Contextual Fear Conditioning Induces Activity-Dependent Gene Expression in the Dorsal but not in the Ventral Hippocampus

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Jha This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7658753/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Feb, 2026 Read the published version in Neurochemical Research → Version 1 posted 4 You are reading this latest preprint version Abstract The hippocampus, a key structure playing an important role in contextual fear-conditioning (CxFC), shows functional specialization along its dorsoventral axis. The dorsal hippocampus is primarily implicated in spatial and contextual processing, whereas the ventral hippocampus is more closely associated with affective and emotional regulation. In this study, we examined whether CxFC selectively engages activity-dependent gene expression in the dorsal hippocampus (DH) but not in the ventral hippocampus (VH). Mice were subjected to CxFC, and freezing behavior across baseline, training, and testing sessions was evaluated. Molecular analyses were conducted to assess the temporal expression patterns of Arc and c-Fos proteins in the DH and VH at 0, 1, 3, and 5 hours on conditioning and post-conditioning days. Fear-conditioned animals displayed a significant increase in freezing behavior during the testing session compared to baseline, indicating strong fear memory retention. Arc expression in the DH showed a time-dependent increase, peaking at the 1st hour and remained highly expressed till the 5th hour on conditioning and post-conditioning days. No significant changes were observed in the VH. Similarly, c-Fos expression in the DH increased significantly at 1st, 3rd, and 5th hours on conditioning and post-conditioning days, while no significant activation was detected in the VH. These findings demonstrate that contextual fear conditioning selectively activates the DH, as evidenced by upregulation of Arc and c-Fos expression. The VH did not show corresponding molecular changes, suggesting a region-specific role of the DH and VH in the consolidation fear memory. Hippocampus Fear Memory Learning Neuronal Activity Protein expression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The hippocampus, a critical brain structure involved in learning and memory, is divided into two principal regions: the dorsal hippocampus (DH) and the ventral hippocampus (VH) [ 1 , 2 ]. Although part of the same structure, the DH and VH regions have discrete anatomical connections and contribute differently to cognitive functions [ 1 – 5 ]. For example, the DH is primarily associated with spatial memory [ 3 ]. It helps in forming and retrieving spatial maps, which are crucial for navigation [ 3 , 6 , 7 ]. Also, the DH contains a high density of place neurons essential for encoding and recalling spatial information [ 8 ]. The DH supports the ability to navigate through an environment by integrating sensory inputs and forming cognitive maps, which are mental representations of physical spaces [ 8 , 9 ]. On the other hand, the VH is more involved in processing emotional aspects of memory through its close interaction with the amygdala [ 10 ]. In addition, the VH plays an essential role in regulating the body’s response to stress through its interaction with the hypothalamic-pituitary-adrenal (HPA) axis [ 11 ]. It has been observed that lesions in the DH typically impair spatial memory and navigation, whereas lesions in the VH affect emotional memory and anxiety-related behaviors [ 12 – 14 ]. Although the DH and VH work together to integrate spatial, contextual, and emotional information, it seems that the DH and VH provide a comprehensive but independent framework for learning and memory [ 15 , 16 ]. The DH and VH possibly play a differential role in contextual fear conditioning [ 17 ]. The DH lesion impairs the ability to form and recall the spatial context of the fear conditioning, leading to a reduced fear response when re-exposed to the context [ 14 ]. In contrast, the VH lesion affects the emotional and stress-related components, altering the intensity of the fear response [ 18 ]. Functional imaging studies show differential DH and VH activation following exposure to a reminder of trauma [ 19 , 20 ]. The DH shows increased activity while encoding the information about the context [ 8 , 21 ]. At the same time, the VH is more active during the expression of the fear response [ 8 ]. The DH is involved in contextual memory, aiding the ability to remember the context in which an event occurs [ 4 ]. Further, the DH connects more extensively with cortical areas involved in cognitive and sensory processing. In contrast, the VH has more substantial connections with subcortical structures related to emotion and motivation [ 8 ]. However, the studies suggest that the VH is essential in gating contextual memory formation through an intra-hippocampal interaction [ 22 ]. The CA1 inputs of the VH convey contextual information to a subset of basolateral amygdala neurons, contributing to the encoding of adaptive fear memory for threat-predictive contexts [ 23 ]. However, despite these distinctions, the specific roles of the DH and VH in contextual fear memory, particularly their differential involvement in memory consolidation and the temporal dynamics of their activation following fear conditioning, remain poorly understood. In this study, we examined the activity-dependent temporal expression of Arc and c-Fos proteins in the DH and VH neurons following contextual fear conditioning at 0, 1, 3, and 5 hours on conditioning and post-conditioning days. Methods Two-month-old male Swiss albino mice (N = 120) were obtained from the University's Central Laboratory Animal Resource (CLAR) facility and housed in the institutional animal facility for one week before the start of the experiments. During this acclimatization period, the mice were maintained under standard laboratory conditions: a temperature-controlled environment (22–24°C), a 12-hour light/dark cycle (lights on at 7:00 AM), and access to food and water ad libitum . All experimental protocols were approved by the Institutional Animal Ethics Committee of Jawaharlal Nehru University, New Delhi (IAEC protocol #36/2018). The study's primary objective was to examine the impact of fear conditioning on the expression of Arc and c-Fos proteins. To this end, mice were randomly assigned to one of two groups: a fear-conditioned group (n = 60), which received foot shocks during the training session in the conditioning chamber, and an unconditioned control group (n = 60), which received no shocks. Contextual Fear Conditioning (CxFC) For CxFC, we followed the protocol published earlier [ 24 , 25 ]. The first two days (Days 1 and 2), the animal was habituated in the neutral chamber for 5 minutes (11:00 − 11:05 AM). On Day 3, the animal's spontaneous freezing behaviour (baseline) was recorded for 5 min through Freeze Frame software (Coulbourn Inc., USA) using a CCTV camera (Sen Tech, USA). We kept light illumination at 20 Lux in the neutral chamber during the habituation and baseline recording (Days 1 − 3). On Day 4, the animal was brought by an unfamiliar person using a different route from the animal colony, and then the animal was placed in the behavioural chamber. A few situational cues (illumination: 80 Lux in the training context and sandalwood fragrance soaked in bedding) were incorporated to differentiate between neutral and shock chambers. The husk bedding used during the fear-conditioning training was used again during the testing. For CxFC training, the animal was given three scrambled foot shocks (0.8mA; 2 s) at 1 min intervals over a 5 min period through the grid floor of the shock chamber using Freeze Frame software (Coulbourn Inc, USA; model# H13-17). For offline analysis, a CCTV camera was used to continuously record the freezing behaviour for 5 min (within a time-matched hour on baseline days). The animal was tested for CxFC on Day 5 in the same chamber with the same cues as the training day, but no foot shock was given. For the complete 5 minutes, the induced freezing was recorded. The changes in the freezing response on the baseline and post-conditioning days were compared statistically. Using the Freeze Frame software, we considered freezing if the animal was not moving at least for 2 sec bout with a fixed 10% threshold of motion index. The percentage of the total time spent freezing was calculated for each animal and compared statistically. The changes in percent freezing response between and within groups on the baseline, training, and testing days were compared statistically. Isolation of the dorsal and ventral hippocampus The fear-conditioned animals were randomly divided into two groups: (a) CxFC training (n = 36) and (b) CxFC testing (n = 24). Animals in both groups were sacrificed at 0, 1, 3, and 5 hours after CxFC training or testing, respectively. Unconditioned control animals (n = 60) were sacrificed at corresponding time points. Each animal was euthanized by cervical dislocation, and the brain was rapidly removed. The hippocampus was dissected between the septal (adjacent to the corpus callosum and dorsal thalamus) and temporal (closer to the amygdala and ventrolateral cortex) regions, to isolate the DH and VH, respectively [ 5 , 24 , 26 ]. The dissected DH and VH tissues were immediately flash-frozen in liquid nitrogen and stored at − 80°C for subsequent western blot analysis (Fig. 1 ). Western Blotting After contextual fear-conditioned training, the hippocampal tissues from 3 animals in each group (CxFC n = 36; control: 36) were pooled and dipped in the ice-cold lysis buffer [RIPA (3 ml/gm), in which phenylmethyl sulfonyl fluoride (PMSF; 1 mM) and protease inhibitor cocktail (PIC; 1:100)] was also added. However, after contextual fear-conditioned testing, the hippocampal tissues from 24 aniamls [(6 animals per groups in CxFC) and (6 animals per groups in control; n = 24)] were taken individually and dipped in the ice-cold lysis buffer [RIPA (3 ml/gm), along with phenylmethyl sulfonyl fluoride (PMSF; 1 mM) and protease inhibitor cocktail (PIC; 1:100)]. The tissue was incubated for 10 min and then homogenized on ice. Tissue lysate was then centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant was collected and stored at ˗80°C for further analysis. Electrophoresis was performed as per the standard protocol. In brief, the total protein content in each sample was quantified using the Bradford assay. Samples for the Western blot were prepared. An equal amount of protein (70 µg per well) was loaded and resolved in 10% SDS-PAGE (Bio-Rad western unit). Proteins were electroblotted onto the PVDF membrane ( Millipore) at 10V for 30 min (Trans Blot semidry, Bio-Rad). At room temperature, the membrane was blocked with 1% BSA in Tris-buffered saline (TBS) for 2 hours. After blocking, the membrane was washed with TBST (TBS with 0.1% Tween-20) and then incubated with primary antibody solution (in TBS) overnight at 4°C. After primary incubation, the membrane was washed four times with TBST and then incubated in the secondary antibody for 2 hours at room temperature. After washing, protein bands were visualized using the ChemiDoc MP (from Bio-Rad) using [Immobilon Forte western HRP substrate (Millipore)]. We used ImageJ software to quantify the intensity of protein bands. Primary antibodies used for western blot analysis were anti-cFos (1:1,000, Abcam, Cat # ab190289), anti-Arc (1:1,000, Sigma-Aldrich, Cat # SAB4200515), and anti-β-actin (1:2,500, Sigma-Aldrich, Cat # A5316). Secondary antibodies used for the western blot analysis were as follows: goat anti-mouse polyclonal HRP-tagged (1:10,000, Abcam, Cat # ab97040), goat anti-rabbit polyclonal HRP-tagged (1:10,000, Abcam, Cat # ab99702). The pre-stained protein ladder (250 kDa, Bio-Rad) was used to identify the desired protein bands. Data Analysis Contextual Fear Conditioning The % freezing responses were calculated using Freeze Frame software, in which we identified a 2-s bout with a 10% threshold of motion index as freezing. The software detected freezing if an animal remained motionless for 2 seconds or more. The percentage of total freezing was calculated in each animal and statistically analyzed using Sigma Plot 12.0 software (Systat, Inc., Chicago, IL, USA). The changes in the percent freezing response between the groups on the baseline, training, and testing days were compared statistically. The data was analyzed between groups using one-way analysis of variance (one-way ANOVA) followed by a Tukey post hoc test. We have also calculated the variance, effect size, and power values. Western Blot Analysis The densitometric analysis was performed in all Western blots using ImageJ software. The absolute intensity of Arc and c-Fos protein bands was normalized with their respective loading control ‘‘β-actin’’ bands in each gel. The normalized values were calculated by dividing the band intensity of each protein by the band intensity of its corresponding β-actin bands. The relative changes in the expression level of Arc and c-Fos proteins in the DH and VH were compared statistically between groups using one-way ANOVA followed by Tukey post hoc test. We further calculated the variance, effect size, and power values. Results The changes in percent freezing in contextual fear-conditioned animals The animals demonstrated a robust freezing response on the testing day, indicating strong fear memory retention (Fig. 2 ). The fear-conditioned animals exhibited a markedly 53.58% increased freezing response during the 5-minute testing period [p < 0.001; F (5, 138) = 327.14] on the testing day. Compared to the control group on the testing day, the CxFC animals exhibited a significant increase in freezing response post-hoc Tukey’s p < 0.001, with a large effect size Cohen’s d = 7.01 and power = 1.00 at α = 0.05. A similar trend was observed on the training day, where the experimental group showed a significantly greater freezing response (21%) compared to baseline controls. This difference was also significant (p < 0.001, F (5,138) = 327.14), with effect size Cohen’s d = 3.34 and statistical power 1.00 at α = 0.05, and Tukey’s post-hoc test p < 0.001 (Fig. 2 ). These findings suggest that the animals formed an association between the training context and the aversive stimulus. The changes in the expression level of Arc proteins in the DH and VH after CxFC Training Our analysis revealed a significant upregulation of Arc protein expression in the DH following contextual fear-conditioned training, as compared to unconditioned control animals (p < 0.001, F (7,16) = 11.32) (Fig. 3 ). In contrast, no statistically significant changes were observed in Arc expression within the VH after CxFC training, indicating a region-specific response to the conditioning paradigm (Fig. 3 ). Post-hoc Tukey’s multiple comparisons test further confirmed a significant increase in Arc protein levels in the DH at both 1 hour (p < 0.001) and 3 hours (p < 0.01) following CxFC training, when compared to the unconditioned control group. Additionally, within the fear-conditioned training groups, Arc expression at 1 hour (p < 0.01), 3 hours (p < 0.01), and 5 hours (p < 0.05) after training was significantly increased compared to the immediate post-conditioning time point (0 hour) (Fig. 3 ). However, Arc protein levels measured at 0 hours and 5 hours after CxFC training were not significantly different from those of the unconditioned controls, suggesting a transient peak in expression at earlier time points. To quantify the magnitude of this response, we also calculated the percent change in Arc protein levels relative to controls. We observed an increase of approximately 121.20% at 1 hour and 73.10% at 3 hours after CxFC training in the DH. In contrast, analysis of the VH revealed no significant changes in Arc protein expression across all time points examined. Expression levels remained comparable between conditioned and unconditioned groups, suggesting a selective involvement of the DH in the consolidation of contextual fear memory via Arc-mediated synaptic plasticity (Fig. 3 ). The changes in the expression level of Arc proteins in the DH and VH after CxFC Testing Similarly, the expression of Arc protein significantly increased in the DH after CxFC testing, relative to unconditioned control animals (p < 0.001, F (7,40) = 27.90). (Fig. 4 ). Conversely, no significant alterations were detected in the VH, indicating that the observed molecular response was region-specific. Post-hoc Tukey’s multiple comparisons further confirmed a robust increase in Arc levels in the DH at 1 hour (p < 0.001), 3 hours (p < 0.001), and 5 hours (p < 0.01) post-testing compared to unconditioned controls. Within the fear-conditioned testing groups, Arc expression at 1 hour (p < 0.01), 3 hours (p < 0.01), and 5 hours (p < 0.05) post-testing was significantly higher than at the immediate testing time point (0 hour). However, Arc expression at 0 hour post-testing did not differ significantly from that of the unconditioned animals, suggesting a transient peak in Arc activity at earlier post-test intervals (Fig. 4 ). Quantitative analysis revealed a substantial increase in Arc expression in the DH, approximately 87.69% at 1 hour, 55.97% at 3 hours, and 41.21% at 5 hours post-testing relative to controls (Fig. 4 ). In contrast, Arc expression in the VH remained unchanged across all examined time points. The results suggest that fear memory retrieval preferentially activates Arc-related plasticity in the dorsal, but not the ventral, hippocampus. The changes in the expression level of c-Fos proteins in the DH and VH after CxFC Training Our findings indicate a significant increase in c-Fos protein expression in the DH of animals subjected to contextual fear conditioning, compared to the unconditioned control group (p < 0.001, F (7,16) = 7.08) (Fig. 5 ). This suggests that fear conditioning induces neural activation in the DH. In contrast, no significant differences were observed in c-Fos expression in the VH between the conditioned and unconditioned groups, indicating a region-specific activation pattern. Post-hoc analysis using Tukey’s test revealed that c-Fos levels in the DH were significantly elevated at 1 hour (p < 0.01) and 3 hours (p < 0.05) after fear conditioning when compared to the unconditioned controls. However, no significant changes in c-Fos expression were observed at the immediate post-conditioning time point (0 hours) or at 5 hours post-conditioning, relative to controls (Fig. 5 ). Quantitative analysis of the percent change further supported these findings. c-Fos expression increased by 61.26% at 1 hour and 76.88% at 3 hours post-conditioning, relative to their respective unconditioned controls, highlighting the temporal dynamics of c-Fos activation in response to fear memory formation (Fig. 5 ). In the case of the VH, our results showed no significant alterations in c-Fos expression across all time points examined. The expression levels remained consistent between conditioned and unconditioned groups, suggesting that the VH is not prominently involved in c-Fos-mediated responses during contextual fear conditioning. The changes in the expression level of c-Fos proteins in the DH and VH after CxFC Testing We further observed that c-Fos protein expression in the DH significantly increased following the retrieval of contextual fear memory, as compared to the unconditioned control group (p < 0.001, F (7,40) = 16.10) (Fig. 6 ). This indicates that memory recall induces neural activation in the DH. In contrast, c-Fos expression levels in the VH did not show significant differences between the conditioned and unconditioned groups, suggesting that the VH may not be involved in memory retrieval processes (Fig. 6 ). Post-hoc Tukey’s analysis revealed that c-Fos levels in the DH were significantly higher at 1 hour (p < 0.001), 3 hours (p < 0.001), and 5 hours (p < 0.05) after the retrieval test compared to unconditioned controls. However, no significant differences were observed at 0 hour post-retrieval, indicating a transient activation window (Fig. 6 ). Further quantitative analysis showed an increase in c-Fos expression by approximately 81.74% at 1 hour, 53.57% at 3 hours, and 37.86% at 5 hours after testing, relative to the corresponding unconditioned controls. These results emphasize the temporally dynamic nature of c-Fos activation in the DH during fear memory recall (Fig. 6 ). In contrast, the VH showed no significant changes across any time point, reinforcing its limited involvement in c-Fos-mediated neuronal activation during the expression of contextual fear memory. Discussion In this study, we aimed to investigate the behavioral and molecular correlates of contextual fear memory formation as well as region- and time-specific expression of the immediate early genes (IEGs) Arc and c-Fos in the DH and VH. Behavioral analysis revealed a robust increase in freezing behavior in the fear-conditioned group on both the training and testing days, with freezing levels reaching 53.58% during the testing period. This substantial increase, supported by a large effect size (Cohen’s d = 7.01) and statistical power, indicates a strong association between the conditioned context and the aversive stimulus. The early increase in freezing behavior observed during the training session (21%) suggests that the animals began associating the context with the aversive stimulus during the acquisition phase. This is in agreement with earlier reports showing that contextual cues are rapidly encoded and can influence behavior even within the initial exposure session [ 13 , 27 , 28 ]. The consistent increase in freezing on training and testing days further supports the notion of successful consolidation of contextual fear memory. Collectively, the behavioral data confirm that the fear conditioning protocol employed in this study was effective in eliciting a reliable and quantifiable memory response. Differential Arc Expression in the Dorsal vs. Ventral Hippocampus In addition to the behavioral data, molecular analyses revealed dynamic and region-specific patterns of Arc protein expression in response to fear conditioning. Arc, a well-established marker of activity-dependent synaptic plasticity, was significantly upregulated in the DH but not in the VH. The highest expression levels were observed at 1st hour post-conditioning, with a remarkable 121.20% increase compared to controls. This was followed by a slightly reduced but significant increase of 73.10% at the 3rd hour. The results also demonstrate a region-specific molecular response, with a robust and transient increase in Arc expression in the DH but not in the VH following contextual fear conditioning testing. Arc levels in the DH showed a pronounced peak at 1 h post-testing (nearly 88% above controls) and remained significantly elevated at 3 h and 5 h, before declining, consistent with Arc’s role as a short-lived immediate-early gene supporting synaptic plasticity and memory consolidation. The absence of significant changes at 0 h suggests that Arc induction requires time-dependent transcriptional and translational processes initiated by memory retrieval. These findings align with previous studies demonstrating that Arc expression is rapidly induced in the hippocampus following learning and is involved in long-term potentiation (LTP), AMPA receptor trafficking, and structural synaptic changes essential for memory consolidation [ 29 , 30 ]. The peak of Arc expression at 1 hour post-conditioning (on training and testing days) suggests that this time point may represent a critical window for activity-dependent plastic changes underlying memory consolidation. Arc levels remained elevated at 3 h and 5 h on both training and testing days, and it is consistent with Arc’s well-established role as a short-lived immediate-early gene that translates neuronal activity into molecular changes underlying synaptic plasticity and memory consolidation. It further suggests that Arc expression is temporally restricted and remains elevated during the early phase of memory formation [ 31 , 32 ]. In contrast, no significant changes in Arc protein levels were detected in the VH at any time point following conditioning. This regional specificity further supports the functional dissociation between the DH and VH in processing contextual vs. emotional aspects of fear. While the DH is heavily implicated in spatial and contextual learning, the VH has been linked to affective regulation and anxiety-like behaviors [ 13 , 27 , 33 , 34 ]. The absence of Arc induction in the VH implies that the mechanisms supporting contextual fear memory are primarily localized to dorsal hippocampal circuits and do not recruit Arc-dependent plasticity in the ventral domain under these experimental conditions [ 1 ]. Conversely, the lack of Arc upregulation in the VH emphasizes the functional dissociation along the hippocampal axis, with the DH preferentially engaged in contextual memory retrieval. At the same time, the VH may contribute to fear processing via Arc-independent pathways. These findings highlight the temporal dynamics and spatial specificity of Arc-mediated plasticity during fear memory retrieval. c-Fos Expression Mirrors Activity-Dependent Neural Engagement Complementing the Arc data, c-Fos expression analyses provided additional insights into the neuronal activation patterns associated with contextual fear memory [ 6 ]. Similar to Arc, c-Fos protein levels were significantly elevated in the DH at 1 and 3 hours on the contextual fear-condition training day. These increases (61.26% and 76.88%, respectively) mirror the temporal pattern of Arc upregulation and suggest that these two IEGs may be co-regulated in response to fear conditioning. Upon retrieval of contextual fear memory elicits a robust yet transient induction of c-Fos expression specifically in the DH, with levels peaking sharply at 1 h and progressively declining thereafter. This temporal profile is consistent with c-Fos’s role as a rapidly activated immediate-early gene that marks neuronal activity associated with synaptic plasticity and memory processing. The lack of changes at 0 h indicates that a short delay is required for activity-dependent transcriptional activation. The gradual decline by 5 h suggests that c-Fos involvement is limited to the early phases of retrieval-induced plasticity. In contrast, the VH did not exhibit any significant modulation, reinforcing the idea that c-Fos-mediated activity is preferentially engaged in dorsal circuits that support contextual memory recall rather than ventral circuits linked more closely to affective regulation. c-Fos is widely used as a marker of neural activity due to its rapid and transient expression following synaptic stimulation [ 35 , 36 ]. The robust elevation observed here supports the involvement of DH neurons in processing and consolidating the fear memory trace. The temporal alignment between c-Fos and Arc upregulation in the DH highlights a coordinated molecular response that is likely essential for initiating downstream plasticity-related pathways. Notably, c-Fos expression did not increase at 0 hour post-conditioning, reinforcing the idea of a narrow time window during which neural circuits undergo maximal activation and plasticity-related gene expression. This is consistent with earlier studies showing that IEG expression peaks within 1–2 hours following behavioral stimulation and returns to baseline thereafter [ 37 , 38 ]. As with Arc, c-Fos expression in the VH remained unchanged across all time points and experimental groups. This again highlights the lack of ventral hippocampal involvement in c-Fos-mediated responses to contextual fear learning. The absence of activation in the VH supports the conclusion that contextual fear memory formation is predominantly mediated by the DH, which is more tightly connected to parahippocampal and cortical structures involved in spatial representation [ 39 ]. Region-Specific Specialization in the Hippocampus The present findings strongly support the notion of functional segregation along the longitudinal axis of the hippocampus, where the DH is preferentially engaged in processing contextual and cognitive components of memory, while the VH is more involved in regulating emotion and stress responses. This functional dichotomy is supported by anatomical studies showing distinct input-output connectivity patterns along the hippocampal axis [ 40 ]. The selective activation of Arc and c-Fos in the DH, but not VH, following contextual fear-conditioning suggesting a region-specific role of the DH and VH in the consolidation fear memory. These results are also consistent with lesion and pharmacological studies showing that disruption of DH activity impairs contextual fear conditioning. At the same time, manipulation of the VH more selectively affects anxiety-like behavior without compromising contextual learning [ 14 , 18 ]. Importantly, the current study uses protein-level analyses of IEGs, providing a molecular basis for the observed behavioral patterns and supporting the idea that synaptic plasticity and memory-related signaling are compartmentalized within hippocampal subregions. Implications for Memory Consolidation Mechanisms The synchronized behavioral and molecular responses observed in this study provide compelling evidence for the DH as a central hub for contextual fear memory encoding and consolidation. The time-dependent upregulation of Arc and c-Fos suggests these genes may operate in tandem to support activity-induced synaptic modifications required for memory storage. Arc is known to regulate AMPA receptor endocytosis and structural remodeling of dendritic spines, while c-Fos is involved in initiating transcriptional cascades that promote long-term cellular changes [ 41 ]. The overlap in their temporal expression profiles post-conditioning suggests a potential synergistic role in orchestrating the early molecular events that stabilize the fear memory trace. The absence of significant changes at 0 and 5 hours post-conditioning may reflect the closure of the molecular window for consolidation, consistent with models proposing that long-term memory formation requires tightly regulated gene expression within specific post-learning time frames. Limitations and Future Directions Despite the strengths of the current study, a few limitations warrant discussion. First, while Arc and c-Fos are widely accepted as markers of neuronal activation and plasticity, they are indirect measures and do not provide spatial resolution at the single-cell level. Future immunohistochemistry or in situ hybridization studies could better localize these proteins within specific hippocampal subfields (e.g., CA1, CA3, dentate gyrus) to further delineate circuit-specific changes. Second, investigating the long-term persistence of these molecular changes beyond the 5-hour window and their relevance to memory retrieval could offer insights into the mechanisms of memory maintenance versus formation. Our findings demonstrate that contextual fear conditioning elicits robust behavioral and molecular responses that are time-dependent and region-specific within the hippocampus. The observed molecular evidence supports the view that the DH as a key locus for encoding contextual fear memories through activity-dependent gene expression. They also offer a foundation for further exploration into the temporal dynamics of memory-related plasticity and the potential for region-specific therapeutic targeting in disorders such as PTSD. Future studies examining cellular specificity, sex differences, and long-term gene expression patterns will be essential to fully elucidate the mechanisms underlying fear memory formation and persistence. Declarations Conflict of Interest: The authors declare no competing interests. Both the authors The authors have no relevant financial or non-financial interests to disclose. Ethics approval: All experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of Jawaharlal Nehru University, New Delhi. IAEC protocol #36/2018; Dated: 21/12/2018. This study was performed in line with the principles of the Declaration of Helsinki. Funding: This work was supported by grants from the Department of Science and Technology-CSRI Grant Numbers: DST-CSRI 2021/136 (G); DST-CSRI/39/2016(G)] funded to Sushil K Jha. We also acknowledge the funding from SERB, DBT-BUILDER, and the Department of Science and Technology-PURSE. Author Contribution YK performed the experiments and analyzed the data, SJ conceived and conceptualized the ideas, designed the experiments, analyzed the data, and finalized the manuscript. 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Front Neurol Neurosci 34:85-94 Ritov G, Ardi Z, Richter-Levin G (2014) Differential activation of amygdala, dorsal and ventral hippocampus following an exposure to a reminder of underwater trauma. Front Behav Neurosci 8:18 Matus-Amat P, Higgins EA, Barrientos RM, Rudy JW (2004) The role of the dorsal hippocampus in the acquisition and retrieval of context memory representations. J Neurosci 24:2431-2439 Fredes F, Silva MA, Koppensteiner P, Kobayashi K, Joesch M, Shigemoto R (2021) Ventro-dorsal Hippocampal Pathway Gates Novelty-Induced Contextual Memory Formation. Current Biology 31:25-38.e25 Kim WB, Cho JH (2020) Encoding of contextual fear memory in hippocampal-amygdala circuit. Nat Commun 11:1382 Kant D, Jha SK (2019) The formation of compensatory contextual fear memory in the absence of dorsal hippocampus does not change sleep architecture. Behav Brain Res 370:111944 Kumar T, Jha SK (2017) Influence of cued-fear conditioning and its impairment on NREM sleep. Neurobiol Learn Mem 144:155-165 Czerniawski J, Ree F, Chia C, Otto T (2012) Dorsal versus ventral hippocampal contributions to trace and contextual conditioning: Differential effects of regionally selective nmda receptor antagonism on acquisition and expression. Hippocampus 22:1528-1539 Jha VM, Jha SK (2020) Sleep: neural optimization as an ultimate function for memory consolidation. In: Sleep: evolution and functions. Springer Singapore Singapore, pp 79-99 Maren S, Hobin JA (2007) Hippocampal regulation of context-dependent neuronal activity in the lateral amygdala. Learn Mem 14:318-324 Shepherd JD, Bear MF (2011) New views of Arc, a master regulator of synaptic plasticity. Nature neuroscience 14:279-284 Plath N, Ohana O, Dammermann B, Errington ML, Schmitz D, Gross C, Mao X, Engelsberg A, Mahlke C, Welzl H, Kobalz U, Stawrakakis A, Fernandez E, Waltereit R, Bick-Sander A, Therstappen E, Cooke SF, Blanquet V, Wurst W, Salmen B, Bösl MR, Lipp HP, Grant SG, Bliss TV, Wolfer DP, Kuhl D (2006) Arc/Arg3.1 is essential for the consolidation of synaptic plasticity and memories. Neuron 52:437-444 Guzowski JF, McNaughton BL, Barnes CA, Worley PF (1999) Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles. Nature neuroscience 2:1120-1124 Bramham CR, Worley PF, Moore MJ, Guzowski JF (2008) The immediate early gene arc/arg3.1: regulation, mechanisms, and function. J Neurosci 28:11760-11767 Jha SK, Jha VM, Jha VM (2019) Sleep, memory and synaptic plasticity. In. Springer Singapore, Jha VM, Jha SK (2020) Sleep: evolution and functions. In. Springer Singapore Singapore:, Mir FA, Jha SK (2021) Locus coeruleus acid-sensing ion channels modulate sleep–wakefulness and state transition from NREM to REM sleep in the rat. Neuroscience Bulletin 37:684-700 Mir FA, Jha SK (2021) Proton pump inhibitor “Lansoprazole” inhibits locus coeruleus’s neuronal activity and increases rapid eye movement sleep. ACS Chemical Neuroscience 12:4265-4274 Radulovic J, Kammermeier J, Spiess J (1998) Relationship between Fos Production and Classical Fear Conditioning: Effects of Novelty, Latent Inhibition, and Unconditioned Stimulus Preexposure. The Journal of Neuroscience 18:7452-7461 Lonergan ME, Gafford GM, Jarome TJ, Helmstetter FJ (2010) Time-dependent expression of Arc and zif268 after acquisition of fear conditioning. Neural Plast 2010:139891 Ranganath C, Ritchey M (2012) Two cortical systems for memory-guided behaviour. Nat Rev Neurosci 13:713-726 Strange BA, Witter MP, Lein ES, Moser EI (2014) Functional organization of the hippocampal longitudinal axis. Nature Reviews Neuroscience 15:655-669 Minatohara K, Akiyoshi M, Okuno H (2015) Role of Immediate-Early Genes in Synaptic Plasticity and Neuronal Ensembles Underlying the Memory Trace. Front Mol Neurosci 8:78 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Feb, 2026 Read the published version in Neurochemical Research → Version 1 posted Editorial decision: Revision requested 22 Sep, 2025 Editor assigned by journal 22 Sep, 2025 Submission checks completed at journal 19 Sep, 2025 First submitted to journal 19 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7658753","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":518985272,"identity":"23bb48fb-62a8-4b12-913a-94ffab02cd73","order_by":0,"name":"Yogendra Kumar","email":"","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":false,"prefix":"","firstName":"Yogendra","middleName":"","lastName":"Kumar","suffix":""},{"id":518985274,"identity":"b842dd6f-110e-4f82-8ba8-5568d95e4bbb","order_by":1,"name":"Sushil K. Jha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYHACNoaEAgYeNmbGxgcMDAeI1WLAIMPP3nzYgHgtDAYMNpI9x9IkiNIiPyP92YMHBgw8BjdyzKp5au7I8TMwP3x0A48WoEpzgwSolts8x54ZSzawGRvn4NMikcMmgdDCdjhxwwEeNml8WkAOg2sp5vlHhBaGGwlmYC0g7zPzthGhxeDMG4gWUCBLzu07bCzZTMAv8u3pzyR/VDDYg6Lyw5tvh+WAeh8+xuswCPgPJpl4QCQzYeUIwPiDFNWjYBSMglEwYgAAp6BIbuKDr0UAAAAASUVORK5CYII=","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":true,"prefix":"","firstName":"Sushil","middleName":"K.","lastName":"Jha","suffix":""}],"badges":[],"createdAt":"2025-09-19 12:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7658753/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7658753/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11064-026-04683-0","type":"published","date":"2026-02-09T15:57:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93583093,"identity":"79057c90-6a25-4b57-b678-578d5a23e9d4","added_by":"auto","created_at":"2025-10-15 10:52:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2868738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design for contextual fear conditioning (CxFC) and molecular analysis. Rats were habituated in the conditioning chamber for two days (Hab 1 \u0026amp; 2), followed by spontaneous behaviour recording as baseline (Bsl) on Day 3. On Day 4, animals were divided into two groups: Control (exposed to the training context without foot shocks) and CxFC (exposed to the training context paired with foot shocks). On Day 5, all animals were re-exposed to the context for memory retrieval testing. Brain tissues were collected at four different test times on training and testing days (0Hr, 1Hr, 3Hr, and 5Hr) for protein analysis. Both dorsal hippocampus (DH) and ventral hippocampus (VH) were dissected to assess region-specific molecular responses (Arc and c-Fos expression).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7658753/v1/0101f8ffa49d9f4b3e2a8fbd.jpg"},{"id":93583096,"identity":"8129bd14-39c4-4d2f-8730-717a9fcc8d9b","added_by":"auto","created_at":"2025-10-15 10:52:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":192209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFreezing behavior during contextual fear conditioning (CxFC). Percentage freezing (mean ± SEM) was measured in control and CxFC animals across three days: baseline, training, and testing days. No significant freezing was observed during baseline in either group. During training, CxFC animals exhibited a significant increase in freezing compared to controls (***p \u0026lt; 0.001). At the testing phase, CxFC animals showed a robust increase in freezing relative to controls (***p \u0026lt; 0.001), confirming successful fear memory retrieval. \u003c/strong\u003eOne-way ANOVA followed by Tukey’s post-hoc test was used for the statistical significance.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7658753/v1/64654940f5bacfda29580165.jpg"},{"id":93584411,"identity":"1a2a1bdf-a3bc-477f-a5f9-00360a033bab","added_by":"auto","created_at":"2025-10-15 11:08:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":761868,"visible":true,"origin":"","legend":"\u003cp\u003eArc protein expression in the dorsal and ventral hippocampus following contextual fear conditioning training. [a] Representative Western blot images showing Arc protein levels in the dorsal hippocampus (DH) and ventral hippocampus (VH) at 0Hr, 1Hr, 3Hr, and 5Hr post-conditioning compared to unconditioned controls and CxFC animals; β-actin served as a loading control. [b] Quantification of Arc protein expression (percent change relative to control) reveals a significant increase in the DH at 1Hr (↑121.2%) and 3Hr (↑73.1%) post-conditioning (p \u0026lt; 0.001 and p \u0026lt; 0.05, respectively). [c] \u0026nbsp;No significant changes were observed in VH across all time points. Data are shown as mean ± SEM; one-way ANOVA followed by Tukey’s post-hoc test was used for statistical significance \u003cstrong\u003e(**= p\u0026lt;0.01 and *** = p \u0026lt; 0.001 represents Tukey’s p).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7658753/v1/097c49ec0603267f2c905a21.jpg"},{"id":93583092,"identity":"744b59b8-df43-40a9-b866-9573392a7e0b","added_by":"auto","created_at":"2025-10-15 10:52:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":705197,"visible":true,"origin":"","legend":"\u003cp\u003eArc protein expression in the dorsal hippocampus (DH) and ventral hippocampus (VH) following contextual fear conditioning (CxFC) testing. [a] Representative Western blot images showing Arc protein expression in DH and VH at 0Hr, 1Hr, 3Hr, and 5Hr post-retrieval in control and CxFC animals; β-actin served as a loading control. [b] Quantitative analysis of Arc protein expression in the DH revealed a significant increase at 1Hr (↑87.69) (***p \u0026lt; 0.001), 3Hr (↑55.97%) (***p \u0026lt; 0.001), and 5Hr (↑41.21%) (**p \u0026lt; 0.01) post-retrieval compared to controls, indicating a transient but robust induction of Arc following memory recall. [c] In contrast, Arc expression in the VH did not differ significantly between CxFC and control groups across any time point, suggesting a region-specific molecular response. Data are presented as mean ± SEM; one-way ANOVA followed by Tukey’s post-hoc test was used for statistical significance \u003cstrong\u003e(**= p\u0026lt;0.01 and *** = p \u0026lt; 0.001 represents Tukey’s p).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7658753/v1/820e5254fc99234d53a34816.jpg"},{"id":93583097,"identity":"cba60b7d-fef1-4ad9-aa81-a6d8a5df07cd","added_by":"auto","created_at":"2025-10-15 10:52:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":710521,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;c-Fos protein expression in the dorsal hippocampus (DH) and ventral hippocampus (VH) following contextual fear conditioning (CxFC) training. [a] Representative Western blot images showing c-Fos expression in DH and VH at 0Hr, 1Hr, 3Hr, and 5Hr post-retrieval in control and CxFC animals; β-actin served as a loading control. [b] Quantitative analysis of c-Fos protein in the DH revealed a significant increase at 1Hr (↑61.26%) (**p \u0026lt; 0.01) and 3Hr (↑76.88%) (*p \u0026lt; 0.05) post-retrieval in CxFC animals compared to controls, indicating activity-dependent induction of c-Fos during memory recall. [c] In contrast, c-Fos expression in the VH did not differ significantly between CxFC and control groups at any time point, suggesting a region-specific regulation of this immediate-early gene. Data are presented as mean ± SEM; one-way ANOVA followed by Tukey’s post-hoc test \u003cstrong\u003e(**= p\u0026lt;0.01 and * = p \u0026lt; 0.05 represents Tukey’s p).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7658753/v1/a22bef1ae0f3448020086c98.jpg"},{"id":93583970,"identity":"1dbea0a9-dc5c-448a-8171-493f51bddd45","added_by":"auto","created_at":"2025-10-15 11:00:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":698659,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in c-Fos protein expression in the dorsal hippocampus (DH) and ventral hippocampus (VH) following contextual fear conditioning (CxFC) testing. [a] Representative Western blot images showing differential expression of c-Fos in the DH and VH at 0Hr, 1Hr, 3Hr, and 5Hr post-retrieval in control and CxFC groups, with β-actin used as a loading control. [b] Quantification of c-Fos expression in the DH revealed a robust increase at 1Hr (↑88.74%) (***p \u0026lt; 0.001), 3Hr (↑53.57%) (***p \u0026lt; 0.001), and 5Hr (↑37.86%) (*p \u0026lt; 0.05) after retrieval in CxFC animals compared to controls, indicating activity-dependent recruitment of the dorsal hippocampus during memory recall. [c] In contrast, c-Fos levels in the VH showed no significant differences between CxFC and control animals across all time points, suggesting a region-specific regulation of c-Fos. Data are expressed as normalized intensity (mean ± SEM). One-way ANOVA followed by Tukey’s post-hoc test was used for the statistical significance \u003cstrong\u003e(***= p\u0026lt;0.001 and * = p \u0026lt; 0.05 represents Tukey’s p).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7658753/v1/c35bf23835c7958e72dc4f06.jpg"},{"id":102786221,"identity":"99b048ac-a801-4302-85a7-e1d643eb0254","added_by":"auto","created_at":"2026-02-16 16:12:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7151018,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7658753/v1/fe1c20ba-fbca-481e-aa98-ea9f75a29a00.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Contextual Fear Conditioning Induces Activity-Dependent Gene Expression in the Dorsal but not in the Ventral Hippocampus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe hippocampus, a critical brain structure involved in learning and memory, is divided into two principal regions: the dorsal hippocampus (DH) and the ventral hippocampus (VH) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although part of the same structure, the DH and VH regions have discrete anatomical connections and contribute differently to cognitive functions [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For example, the DH is primarily associated with spatial memory [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It helps in forming and retrieving spatial maps, which are crucial for navigation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Also, the DH contains a high density of place neurons essential for encoding and recalling spatial information [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The DH supports the ability to navigate through an environment by integrating sensory inputs and forming cognitive maps, which are mental representations of physical spaces [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. On the other hand, the VH is more involved in processing emotional aspects of memory through its close interaction with the amygdala [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition, the VH plays an essential role in regulating the body\u0026rsquo;s response to stress through its interaction with the hypothalamic-pituitary-adrenal (HPA) axis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It has been observed that lesions in the DH typically impair spatial memory and navigation, whereas lesions in the VH affect emotional memory and anxiety-related behaviors [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Although the DH and VH work together to integrate spatial, contextual, and emotional information, it seems that the DH and VH provide a comprehensive but independent framework for learning and memory [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe DH and VH possibly play a differential role in contextual fear conditioning [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The DH lesion impairs the ability to form and recall the spatial context of the fear conditioning, leading to a reduced fear response when re-exposed to the context [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In contrast, the VH lesion affects the emotional and stress-related components, altering the intensity of the fear response [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Functional imaging studies show differential DH and VH activation following exposure to a reminder of trauma [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The DH shows increased activity while encoding the information about the context [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. At the same time, the VH is more active during the expression of the fear response [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The DH is involved in contextual memory, aiding the ability to remember the context in which an event occurs [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Further, the DH connects more extensively with cortical areas involved in cognitive and sensory processing. In contrast, the VH has more substantial connections with subcortical structures related to emotion and motivation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the studies suggest that the VH is essential in gating contextual memory formation through an intra-hippocampal interaction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The CA1 inputs of the VH convey contextual information to a subset of basolateral amygdala neurons, contributing to the encoding of adaptive fear memory for threat-predictive contexts [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, despite these distinctions, the specific roles of the DH and VH in contextual fear memory, particularly their differential involvement in memory consolidation and the temporal dynamics of their activation following fear conditioning, remain poorly understood. In this study, we examined the activity-dependent temporal expression of Arc and c-Fos proteins in the DH and VH neurons following contextual fear conditioning at 0, 1, 3, and 5 hours on conditioning and post-conditioning days.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eTwo-month-old male Swiss albino mice (N\u0026thinsp;=\u0026thinsp;120) were obtained from the University's Central Laboratory Animal Resource (CLAR) facility and housed in the institutional animal facility for one week before the start of the experiments. During this acclimatization period, the mice were maintained under standard laboratory conditions: a temperature-controlled environment (22\u0026ndash;24\u0026deg;C), a 12-hour light/dark cycle (lights on at 7:00 AM), and access to food and water \u003cem\u003ead libitum\u003c/em\u003e. All experimental protocols were approved by the Institutional Animal Ethics Committee of Jawaharlal Nehru University, New Delhi (IAEC protocol #36/2018). The study's primary objective was to examine the impact of fear conditioning on the expression of Arc and c-Fos proteins. To this end, mice were randomly assigned to one of two groups: a fear-conditioned group (n\u0026thinsp;=\u0026thinsp;60), which received foot shocks during the training session in the conditioning chamber, and an unconditioned control group (n\u0026thinsp;=\u0026thinsp;60), which received no shocks.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eContextual Fear Conditioning (CxFC)\u003c/h2\u003e\u003cp\u003eFor CxFC, we followed the protocol published earlier [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The first two days (Days 1 and 2), the animal was habituated in the neutral chamber for 5 minutes (11:00\u0026thinsp;\u0026minus;\u0026thinsp;11:05 AM). On Day 3, the animal's spontaneous freezing behaviour (baseline) was recorded for 5 min through Freeze Frame software (Coulbourn Inc., USA) using a CCTV camera (Sen Tech, USA). We kept light illumination at 20 Lux in the neutral chamber during the habituation and baseline recording (Days 1\u0026thinsp;\u0026minus;\u0026thinsp;3). On Day 4, the animal was brought by an unfamiliar person using a different route from the animal colony, and then the animal was placed in the behavioural chamber. A few situational cues (illumination: 80 Lux in the training context and sandalwood fragrance soaked in bedding) were incorporated to differentiate between neutral and shock chambers. The husk bedding used during the fear-conditioning training was used again during the testing. For CxFC training, the animal was given three scrambled foot shocks (0.8mA; 2 s) at 1 min intervals over a 5 min period through the grid floor of the shock chamber using Freeze Frame software (Coulbourn Inc, USA; model# H13-17). For offline analysis, a CCTV camera was used to continuously record the freezing behaviour for 5 min (within a time-matched hour on baseline days). The animal was tested for CxFC on Day 5 in the same chamber with the same cues as the training day, but no foot shock was given. For the complete 5 minutes, the induced freezing was recorded. The changes in the freezing response on the baseline and post-conditioning days were compared statistically. Using the Freeze Frame software, we considered freezing if the animal was not moving at least for 2 sec bout with a fixed 10% threshold of motion index. The percentage of the total time spent freezing was calculated for each animal and compared statistically. The changes in percent freezing response between and within groups on the baseline, training, and testing days were compared statistically.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIsolation of the dorsal and ventral hippocampus\u003c/h3\u003e\n\u003cp\u003eThe fear-conditioned animals were randomly divided into two groups: (a) CxFC training (n\u0026thinsp;=\u0026thinsp;36) and (b) CxFC testing (n\u0026thinsp;=\u0026thinsp;24). Animals in both groups were sacrificed at 0, 1, 3, and 5 hours after CxFC training or testing, respectively. Unconditioned control animals (n\u0026thinsp;=\u0026thinsp;60) were sacrificed at corresponding time points. Each animal was euthanized by cervical dislocation, and the brain was rapidly removed. The hippocampus was dissected between the septal (adjacent to the corpus callosum and dorsal thalamus) and temporal (closer to the amygdala and ventrolateral cortex) regions, to isolate the DH and VH, respectively [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The dissected DH and VH tissues were immediately flash-frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cp\u003eAfter contextual fear-conditioned training, the hippocampal tissues from 3 animals in each group (CxFC n\u0026thinsp;=\u0026thinsp;36; control: 36) were pooled and dipped in the ice-cold lysis buffer [RIPA (3 ml/gm), in which phenylmethyl sulfonyl fluoride (PMSF; 1 mM) and protease inhibitor cocktail (PIC; 1:100)] was also added. However, after contextual fear-conditioned testing, the hippocampal tissues from 24 aniamls [(6 animals per groups in CxFC) and (6 animals per groups in control; n\u0026thinsp;=\u0026thinsp;24)] were taken individually and dipped in the ice-cold lysis buffer [RIPA (3 ml/gm), along with phenylmethyl sulfonyl fluoride (PMSF; 1 mM) and protease inhibitor cocktail (PIC; 1:100)]. The tissue was incubated for 10 min and then homogenized on ice. Tissue lysate was then centrifuged at 10,000 rpm for 30 min at 4\u0026deg;C. The supernatant was collected and stored at ˗80\u0026deg;C for further analysis. Electrophoresis was performed as per the standard protocol. In brief, the total protein content in each sample was quantified using the Bradford assay. Samples for the Western blot were prepared. An equal amount of protein (70 \u0026micro;g per well) was loaded and resolved in 10% SDS-PAGE (Bio-Rad western unit). Proteins were electroblotted onto the PVDF membrane ( Millipore) at 10V for 30 min (Trans Blot semidry, Bio-Rad). At room temperature, the membrane was blocked with 1% BSA in Tris-buffered saline (TBS) for 2 hours. After blocking, the membrane was washed with TBST (TBS with 0.1% Tween-20) and then incubated with primary antibody solution (in TBS) overnight at 4\u0026deg;C. After primary incubation, the membrane was washed four times with TBST and then incubated in the secondary antibody for 2 hours at room temperature. After washing, protein bands were visualized using the ChemiDoc MP (from Bio-Rad) using [Immobilon Forte western HRP substrate (Millipore)]. We used ImageJ software to quantify the intensity of protein bands. Primary antibodies used for western blot analysis were anti-cFos (1:1,000, Abcam, Cat # ab190289), anti-Arc (1:1,000, Sigma-Aldrich, Cat # SAB4200515), and anti-β-actin (1:2,500, Sigma-Aldrich, Cat # A5316). Secondary antibodies used for the western blot analysis were as follows: goat anti-mouse polyclonal HRP-tagged (1:10,000, Abcam, Cat # ab97040), goat anti-rabbit polyclonal HRP-tagged (1:10,000, Abcam, Cat # ab99702). The pre-stained protein ladder (250 kDa, Bio-Rad) was used to identify the desired protein bands.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003eContextual Fear Conditioning\u003c/h2\u003e\u003cp\u003eThe % freezing responses were calculated using Freeze Frame software, in which we identified a 2-s bout with a 10% threshold of motion index as freezing. The software detected freezing if an animal remained motionless for 2 seconds or more. The percentage of total freezing was calculated in each animal and statistically analyzed using Sigma Plot 12.0 software (Systat, Inc., Chicago, IL, USA). The changes in the percent freezing response between the groups on the baseline, training, and testing days were compared statistically. The data was analyzed between groups using one-way analysis of variance (one-way ANOVA) followed by a Tukey post hoc test. We have also calculated the variance, effect size, and power values.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e\u003cp\u003eThe densitometric analysis was performed in all Western blots using ImageJ software. The absolute intensity of Arc and c-Fos protein bands was normalized with their respective loading control \u0026lsquo;\u0026lsquo;β-actin\u0026rsquo;\u0026rsquo; bands in each gel. The normalized values were calculated by dividing the band intensity of each protein by the band intensity of its corresponding β-actin bands. The relative changes in the expression level of Arc and c-Fos proteins in the DH and VH were compared statistically between groups using one-way ANOVA followed by Tukey post hoc test. We further calculated the variance, effect size, and power values.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eThe changes in percent freezing in contextual fear-conditioned animals\u003c/h2\u003e\u003cp\u003eThe animals demonstrated a robust freezing response on the testing day, indicating strong fear memory retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The fear-conditioned animals exhibited a markedly 53.58% increased freezing response during the 5-minute testing period [p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; F\u003csub\u003e(5, 138)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;327.14] on the testing day. Compared to the control group on the testing day, the CxFC animals exhibited a significant increase in freezing response post-hoc Tukey\u0026rsquo;s p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, with a large effect size Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;7.01 and power\u0026thinsp;=\u0026thinsp;1.00 at α\u0026thinsp;=\u0026thinsp;0.05. A similar trend was observed on the training day, where the experimental group showed a significantly greater freezing response (21%) compared to baseline controls. This difference was also significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, F\u003csub\u003e(5,138)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;327.14), with effect size Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;3.34 and statistical power 1.00 at α\u0026thinsp;=\u0026thinsp;0.05, and Tukey\u0026rsquo;s post-hoc test p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings suggest that the animals formed an association between the training context and the aversive stimulus.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eThe changes in the expression level of Arc proteins in the DH and VH after CxFC Training\u003c/span\u003e\u003c/p\u003e\u003cp\u003eOur analysis revealed a significant upregulation of Arc protein expression in the DH following contextual fear-conditioned training, as compared to unconditioned control animals (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, F\u003csub\u003e(7,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.32) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, no statistically significant changes were observed in Arc expression within the VH after CxFC training, indicating a region-specific response to the conditioning paradigm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePost-hoc Tukey\u0026rsquo;s multiple comparisons test further confirmed a significant increase in Arc protein levels in the DH at both 1 hour (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 3 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) following CxFC training, when compared to the unconditioned control group. Additionally, within the fear-conditioned training groups, Arc expression at 1 hour (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 3 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and 5 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after training was significantly increased compared to the immediate post-conditioning time point (0 hour) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, Arc protein levels measured at 0 hours and 5 hours after CxFC training were not significantly different from those of the unconditioned controls, suggesting a transient peak in expression at earlier time points.\u003c/p\u003e\u003cp\u003eTo quantify the magnitude of this response, we also calculated the percent change in Arc protein levels relative to controls. We observed an increase of approximately 121.20% at 1 hour and 73.10% at 3 hours after CxFC training in the DH. In contrast, analysis of the VH revealed no significant changes in Arc protein expression across all time points examined. Expression levels remained comparable between conditioned and unconditioned groups, suggesting a selective involvement of the DH in the consolidation of contextual fear memory via Arc-mediated synaptic plasticity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eThe changes in the expression level of Arc proteins in the DH and VH after CxFC Testing\u003c/span\u003e\u003c/p\u003e\u003cp\u003eSimilarly, the expression of Arc protein significantly increased in the DH after CxFC testing, relative to unconditioned control animals (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, F\u003csub\u003e(7,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;27.90). (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Conversely, no significant alterations were detected in the VH, indicating that the observed molecular response was region-specific. Post-hoc Tukey\u0026rsquo;s multiple comparisons further confirmed a robust increase in Arc levels in the DH at 1 hour (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 3 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 5 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) post-testing compared to unconditioned controls. Within the fear-conditioned testing groups, Arc expression at 1 hour (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 3 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and 5 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) post-testing was significantly higher than at the immediate testing time point (0 hour). However, Arc expression at 0 hour post-testing did not differ significantly from that of the unconditioned animals, suggesting a transient peak in Arc activity at earlier post-test intervals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eQuantitative analysis revealed a substantial increase in Arc expression in the DH, approximately 87.69% at 1 hour, 55.97% at 3 hours, and 41.21% at 5 hours post-testing relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, Arc expression in the VH remained unchanged across all examined time points. The results suggest that fear memory retrieval preferentially activates Arc-related plasticity in the dorsal, but not the ventral, hippocampus.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eThe changes in the expression level of c-Fos proteins in the DH and VH after CxFC Training\u003c/span\u003e\u003c/p\u003e\u003cp\u003eOur findings indicate a significant increase in c-Fos protein expression in the DH of animals subjected to contextual fear conditioning, compared to the unconditioned control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, F\u003csub\u003e(7,16)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.08) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This suggests that fear conditioning induces neural activation in the DH. In contrast, no significant differences were observed in c-Fos expression in the VH between the conditioned and unconditioned groups, indicating a region-specific activation pattern. Post-hoc analysis using Tukey\u0026rsquo;s test revealed that c-Fos levels in the DH were significantly elevated at 1 hour (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 3 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after fear conditioning when compared to the unconditioned controls. However, no significant changes in c-Fos expression were observed at the immediate post-conditioning time point (0 hours) or at 5 hours post-conditioning, relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eQuantitative analysis of the percent change further supported these findings. c-Fos expression increased by 61.26% at 1 hour and 76.88% at 3 hours post-conditioning, relative to their respective unconditioned controls, highlighting the temporal dynamics of c-Fos activation in response to fear memory formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the case of the VH, our results showed no significant alterations in c-Fos expression across all time points examined. The expression levels remained consistent between conditioned and unconditioned groups, suggesting that the VH is not prominently involved in c-Fos-mediated responses during contextual fear conditioning.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eThe changes in the expression level of c-Fos proteins in the DH and VH after CxFC Testing\u003c/span\u003e\u003c/p\u003e\u003cp\u003eWe further observed that c-Fos protein expression in the DH significantly increased following the retrieval of contextual fear memory, as compared to the unconditioned control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, F\u003csub\u003e(7,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;16.10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This indicates that memory recall induces neural activation in the DH. In contrast, c-Fos expression levels in the VH did not show significant differences between the conditioned and unconditioned groups, suggesting that the VH may not be involved in memory retrieval processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePost-hoc Tukey\u0026rsquo;s analysis revealed that c-Fos levels in the DH were significantly higher at 1 hour (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 3 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 5 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after the retrieval test compared to unconditioned controls. However, no significant differences were observed at 0 hour post-retrieval, indicating a transient activation window (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Further quantitative analysis showed an increase in c-Fos expression by approximately 81.74% at 1 hour, 53.57% at 3 hours, and 37.86% at 5 hours after testing, relative to the corresponding unconditioned controls. These results emphasize the temporally dynamic nature of c-Fos activation in the DH during fear memory recall (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast, the VH showed no significant changes across any time point, reinforcing its limited involvement in c-Fos-mediated neuronal activation during the expression of contextual fear memory.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we aimed to investigate the behavioral and molecular correlates of contextual fear memory formation as well as region- and time-specific expression of the immediate early genes (IEGs) Arc and c-Fos in the DH and VH. Behavioral analysis revealed a robust increase in freezing behavior in the fear-conditioned group on both the training and testing days, with freezing levels reaching 53.58% during the testing period. This substantial increase, supported by a large effect size (Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;7.01) and statistical power, indicates a strong association between the conditioned context and the aversive stimulus.\u003c/p\u003e\u003cp\u003eThe early increase in freezing behavior observed during the training session (21%) suggests that the animals began associating the context with the aversive stimulus during the acquisition phase. This is in agreement with earlier reports showing that contextual cues are rapidly encoded and can influence behavior even within the initial exposure session [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The consistent increase in freezing on training and testing days further supports the notion of successful consolidation of contextual fear memory. Collectively, the behavioral data confirm that the fear conditioning protocol employed in this study was effective in eliciting a reliable and quantifiable memory response.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDifferential Arc Expression in the Dorsal vs. Ventral Hippocampus\u003c/h2\u003e\u003cp\u003eIn addition to the behavioral data, molecular analyses revealed dynamic and region-specific patterns of Arc protein expression in response to fear conditioning. Arc, a well-established marker of activity-dependent synaptic plasticity, was significantly upregulated in the DH but not in the VH. The highest expression levels were observed at 1st hour post-conditioning, with a remarkable 121.20% increase compared to controls. This was followed by a slightly reduced but significant increase of 73.10% at the 3rd hour. The results also demonstrate a region-specific molecular response, with a robust and transient increase in Arc expression in the DH but not in the VH following contextual fear conditioning testing. Arc levels in the DH showed a pronounced peak at 1 h post-testing (nearly 88% above controls) and remained significantly elevated at 3 h and 5 h, before declining, consistent with Arc\u0026rsquo;s role as a short-lived immediate-early gene supporting synaptic plasticity and memory consolidation. The absence of significant changes at 0 h suggests that Arc induction requires time-dependent transcriptional and translational processes initiated by memory retrieval. These findings align with previous studies demonstrating that Arc expression is rapidly induced in the hippocampus following learning and is involved in long-term potentiation (LTP), AMPA receptor trafficking, and structural synaptic changes essential for memory consolidation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe peak of Arc expression at 1 hour post-conditioning (on training and testing days) suggests that this time point may represent a critical window for activity-dependent plastic changes underlying memory consolidation. Arc levels remained elevated at 3 h and 5 h on both training and testing days, and it is consistent with Arc\u0026rsquo;s well-established role as a short-lived immediate-early gene that translates neuronal activity into molecular changes underlying synaptic plasticity and memory consolidation. It further suggests that Arc expression is temporally restricted and remains elevated during the early phase of memory formation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast, no significant changes in Arc protein levels were detected in the VH at any time point following conditioning. This regional specificity further supports the functional dissociation between the DH and VH in processing contextual vs. emotional aspects of fear. While the DH is heavily implicated in spatial and contextual learning, the VH has been linked to affective regulation and anxiety-like behaviors [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The absence of Arc induction in the VH implies that the mechanisms supporting contextual fear memory are primarily localized to dorsal hippocampal circuits and do not recruit Arc-dependent plasticity in the ventral domain under these experimental conditions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Conversely, the lack of Arc upregulation in the VH emphasizes the functional dissociation along the hippocampal axis, with the DH preferentially engaged in contextual memory retrieval. At the same time, the VH may contribute to fear processing via Arc-independent pathways. These findings highlight the temporal dynamics and spatial specificity of Arc-mediated plasticity during fear memory retrieval.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ec-Fos Expression Mirrors Activity-Dependent Neural Engagement\u003c/h2\u003e\u003cp\u003eComplementing the Arc data, c-Fos expression analyses provided additional insights into the neuronal activation patterns associated with contextual fear memory [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Similar to Arc, c-Fos protein levels were significantly elevated in the DH at 1 and 3 hours on the contextual fear-condition training day. These increases (61.26% and 76.88%, respectively) mirror the temporal pattern of Arc upregulation and suggest that these two IEGs may be co-regulated in response to fear conditioning. Upon retrieval of contextual fear memory elicits a robust yet transient induction of c-Fos expression specifically in the DH, with levels peaking sharply at 1 h and progressively declining thereafter. This temporal profile is consistent with c-Fos\u0026rsquo;s role as a rapidly activated immediate-early gene that marks neuronal activity associated with synaptic plasticity and memory processing. The lack of changes at 0 h indicates that a short delay is required for activity-dependent transcriptional activation. The gradual decline by 5 h suggests that c-Fos involvement is limited to the early phases of retrieval-induced plasticity. In contrast, the VH did not exhibit any significant modulation, reinforcing the idea that c-Fos-mediated activity is preferentially engaged in dorsal circuits that support contextual memory recall rather than ventral circuits linked more closely to affective regulation. c-Fos is widely used as a marker of neural activity due to its rapid and transient expression following synaptic stimulation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The robust elevation observed here supports the involvement of DH neurons in processing and consolidating the fear memory trace.\u003c/p\u003e\u003cp\u003eThe temporal alignment between c-Fos and Arc upregulation in the DH highlights a coordinated molecular response that is likely essential for initiating downstream plasticity-related pathways. Notably, c-Fos expression did not increase at 0 hour post-conditioning, reinforcing the idea of a narrow time window during which neural circuits undergo maximal activation and plasticity-related gene expression. This is consistent with earlier studies showing that IEG expression peaks within 1\u0026ndash;2 hours following behavioral stimulation and returns to baseline thereafter [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs with Arc, c-Fos expression in the VH remained unchanged across all time points and experimental groups. This again highlights the lack of ventral hippocampal involvement in c-Fos-mediated responses to contextual fear learning. The absence of activation in the VH supports the conclusion that contextual fear memory formation is predominantly mediated by the DH, which is more tightly connected to parahippocampal and cortical structures involved in spatial representation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRegion-Specific Specialization in the Hippocampus\u003c/h2\u003e\u003cp\u003eThe present findings strongly support the notion of functional segregation along the longitudinal axis of the hippocampus, where the DH is preferentially engaged in processing contextual and cognitive components of memory, while the VH is more involved in regulating emotion and stress responses. This functional dichotomy is supported by anatomical studies showing distinct input-output connectivity patterns along the hippocampal axis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The selective activation of Arc and c-Fos in the DH, but not VH, following contextual fear-conditioning suggesting a region-specific role of the DH and VH in the consolidation fear memory.\u003c/p\u003e\u003cp\u003eThese results are also consistent with lesion and pharmacological studies showing that disruption of DH activity impairs contextual fear conditioning. At the same time, manipulation of the VH more selectively affects anxiety-like behavior without compromising contextual learning [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Importantly, the current study uses protein-level analyses of IEGs, providing a molecular basis for the observed behavioral patterns and supporting the idea that synaptic plasticity and memory-related signaling are compartmentalized within hippocampal subregions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eImplications for Memory Consolidation Mechanisms\u003c/h2\u003e\u003cp\u003eThe synchronized behavioral and molecular responses observed in this study provide compelling evidence for the DH as a central hub for contextual fear memory encoding and consolidation. The time-dependent upregulation of Arc and c-Fos suggests these genes may operate in tandem to support activity-induced synaptic modifications required for memory storage. Arc is known to regulate AMPA receptor endocytosis and structural remodeling of dendritic spines, while c-Fos is involved in initiating transcriptional cascades that promote long-term cellular changes [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The overlap in their temporal expression profiles post-conditioning suggests a potential synergistic role in orchestrating the early molecular events that stabilize the fear memory trace. The absence of significant changes at 0 and 5 hours post-conditioning may reflect the closure of the molecular window for consolidation, consistent with models proposing that long-term memory formation requires tightly regulated gene expression within specific post-learning time frames.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eLimitations and Future Directions\u003c/h2\u003e\u003cp\u003eDespite the strengths of the current study, a few limitations warrant discussion. First, while Arc and c-Fos are widely accepted as markers of neuronal activation and plasticity, they are indirect measures and do not provide spatial resolution at the single-cell level. Future immunohistochemistry or in situ hybridization studies could better localize these proteins within specific hippocampal subfields (e.g., CA1, CA3, dentate gyrus) to further delineate circuit-specific changes. Second, investigating the long-term persistence of these molecular changes beyond the 5-hour window and their relevance to memory retrieval could offer insights into the mechanisms of memory maintenance versus formation.\u003c/p\u003e\u003cp\u003eOur findings demonstrate that contextual fear conditioning elicits robust behavioral and molecular responses that are time-dependent and region-specific within the hippocampus. The observed molecular evidence supports the view that the DH as a key locus for encoding contextual fear memories through activity-dependent gene expression. They also offer a foundation for further exploration into the temporal dynamics of memory-related plasticity and the potential for region-specific therapeutic targeting in disorders such as PTSD. Future studies examining cellular specificity, sex differences, and long-term gene expression patterns will be essential to fully elucidate the mechanisms underlying fear memory formation and persistence.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest:\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests. Both the authors The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics approval:\u003c/h2\u003e\u003cp\u003eAll experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of Jawaharlal Nehru University, New Delhi. IAEC protocol #36/2018; Dated: 21/12/2018. This study was performed in line with the principles of the Declaration of Helsinki.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis work was supported by grants from the Department of Science and Technology-CSRI Grant Numbers: DST-CSRI 2021/136 (G); DST-CSRI/39/2016(G)] funded to Sushil K Jha. We also acknowledge the funding from SERB, DBT-BUILDER, and the Department of Science and Technology-PURSE.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYK performed the experiments and analyzed the data, SJ conceived and conceptualized the ideas, designed the experiments, analyzed the data, and finalized the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll the data used in the current study will be available from the corresponding and co-author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTripathi S, Verma A, Jha SK (2020) Training on an appetitive trace-conditioning task increases adult hippocampal neurogenesis and the expression of Arc, Erk and CREB proteins in the dorsal hippocampus. Frontiers in Cellular Neuroscience 14:89\u003c/li\u003e\n\u003cli\u003eJha VM, Jha SK, Jha VM, Jha SK (2020) Sleep Loss: What Does It Do to Our Brain and Body? 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Front Mol Neurosci 8:78\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Hippocampus, Fear Memory, Learning, Neuronal Activity, Protein expression","lastPublishedDoi":"10.21203/rs.3.rs-7658753/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7658753/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe hippocampus, a key structure playing an important role in contextual fear-conditioning (CxFC), shows functional specialization along its dorsoventral axis. The dorsal hippocampus is primarily implicated in spatial and contextual processing, whereas the ventral hippocampus is more closely associated with affective and emotional regulation. In this study, we examined whether CxFC selectively engages activity-dependent gene expression in the dorsal hippocampus (DH) but not in the ventral hippocampus (VH). Mice were subjected to CxFC, and freezing behavior across baseline, training, and testing sessions was evaluated. Molecular analyses were conducted to assess the temporal expression patterns of Arc and c-Fos proteins in the DH and VH at 0, 1, 3, and 5 hours on conditioning and post-conditioning days. Fear-conditioned animals displayed a significant increase in freezing behavior during the testing session compared to baseline, indicating strong fear memory retention. Arc expression in the DH showed a time-dependent increase, peaking at the 1st hour and remained highly expressed till the 5th hour on conditioning and post-conditioning days. No significant changes were observed in the VH. Similarly, c-Fos expression in the DH increased significantly at 1st, 3rd, and 5th hours on conditioning and post-conditioning days, while no significant activation was detected in the VH. These findings demonstrate that contextual fear conditioning selectively activates the DH, as evidenced by upregulation of Arc and c-Fos expression. The VH did not show corresponding molecular changes, suggesting a region-specific role of the DH and VH in the consolidation fear memory.\u003c/p\u003e","manuscriptTitle":"Contextual Fear Conditioning Induces Activity-Dependent Gene Expression in the Dorsal but not in the Ventral Hippocampus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 10:51:57","doi":"10.21203/rs.3.rs-7658753/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-22T22:23:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-22T14:06:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-20T02:33:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurochemical Research","date":"2025-09-19T12:38:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"08800a37-15af-4a6b-89d0-da413978cff0","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:09:48+00:00","versionOfRecord":{"articleIdentity":"rs-7658753","link":"https://doi.org/10.1007/s11064-026-04683-0","journal":{"identity":"neurochemical-research","isVorOnly":false,"title":"Neurochemical Research"},"publishedOn":"2026-02-09 15:57:56","publishedOnDateReadable":"February 9th, 2026"},"versionCreatedAt":"2025-10-15 10:51:57","video":"","vorDoi":"10.1007/s11064-026-04683-0","vorDoiUrl":"https://doi.org/10.1007/s11064-026-04683-0","workflowStages":[]},"version":"v1","identity":"rs-7658753","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7658753","identity":"rs-7658753","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}