Increased glucocorticoid signaling during
cued fear conditioning training impairs
long term fear memory retrieval
Bachelor ProjectBachelor Psychobiology Priya Gajadien
Student ID: 11232323
Student mail: priya.gajadien@student.uva.nl Supervisor: Dhr. Dr. H. J. Krugers
Faculty of Science
Swammerdam Institute for Life Sciences Structural and Functional Plasticity University of Amsterdam
Institutional supervisor: Dhr. A.A.D. Brosens (Msc) Faculty of Science
Swammerdam Institute for Life Sciences Structural and Functional Plasticity University of Amsterdam
ABSTRACT
AbstractStressful experiences are thought to be remembered for a longer time compared to non-stressful experiences. However, preliminary data showed in mice that retrieval of long term memory after using cued fear conditioning paradigm is impaired after acute corticosterone injection. It is not exactly known yet what the role is of stress on long term memory. A hypothesis is that increased corticosterone signalling during fear conditioning training lead to less freezing behaviour based on long term. For this reason it is also expected to observe a decrease of corticosterone levels and immediate early genes, such as Arc and c-Fos, which play a role in memory formation. In this study male adult transgenic Arc::dVenus mice were recruited to undergo a cued fear conditioning paradigm whereby retrieval of the fear memory was determined after 28 days after learning. To assess the freezing behaviour, corticosterone levels and quantity of the immediate early genes (IEGs), Arc and c-Fos protein are determined using immunohistochemistry. This study found an impairment of retrieval of long term fear memory by an increase of glucocorticoids during training. However this study did not find an effect in the corticosterone levels and IEGs of mice in the retrieval and non-retrieval control group that were treated with either corticosterone or vehicle acutely after fear conditioning. From these results it can be concluded that increased corticosterone signalling during fear conditioning training results in impaired expression of long term fear memory without affecting plasma corticosterone levels and the number of Arc::dVenus and c-Fos positive cells
in the dentate gyrus.
Keywords:
Cued fear conditioning paradigm; long-term fear memory; freezing behaviour; stress; corticosterone; Arc::dVenus; c-Fos; immunohistochemistry; neuronal correlations; transgenic mice
TABLE OF CONTENTS
1. Introduction 5
2. Material and methods 7 2.1. Subjects 7 2.2. Behavioural procedure 7 2.2.1. Drug treatment 8 2.2.2. Behavioural analysis 8 2.3. Corticosterone assay 8 2.4. Immunohistochemistry 8 2.4.1. Arc::dVenus 9 2.4.2. c-Fos 9 2.5. Statistical analysis 9 3. Results 10 3.1. Behaviour 10 3.2. Corticosterone assay 10 ` 3.3. Immunohistochemistry 11 3.3.1. Arc::dVenus 11 3.3.2. c-Fos 12
3.3.3. Colocalization Arc::dVenus and c-Fos 13
4. Discussion 15
5. References 17
LIST OF ABBREVIATIONS
Arc Activity-regulated cytoskeleton-associated protein
BSA Bovine serum albumin
ChAT Choline Acetyltransferase
CS Conditioned stimulus CORT Corticosterone DG Dentate gyrus EtOH Ethanol GC Glucocorticoid HPA Hypothalamic-pituitary-adrenal
IEG Immediate early genes
OD Optical density
US Unconditioned stimulus
PB Phosphate buffer
PBS Phosphate buffer saline
PBST Phosphate buffer saline Triton X-100
1. INTRODUCTION
Stressful events are thought to be remembered for a long time when comparing to non-stressful events (Joëls, Pu, Wieger, Oitzl & Krugers, 2016). Experiences of stress and emotionally arousing events induce the release of stress hormones, such as glucocorticoids (GCs). The release of GCs can affect memory processing of that experience (Joëls & Baram, 2009). Previous studies showed that memory is encoded by small populations of cells (engrams) and corticosterone and may increase the activity and size of those neuronal ensembles (Josselyn, Köhler & Frankland, 2015). However, preliminary data showed that long term memory is inversely affected by GCs as fear memory has been shown to be significantly decreased after 28 days.
Formation of long term fear memory is mediated by rapid molecular cascades that play an important role in synaptic plasticity (Duvarci, Nader & LeDoux, 2008; Helmstetter, Parsons & Gafford, 2008). One of the proteins that is involved in these molecular cascades is an activity-regulated cytoskeleton-associated protein (Arc). Arc is part of the immediate early genes (IEGs) family and encodes a protein that is critical for memory consolidation (Guzowski, McNaughton, Barnes & Worley, 1999). Previous studies showed that Arc expression occurs biphasic with the first wave of expression 15-30 min after training and the second wave after 1-3 hours (Nakayama et al, 2015).
Whereas Arc is strongly involved in the synaptic plasticity which plays a role in memory formation, there are also other IEGs involved by the retrieval of memory. For instance, c-Fos is an IEG that has been long known as a marker of neural activity and neuronal circuits and had been used to determine the neuronal circuits underlying the behavioural responses induced by stress (Sagar, Sharp & Curran, 1988). When stress occurs as an extracellular stimulus, c-Fos gene evokes very rapid transcription. This gene is transiently expressed in neurons after stimulation and provides detectable amounts of its product and peaks between 30 and 90 minutes (Strekalova et al., 2003; Meyza, Boguszewski, Nikolaev & Zagrodzka, 2007). The c-Fos protein that is formed due to this transcriptional activity is detectable for at least a few hours marking the stimulated neurons and gradually disappear from the cell nucleus by 4-6 hours after treatment (Pezzone, Lee, Hoffman & Rabin, 1992).
However Arc and c-Fos play an important role in synaptic plasticity and neuronal activation, these immediate early genes could be used as readout for the neuronal engrams since previous studies found evidence from electrophysiological and IEG imaging experiments that a small portion of neurons within a region encodes any one memory (Guzowski et al., 2019; Vazdarjanova et al, 2006; Reijmers, Perkins, Matsuo & Mayford, 2007). Because the hippocampus is involved during memory formation it is predicted that the neuronal ensembles are present in that area. (Scoville & Milner, 1957; Josselyn et al., 2015). This seems particularly true in the dentate gyrus (DG) layer of the hippocampus which exhibits strikingly sparse activity patterns during experiences. Besides that, previous studies identified transcriptional changes in the DG neurons that predicted the reactivation of engram cells. For this reason, activated DG nuclei contains stronger transcriptional activity than other
hippocampal nuclei (Ramirez-Amaya, Angulo-Perkins, Chawla, Barnes & Rosi, 2013). Therefore it would be crucial to examine the IEGs in the DG of the hippocampus during the retrieval of long term fear memory. The goal of this study is to focus on the effects of increased glucocorticoids signaling on long term fear memory.
In the present study the quantity of Arc and c-Fos protein was examined in the DG during retrieval after using cued fear conditioning paradigm. To measure the quantity of Arc, this protein could be visualized by the reporter gene dVenus. Previous studies demonstrated the value of Arc::dVenus transgenic mice for monitoring the dynamics of Arc gene expression in the DG (Eguchi & Yamaguchi, 2008). In this study male adult transgenic Arc::dVenus mice were used for measuring the amount of Arc+ cells. It is expected that Arc and c-Fos levels will be decreased in the DG induced by alterations of the corticosterone levels. To make sure corticosterone is associated in this context, blood levels will be measured. To test this hypothesis immunohistochemistry on the IEGs and a corticosterone assay were performed. This will provide more information about the relationship between corticosterone on IEGs on long term fear memory.
2. MATERIALS & METHODS
2.1 Subjects
In this study male adult C57Bl/6J transgenic Arc::dVenus mice (postnatal weeks 8-12) were housed in groups of 2-3 mice per cage under standard housing conditions (temperature 20-22 °C, 40-60% humidity) on a 12 hours light/dark cycle with standard chow and water available ad libitum. These mice were individually housed for 7 days before the start of the experiments. The experiments were performed during the light phase between 8:00 AM and 8:00 PM. All experiments were conducted under the EU directive 2010/63/EU for animal experiments and were approved by the animal welfare committee of the University of Amsterdam.
2.2 Behavioural procedure
Cued fear conditioning was performed in a chamber provided with a stainless steel grid floor connected to a shock generator. The whole fear conditioning paradigm consisted of two phases: training and retrieval phase. In the training phase, all mice were trained in a fear conditioning paradigm with a foot shock of 0.2 mA for 2 seconds. First, they were placed in the conditioning chamber (A) for 180 seconds, followed by a series of three co-terminating presentations of a tone condition stimulus (CS) (30 s, 2.8 kHz, 82 dB) and the foot shock unconditioned stimulus (US). Between the tone-shock presentation, there was an inter-stimulus interval of 60 s. After this fear conditioning paradigm the mice were then returned to their home cages. Twenty-eight days after the training phase the mice underwent the retrieval phase which constituted of a novel chamber (B). In this retrieval phase mice were presented with the same tone as in the training phase after 180 seconds and kept in chamber B for the rest 90 seconds. Non-retrieval control mice remained in their home cage for 28 days after training.
Figure 1. Behaviour procedure. This figure shows the fear conditioning paradigm that was used
in this study. Context A represents the training phase whereas context B represents the retrieval phase. After the training phase mice were injected with corticosterone (cort) or saline.
2.2.1. Drug treatment
In this study a retrieval and non-retrieval control group were treated with either corticosterone (n=8) or vehicle (n=8) acutely after fear conditioning. Corticosterone (Sigma) (16 mg/ml dissolved in 99.9% EtOH and diluted 40x in saline; final dose: 2 mg/kg, injection volume: 5 μl/g body weight) or vehicle (99.9% EtOH and diluted 40x in saline: injection volume 5 μl/g body weight) was injected intraperitoneally immediately following fear conditioning.
2.2.2. Behavioural analysis
Each session was video-recorded for manual scoring of freezing. Fear memory was assessed and expressed as the percentage of time the animal remained frozen. Freezing was defined as the absence of all movements, except those related to breathing. Freezing behaviour was determined by measuring the time of freezing during the training phase and retrieval phase. In the training phase shock of 0.2 mA was given in a three-time window; the first one was given on the third minute followed by the second tone after one and a half minutes and the third tone one and a half minutes after the second tone. Only the first 30 seconds from the tone were measured. After measuring the time of freezing during these time windows the percentage freezing behaviour of each 30 seconds was calculated. These percentages were further used for data analysis.
2.3 Corticosterone levels
For the corticosterone assay 20 µl standard, control and experimental samples were mixed with 200 µl enzyme conjugate. After mixing for 10 seconds the samples were incubated for 60 minutes on 20 °C. Then the wells were 3 times rinsed with diluted wash solution (400 µl per well) and mixed in 100 µl substrate solution to start the enzymatic reaction. This had to incubate for 15 minutes at 20 °C. This enzymatic reaction was terminated by adding 50 µl of stop solution. Then the optical density (OD) was read at 450
±
10 nm with a microtiter plate reader within 10 minutesafter adding the stop solution.
2.4 Immunohistochemistry
In this study the quantity was measured for Arc::dVenus and c-Fos. Arc protein was crucial in this study to encode synaptic plasticity whereas c-Fos represents the retrieval of the fear memory after 28 days. Mice were sacrificed by decapitation after 90 minutes after the behavioural procedure and brains were dissected and post-fixed in 4% paraformaldehyde in 0.1 PB for 48 hours. Then the brain hemi sections were transferred to 15% sucrose for 4 hours and then 30% sucrose for 24 hours. Coronal sections of 40 micro meter of the left hemisphere spanning the anterior-posterior extent of the whole brain were sectioned on a microtome and stored at – 20 °C until staining. Using only the left hemisphere was needed because of the lateralization of Arc protein synthesis in the left hemisphere during memory consolidation (Ingberg, Elkobi, Edri & Rosenblum, 2013). The fluorescence intensity for both techniques was used to identify Arc::dVenus and c-Fos cells in the dentate gyrus granule cell layer in
sections containing either anterior
-2.80 to -6.80 m posterior to bregma. The immunohistochemistry of c-Fos was in fact a double staining with choline acetyl transferase (ChAT) that was used to distinguish the amygdala in the lateral and basolateral part. This was crucial to analyse the amygdala.
2.4.1 Arc::dVenus
To assess the Arc::dVenus+ cells in the dentate gyrus, dVenus fluorescence could be analysed immediately without performing the immunohistochemistry. Brain sections were washed with PB (0,1 M, pH= 7.4) 3 times for 10 minutes. After the washing steps, the sections were coverslipped in DAPI mounting medium. Fluorescence intensity was used to identify Arc::dVenus cells in the dentate gyrus granule cell layer in sections containing either anterior -2.80 to -6.80 m posterior to bregma.
2.4.2 c-Fos
For the c-Fos immunohistochemistry, brain sections were washed 5 times in PBS for 10 minutes. After the washing steps, the sections were blocked for 30 minutes at room temperature with 1% BSA in 0.2 % PBST (0.1 M PB with 0.9% saline, 0.2% Triton X-100). Subsequently, the primary antibodies 1:500 mouse anti c-fos, sc-52, Lot # i1019 (Santa Cruz Biotechnology) and 1:200 polyclonal goat anti-ChAT, AB144P (Merck) were diluted in blocking mix. After these step, the sections with the primary antibodies were incubated for the first hour on 20 °C and overnight at 4 °C. The next day the sections were washed 3x with 0.1 M PBS (pH=7.4) for 10 minutes. Then the sections were incubated with secondary antibodies, donkey anti-mouse (for c-Fos – A31571 diluted in 1:500 0.2% PBST) and donkey anti goat 594 (1:500 for ChAT) for 2 hours on 20 °C. After incubating the samples were washed again 3 times with 0.1 M PBS again. After the washing steps, the sections were cover slipped in DAPI mounting medium.
Fluorescence intensity was used to identify c-Fos protein-expressing. Besides c-Fos, the sections also contained Arc::dVenus as described in the previous method section. The slides were examined under the light microscope (Nikon eclipse) with the DAPI, GFP, CY5 and TxRED channels. DAPI represents all cells in the tissue, GFP encodes the Arc protein, CY5 was used to represents c-Fos and TxRED represented ChAT. For imaging of the proteins different levels of exposure, gain and intensity for the different channels were settled; the DAPI the exposure time was 500 ms, the gain was settled on 1x and the intensity was 50%, GFP had an exposure time of 1 s, a gain of 1.8x and an intensity of 28, the CY5 had an exposure time of 1 s, a gain of 4.1x and an intensity of 100 and TxRED for ChAT had an exposure time of 1 s , a gain of 1x and an intensity of 100.
2.5. Statistical analysis
Fear memory was determined as the percentage of time the animal remained frozen. After determining the time of freezing the percentage freezing behaviour of each 30 seconds was calculated. These percentages were further used for data analysis. The OD for measuring the corticosterone levels were determined by reading the absorbance endpoint on a wavelength of 450 nm. These values were plotted in a bar graph and further used for data analysis. For the immunohistochemistry means of the cells per nm2 was calculated per animal in all conditions with ImageJ. The data was
and whether those animals had a retrieval of their fear memory or not. Subsequently, the data was displayed in a bar plot. Hereby the independent variable were the four different conditions and the dependent variable was the mean of the cells per mm2.
Further analysis of all data of the experiments was determined using R-studio and GraphPad. The normality was checked using the Shapiro-Wilk test and the equality of variances was checked using a Levene’s test. Subsequently, the means were compared using a two-way ANOVA. A baseline correction was then applied to the trials and each trial was scanned for outliers.
3. RESULTS
3.1 Behaviour
To test the retrieval of fear memory the retrieval mice were placed in context B (figure 1). The percentage freezing during the retrieval phase was measured on different time points (figure 2A). During the interval training the baseline freezing is comparable before the tone was given. On 210 seconds the mice who were injected with corticosterone showed less freezing in percentages (n=8, mean = 13.5%) compared to the vehicle mice (n= 8, mean= 35.1%) The behavioural analysis showed significant differences in freezing between the mice who were injected with corticosterone and the vehicle mice (F(1, 63) = 10.11, p= 0.023). Also the boxplot in figure 2B showed significant differences between the vehicle and corticosterone group (t= 2.526, df= 14, p= 0.0242). Figure 2 represents that corticosterone injected
mice showed significantly more freezing behaviour compared to the vehicle mice when a the tone was given.
Figure 2. Increased glucocorticoid signalling during training impairs retrieval of fear memory. This figure represents the graphs associated with the fear conditioning paradigm. A) This
graph represents the percentages of freezing during different time windows. The black lines and dots represent the vehicle mice whereas the red lines and dots represent the mice that were injected with corticosterone. By 210 seconds the manual tone was given. Corticosterone mice
showed less freezing behaviour (n=8, mean = 13.5%) compared to the vehicle mice (n= 8, mean= 35.1%) during the retrieval of fear memory by 210 seconds. (p = 0.0023. B) This boxplot displays the significant differences in the freezing behaviour between the vehicle and corticosterone mice when the manual tone was given. Statistical analysis was performed with two-way ANOVA for multiple comparisons, a post-hoc Bonferroni for comparison of the means of two independent groups and Pearson correlation coefficient to assess covariance of 2 variables. Data were presented as mean ± SEM; P<0.05 was deemed statistically significant. (p = 0.0242).
3.2 Corticosterone assay
Blood was collected to test the corticosterone levels in each mice. Figure 3 showed the results of the corticosterone levels between the cort- and vehicle treated retrieval and non-retrieval groups. The data of the corticosterone assay show that there were no differences in plasma corticosterone levels between the experimental groups (p=0.6345).
Figure 3. Similar levels of corticosterone between cort- and vehicle-treated retrieval and non-retrieval groups. This figure represents the plasma corticosterone levels in ng/ml in
experimental conditions, no retrieval-vehicle (NR-VEH) no retrieval-corticosterone (NR-CORT), retrieval-vehicle (R-VEH) and retrieval-corticosterone (R-CORT). The black dots represent the corticosterone levels in each animal. Statistical analysis was performed with two-way ANOVA for multiple comparisons, a post-hoc Bonferroni for comparison of the means of two independent groups and Pearson correlation coefficient to assess covariance of 2 variables.
3.3 Immunohistochemistry
Fluorescence intensity of the dVenus reporter gene under the Arc promotor was used to identify Arc::dVenus cells in the dentate gyrus granule cell layer. After counting the cells with ImageJ the mean of cells were calculated per mm2. The calculated values
were plotted in bar graph shown in figure 4. This figure shows the means of cells per mm2 between the the cort- and vehicle treated retrieval and non-retrieval groups in
the anterior, posterior and whole dentate gyrus. The data for the Shapiro test for normality and the Levene’s test for equal variances were passed (p>0.05), therefore a two way ANOVA could be used. This ANOVA showed that the effect of the condition on whether the mice were injected with corticosterone or saline and whether used for the retrieval of memory or not was not significant (F(3, 21) = 0.5038, p= 0.6838) in the whole dentate gyrus (anterior + posterior) (Figure 4). Besides the whole dentate gyrus, also the anterior and posterior dentate gyrus separately were included for the analysis. The anterior dentate gyrus showed no significant differences in Arc::dVenus+ cells between the experimental groups (F(3, 21) = 0.5791, p= 0.6352) (Figure 4A). The posterior dentate gyrus also showed no significant differences in Arc::dVenus cells between the experimental groups (F(1, 22) = 0.1055, p= 0.7484) (Figure 4B). This analysis showed no effect in Arc::dVenus+ cells between experimental groups.
Figure 4. Similar amount of Arc::dVenus+ cells per mm2 between cort- and
vehicle-treated retrieval and non-retrieval groups. This figure represents the bar plots of
Arc::dVenus+ cells per in the dentate gyrus in experimental conditions; no retrieval-vehicle (NR-VEH) no retrieval-corticosterone (NR-CORT), retrieval-vehicle (R-(NR-VEH) and retrieval-corticosterone (R-CORT). The black dots represent the means of the amount of Arc::dVenus+ cells in each animal. Statistical analysis was performed with two-way ANOVA for multiple comparisons, a post-hoc Bonferroni for comparison of the means of two independent groups and Pearson correlation coefficient to assess covariance of 2 variables. A) This bar graph represents the amount of Arc::dVenus+ cells per mm2 in the anterior dentate gyrus in experimental conditions. B) This bar
graph represents the amount of Arc::dVenus+ cells per mm2 in the posterior dentate gyrus in
experimental conditions. C) This bar graph represents the amount of Arc::dVenus+ cells per mm2 in
the whole dentate gyrus in experimental conditions.
3.3.2 c-Fos immunohistochemistry
With c-Fos staining for the immunohistochemistry, c-Fos+ cells in the dentate gyrus granule cell layer were measured. After counting the cells with ImageJ the mean of cells were calculated per mm2. The calculated values were plotted in bar graph
shown in figure 5. This figure shows the means of cells per mm2 between the the
cort- and vehicle treated retrieval and non-retrieval groups in the anterior, posterior and whole dentate gyrus. The data for the Shapiro test for normality and the Levene’s test for equal variances were passed (p>0.05), therefore a two way ANOVA could be used. This ANOVA showed that the effect of the condition whether the mice were injected with corticosterone or saline and whether used for the retrieval of memory or not was not significant (F(3, 21) = 1.231, p= 0.3233) in the whole dentate gyrus (anterior + posterior). Besides the whole dentate gyrus, also the anterior and posterior dentate gyrus separately were included for the analysis. The anterior dentate gyrus showed no significant differences in c-Fos+ cells between the experimental groups (F(3, 21) = 0.7029, p= 0.5609). The posterior dentate gyrus also showed no significant differences in c-Fos+ cells between the experimental groups (F(1,20) = 0.004066, p= 0.9488). These analysis showed no effect in c-Fos+ cells between experimental groups.
Figure 5. Similar amount of c-Fos+ cells per mm2 between cort- and vehicle-treated
retrieval and non-retrieval groups. This figure represents the bar plots of c-Fos+ cells per in the
dentate gyrus in experimental conditions; no retrieval-vehicle (NR-VEH) no retrieval-corticosterone (NR-CORT), retrieval-vehicle (R-VEH) and retrieval-corticosterone (R-CORT). The black dots represent the means of the amount of c-Fos+ cells in each animal. Statistical analysis was performed with two-way ANOVA for multiple comparisons, a post-hoc Bonferroni for comparison of the means of two independent groups and Pearson correlation coefficient to assess covariance of 2 variables. A) This bar graph represents the amount of c-Fos+ cells per mm2 in the anterior dentate gyrus in
experimental conditions. B) This bar graph represents the amount of c-Fos+ cells per mm2 in the
posterior dentate gyrus in experimental conditions. C) This bar graph represents the amount of c-Fos+ cells per mm2 in the whole dentate gyrus in experimental conditions.
3.3.2 Co-localization Arc::dVenus & c-Fos immunohistochemistry
Besides the analysis of Arc::dVenus and c-Fos, also the co-localisation of both proteins are determined. After counting the cells with ImageJ the mean of cells were calculated per mm2. The calculated values were plotted in bar graph shown in figure6. This figure shows the means of cells per mm2 between the the cort- and vehicle
treated retrieval and non-retrieval groups in the anterior, posterior and whole dentate gyrus. The data for the Shapiro test for normality and the Levene’s test for equal variances were passed (p>0.05), therefore a two way ANOVA could be used. This ANOVA showed that the effect of condition whether the mice the condition whether
the mice were injected with corticosterone or saline and whether used for the retrieval of memory or not was not significant (F(3, 21) = 0.4518 , p= 0.7187) in the whole dentate gyrus. Besides the whole dentate gyrus, also the anterior and posterior dentate gyrus separately were included for the measurements. The anterior dentate gyrus showed no significant differences in the colocalization of Arc::dVenus+ and c-Fos+ cells between the experimental groups (F(1, 19) = 2.264, p= 0.1488). The posterior dentate gyrus also showed no significant differences in the colocalization of Arc::dVenus+ and c-Fos cells between the experimental groups (F(1, 9) = 1.549, p= 0.2447). These analysis showed no effect in the colocalization of Arc::dVenus+ and c-Fos+ cells between the experimental conditions.
Figure 6. Similar amount of colocalised Arc::dVenus+ and c-Fos+ cells per mm2 between
cort- and vehicle-treated retrieval and non-retrieval groups This figure represents the bar
plots of the colocalization of Arc::dVenus+ and c-Fos+ cells per in the dentate gyrus in experimental conditions; no retrieval-vehicle (NR-VEH) no retrieval-corticosterone (NR-CORT), retrieval-vehicle (R-VEH) and retrieval-corticosterone (R-CORT). The black dots represent the means of the amount of the colocalization of Arc::dVenus+ and c-Fos+ cells in each animal. Statistical analysis was performed with two-way ANOVA for multiple comparisons, a post-hoc Bonferroni for comparison of the means of two independent groups and Pearson correlation coefficient to assess covariance of 2 variables. A) This bar graph represents the amount of the colocalization of Arc::dVenus+ and c-Fos+ cells per mm2 in the anterior dentate gyrus in experimental conditions. B) This bar graph
represents the amount of the colocalization of Arc::dVenus+ and c-Fos+ cells cells per mm2 in the
posterior dentate gyrus in experimental conditions. C) This bar graph represents the amount the colocalization of Arc::dVenus+ and c-Fos+ cells per mm2 in the whole dentate gyrus in experimental
4. DISCUSSION
This study found in impairment on long term fear memory retrieval in mice treated with corticosterone compared to mice treated with vehicle. However, this study did not find an effect on corticosterone levels and the quantity of IEGs Arc::dVenus+ and c-Fos+ cells in the retrieval and non-retrieval control group that were treated with either corticosterone or vehicle acutely after fear conditioning. Therefore it can be concluded that increased corticosterone signalling during fear conditioning training results in impaired expression of long term fear memory without affecting plasma corticosterone levels and the number of Arc::dVenus and c-Fos positive cells in the dentate gyrus.
This is not in line with the expectation that these neuronal correlates decrease in their quantity when long term fear memory is thought to be impaired. However, this study found a decrease in freezing behaviour in mice acutely injected with corticosterone after training. This is in line with the preliminary data that showed an impairment of long term memory after experiencing stress. When animals are exposed to stress the HPA-axis becomes active which leads to a strong release of glucocorticoids that affect memory processing in the hippocampus (Joëls et al., 2009). When this occurs, corticosterone levels increase during retrieval of short term fear memory. However, in the case of long term memory, the corticosterone levels are expected to decrease. This is not found in this study. Because of the fact that this study did not find differences between the experimental groups, it could be indicated that corticosterone levels are not underlying to behavioural effects as observed.
Due to the preliminary study that showed an impairment of the long term memory by retrieval, it was also expected to see an effect of c-Fos+ cells since they are suggested to be involved in the synaptic plasticity and neuronal activity in the neuronal engram. The data of this study showed no significant differences in c-Fos+ cells in the dentate gyrus. This IEG is thought to be involved in making the neuronal engram more active. However, this was not found in this study. An explanation for these result could be that the sample size for the immunohistochemistry based on these proteins were too small. For this study, each condition contained 8 mice. For the dentate gyrus a larger sample size of brain sections was needed to provide more reliable results of the Arc::dVenus+ and c-Fos+ quantity. Besides this, only the left
hemisphere was included in the study, because of lateralization of the Arc::dVenus+ and c-Fos+ cells in the left hippocampus (Ingberg et al., 2013).
It was not expected to find an effect of long term fear memory on Arc::dVenus. It is known that dVenus has a fast degradation whereby the fluorescence intensity halves 8 hours after stimulation. For this reason, dVenus could not be used as a marker of the engram that was formed following training (Eguchi et al., 2008). Due to the window of the dVenus fluorescence expression, the Arc:dVenus+ cells that were shown in the dentate gyrus could not be linked to the training phase. The Arc::dVenus+ cells shown in the imaging pictures may possibly be linked to the basal activity of the cell itself. Therefore the co-localization between Arc::dVenus and c-Fos could not be directly linked to reactivation of the neuronal engram that was formed during training. Moreover, to measure direct co-localization it is crucial to do this with the confocal microscopy instead of the Nikon eclipse.
Taken together, this study showed no differences in Arc::dVenus+ and c-Fos+ levels in the dentate gyrus between the cort- and vehicle treated retrieval and non-retrieval groups. For this reason this is could be an indication that the dentate gyrus not directly plays a role in the behavioural effects as examined in this study.
It could be possible that not only the hippocampus is involved in memory formation, but also other areas of the brain. It is known that memories are thought to be initially stored within the hippocampal-enthorhinal cortex network, but over time become independent of the hippocampus. The memory that first is represented in the hippocampus become mostly engrained in cortical networks where the memory is consolidated for a permanent storage (Müller & Pilzecker, 1900; Bontempi, Laurent-Demir, Destrade & Jaffard, 1999; Takehara, Kawahara & Kirino, 2003; Cui et al., 2004; Hayashi et al., 2004). Previous studies showed that reactivation of the consolidated memories after cued fear conditioning induces protein-synthesis dependent cascades in the amygdala that is needed for the reconsolidation after retrieval (Nader, Schafe & Le Doux, 2000). It is suggested that c-Fos is involved in those molecular processes in the basal and lateral nuclei of the amygdala. Previous research found an increased number of c-Fos neurons in the lateral and basolateral amygdala (Scicli, Petrovich, Swanson & Thompson, 2004). Also, other research found that Arc is required for memory consolidation of fear conditioning in the lateral amygdala (Ploski et al., 2008). However c-Fos induction was not observed in the hippocampus (Hall et al, 2001). This is line with the finding that the hippocampus only inferences with contextual fear conditioning whereas the amygdala is involved with contextual as well as cued fear conditioning (Philips & Le Doux, 1992). In this study cued fear conditioning paradigm was used. Therefore an effect of Arc::dVenus and c-Fos positive cells between the cort- and vehicle treated retrieval and non-retrieval groups in the amygdala could be expected.
For further research it would be crucial to replicate the experimental procedures of this study including other areas in the brain, for instance amygdala (as described), the prefrontal cortex and auditory cortex since these areas also are associated with (auditory) cued fear conditioning (Song, Boatman, Jung, Kim, 2010). However this study did not find neuronal correlates of the behaviour effect observed in the experiment, it is possible that Arc and c-Fos may contribute to memory formation in the amygdala and other parts in the brain. For now, this study showed impaired expression of long term fear memory by increased corticosterone signalling.
This was in line with the preliminary data as described. Finally, these results give us more insight about the effects of stress on long term retrieval memory without affecting the plasma corticosterone levels and the quantity of Arc::dVenus and c-Fos positive cells in the dentate gyrus which take us to the next step to examine these parameters in the amygdala.
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Figure A. Preliminary data of S.L.Lesuis. This figure shows the preliminary data of the impaired
long term fear memory in corticosterone treated mice compare to vehicle treated mice after 28 days. A) A display of the behavioural procedure. B) Percentage of freezing during different time points over 3 minutes in context A after 14 days. C) Percentage of freezing in the retrieval phase in context B after 28 days of the training phase. D) Percentage of freezing during different time points over 3 minutes in context A after 28 days. E) Percentage of freezing in the retrieval context B after 28 days of the training phase.
