Context-dependent Fear Conditioning Research Articles

Neural correlates of context-dependent extinction recall in social anxiety disorder: relevance of intrusions in response to aversive social experiences.

There are phenomenological similarities between social anxiety disorder (SAD) and posttraumatic stress disorder, such as a provoking aversive event, posttraumatic stress symptoms (e.g. intrusions) in response to these events and deficient (context-dependent) fear conditioning processes. This study investigated the neural correlates of context-dependent extinction recall and fear renewal in SAD, specifically in patients with intrusions in response to an etiologically relevant aversive social event. During functional magnetic resonance imaging a two-day context-dependent fear conditioning paradigm was conducted in 54 patients with SAD and 54 healthy controls (HC). This included fear acquisition (context A) and extinction learning (context B) on one day, and extinction recall (context B) as well as fear renewal (contexts C and A) one day later. The main outcome measures were blood oxygen level-dependent responses in regions of interest and skin conductance responses. Patients with SAD showed reduced differential conditioned amygdala activation during extinction recall in the safe extinction context and during fear renewal in the acquisition context compared to HC. Patients with clinically relevant intrusions moreover exhibited hypoactivation of the ventromedial prefrontal cortex (vmPFC) during extinction learning, extinction recall, and fear renewal in a novel context, while amygdala activation more strongly decreased during extinction learning and increased during fear renewal in the acquisition context compared with patients without intrusions. Our study provides first evidence that intrusions in SAD are associated with similar deficits in context-dependent regulation of conditioned fear via the vmPFC as previously demonstrated in posttraumatic stress disorder.

Decontextualized fear memories? Stronger conditioned fear responses during extinction learning and extinction recall in a safe context predict the development of long-term analog intrusions.

Difficulties in the context-dependent modulation of conditioned fear are known for posttraumatic stress disorder and may explain the occurrence of intrusive memories in safe contexts. The current study therefore investigated if reduced context-dependent modulation of conditioned fear and its underlying neural circuitry constitute risk factors for the development of analog intrusions in response to an experimental trauma. Eighty-five healthy women participated in the trauma film paradigm to investigate the development of analog intrusions as well as explicit memory for an experimental trauma after one week and three months, respectively. Before, participants underwent a context-dependent fear conditioning paradigm during functional magnetic resonance imaging with fear acquisition in context A and extinction training in context B on a first day, as well as extinction recall in context B and fear renewal in a novel context C one day later. Skin conductance responses (SCRs) and blood oxygen level dependent responses were main outcome measures. In addition to stronger fear acquisition in context A, stronger conditioned fear responses in the safe context B, as indicated by stronger conditioned SCRs or stronger activation of fear expressing regions during extinction learning and recall, predicted the development of long-term analog intrusions. Stronger fear responses in safe and danger contexts were risk factors for the development of long-term analog intrusions and point to decontextualized fear memories and difficulties in the context-dependent modulation of conditioned fear. Altered fear conditioning processes and reduced storage of contextual information may cause the occurrence of fear independent of context.

Behavioral pattern separation is associated with neural and electrodermal correlates of context-dependent fear conditioning

Hippocampus-dependent pattern separation is considered as a relevant factor for context discrimination and might therefore impact the contextual modulation of conditioned fear. However, the association between pattern separation and context-dependent fear conditioning has not been investigated so far. In the current study, 72 healthy female students completed the Mnemonic Similarity Task, a measure of behavioral pattern separation, in addition to a context-dependent fear conditioning paradigm during functional magnetic resonance imaging. The paradigm included fear acquisition in context A and extinction training in context B on a first day, as well as retrieval testing of the fear and extinction memories in the safe context B (extinction recall) and a novel context C (fear renewal) one day later. Main outcome measures comprised skin conductance responses (SCRs) and blood oxygen level-dependent responses in brain regions of the fear and extinction circuit. Regarding retrieval testing, pattern separation did not correlate with extinction recall, but with stronger dorsal anterior cingulate cortex activation and conditioned SCRs (trend) during fear renewal, indicating a stronger retrieval of the fear memory trace. Our findings suggest that behavioral pattern separation ability seems to be important for context-dependent fear modulation, which is impaired in patients with posttraumatic stress disorder.

Tagging active neurons by soma-targeted Cal-Light

Verifying causal effects of neural circuits is essential for proving a direct circuit-behavior relationship. However, techniques for tagging only active neurons with high spatiotemporal precision remain at the beginning stages. Here we develop the soma-targeted Cal-Light (ST-Cal-Light) which selectively converts somatic calcium rise triggered by action potentials into gene expression. Such modification simultaneously increases the signal-to-noise ratio of reporter gene expression and reduces the light requirement for successful labeling. Because of the enhanced efficacy, the ST-Cal-Light enables the tagging of functionally engaged neurons in various forms of behaviors, including context-dependent fear conditioning, lever-pressing choice behavior, and social interaction behaviors. We also target kainic acid-sensitive neuronal populations in the hippocampus which subsequently suppress seizure symptoms, suggesting ST-Cal-Light’s applicability in controlling disease-related neurons. Furthermore, the generation of a conditional ST-Cal-Light knock-in mouse provides an opportunity to tag active neurons in a region- or cell-type specific manner via crossing with other Cre-driver lines. Thus, the versatile ST-Cal-Light system links somatic action potentials to behaviors with high temporal precision, and ultimately allows functional circuit dissection at a single cell resolution.

Synaptic activity-dependent changes in the hippocampal palmitoylome.

Dynamic protein S-palmitoylation is critical for neuronal function, development, and synaptic plasticity. Synaptic activity-dependent changes in palmitoylation have been reported for a small number of proteins. Here, we characterized the palmitoylome in the hippocampi of male mice before and after context-dependent fear conditioning. Of the 121 differentially palmitoylated proteins identified, just over half were synaptic proteins, whereas others were associated with metabolic functions, cytoskeletal organization, and signal transduction. The synapse-associated proteins generally exhibited increased palmitoylation after fear conditioning. In contrast, most of the proteins that exhibited decreased palmitoylation were associated with metabolic processes. Similar results were seen in cultured rat hippocampal neurons in response to chemically induced long-term potentiation. Furthermore, we found that the palmitoylation of one of the synaptic proteins, plasticity-related gene-1 (PRG-1), also known as lipid phosphate phosphatase-related protein type 4 (LPPR4), was important for synaptic activity-induced insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynaptic membrane. The findings identify proteins whose dynamic palmitoylation may regulate their role in synaptic plasticity, learning, and memory.

The association between fear extinction, the ability to accomplish exposure and exposure therapy outcome in specific phobia

Great interest exists in maximizing exposure therapy efficacy in anxiety disorders. At the same time, reduced frequency and shortened duration of exposure sessions are required to meet the specific regularities in routine care settings. Extinction has emerged as the key mechanism of exposure treatment in anxiety disorders. Examining exposure treatment processes from the perspective of extinction learning might provide novel insights into variability in exposure treatment duration and outcome. The present study sought to examine the functional link between fear extinction, the ability to accomplish exposure in a predetermined time and exposure therapy outcome in specific phobia. Treatment-seeking individuals (N = 53) with spider phobia underwent a context-dependent fear conditioning paradigm prior to a standardized exposure. Spider-phobic participants who were able to complete exposure within the pre-determined time (i.e., completers) showed a more pronounced short- and long-term exposure therapy benefit. In the fear conditioning task, a more pronounced decline in CS-US contingency ratings during extinction (retrieval) was found in completers relative to non-completers. The failure to further extinguish US expectancy to the CSs in non-completers might offer a potential mechanistic explanation why non-completers have difficulties to accomplish all exposure steps in a fixed time and show less pronounced treatment gains. Our findings bear specific implications for the implementation of exposure treatment to routine care settings.

Fear Without Context: Acute Stress Modulates the Balance of Cue-Dependent and Contextual Fear Learning

During a threatening encounter, people can learn to associate the aversive event with a discrete preceding cue or with the context in which the event took place, corresponding to cue-dependent and context-dependent fear conditioning, respectively. Which of these forms of fear learning prevails has critical implications for fear-related psychopathology. We tested here whether acute stress may modulate the balance of cue-dependent and contextual fear learning. Participants (N = 72) underwent a stress or control manipulation 30 min before they completed a fear-learning task in a virtual environment that allowed both cued and contextual fear learning. Results showed equally strong cue- and context-dependent fear conditioning in the control group. Stress, however, abolished contextual fear learning, which was directly correlated with the activity of the stress hormone cortisol, and made cue-dependent fear more resistant to extinction. These results are the first to show that stress favors cue-dependent over contextual fear learning.

Expression and Function of Neuron-Glia-Related Cell Adhesion Molecule (NrCAM) in the Amygdalar Pathway.

Neuron-Glia related cell adhesion molecule (NrCAM) is a candidate autism risk factor that promotes axon guidance through cytoskeletal linkages in developing brain but its role in limbic circuitry has not been investigated. In situ hybridization (ISH) and immunofluorescence staining showed that NrCAM is expressed in the developing amygdalar pathway of mouse embryos during outgrowth of projections in the stria terminalis, a major limbic tract that interconnects the central amygdala (CeA) with key targets in the bed nucleus of the stria terminalis (BNST). Analysis of fiber tracts in NrCAM mutant mice by Neurofilament protein immunohistochemistry showed pronounced defasciculation and misprojection of fibers in the ST. The defasciculation phenotype may result from impairment in NrCAM homophilic inter-axonal adhesion or axon repulsion from the secreted ligand Semaphorin 3F, which is expressed in limbic areas in proximity to the ST. Behavioral testing indicated that NrCAM null mice were impaired in context-dependent fear conditioning, in accord with altered amygdala-BNST connectivity, but displayed normal cued (tone-shock) conditioning. Results are consistent with the novel finding that NrCAM mediates fasciculation of axon fibers in the ST important for proper amygdalar-BNST circuitry and response to contextual fear conditioning.

Genetic Model to Study the Co-Morbid Phenotypes of Increased Alcohol Intake and Prior Stress-Induced Enhanced Fear Memory.

Posttraumatic Stress Disorder (PTSD) is a complex illness, frequently co-morbid with depression, caused by both genetics, and the environment. Alcohol Use Disorder (AUD), which also co-occurs with depression, is often co-morbid with PTSD. To date, very few genes have been identified for PTSD and even less for PTSD comorbidity with AUD, likely because of the phenotypic heterogeneity seen in humans, combined with each gene playing a relatively small role in disease predisposition. In the current study, we investigated whether a genetic model of depression-like behavior, further developed from the depression model Wistar Kyoto (WKY) rat, is a suitable vehicle to uncover the genetics of co-morbidity between PTSD and AUD. The by-now inbred WKY More Immobile (WMI) and the WKY Less Immobile (WLI) rats were generated from the WKY via bidirectional selective breeding using the forced swim test, a measure of despair-like behavior, as the functional selector. The colonies of the WMIs that show despair-like behavior and the control strain showing less or no despair-like behavior, the WLI, are maintained with strict inbreeding over 40 generations to date. WMIs of both sexes intrinsically self-administer more alcohol than WLIs. Alcohol self-administration is increased in the WMIs without sucrose fading, water deprivation or any prior stress, mimicking the increased voluntary alcohol-consumption of subjects with AUD. Prior Stress-Enhanced Fear Learning (SEFL) is a model of PTSD. WMI males, but not females, show increased SEFL after acute restraint stress in the context-dependent fear conditioning paradigm, a sexually dimorphic pattern similar to human data. Plasma corticosterone differences between stressed and not-stressed WLI and WMI male and female animals immediately prior to fear conditioning predict SEFL results. These data demonstrate that the WMI male and its genetically close, but behaviorally divergent control the WLI male, would be suitable for investigating the underlying genetic basis of comorbidity between SEFL and alcohol self-administration.

Calcium sensor regulation of the CaV2.1 Ca2+ channel contributes to long-term potentiation and spatial learning

Many forms of short-term synaptic plasticity rely on regulation of presynaptic voltage-gated Ca2+ type 2.1 (CaV2.1) channels. However, the contribution of regulation of CaV2.1 channels to other forms of neuroplasticity and to learning and memory are not known. Here we have studied mice with a mutation (IM-AA) that disrupts regulation of CaV2.1 channels by calmodulin and related calcium sensor proteins. Surprisingly, we find that long-term potentiation (LTP) of synaptic transmission at the Schaffer collateral-CA1 synapse in the hippocampus is substantially weakened, even though this form of synaptic plasticity is thought to be primarily generated postsynaptically. LTP in response to θ-burst stimulation and to 100-Hz tetanic stimulation is much reduced. However, a normal level of LTP can be generated by repetitive 100-Hz stimulation or by depolarization of the postsynaptic cell to prevent block of NMDA-specific glutamate receptors by Mg2+ The ratio of postsynaptic responses of NMDA-specific glutamate receptors to those of AMPA-specific glutamate receptors is decreased, but the postsynaptic current from activation of NMDA-specific glutamate receptors is progressively increased during trains of stimuli and exceeds WT by the end of 1-s trains. Strikingly, these impairments in long-term synaptic plasticity and the previously documented impairments in short-term synaptic plasticity in IM-AA mice are associated with pronounced deficits in spatial learning and memory in context-dependent fear conditioning and in the Barnes circular maze. Thus, regulation of CaV2.1 channels by calcium sensor proteins is required for normal short-term synaptic plasticity, LTP, and spatial learning and memory in mice.

Dynamic methylation driven by neuronal activity in hippocampal neurons impacts complex behavior

Epigenetic processes are well-known to play critical roles in learning and memory. Among epigenetic processes, accumulating data suggests that DNA methylation in particular is a critical determinant of learning and memory. In vitro data have suggested that DNA methyltransferase inhibitors can trigger DNA demethylation and subsequent gene expression of the brain-derived neurotrophic factor gene in an activity dependent manner. To examine if these processes occur in vivo, we chronically infused DNMT inhibitors into the hippocampus and examined the impact on behavior. We find that chronic DNMT inhibition in the hippocampus results in increased anxiety-related behavior and deficits in context-dependent fear conditioning accompanied by an increase in BDNF expression. Gene expression changes were blocked by pretreatment with the NMDA receptor antagonist AP5, suggesting that DNMT inhibition enhances gene expression in an activity-dependent manner and that, conversely, the behavior deficits and abnormal gene expression are facilitated by NMDA receptor activity. Open image in new window

Dissecting the phenotypes of Dravet syndrome by gene deletion.

Neurological and psychiatric syndromes often have multiple disease traits, yet it is unknown how such multi-faceted deficits arise from single mutations. Haploinsufficiency of the voltage-gated sodium channel Nav1.1 causes Dravet syndrome, an intractable childhood-onset epilepsy with hyperactivity, cognitive deficit, autistic-like behaviours, and premature death. Deletion of Nav1.1 channels selectively impairs excitability of GABAergic interneurons. We studied mice having selective deletion of Nav1.1 in parvalbumin- or somatostatin-expressing interneurons. In brain slices, these deletions cause increased threshold for action potential generation, impaired action potential firing in trains, and reduced amplification of postsynaptic potentials in those interneurons. Selective deletion of Nav1.1 in parvalbumin- or somatostatin-expressing interneurons increases susceptibility to thermally-induced seizures, which are strikingly prolonged when Nav1.1 is deleted in both interneuron types. Mice with global haploinsufficiency of Nav1.1 display autistic-like behaviours, hyperactivity and cognitive impairment. Haploinsufficiency of Nav1.1 in parvalbumin-expressing interneurons causes autistic-like behaviours, but not hyperactivity, whereas haploinsufficiency in somatostatin-expressing interneurons causes hyperactivity without autistic-like behaviours. Heterozygous deletion in both interneuron types is required to impair long-term spatial memory in context-dependent fear conditioning, without affecting short-term spatial learning or memory. Thus, the multi-faceted phenotypes of Dravet syndrome can be genetically dissected, revealing synergy in causing epilepsy, premature death and deficits in long-term spatial memory, but interneuron-specific effects on hyperactivity and autistic-like behaviours. These results show that multiple disease traits can arise from similar functional deficits in specific interneuron types.

Inducible forebrain-specific ablation of the transcription factor Creb during adulthood induces anxiety but no spatial/contextual learning deficits.

The cyclic AMP (cAMP)-response element binding protein (CREB) is an activity-dependent transcription factor playing a role in synaptic plasticity, learning and memory, and emotional behavior. However, the impact of Creb ablation on rodent behavior is vague as e.g., memory performance of different Creb mutant mice depends on the specific type of mutation per se but additionally on the background and learning protocol differences. Here we present the first targeted ablation of CREB induced during adulthood selectively in principal forebrain neurons in a pure background strain of C57BL/6 mice. All hippocampal principal neurons exhibited lack of CREB expression. Mutant mice showed a severe anxiety phenotype in the openfield and novel object exploration test as well as in the Dark-Light Box Test, but unaltered hippocampus-dependent long-term memory in the Morris water maze and in context dependent fear conditioning. On the molecular level, CREB ablation led to CREM up regulation in the hippocampus and frontal cortex which may at least in part compensate for the loss of CREB. BDNF, a postulated CREB target gene, was down regulated in the frontal lobe but not in the hippocampus; neurogenesis remained unaltered. Our data indicate that in the adult mouse forebrain the late onset of CREB ablation can, in case of memory functionality, be compensated for and is not essential for memory consolidation and retrieval during adulthood. In contrast, the presence of CREB protein during adulthood seems to be pivotal for the regulation of emotional behavior.

The hypocretin/orexin system mediates the extinction of fear memories.

Anxiety disorders are often associated with an inability to extinguish learned fear responses. The hypocretin/orexin system is involved in the regulation of emotional states and could also participate in the consolidation and extinction of aversive memories. Using hypocretin receptor-1 and hypocretin receptor-2 antagonists, hypocretin-1 and hypocretin-2 peptides, and hypocretin receptor-1 knockout mice, we investigated the role of the hypocretin system in cue- and context-dependent fear conditioning and extinction. Hypocretins were crucial for the consolidation of fear conditioning, and this effect was mainly observed in memories with a high emotional component. Notably, after the acquisition of fear memory, hypocretin receptor-1 blockade facilitated fear extinction, whereas hypocretin-1 administration impaired this extinction process. The extinction-facilitating effects of the hypocretin receptor-1 antagonist SB334867 were associated with increased expression of cFos in the basolateral amygdala and the infralimbic cortex. Intra-amygdala, but neither intra-infralimbic prefrontal cortex nor intra-dorsohippocampal infusion of SB334867 enhanced fear extinction. These results reveal a key role for hypocretins in the extinction of aversive memories and suggest that hypocretin receptor-1 blockade could represent a novel therapeutic target for the treatment of diseases associated with inappropriate retention of fear, such as post-traumatic stress disorder and phobias.

Palmitoylation of δ-catenin by DHHC5 mediates activity-induced synapse plasticity

Synaptic cadherin adhesion complexes are known to be key regulators of synapse plasticity. However, the molecular mechanisms that coordinate activity-induced modifications in cadherin localization and adhesion and subsequent changes in synapse morphology and efficacy, remain unanswered. We demonstrate that the intracellular cadherin binding protein, δ-catenin, is transiently palmitoylated by DHHC5 following enhanced synaptic activity, and that palmitoylation increases δ-catenin/cadherin interactions at synapses. Both the palmitoylation of δ-catenin and its binding to cadherin are required for activity-induced stabilization of N-cadherin at synapses, the enlargement of postsynaptic spines, as well as insertion of GluA1 and GluA2 subunits into the synaptic membrane and the concomitant increase in mEPSC amplitude. Importantly, context-dependent fear conditioning in mice results in increased δ-catenin palmitoylation as well as increased δ-catenin/cadherin associations at hippocampal synapses. Together, this suggests a role for palmitoylated δ-catenin in coordinating activity-dependent changes in synaptic adhesion molecules, synapse structure, and receptor localization that are involved in memory formation.

Evolutionary genetic analyses of MEF2C gene: Implications for learning and memory in Homo sapiens

MEF2C facilitates context-dependent fear conditioning (CFC) which is a salient aspect of hippocampus-dependent learning and memory. CFC might have played a crucial role in human evolution because of its advantageous influence on survival of species. In this study, we analyzed 23 orthologous mammalian gene sequences of MEF2C gene to examine the evidence for positive selection on this gene in Homo sapiens using Phylogenetic Analysis by Maximum Likelihood (PAML) and HyPhy software. Both PAML Bayes Empirical Bayes (BEB) and HyPhy Fixed Effects Likelihood (FEL) analyses supported significant positive selection on 4 codon sites in H. sapiens. Also, haplotter analysis revealed significant ongoing positive selection on this gene in Central European population. The study findings suggest that adaptive selective pressure on this gene might have influenced human evolution. Further research on this gene might unravel the potential role of this gene in learning and memory as well as its pathogenetic effect in certain hippocampal disorders with evolutionary basis like schizophrenia.

Lithium reduced neural progenitor apoptosis in the hippocampus and ameliorated functional deficits after irradiation to the immature mouse brain

Lithium was recently shown to inhibit apoptosis and promote survival of neural progenitor cells after hypoxia-ischemia in the immature rat brain. Our aim was to evaluate the effects of lithium on cell death and proliferation in the hippocampus after irradiation (IR) to the immature brain. Male mice were injected with 2 mmol/kg lithium chloride i.p. on postnatal day 9 (P9) and additional lithium injections, 1 mmol/kg, were administered at 24 h intervals for up to 7 days. BrdU was injected 4 h after lithium injections on P9 and P10. The left hemisphere received a single dose of 8 Gy (MV photons) on P11. The animals were euthanized 6 h or 7 weeks after IR. The number of BrdU-labeled cells in the subgranular zone (SGZ) of the granule cell layer (GCL) 6h after IR was 24% higher in the lithium-treated mice. The number of proliferating, phospho-histone H3-positive cells in the SGZ 7 weeks after IR was 59% higher in the lithium group, so the effect was long-lasting. The number of apoptotic cells in the SGZ 6 h after IR was lower in the lithium group, as judged by 3 different parameters, pyknosis, staining for active caspase-3 and TUNEL. Newly formed cells (BrdU-labeled 1 or 2 days before IR) showed the greatest degree of protection, as judged by 50% fewer TUNEL-positive cells, whereas non-BrdU-labeled cells showed 38% fewer TUNEL-positive cells 6 h after IR. Consequently, the growth retardation of the GCL was less pronounced in the lithium group. The number and size of microglia in the DG were also lower in the lithium group, indicating reduced inflammation. Learning was facilitated after lithium treatment, as judged by improved context-dependent fear conditioning, and improved place learning, as judged by assessment in the IntelliCage platform. In summary, lithium administration could decrease IR-induced neural progenitor cell apoptosis in the GCL of the hippocampus and ameliorate learning impairments. It remains to be shown if lithium can be used to prevent the debilitating cognitive late effects seen in children treated with cranial radiotherapy.

Can zebrafish learn spatial tasks? An empirical analysis of place and single CS–US associative learning

The zebrafish may be an ideal tool with which genes underlying learning and memory can be identified and functionally investigated. From a translational viewpoint, relational learning and episodic memory are particularly important as their impairment is the hallmark of prevalent human neurodegenerative diseases. Recent reports suggest that zebrafish are capable of solving complex relational-type associative learning tasks, namely spatial learning tasks. However, it is not known whether good performance in these tasks was truly based upon relational learning or upon a single CS–US association. Here we study whether zebrafish can find a rewarding stimulus (sight of conspecifics) based upon a single associative cue or/and upon the location of the reward using a method conceptually similar to ‘context and cue dependent fear conditioning’ employed with rodents. Our results confirm that zebrafish can form an association between a salient visual cue and the rewarding stimulus and at the same time they can also learn where the reward is presented. Although our results do not prove that zebrafish form a dynamic spatial map of their surroundings and use this map to locate their reward, they do show that these fish perform similarly to rodents whose hippocampal function is unimpaired. These results further strengthen the notion that complex cognitive abilities exist in the zebrafish and thus they may be analyzed using the excellent genetic tool set developed for this simple vertebrate.

Na+–Ca2+Exchanger (NCX3) Knock-Out Mice Display an Impairment in Hippocampal Long-Term Potentiation and Spatial Learning and Memory

Long-term potentiation (LTP) depends on the coordinated regulation of an ensemble of proteins related to Ca(2+) homeostasis, including Ca(2+) transporters. One of the major players in the regulation of intracellular Ca(2+) ([Ca(2+)](i)) homeostasis in neurons is the sodium/calcium exchanger (NCX), which represents the principal mechanism of Ca(2+) clearance in the synaptic sites of hippocampal neurons. Because NCX3, one of the three brain isoforms of the NCX family, is highly expressed in the hippocampal subfields involved in LTP, we hypothesized that it might represent a potential candidate for LTP modulation. To test this hypothesis, we first examined the effect of ncx3 gene ablation on NCX currents (I(NCX)) and Ca(2+) homeostasis in hippocampal neurons. ncx3(-/-) neurons displayed a reduced I(NCX), a higher basal level of [Ca(2+)](i), and a significantly delayed clearance of [Ca(2+)](i) following depolarization. Furthermore, measurement of field EPSPs, recorded from the CA1 area, revealed that ncx3(-/-) mice had an impaired basal synaptic transmission. Moreover, hippocampal slices from ncx3(-/-) mice exhibited a worsening in LTP compared with congenic ncx3(+/+). Consistently, immunohistochemical and immunoblot analysis indicated that in the hippocampus of ncx3(-/-) mice both Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) expression and the phosphoCaMKIIα/CaMKIIα ratio were significantly reduced compared with ncx3(+/+). Interestingly, ncx3(-/-) mice displayed a reduced spatial learning and memory performance, as revealed by the novel object recognition, Barnes maze, and context-dependent fear conditioning assays. Collectively, our findings demonstrate that the deletion of the ncx3 gene in mice has detrimental consequences on basal synaptic transmission, LTP regulation, spatial learning, and memory performance.

Effects of transient global ischaemia on freezing behaviour and activity in a context-dependent fear conditioning task – Implications for memory investigations

Transient global ischaemia induces cell death in the CA1 layer of the hippocampus. To evaluate the functional consequences, we performed context-dependent fear conditioning. Ischaemia was induced by 2-vessel-occlusion (2VO) in gerbils. On day 6 post ischaemia or sham procedures (in control group) gerbils were placed in a test chamber and after 3min adaption time exposed to foot-shocks (training session). On the next day the animals were placed in the same test chamber without foot-shocks (test session). As a parameter for memory performance we used the standard method of measuring the total freezing time via a cumulative time-sampling procedure during the test session. We found a significant longer total freezing time in control animals than in ischaemic animals. In addition, however, we applied a more detailed analysis of (i) quantifying the number of freezing bouts, (ii) the average duration of single freezing bouts, (iii) the activity pattern during the training and test situation and (iv) we differentially evaluated all the single time segments of the experiment. These analyses revealed that although maintenance of freezing (duration of freezing bout) was significantly lower in ischaemic animals compared to controls, the initiation of freezing (number of freezing bouts) was not significantly different between the two groups during the test session. The activity scores of ischaemic and non-ischaemic gerbils were similar during the adaption time of the training session. The foot-shock, however, induced a significantly different pattern of behaviour in the ischaemic animals, which was selectively reproduced during the test session. In conclusion, ischaemic gerbils reacted to a fearsome thread with a behavioural pattern different from unlesioned animals and they revealed this specific foot-shock induced behaviour again during the test session. This indicated that CA1 hippocampal death did not interrupt memory performance but changed expression of fear. Therefore, measuring duration of freezing and the activity score seems to be not applicable for quantitative comparisons of memory deficits after 2VO in gerbils in a context-dependent fear conditioning task. Our results indicate, however, that initiation of freezing (number of freezing bouts) may be a more suitable parameter comparing gerbils with and without CA1 damage.