Role In Fear Conditioning Research Articles

Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to fast synaptic transmission and have roles in fear conditioning and nociception. Apart from activation at low pH, ASIC1a also undergoes several types of desensitization, including acute desensitization, which terminates activation; steady-state desensitization, which occurs at sub-activating proton concentrations and limits subsequent activation; and tachyphylaxis, which results in a progressive decrease in response during a series of activations. Structural insights from a desensitized state of ASIC1 have provided great spatial detail, but dynamic insights into conformational changes in different desensitizing conditions are largely missing. Here, we use electrophysiology and voltage-clamp fluorometry to follow the functional changes of the pore along with conformational changes at several positions in the extracellular and upper transmembrane domain via cysteine-labeled fluorophores. Acute desensitization terminates activation in wild type, but introducing an N414K mutation in the β11-12 linker of mouse ASIC1a interfered with this process. The mutation also affected steady-state desensitization and led to pronounced tachyphylaxis. Although the extracellular domain of this mutant remained sensitive to pH and underwent pH-dependent conformational changes, these conformational changes did not necessarily lead to desensitization. N414K-containing channels also remained sensitive to a known peptide modulator that increases steady-state desensitization, indicating that the mutation only reduced, but not precluded, desensitization. Together, this study contributes to our understanding of the fundamental properties of ASIC1a desensitization, emphasizing the complex interplay between the conformational changes of the extracellular domain and the pore during channel activation and desensitization.

P105 Women with greater post-traumatic stress symptom severity and less disrupted sleep have improved fear extinction recall compared to men

Abstract Sleep has been found to play a key role in fear conditioning, extinction learning and extinction recall, the paradigm modelling the development, maintenance and treatment of anxiety disorders including post-traumatic stress disorder (PTSD), which are disorders commonly associated with sleep disturbances. Previous studies examining associations between sleep and fear or extinction processes predominately focused on objectively measured sleep architecture using polysomnography, and the majority of the research has been done in healthy populations. To date, little research has focused on subjective sleep measures and particularly in clinical populations including PTSD, which often experience subjectively poor sleep. Here we investigated whether subjective sleep disturbance, sleep onset latency, wake after sleep onset or sleep efficiency were related to fear conditioning, extinction learning or extinction recall in a large sample of individuals with a range of PTSD symptom severity and healthy controls. Overall, we did not find that sleep was associated with fear conditioning extinction learning or extinction recall. However, emerging evidence highlighting the critical role of sex in PTSD risk, fear conditioning processes, as well as in the sleep-fear conditioning relationship. Exploratory analyses examining sex-specific effects found that shorter sleep onset latency and greater sleep efficiency during the previous night were associated with improved extinction recall in women with higher PTSD symptom severity. This suggests that less time falling asleep and longer time asleep while in bed may be protective against the commonly observed impaired extinction recall in PTSD in highly symptomatic women, but is not relevant in men.

Sex differences in the effect of subjective sleep on fear conditioning, extinction learning, and extinction recall in individuals with a range of PTSD symptom severity

Sleep has been found to play a key role in fear conditioning, extinction learning and extinction recall, and sleep disturbances are linked to many mental disorders including post-traumatic stress disorder (PTSD). Previous studies examining associations between sleep and fear or extinction processes primarily focused on objectively measured sleep architecture. Little research has so far focused on subjective sleep measures and particularly in clinical populations, which often experience subjectively poor sleep, including PTSD. Here we investigated whether subjective sleep disturbance, sleep onset latency, wake after sleep onset or sleep efficiency were related to fear conditioning, extinction learning or extinction recall in a large sample of individuals with a range of PTSD symptom severity (n = 248). Overall, we did not find that subjective sleep was associated with fear conditioning or extinction processes. However, exploratory analyses examining the moderating effect of sex found that shorter sleep onset latency and greater sleep efficiency were associated with improved extinction recall in women with higher PTSD symptom severity. This suggests that less time falling asleep and longer time asleep while in bed may be protective in highly symptomatic women against the commonly observed impaired extinction recall in PTSD. More studies are needed to explore sex-specific effects further.

It Is All in the Right Amygdala: Increased Synaptic Plasticity and Perineuronal Nets in Male, But Not Female, Juvenile Rat Pups after Exposure to Early-Life Stress.

Early-life stress (ELS) is associated with increased vulnerability to mental disorders. The basolateral amygdala (BLA) plays a critical role in fear conditioning and is extremely sensitive to ELS. Using a naturalistic rodent model of ELS, the limited bedding paradigm (LB) between postnatal days 1-10, we previously documented that LB male, but not female preweaning rat pups display increased BLA neuron spine density paralleled with enhanced evoked synaptic responses and altered BLA functional connectivity. Since ELS effects are often sexually dimorphic and amygdala processes exhibit hemispheric asymmetry, we investigated changes in synaptic plasticity and neuronal excitability of BLA neurons in vitro in the left and right amygdala of postnatal days 22-28 male and female offspring from normal bedding or LB mothers. We report that LB conditions enhanced synaptic plasticity in the right, but not the left BLA of males exclusively. LB males also showed increased perineuronal net density, particularly around parvalbumin (PV) cells, and impaired fear-induced activity of PV interneurons only in the right BLA. Action potentials fired from right BLA neurons of LB females displayed slower maximal depolarization rates and decreased amplitudes compared with normal bedding females, concomitant with reduced NMDAR GluN1 subunit expression in the right BLA. In LB males, reduced GluA2 expression in the right BLA might contribute to the enhanced LTP. These findings suggest that LB differentially programs synaptic plasticity and PV/perineuronal net development in the left and right BLA. Furthermore, our study demonstrates that the effects of ELS exposure on BLA synaptic function are sexually dimorphic and possibly recruiting different mechanisms.SIGNIFICANCE STATEMENT Early-life stress (ELS) induces long-lasting consequences on stress responses and emotional regulation in humans, increasing vulnerability to the development of psychopathologies. The effects of ELS in a number of brain regions, including the amygdala, are often sexually dimorphic, and have been reproduced using the rodent limited bedding paradigm of early adversity. The present study examines sex differences in synaptic plasticity and cellular activation occurring in the developing left and right amygdala after limited bedding exposure, a phenomenon that could shape long-term emotional behavioral outcomes. Studying how ELS selectively produces effects in one amygdala hemisphere during a critical period of brain development could guide further investigation into sex-dependent mechanisms and allow for more targeted and improved treatment of stress-and emotionality-related disorders.

Rho GTPases in the Amygdala-A Switch for Fears?

Fear is a fundamental evolutionary process for survival. However, excess or irrational fear hampers normal activity and leads to phobia. The amygdala is the primary brain region associated with fear learning and conditioning. There, Rho GTPases are molecular switches that act as signaling molecules for further downstream processes that modulate, among others, dendritic spine morphogenesis and thereby play a role in fear conditioning. The three main Rho GTPases—RhoA, Rac1, and Cdc42, together with their modulators, are known to be involved in many psychiatric disorders that affect the amygdala′s fear conditioning mechanism. Rich2, a RhoGAP mainly for Rac1 and Cdc42, has been studied extensively in such regard. Here, we will discuss these effectors, along with Rich2, as a molecular switch for fears, especially in the amygdala. Understanding the role of Rho GTPases in fear controlling could be beneficial for the development of therapeutic strategies targeting conditions with abnormal fear/anxiety-like behaviors.

NMDA receptors in the CeA and BNST differentially regulate fear conditioning to predictable and unpredictable threats

Considerable work demonstrates that Pavlovian fear conditioning depends on N-methyl-D-aspartate (NMDA) receptor-dependent plasticity within the amygdala. In addition, the bed nucleus of the stria terminalis (BNST) has also been implicated in fear conditioning, particularly in the expression of fear to poor predictors of threat. We recently found that the expression of backward (BW) fear conditioning, in which an auditory conditioned stimulus (CS) follows a footshock unconditioned stimulus (US), requires the BNST; the expression of forward (FW) fear conditioning was not disrupted by BNST inactivation. However, whether NMDA receptors within the BNST contribute to the acquisition of fear conditioning is unknown. Moreover, the central nucleus of the amygdala (CeA), which has extensive connections with the BNST, is critically involved in FW conditioning, however whether it participates in BW conditioning has not been explored. Here we test the specific hypothesis that the CeA and the BNST mediate the acquisition of FW and BW fear conditioning, respectively. Adult female and male rats were randomly assigned to receive bilateral infusions of the NMDA receptor antagonist, D,L-2-amino-5-phosphonovalerate (APV), into the CeA or BNST prior to FW or BW fear conditioning. We found that intra-CeA APV impaired the acquisition of both FW and BW conditioning, whereas intra-BNST APV produced selective deficits in BW conditioning. Moreover, APV in the BNST significantly reduced contextual freezing, whereas CeA NMDA receptor antagonism impeded early but not long-lasting contextual fear. Collectively, these data reveal that CeA and BNST NMDA receptors have unique roles in fear conditioning.

The Neurochemical Effects of Prazosin Treatment on Fear Circuitry in a Rat Traumatic Stress Model.

ObjectiveThe timing of administration of pharmacologic agents is crucial in traumatic stress since they can either potentiate the original fear memory or may cause fear extinction depending on the phase of fear conditioning. Brain noradrenergic system has a role in fear conditioning. Data regarding the role of prazosin in traumatic stress are controversial.MethodsIn this study, we examined the effects of prazosin and the noradrenergic system in fear conditioning in a predator stress rat model. We evaluated the direct or indirect effects of stress and prazosin on noradrenaline (NA), gamma-aminobuytyric acid (GABA), glutamate, glycine levels and choline esterase activity in the amygdaloid complex, the dorsal hippocampus, the prefrontal cortex and the rostral pons.ResultsOur results demonstrated that prazosin might alleviate defensive behaviors and traumatic stress symptoms when given during the traumatic cue presentation in the stressed rats. However prazosin administration resulted in higher anxiety levels in non stressed rats when the neutral cue was presented.ConclusionPrazosin should be used in PTSD with caution because prazosin might exacerbate anxiety in non-traumatized subjects. However prazosin might as well alleviate stress responses very effectively. Stress induced changes included increased NA and GABA levels in the amygdaloid complex in our study, attributing noradrenaline a possible inhibitory role on fear acquisition. Acetylcholine also has a role in memory modulation in the brain. We also demonstrated increased choline esterase acitivity. Cholinergic modulation might be another target for indirect prazosin action which needs to be further studied.

Characteristic Alteration of Subcortical Nuclei Shape in Medication-Free Patients with Obsessive-Compulsive Disorder

Background: Subcortical nuclei are important components in the pathology model of obsessive-compulsive disorder (OCD); however, subregions of these structures subserve different functions that may distinctively contribute to OCD symptoms. A subregional-level profile of structural abnormalities of the basal ganglia in OCD is needed for a further understanding of the disorder. Methods: A total of 83 medication-free, non-comorbid OCD patients and 93 age- and sex-matched healthy controls were recruited, and high-resolution T1-weighted MR images were obtained for all participants. The volume and shape of the subcortical nuclei (including the nucleus accumbens, amygdala, caudate, pallidum, putamen and thalamus) were quantified and compared with an automated parcellation approach and vertex-wise shape analysis using FSL-FIRST software. Sex differences in these measurements were also explored with an exploratory subgroup analysis. Results: Volumetric analysis showed no significant differences between patients and healthy control subjects. Relative to healthy control subjects, the OCD patients showed an expansion of the lateral amygdala (right hemisphere) and right pallidum. These deformities were associated with illness duration and symptom severity of OCD. Exploratory subgroup analysis by sex revealed amygdala deformity in male patients and caudate deformity in female patients. Conclusions: The lateral amygdala may contribute to OCD pathology by its role in fear conditioning, and deformity in the subregion of the pallidum suggests a disrupted punishment/reinforcement learning pathway. Our study not only provides deeper insight into how these structures contribute to OCD symptoms by revealing these subregional-level deformities but also suggests that gender effects may be important in OCD studies. Funding Statement: This study was supported by the National Natural Science Foundation (Grant Nos. 81671669 and 81171488) and Youth Technology Grant of Sichuan Province (No. 2017JQ0001). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: This study was approved by the Research Ethics Committee of West China Hospital, Sichuan University, and fully informed written consent was obtained from each participant.

Inhibition of Diacylglycerol Lipase Impairs Fear Extinction in Mice.

Elucidating the underlying molecular mechanisms regulating fear and extinction learning may offer insights that can lead to novel treatments for debilitating anxiety and trauma-related disorders including posttraumatic stress disorder. The endocannabinoid (eCB) system is a retrograde inhibitory signaling pathway involved in regulating central responses to stress. The eCB 2-arachidonoylglycerol (2-AG) has recently been proposed to serve as a homeostatic signal mitigating adverse effects of stress exposure, however, less well understood is 2-AG’s role in fear learning and fear extinction. In this study, we have sought to explore 2-AG’s role in fear conditioning and fear extinction by disrupting 2-AG synthesis utilizing the DAGL inhibitor (DO34) and DAGLα knock-out mice (DAGLα−/−). We found that DAGLα−/− mice, and male and female C57B6/J mice treated with DO34, exhibited impairment in extinction learning in an auditory cue fear-conditioning paradigm. DO34 did not increase unconditioned freezing. Interestingly, inhibition of fatty-acid amide hydrolase was not able to restore normal fear extinction in DO34-treated mice suggesting increased Anandamide cannot compensate for deficient 2-AG signaling in the regulation of fear extinction. Moreover, augmentation of CB1R signaling with tetrahydrocannabinol also failed to restore normal fear extinction in DO34-treated mice. Overall, these data support the hypothesis that DAGLα plays an important role in fear extinction learning. Although genetic and pharmacological disruption of DAGL activity causes widespread lipidomic remodeling, these data combined with previous studies putatively suggest that deficient 2-AG signaling could be a susceptibility endophenotype for the development of trauma-related psychiatric disorders.

Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish

BackgroundFear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala by anatomical and ablation studies, showing a role in conditioned avoidance response. However, the neuronal populations required for a conditioned avoidance response via the Dm have not been functionally or genetically defined.ResultsWe aimed to identify the neuronal population essential for fear conditioning through a genetic approach in zebrafish. First, we performed large-scale gene trap and enhancer trap screens, and created transgenic fish lines that expressed Gal4FF, an engineered version of the Gal4 transcription activator, in specific regions in the brain. We then crossed these Gal4FF-expressing fish with the effector line carrying the botulinum neurotoxin gene downstream of the Gal4 binding sequence UAS, and analyzed the double transgenic fish for active avoidance fear conditioning. We identified 16 transgenic lines with Gal4FF expression in various brain areas showing reduced performance in avoidance responses. Two of them had Gal4 expression in populations of neurons located in subregions of the Dm, which we named 120A-Dm neurons. Inhibition of the 120A-Dm neurons also caused reduced performance in Pavlovian fear conditioning. The 120A-Dm neurons were mostly glutamatergic and had projections to other brain regions, including the hypothalamus and ventral telencephalon.ConclusionsHerein, we identified a subpopulation of neurons in the zebrafish Dm essential for fear conditioning. We propose that these are functional equivalents of neurons in the mammalian pallial amygdala, mediating the conditioned stimulus–unconditioned stimulus association. Thus, the study establishes a basis for understanding the evolutionary conservation and diversification of functional neural circuits mediating fear conditioning in vertebrates.

Neural correlates of subjective CS/UCS association in appetitive conditioning.

Explicit knowledge of conditioned stimulus (CS)/unconditioned stimulus (UCS) associations is proposed as important factor in classical conditioning. However, while previous studies have focused on its roles in fear conditioning, it has been neglected in the context of appetitive conditioning. The present functional magnetic resonance study aimed to investigate neural activation and functional connectivity linked to subjective CS/UCS association in appetitive conditioning. In total, 85 subjects participated in an appetitive acquisition procedure in which a neutral stimulus (CS+) was paired with a monetary reward, while another neutral stimulus (CS-) was never paired with the reward. Directly afterwards, subjective CS/UCS association was assessed by measuring the extent to which the CS+ was thought to be associated with the UCS compared to the CS-. Close relationships were established between subjective CS/UCS association and activations in the primary visual cortex (V1) during the early phase of conditioning and in the striatum during the late conditioning phase. In addition, we observed inverse relationships between subjective CS/UCS association and both V1/ventromedial prefrontal cortex (vmPFC) and striatal/vmPFC connectivity. The results suggest the involvement of decoupling vmPFC connectivity in reward learning in general and the roles of attentional processes in the formation of the subjective CS/UCS association during the early phase and reward prediction during the late phase of appetitive conditioning.

Basolateral Amygdala Neurons Maintain Aversive Emotional Salience.

BLA neurons serve a well-accepted role in fear conditioning and fear extinction. However, the specific learning processes related to their activity at different times during learning remain poorly understood. We addressed this using behavioral tasks isolating distinct aspects of fear learning in male rats. We show that brief optogenetic inhibition of BLA neurons around moments of aversive reinforcement or nonreinforcement causes reductions in the salience of conditioned stimuli, rendering these stimuli less able to be learned about and less able to control fear or safety behaviors. This salience reduction was stimulus-specific, long-lasting, and specific to learning about, or responding to, the same aversive outcome, precisely the goals of therapeutic interventions in human anxiety disorders. Our findings identify a core learning process disrupted by brief BLA optogenetic inhibition. They show that a primary function of the unconditioned stimulus-evoked activity of BLA neurons is to maintain the salience of conditioned stimuli that precede it. This maintenance of salience is a necessary precursor for these stimuli to gain and maintain control over fear and safety behavior.SIGNIFICANCE STATEMENT The amygdala is essential for learning to fear and learning to reduce fear. However, the specific roles served by activity of different amygdala neurons at different times during learning is poorly understood. We used behavioral tasks isolating distinct aspects of learning in rats to show that brief optogenetic inhibition of BLA neurons around moments of reinforcement or nonreinforcement disrupts maintenance of conditioned stimulus salience. This causes a stimulus-specific and long-lasting deficit in the ability of the conditioned stimulus to be learned about or control fear responses. These consequences are the precisely goals of therapeutic interventions in human anxiety disorders. Our findings identify a core learning process disrupted by brief BLA optogenetic inhibition.

Calcium signalling in medial intercalated cell dendrites and spines.

Dendritic and spine calcium imaging in combination with electrophysiology in acute slices revealed that in medial intercalated cells of the amygdala: Action potentials back-propagate into the dendritic tree, but due to the presence of voltage-dependent potassium channels, probably Kv4.2 channels, attenuate over distance. A mixed population of AMPA receptors with rectifying and linear I-V relations are present at individual spines of a single neuron. Decay kinetics and pharmacology suggest tri-heteromeric NMDA receptors at basolateral-intercalated cell synapses. NMDA receptors are the main contributors to spine calcium entry in response to synaptic stimulation. Calcium signals in response to low- and high-frequency stimulation, and in combination with spontaneous action potentials are locally restricted to the vicinity of active spines. Together, these data show that calcium signalling in these GABAergic neurons is tightly controlled and acts as a local signal. The amygdala plays a central role in fear conditioning and extinction. The medial intercalated (mITC) neurons are GABAergic cell clusters interspaced between the basolateral (BLA) and central amygdala (CeA). These neurons are thought to play a key role in fear and extinction, controlling the output of the CeA by feed-forward inhibition. BLA to mITC cell inputs are thought to undergo synaptic plasticity, a mechanism underlying learning, which is mediated by NMDA receptor-dependent mechanisms that require changes in cytosolic calcium. Here, we studied the electrical and calcium signalling properties of mITC neurons in GAD67-eGFP mice using whole-cell patch clamp recordings and two-photon calcium imaging. We show that action potentials back-propagate (bAP) into dendrites, and evoke calcium transients in both the shaft and the dendritic spine. However, bAP-mediated calcium rises in the dendrites attenuate with distance due to shunting by voltage-gated potassium channels. Glutamatergic inputs make dual component synapses on spines. At these synapses, postsynaptic AMPA receptors can have linear or rectifying I-V relationships, indicating that some synapses express GluA2-lacking AMPA receptors. Synaptic NMDA receptors had intermediate decay kinetics, and were only partly blocked by GuN2B selective blockers, indicating these receptors are GluN1/GluN2A/GluN2B trimers. Low- or high-frequency synaptic stimulation raised spine calcium, mediated by calcium influx via NMDA receptors, was locally restricted and did not invade neighbouring spines. Our results show that in mITC neurons, postsynaptic calcium is tightly controlled, and acts as a local signal.

Altered activity of the medial prefrontal cortex and amygdala during acquisition and extinction of an active avoidance task.

Altered medial prefrontal cortex (mPFC) and amygdala function is associated with anxiety-related disorders. While the mPFC-amygdala pathway has a clear role in fear conditioning, these structures are also involved in active avoidance. Given that avoidance perseveration represents a core symptom of anxiety disorders, the neural substrate of avoidance, especially its extinction, requires better understanding. The present study was designed to investigate the activity, particularly, inhibitory neuronal activity in mPFC and amygdala during acquisition and extinction of lever-press avoidance in rats. Neural activity was examined in the mPFC, intercalated cell clusters (ITCs) lateral (LA), basal (BA) and central (CeA) amygdala, at various time points during acquisition and extinction, using induction of the immediate early gene product, c-Fos. Neural activity was greater in the mPFC, LA, BA, and ITC during the extinction phase as compared to the acquisition phase. In contrast, the CeA was the only region that was more activated during acquisition than during extinction. Our results indicate inhibitory neurons are more activated during late phase of acquisition and extinction in the mPFC and LA, suggesting the dynamic involvement of inhibitory circuits in the development and extinction of avoidance response. Together, these data start to identify the key brain regions important in active avoidance behavior, areas that could be associated with avoidance perseveration in anxiety disorders.

Sex differences in the expression of MOR in areas critical for fear conditioning and extinction learning (LB557)

The mu opioid receptor (MOR) has a significant role in fear conditioning and extinction learning. Communication between amygdala, periaqueductal gray (PAG) and medial prefrontal cortex (mPFC) regulates fear conditioning and extinction. Fear conditioned female rats during the metaestrus (M, low hormones) stage of the estrous cycle, showed impaired extinction while rats during the proestrus (P, high hormones) stage showed normal extinction learning. We aim to elucidate how opioid modulators alter MOR expression during fear conditioning and extinction in both sexes. Female rats on M or P stages and males rats underwent auditory fear conditioning and immediately received MOR agonist morphine, or antagonist naloxone or saline. The next day rats received extinction trials. Western blots were used to measure MOR expression. In the PAG, female rats showed no change with either treatment. In male rats morphine produced an increase in MOR expression while naloxone had an opposite effect. In the PFC, morphine during P decreased MOR expression while naloxone had no effect. Increased MOR expression was observed in the amygdala of male rats after either morphine or naloxone treatments, while in females an increase in MOR was only observed during P. Our data portraits sex differences in MOR expression in the fear circuitry, which might be responsible for the persistence of fear responses during low ovarian hormone stages.Grant Funding Source: Supported by Molecular and Genomics core RR003050, Behavioral Core Facilities MD007579 , HPD335698 Nova Southeastern University to Edwin Santini and Dinah Ramos‐Ortolaza

Fimbria–fornix and entorhinal cortex differential contribution to contextual and cued fear conditioning consolidation in rats

The Fimbria–Fornix (FF) and Entorhinal Cortex (EC) are the primary interfaces between the hippocampus and, respectively, subcortical structures and cortical areas. Their mnemonic role has been repeatedly proposed. In order to investigate their role in fear conditioning, FF and EC were subjected to bilateral fully reversible tetrodotoxin (TTX) inactivation during consolidation in adult male Wistar rats that had undergone training for fear conditioning to an acoustic stimulus (CS) and context. TTX was stereotaxically injected into animals of different groups at increasing post-acquisition delays. Memory was assessed as conditioned freezing duration measured during retention testing, performed 72 and 96h after TTX administration in a counterbalanced manner. The results showed that FF inactivation, performed immediately after conditioning, did not disrupt consolidation of either contextual or auditory fear memory. On the contrary, EC inactivation performed at the same time was followed by both contextual and CS fear response retention impairment. EC inactivation performed 1.5h post-acquisition impaired only contextual fear response retention. EC inactivation performed 24h after acquisition training had no effect on the consolidation process. The present findings show a clearly different role of FF and EC in fear conditioning consolidation in the rat. The results are discussed in relation to their known connections with the hippocampus.

D-serine enhances extinction of auditory cued fear conditioning via ERK1/2 phosphorylation in mice

Several lines of evidence suggest that the N-methyl-D-aspartate (NMDA) receptor plays a significant role in fear conditioning and extinction. However, our knowledge of the role of d-serine, an endogenous ligand for the glycine site of the NMDA receptor, in fear extinction is quite limited compared to that of d-cycloserine, an exogenous partial agonist for the same site. In the current study, we examined the effects of d-serine on fear extinction and phosphorylation of extracellular signal-regulated kinase (ERK) in the hippocampus, basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) during the process of fear extinction. Systemic administrations of d-serine (2.7 g/kg, i.p.) with or without the ERK inhibitor SL327 (30 mg/kg, i.p.) to C57BL/6 J mice were performed before fear extinction in a cued fear conditioning and extinction paradigm. Cytosolic and nuclear ERK 1/2 phosphorylation in the hippocampus, BLA, and mPFC were measured 1 h after extinction (E1h), 24 h after extinction (E24h), and 1 h after recall (R1h) by Western blotting. We found that d-serine enhanced the extinction of fear memory, and the effects of d-serine were reduced by the ERK phosphorylation inhibitor SL327. The Western blot analyses showed that d-serine significantly increased cytosolic ERK 2 phosphorylation at E1h in the hippocampus and cytosolic ERK 1/2 phosphorylation at R1h in the BLA. The present study suggested that d-serine might enhance fear extinction through NMDA receptor-induced ERK signaling in mice, and that d-serine has potential clinical importance for the treatment of anxiety disorders.

Involvement of the prelimbic prefrontal cortex on cannabidiol-induced attenuation of contextual conditioned fear in rats

Rationale Systemic administration of cannabidiol (CBD), a non-psychotomimetic component of Cannabis sativa, is able to attenuate cardiovascular and behavioral (freezing) changes induced by re-exposure to a context that had been previously paired with footshocks. The brain sites mediating this effect, however, remain unknown. The medial prefrontal cortex (mPFC) has been related to contextual fear conditioning. Objectives (1) To verify, using c-Fos immunocytochemistry, if the mPFC is involved in the attenuation of contextual fear induced by systemic administration of CBD; (2) to investigate if direct microinjections of CBD into mPFC regions would also attenuate contextual fear. Results Confirming previous results systemic administration of CBD (10 mg/kg) decreased contextual fear and associated c-Fos expression in the prefrontal cortex (prelimbic and infralimbic regions). The drug also attenuated c-Fos expression in the bed nucleus of the stria terminalis (BNST). Direct CBD (30 nmol) microinjection into the PL prefrontal cortex reduced freezing induced by re-exposure to the aversively conditioned context. In the infralimbic (IL) prefrontal cortex, however, CBD (30 nmol) produced an opposite result, increasing the expression of contextual fear conditioning. This result was confirmed by an additional experiment where the conditioning session was performed under a less aversive protocol. Conclusion These results suggest that the PL prefrontal cortex may be involved in the attenuation of contextual fear induced by systemic injection of CBD. They also support the proposition that the IL and PL play opposite roles in fear conditioning. A possible involvement of the BNST in CBD effects needs to be further investigated.

Bidirectional modulation of classical fear conditioning in mice by 5-HT 1A receptor ligands with contrasting intrinsic activities

5-HT 1A receptors are implicated in the modulation of cognitive processes including encoding of fear learning. However, their exact role has remained unclear due to contrasting contributions of pre- vs. postsynaptic 5-HT 1A receptors. Therefore, their role in fear conditioning was studied in mice using the selective ligand S15535, which fully activates 5-HT 1A autoreceptors, yet only weakly stimulates their postsynaptic counterparts. The effects of S15535 were compared to those of the full agonist 8-OH-DPAT and the selective antagonist NAD-299. 8-OH-DPAT dose-dependently (0.01–0.5 mg/kg) and markedly impaired both context- and tone-dependent fear conditioning, as determined by complementary measures of inactivity and freezing. 8-OH-DPAT-mediated impairments were blocked by pre-injection of the selective 5-HT 1A antagonist WAY100635. S15535 (0.01–5.0 mg/kg) mimicked 8-OH-DPAT in predominantly impairing conditioned contextual fear, though with smaller effect size than 8-OH-DPAT, consistent with lower efficacy at postsynaptic 5-HT 1A receptors. Furthermore, S15535 (1.0 mg/kg) tended to attenuate the impairment of fear conditioning by 8-OH-DPAT (0.3 mg/kg). In contrast, NAD-299 (0.3 and 1 mg/kg) facilitated contextual freezing. WAY100635 (0.3 mg/kg) prevented the impairment of contextual fear by S15535 (1 and 5 mg/kg), underpinning the role of 5-HT 1A receptors in the actions of S15535. Collectively, these data indicate that 5-HT 1A receptor ligands modulate fear conditioning in a bidirectional manner: activation of postsynaptic 5-HT 1A sites exerts an inhibitory influence, whereas their blockade promote facilitation of fear conditioning. The results with S15535 underscore the importance of ligand efficacy in determining the actions of 5-HT 1A receptor ligands in fear conditioning and other models of cognitive function.

Lack of association between the amiloride-sensitive cation channel 2 (ACCN2) gene and anxiety spectrum disorders

Ion channels are involved in a wide range of central nervous system functions and have been implicated in several neuropsychiatric disorders. Rodent studies suggest that the acid-sensing ion channel, ASIC1, may play a role in fear conditioning, a model for human anxiety disorders. In this study, we examined, for the first time, the human analog of ASIC1, the amiloride-sensitive cation channel 2 (ACCN2) gene, for its association with genetic risk across a range of anxiety spectrum phenotypes. Using multivariate structural equation modeling, we selected twin pairs scoring at the extremes of a latent genetic risk factor that underlies susceptibility to neuroticism, major depression, generalized anxiety disorder, panic disorder, agoraphobia, and social phobia, from the population-based Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. One member from each selected pair for whom DNA was available was entered into a 2-stage, case-control association study for the ACCN2 gene. In the resulting sample of 589 cases and 539 controls, a total of seven single nucleotide polymorphisms that represented the major allelic variation across the ACCN2 locus were screened in stage 1, the positive results of which were tested for replication in stage 2. Although several markers or haplotypic combinations met threshold significance criteria in stage 1, their association was not replicated in stage 2. Post hoc analyses did not reveal significant association to the specific psychiatric phenotypes. Although the ACCN2 gene may play a role in rodent fear conditioning, we could not detect association with genetic risk shared among human anxiety spectrum disorders.