsp, CA1, CA2 and CA3 were compared in VWR rats and SED rats of both cohorts, this is also done for the ventral side. The reason for this is to see if the possible increase in ∆FosB-positive neurons is location-dependent. The DGip had no data in the ventral side, so no analysis could be performed on this subregion.
Results
Cohort 1∆FosB-positive neurons for every subregion of the hippocampus were compared between SED and VWR rats from cohort 1 (Figure 1a-e). There was no significant diference found between the SED and VWR rats in the CA3 (p=0.1442; figure 1e). There is an increased trend seen in the DGsp (p=0.07118; figure 1b) and the subregion CA1 (p=0.05286; figure 1c) for VWR rats compared with SED rats. VWR rats had a significant higher average number of ∆FosB-positive neurons in the DGip (p=0.01065; figure 1a) and in the CA2 (p=0.043; figure 1d) than SED controls. Additionally, Figure 1f shows that VWR rats had a significant higher average number of ∆FosB-positive neurons in the hippocampus in general (p=0.03195).
Cohort 2
Furthermore, SED and VWR rats from cohort 2 were compared for every subregion of the
hippocampus. Figure 2a-e show the results of the subregions of cohort 2. There were no significant diferences found in the average number of ∆FosB-positive neurons between the SED and VWR rats in the DGip (p=0.9445; figure 2a ), the DGsp (p=0.6674; figure 2b), the CA1 (p=0.8879; figure 2c) and the CA3 (p=0.4632; figure 2e ). Figure 2f shows that there was no significant diference in the average number of ∆FosB-positive neurons in the hippocampus in general (p=0.6734) of cohort 2. However, solely one subregion of the hippocampus in cohort 2, the CA2, had a significant diference in the average number of ∆FosB-positive neurons between the VWR and SED rats. Namely, SED rats had a significant higher average number of ∆FosB-positive neurons than VWR rats (p=0.04387; figure 2d ).
Cohorts combined
Cohorts 1 and 2 were combined, because this increased the power. SED and VWR rats from cohort 1 and 2 were compared for every subregion of the hippocampus. Figure 3a-e show the results of the subregions of the hippocampus. There was no significant diference between the average number of ∆FosB-positive neurons of the SED and VWR rats for the DGsp (p=0.3348; figure 3b), the CA2
(p=0.3957; figure 3d), the CA3 (p=0.424; figure 3e) and the hippocampus in general (p=0.1030; Figure 3f). However, VWR rats had a significant higher average number of ∆FosB-positive neurons in the DGip (p=0.03929; figure 3a) and in the CA1 (p=0.04793; figure 3c) than SED rats.
a b
c d
e f
Figure 1: Data cohort 1. (a-e) The average number of ∆FosB-positive neurons for the SED and VWR rats per subregion of the hippocampus for cohort 1. (f) The average number of ∆FosB-positive neurons for the SED and VWR rats for the subregions combined for cohort 1.
a b
c d
e f
Figure 2: Data cohort 2. (a-e) The average number of ∆FosB-positive neurons for the SED and VWR rats per subregion of the hippocampus for cohort 2. (f) The average number of ∆FosB-positive neurons for the SED and VWR rats for the subregions combined for cohort 2.
a b
c d
e f
Figure 3: Data cohorts 1 and 2 combined. (a-e) The average number of ∆FosB-positive neurons for the SED and VWR rats per subregion of the hippocampus. (f) The average number of ∆FosB-positive neurons for the SED and VWR rats for the subregions combined.
Furthermore, in this study is investigated if there is a significant diference of ∆FosB-positive neurons between SED and VWR rats of the combined cohorts for the dorsal parts of the subregions of the hippocampus and ventral parts for a few bregma’s. The following figure (figure 4) shows the
hippocampus which is marked in yellow and explains the definition of dorsal and ventral subregions:
Figure 4: Location of the hippocampus subregions. CA1, CA2 and CA3 have a dorsal and ventral side at bregma -4.36 till around bregma -5.28. The subregion DGsp has a dorsal and ventral side at bregma -5.40 till -5.64 (not shown in figure).
CA1 dorsal and ventral difference between the SED and VWR rats
For bregma -4.56 there was no significant diference in the average number of ∆FosB-positive neurons between the SED and VWR rats for the dorsal CA1 (p=0,8303; figure 5a) and the ventral CA1 (p=0,2658; figure 5b ). See supplementary A-m for the appearance of the CA1 for this bregma.
a b
Figure 5: CA1 dorsal and ventral difference between the SED and VWR rats at bregma -4.56. (a) Dorsal CA1. (b) ventral CA1.
For bregma -4.80 there was also no significant diference between the SED and VWR rats for the dorsal CA1 (p=0,8691; figure 6a ). However, for the ventral CA1 the VWR rats had a significant higher average number of ∆FosB-positive neurons than the SED rats (p=0,04335; figure 6b). See
supplementary A-o for the appearance of the CA1 for this bregma.
a b
Figure 6: CA1 dorsal and ventral difference between the SED and VWR rats at bregma -4.80. (a) Dorsal CA1. (b) ventral CA1.
CA2 dorsal and ventral difference between the SED and VWR rats
For bregma -4.68 there was no significant diference in the average number of ∆FosB-positive neurons between the SED and VWR rats for the dorsal CA2 (p=0,3684; figure 7a) and the ventral CA2 (p=0,9877; figure 7b). See supplementary A-n for the appearance of the CA2 for this bregma.
a b
Figure 7: CA2 dorsal and ventral difference between the SED and VWR rats. (a) Dorsal CA2. (b) ventral CA2.
CA3 dorsal and ventral difference between the SED and VWR rats
For bregma -4.44 there was no significant diference in the average number of ∆FosB-positive neurons between the SED and VWR rats for the dorsal CA3 (p=0,2010; figure 8a) and the ventral CA3 (p=0,2945; figure 8b). See supplementary A-l for the appearance of the CA3 for this bregma.
a b
DGsp dorsal and ventral difference between the SED and VWR rats
For bregma -5.64 there was also no significant diference in the average number of ∆FosB-positive neurons between the SED and VWR rats for the dorsal DGsp (p=0,9516; figure 9a) and the ventral DGsp (p=0,5233; figure 9b). See supplementary A-v for the appearance of the DGsp for this bregma.
a b
Figure 9: DGsp dorsal and ventral difference between the SED and VWR rats. (a) Dorsal DGsp. (b) ventral DGsp.
Correlations between total running distance and average number of ∆FosB-positive neurons for the significant subregions in VWR rats
Cohort 1
There is no significant correlation between the total running distance and the average number of ∆FosB-positive neurons for the significant subregions; DGip (p=0.4597, figure 10a), CA2 (p=0.5992, figure 10b) and the subregions combined (p=0.7006, figure 10c) in the VWR rats.
a b 30000 35000 40000 45000 50000 55000 60000 650000 50 100 150 200 250 300 f(x) = 0 x + 38.64 R² = 0.19
Regression DGip-distance of cohort 1
Total running distance (m)
A ve ra ge n u m b e r o f ∆ Fo sB -p o si ti ve n eu ro n s 30000350004000045000500005500060000650000 10 20 30 40 50 60 70 f(x) = 0 x + 3.16 R² = 0.1
Regression CA2-distance of cohort 1
Total running distance (m)
A ve ra ge n u m b e r o f ∆ Fo sB -p o si ti ve n e u ro ns c 30000 35000 40000 45000 50000 55000 60000 650000 50 100 150 200 f(x) = 0 x + 53.76 R² = 0.06
Regression whole hippocampus-distance of cohort 1
Total running distance (m)
A ve ra ge n u m b er o f ∆ Fo sB -p o si ti ve n e u ro n s
Figure 10: Correlations between total running distance and average number of ∆FosB-positive neurons for cohort 1. (a) Regression analysis between total running distance in meters and average number of ∆FosB-positive neurons for DGip. R= 0.4389, R2=0.1926, p=0.4597. (b) Regression analysis between total running distance in meters and average number of ∆FosB-positive neurons for subregion CA2. R=0.3204, R2=0.1026, p=0.5992. (c) Regression analysis between total running distance in meters and average number of ∆FosB-positive neurons for all the subregions combined. R=0.2374, R2=0.0564, p=0.7006.
Cohort 2
There is no significant correlation between the total running distance and the average number of ∆FosB-positive neurons for the significant subregion CA2 (p=0.2749; figure 11) in the VWR rats.
25000 35000 45000 55000 65000 75000 850000 2 4 6 8 10 12 f(x) = 0 x + 2.44 R² = 0.29
Regression CA2-distance of cohort 2
Total running distance (m)
A ve ra ge n u m b e r o f ∆ Fo sB -p o si ti ve n e u ro ns
Figure 11. Correlations between total running distance and average number of ∆FosB-positive neurons for cohort 2. Regression analysis between total running distance in meters and average number of ∆FosB-positive neurons for CA2. R= 0.5342, R2=0.2854, p=0.2749.
Cohorts combined
For the cohorts combined, there is also no significant correlation between the total running distance and the average number of ∆FosB-positive neurons for the significant subregions DGip (p=0.7538; figure 12a) and CA1 (p=0.2938; figure 12b) in the VWR rats.
a b 20000 30000 40000 50000 60000 70000 800000 50 100 150 200 250 300 f(x) = 0 x + 98.43 R² = 0.01
Regression DGip-distance of cohorts combined
Total running distance (m)
A ve ra ge n u m b e r o f ∆ Fo sB -p o si ti ve n e u ro ns 20000 30000 40000 50000 60000 70000 800000 50 100 150 200 f(x) = 0 x + 17.22 R² = 0.12
Regression CA1-distance of cohorts combined
Total running distance (m)
A ve ra ge n u m b e r o f ∆ Fo sB -p o si ti ve n eu ro n s
Figure 12. Correlations between total running distance and average number of ∆FosB-positive neurons for the cohorts combined. (a) Regression analysis between total running distance in meters and average number of ∆FosB-positive neurons for DGip. R= 0.1072, R2=0.01149, p=0.7538. (b) Regression analysis between total running distance in meters and average number of ∆FosB-positive neurons for CA1. R=0.3483, R2=0.1213, p=0.2938
Discussion
This study combined two previous cohorts to investigate if there is a diference in the average number of ∆FosB-positive neurons between voluntary wheel running rats and sedentary rats in the (subregions of) the hippocampus. This was also done per cohort. And last, there was also investigated if there is a diference between the dorsal and ventral side of a few subregions in the increase of the average number of ∆FosB-positive neurons between VWR rats and SED rats.
For cohort 1, the DGsp, CA1, CA3 had an increasing trend for the VWR group compared with the SED group. The subregions DGip and CA2 had significantly a higher average number of ∆FosB-positive neurons in the VWR rats than in the SED control rats. Furthermore, the hippocampus as a whole has a significant higher average number of ∆FosB-positive neurons in the VWR rats than in the SED rats. For cohort 2, only the subregion CA2 had significantly a higher average number of ∆FosB-positive neurons in the SED rats than in the VWR control rats. This is against the expectations. An explanation for this unexpected result could be that there is not corrected for the surface of the ROIs, however it could also be the case that the biology of these rats is diferent, for instance a rat could have a higher basic level of ∆FosB than other rats. The DGip, DGsp, CA1, CA3 and the hippocampus as a whole did not have a significant diference between the two groups of rats. This could have a technical cause or could be the result of the small sample size.
Cohort 1 and 2 show very diferent results, a reason for this could be the diferent ways of obtaining the images of the slices. Cohort 1 used a microscope and this could result in more manual flaws. Cohort 2 used a scanner. These diferent methods could result in a diference in observing signal. Furthermore, both cohorts had a diferent volume of reagents used for the staining, this means that there is more volume reagent per tissue in cohort 2, which could lead to a saturation efect in this cohort. Additionally, diferent researchers conducted the methods, so methodological changes could also be present. This was unavoidable, because it was only feasible to run one cohort at the time. To reduce the bias of a low power because of a small sample size, cohorts 1 and 2 were combined. Now the experimental group (VWR) existed of eleven rats and the control group (SED) existed of twelve rats. The subregions DGsp, CA2 and CA3 and the hippocampus in general did not have a significant diference between the two groups. However, the subregions DGip and CA1 had a significant higher average number of ∆FosB-positive neurons in VWR rats than in SED controls. This significant result of the DGip and the CA1 is very similar with what the researchers Nishijima, Kawakami and Kita (2013) found, this increases the research-validity.
A reason for the little amount of subregions with a significant result could be that the running distance of the sedentary rats were not measured, so there is also a possibility that the rats started moving more and perhaps run in their cages out of boredom.
For all the bregma’s, except bregma -4.80, there was no significant diference in the dorsal and ventral subregions, this could mean that VWR does not have a diferent efect within the subregions. For bregma -4.80, there was also no significant diference between the SED and VWR rats for the dorsal CA1. However, for the ventral CA1 of bregma -4.80, the VWR rats had a significant higher average number of ∆FosB-positive neurons than the SED rats. This could imply that VWR has a better efect on ∆FosB-induction in the ventral CA1 than in the dorsal CA1.
Furthermore, the results showed no significant correlation between the total running distance and the average number of ∆FosB-positive neurons for the significant subregions per cohort and for the cohorts combined. So, the data do not suggest that the more distance run, the higher the average
number of ∆FosB-positive neurons was. This could be explained by the fact that at one point a steady state of ∆FosB accumulation is reached in the four weeks of VWR, so that ∆FosB accumulation is perhaps no longer equal to the running distance.
The dentate gyrus is important for information processing, which results in the production of episodic memories (Amaral, Scharfman & Lavenex, 2007). The CA1 is important for spatial learning (Moser, Trommald & Andersen, 1994). Interestingly, physical activity training increases spatial memory in older adults (Erickson et al., 2011) and mice (van Praag, Christie, Sejnowski, & Gage, 1999). As ∆FosB
alters neuronal function (Eagle et al., 2015), this data suggest that exercise-induction of ∆FosB in the dentate gyrus could help by processing daily events, which could possibly lead to a reduction of the negative efects of high levels of stress (for instance processing trauma). Furthermore, exercise-induction of ∆FosB in the CA1 could underlie the increase in spatial memory which is increased during VWR (van Praag, Christie, Sejnowski, & Gage, 1999).
A limitation of the current study is that the number of ∆FosB-positive neurons were not corrected for ROI size. In future analyses, the number of ∆FosB-positive neurons per brain region needs to be normalized for ROI size, to reduce the potential efect of physical activity training on hippocampal size in rats (Erickson et al., 2011; Sierakowiak et al., 2015).
To functionally test the efects of exercise-induction of ∆FosB in the DG or in the CA1, a follow-up study would be to inject a virus in a specific subregion of the hippocampus which will impair the ∆FosB function during VWR, and then assess what the efects on stress-related behavior or cognition (eg spatial learning and episodic memory) are. To discover if exercise-induction of ∆FosB in the hippocampus in fact has a positive efect on spatial learning and episodic memory as suggested above.
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Appendix A
Rat brain atlas images of the hippocampus per bregma (-3.12 till -5.64). The hippocampus is marked in yellow in a few bregma’s.
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