Auditory-cued Fear Conditioning Research Articles

Infralimbic activity during REM sleep facilitates fear extinction memory

Rapid eye movement (REM) sleep is known to facilitate fear extinction and play a protective role against fearful memories.1,2 Consequently, disruption of REM sleep after a traumatic event may increase the risk for developing PTSD.3,4 However, the underlying mechanisms by which REM sleep promotes extinction of aversive memories remain largely unknown. The infralimbic cortex (IL) is a key brain structure for the consolidation of extinction memory.5 Using calcium imaging, we found in mice that most IL pyramidal neurons are intensively activated during REM sleep. Optogenetically suppressing the IL specifically during REM sleep within a 4-h window after auditory-cued fear conditioning impaired extinction memory consolidation. In contrast, REM-specific IL inhibition after extinction learning did not affect the extinction memory. Whole-cell patch-clamp recordings demonstrated that inactivating IL neurons during REM sleep depresses their excitability. Together, our findings suggest that REM sleep after fear conditioning facilitates fear extinction by enhancing IL excitability and highlight the importance of REM sleep in the aftermath of traumatic events for protecting against traumatic memories.

Restoration of brain dystrophin using tricyclo-DNA ASOs restores neurobehavioral deficits in DMD mice

Restoration of brain dystrophin using tricyclo-DNA ASOs restores neurobehavioral deficits in DMD mice

Psilocybin facilitates fear extinction in mice by promoting hippocampal neuroplasticity.

Posttraumatic stress disorder (PTSD) and depression are highly comorbid. Psilocybin exerts substantial therapeutic effects on depression by promoting neuroplasticity. Fear extinction is a key process in the mechanism of first-line exposure-based therapies for PTSD. We hypothesized that psilocybin would facilitate fear extinction by promoting hippocampal neuroplasticity. First, we assessed the effects of psilocybin on percentage of freezing time in an auditory cued fear conditioning (FC) and fear extinction paradigm in mice. Psilocybin was administered 30 min before extinction training. Fear extinction testing was performed on the first day; fear extinction retrieval and fear renewal were tested on the sixth and seventh days, respectively. Furthermore, we verified the effect of psilocybin on hippocampal neuroplasticity using Golgi staining for the dendritic complexity and spine density, Western blotting for the protein levels of brain derived neurotrophic factor (BDNF) and mechanistic target of rapamycin (mTOR), and immunofluorescence staining for the numbers of doublecortin (DCX)- and bromodeoxyuridine (BrdU)-positive cells. A single dose of psilocybin (2.5 mg/kg, i.p.) reduced the increase in the percentage of freezing time induced by FC at 24 h, 6th day and 7th day after administration. In terms of structural neuroplasticity, psilocybin rescued the decrease in hippocampal dendritic complexity and spine density induced by FC; in terms of neuroplasticity related proteins, psilocybin rescued the decrease in the protein levels of hippocampal BDNF and mTOR induced by FC; in terms of neurogenesis, psilocybin rescued the decrease in the numbers of DCX- and BrdU-positive cells in the hippocampal dentate gyrus induced by FC. A single dose of psilocybin facilitated rapid and sustained fear extinction; this effect might be partially mediated by the promotion of hippocampal neuroplasticity. This study indicates that psilocybin may be a useful adjunct to exposure-based therapies for PTSD and other mental disorders characterized by failure of fear extinction.

Immunization with a heat-killed preparation of Mycobacterium vaccae NCTC 11659 enhances auditory-cued fear extinction in a stress-dependent manner

Stress-related psychiatric disorders including anxiety disorders, mood disorders, and trauma and stressor-related disorders, such as posttraumatic stress disorder (PTSD), affect millions of people world-wide each year. Individuals with stress-related psychiatric disorders have been found to have poor immunoregulation, increased proinflammatory markers, and dysregulation of fear memory. The “Old Friends” hypothesis proposes that a lack of immunoregulatory inputs has led to a higher prevalence of inflammatory disorders and stress-related psychiatric disorders, in which inappropriate inflammation is thought to be a risk factor. Immunization with a soil-derived saprophytic bacterium with anti-inflammatory and immunoregulatory properties, Mycobacterium vaccae NCTC 11659, can lower proinflammatory biomarkers, increase stress resilience, and, when given prior to or after fear conditioning in a rat model of fear-potentiated startle, enhance fear extinction. In this study, we investigated whether immunization with heat-killed M. vaccae NCTC 11659 would enhance fear extinction in contextual or auditory-cued fear conditioning paradigms and whether M. vaccae NCTC 11659 would prevent stress-induced exaggeration of fear expression or stress-induced resistance to extinction learning. Adult male Sprague Dawley rats were immunized with M. vaccae NCTC 11659 (subcutaneous injections once a week for three weeks), and underwent either: Experiment 1) one-trial contextual fear conditioning; Experiment 2) two-trial contextual fear conditioning; Experiment 3) stress-induced enhancement of contextual fear conditioning; Experiment 4) stress-induced enhancement of auditory-cued fear conditioning; or Experiment 5) stress-induced enhancement of auditory-cued fear conditioning exploring short-term memory. Immunizations with M. vaccae NCTC 11659 had no effect on one- or two-trial contextual fear conditioning or contextual fear extinction, with or without exposure to inescapable stress. However, inescapable stress increased resistance to auditory-cued fear extinction. Immunization with M. vaccae NCTC 11659 prevented the stress-induced increase in resistance to auditory-cued fear extinction learning. Finally, in an auditory-cued fear conditioning paradigm exploring short-term memory and fear acquisition, immunization with M. vaccae did not prevent fear acquisition, either with or without exposure to inescapable stress, consistent with the hypothesis that M. vaccae NCTC 11659 has no effect on fear acquisition but enhances fear extinction. These data are consistent with the hypothesis that increased immunoregulation following immunization with M. vaccae NCTC 11659 promotes stress resilience, in particular by preventing stress-induced resistance to fear extinction, and may be a potential therapeutic intervention for trauma- and stressor-related disorders such as PTSD.

Adolescent Stress Confers Resilience to Traumatic Stress Later in Life: Role of the Prefrontal Cortex

BackgroundAdolescent brains are sensitive to stressors. However, under certain circumstances, developmental stress can promote an adaptive phenotype, allowing individuals to cope better with adverse situations in adulthood, thereby contributing to resilience. MethodsSprague Dawley rats (50 males, 48 females) were subjected to adolescent chronic variable stress (adol CVS) for 2 weeks at postnatal day 45. At postnatal day 85, a group was subjected to single prolonged stress (SPS). After a week, animals were evaluated in an auditory-cued fear conditioning paradigm, and neuronal recruitment during reinstatement was assessed by Fos expression. Patch clamp electrophysiology (17–35 cells/group) was performed in male rats to examine physiological changes associated with resilience. ResultsAdol CVS blocked fear potentiation evoked by SPS. We observed that SPS impaired extinction (males) and enhanced reinstatement (both sexes) of the conditioned freezing response. Prior adol CVS prevented both effects. SPS effects were associated with a reduction of infralimbic (IL) cortex neuronal recruitment after reinstatement in males and increased engagement of the central amygdala in females, both also prevented by adol CVS, suggesting different neurocircuits involved in generating resilience between sexes. We explored the mechanism behind reduced IL recruitment in males by studying the intrinsic excitability of IL pyramidal neurons. SPS reduced excitability of IL neurons, and prior adol CVS prevented this effect. ConclusionsOur data indicate that adolescent stress can impart resilience to the effects of traumatic stress on neuroplasticity and behavior. Our data provide a mechanistic link behind developmental stress-induced behavioral resilience and prefrontal (IL) cortical excitability in males.

D1 Receptor Mediated Dopaminergic Neurotransmission Facilitates Remote Memory of Contextual Fear Conditioning.

Dopaminergic neurotransmission via dopamine D1 receptors (D1Rs) is considered to play an important role not only in reward-based learning but also in aversive learning. The contextual and auditory cued fear conditioning tests involve the processing of classical fear conditioning and evaluates aversive learning memory. It is possible to evaluate aversive learning memory in two different types of neural transmission circuits. In addition, when evaluating the role of dopaminergic neurotransmission via D1R, to avoid the effects in D1R-mediated neural circuitry alterations during development, it is important to examine using mice who D1R expression in the mature stage is suppressed. Herein, we investigated the role of dopaminergic neurotransmission via D1Rs in aversive memory formation in contextual and auditory cued fear conditioning tests using D1R knockdown (KD) mice, in which the expression of D1Rs could be conditionally and reversibly controlled with doxycycline (Dox) treatment. For aversive memory, we examined memory formation using recent memory 1 day after conditioning, and remote memory 4 weeks after conditioning. Furthermore, immunostaining of the brain tissues of D1RKD mice was performed after aversive footshock stimulation to investigate the distribution of activated c-Fos, an immediate-early gene, in the hippocampus (CA1, CA3, dentate gyrus), striatum, amygdala, and prefrontal cortex during aversive memory formation. After aversive footshock stimulation, immunoblotting was performed using hippocampal, striatal, and amygdalar samples from D1RKD mice to investigate the increase in the amount of c-Fos and phosphorylated SNAP-25 at Ser187 residue. When D1R expression was suppressed using Dox, behavioral experiments revealed impaired contextual fear learning in remote aversion memory following footshock stimulation. Furthermore, expression analysis showed a slight increase in the post-stimulation amount of c-Fos in the hippocampus and striatum, and a significant increase in the amount of phosphorylated SNAP-25 in the hippocampus, striatum, and prefrontal cortex before and after stimulation. These findings indicate that deficiency in D1R-mediated dopaminergic neurotransmission is an important factor in impairing contextual fear memory formation for remote memory.

Learning-induced biases in the ongoing dynamics of sensory representations predict stimulus generalization.

Sensory stimuli have long been thought to be represented in the brain as activity patterns of specific neuronal assemblies. However, we still know relatively little about the long-term dynamics of sensory representations. Using chronic invivo calcium imaging in the mouse auditory cortex, we find that sensory representations undergo continuous recombination, even under behaviorally stable conditions. Auditory cued fear conditioning introduces a bias into these ongoing dynamics, resulting in a long-lasting increase in the number of stimuli activating the same subset of neurons. This plasticity is specific for stimuli sharing representational similarity to the conditioned sound prior to conditioning and predicts behaviorally observed stimulus generalization. Our findings demonstrate that learning-induced plasticity leading to a representational linkage between the conditioned stimulus and non-conditioned stimuli weaves into ongoing dynamics of the brain rather than acting on an otherwise static substrate.

Male, but not female, Sprague Dawley rats display enhanced fear learning following acute ethanol withdrawal (hangover)

The present studies investigated the effects of withdrawal from a single binge-like dose of ethanol (hangover) on fear conditioning in male and female Sprague Dawley rats. In Experiment 1, males and females were given 0 or 3.5 g/kg ethanol intraperitoneally (i.p.) and then conditioned to contextual fear 24 h post injection. Withdrawal from acute ethanol enhanced expression of the conditioned freezing response in males, but not in females. Experiment 2 demonstrated that in males, withdrawal from acute ethanol administered 24 h prior to conditioning enhanced contextual fear conditioning, but not auditory-cued fear conditioning. In Experiment 3, male and female rats were given 3.5 g/kg ethanol, and blood ethanol concentrations (BECs) were assessed at various time points for determination of ethanol clearance. Female rats cleared ethanol at a higher rate than males, with 10 h required for females and 14 for males to eliminate ethanol from their systems. Because females cleared ethanol faster than males, in Experiment 4, females were conditioned 18 h after ethanol administration to keep the interval between ethanol clearance and fear conditioning similar to that of males. Withdrawal from acute ethanol given 18 h prior to conditioning did not affect both contextual and auditory-cued fear conditioning in females. In summary, these results highlight sex differences in the impact of withdrawal from acute ethanol (hangover) on fear learning; suggesting that males are more sensitive to hangover-associated enhancement of negative affect than females.

Rapid nucleus-scale reorganization of chromatin in neurons enables transcriptional adaptation for memory consolidation.

The interphase nucleus is functionally organized in active and repressed territories defining the transcriptional status of the cell. However, it remains poorly understood how the nuclear architecture of neurons adapts in response to behaviorally relevant stimuli that trigger fast alterations in gene expression patterns. Imaging of fluorescently tagged nucleosomes revealed that pharmacological manipulation of neuronal activity in vitro and auditory cued fear conditioning in vivo induce nucleus-scale restructuring of chromatin within minutes. Furthermore, the acquisition of auditory fear memory is impaired after infusion of a drug into auditory cortex which blocks chromatin reorganization in vitro. We propose that active chromatin movements at the nucleus scale act together with local gene-specific modifications to enable transcriptional adaptations at fast time scales. Introducing a transgenic mouse line for photolabeling of histones, we extend the realm of systems available for imaging of chromatin dynamics to living animals.

Clmp Regulates AMPA and Kainate Receptor Responses in the Neonatal Hippocampal CA3 and Kainate Seizure Susceptibility in Mice

Synaptic adhesion molecules regulate synapse development through trans-synaptic adhesion and assembly of diverse synaptic proteins. Many synaptic adhesion molecules positively regulate synapse development; some, however, exert negative regulation, although such cases are relatively rare. In addition, synaptic adhesion molecules regulate the amplitude of post-synaptic receptor responses, but whether adhesion molecules can regulate the kinetic properties of post-synaptic receptors remains unclear. Here we report that Clmp, a homophilic adhesion molecule of the Ig domain superfamily that is abundantly expressed in the brain, reaches peak expression at a neonatal stage (week 1) and associates with subunits of AMPA receptors (AMPARs) and kainate receptors (KARs). Clmp deletion in mice increased the frequency and amplitude of AMPAR-mediated miniature excitatory post-synaptic currents (mEPSCs) and the frequency, amplitude, and decay time constant of KAR-mediated mEPSCs in hippocampal CA3 neurons. Clmp deletion had minimal impacts on evoked excitatory synaptic currents at mossy fiber-CA3 synapses but increased extrasynaptic KAR, but not AMPAR, currents, suggesting that Clmp distinctly inhibits AMPAR and KAR responses. Behaviorally, Clmp deletion enhanced novel object recognition and susceptibility to kainate-induced seizures, without affecting contextual or auditory cued fear conditioning or pattern completion-based contextual fear conditioning. These results suggest that Clmp negatively regulates hippocampal excitatory synapse development and AMPAR and KAR responses in the neonatal hippocampal CA3 as well as object recognition and kainate seizure susceptibility in mice.

REM sleep promotes experience-dependent dendritic spine elimination in the mouse cortex

In many parts of the nervous system, experience-dependent refinement of neuronal circuits predominantly involves synapse elimination. The role of sleep in this process remains unknown. We investigated the role of sleep in experience-dependent dendritic spine elimination of layer 5 pyramidal neurons in the visual (V1) and frontal association cortex (FrA) of 1-month-old mice. We found that monocular deprivation (MD) or auditory-cued fear conditioning (FC) caused rapid spine elimination in V1 or FrA, respectively. MD- or FC-induced spine elimination was significantly reduced after total sleep or REM sleep deprivation. Total sleep or REM sleep deprivation also prevented MD- and FC-induced reduction of neuronal activity in response to visual or conditioned auditory stimuli. Furthermore, dendritic calcium spikes increased substantially during REM sleep, and the blockade of these calcium spikes prevented MD- and FC-induced spine elimination. These findings reveal an important role of REM sleep in experience-dependent synapse elimination and neuronal activity reduction.

Persistence of Fear Memory Depends on a Delayed Elevation of BAF53b and FGF1 Expression in the Lateral Amygdala.

Endurance represents a highly adaptive function of fear memory and a major cause of maladaptive fear- and anxiety-related mental disorders. However, less is known about the mechanisms underlying the persistence of fear memory. The epigenetic gene regulation recently emerged as an important mechanism for memory persistence. In the previous study, we found that BAF53b, a neuron-specific subunit of BAF chromatin remodeling complex, is induced after auditory cued fear conditioning in the lateral amygdala (LA) and is crucial for recent fear memory formation. In this study using mice of both sexes, we report a delayed induction of BAF53b in the LA 48 h after auditory fear conditioning and its critical role for the persistence of established fear memory. To specifically block the delayed but not the early induced BAF53b function, we used a postlearning knock-down method based on RNAi. The transient knockdown of Baf53b using siRNA in the lateral amygdala 24 h after cued fear conditioning led to specific impairment of remote but not recent memory retrieval. RNA-sequencing analyses identified fibroblast growth factor 1 (FGF1) as a candidate downstream effector. Consistently, postlearning administration of FGF1 peptide rescued memory persistence in Baf53b knock-down mice. These results demonstrate the crucial role of BAF53b and FGF1 in persistent retention of fear memory, giving insights into how fear memory persistently stored through consolidation processes and suggest candidate target for treating mental disorders related to traumatic memory.SIGNIFICANCE STATEMENT It is still unclear how once consolidated memory persists over time. In this study, we report the delayed induction of nucleosome remodeling factor BAF53b in the lateral nucleus of amygdala after fear learning and its crucial role for persistence of established memory beyond 24 h after learning. Our data link the regulation of BAF53b and fibroblast growth factor 1 expression in the amygdala to fear memory persistence. Results from this study open a new direction to understand the time-dependent continuous consolidation processes potentially by a nucleosome-remodeling mechanism enabling long-lasting memory formation and give insights into how to treat mental disorders caused by enduring traumatic memory.

A critical role of hippocampus for formation of remote cued fear memory

A unique feature of fear memory is its persistence that is highly relevant to fear and anxiety-related mental disorders. Recurrent reactivation of neural representations acquired from a traumatic event is thought to contribute to the indelibility of fear memory. Given a well-established role of hippocampus for memory reactivation, hippocampus is likely involved in consolidation process of fear memory. However, evidence suggests that formation of fear memory to a discrete sensory cue is hippocampus-independent. Here, using a pharmacological reversible inactivation of dorsal hippocampus in auditory cued fear conditioning by local infusion of muscimol, we demonstrate in mice that hippocampus is critical for remote memory formation of learned fear to the discrete sensory cue. Muscimol infusion before conditioning did not affect formation of recent auditory fear memory as previously reported. Same muscimol infusion, however, impaired remote auditory fear memory. Muscimol infusion before remote test of auditory fear memory did not affect memory retrieval, indicating hippocampus is not a brain site for storage of remote cued fear memory. Moreover, memory reactivation enforced by re-exposure to the conditioned tone could compensate for hippocampal inactivation, as memory-reactivated mice showed normal remote auditory fear memory despite hippocampal inactivation. Our findings support that hippocampus may have a general role for consolidation of remote associative memory through reactivation of memory trace, giving an insight into how learned fear persists over time.

Cued Fear Conditioning in Carioca High- and Low-Conditioned Freezing Rats.

Anxiety disorders (AD) comprise a broad range of psychiatric conditions, including general anxiety (GAD) and specific phobias. For the last decades, the use of animal models of anxiety has offered important insights into the understanding of the association between these psychopathologies. Here, we investigate whether Carioca high- and low-conditioned freezing rats (CHF and CLF, respectively), a GAD animal model of anxiety, show similar high- and low-freezing behavioral phenotypes for cued auditory fear conditioning. Adult CHF (n = 16), CLF (n = 16) and normal age-matched Wistar rats (control, CTL, n = 16) were tested in a classical auditory-cued fear conditioning paradigm over 3 days (Tone + Shock and Tone only groups, n = 8 per treatment). Freezing responses were measured and used as evidence of fear conditioning. Overall, both CHF and CLF rats, as well as CTL animals displayed fear conditioning to the auditory CS. However, CLF animals showed a rapid extinction to the auditory conditioned stimulus compared to CHF and CTL rats. We discuss these findings in the context of the behavioral and neuronal differences observed in rodent lines of high and low anxiety traits.

Fear conditioning and extinction induce opposing changes in dendritic spine remodeling and somatic activity of layer 5 pyramidal neurons in the mouse motor cortex

Multiple brain regions including the amygdala and prefrontal cortex are crucial for modulating fear conditioning and extinction. The primary motor cortex is known to participate in the planning, control, and execution of voluntary movements. Whether and how the primary motor cortex is involved in modulating freezing responses related to fear conditioning and extinction remains unclear. Here we show that inactivation of the mouse primary motor cortex impairs both the acquisition and extinction of freezing responses induced by auditory-cued fear conditioning. Fear conditioning significantly increases the elimination of dendritic spines on apical dendrites of layer 5 pyramidal neurons in the motor cortex. These eliminated spines are further apart from each other than expected from random distribution along dendrites. On the other hand, fear extinction causes the formation of new spines that are located near the site of spines eliminated previously after fear conditioning. We further show that fear conditioning decreases and fear extinction increases somatic activities of layer 5 pyramidal neurons in the motor cortex respectively. Taken together, these findings indicate fear conditioning and extinction induce opposing changes in synaptic connections and somatic activities of layer 5 pyramidal neurons in the primary motor cortex, a cortical region important for the acquisition and extinction of auditory-cued conditioned freezing responses.

Fear extinction reverses dendritic spine formation induced by fear conditioning in the mouse auditory cortex

Fear conditioning-induced behavioral responses can be extinguished after fear extinction. While fear extinction is generally thought to be a form of new learning, several lines of evidence suggest that neuronal changes associated with fear conditioning could be reversed after fear extinction. To better understand how fear conditioning and extinction modify synaptic circuits, we examined changes of postsynaptic dendritic spines of layer V pyramidal neurons in the mouse auditory cortex over time using transcranial two-photon microscopy. We found that auditory-cued fear conditioning induced the formation of new dendritic spines within 2 days. The survived new spines induced by fear conditioning with one auditory cue were clustered within dendritic branch segments and spatially segregated from new spines induced by fear conditioning with a different auditory cue. Importantly, fear extinction preferentially caused the elimination of newly formed spines induced by fear conditioning in an auditory cue-specific manner. Furthermore, after fear extinction, fear reconditioning induced reformation of new dendritic spines in close proximity to the sites of new spine formation induced by previous fear conditioning. These results show that fear conditioning, extinction, and reconditioning induce cue- and location-specific dendritic spine remodeling in the auditory cortex. They also suggest that changes of synaptic connections induced by fear conditioning are reversed after fear extinction.

Glucocorticoid receptor-mediated amygdalar metaplasticity underlies adaptive modulation of fear memory by stress

Glucocorticoid receptor (GR) is crucial for signaling mediated by stress-induced high levels of glucocorticoids. The lateral nucleus of the amygdala (LA) is a key structure underlying auditory-cued fear conditioning. Here, we demonstrate that genetic disruption of GR in the LA (LAGRKO) resulted in an auditory-cued fear memory deficit for strengthened conditioning. Furthermore, the suppressive effect of a single restraint stress (RS) prior to conditioning on auditory-cued fear memory in floxed GR (control) mice was abolished in LAGRKO mice. Optogenetic induction of long-term depression (LTD) at auditory inputs to the LA reduced auditory-cued fear memory in RS-exposed LAGRKO mice, and in contrast, optogenetic induction of long-term potentiation (LTP) increased auditory-cued fear memory in RS-exposed floxed GR mice. These findings suggest that prior stress suppresses fear conditioning-induced LTP at auditory inputs to the LA in a GR-dependent manner, thereby protecting animals from encoding excessive cued fear memory under stress conditions.

Cued fear memory generalization increases over time.

Fear memory is a highly stable and durable form of memory, even over vast (remote) time frames. Nevertheless, some elements of fear memory can be forgotten, resulting in generalization. The purpose of this study is to determine how cued fear memory generalizes over time and measure underlying patterns of cortico-amygdala synaptic plasticity. We established generalization gradients at recent (1-d) and remote (30-d) retention intervals following auditory cued fear conditioning in adult male C57BL/6 mice. Results revealed a flattening of the generalization gradient (increased generalization) that was dissociated from contextual fear generalization, indicating a specific influence of time on cued fear memory performance. This effect reversed after a brief exposure to the novel stimulus soon after learning. Measurements from cortico-amygdala imaging of the activity-regulated cytoskeletal Arc/arg 3.1 (Arc) protein using immunohistochemistry after cued fear memory retrieval revealed a stable pattern of Arc expression in the dorsolateral amygdala, but temporally dynamic expression in the cortex. Over time, increased fear memory generalization was associated with a reduction in Arc expression in the agranular insular and infralimbic cortices while discrimination learning was associated with increased Arc expression in the prelimbic cortex. These data identify the dorsolateral amygdala, medial prefrontal, and insular cortices as loci for synaptic plasticity underlying cued fear memory generalization over time.

NMDA receptors in the avian amygdala and the premotor arcopallium mediate distinct aspects of appetitive extinction learning

Extinction learning is an essential mechanism that enables constant adaptation to ever-changing environmental conditions. The underlying neural circuit is mostly studied with rodent models using auditory cued fear conditioning. In order to uncover the variant and the invariant neural properties of extinction learning, we adopted pigeons as an animal model in an appetitive sign-tracking paradigm. The animals firstly learned to respond to two conditioned stimuli in two different contexts (CS-1 in context A and CS-2 in context B), before conditioned responses to the stimuli were extinguished in the opposite contexts (CS-1 in context B and CS-2 in context A). Subsequently, responding to both stimuli was tested in both contexts. Prior to extinction training, we locally injected the N-methyl-d-aspartate receptor (NMDAR) antagonist 2-Amino-5-phosphonovaleric acid (APV) in either the amygdala or the (pre)motor arcopallium to investigate their involvement in extinction learning. Our findings suggest that the encoding of extinction memory required the activation of amygdala, as visible by an impairment of extinction acquisition by concurrent inactivation of local NMDARs. In contrast, consolidation and subsequent retrieval of extinction memory recruited the (pre)motor arcopallium. Also, the inactivation of arcopallial NMDARs induced a general motoric slowing during extinction training. Thus, our results reveal a double dissociation between arcopallium and amygdala with respect to acquisition and consolidation of extinction, respectively. Our study therefore provides new insights on the two key components of the avian extinction network and their resemblance to the data obtained from mammals, possibly indicating a shared neural mechanism underlying extinction learning shaped by evolution.

S191 Cerebellar contributions to fear behaviour

Various regions of the midbrain and cerebrum are known to play an important role in fear behaviour. This talk will present evidence that the cerebellum should also be added to this network. Electrophysiological, lesioning and behavioural techniques in rats were used to study interactions between the cerebellum and the midbrain periaqueductal grey (PAG). Freezing was used as a behavioural measure to quantify fear before and after localized cerebellar lesions and was initiated innately e.g. via the predator odour test, or learned via auditory cued aversive foot shock fear conditioning. Electrodes were also implanted into the PAG and cerebellar nuclei, and neuronal activity was recorded during these fear paradigms. Our research has shown that a powerful physiological connection exists between the ventrolateral PAG and vermal lobule VIII (pyramis) of the cerebellum. Lesioning the pyramis disrupts innate and fear-conditioned freezing behaviour. In response to auditory cued fear conditioning there is an increase in beta and gamma local field potential activity in the cerebellar nuclei during presentation of the conditioned tone. Further work is needed to understand how cerebellar computations play a role in the affective and motor aspects of fear evoked freezing behaviour. The cerebellar- PAG connection is a critical component of the neural network subserving emotionally related freezing behaviour. These results highlight a role for the cerebellum in the neural networks underlying fear behaviour, raising the possibility of a new therapeutic target for fear related disorders such as post-traumatic stress disorder.