Fear Conditioning In Mice Research Articles

Characterization of NMDAR-Independent Learning in the Hippocampus

It is currently thought that memory formation requires the activation of NMDA receptors (NMDARs) in the hippocampus. However, recent studies indicate that these receptors are not necessary for all forms of learning. The current experiments examine this issue using context fear conditioning in mice. First, we show that context fear can be acquired without NMDAR activation in previously trained animals. Mice trained in one environment (context A) are subsequently able to learn about a second environment (context B) in the presence of NMDAR antagonists. Second, we demonstrate that NMDAR-independent learning requires the hippocampus and is dependent on protein synthesis. However, unlike NMDAR-dependent learning, it is not contingent on the expression of activity-regulated cytoskeleton-associated protein (Arc). Lastly, we present data that suggests NMDAR-independent learning is only observed when recently stimulated neurons are reactivated during conditioning. These data suggest that context fear conditioning modifies synaptic plasticity mechanisms in the hippocampus and allows subsequent learning to occur in the absence of NMDAR activation.

Facilitating actions of an AMPA receptor potentiator upon extinction of contextually conditioned fear response in stressed mice

Extinction of conditioned fear response is thought to be a biological process underlying exposure therapy for anxiety disorders. We have previously reported that an AMPA receptor potentiator, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluorophenoxyacetamide (PEPA), facilitates extinction of fear memory formed through contextual fear conditioning in mice that had never been exposed to experimental stress. On the other hand, recent findings suggest that the fear extinction is impaired in stressed rats or mice. The purpose of the present study was to examine whether PEPA facilitates impaired extinction of fear in stressed mice. For this purpose, mice were applied stress (a 2 h restraint, a 20 min forced swim, and ether inhalation), and contextual fear conditioning was carried out 7 days later. After 1–3 days of conditioning, mice were re-exposed to the context for 6 min, and behavioral freezing response was measured. The time mice spent frozen decreased following every extinction session, and the decrease was remarkably slower in the stressed mice than in control non-stressed mice. PEPA (3, 10, 30 mg/kg body weight) or vehicle was intraperitoneally administered into stressed mice once before the first extinction session. The significant decrease of the freezing response in the extinction sessions was only seen in the 30 mg/kg PEPA-administered stressed mice, compared with vehicle-administered stressed mice. A similar extent of decrease in the freezing response in the extinction sessions was observed in the PEPA-administered (30 mg/kg) and d-cycloserine-administered (30 mg/kg) mice. These results suggest that PEPA facilitates extinction of contextual fear in stressed mice.

Rapid Changes in the Light/Dark Cycle Disrupt Memory of Conditioned Fear in Mice

BackgroundCircadian rhythms govern many aspects of physiology and behavior including cognitive processes. Components of neural circuits involved in learning and memory, e.g., the amygdala and the hippocampus, exhibit circadian rhythms in gene expression and signaling pathways. The functional significance of these rhythms is still not understood. In the present study, we sought to determine the impact of transiently disrupting the circadian system by shifting the light/dark (LD) cycle. Such “jet lag” treatments alter daily rhythms of gene expression that underlie circadian oscillations as well as disrupt the synchrony between the multiple oscillators found within the body.Methodology/Principal FindingsWe subjected adult male C57Bl/6 mice to a contextual fear conditioning protocol either before or after acute phase shifts of the LD cycle. As part of this study, we examined the impact of phase advances and phase delays, and the effects of different magnitudes of phase shifts. Under all conditions tested, we found that recall of fear conditioned behavior was specifically affected by the jet lag. We found that phase shifts potentiated the stress-evoked corticosterone response without altering baseline levels of this hormone. The jet lag treatment did not result in overall sleep deprivation, but altered the temporal distribution of sleep. Finally, we found that prior experience of jet lag helps to compensate for the reduced recall due to acute phase shifts.Conclusions/SignificanceAcute changes to the LD cycle affect the recall of fear-conditioned behavior. This suggests that a synchronized circadian system may be broadly important for normal cognition and that the consolidation of memories may be particularly sensitive to disruptions of circadian timing.

Effects of 860 MHz microwave on context conditioned fear in mice

Objective To investigate the effects of 860MHz microwave on the formation and extinction of context conditioned fear in mice. Methods The mice were exposed to 860MHz continuous microwave ( power density were 380 μW/cm2 or 550 μW/cm2, respectively) for 30 min or 2 h, which then were divided in to 5 groups.Each group consisted of 15 animals. Footshocks were used to induce context conditioned fear by 75 voltages. The frequency and time of freezing after irradiation were investigated. Results When 24 h after foot shocking, the values of freezing time: control group was 2.31 ±4. 17 , two groups of the microwave irradiation 2 h were 3.93 ±6.99 and 2.47 ± 3.34, the Nemenyi test results (P = 0.004): control group was 32.63333, while two groups of the microwave irradiation 2 h were 52.46667 and 39.76667; and the values of freezing frequency: control group was 0.73 ± 1.16 , two groups of the microwave irradiation 2 h were 0.86 ± 1.41 and 1.07 ± 1.16, the Nemenyi test results of (P=0. 014): control group was 33. 26667, while two groups of the microwave irradiation 2 h were 50. 76667 and 40.90000. Conclusion The mice receiving relatively longer period of microwave irradiation showed more stable memory of the context conditioned fear. Key words: Microwave; Context conditioned fear; Freezing

The type IV phosphodiesterase inhibitor rolipram disturbs expression and extinction of conditioned fear in mice

Recent studies suggest that intracellular signaling pathways involving cyclic adenosine monophosphate (cAMP) may be related to fear processing and long-term memory formation. The type IV phosphodiesterase (PDE4) inhibitor rolipram prevents breakdown of cAMP, enhances long-term memory and may reduce anxiety. In the present study we investigated the role of rolipram in the expression (0, 0.2, or 1 mg/kg), acquisition (0, 0.03, 0.2 or 1 mg/kg), and extinction (0, 0.03, 0.2, 1 mg/kg) of fear using a fear-potentiated startle paradigm in mice. It was shown that rolipram reduced the expression (Experiment 1), did not influence acquisition (Experiment 2) and disturbed between-session extinction (Experiments 3 and 4) of fear responses to conditioned tones. Because within-session extinction was not impaired by rolipram and because low (i.e. 0.03 and 0.2 mg/kg) doses strongly affected extinction but not expression of fear, these findings suggest that the effect of rolipram on extinction is not directly dependent on its effect on fear expression. Taken together, these experiments indicate that preventing breakdown of cAMP interferes with the expression and extinction consolidation of conditioned fear.

Changes in microtubule turnover accompany synaptic plasticity and memory formation in response to contextual fear conditioning in mice

Synaptic plasticity plays a crucial role in learning, memory, and cognitive disorders. Cytoskeletal reorganization underlies neuronal synaptic plasticity, but little is known about the regulation of cytoskeletal dynamics in living animals. We used stable isotope labeling to measure the turnover of tubulin in defined microtubule (MT) populations in murine brain. Neuronal MTs generally exhibited low turnover rates in vivo. Basal turnover was highest in tau-associated MTs, intermediate in microtubule-associated protein 2 (MAP2)–associated MTs, and lowest in cold-stable MTs. Labeling of MTs in mature neurons in cell culture yielded similar turnover results. Intracerebroventricular glutamate injection stimulated, via N-methyl-d-aspartic acid receptors, label incorporation (turnover) in cold-stable, tau-associated, and MAP2-associated MTs, the last of which was shown to be dependent on cyclic adenosine-3′, 5′-monophosphorothioate–protein kinase A. Contextual fear conditioning, a hippocampus-mediated form of memory formation, was accompanied by increased turnover of hippocampal MAP2-associated and cold-stable MTs. Treatment with the MT-depolymerizing drug nocodazole reversed the conditioning-induced increase in label incorporation in MAP2-associated MTs, reduced dendritic spine density, and impaired memory formation. The effects of nocodazole on MT turnover were prevented by the MT-stabilizing agent Taxol (Sigma-Aldrich, St. Louis, MO, USA) and by brain-derived nerve growth factor, both of which also restored dendritic spine density and memory formation in this model. In conclusion, these results suggest that changes in hippocampal MT turnover are required for, and are a biomarker of, the synaptic plasticity that is involved in memory formation.

C-fos expression and accelerated visual cued fear conditioning in mice with visual input directed to the auditory thalamus

C-fos expression and accelerated visual cued fear conditioning in mice with visual input directed to the auditory thalamus

Neural Cell Adhesion Molecule-Associated Polysialic Acid Regulates Synaptic Plasticity and Learning by Restraining the Signaling through GluN2B-Containing NMDA Receptors

The neural cell adhesion molecule (NCAM) is the predominant carrier of alpha2,8 polysialic acid (PSA) in the mammalian brain. Abnormalities in PSA and NCAM expression are associated with schizophrenia in humans and cause deficits in hippocampal synaptic plasticity and contextual fear conditioning in mice. Here, we show that PSA inhibits opening of recombinant NMDA receptors composed of GluN1/2B (NR1/NR2B) or GluN1/2A/2B (NR1/NR2A/NR2B) but not of GluN1/2A (NR1/NR2A) subunits. Deficits in NCAM/PSA increase GluN2B-mediated transmission and Ca(2+) transients in the CA1 region of the hippocampus. In line with elevation of GluN2B-mediated transmission, defects in long-term potentiation in the CA1 region and contextual fear memory in NCAM/PSA-deficient mice are abrogated by application of a GluN2B-selective antagonist. Furthermore, treatment with the glutamate scavenger glutamic-pyruvic transaminase, ablation of Ras-GRF1 (a mediator of GluN2B signaling to p38 MAPK), or direct inhibition of hyperactive p38 MAPK can restore impaired synaptic plasticity in brain slices lacking PSA/NCAM. Thus, PSA carried by NCAM regulates plasticity and learning by inhibition of the GluN2B-Ras-GRF1-p38 MAPK signaling pathway. These findings implicate carbohydrates carried by adhesion molecules in modulating NMDA receptor signaling in the brain and demonstrate reversibility of cognitive deficits associated with ablation of a schizophrenia-related adhesion molecule.

Pharmacological modulators of nitric oxide signaling and contextual fear conditioning in mice

Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is a retrograde neuronal messenger that participates in synaptic plasticity, including late-phase long-term potentiation (LTP) and long-term memory (LTM) formation. Our recent studies have shown that nNOS knockout (KO) mice have a severe deficit in contextual fear conditioning compared to wild type (WT) counterparts (Kelley et al. 2009). Given the role of the nNOS gene in fear conditioning, we investigated whether systemic administration of modulators of NO signaling affect the formation of contextual and cued fear memories and the effects of these modulators on cyclic 3'5'-guanosine monophosphate (cGMP) levels in the hippocampus and amygdala. The preferential nNOS inhibitor S-methyl-L-thiocitrulline (SMTC; 10-200 mg/kg) was administered (IP) to WT mice, and the NO donor molsidomine (10 mg/kg) was administered (IP) to nNOS KO mice either 30 min pretraining or immediately posttraining. Pretraining SMTC administration to WT mice impaired both short- and long-term memories of contextual (36% inhibition) but not cued fear conditioning. Pretraining molsidomine administration to nNOS KO mice improved their deficit in short- and long-term memories of contextual fear conditioning (46% increase). Posttraining drug administration had no effect on WT and nNOS KO mice. The systemic administration of SMTC dose-dependently decreased cGMP concentrations down to 25% of control, while molsidomine increased cGMP concentration (three- and five-fold) in amygdala and hippocampus, respectively. These findings suggest that neuronal NO and its downstream second messenger cGMP are important for acquisition and subsequent consolidation of LTM of contextual fear conditioning.

Increased excitability in lateral amygdala neurons in fear-conditioned mice

Increased excitability in lateral amygdala neurons in fear-conditioned mice

Enhancement of acoustic prepulse inhibition by contextual fear conditioning in mice is maintained even after contextual fear extinction

Prepulse inhibition (PPI) of the acoustic startle response is one of the few and major paradigms for investigating sensorimotor gating systems in humans and rodents in a similar fashion. PPI deficits are observed not only in patients with schizophrenia, but also in patients with anxiety disorders. Previous studies have shown that PPI in rats can be enhanced by auditory fear conditioning. In this study, we evaluated the effects of contextual fear conditioning (FC) for six times a day and fear extinction (FE) for seven days on PPI in mice. C57BL/6J mice (male, 8-12 weeks) were divided into three groups; no-FC (control), FC and FC + FE. We measured PPI at the following three time points, (1) baseline before FC, (2) after FC, and (3) after FE. The results showed that PPI was increased after FC. Moreover, the enhanced PPI following FC was observed even after FE with decreased freezing behaviors. These results suggested contextual fear conditioning could enhance acoustic PPI, and that contextual fear extinction could decrease freezing behaviors, but not acoustic PPI.

S.15.01 Heart rate dynamics during conditioned fear in mice and its implications for affective disorders

S.15.01 Heart rate dynamics during conditioned fear in mice and its implications for affective disorders

Pharmacological Discrimination of Extinction and Reconsolidation of Contextual Fear Memory by a Potentiator of AMPA Receptors

Conditioned fear memory, once formed through fear conditioning, is modulated by reexposure of individuals to a conditioned stimulus. The reexposure reactivates the fear memory, which induces reconsolidation of the memory first, and then extinction of the fear response. Both attenuating the former and facilitating the latter are effective in reducing the fear response, and these findings are potentially translatable to the enhancement of exposure therapy for complex anxiety disorders. Currently, there is no drug that is established to modulate either reconsolidation or extinction selectively, which are thought to be independent processes. Here, we report that an extinction-facilitating AMPA potentiator, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA), does not act on the reconsolidation of fear memory formed by contextual fear conditioning in mice. The freezing rates observed in contextually conditioned mice following short reexposure (3 min) to the context were not influenced by intraperitoneal or intra-amygdala administration of PEPA. The same short reexposure to the context enhanced freezing responses in mice that were similarly administered D-cycloserine (DCS), a drug that facilitates both extinction and reconsolidation, and this enhancement of freezing responses in mice intraperitoneally administered DCS was abolished by propranolol, a drug that suppresses reconsolidation. At the same doses used in the short reexposure experiments, PEPA and DCS facilitated extinction of the fear response induced by long reexposure to the context and suppressed reinstatement of the conditioned fear memory. PEPA and DCS did not affect reextinction. These results suggest that PEPA acts on extinction of contextual fear memory without having detectable influences on its reconsolidation.

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.

Transcriptional regulation of the murine Presenilin-2 gene reveals similarities and differences to its human orthologue

Inherited Presenilin-2 mutations cause familial Alzheimer's disease, and its regulation may play a role in sporadic cases. The human Presenilin-2 (PSEN2) regulatory region includes two separate promoters modulated by Egr-1, a transcription factor involved in learning and memory. To enable in-vivo analysis of Presenilin-2 regulation, we characterized the murine Presenilin-2 (Psen2) promoter. We identified novel Psen2 Transcription start sites (TSSs) 10 kb upstream of previously reported sites, along with two new alternatively transcribed exons (1A, and 1BC) in the 5′ untranslated region. Transcripts initiating in Exon 1A are ubiquitous, whereas exon 1BC-initiated transcripts are non-neuronal. Only the sequence surrounding exon 1A, which includes homologous sequences to the human PSEN2 promoter, harbored significant promoter activity. Sequences upstream of exon 1A and a downstream enhancer were specifically important in neuronal cells, but similar to the human promoter, the murine promoter was characteristic of a housekeeping gene, and its activity depended on Sp1 binding. Egr-1 did not bind the murine promoter. Egr-1 over-expression and down-regulation, as well as in-vivo examination of Egr-1 and Psen2 expression during fear conditioning in mice, showed that Egr-1 does not regulate the murine Psen2 promoter. Differential Psen2 regulation in human and mouse has implications for Alzheimer disease mouse models.

Impairments in fear conditioning in mice lacking the nNOS gene

The fear conditioning paradigm is used to investigate the roles of various genes, neurotransmitters, and substrates in the formation of fear learning related to contextual and auditory cues. In the brain, nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) functions as a retrograde neuronal messenger that facilitates synaptic plasticity, including the late phase of long-term potentiation (LTP) and formation of long-term memory (LTM). Evidence has implicated NO signaling in synaptic plasticity and LTM formation following fear conditioning, yet little is known about the role of the nNOS gene in fear learning. Using knockout (KO) mice with targeted mutation of the nNOS gene and their wild-type (WT) counterparts, the role of NO signaling in fear conditioning was investigated. Plasma levels of the stress hormone corticosterone were measured to determine the relationship between physiological and behavioral response to fear conditioning. Contextual fear learning was severely impaired in male and female nNOS KO mice compared with WT counterparts; cued fear learning was slightly impaired in nNOS KO mice. Sex-dependent differences in both contextual and cued fear learning were not observed in either genotype. Deficits in contextual fear learning in nNOS KO mice were partially overcome by multiple trainings. A relationship between increase in plasma corticosterone levels following footshock administration and the magnitude of contextual, but not cued freezing was also observed. Results suggest that the nNOS gene contributes more to optimal contextual fear learning than to cued fear learning, and therefore, inhibition of the nNOS enzyme may ameliorate context-dependent fear response.

Social modeling of conditioned fear in mice by non-fearful conspecifics

Social interactions with conspecifics markedly alter the neuroendocrine, behavioral and emotional responses to stressful events. Some of these effects involve observational learning and result in lasting changes of fear-motivated behavior. While most evidence reveals increased fearfulness after observation of fearful demonstrators (models) in a number of species, a few reports from human and non-human primates indicate that observational learning can also attenuate some forms of fear. In the present study, we set out to determine the effects of social modeling and observational learning on fear conditioning in the mouse. Observers were pre-exposed to a novel context in the presence of fearful (F group) or non-fearful (NF group) demonstrators. Mice of the F group acquired control levels of conditioned fear. On the other hand, mice of the NF group exhibited profound and persistent reduction of fear. The decrease of fear in NF observers was most likely due to context-specific impairments of fear conditioning, as revealed by selective effects on long- but not short-term contextual fear memory, and normal fear conditioning in response to a novel context or cue. The effect was lasting, but constrained by the shock intensity. Attenuation of fear conditioning resulting from interactions with non-fearful conspecifics was largely, but not entirely, mediated by vicarious learning. These findings identify an important social buffering process serving to prevent a lasting induction of fear in response to isolated, moderately intense stressful events.

Neonatal Exposure to Sevoflurane Induces Abnormal Social Behaviors and Deficits in Fear Conditioning in Mice

Neonatal exposure to anesthetics that block N-methyl-D-aspartate receptors and/or hyperactivate gamma-aminobutyric acid type A receptor has been shown to cause neuronal degeneration in the developing brain, leading to functional deficits later in adulthood. The authors investigated whether exposure of neonatal mice to inhaled sevoflurane causes deficits in social behavior as well as learning disabilities. Six-day-old C57BL/6 mice were exposed to 3% sevoflurane for 6 h. Activated cleaved caspase-3 immunohistochemical staining was used for detection of apoptosis. Cognitive functions were tested by pavlovian conditioned fear test. Social behavior was tested by social recognition and interaction tests. Neonatal exposure to sevoflurane significantly increased the number of apoptotic cells in the brain immediately after anesthesia. It caused persistent learning deficits later in adulthood as evidenced by decreased freezing response in both contextual and cued fear conditioning. The social recognition test demonstrated that mice with neonatal exposure to sevoflurane did not develop social memory. Furthermore, these mice showed decreased interactions with a social target compared with controls in the social interaction test, indicating a social interaction deficit. The authors did not attribute these abnormalities in social behavior to impairments of general interest in novelty or olfactory sensation, because they did not detect significant differences in the test for novel inanimate object interaction or for olfaction. This study shows that exposure of neonatal mice to inhaled sevoflurane could cause not only learning deficits but also abnormal social behaviors resembling autism spectrum disorder.

Enhancement of prepulse inhibition by fear conditioning in mice

Enhancement of prepulse inhibition by fear conditioning in mice

Gender-dependent Association of a Functional NGF Polymorphism with Anxiety-related Personality Traits

Nerve growth factor (NGF) has been shown to be involved in anxiety behaviour and the expression of conditioned fear in mice. We have tested a total of 337 (age: 39.2 +/- 14.6 years) unrelated subjects of German descent (166 males; 171 females) who were carefully screened for psychiatric health. The self-ratable State-Trait Anxiety Inventory, which enables anxiety to be quantified as a comparatively stable personality trait was applied and a recently described non-synonymous NGF SNP (rs6330,c.104C > T,p.Ala35Val) was examined. In the trait-related anxiety score, a significant gender-dependent effect of the genotype was observed (F=4.580, df=2, p=0.011) with higher levels of trait anxiety in females with C/C genotype when compared to females with T/T and C/T genotypes and an opposite effect in males. Our findings support the hypothesis that anxiety relates to a genetic variation of NGF and that genes modulate behaviour in a gender-dependent manner. Since anxiety and attention deficit hyperactivity disorder are related to NGF, the present results may represent a common biological link for the gender-specific occurrence and comorbidity of the diseases.