Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.     

Induction of immediate early genes in the mouse auditory cortex after auditory cued fear conditioning to complex sounds

Immediate early genes (IEGs) are widely used as markers to delineate neuronal circuits because they show fast and transient expression induced by various behavioral paradigms. In this study, we investigated the expression of the IEGs c-fos and Arc in the auditory cortex of the mouse after auditory cued fear conditioning using quantitative polymerase chain reaction and microarray analysis. To test for the specificity of the IEG induction, we included several control groups that allowed us to test for factors other than associative learning to sounds that could lead to an induction of IEGs. We found that both c-fos and Arc showed strong and robust induction after auditory fear conditioning. However, we also observed increased expression of both genes in any control paradigm that involved shocks, even when no sounds were presented. Using mRNA microarrays and comparing the effect of the various behavioral paradigms on mRNA expression levels, we did not find genes being selectively upregulated in the auditory fear conditioned group. In summary, our results indicate that the use of IEGs to identify neuronal circuits involved specifically in processing of sound cues in the fear conditioning paradigm can be limited by the effects of the aversive unconditional stimulus and that activity levels in a particular primary sensory cortical area can be strongly influenced by stimuli mediated by other modalities.     

Estrogen facilitates fear conditioning and increases corticotropin-releasing hormone mRNA expression in the central amygdala in female mice

Estrogens exert important actions on fear and anxiety-like behavior both in humans and non-human animals. Currently, the mechanisms underlying estrogenic modulation of fear are not known. However, evidence suggests that estrogens may exert their influence on fear within the amygdala. The purpose of the present study was to examine effects of estrogen on fear conditioning. Specifically, the present study examined whether long-term estrogen treatment in ovariectomized female mice via Silastic capsule implantation would facilitate both contextual and cued fear conditioning. In a separate set of experiments, we then examined whether estrogen treatment in ovariectomized female mice would modulate corticotropin-releasing hormone (CRH) gene expression within the amygdala. Long-term estrogen treatment facilitated both contextual and cued fear. Ovariectomized mice treated with estrogen froze significantly more to a context as well as to a discrete auditory cue. In addition, estrogen treatment significantly increased CRH mRNA expression within the central nucleus of the amygdala as measured by in situ hybridization and quantitative PCR. These data raise the possibility that estrogens could influence fear responses in females through their actions in the amygdala.     

Fear-conditioning mechanisms associated with trait vulnerability to anxiety in humans

Investigations of fear conditioning in rodents and humans have illuminated the neural mechanisms underlying cued and contextual fear. A critical question is how personality dimensions such as trait anxiety act through these mechanisms to confer vulnerability to anxiety disorders, and whether humans' ability to overcome acquired fears depends on regulatory skills not characterized in animal models. In a neuroimaging study of fear conditioning in humans, we found evidence for two independent dimensions of neurocognitive function associated with trait vulnerability to anxiety. The first entailed increased amygdala responsivity to phasic fear cues. The second involved impoverished ventral prefrontal cortical (vPFC) recruitment to downregulate both cued and contextual fear prior to omission (extinction) of the aversive unconditioned stimulus. These two dimensions may contribute to symptomatology differences across anxiety disorders; the amygdala mechanism affecting the development of phobic fear and the frontal mechanism influencing the maintenance of both specific fears and generalized anxiety.     

Expression of freezing and fear‐potentiated startle during sustained fear in mice

Fear‐potentiated acoustic startle paradigms have been used to investigate phasic and sustained components of conditioned fear in rats and humans. This study describes a novel training protocol to assess phasic and sustained fear in freely behaving C57BL/6J mice, using freezing and/or fear‐potentiated startle as measures of fear, thereby, if needed, allowing in vivo application of various techniques, such as optogenetics, electrophysiology and pharmacological intervention, in freely behaving animals. An auditory Pavlovian fear conditioning paradigm, with pseudo‐randomized conditioned–unconditioned stimulus presentations at various durations, is combined with repetitive brief auditory white noise burst presentations during fear memory retrieval 24 h after fear conditioning. Major findings are that (1) a motion sensitive platform built on mechano‐electrical transducers enables measurement of startle responses in freely behaving mice, (2) absence or presence of startle stimuli during retrieval as well as unpredictability of a given threat determine phasic and sustained fear response profiles and (3) both freezing and startle responses indicate phasic and sustained components of behavioral fear, with sustained freezing reflecting unpredictability of conditioned stimulus (CS)/unconditioned stimulus (US) pairings. This paradigm and available genetically modified mouse lines will pave the way for investigation of the molecular and neural mechanisms relating to the transition from phasic to sustained fear.     

Human fear acquisition deficits in relation to genetic variants of the corticotropin‐releasing hormone receptor 1 and the serotonin transporter — revisited

We recently showed that a genetic polymorphism (rs878886) in the human corticotropin‐releasing hormone receptor 1 (CRHR1) is associated with reduced fear‐conditioned responses to a threat cue. This is a potentially important finding considering that the failure to acquire fear contingencies can leave an individual in a maladaptive state of more generalized anxiety. Consistent with that idea, the CRHR1‐dependent fear acquisition deficit translated into heightened contextual anxiety when taking genetic variability within the serotonin transporter long polymorphic region (5‐HTTLPR) into account. To replicate our previous findings, we conducted a replication study in 224 healthy medication‐free human subjects using the exact same cue and context virtual reality fear‐conditioning procedure as in study by Heitland et al. (2013). In the replication study, consistent with the original findings, CRHR1 rs878886 G‐allele carriers showed reduced acquisition of cue‐specific fear‐conditioned responses compared with C/C homozygotes. Also, in this larger sample the cue acquisition deficit of G‐allele carriers translated into heightened contextual anxiety, even independent of 5‐HTT gene variation. In contrast to our earlier findings, there was an additional interaction effect of CRHR1 rs878886 and the triallelic 5‐HTTLPR/rs25531 variant on cued fear acquisition. In summary, this study replicated the initially reported association of the CRHR1 rs878886 G‐allele with cued fear acquisition deficits, albeit with a different pattern of results regarding the interaction with 5‐HTT variation. This further supports the notion that the human corticotropin‐releasing hormone plays a role in the acquisition of fears.     

Altered conditioned fear behavior in glutamate decarboxylase 65 null mutant mice

We investigated the involvement of the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and GAD65-mediated γ-aminobutyric acid (GABA) synthesis in the formation and expression of Pavlovian fear memory. To this end, behavioral, endocrine and autonomic parameters were examined during conditioned fear retrieval of mice with targeted ablation of the GAD65 gene (GAD65^–/– mice). These mutant mice were found to display specific fear behavior (freezing, escape), as well as autonomic (increased defecation) and endocrine activation (increased plasma corticosterone) during fear memory retrieval. However, freezing was reduced and flight and escape behavior were increased in GAD65^–/– mice compared to their wild type and heterozygous littermates, while corticosterone levels and defecation rates did not differ between genotypes. Active defensive behavior of GAD65^–/– mice was observed during both auditory cued and contextual retrieval of fear memory, as well as immediately after conditioning. These data indicate a selectively altered behavioral fear response in GAD65^–/– mice, most likely due to deficits in threat estimation or the elicitation of appropriate conditioned fear behavior, and suggest that GAD65 is a genetic determinant of conditioned fear behavior. GAD65^–/– mice provide a valuable tool to further dissect the GABAergic mechanisms involved in fear and anxiety and to model GABA-related neurological and psychiatric disorders.     

Increased fear learning,spatial learning as well as neophobia in Rgs2−/− mice

Anxiety disorders result from a complex interplay of genetic and environmental factors such as stress. On the level of cellular signaling, regulator of G protein signaling 2 (Rgs2) has been implicated in human and rodent anxiety. However, there is limited knowledge about the role of Rgs2 in fear learning and reactivity to stress. In this study, Rgs2^?/? mice showed increased fear learning, male mice displayed increased contextual and cued fear learning, while females showed selectively enhanced cued fear learning. Male Rgs2^?/? mice displayed increased long‐term‐contextual fear memory, but increased cued fear extinction. Learning in spatial non‐aversive paradigms was also increased in Rgs2^?/? mice. Female, but not male mice show increased spatial learning in the Barnes maze, while male mice showed enhanced place preference in the IntelliCage, rendering enhanced cognitive function non‐specific for aversive stimuli. Consistent with the previous results, Rgs2 deletion resulted in increased innate anxiety, including neophobic behavior expressed as hypolocomotion, in three different tests based on the approach‐avoidance conflict. Acute electric foot shock stress provoked hypolocomotion in several exploration‐based tests, suggesting fear generalization in both genotypes. Rgs2 deletion was associated with reduced monoaminergic neurotransmitter levels in the hippocampus and prefrontal cortex and disturbed corresponding GPCR expression of the adrenergic, serotonergic, dopaminergic and neuropeptide Y system. Taken together, Rgs2 deletion promotes improved cognitive function as well as increased anxiety‐like behavior, but has no effect on acute stress reactivity. These effects may be related to the observed disruption of the monoaminergic systems.     

Oxytocin receptor activation does not mediate associative fear deficits in a Williams Syndrome model

Williams Syndrome results in distinct behavioral phenotypes, which include learning deficits, anxiety, increased phobias and hypersociability. While the underlying mechanisms driving this subset of phenotypes is unknown, oxytocin (OT) dysregulation is hypothesized to be involved as some studies have shown elevated blood OT and altered OT receptor expression in patients. A “Complete Deletion” (CD) mouse, modeling the hemizygous deletion in Williams Syndrome, recapitulates many of the phenotypes present in humans. These CD mice also exhibit impaired fear responses in the conditioned fear task. Here, we address whether OT dysregulation is responsible for this impaired associative fear memory response. We show direct delivery of an OT receptor antagonist to the central nervous system did not rescue the attenuated contextual or cued fear memory responses in CD mice. Thus, increased OT signaling is not acutely responsible for this phenotype. We also evaluated OT receptor and serotonin transporter availability in regions related to fear learning, memory and sociability using autoradiography in wild type and CD mice. While no differences withstood correction, we identified regions that may warrant further investigation. There was a nonsignificant decrease in OT receptor expression in the lateral septal nucleus and nonsignificant lowered serotonin transporter availability in the striatum and orbitofrontal cortex. Together, these data suggest the fear conditioning anomalies in the Williams Syndrome mouse model are independent of any alterations in the oxytocinergic system caused by deletion of the Williams locus.     

Drug-induced conditioned place preference and aversion in mice

This protocol describes the equipment and methods used to establish conditioned place preference (CPP) or aversion (CPA). Place conditioning is a form of Pavlovian conditioning routinely used to measure the rewarding or aversive motivational effects of objects or experiences (e.g., abused drugs). Here, we present a place conditioning procedure that has been used extensively to study the motivational effects of ethanol and other abused drugs in mice. This protocol involves three phases: (i) habituation (or a pretest), (ii) conditioning of an association between the drug and a tactile or visual stimulus and (iii) a test that offers a choice between the drug-associated cue and a neutral cue. If the drug has motivational significance, mice will spend significantly more time (CPP) or less time (CPA) in proximity to the drug-associated cue. Potential problems in the design and interpretation of place conditioning studies are discussed. A typical experiment lasts 2 weeks.     

Behavioral determination of stimulus pair discrimination of auditory acoustic and electrical stimuli using a classical conditioning and heart-rate approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants and auditory midbrain implants. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices. Several techniques such as reward-based operant conditioning, conditioned avoidance, or classical fear conditioning have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed). Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.     

视觉功能减弱增强小鼠听觉恐惧条件反射

作为一种高级认知活动,视觉功能减弱是否影响听觉恐惧条件化学习目前还不清楚.本文以突变体rd/rd、cl/cl小鼠为视觉功能减弱组,研究视觉功能减弱是否对听觉巴甫洛夫条件化恐惧反应有影响.在恐惧条件化、恐惧消退和消除记忆再现阶段记录了僵直行为.研究结果表明,视觉功能的减弱更有利于小鼠听觉恐惧条件化的建立.文中讨论了出现此...     

Altered synaptic plasticity and behavioral abnormalities in CNGA3‐deficient mice

The role of the cyclic nucleotide‐gated (CNG) channel CNGA3 is well established in cone photoreceptors and guanylyl cyclase‐D‐expressing olfactory neurons. To assess a potential function of CNGA3 in the mouse amygdala and hippocampus, we examined synaptic plasticity and performed a comparative analysis of spatial learning, fear conditioning and step‐down avoidance in wild‐type mice and CNGA3 null mutants (CNGA3^?/?). CNGA3^?/? mice showed normal basal synaptic transmission in the amygdala and the hippocampus. However, cornu Ammonis (CA1) hippocampal long‐term potentiation (LTP) induced by a strong tetanus was significantly enhanced in CNGA3^?/? mice as compared with their wild‐type littermates. Unlike in the hippocampus, LTP was not significantly altered in the amygdala of CNGA3^?/? mice. Enhanced hippocampal LTP did not coincide with changes in hippocampus‐dependent learning, as both wild‐type and mutant mice showed a similar performance in water maze tasks and contextual fear conditioning, except for a trend toward higher step‐down latencies in a passive avoidance task. In contrast, CNGA3^?/? mice showed markedly reduced freezing to the conditioned tone in the amygdala‐dependent cued fear conditioning task. In conclusion, our study adds a new entry on the list of physiological functions of the CNGA3 channel. Despite the dissociation between physiological and behavioral parameters, our data describe a so far unrecognized role of CNGA3 in modulation of hippocampal plasticity and amydgala‐dependent fear memory.     

Localization and behaviors in null mice suggest that ASIC1 and ASIC2 modulate responses to aversive stimuli

Acid‐sensing ion channels (ASICs) generate H⁺‐gated Na⁺ currents that contribute to neuronal function and animal behavior. Like ASIC1, ASIC2 subunits are expressed in the brain and multimerize with ASIC1 to influence acid‐evoked currents and facilitate ASIC1 localization to dendritic spines. To better understand how ASIC2 contributes to brain function, we localized the protein and tested the behavioral consequences of ASIC2 gene disruption. For comparison, we also localized ASIC1 and studied ASIC1^?/? mice. ASIC2 was prominently expressed in areas of high synaptic density, and with a few exceptions, ASIC1 and ASIC2 localization exhibited substantial overlap. Loss of ASIC1 or ASIC2 decreased freezing behavior in contextual and auditory cue fear conditioning assays, in response to predator odor and in response to CO₂ inhalation. In addition, loss of ASIC1 or ASIC2 increased activity in a forced swim assay. These data suggest that ASIC2, like ASIC1, plays a key role in determining the defensive response to aversive stimuli. They also raise the question of whether gene variations in both ASIC1 and ASIC2 might affect fear and panic in humans .     

Mice lacking synapsin III show abnormalities in explicit memory and conditioned fear

Synapsin III is a neuron‐specific phosphoprotein that plays an important role in synaptic transmission and neural development. While synapsin III is abundant in embryonic brain, expression of the protein in adults is reduced and limited primarily to the hippocampus, olfactory bulb and cerebral cortex. Given the specificity of synapsin III to these brain areas and because it plays a role in neurogenesis in the dentate gyrus, we investigated whether it may affect learning and memory processes in mice. To address this point, synapsin III knockout mice were examined in a general behavioral screen, several tests to assess learning and memory function, and conditioned fear. Mutant animals displayed no anomalies in sensory and motor function or in anxiety‐ and depressive‐like behaviors. Although mutants showed minor alterations in the Morris water maze, they were deficient in object recognition 24 h and 10 days after training and in social transmission of food preference at 20 min and 24 h. In addition, mutants displayed abnormal responses in contextual and cued fear conditioning when tested 1 or 24 h after conditioning. The synapsin III knockout mice also showed aberrant responses in fear‐potentiated startle. As synapsin III protein is decreased in schizophrenic brain and because the mutant mice do not harbor obvious anatomical deficits or neurological disorders, these mutants may represent a unique neurodevelopmental model for dissecting the molecular pathways that are related to certain aspects of schizophrenia and related disorders.     

SERT and uncertainty: serotonin transporter expression influences information processing biases for ambiguous aversive cues in mice

The long allele variant of the serotonin transporter (SERT, 5‐HTT) gene‐linked polymorphic region (5‐HTTLPR) is associated with higher levels of 5‐HTT expression and reduced risk of developing affective disorders. However, little is known about the mechanisms underlying this protective effect. One hypothesis is that 5‐HTT expression influences aversive information processing, with reduced negative cognitive bias present in those with higher 5‐HTT expression. Here we investigated this hypothesis using genetically‐modified mice and a novel aversive learning paradigm. Mice with high levels of 5‐HTT expression (5‐HTT over‐expressing, 5‐HTTOE mice) and wild‐type mice were trained to discriminate between three distinct auditory cues: one cue predicted footshock on all trials (CS+); a second cue predicted the absence of footshock (CS?); and a third cue predicted footshock on 20% of trials (CS20%), and was therefore ambiguous. Wild‐type mice exhibited equivalently high levels of fear to the CS+ and CS20% and minimal fear to the CS?. In contrast, 5‐HTTOE mice exhibited high levels of fear to the CS+ but minimal fear to the CS? and the CS20%. This selective reduction in fear to ambiguous aversive cues suggests that increased 5‐HTT expression reduces negative cognitive bias for stimuli with uncertain outcomes.     

Updating fearful memories with extinction training during reconsolidation: a human study using auditory aversive stimuli

Learning to fear danger in the environment is essential to survival, but dysregulation of the fear system is at the core of many anxiety disorders. As a consequence, a great interest has emerged in developing strategies for suppressing fear memories in maladaptive cases. Recent research has focused in the process of reconsolidation where memories become labile after being retrieved. In a behavioral manipulation, Schiller et al., (2010) reported that extinction training, administrated during memory reconsolidation, could erase fear responses. The implications of this study are crucial for the possible treatment of anxiety disorders without the administration of drugs. However, attempts to replicate this effect by other groups have been so far unsuccessful. We sought out to reproduce Schiller et al., (2010) findings in a different fear conditioning paradigm based on auditory aversive stimuli instead of electric shock. Following a within-subject design, participants were conditioned to two different sounds and skin conductance response (SCR) was recorded as a measure of fear. Our results demonstrated that only the conditioned stimulus that was reminded 10 minutes before extinction training did not reinstate a fear response after a reminder trial consisting of the presentation of the unconditioned stimuli. For the first time, we replicated Schiller et al., (2010) behavioral manipulation and extended it to an auditory fear conditioning paradigm.     

ChAT::Cre transgenic rats show sex-dependent altered fear behaviors,ultrasonic vocalizations and cholinergic marker expression

The cholinergic system is a critical regulator of Pavlovian fear learning and extinction. As such, we have begun investigating the cholinergic system's involvement in individual differences in cued fear extinction using a transgenic ChAT::Cre rat model. The current study extends behavioral phenotyping of a transgenic ChAT::Cre rat line by examining both freezing behavior and ultrasonic vocalizations (USVs) during a Pavlovian cued fear learning and extinction paradigm. Freezing, 22 kHz USVs, and 50 kHz USVs were compared between male and female transgenic ChAT::Cre+ rats and their wildtype (Cre-) littermates during fear learning, contextual and cue-conditioned fear recall, cued fear extinction, and generalization to a novel tone. During contextual and cued fear recall ChAT::Cre+ rats froze slightly more than their Cre- littermates, and displayed significant sex differences in contextual and cue-conditioned freezing, 22 kHz USVs, and 50 kHz USVs. Females showed more freezing than males in fear recall trials, but fewer 22 kHz distress calls during fear learning and recall. Females also produced more 50 kHz USVs during exposure to the testing chambers prior to tone (or shock) presentation compared with males, but this effect was blunted in ChAT::Cre+ females. Corroborating previous studies, ChAT::Cre+ transgenic rats overexpressed vesicular acetylcholine transporter immunolabeling in basal forebrain, striatum, basolateral amygdala, and hippocampus, but had similar levels of acetylcholinesterase and numbers of ChAT+ neurons as Cre- rats. This study suggests that variance in behavior between ChAT::Cre+ and wildtype rats is sex dependent and advances theories that distinct neural circuits and processes regulate sexually divergent fear responses.     

A new model of Pde4d deficiency: genetic knock-down of PDE4D enzyme in rats produces an antidepressant phenotype without spatial cognitive effects

Phosphodiesterases (PDEs) are a superfamily of intracellular second messenger cyclic nucleotide hydrolyzing enzymes composed of 12 families. The Pde4 family has been implicated in depression and cognition, and PDE4 inhibitors have been evaluated as antidepressants and possible cognitive enhancers. Pde4d(-/-) mice show an antidepressant phenotype and learning enhancement on some tests, but not others as do mice treated with PDE4 inhibitors. Here, we report for the first time the behavioral phenotype of a new Pde4d knock-down (KD) rat model of PDE4D deficiency. Consistent with other data on PDE4D deficiency, Pde4d KD rats showed depression resistance in the Porsolt forced swim test and hyperreactivity of the acoustic startle response with no differential response on prepulse inhibition, suggesting no sensorimotor gating defect. Pde4d KD rats also exhibited a small exploratory activity reduction but no difference following habituation, and no enhanced spatial learning or reference memory in the Morris water maze. A selective improvement in route-based learning in the Cincinnati water maze was seen as well as enhanced contextual and cued fear conditioning and a more rapid rate of cued extinction from their higher freezing level that declined to wild-type (WT) levels only after ～20 extinction trials. The rat model confirms Pde4d's role in depression but not in spatial learning or memory enhancement and shows for the first time higher fear conditioning and altered extinction compared with controls. The new model provides a tool by which to better understand the role of PDE4D in neuropsychiatric disorders and for the development of alternate treatment approaches.