Footshock Intensity Research Articles

Fear generalization modulated by shock intensity and protein synthesis inhibitor.

Maladaptive fear responses, including sensitized threat reactions and overgeneralization, contribute to anxiety disorders such as generalized anxiety disorder and post-traumatic stress disorder. Although stress intensity influences the generation and extent of these maladaptive fears, the underlying mechanisms remain unclear. The present study examined whether varying footshock stress intensity and inhibition of protein synthesis have differential effect on fear sensitization and generalization in mice. Mice were subjected to a classic fear conditioning protocol involving five different levels of footshock intensities. Prior to fear acquisition, the protein synthesis inhibitor cycloheximide (CHX) was administered intraperitoneally. Fear sensitization to white noise and fear generalization to tones with frequencies differing from the conditioned tone were assessed at either 2 or 4 days after fear acquisition. The results showed that, although varying shock intensities (except the lowest) led to a similar pattern of increased freezing during auditory cues in fear acquisition, the extent of both fear sensitization and generalization increased with the intensity of the footshock in the following days. As shock intensities increased, there was a proportional rise in sensitized fear to white noise and generalized freezing to tones with frequencies progressively closer to the conditioned stimulus. Mildest shocks did not induce discriminative conditioned fear memory, whereas the most intense shocks led to pronounced fear generalization. Administration of CHX before fear acquisition did not affect sensitized fear but reduced generalization of freezing to tones dissimilar from the conditioned stimulus in the group exposed to the most intense shock. Our results suggest that maladaptive fear responses elicited by varying stress intensities exhibit distinct characteristics. The effect of CHX to prevent overgeneralization without affecting discriminative fear memory points to potential therapeutic approaches for fear-related disorders, suggesting the possibility of mitigating overgeneralization while preserving necessary fear discrimination.

Salience signaling and stimulus scaling of ventral tegmental area glutamate neuron subtypes.

Ventral tegmental area (VTA) glutamatergic neurons participate in reward, aversion, drug-seeking, and stress. Subsets of VTA VGluT2+ neurons are capable of co-transmitting glutamate and GABA (VGluT2+VGaT+ neurons), transmitting glutamate without GABA (VGluT2+VGaT- neurons), or co-transmitting glutamate and dopamine (VGluT2+TH+ neurons), but whether these molecularly distinct subpopulations show behavior-related differences is not wholly understood. We identified that neuronal activity of each VGluT2+ subpopulation is sensitive to reward value but signaled this in different ways. The phasic maximum activity of VGluT2+VGaT+ neurons increased with sucrose concentration, whereas VGluT2+VGaT- neurons increased maximum and sustained activity with sucrose concentration, and VGluT2+TH+ neurons increased sustained but not maximum activity with sucrose concentration. Additionally, VGluT2+ subpopulations signaled consummatory preferences in different ways. VGluT2+VGaT- neurons and VGluT2+TH+ neurons showed a signaling preference for a behaviorally-preferred fat reward over sucrose, but in temporally-distinct ways. In contrast, VGluT2+VGaT+ neurons uniquely signaled a less behaviorally-preferred sucrose reward compared with fat. Further experiments suggested that VGluT2+VGaT+ consummatory reward-related activity was related to sweetness, partially modulated by hunger state, and not dependent on caloric content or behavioral preference. All VGluT2+ subtypes increased neuronal activity following aversive stimuli but VGluT2+VGaT+ neurons uniquely scaled their magnitude and sustained activity with footshock intensity. Optogenetic activation of VGluT2+VGaT+ neurons during low intensity footshock enhanced fear-related behavior without inducing place preference or aversion. We interpret these data such that VTA glutamatergic subpopulations signal different elements of rewarding and aversive experiences and highlight the unique role of VTA VGluT2+VGaT+ neurons in enhancing the salience of behavioral experiences.

Enhancing Associative Learning in Rats With a Computationally Designed Training Protocol

BackgroundLearning requires the activation of protein kinases with distinct temporal dynamics. In Aplysia, nonassociative learning can be enhanced by a computationally designed learning protocol with intertrial intervals (ITIs) that maximize the interaction between fast-activated PKA (protein kinase A) and slow-activated ERK (extracellular signal-regulated kinase). Whether a similar strategy can enhance associative learning in mammals is unknown. MethodsWe simulated 1000 training protocols with varying ITIs to predict an optimal protocol based on empirical data for PKA and ERK dynamics in rat hippocampus. Adult male rats received the optimal protocol or control protocols in auditory fear conditioning and fear extinction experiments. Immunohistochemistry was performed to evaluate pCREB (phosphorylated cAMP response element binding)\\protein levels in brain regions that have been implicated in fear acquisition. ResultsRats exposed to the optimal conditioning protocol with irregular ITIs exhibited impaired extinction memory acquisition within the session using a standard footshock intensity, and stronger fear memory retrieval and spontaneous recovery with a weaker footshock intensity, compared with rats that received massed or spaced conditioning protocols with fixed ITIs. Rats exposed to the optimal extinction protocol displayed improved extinction of contextual fear memory and reduced spontaneous recovery compared with rats that received standard extinction protocols. Moreover, the optimal conditioning protocol increased pCREB levels in the dentate gyrus of the dorsal hippocampus, suggesting enhanced induction of long-term potentiation. ConclusionsThese findings demonstrate that a computational model–driven behavioral intervention can enhance associative learning in mammals and may provide insight into strategies to improve cognition in humans.

Characterization of deconditioning-update on fear memory attenuation

Fear memory expression can be attenuated by updating the footshock perception during the plastic state induced by retrieval, from a strong unconditioned stimulus to a very weak one through deconditioning. In this process, the original fear association of the conditioned stimulus with the footshock is substituted by an innocuous stimulus and the animals no longer express a fear response. In the present study, we explore the boundaries of this deconditioning-update strategy by the characterization of this phenomenon. We found that there is an optimal mismatch between the footshock intensity delivered in the training and in the reactivation. Likewise, we characterized the temporal window that the protocol is efficient in hindering fear response. Our findings contribute to a better understanding of the limits in which deconditioning acts in attenuating fear memory, so that an optimized protocol using this strategy can be planned in order to deal with emotional disorders.

Intense inhibitory avoidance training increases nuclear-phosphorylated glucocorticoid receptors in neurons of CA1 of hippocampus and ventral caudate putamen

Corticosterone (CORT), the principal glucocorticoid in rodents, is released after stressful experiences such as training with high foot-shock intensities in the inhibitory avoidance task (IA). CORT reaches the glucocorticoid receptor (GR) located in almost all brain cells; the GR is subsequently phosphorylated at serine 232 (pGRser232). This has been reported as an indicator of ligand-dependent activation of the GR, as well as a requirement for its translocation into the nucleus for its transcription factor activity. The GR is present in the hippocampus with a high concentration in CA1 and dentate gyrus (DG), and a smaller proportion in CA3, and sparsely present in the caudate putamen (CPu); both structures are involved in memory consolidation of IA. To study the participation of CORT in IA, we quantified the ratio of pGR-positive neurons in both dorsal hippocampus (CA1, CA3 and DG) and dorsal and ventral regions of CPu of rats trained in IA, using different foot-shock intensities. Brains were dissected 60 min after training for immunodetection of pGRser232 positive cells. The results show that the groups trained with 1.0 and 2.0 mA had higher retention latencies than the 0.0 mA or 0.5 mA groups. An increase in the ratio of pGR-positive neurons was found in CA1 and ventral region of CPu only for the 2.0 mA trained group. These findings suggest that activation of GRs in CA1 and ventral CPu is involved in the consolidation of a stronger memory of IA, possibly through the modulation of gene expression.

Early life adversity ablates sex differences in active versus passive threat responding in mice

Early life adversity (ELA) heightens the risk for anxiety disorders (which are characterized by heightened fear and avoidance behaviors), with females being twice as likely as males to develop pathology. Pavlovian fear conditioning tasks have been used to study possible mechanisms supporting endophenotypes of pathology. Identification of sex and ELA selective effects on the nature of behavioral responding in these paradigms may provide a unique window into coping strategies in response to learned fear to guide more mechanistic studies. The goals of this study were two-fold; First, to test if male and female mice employed different coping strategies in response to threat learning using different conditioning parameters (low, medium, and high intensity foot shocks). Second, to test if ELA in the form of limited bedding and nesting (LBN) altered the behavioral response of mice to conditioning. Mice received 6 tone/foot-shock pairings at one of three different foot-shock intensities (0.35 mA; 0.57 mA; 0.7 mA). Freezing, darting, and foot-shock reactivity were measured across trials. During conditioning, control-reared female mice exhibited significantly higher rates of darting behavior compared to control males at nearly all shock intensities tested. LBN rearing decreased the proportion of darting females to levels observed in males. Thus, ELA in the form of LBN significantly diminished the recruitment of active versus passive coping strategies in female mice but did not generally change male responding. Additional work will be required to understand the neural basis of these behavioral effects. Findings extending from this work have the potential to shed light on how ELA impacts trajectories of regional brain development with implications for sex-selective risk for behavioral endophenotypes associated with pathology and possibly symptom presentation.

Evaluating Compulsive‐Like Drug‐Taking Using Signaled and Unsignaled Footshock Procedures in Male and Female Rats Self‐Administering MDPV

A subset of rats that self‐administer synthetic cathinones, such as 3,4‐methylenedioxypyrovalerone (MDPV), develop high levels of drug intake and patterns of responding similar to the compulsive, binge‐like patterns of cathinone (e.g., “bath salts”) use reported in humans. Though high‐responder rats exhibit some behaviors thought to be related to substance use disorder, the effects of punished responding in these rats were unknown. These studies evaluated whether (1) high‐responder rats were less sensitive to footshock‐punished responding than low‐responder rats using both unsignaled and signaled footshock procedures; (2) whether sensitivity to footshock was dependent on the self‐administered dose of MDPV. Inhibition curves for unsignaled footshock (0.05‐0.9 mA, increasing across sessions) were evaluated in forty adult Sprague Dawley rats self‐administering 0.01, 0.032, and 0.1 mg/kg/infusion MDPV under a FR5 schedule of reinforcement; footshocks were paired with ~50% of drug infusions. Rats were then trained on a signaled footshock procedure, in which a change in stimulus conditions signaled that the next infusion would be paired with a footshock (intensities individualized to each rat’s IC50). Rats could avoid this footshock by withholding responding for 30‐sec. After rats learned the signaled shock procedure, inhibition curves were generated using a range of footshock intensities (0.05‐0.9 mA). High‐ and low‐responder rats were similarly sensitive to unsignaled punishment by footshock (mean IC50s‐ high‐responders: 0.30mA; low‐responders: 0.36mA). However, high‐responder rats (mean: 45mA) received significantly more total current than low‐responder rats (mean: 18mA). Compared to low‐responder rats, high‐responder rats respond more in the presence of the signal indicating impending shock and avoid a smaller proportion of shocks. This suggests high‐responder rats may be less able to adapt their behavior in response to a stimulus associated with punishment. These studies suggest that the high levels of drug‐taking observed in the high‐responder rats reflects a compulsive‐like pattern of responding, which may be related to continued use despite adverse consequence, a DSM symptom of substance use disorder.

A Procedure to Study Stress-induced Relapse of Heroin Seeking after Punishment-imposed Abstinence.

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.

Increased responsiveness to punishment of cocaine self-administration after experience with high punishment.

One behavioral feature of drug addiction is continued drug use despite awareness that this causes negative consequences. Attempts to model this feature in animals typically involve punishing drug self-administration with electrical footshock to identify individuals whose drug use is differently suppressed by punishment. Here we sought to further study individual responsiveness of drug use to punishment in rats self-administering intravenous cocaine. Rats were first trained during several weeks to self-administer cocaine under a fixed-ratio 3 schedule of reinforcement. Then, their self-administration behavior was punished with increasing intensity of footshock (i.e., from 0.1 mA to 0.9 mA, every 30 min). With increasing intensity of punishment, rats first continued to self-administer cocaine before eventually stopping near completely. When retested, however, drug use became more responsive to punishment and was suppressed by a low and initially ineffective footshock intensity (i.e., 0.1 mA). This increase in responsiveness to punishment was seen in all individuals tested, albeit with varying degrees, and was acquired after one single experience with an intensity of punishment that near completely suppressed drug self-administration. Mere passive, non-contingent exposure to the same intensity, however, had no such effect. Once acquired, increased responsiveness to punishment persisted during at least one month when rats were tested every week, but not every day. Finally, increased responsiveness to punishment was not observed after exposure to a non-painful form of punishment (i.e., histamine). Overall, this study reveals that initial responsiveness of drug use to punishment can change rapidly and persistently with experience. We discuss several possible mechanisms that may account for this change in punishment responsiveness and also draw some of the implications and future perspectives for research on animal models of compulsion-like behavior.

Exposure to footshock stress downregulates antioxidant genes and increases neuronal apoptosis in an Aβ(1–42) rat model of Alzheimer's disease

Post-traumatic stress disorder (PTSD) is a neuropsychiatric disorder that develops from exposure to trauma, mostly when normal psychological mechanisms fail. Studies have shown that people who have PTSD are susceptible to developing dementia, mostly Alzheimer's disease (AD), suggesting common underlying risk factors in the comorbidity. However, data elucidating links between these conditions is scarce. Here we show that footshock stress exacerbates AD-like pathology. To induce a trauma-like condition, the rats were exposed to multiple intense footshocks followed by a single reminder. This was followed by bilateral intrahippocampal lesions with amyloid-beta (Aβ) (1–42), to model AD-like pathology. We found that footshocks increased anxiety behavior and impaired fear memory extinction in Aβ(1–42) lesioned rats. We also found a reduced expression of nuclear factor erythroid 2-related factor 2 (Nrf2), NAD (P) H: quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and an increased expression of Kelch-like ECH-associated protein 1 (Keap1) in the amygdala and hippocampus. Furthermore, oxidative stress level was sustained, which was associated with increased apoptosis in the amygdala and hippocampus.Our finding suggests that AD-like pathology can induce oxidative changes in the amygdala and hippocampus, which can be exaggerated by footshock stress.

Corticosterone differentially modulates time-dependent fear generalization following mild or moderate fear conditioning training in rats

Stressful and emotionally arousing experiences create strong memories that seem to lose specificity over time. It is uncertain, however, how the stress system contributes to the phenomenon of time-dependent fear generalization. Here, we investigated whether post-training corticosterone (CORT-HBC) injections, given after different training intensities, affect contextual fear memory specificity at several time points. We trained male Wistar rats on the contextual fear conditioning (CFC) task using two footshock intensities (mild CFC, 3 footshocks of 0.3 mA, or moderate CFC, 3x 0.6 mA) and immediately after the training session we administered CORT-HBC systemically. We first tested the animals in a novel context and then in the training context at different intervals following training (2, 14, 28 or 42 days). By measuring freezing in the novel context and then contrasting freezing times shown in both contexts, we inferred contextual fear generalization for each rat, classifying them into Generalizers or Discriminators. Following mild CFC training, the glucocorticoid injection promoted an accurate contextual memory at the recent time point (2 days), and increase the contextual memory accuracy 28 days after training. In contrast, after the moderate CFC training, CORT-HBC facilitated contextual generalization at 14 days, compared to the control group that maintained contextual discrimination at this timepoint. For this training intensity, however, CORT-HBC did not have any effect on recent memory specificity. These findings indicate that treatment with CORT-HBC immediately after the encoding of mild or moderately arousing experiences may differentially modulate memory consolidation and time-dependent fear generalization.

Recruitment of neurons in basolateral amygdala after intense training produces a stronger memory trace

Typical amnestic treatments are ineffective when administered to subjects trained in aversively-motivated tasks using relatively high foot-shock intensities. This effect has been found when treatments that disrupt neuronal activity are administered to different regions of the brain, including the amygdala. However, the molecular mechanisms induced by this intense training are unknown. We made a detailed mapping of c-Fos-expressing neurons in four regions of the amygdala after moderate and intense one-trial inhibitory avoidance training. Rats were sacrificed 90 min after training or after appropriate control procedures, and their brains were prepared for immunohistochemical c-Fos protein detection in the central, lateral, and in the anterior and posterior parts of the basolateral amygdaloid nucleus. We found a high percentage of neurons expressing c-Fos in the anterior part of the basolateral nucleus after moderate training, and this percentage increased further after intense training. Moderate and intense training did not induce changes in c-Fos expression in the other explored amygdaloid regions. These results show that inhibitory avoidance training produces a localized expression of c-Fos in the basolateral anterior nucleus of the amygdala, which is dependent upon the intensity of training, and indicate that synaptic plastic changes in this region may be required for the formation of memory of moderate and intense aversive learning.

A projection from the paraventricular nucleus of the thalamus to the shell of the nucleus accumbens contributes to footshock stress-induced social avoidance

The paraventricular nucleus of the thalamus (PVT) is an area of the dorsal midline thalamus that contributes to footshock induced anxiety. The PVT sends a dense projection to the shell of the nucleus accumbens (NAcSh) and the present study explored if this projection is involved in the behavioral changes produced by a single exposure of rats to inescapable footshocks. The inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) hM4Di was transduced in PVT neurons that project to the NAcSh. Rats were exposed to an episode of moderately intense footshock (1.5 mA × 2 s × 5) and assigned to either high-responder (HR) or low-responder groups (LR) according to their level of fear generalization 24 h later. The effect of chemogenetic inhibition of the PVT-NAcSh projection on anxiety- and fear-like behaviors was assessed at approximately 2 weeks post-footshock. HR showed a higher level of social avoidance compared to non-shocked animals and LR. The elevated level of social avoidance was attenuated in the HR treated with the hM4Di agonist clozapine (0.01 mg/kg, i.p.) or clozapine N-oxide (CNO) administrations in the NAcSh while avoidance of open spaces and contextual fear expression were not affected. Analysis of protein product of the early to immediate gene cfos indicated that these effects were mediated by dynorphin neurons in the NAcSh. This study provides evidence for a role of a projection from the PVT to the NAcSh in stress-induced social avoidance independent of anxiety to non-social stimuli and contextual fear mechanisms.

Imbalance in cerebral protein homeostasis: Effects on memory consolidation

The long-standing hypothesis that memory consolidation is dependent upon de novo protein synthesis is based primarily on the amnestic effects of systemic administration of protein synthesis inhibitors (PSIs). Early experiments on mice showed that PSIs produced interference with memory consolidation that was dependent on the doses of PSIs, on the interval between drug injection and training, and, importantly, on the degree and duration of protein synthesis inhibition in the brain. Surprisingly, there is a conspicuous lack of information regarding the relationship between the duration of protein synthesis inhibition produced by PSIs and memory consolidation in the rat, one of the species most widely used to study memory processes. We found that, in the male rat, a single injection of cycloheximide, a commonly used PSI, produced a significant imbalance in protein homeostasis: an early inhibition of protein synthesis that lasted for at least one hour, followed by hyperproduction of proteins that lasted three days. We evaluated memory consolidation of inhibitory avoidance trained with either low or high intensity of foot-shock at the peaks of protein synthesis inhibition and protein hyperproduction. We found that, independent of the moment of training, the low-foot-shock groups showed amnesia, while the high-foot-shock groups displayed optimal memory performance. These results indicate that memory consolidation of relatively weak training is impaired by the inhibition or hyperproduction of protein synthesis, and that intense training overcomes this dysregulation of protein homeostasis allowing for memory formation probably through non-genomic mechanisms.

Differential Phosphorylation of the Glucocorticoid Receptor in Hippocampal Subregions Induced by Contextual Fear Conditioning Training.

Aversive events induce the release of glucocorticoid stress hormones that facilitate long-term memory consolidation, an effect that depends on the activation of glucocorticoid receptors (GRs). GRs are distributed widely in the hippocampus. The dorsal region of the hippocampus has been related to cognitive functions and the ventral region to stress and emotion. GR acts as a transcription factor which after hormone binding becomes phosphorylated, affecting its cellular distribution and transcriptional activity. Two functionally well-described GR phosphorylation sites are serine 232 (pSer232), which enhances gene expression, and serine 246 (pSer246), having the opposite effect. Since gene expression is one of the plastic mechanisms needed for memory consolidation, we investigated if an aversive learning task would induce GR phosphorylation in the dorsal (DH) and the ventral (VH) hippocampus. We trained rats in contextual fear conditioning (CFC) using different foot-shock intensities (0.0, 0.5, or 1.5 mA). One subgroup of animals trained with each intensity was sacrificed 15 min after training and blood was collected to quantify corticosterone (CORT) levels in serum. Another subgroup was sacrificed 1 h after training and brains were collected to evaluate the immunoreactivity (IR) to GR, pSer232 and pSer246 by SDS-PAGE/Western blot in DH and VH, and by immunohistochemistry in dorsal and ventral CA1, CA2, CA3, and dentate gyrus (DG) hippocampal regions. The conditioned freezing response increased in animals trained with 0.5 and 1.5 mA during training and extinction sessions. The degree of retention and CORT levels were directly related to the intensity of the foot-shock. Although total GR-IR remained unaffected after conditioning, we observed a significant increase of pSer246-IR in the dorsal region of CA1 and in both dorsal and ventral DG. The only region in which pSer232-IR was significantly elevated was ventral CA3. Our results indicate that fear conditioning training is related to GR phosphorylation in specific subregions of the hippocampus, suggesting that its transcriptional activity for gene expression is favored in ventral CA3, whereas its repressor activity for gene-silencing is increased in dorsal CA1 and in both dorsal and ventral DG.

A novel stress re-stress model: modification of re-stressor cue induces long-lasting post-traumatic stress disorder-like symptoms in rats

Background: Post-traumatic stress disorder (PTSD) develops after exposure to a severe traumatic event. Stress re-stress (SRS) model of PTSD using forced swim as a re-stress shows hypocortisolism, an index of hypothalamic–pituitary–adrenal (HPA) axis dysfunction. Despite this, the SRS model lacks development of hyperarousal and intrusive memory which are prominent symptoms of PTSD.Aim: Developing a model which shows more clinical similarities to PTSD. We used intense foot-shock (FS) (2 mA for 10 sec) instead of the forced-swim test (FST) as a re-stress cue.Methods: The stress session was performed on D-2 except control group. The FST group rats were forced to swim for 20 min, and FS group rats were exposed to foot shock of 2 mA for 10 sec. The re-stress sessions of FST and FS were performed on D-8, 14, 20, 26 and 32. The freezing behavior was evaluated after the FS re-stress cue, and remaining behavioral assessments such as anxiety and depressive-like behavior, and cognitive dysfunction were performed on last day of the experiment.Results: The FS-induced PTSD-like (anxiety and depressive-like behavior, and cognitive dysfunction) symptoms and a decrease in plasma corticosterone were more significant compared to FST. The hyperarousal and intrusive memory induced by FS were additional symptoms in the SRS model. Treatment with paroxetine significantly attenuated FST and FS-induced behavioral deficits in PTSD rats.Conclusion: FS as a re-stress model shows prominent PTSD-like symptoms with HPA axis dysfunction compared to FST.

Contribution of the prefrontal cortex and basolateral amygdala to behavioral decision-making under reward/punishment conflict.

Control of reward-seeking behavior under conditions of punishment is an important function for survival. We designed a task in which rats could choose to either press a lever and obtain a food pellet accompanied by a footshock or refrain from pressing the lever to avoid footshock, in response to tone presentation. In the task, footshock intensity steadily increased, and the task was terminated when the lever press probability reached < 25% (last intensity). Rats were trained until the last intensity was stable. Subsequently, we investigated the effects of the pharmacological inactivation of the ventromedial prefrontal cortex (vmPFC), lateral orbitofrontal cortex (lOFC), and basolateral amygdala (BLA) on task performance. Bilateral inactivation of the vmPFC, lOFC, and BLA did not alter lever press responses at the early stage of the task. The number of lever presses increased following vmPFC and BLA inactivation but decreased following lOFC inactivation during the later stage of the task. The last intensity was elevated by vmPFC or BLA inactivation but lowered by lOFC inactivation. Disconnection of the vmPFC-BLA pathway induced behavioral alterations that were similar to vmPFC or BLA inactivation. Inactivation of any regions did not alter footshock sensitivity and anxiety levels. Our results demonstrate a strong role of the vmPFC and BLA and their interactions in reward restraint to avoid punishment and a prominent role of the lOFC in reward-seeking under reward/punishment conflict situations.

Inhibition of transcription and translation in dorsal hippocampus does not interfere with consolidation of memory of intense training.

Findings of several experiments indicate that many treatments that typically interfere with memory consolidation are ineffective in preventing or attenuating memory induced by intense training. As extensive evidence suggests that the consolidation of newly acquired memories requires gene expression and de novo protein synthesis the present study investigated whether intense training prevents consolidation impairment induced by blockers of mRNA and protein synthesis. Rats were given a single inhibitory training trial using a moderate (1.0 mA) or a relatively intense (2.0 mA) foot-shock. Bilateral hippocampal infusions of the mRNA synthesis blocker DRB (10, 40 or 80 ng/0.5 µL/hemisphere) or the protein synthesis inhibitor anisomycin (ANI), an inhibitor de novo protein synthesis (15.62, 31.25, or 62.50 µg/0.5 µL/hemisphere) were administered 15 min prior to training. Retention was measured at 30 min or 48 h following training. DRB and ANI impaired memory of moderate training in a dose-dependent manner without affecting short-term memory. In contrast, memory consolidation was not impaired in the groups trained with 2.0 mA. The findings showed that: (1) inhibitors of transcription and translation in the hippocampus impair the consolidation of memory of inhibitory avoidance learning induced by moderate levels of aversive stimulation and (2) blocking of mRNA and protein synthesis does not prevent the consolidation of memory induced by relatively high levels of aversive stimulation. These findings do not support the hypothesis that gene expression and de novo protein synthesis are necessary steps for long-term memory formation as memory was not impaired if intense foot-shock was used in training.

Relationship between footshock intensity, post-training corticosterone release and contextual fear memory specificity over time

Overgeneralized fear has long been implicated in generalized anxiety and post-traumatic stress disorder, however, time-dependent mechanisms underlying memory retrieval are still not completely understood. Previous studies have revealed that stronger fear conditioning training protocols are associated with both increased post-training corticosterone (CORT) levels and fear responses at later retrieval tests. Here we used contextual fear conditioning (CFC) to investigate the relationship between post-training CORT levels and memory specificity in different retrieval timepoints. Wistar rats were exposed to CFC training with increasing footshock intensities (0.3, 0.6 or 1.0mA) and had their blood collected 30 min afterwards to measure post-training plasma CORT. After 2, 14 or 28 days, rats were tested for memory specificity either in the training or in the novel context. Regression analysis was used to verify linear and non-linear interactions between CORT levels and freezing. Higher footshock intensities increased post-training CORT levels and freezing times during tests in all timepoints. Moreover, stronger trainings elicited faster memory generalization, which was associated with higher CORT levels during memory consolidation. The 0.3mA training maintained memory specificity up to 28 days. Additionally, linear regressions suggest that the shift from specific to generalized memories is underway at 14 days after training. These results are consistent with the hypotheses that stronger training protocols elicit a faster generalization rate, and that this process is associated with increased post-training CORT release.

Microglial cell hyper-ramification and neuronal dendritic spine loss in the hippocampus and medial prefrontal cortex in a mouse model of PTSD

Few animal models exist that successfully reproduce several core associative and non-associative behaviours relevant to post-traumatic stress disorder (PTSD), such as long-lasting fear reactions, hyperarousal, and subtle attentional and cognitive dysfunction. As such, these models may lack the face validity required to adequately model pathophysiological features of PTSD such as CNS grey matter loss and neuroinflammation. Here we aimed to investigate in a mouse model of PTSD whether contextual fear conditioning associated with a relatively high intensity footshock exposure induces loss of neuronal dendritic spines in various corticolimbic brain regions, as their regression may help explain grey matter reductions in PTSD patients. Further, we aimed to observe whether these changes were accompanied by alterations in microglial cell number and morphology, and increased expression of complement factors implicated in the mediation of microglial cell-mediated engulfment of dendritic spines. Adult male C57Bl6J mice were exposed to a single electric footshock and subsequently underwent phenotyping of various PTSD-relevant behaviours in the short (day 2–4) and longer-term (day 29–31). 32 days post-exposure the brains of these animals were subjected to Golgi staining of dendritic spines, microglial cell Iba-1 immunohistochemistry and immunofluorescent staining of the complement factors C1q and C4. Shock exposure promoted a lasting contextual fear response, decreased locomotor activity, exaggerated acoustic startle responses indicative of hyperarousal, and a short-term facilitation of sensorimotor gating function. The shock triggered loss of dendritic spines on pyramidal neurons was accompanied by increased microglial cell number and complexity in the medial prefrontal cortex and dorsal hippocampus, but not in the amygdala. Shock also increased expression of C1q in the pyramidal layer of the CA1 region of the hippocampus but not in other brain regions. The present study further elaborates on the face and construct validity of a mouse model of PTSD and provides a good foundation to explore potential molecular interactions between microglia and dendritic spines.