Social defeat: impact on fear extinction and amygdala-prefrontal cortical theta synchrony in 5-HTT deficient mice

Emotions, such as fear and anxiety, can be modulated by both environmental and genetic factors. One genetic factor is for example the genetically encoded variation of the serotonin transporter (5-HTT) expression. In this context, the 5-HTT plays a key role in the regulation of central 5-HT neurotransmission, which is critically involved in the physiological regulation of emotions including fear and anxiety. However, a systematic study which examines the combined influence of environmental and genetic factors on fear-related behavior and the underlying neurophysiological basis is missing. Therefore, in this study we used the 5-HTT-deficient mouse model for studying emotional dysregulation to evaluate consequences of genotype specific disruption of 5-HTT function and repeated social defeat for fear-related behaviors and corresponding neurophysiological activities in the lateral amygdala (LA) and infralimbic region of the medial prefrontal cortex (mPFC) in male 5-HTT wild-type (+/+), homo- (-/-) and heterozygous (+/-) mice. Naive males and experienced losers (generated in a resident-intruder paradigm) of all three genotypes, unilaterally equipped with recording electrodes in LA and mPFC, underwent a Pavlovian fear conditioning. Fear memory and extinction of conditioned fear was examined while recording neuronal activity simultaneously with fear-related behavior. Compared to naive 5-HTT+/+ and +/- mice, 5-HTT-/- mice showed impaired recall of extinction. In addition, 5-HTT-/- and +/- experienced losers showed delayed extinction learning and impaired recall of extinction. Impaired behavioral responses were accompanied by increased theta synchronization between the LA and mPFC during extinction learning in 5-HTT-/- and +/- losers. Furthermore, impaired extinction recall was accompanied with increased theta synchronization in 5-HTT-/- naive and in 5-HTT-/- and +/- loser mice. In conclusion, extinction learning and memory of conditioned fear can be modulated by both the 5-HTT gene activity and social experiences in adulthood, accompanied by corresponding alterations of the theta activity in the amygdala-prefrontal cortex network.     

Age-related deficits in neuronal physiology and cognitive function are recapitulated in young mice overexpressing the L-type calcium channel,CaV1.3

The calcium dysregulation hypothesis of brain aging posits that an age-related increase in neuronal calcium concentration is responsible for alterations in a variety of cellular processes that ultimately result in learning and memory deficits in aged individuals. We previously generated a novel transgenic mouse line, in which expression of the L-type voltage-gated calcium, Ca_V1.3, is increased by ~50% over wild-type littermates. Here, we show that, in young mice, this increase is sufficient to drive changes in neuronal physiology and cognitive function similar to those observed in aged animals. Specifically, there is an increase in the magnitude of the postburst afterhyperpolarization, a deficit in spatial learning and memory (assessed by the Morris water maze), a deficit in recognition memory (assessed in novel object recognition), and an overgeneralization of fear to novel contexts (assessed by contextual fear conditioning). While overexpression of Ca_V1.3 recapitulated these key aspects of brain aging, it did not produce alterations in action potential firing rates, basal synaptic communication, or spine number/density. Taken together, these results suggest that increased expression of Ca_V1.3 in the aged brain is a crucial factor that acts in concert with age-related changes in other processes to produce the full complement of structural, functional, and behavioral outcomes that are characteristic of aged animals.     

Impaired Extinction of Learned Contextual Fear Memory in Early Growth Response 1 Knockout Mice

Inductive expression of early growth response 1 (Egr-1) in neurons is associated with many forms of neuronal activity. However, only a few Egr-1 target genes are known in the brain. The results of this study demonstrate that Egr-1 knockout (KO) mice display impaired contextual extinction learning and normal fear acquisition relative to wild-type (WT) control animals. Genome-wide microarray experiments revealed 368 differentially expressed genes in the hippocampus of Egr-1 WT exposed to different phases of a fear conditioning paradigm compared to gene expression profiles in the hippocampus of KO mice. Some of genes, such as serotonin receptor 2C (Htr2c), neuropeptide B (Npb), neuronal PAS domain protein 4 (Npas4), NPY receptor Y1 (Npy1r), fatty acid binding protein 7 (Fabp7), and neuropeptide Y (Npy) are known to regulate processing of fearful memories, and promoter analyses demonstrated that several of these genes contained Egr-1 binding sites. This study provides a useful list of potential Egr-1 target genes which may be regulated during fear memory processing.     

Superior working memory and behavioural habituation but diminished psychomotor coordination in mice lacking the ecto-5′-nucleotidase (CD73) gene

Adenosine is an important neuromodulator in the central nervous system involved in the regulation of wakefulness, sleep, learning and memory, fear and anxiety as well as motor functions. Extracellular adenosine is synthesized by the cell-surface ectoenzyme ecto-5′-nucleotidase (CD73) from 5′-adenosine monophosphate. While CD73 is widely expressed throughout the mammalian brain, its specific role for behaviour is poorly understood. We examined spatial working memory, emotional responses, motor coordination and motor learning as well as behavioural habituation in mice with a targeted deletion of CD73. CD73 knockout (CD73?/?) mice exhibit enhanced spatial working memory in the Y-maze and enhanced long-term behavioural habituation in the open field. Furthermore, impaired psychomotor coordination on the accelerating rotarod was found in CD73?/? mice. No changes in motor learning and/or anxiety-like behaviour were evident in CD73?/? mice. Our data provide evidence for a role of CD73 in the regulation of learning and memory and psychomotor coordination. Our results might be important for the evaluation of adenosine neuromodulators as possible treatments to ameliorate cognitive and motor deficits associated with neurodegenerative diseases.     

A mouse model of Bardet-Biedl Syndrome has impaired fear memory,which is rescued by lithium treatment

Primary cilia are microtubule-based organelles present on most cells that regulate many physiological processes, ranging from maintaining energy homeostasis to renal function. However, the role of these structures in the regulation of behavior remains unknown. To study the role of cilia in behavior, we employ mouse models of the human ciliopathy, Bardet-Biedl Syndrome (BBS). Here, we demonstrate that BBS mice have significant impairments in context fear conditioning, a form of associative learning. Moreover, we show that postnatal deletion of BBS gene function, as well as congenital deletion, specifically in the forebrain, impairs context fear conditioning. Analyses indicated that these behavioral impairments are not the result of impaired hippocampal long-term potentiation. However, our results indicate that these behavioral impairments are the result of impaired hippocampal neurogenesis. Two-week treatment with lithium chloride partially restores the proliferation of hippocampal neurons which leads to a rescue of context fear conditioning. Overall, our results identify a novel role of cilia genes in hippocampal neurogenesis and long-term context fear conditioning.     

Focus: Rare Disease: Oxytocin and Oxytocin Receptor Gene Regulation in Williams Syndrome: A Systematic Review

Williams Syndrome (WS) is a rare genetic multisystem disorder that occurs because of a deletion of approximately 25 genes in the 7q11.23 chromosome region. This causes dysmorphic facial appearances, multiple congenital cardiovascular defects, delayed motor skills, and abnormalities in connective tissues and the endocrine system. The patients are mostly diagnosed with mild to moderate mental retardation, however, they have a hyper sociable, socially dis-inhibited, and outgoing personality, empathetic behavior, and are highly talkative. Oxytocin (OT), a neuropeptide synthesized at the hypothalamus, plays an important role in cognition and behavior, and is thought to be affecting WS patients’ attitudes at its different amounts. Oxytocin receptor gene (OXTR), on chromosome 3p25.3, is considered regulating oxytocin receptors, via which OT exerts its effect. WS is a crucial disorder to understand gene, hormone, brain, and behavior associations in terms of sociality and neuropsychiatric conditions. Alterations to the WS gene region offer an opportunity to deepen our understandings of autism spectrum disorder, schizophrenia, anxiety, or depression. We aim to systematically present the data available of OT/OXTR regulation and expression, and the evidence for whether these mechanisms are dysregulated in WS. These results are important, as they predict strong epigenetic control over social behavior by methylation, single nucleotide polymorphisms, and other alterations. The comparison and collaboration of these studies may help to establish a better treatment or management approach for patients with WS if backed up with future research.     

Variability in empathic fear response among 11 inbred strains of mice

Empathy is an important emotional process that involves the ability to recognize and share emotions with others. We have previously developed an observational fear learning (OFL) behavioral assay to measure empathic fear in mice. In the OFL task, a mouse is conditioned for context‐dependent fear when it observes a conspecific demonstrator receiving aversive stimuli. In the present study, by comparing 11 different inbred mouse strains that are commonly used in the laboratory, we found that empathic fear response was highly variable between different strains. Five strains – C57BL/6J, C57BL/6NTac, 129S1/SvImJ, 129S4/SvJae and BTBR T⁺ Itpr3^tf/J – showed observational fear (OF) responses, whereas AKR/J, BALB/cByJ, C3H/HeJ, DBA/2J, FVB/NJ and NOD/ShiLtJ mice exhibited low empathic fear response. Importantly, day 2 OF memory was significantly correlated with contextual memory in the classical fear conditioning among the 11 strains. Innate differences in anxiety, locomotor activity, sociability and preference for social novelty were not significantly correlated with OFL. Interestingly, early adolescent C57BL/6J mice exhibited an increase in acquisition of OF. The level of OFL in C57BL/6J strain was not affected by sex or strains of the demonstrator. Taken together, these data strongly suggest that there are naturally occurring OFL‐specific genetic variations modulating empathic fear behaviors in mice. The identification of causal genes may uncover novel genetic pathways and underlying neural mechanisms that modulate empathic fear and, ultimately, provide new targets for therapeutic intervention in human mental disorders associated with impaired empathy.     

Hippocampal knockdown of α2 nicotinic or M1 muscarinic acetylcholine receptors in C57BL/6J male mice impairs cued fear conditioning

Acetylcholine (ACh) signaling in the hippocampus is important for behaviors related to learning, memory and stress. In this study, we investigated the role of two ACh receptor subtypes previously shown to be involved in fear and anxiety, the M1 mAChR and the α2 nAChR, in mediating the effects of hippocampal ACh on stress‐related behaviors. Adeno‐associated viral vectors containing short‐hairpin RNAs targeting M1 or α2 were infused into the hippocampus of male C57BL/6J mice, and behavior in a number of paradigms related to stress responses and fear learning was evaluated. There were no robust effects of hippocampal M1 mAChR or α2 nAChR knockdown (KD) in the light/dark box, tail suspension, forced swim or novelty‐suppressed feeding tests. However, effects on fear learning were observed in both KD groups. Short term learning was intact immediately after training in all groups of mice, but both the M1 and α2 hippocampal knock down resulted in impaired cued fear conditioning 24 h after training. In addition, there was a trend for a deficit in contextual memory the M1 mAChR KD group 24 h after training. These results suggest that α2 nicotinic and M1 muscarinic ACh receptors in the hippocampus contribute to fear learning and could be relevant targets to modify brain circuits involved in stress‐induced reactivity to associated cues.     

Behavioral profiles of inbred strains on novel olfactory,spatial and emotional tests for reference memory in mice

Studying the behavior of genetic background strains provides important information for the design and interpretation of cognitive phenotypes in mutant mice. Our experiments examined the performance of three commonly used strains (C57BL/6J, 129S6, DBA/2J) on three behavioral tests for learning and memory that measure very different forms of memory, and for which there is a lack of data on strain differences. In the social transmission of food preference test (STFP) all three strains demonstrated intact memory for an odor-cued food that had been sampled on the breath of a cagemate 24 hours previously. While C57BL/6J and 129S6 mice showed good trace fear conditioning, DBA/2J mice showed a profound deficit on trace fear conditioning. In the Barnes maze test for spatial memory, the 129S6 strain showed poor probe trial performance, relative to C57BL/6J mice. Comparison of strains for open field exploratory activity and anxiety-like behavior suggests that poor Barnes maze performance reflects low exploratory behavior, rather than a true spatial memory deficit, in 129S6 mice. This interpretation is supported by good Morris water maze performance in 129S6 mice. These data support the use of a C57BL/6J background for studying memory deficits in mutant mice using any of these tasks, and the use of a 129S6 background in all but the Barnes maze. A DBA/2J background may be particularly useful for investigating the genetic basis of emotional memory using fear conditioning.     

LIMK1 and CLIP-115: linking cytoskeletal defects to Williams syndrome

Williams Syndrome is a developmental disorder that is characterized by cardiovascular problems, particular facial features and several typical behavioral and neurological abnormalities. In Williams Syndrome patients, a heterozygous deletion is present of a region on chromosome 7q11.23 (the Williams Syndrome critical region), which spans approximately 20 genes. Two of these genes encode proteins that regulate dynamic aspects of the cytoskeleton of the cell, either via the actin filament system (LIM kinase 1, or LIMK1), or through the microtubule network (cytoplasmic linker protein of 115 kDa, or CLIP-115). The recent findings that knockout mice lacking LIMK1 or CLIP-115 have distinct neurological and behavioural phenotypes, indicates that cytoskeletal defects might play a role in the development of neurological symptoms in Williams Syndrome patients. In this review, we discuss the properties of LIMK and CLIP family proteins, their function in the regulation of the actin and microtubule cytoskeletal systems, respectively, and the relationship with neurodevelopmental aspects of Williams Syndrome.     

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.     

Angiotensin type 1a receptors on corticotropin‐releasing factor neurons contribute to the expression of conditioned fear1

Although generally associated with cardiovascular regulation, angiotensin II receptor type 1a (AT_1aR) blockade in mouse models and humans has also been associated with enhanced fear extinction and decreased post‐traumatic stress disorder (PTSD) symptom severity, respectively. The mechanisms mediating these effects remain unknown, but may involve alterations in the activities of corticotropin‐releasing factor (CRF)‐expressing cells, which are known to be involved in fear regulation. To test the hypothesis that AT_1aR signaling in CRFergic neurons is involved in conditioned fear expression, we generated and characterized a conditional knockout mouse strain with a deletion of the AT_1aR gene from its CRF‐releasing cells (CRF‐AT_1aR^(?/?)). These mice exhibit normal baseline heart rate, blood pressure, anxiety and locomotion, and freeze at normal levels during acquisition of auditory fear conditioning. However, CRF‐AT_1aR^(?/?) mice exhibit less freezing than wild‐type mice during tests of conditioned fear expression—an effect that may be caused by a decrease in the consolidation of fear memory. These results suggest that central AT_1aR activity in CRF‐expressing cells plays a role in the expression of conditioned fear, and identify CRFergic cells as a population on which AT₁R antagonists may act to modulate fear extinction.     

Schizophrenia-relevant behaviors in a genetic mouse model of constitutive Nurr1 deficiency

Nurr1 (NR4A2) is an orphan nuclear receptor highly essential for the dopaminergic development and survival. Altered expression of Nurr1 has been suggested as a potential genetic risk factor for dopamine-related brain disorders, including schizophrenia. In support of this, recent experimental work in genetically modified mice shows that mice with a heterozygous constitutive deletion of Nurr1 show a facilitation of the development of schizophrenia-related behavioral abnormalities. However, the behavioral characterization of this Nurr1-deficient mouse model remains incomplete. This study therefore used a comprehensive behavioral test battery to evaluate schizophrenia-relevant phenotypes in Nurr1-deficient mice. We found that these mice displayed increased spontaneous locomotor activity and potentiated locomotor reaction to systemic treatment with the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, dizocilpine (MK-801). In addition, male but not female Nurr1-deficient mice showed significant deficits in the prepulse inhibition and prepulse-elicited reactivity. However, Nurr1 deletion did not induce overt abnormalities in other cardinal behavioral and cognitive functions known to be impaired in schizophrenia, including social interaction and recognition, spatial recognition memory or discrimination reversal learning. Our findings thus suggest that heterozygous constitutive deletion of Nurr1 results in a restricted phenotype characteristic of schizophrenia symptomatology, which primarily relates to motor activity, sensorimotor gating and responsiveness to the psychomimetic drug MK-801. This study further emphasizes a critical role of altered dopaminergic development in the precipitation of specific brain dysfunctions relevant to human psychotic disorder.     

5-HT(1A) receptor mutant mice exhibit enhanced tonic, stress-induced and fluoxetine-induced serotonergic neurotransmission

Mutant mice that lack serotonin(1A) receptors exhibit enhanced anxiety-related behaviors, a phenotype that is hypothesized to result from impaired autoinhibitory control of midbrain serotonergic neuronal firing. Here we examined the impact of serotonin(1A) receptor deletion on forebrain serotonin neurotransmission using in vivo microdialysis in the frontal cortex and ventral hippocampus of serotonin(1A) receptor mutant and wild-type mice. Baseline dialysate serotonin levels were significantly elevated in mutant animals as compared with wild-types both in frontal cortex (mutant = 0.44 +/- 0.05 n M; wild-type = 0.28 +/- 0.03 n M) and hippocampus (mutant = 0.46 +/- 0.07 n M; wild-type = 0.27 +/- 0.04 n M). A stressor known to elicit enhanced anxiety-like behaviors in serotonin(1A) receptor mutants increased dialysate 5-HT levels in the frontal cortex of mutant mice by 144% while producing no alteration in cortical 5-HT in wild-type mice. There was no phenotypic difference in the effect of this stressor on serotonin levels in the hippocampus. Fluoxetine produced significantly greater increases in dialysate 5-HT content in serotonin(1A) receptor mutants as compared with wild-types, with two- and three-fold greater responses being observed in the hippocampus and frontal cortex, respectively. This phenotypic effect was mimicked in wild-types by pretreatment with the serotonin(1A) antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide (p-MPPI). These results indicate that deletion of central serotonin(1A) receptors results in a tonic disinhibition of central serotonin neurotransmission, with a greater dysregulation of serotonin release in the frontal cortex than ventral hippocampus under conditions of stress or increased interstitial serotonin levels.     

Creatine transporter (CrT; Slc6a8) knockout mice as a model of human CrT deficiency

Mutations in the creatine (Cr) transporter (CrT; Slc6a8) gene lead to absence of brain Cr and intellectual disabilities, loss of speech, and behavioral abnormalities. To date, no mouse model of CrT deficiency exists in which to understand and develop treatments for this condition. The purpose of this study was to generate a mouse model of human CrT deficiency. We created mice with exons 2–4 of Slc6a8 flanked by loxP sites and crossed these to Cre:CMV mice to create a line of ubiquitous CrT knockout expressing mice. Mice were tested for learning and memory deficits and assayed for Cr and neurotransmitter levels. Male CrT^−/y (affected) mice lack Cr in the brain and muscle with significant reductions of Cr in other tissues including heart and testes. CrT^−/y mice showed increased path length during acquisition and reversal learning in the Morris water maze. During probe trials, CrT^−/y mice showed increased average distance from the platform site. CrT^−/y mice showed reduced novel object recognition and conditioned fear memory compared to CrT^+/y. CrT^−/y mice had increased serotonin and 5-hydroxyindole acetic acid in the hippocampus and prefrontal cortex. Ubiquitous CrT knockout mice have learning and memory deficits resembling human CrT deficiency and this model should be useful in understanding this disorder.     

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.     

Retrieval of context-associated memory is dependent on the Ca(v)3.2 T-type calcium channel

Among all voltage-gated calcium channels, the T-type Ca²⁺ channels encoded by the Ca_v3.2 genes are highly expressed in the hippocampus, which is associated with contextual, temporal and spatial learning and memory. However, the specific involvement of the Ca_v3.2 T-type Ca²⁺ channel in these hippocampus-dependent types of learning and memory remains unclear. To investigate the functional role of this channel in learning and memory, we subjected Ca_v3.2 homozygous and heterozygous knockout mice and their wild-type littermates to hippocampus-dependent behavioral tasks, including trace fear conditioning, the Morris water-maze and passive avoidance. The Ca_v3.2 ^−/− mice performed normally in the Morris water-maze and auditory trace fear conditioning tasks but were impaired in the context-cued trace fear conditioning, step-down and step-through passive avoidance tasks. Furthermore, long-term potentiation (LTP) could be induced for 180 minutes in hippocampal slices of WTs and Ca_v3.2 ^+/− mice, whereas LTP persisted for only 120 minutes in Ca_v3.2 ^−/− mice. To determine whether the hippocampal formation is responsible for the impaired behavioral phenotypes, we next performed experiments to knock down local function of the Ca_v3.2 T-type Ca²⁺ channel in the hippocampus. Wild-type mice infused with mibefradil, a T-type channel blocker, exhibited similar behaviors as homozygous knockouts. Taken together, our results demonstrate that retrieval of context-associated memory is dependent on the Ca_v3.2 T-type Ca²⁺ channel.     

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.