Unsignaled Shock Research Articles

Sex difference in the facilitation of fear learning by prior fear conditioning

There is now ample evidence that the strength and underlying mechanisms of memory formation can be drastically altered by prior experience. However, the prior work using rodent models on this topic has used only males as subjects, and as a result, we do know whether or not the effects of prior experience on subsequent learning are similar in both sexes. As a first step towards addressing this shortcoming, rats of both sexes were given auditory fear conditioning, or fear conditioning with unsignaled shocks, followed an hour or a day later by a single pairing of light and shock. Fear memory for each experience was assessed by measuring freezing behavior to the auditory cue and fear-potentiated startle to the light. Results showed that males trained with auditory fear conditioning showed facilitated learning to the subsequent visual fear conditioning session when the two training sessions were separated by one hour or one day. Females showed evidence of facilitation in rats given auditory conditioning when they were spaced by an hour but not when they were spaced by one day. Contextual fear conditioning did not support the facilitation of subsequent learning under any conditions. These results indicate that the mechanism by which prior fear conditioning facilitates subsequent learning differs between sexes, and they set the stage for mechanistic studies to understand the neurobiological basis of this sex difference.

Role of dorsal hippocampal muscarinic receptor activity in acquisition and retention of single- versus multiple-trial contextual fear conditioning in adolescent rats.

The present study examined the effects of the muscarinic acetylcholine receptor (mAChR) antagonist, scopolamine, on standard contextual fear conditioning (sCFC). It compared effects of the drug on acquisition (post-shock freezing) versus 24-hr retention of a context-shock association acquired after one or three pairings of a context with unsignaled shock. During single-trial sCFC, systemic scopolamine (0.5 mg/kg, i.p.) prior to training abolished both post-shock and retention freezing (Experiment 1). This same injection during multiple-trial sCFC also abolished post-shock freezing and impaired 24-hr retention freezing (Experiment 2). These results indicate that cholinergic signaling mediates both acquisition and 24-hr retention of a context-shock association across different trial parameters. Experiment 3 further explored these effects by infusing scopolamine (35 μg per side) into the dorsal hippocampus (dHPC) prior to training in single versus multiple-trial sCFC. This infusion spared post-shock but abolished retention test freezing in single-trial sCFC (Experiment 3A), and had no effect on multiple-trial sCFC (Experiment 3B). The current findings suggest that brain-wide cholinergic signaling mediates acquisition and retention of single-trial sCFC. Despite this, while muscarinic cholinergic signaling in the dHPC does mediate retention of single-trial sCFC, it is not required for acquisition of either variant, or retention of multiple-trial sCFC. These findings also rule out impaired sensory processing of contextual cues as a mechanism of impaired context learning by dHPC scopolamine. The results are discussed in relation to the role of cholinergic function across multiple brain memory systems in elemental versus configural forms of contextual fear conditioning (Fanselow, 2010; Rudy, 2009). (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Contributions of postrhinal and perirhinal cortex to contextual information processing.

The role of the postrhinal cortex (POR) and the perirhinal cortex (PER) in processing relational or contextual information was examined with Pavlovian fear conditioning. Rats with electrolytic or neurotoxic lesions of the POR or PER were tested in 2 contextual fear conditioning paradigms. In Experiment 1, electrolytic lesions of the POR or PER produced impairments in contextual fear conditioning but not in conditioning to a phasic auditory conditioned stimulus. Neurotoxic lesions of the POR or PER likewise resulted in anterograde (Experiment 2) and retrograde (Experiment 3) deficits in fear conditioning to the training context in an unsignaled shock paradigm. The results suggest that operations performed on sensory information by the POR and PER are necessary to support contextual learning. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Impaired contextual fear conditioning in RasGRF2 mutant mice is likely Ras-ERK-dependent

Ras/Raf/MEK/ERK (Ras-ERK) signaling has been shown to play an important role in fear acquisition. However, little information is known regarding the mechanisms that contribute to the regulation of this pathway in terms of the learning of conditioned fears. Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) is one of two guanine nucleotide exchange factors (GEF) that regulates the Ras-ERK signaling pathway in a Ca2+-dependent manner via control of the cycling of Ras isoforms between an inactive and active state. Here we sought to determine the role of RasGRF2 on contextual fear conditioning in RasGRF2 knockout (KO) and their wild type (WT) counterparts. Male KO and WT mice underwent a single session of contextual fear conditioning (12 min, 4 unsignaled shocks), followed by either daily 12-min retention trials or the molecular analysis of Ras activation and pERK1/2 activity. KO mice showed an impaired acquisition of contextual fear, as demonstrated by reduced freezing during fear conditioning and 24-hr retention tests relative to WT mice. Ras analysis following fear conditioning demonstrated a reduction in Ras activation in the hippocampus as well as a reduction in pERK1/2 in the CA1 region of the hippocampus in KO mice, suggesting that the decrease in fear conditioning in KO mice is at least in part due to the impairment of Ras-ERK signaling in the hippocampus during learning. These data indicate a role for RasGRF2 in contextual fear conditioning in mice that may be Ras-ERK-dependent.

Reducing negative stimulus valence does not attenuate the return of fear: Two counterconditioning experiments

Exposure-based treatment for anxiety disorders is effective for many patients, but relapse is not uncommon. One predictor of the return of fear is the negative valence of fear-relevant stimuli. The aim of the current experiments was to examine whether counterconditioning with positive film clips reduces this negative stimulus valence as well as the return of fear, compared to standard extinction training and to an extinction training with non-contingent exposure to the positive film clips. Participants were 87 students in Experiment 1 (three-day paradigm), and 90 students in Experiment 2 (one-day paradigm). They first underwent a differential acquisition phase, in which one of three pictures was paired with an electric shock. They were then randomly allocated to one of the three intervention groups. Afterwards, they underwent a test phase in which pictures were presented without shock (to measure spontaneous recovery of fear), which was followed by unsignaled shocks to induce reinstatement of extinguished fear. Outcome variables were self-reported stimulus valence, shock expectancy, skin conductance, and fear-potentiated startle. In both experiments, counterconditioning decreased negative stimulus valence, relative to the other interventions, but it did not reduce spontaneous fear recovery or fear reinstatement. Overall, our findings do not support the notion that counterconditioning reduces return of fear.

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Relapse of extinguished fear after exposure to a dangerous context is mitigated by testing in a safe context.

Aversive events can trigger relapse of extinguished fear memories, presenting a major challenge to the long-term efficacy of therapeutic interventions. Here, we examined factors regulating the relapse of extinguished fear after exposure of rats to a dangerous context. Rats received unsignaled shock in a distinct context (“dangerous” context) 24 h prior to auditory fear conditioning in another context. Fear to the auditory conditioned stimulus (CS) was subsequently extinguished either in the conditioning context (“ambiguous” context) or in a third novel context (“safe” context). Exposure to the dangerous context 30 min before a CS retention test caused relapse to the CS in the ambiguous and safe test contexts relative to nonextinguished controls. When rats were tested 24 h later (with or without short-term testing), rats tested in the ambiguous context continued to exhibit relapse, whereas rats tested in the safe context did not. Additionally, exposure of rats to the conditioning context—in place of the unsignaled shock context—did not result in relapse of fear to the CS in the safe testing context. Our work highlights the vulnerabilities of extinction recall to interference, and demonstrates the importance of context associations in the relapse of fear after extinction.

Enhanced extinction of contextual fear conditioning in ClockΔ19 mutant mice.

Clock genes have been implicated in several disorders, such as schizophrenia, bipolar disorder, autism spectrum disorders, and drug dependence. However, few studies to date have examined the role of clock genes in fear-related behaviors. The authors used mice with the ClockΔ19 mutation to assess the involvement of this gene in contextual fear conditioning. Male wild-type (WT) and ClockΔ19 mutant mice underwent a single session of contextual fear conditioning (12 min, 4 unsignaled shocks), followed by daily 12-min retention trials. There were no differences between mutant and WT mice in the acquisition of contextual fear, and WT and mutant mice demonstrated similar freezing during the first retention session. However, extinction of contextual fear was accelerated in mutant mice across the remaining retention sessions, as compared to WT mice, suggesting a role for Clock in extinction following aversive learning. Because the ClockΔ19 mutation has previously been demonstrated to result in an increase in dopamine signaling, the authors confirmed the role of dopamine in extinction learning using preretention session administration of a low dose of the dopamine transport reuptake inhibitor modafinil (0.75 mg/kg), which resulted in decreased freezing across retention sessions. These findings are consistent with an emerging portrayal of the importance of Clock genes in noncircadian functions, as well as the important role of dopamine in extinction learning.

Exposure to the context and removing the unpredictability of the US: Two methods to reduce contextual anxiety compared

Chronic anxiety may differ from cued fear and hence require other treatment strategies. In a human fear conditioning paradigm, chronic anxiety to the experimental context was experimentally induced by presenting unpredictable shocks. Two methods to reduce chronic anxiety were tested and compared. First, in parallel with the standard extinction procedure, participants were exposed to the anxiety-eliciting context in the absence of shocks (context-exposure group). Second, an alternative procedure was tested in which the previously unpredictable shocks were now signaled by a specific cue (signaled group). A control group continued to receive unsignaled shocks. Results indicated that chronic contextual anxiety, as measured by fear-potentiated startle and US-expectancy ratings, was equally reduced in the context-exposure group as in the signaled group compared with the control group. When applied to the treatment of, for example, panic disorder, these findings support the idea that exposure to the context in which the unpredictable panic attacks occurred and making unpredictable panic attacks predictable, are both valuable methods in order to reduce chronic anxiety.

Male and female Fmr1 knockout mice on C57 albino background exhibit spatial learning and memory impairments

Impaired spatial learning is a prominent deficit in fragile X syndrome (FXS). Previous studies using the Fmr1 knockout (KO) mouse model of FXS have not consistently reported a deficit in spatial learning. Fmr1 KO mice bred onto an albino C57BL/6J-Tyr(c-Brd) background showed significant deficits in several primary measures of performance during place navigation and probe trials in the Morris water maze. Fmr1 KO mice were also impaired during a serial reversal version of the water maze task. We examined fear conditioning as an additional cognitive screen. Knockout mice exhibited contextual memory deficits when trained with unsignaled shocks; however, deficits were not found in a separate group of KO mice trained with signaled shocks. No potentially confounding genotypic differences in locomotor activity were observed. A decreased anxiety-like profile was apparent in the open field, as others have noted, and also in the platform test. Also as previously reported, startle reactivity to loud auditory stimuli was decreased, prepulse inhibition and social interaction increased in KO mice. Female Fmr1 KO mice were tested along with male KO mice in all assays, except for social interaction. The female and male KO exhibited very similar impairments indicating that sex does not generally drive the behavioral symptoms of the disorder. Our results suggest that procedural factors, such as the use of albino mice, may help to reliably detect spatial learning and memory impairments in both sexes of Fmr1 KO mice, making it more useful for understanding FXS and a platform for evaluating potential therapeutics.

Strain difference in the effect of infralimbic cortex lesions on fear extinction in rats.

The infralimbic division of the medial prefrontal cortex (IL) has been implicated in the consolidation and retention of extinction memories. However, the effects of IL lesions on the retention of extinction memory are inconsistent. In the present experiments, we examined whether rat strain influences the effects of IL lesions on extinction. In Experiment 1, Sprague-Dawley (SD) or Long-Evans (LE) rats received a standard auditory fear conditioning procedure, which was followed by an extinction session; freezing served as the index of conditional fear. Our results reveal that focal IL lesions impair the retention of extinction in SD, but not LE rats. In addition to the strain difference in sensitivity to IL lesions, LE rats exhibited significantly higher levels of contextual fear before the outset of extinction training than SD rats. In a second experiment we thus examined whether contextual fear influenced the sensitivity of extinction to IL lesions in LE rats. LE rats received the same conditioning as in Experiment 1, and then were either merely exposed to a novel context or administered unsignaled shocks in that context, followed by extinction and test sessions. Our results reveal that LE rats with IL lesions showed normal extinction regardless of the levels of contextual fear manifest before extinction. Thus, we conclude that rat strain is an important variable that influences the role of infralimbic cortex in fear extinction.

Central CRF receptor antagonist α-helical CRF9-41 blocks reinstatement of extinguished fear: The role of the bed nucleus of the stria terminalis.

The present experiments assessed the necessity of central CRF in reinstatement of extinguished fear. Using the fear-potentiated startle procedure, rats were given light-shock pairings (fear conditioning) followed by light-alone extinction training. Rats were then given unsignaled shocks to reinstate fear to the light conditioned stimulus (CS). Intracerebroventricular administration of the CRF antagonist a-Helical CRF9-41 prior to reinstatement training dose-dependently prevented reinstatement. Further, a-Helical CRF9-41 administration prior to reinstatement training or the test for reinstatement of fear to the extinguished CS prevented reinstatement at both treatment times, suggesting that CRF activity is critical for this type of return of fear to an extinguished CS. The abolition of reinstatement by drug administration was not due to state-dependent learning, as rats treated with the drug prior to both reinstatement training or testing also failed a-Helical CRF9-41 in the bed nucleus of the stria terminalis suggested that this area is a site at which central CRF is involved in this form of relapse.

Dynamic amino acid increases in the basolateral amygdala during acquisition and expression of conditioned fear

Glutamate and gamma-aminobutyric acid (GABA) release in the amygdala are thought to be crucial for the acquisition and expression of fear memories, but the time course of amino acid changes during conditioning is unknown. We used rapid-sampling microdialysis with 14 s temporal resolution to address this issue. During auditory fear conditioning, large, rapid and transient increases in glutamate and GABA were detected, but only during the first noise-shock pairing. In contrast, rats receiving unsignaled shocks during contextual fear conditioning showed no changes in GABA and less glutamate release for the initial shock, but increased glutamate release during later shocks. Expression of conditioned fear to either a white noise or the context previously paired with shock produced similar rapid and transient increases in many amino acids in the amygdala. These experiments demonstrate glutamate and GABA levels in the amygdala are differentially modulated during auditory and contextual fear learning, and are transiently increased during the expression of fear memories.

Blockade of cannabinoid CB 1 receptors alters contextual learning and memory

The endocannabinoid system appears to have an important role in specific aspects of learning and memory, yet there has been no systematic study of the role of cannabinoid receptors in contextual fear conditioning. The present study examined the effects of cannabinoid CB 1 receptor blockade on the acquisition, consolidation, and expression of contextual fear using the selective cannabinoid CB 1 receptor antagonist AM251. AM251 produced a decrease in the expression of contextual fear when administered prior to training, testing, or both. This effect was observed when footshock was signaled by an auditory cue but not in an unsignaled shock version of the task. Moreover, blocking cannabinoid CB 1 receptors had no effect on consolidation of contextual memory regardless of the conditioning paradigm. These data indicate that inhibition of cannabinoid CB 1 receptors produces specific deficits in processing contextual information and that the effects of CB 1 antagonists on contextual learning may differ from effects on other types of learning.

Role of the basolateral amygdala in the storage of fear memories across the adult lifetime of rats.

The basolateral amygdala (BLA) is intimately involved in the development of conditional fear. Converging lines of evidence support a role for this region in the storage of fear memory but do not rule out a time-limited role in the memory consolidation. To examine this issue, we assessed the stability of BLA contribution to fear memories acquired across the adult lifetime of rats. Fear conditioning consisted of 10 tone-shock pairings in one context (remote memory), followed 16 months later by 10 additional tone-shock pairings with a novel tone in a novel context (recent memory). Twenty-four hours after recent training, rats were given NMDA or sham lesions of the BLA. Contextual and tone freezing were independently assessed in individual test sessions. Sham-lesioned rats showed high and comparable levels of freezing across all context and tone tests. In contrast, BLA-lesioned rats displayed robust freezing deficits across both recent and remote tests. Subsequent open-field testing revealed no effects of BLA lesions on activity patterns in a dark open field or during bright light exposure. Lesioned rats were able to reacquire normal levels of context-specific freezing after an overtraining procedure (76 unsignaled shocks). Together, these findings indicate that BLA lesions do not disrupt freezing behavior by producing hyperactivity, an inability to suppress behavior, or an inability to freeze. Rather, the consistent pattern of freezing deficits at both training-to-lesion intervals supports a role for the BLA in the permanent storage of fear memory.

Morphine promotes autonomic learning.

Morphine (0.01-10 mg/kg) promoted rapid autonomic learning of discriminative, Pavlovian conditioned heart rate decelerations to tone signals in male and female rabbits, and the higher doses (1-10 mg/kg) promoted decelerative heart rate orienting reflexes to novel tones. Morphine dose dependently reduced heart rate acceleration to signaled shock but had no effect on heart rate acceleration to unsignaled shock. Morphine did not impair retention of cardiac conditioned reflexes, and its U-shaped dose effect, increasing conditioned heart rate discrimination early in training, reappeared in extinction. The authors propose that morphine promotes autonomic learning of preparatory, compensatory reflexes to signaled stressors that reduce their stressful effects. This action may mimic the normal, adaptive function of an endogenous messenger released by the Pavlovian contingency.

Impairment of conditioned contextual fear of C57BL/6J mice by intracerebral injections of the NMDA receptor antagonist APV

The effects of acute injections of the NMDA receptor antagonist APV on one-trial fear conditioning of C57BL/6J mice were investigated in a time, dose (0.4–3.2 μg) and region-specific manner. Conditioned fear was determined by the assessment of freezing and the computer-controlled measurement of inactivity. Additionally, conditioned heart rate responses were evaluated in the tone-dependent memory test performed in the home cage with the help of implanted ECG transmitters. Injections of APV into the dorsal hippocampus (i.h.) 15 min before training, impaired dose-dependent contextual fear conditioning without discrimination between contextual foreground (unsignaled shock) and background (signaled shock) conditioning as tested 24 h after training. Short-term memory analyzed 1 h after training was also impaired. The observation that a smaller dose of APV was required for intracerebroventricular than for i.h. injection to achieve a similar memory impairment, pointed to the involvement of extrahippocampal NMDA receptors. APV treatment did not affect conditioned tone-dependent fear as indicated by unchanged freezing and heart rate responses of the freely moving mice. The results indicated the contribution of hippocampal and extrahippocampal NMDA receptors to multisensory contextual information processing during acquisition.

Contributions of postrhinal and perirhinal cortex to contextual information processing.

The role of the postrhinal cortex (POR) and the perirhinal cortex (PER) in processing relational or contextual information was examined with Pavlovian fear conditioning. Rats with electrolytic or neurotoxic lesions of the POR or PER were tested in 2 contextual fear conditioning paradigms. In Experiment 1, electrolytic lesions of the POR or PER produced impairments in contextual fear conditioning but not in conditioning to a phasic auditory conditioned stimulus. Neurotoxic lesions of the POR or PER likewise resulted in anterograde (Experiment 2) and retrograde (Experiment 3) deficits in fear conditioning to the training context in an unsignaled shock paradigm. The results suggest that operations performed on sensory information by the POR and PER are necessary to support contextual learning.

Delta-opioid stimulation by BW373U86 promotes autonomic reactivity to stressors and alters attention-related cardiac responses in rabbits.

The nonpeptide delta-opioid agonist BW373U86 (3 to 300 micrograms/kg) was tested in rabbits for effects on heart rate, cardiac orienting and Pavlovian conditioned responses to tones, and unconditioned cardiac and somatomotor responses to signaled and unsignaled shocks. BW373U86 did not alter shock-evoked somatomotor reflexes and had few effects on the development or retention of Pavlovian conditioned heart rate discrimination. However, BW373U86 appeared to modulate cardiac conditioning indirectly, by facilitating sympathetic reflexes evoked by the signaled stressor, and the dose effect was U-shaped within the dose range tested. The pronounced tachycardiac effect of BW373U86 was completely blocked, or rapidly reversed, by the selective delta-opiate antagonist naltrindole. BW373U86 was more potent in increasing signaled than unsignaled shock-evoked tachycardia, suggesting release of an endogenous substance (e.g., a delta-opioid) because of the Pavlovian conditioning contingency.

Neurotoxic lesions of the dorsal hippocampus and Pavlovian fear conditioning in rats

Electrolytic lesions of the dorsal hippocampus (DH) produce deficits in both the acquisition and expression of conditional fear to contextual stimuli in rats. To assess whether damage to DH neurons is responsible for these deficits, we performed three experiments to examine the effects of neurotoxic N-methyl- d-aspartate (NMDA) lesions of the DH on the acquisition and expression of fear conditioning. Fear conditioning consisted of the delivery of signaled or unsignaled footshocks in a novel conditioning chamber and freezing served as the measure of conditional fear. In Experiment 1, posttraining DH lesions produced severe retrograde deficits in context fear when made either 1 or 28, but not 100, days following training. Pretraining DH lesions made 1 week before training did not affect contextual fear conditioning. Tone fear was impaired by DH lesions at all training-to-lesion intervals. In Experiment 2, posttraining (1 day), but not pretraining (1 week), DH lesions produced substantial deficits in context fear using an unsignaled shock procedure. In Experiment 3, pretraining electrolytic DH lesions produced modest deficits in context fear using the same signaled and unsignaled shock procedures used in Experiments 1 and 2, respectively. Electrolytic, but not neurotoxic, lesions also increased pre-shock locomotor activity. Collectively, this pattern of results reveals that neurons in the DH are not required for the acquisition of context fear, but have a critical and time-limited role in the expression of context fear. The normal acquisition and expression of context fear in rats with neurotoxic DH lesions made before training may be mediated by conditioning to unimodal cues in the context, a process that may rely less on the hippocampal memory system.