Abstract
Analysis of expression of the immediate early gene c-Fos in neuronal populations is a commonly used method to assess changes in neuronal activity due to various factors of interest. However, different levels of c-Fos have been observed between control animals across studies. The present investigation assessed whether such differences could reflect different behavioral or physiological states in housing conditions that are typically considered naïve controls. Specifically, we assessed c-Fos expression in 19 brain regions in male C57BL6/J mice that were housed either socially (in groups of 4/cage) or individually. c-Fos expression was compared with socially-housed mice under either normal or reverse light conditions to assess the effect of light cycle on neuronal activity. We identified three main patterns of differences between groups. Light, but not social housing conditions, influenced c-Fos expression in the suprachiasmatic nucleus of hypothalamus and the dentate gyrus. A large number of brain regions across cortex, hypothalamus, ventral striatum and midbrain showed increased activity during the dark phase of circadian cycle only in the social, but not individual, housing. Finally, activity in the amygdala appeared to be induced by social housing conditions only during the dark phase of circadian cycle. Taken together, our experiment identified differential regulation of c-Fos expression by basal housing conditions and circadian phase. It also indicates that despite the well-known habituation of c-Fos expression to repeated stimulation, this expression is sensitive to basal housing conditions. This sensitivity needs to be taken into account when analyzing c-Fos data in various studies.
Keywords: inducible transcription factor, Fos, circadian cycle, stress, housing conditions, immunohistochemistry
Introduction
More than thirty years have passed since expression of c-Fos and immediate early genes (IEGs) were introduced as a marker allowing mapping activated neurons across animal brains (Morgan and Curran, 1988; 1989; Winston et al., 1990). Despite the frequent perception that IEGs are not ideal markers of neural activity and that such analyses will pass with the development of better techniques, this methodology has maintained its popularity. For example, a PubMed search performed on papers published using keywords “c-fos” and “brain OR neuron” identified 533 papers published in 2003, 578 papers published in 2008, 599 papers published in 2013 and 559 papers published in 2018, demonstrating stable use of this methodology.
Briefly, the focus on IEGs in neuroscience is based on their rapid induction upon stimulation of neurons leading to neuronal depolarization (Morgan and Curran, 1988). The induction is transient, such that detection of neurons expressing IEGs suggests their depolarization at a specific timeframe prior to animal euthanasia (Ikeda et al., 1994; Cullinan et al., 1995). The timecourse of induction following neuronal activation is most extensively studied for the IEG c-Fos. Thus, c-Fos protein can be detected in neurons by immunohistochemistry (IHC) 1–4 hours following depolarization. While exceptions to this timecourse have been noted early during development of this methodology and not all neurons do express c-Fos following depolarization (Herdegen and Leah, 1998; Kovacs, 2008), the reliability and relative ease of c-Fos detection via IHC has ensured the frequent use of this technique in neuroscience.
The frequent use of c-Fos IHC to detect or confirm changes in neuronal activity relies not only on rapid and transient induction of this IEG after many stimuli affecting neural activity, but also on the relatively low level of its expression in brains of control animals (Hughes et al., 1992). Moreover, c-Fos expression has been shown to habituate with repeated exposure of animals to homotypic stimuli, allowing reliable detection of c-Fos induction following exposure to novel stimuli (Melia et al., 1994). While such low levels are reliably observed across hundreds of c-Fos studies, some papers report relatively high basal c-Fos levels even in control subjects (Zheng et al., 2002; Ryabinin et al., 2003).
High c-Fos levels could be due to attempts to artificially increase c-Fos counts (for example, by counting weakly-stained cells or counting cells that are out of the focus plane). Such technical reasons of varied basal expression levels across studies might not influence the interpretation of c-Fos experiments. However, we hypothesized that the varied basal levels of c-Fos across studies could also be due to differences in housing conditions. Agonistic interactions engage medial prefrontal cortex, resulting in differences in c-Fos expression (Wang et al., 2011). Therefore, it is possible that baseline IEG levels could be different between individually- and socially-housed mice. In addition, these levels could vary across different phases of circadian cycle due to differences in animal activity between these phases. Indeed, activity of the suprachiasmatic nucleus is involved in regulation of circadian activity rhythms (Welsh et al., 2010), and levels of of c-Fos in this region could change at different phases of the circadian cycle. In other words, the differences in baseline c-Fos could be reflective of differences in behavior of animals in these varied conditions, and these biological causes of differences in c-Fos levels could affect the interpretation of IEG studies.
To test this hypothesis, we compared c-Fos levels in several brain regions by IHC in “control” male mice euthanized at three standard conditions: socially housed, euthanized during the day (light) phase of circadian cycle, socially housed, euthanized during the night (dark) phase of circadian cycle, and single-housed, euthanized during the dark phase of circadian cycle. We report significant differences in c-Fos levels across these housing conditions confirming our hypothesis that housing conditions and times of sample collection affect levels of expression of this IEG across different brain regions.
Methods
Animals
Male C57BL/6J mice arrived from Jackson Laboratories at 7–8 weeks of age. They were housed in the main colony room at 4 per cage for one week at 12/12 light:dark cycle (lights on at 6:00 am) in standard “shoebox” cages (18.4 cm W × 29.2 cm D × 12.7 cm H) in a temperature (20–22°C)- and humidity-controlled environment with food (LabDiet 5001; LabDiet) and water ad libitum. Following one week, animals were split into three groups and housed under three different conditions (see below). All protocols were approved by the Oregon Health & Science University animal care and use committee and performed within the National Institutes for Health Guidelines for the Care and Use of Laboratory Animals, as well as the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research.
Experimental Groups
There were 3 groups in the study (8 mice per group). Group 1 (Social/Light) mice were kept in the main colony room along with other mice from three different laboratories for another six days before euthanasia. Group 2 (Social/Dark) mice were moved into a different room with a shifted (reversed) light:dark cycle (12:12, lights on at 20:00) and continued to be kept in group-housed conditions for six days until euthanasia. Group 3 (Individual/Dark) mice were moved into the same room as Group 2, but were single-housed (12:12 reversed light/dark cycle, lights on at 20:00) for six days until euthanasia.
Tissue processing
On the test day starting at 10:00 am, animals (in staggered cohorts with 4 mice at a time) were taken to a neighboring room and quickly euthanized by a CO2 overdose. Brains were dissected and placed into 2% paraformaldehyde in phosphate-buffered saline (PBS). Three experimenters participated in the dissections, so it took approximately 1 minute to dissect a single brain. Therefore, the time between the first and last dissection was less than half an hour. We use the immersion technique for fixation instead of perfusion because it allows quicker dissection times and processing more brains at a time, which decreases batch effects. At 20 hours post-immersion, the solution was switched to 20% sucrose/PBS for cryopreservation. The next day the cryopreservation solution was switched to 30% sucrose/PBS, in which the brains were stored prior to sectioning. Brains were sectioned at 35 μm. Floating brain slices were collected into PBS and stored in a PBS/sodium azide solution.
Immunohistochemistry
Brain slices containing 19 brain regions of interest were selected for c-Fos immunohiostochamical analysis using standard avidin-biotin-diaminobenzidine protocols (Bachtell RK et al., 2002;Ryabinin AE, 2000). Specifically, the sections were washed in PBS and incubated for 15 minutes with 0.3% hydrogen peroxide/PBS to inhibit endogenous peroxidase activity. Non-specific binding was eliminated by a 4-hour incubation with normal goat serum (Vector Laboratories, Burlingame CA, 1:10 dilution in PBS/Triton-100). Next, the slices were incubated overnight with the primary rabbit polyclonal anti-c-Fos antibody (F7799, Millipore-Sigma, St Louis, MO, 1:5000 in PBS/Triton-100/Bovine Serum Albumin). This was followed by an hour-long incubation with biotinylated goat secondary antibodies (Vector Laboratories, Burlingame CA, 1:200 in PBS/Triton-100). One-hour incubation with ABC solution (Vector Laboratories, Burlingame CA) was used to detect the secondary antibodies. All incubations were interspersed by 3 washes with PBS. The formed horseradish peroxidase-containing complexes were visualized following a 3-minute incubation with the 3,3’-diaminobenzidine tetrahydrochloride substrate solution (Pierce DAB Substrate kit, ThermoFisher Scientific, Waltham, MA). The reaction was stopped by 2 washes in water, and slices were mounted on microscopic slides.
Quantitation and statistical analysis
We analyzed c-Fos levels by counting positive cells in 19 brain regions either relevant to cognition/memory (hippocampal and cortical areas), emotion/stress (amygdala, ventral striatum, paraventricular nucleus of hypothalamus, centrally-projecting Edinger-Westphal nucleus) or circadian cycles (suprachiasmatic nucleus). Brain regions were defined using the Mouse Brain Atlas parameters (Paxinos G, 2008). Two brain slices best matching in position across all animals were selected for counting for each brain region for each animal. Immuno-positive cells on the left and right sides were counted separately for each animal resulting in 4 counts per brain region for most animals. For a few animals, one of the sides was damaged during tissue processing, resulting in 3 counts for some of the brain regions for the animal. Since the centrally-projecting Edinger-Westphal nucleus is a unilateral brain region, counts were obtained across 3 brain slices. The counts were performed manually for most brain regions as the number of cells was low. To avoid miscounting, for the two cortical brain regions where large numbers of cells exhibited Fos-ir, semi-automatic cell counting was done using ImageJ (ImageJ, NIH, RRID:SCR_003070) (Schneider et al., 2012). For these two brain regions, each microphotograph was cropped to contain only the region of interest and exclude visual artifacts (for example, edges). Next, threshold and particle size filters were used to generate an image containing only cell nucleus-sized particles that would be counted if the cells would be counted manually. Finally, the automated counting feature of the software was used to generate the cell count. The number of c-Fos-positive cells per each brain side per animal was averaged resulting in a single number that was used in statistical analysis. Normality was assessed using the D’Augostino and Pearson omnibus normality test. As many of the expression values in these basal conditions equaled or approached zero, the distribution of vast majority of data was non-normal. Therefore, the non-parametric Kruskal-Wallis test followed by post-hoc Dunn’s test for multiple comparisons was utilized in analysis. P-values less than 0.05 were considered significant.
Results
c-Fos immunoreactivity appeared as dark nuclear staining. Cytoplasmic staining, indicative of non-specific staining, was negligible. There was no evidence of any positive capillary staining that could obscure analysis of c-Fos. As expected, the majority of brain regions did not show high levels of c-Fos immunoreactivity under any of the conditions (Table 1). Still, significant differences between groups were detected in a number of brain regions. Statistical results along with specific Bregma positions for analyzed brain regions are presented in Table 1. The overall pattern of responses could be clustered in two main categories: 1) responding to the circadian phase, but not housing conditions, and 2) responding to both circadian phase and housing condition.
Table 1. Descriptive statistics of c-Fos-IR in brain regions.
Statistically significant differences were determined by Kruskal-Wallis test, followed by Dunn’s test for multiple comparisons: a – significantly different from Social lights on, b- significantly different from Social lights off, p<0.05), double letters indicate p<0.01, triple letters indicate p<0.001. Brain regions with significant differences between groups are italicized. IQR = interquartile range.
| Brain region | Bregma Level (mm) | Kruskal-Wallis (df = 2) | Social, lights on (median , 25% – 75% IQR) | Social, lights off (median , 25% – 75% IQR) | Individual, lights off (median , 25% –75% IQR) |
|---|---|---|---|---|---|
| Cortex: | |||||
| Hippocampus: | |||||
| Amygdala: | |||||
| Striatum: | |||||
| Hypothalamus: | |||||
| Midbrain: | |||||
Regions responsive to circadian time, but not housing
The suprachiasmatic nucleus (SCN) showed a robust induction of c-Fos in the Social/Light group compared to Social/Dark and Individual/Dark groups (Figure 1, Table 1). Posthoc analyses confirmed these differences (p=0.011 and p=0.0003, correspondingly). In the dentate gyrus (DG), c-Fos levels were significantly higher in the Social/Light group than the Individual/Dark group (p=0.003), although no significant difference was found between Social/Light and Social/Dark (p=0.059). Thus, both the ventral dentate gyrus had significantly higher levels of c-Fos in the Social/Light group versus the Social/Dark (p=0.0042) and versus the Individual/Dark groups (p=0.0007). In contrast, the dorsal CA3 and the ventral CA1 regions showed induction of c-Fos in the Social/Dark versus the Social/Light condition (dorsal CA3: p<0.0001, ventral CA1: p=0.0006) and in the Individual/Dark versus the Social/Light condition (dorsal CA3: p=0.0046, ventral CA1: p=0.006).
Figure 1. c-Fos immunoractivity in brain regions responsive to circadian time, but not housing.
A-C: Representative microphotograph of suprachiasmatic nucleus from animals of the Social/lights on group (A), Social/lights off group (B), Individual/lights off group (C). D-F: Quantitative analysis. D: Suprachiasmatic nucleus. E: Dorsal dentate gyrus. F: Ventral dentate gyrus. Stars indicate levels of significant difference: *- p<0.05; **- p<0.01, ***- p<0.001, ****- p<0.0001.
Regions responsive to both circadian and housing conditions.
All analyzed cortical brain regions showed an induction of c-Fos in the socially housed animals during the dark phase compared to light phase, but this induction was not observed in the dark phase in individually-housed animals (Figure 2). Specifically, the prelimbic cortex (PrL), the infralimbic cortex (IL), the anterior cingulate (Cg1) and the motor cortex (M2) all showed significantly higher levels in the Social/Dark versus the Social/Light group (p=0.0009, p=0.0011, p=0.0013, p=0.0016, respectively), and the M2 showed significantly higher levels in the Social/Dark versus Individual/Dark groups (p=0.029). The difference between the latter groups did not reach statistical significance in PRL, IL, or Cg1 (p=0.066, p=0.27, p=0.16, respectively).
Figure 2. c-Fos immunoractivity in brain regions responsive to both circadian and housing conditions.
A-C: Representative microphotograph of anterior cingulate (Cg1) and Motor cortex (M2) from animals of the Social/lights on group (A), Social/lights off group (B), Individual/lights off group (C). D-F. Quantitative analysis. D: Anterior Cingulate (Cg1). E: Motor Cortex (M2). F: Anterior Hypothalamus. Stars indicate levels of significant difference: *- p<0.05; **- p<0.01, ***- p<0.001, ****- p<0.0001.
A similar pattern of differences was also observed in some hypothalamic, striatal and midbrain areas. Thus, the Social/Dark animals showed higher levels of expression than the Social/Light mice in the anterior hypothalamus (AH, p=0.023), the shell of nucleus accumbens (NACS, p=0.0044) and the Edinger-Westphal nucleus (EW, p=0.032). There was also a significantly higher expression in the Social/Dark group than in the Individual/Dark group in the NACS (p=0.0038). The AH did not show a significant difference in c-Fos levels between the Social/Dark and Individual/Dark groups (p=0.071). This difference also did not reach a statistical significance for the EW (p=0.28), but there was also no statistical difference in this measure between the Social/Light and Individual/Dark mice (p>0.99) across AH, NACS and EW, suggesting that these brain regions are regulated by baseline conditions similarly to the cortical regions.
Two regions of the extended amygdala also suggested a regulation by both the light phase of circadian cycle and housing, but this regulation appeared different from the one described for the regions above (Figure 3). Thus, in the basolateral amygdala (BLA) and central amygdala (CeA), there was a significant difference in c-Fos levels only between the Social/Light and Individual/Dark groups (p=0.012 and p=0.0027, respectively), but not between the Social/Light versus Social/Dark (p=0.24 and p=0.54, respectively) or between Social/Dark and Individual/Dark groups (p=0.80 and p=0.14, respectively), suggesting synergic activation of activity in amygdala by light and individual housing.
Figure 3. c-Fos immunoractivity in stress-sensitive brain areas.
A-C: Representative microphotograph of amygdala from animals of the Social/lights on group (A), Social/lights off group (B), Individual/lights off group (C). D-F. Quantitative analysis. D: Basolateral amygdala. E: Central Nucelus of Amygdala F: Paraventricular Nucleus of Amygdala. Stars indicate levels of significant difference: *- p<0.05; **- p<0.01, ***-p<0.001, ****- p<0.0001.
Discussion
Our experiments detected differences in c-Fos levels between groups that are typically considered control or baseline conditions in many experiments. These findings might explain why different levels have been detected between control groups across different studies (Zheng et al., 2002; Ryabinin et al., 2003). Interestingly, c-Fos induction in many brain regions is known to habituate with repeated homotypic stimulation (Melia et al., 1994; Ryabinin et al., 1999). Therefore, much of the observed c-Fos following robust behavioral and pharmacological stimuli is presumed to be not only due to direct stimulation by these stimuli, but also by the novelty of this stimulation. Consequently, it could be expected that repeated exposure to standard housing conditions would result in non-existent basal c-Fos levels across the brain. Indeed, in the majority of analyzed brain regions in the current study the numbers of c-Fos-positive cells are relatively low. Nevertheless, significant differences between groups are detected, suggesting that this habituation is not complete or that there is still an element of novelty even under standard housing conditions - for example, due to fluctuations in external stimuli and complexity of interactions in social groups.
Importantly, our experiments were designed to maximally avoid potential random fluctuations between separate cohorts of animals. Specifically, the samples were collected from animals simultaneously and processed for c-Fos immunohistochemistry as a single batch. Therefore, differences in c-Fos level between groups are not reflecting technical variability but are due to biological differences between the groups. We interpret these differences as reflecting different basal states of the animals, which in turn are accompanied by different behavioral repertoires and selective activities in brain regions involved in these behaviors.
Specifically, most brain regions in animals in the Social/Light condition show very low c-Fos levels. This observation was expected, as this circadian phase is characterized by low locomotor and exploratory activity levels (Bains et al., 2016). In fact, it is possible that at least some of the animals in this group were asleep shortly before euthanasia. It is not surprising that, in contrast to majority of other brain regions, the suprachiasmatic nucleus showed high levels of c-Fos as this brain region is the master regulator of circadian rhythms and is activated by light (Welsh et al., 2010) . On the other hand, it might seem surprising that the DG also showed higher levels of activity in this group of animals. This observation is not without precedent, as previous studies performed during the light phase of circadian cycle detected inhibitory effects of alcohol in mice and rats (Ryabinin, 1998), which is only possible if there are reasonably high basal levels of c-Fos. Moreover, previous studies indicate that expression of clock-related genes is differentially regulated during the circadian cycle in the SCN, DG and CeA (Harbour et al., 2014). The DG and CeA do not constitute major projection sites of the SCN. Therefore, it is not clear whether activity in the DG or lack of activity in the CeA is dependent on SCN.
In contrast to these brain regions, the majority of brain regions showing significant differences between groups had higher levels of activity in the Social/Dark group versus the Social/Light group. This observation is predictable as the specific time when these animals were euthanized (2 hours after the onset of dark), is known as the peak of their activity (Bains et al., 2016). Therefore, the higher c-Fos activity in this group could reflect higher levels of sensory stimulation due to ambient light, higher exploratory activity, higher intensity of social interactions, as well as peak food and water consumption. Since there was no statistical difference between the Social/Dark and Individual/Dark condition, but a significant difference from the Social/Light group in the dorsal CA3 and ventral CA1, we presume that induction of c-Fos in these areas are associated with higher sensory stimulation.
Importantly, most of the brain regions showing increased c-Fos induction during the Dark phase also showed modulation by the social versus individual housing conditions. Individual housing in known to be a stressor in most animals (Berry et al., 2012; Ieraci et al., 2016; Manouze et al., 2019). Interestingly, we did not observe an induction of c-Fos in the paraventricular nucleus of hypothalamus (Fig 3, Table 1), suggesting that individual housing did not result in a major activation of the hypothalamic-pituitary adrenal axis. Nevertheless, there was an induction of c-Fos in the BLA and CeA, areas well known to be associated with anxiety-related behaviors (Tye et al., 2011), in the Individual/Dark versus Social/Light groups of animals. However, the difference between Individual/Dark and Social/Dark groups was not significant - a finding suggesting that either both non-stressful sensory stimulation and isolation stress activate these regions or that these regions are also sensitive to mild stressors resulting from social interactions.
Finally, a large group of regions across cortical areas, hypothalamus, striatum and midbrain (PrL, IL, Cg1, M2, AH, NACS, EW) showed a significant induction in the Social/Dark group, but not in the Individual/Dark group. It is possible that isolation stress directly inhibits neural activity in these brain regions. However, it appears more likely that these brain areas are responsive to stimulation because of social interactions in the Social/Dark group that are absent in the Individual/Dark group. Cortical and striatal areas are well known to be involved in complex regulation of social interactions (Ko, 2017). The AH is also known to be involved in social behaviors across rodent species (Delville et al., 2000). The anatomically defined EW assessed in this study corresponds to the centrally projecting EW, a peptidergic brain with multiple central projections region sensitive to environmental stressors and pharmacological agents (Kozicz et al., 2011). A study in prairie voles suggests that it could also be involved in regulation of social affiliation (Walcott and Ryabinin, 2019). Together, a large number of brain regions regulated by basal social housing conditions is in agreement with a wide neuronal network required for regulation of social behavior.
Overall, we believe our findings will be useful for other investigators analyzing the response of specific brain regions to the stimulus of interest as this response will depend on the circadian time of analysis and housing conditions. Our study has several limitations we need to note. First, we only tested adult male C57BL/6J mice. It is possible that basal c-Fos levels will vary dependent on sex, age and strains of animals in some of the brain regions. For example, “basal” activity could be different between males and females in brain regions regulating social hierarchies, between adolescent and adult animals in brain regions involved in cognitive processing, and in different strains known to differ in sensory acuity. Second, we did not include an Individual/Light group in this study as our focus was on testing whether differences in baseline c-Fos levels exist, not on identifying specific factors contributing to these differences. Of note, two-factorial design of experiments would not help distinguishing effects of housing versus light in our study as data under these basal conditions are not distributed normally. Also, our list of analyzed brain regions is not exhaustive. It is also possible that other patterns of differences between these groups can be observed in brain regions we have not analyzed. Our findings, thus, serve only as illustrations of specific patterns. We believe this is a reasonable approach as it impossible to analyze all brain regions and, even then, there could be differences in c-Fos response in subpopulations of neurons within brain regions. Finally, our study focused on c-Fos, and it is possible that other markers of neuronal activity would identify other brain regions differentially responsive to the studied circumstances. Nevertheless, our experiments demonstrated lack of habituation of c-Fos responses to homotypic housing and lighting conditions and identified differential patterns of activities between brain regions under different presumably basal “control” conditions. Our results illustrate that analysis of potential low levels of IEG expression in these control conditions can be informative.
Highlights:
Housing conditions and circadian time of analysis affect basal c-Fos expression in untreated “control” mice.
Basal c-Fos level in suprachiasmatic nucleus and dentate gyrus is regulated by the circadian time of analysis.
In many brain regions, social housing affects basal c-Fos levels only during the dark phase of circadian cycle.
In regions of amygdala, individual housing leads to increased c-Fos levels only during the dark phase of circadian cycle.
Acknowledgements
This work was supported by NIH Grants R21 AA025548 and RO1 AA025024. We thank Alfredo Zuniga for assistance with sample collection.
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
References
- Bachtell RK, Tsivkovskaia NO, Ryabinin AE (2002), Alcohol-induced c-Fos expression in the Edinger-Westphal nucleus: pharmacological and signal transduction mechanisms. J Pharmacol Exp Ther 302:516–524. [DOI] [PubMed] [Google Scholar]
- Bains RS, Cater HL, Sillito RR, Chartsias A, Sneddon D, Concas D, Keskivali-Bond P, Lukins TC, et al. (2016), Analysis of Individual Mouse Activity in Group Housed Animals of Different Inbred Strains using a Novel Automated Home Cage Analysis System. Front Behav Neuroscie 10:106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berry A, Bellisario V, Capoccia S, Tirassa P, Calza A, Alleva E, Cirulli F (2012), Social deprivation stress is a triggering factor for the emergence of anxiety- and depression-like behaviours and leads to reduced brain BDNF levels in C57BL/6J mice. Psychoneuroendocrinol 37:762–772. [DOI] [PubMed] [Google Scholar]
- Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ (1995), Pattern and time course of immediate early gene expression in rat brain following acute stress. Neurosci 64:477–505. [DOI] [PubMed] [Google Scholar]
- Delville Y, De Vries GJ, Ferris CF (2000), Neural connections of the anterior hypothalamus and agonistic behavior in golden hamsters. Brain Behav Evol 55:53–76. [DOI] [PubMed] [Google Scholar]
- Harbour VL, Weigl Y, Robinson B, Amir S (2014), Phase differences in expression of circadian clock genes in the central nucleus of the amygdala, dentate gyrus, and suprachiasmatic nucleus in the rat. PloS One 9:e103309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Herdegen T, Leah JD (1998), Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res Brain Res Rev 28:370–490. [DOI] [PubMed] [Google Scholar]
- Hughes P, Lawlor P, Dragunow M (1992), Basal expression of Fos, Fos-related, Jun, and Krox 24 proteins in rat hippocampus. Brain Res Mol Brain Res 13:355–357. [DOI] [PubMed] [Google Scholar]
- Ieraci A, Mallei A, Popoli M (2016), Social Isolation Stress Induces Anxious-Depressive-Like Behavior and Alterations of Neuroplasticity-Related Genes in Adult Male Mice. Neural Plast 2016:6212983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ikeda J, Nakajima T, Osborne OC, Mies G, Nowak TS Jr. (1994), Coexpression of c-fos and hsp70 mRNAs in gerbil brain after ischemia: induction threshold, distribution and time course evaluated by in situ hybridization. Brain Res Mol Brain Res 26:249–258. [DOI] [PubMed] [Google Scholar]
- Ko J (2017), Neuroanatomical Substrates of Rodent Social Behavior: The Medial Prefrontal Cortex and Its Projection Patterns. Front Neural Circuits 11:41. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kovacs KJ (2008), Measurement of immediate-early gene activation- c-fos and beyond. J Neuroendocrinol 20:665–672. [DOI] [PubMed] [Google Scholar]
- Kozicz T, Bittencourt JC, May PJ, Reiner A, Gamlin PD, Palkovits M, Horn AK, Toledo CA, et al. (2011), The Edinger-Westphal nucleus: a historical, structural, and functional perspective on a dichotomous terminology. J Comp Neurol 519:1413–1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Manouze H, Ghestem A, Poillerat V, Bennis M, Ba-M’hamed S, Benoliel JJ, Becker C, Bernard C (2019), Effects of Single Cage Housing on Stress, Cognitive, and Seizure Parameters in the Rat and Mouse Pilocarpine Models of Epilepsy. eNeuro 6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Melia KR, Ryabinin AE, Schroeder R, Bloom FE, Wilson MC (1994), Induction and habituation of immediate early gene expression in rat brain by acute and repeated restraint stress. J Neurosci 14:5929–5938. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morgan JI, Curran T (1988), Calcium as a modulator of the immediate-early gene cascade in neurons. Cell Calcium 9:303–311. [DOI] [PubMed] [Google Scholar]
- Morgan JI, Curran T (1989), Calcium and proto-oncogene involvement in the immediate-early response in the nervous system. Ann NY Acad Sci 568:283–290. [DOI] [PubMed] [Google Scholar]
- Paxinos G FK (2008) The Mouse Brain in Stereotaxic Coordinates. San Diego: Academic. [Google Scholar]
- Ryabinin AE (1998), Role of hippocampus in alcohol-induced memory impairment: implications from behavioral and immediate early gene studies. Psychopharmacol 139:34–43. [DOI] [PubMed] [Google Scholar]
- Ryabinin AE (2000), ITF mapping after drugs of abuse: pharmacological versus perceptional effects. Acta Neurobiol Exp (Wars) 60:547–555. [DOI] [PubMed] [Google Scholar]
- Ryabinin AE, Galvan-Rosas A, Bachtell RK, Risinger FO (2003), High alcohol/sucrose consumption during dark circadian phase in C57BL/6J mice: involvement of hippocampus, lateral septum and urocortin-positive cells of the Edinger-Westphal nucleus. Psychopharmacol 165:296–305. [DOI] [PubMed] [Google Scholar]
- Ryabinin AE, Wang YM, Finn DA (1999), Different levels of Fos immunoreactivity after repeated handling and injection stress in two inbred strains of mice. Pharm Biochem Behav 63:143–151. [DOI] [PubMed] [Google Scholar]
- Schneider CA, Rasband WS, Eliceiri KW (2012), NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tye KM, Prakash R, Kim SY, Fenno LE, Grosenick L, Zarabi H, Thompson KR, Gradinaru V, et al. (2011), Amygdala circuitry mediating reversible and bidirectional control of anxiety. Nature 471:358–362. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walcott AT, Ryabinin AE (2019), Effects of Alcohol Consumption on Pair Bond Maintenance and Potential Neural Substrates in Female Prairie Voles. Alcohol Alcohol 54:353–360. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang F, Zhu J, Zhu H, Zhang Q, Lin Z, Hu H (2011), Bidirectional control of social hierarchy by synaptic efficacy in medial prefrontal cortex. Science 334:693–697. [DOI] [PubMed] [Google Scholar]
- Welsh DK, Takahashi JS, Kay SA (2010), Suprachiasmatic nucleus: cell autonomy and network properties. Annu Rev Physiol 72:551–577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Winston SM, Hayward MD, Nestler EJ, Duman RS (1990), Chronic electroconvulsive seizures down-regulate expression of the immediate-early genes c-fos and c-jun in rat cerebral cortex. J Neurochem 54:1920–1925. [DOI] [PubMed] [Google Scholar]
- Zheng H, Li YF, Weiss M, Mayhan WG, Patel KP (2002), Neuronal expression of fos protein in the forebrain of diabetic rats. Brain research 956:268–275. [DOI] [PubMed] [Google Scholar]