Enhanced anxiety, depressive-like behaviour and impaired recognition memory in mice with reduced expression of the vesicular glutamate transporter 1 (VGLUT1)

Three isoforms of a vesicular glutamate transporter (VGLUT1-3) have been identified. Of these, VGLUT1 is the major isoform in the cerebral cortex and hippocampus where it is selectively located on synaptic vesicles of excitatory glutamatergic terminals. Variations in VGLUT1 expression levels have a major impact on the efficacy of glutamate synaptic transmission. Given evidence linking alterations in glutamate neurotransmission to various neuropsychiatric disorders, we investigated the possible influence of a down-regulation of VGLUT1 transporter on anxiety, depressive-like behaviour and learning. The behavioural phenotype of VGLUT1-heterozygous mice (C57BL/6) was compared to wild-type (WT) littermates. Moreover, VGLUT1-3 expression, hippocampal excitatory terminal ultrastructure and neurochemical phenotype were analysed. VGLUT1-heterozygous mice displayed normal spontaneous locomotor activity, increased anxiety in the light-dark exploration test and depressive-like behaviour in the forced swimming test: no differences were shown in the elevated plus-maze model of anxiety. In the novel object recognition test, VGLUT1(+/-) mice showed normal short-term but impaired long-term memory. Spatial memory in the Morris water maze was unaffected. Western blot analysis confirmed that VGLUT1 heterozygotes expressed half the amount of transporter compared to WT. In addition, a reduction in the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35-45% reduction in GABA in the frontal cortex and the hippocampus were observed in the mutant mice. These observations suggest that a VGLUT1-mediated presynaptic alteration of the glutamatergic synapses, in specific brain regions, leads to a behavioural phenotype resembling certain aspects of psychiatric and cognitive disorders.     

Traveling in the dark: the legibility of a regular and predictable structure of the environment extends beyond its borders

The Lsamp gene gives rise to limbic system-associated membrane protein (LAMP), which is expressed on the surface of somata and proximal dendrites of neurons. Lsamp-deficient mice have been shown to be slightly hyperactive in novel environments and less anxious, and they display alterations in swimming speed, fear reaction, fear conditioning and social behaviour. In human studies, links between the LSAMP gene and several psychiatric disorders have been found and LSAMP has been established as a tumour suppressor gene. To study the impact of environmental manipulations on the phenotype, we exposed male Lsamp-deficient mice to environmental enrichment (EE), a technique that has often been shown to abolish phenotypic deviations in knockout mice, and to social isolation, a stressful manipulation, after which all the mice were tested in a behavioural battery. EE abolished differences between the genotypes in body weight and anogenital sniffing, a behaviour related to aggressiveness, and amplified the anxiolytic-like phenotype of Lsamp-deficient mice both in the plus maze and motility box. Isolation abolished differences between the genotypes in body weight and anxiety and amplified the differences in swimming speed and anogenital sniffing. EE and isolation failed to modify the results as compared to standard housing in whisker trimming, locomotor activity, marble burying and corticosterone levels. In conclusion, Lsamp-deficient mice were less sensitive to isolation stress than their wild-type littermates. Lack of LAMP protein seemingly leads to a deterioration in the ability to adapt to novel stressful environments and stimuli.     

Genetic deletion of Lsamp causes exaggerated behavioral activation in novel environments

The limbic system-associated membrane protein (LAMP) is a GPI-anchored cell adhesion molecule expressed heavily in limbic and limbic-associated regions of the developing and adult brain. Experimental studies show that LAMP promotes the growth of limbic neurons and guides the projections of limbic fibers. In order to examine the functional consequences of disrupting limbic circuit assembly, we generated a mouse line in which the Lsamp gene encoding LAMP was deleted. Basic neuroanatomical organization and sensory and motor development are normal in Lsamp(-/-) mice. The most profound change in behavior in both male and female Lsamp(-/-) mice is a heightened reactivity to novelty exhibited in several behavioral tests. Lsamp(-/-) mice display hyperactivity in a novel arena and both sexes habituate to the same activity levels as their wild type littermates, but at different rates. In the elevated plus maze, Lsamp(-/-) mice exhibit increased total arm entries, with a bias towards the open arms; they spend more time in the open arms and have a substantial increase in the amount of risk assessment in unprotected areas of the maze. In the y-maze, Lsamp(-/-) mice exhibit characteristic hyperactivity and a decreased level of spontaneous alternation during the period when their novelty-induced hyperactivity is at its peak. We hypothesize that Lsamp(-/-) mice may not simply exhibit a decrease in anxiety, but may have a heightened, and possibly maladaptive, response to novel environmental stressors. Genetic deletion of Lsamp may thus cause circumscribed changes in the fine connectivity of specific circuits that underlie these behaviors.     

Association of fear auras with mood and anxiety disorders after temporal lobectomy

PURPOSE: Epilepsy has been associated with increased occurrence of behavioral disorders. Auras reflect abnormal stimulation of brain areas in close proximity to regions from which clinical seizures originate. The purpose of our study was to investigate whether fear auras are associated with a higher rate of mood and anxiety disorders before and 1 year after temporal lobectomy. METHODS: Twenty-two patients with fear auras were compared with matched groups with other auras and no auras. Neurologic and neuropsychological evaluations before, 1-2 months after, and 1 year after temporal lobectomy were reviewed for mood and anxiety disorders and psychotropic medication treatment. A logistic regression model examined effects of patient group and psychiatric status on postoperative psychiatric status. RESULTS: The majority of patients in the three groups experienced mood and anxiety disorders before surgery. Mood and anxiety disorders declined in the control, but not in the fear aura group after surgery. Presence of auras at 1 year after surgery was not related to psychiatric outcome. Postoperative mood and anxiety disorders were more common in patients with persistence of seizures and in those in the fear group who were seizure free. The minority of patients in all groups underwent psychotropic treatment before surgery, but the majority with fear auras underwent treatment after surgery. CONCLUSIONS: Postoperative mood and anxiety disorders were more common in fear aura patients after temporal lobectomy, in particular, if seizure free. Possible mechanisms include the role of the amygdala in fear conditioning, the concepts of forced normalization, and kindling.     

Nocturnal hyperactivity, increased social novelty preference and delayed extinction of fear responses in post-weaning socially isolated mice

When rodents are reared in isolation from young age onwards, they manifest a number of behavioural alterations in adulthood. Since some of these alterations resemble symptoms of psychiatric disorders, the post-weaning social isolation (ISO) manipulation is often applied to create rodent models of these disorders. In rats, ISO effects have been thoroughly characterised, but in mice they are less well documented. Therefore, we further evaluated behaviour of adult ISO mice with a test battery that focussed on abnormalities relevant to schizophrenia. We found that ISO mice were hyperactive during the dark phase. Also, ISO mice showed alterations in magnitude, habituation and prepulse-inhibition of the acoustic startle reflex, increased anxiety, increased social preference and changes in extinction of fear responses. We did not observe increased sensitivity to locomotor-activating effects of amphetamine. It is concluded that ISO of mice might serve as a useful model to test further hypotheses regarding pathogenesis occurring at specific developmental timeframes.     

Reduced anxiety in the mice expressing mutant (N141I) presenilin 2

Alzheimer's disease (AD) is characterized by progressive cognitive impairment. The effect of presenilin 1 (PS1) and PS2 mutation on cognition has been well characterized in a variety of transgenic mice. However, noncognitive behaviors have not been considered in these mice. In the present study, we found that transgenic mice expressing mutant PS2 (N141I) displayed decreased anxiety behavior determined by the elevated plus maze test and the light dark box test. However, these mice showed biphasic ambulatory activity (hyperactivity followed by hypoactivity) in an open field test. Correlated well with the reduced anxiety, expression of GABA(A)alpha(1) receptor was higher whereas c-Fos was lower in the cortex, hippocampus, and amygdala of the mice expressing PS2 mutation than those of the wild-type PS2 or nontransgenic control mice. These data indicate that PS2 mutation causes reduction of anxiety, and this effect may be related to the change of the expression of GABA(A)alpha(1) receptor and c-Fos. These findings could be useful in the understanding and the treatment of AD patients.     

Targeted gene mutation approaches to the study of anxiety-like behavior in mice

Studying the behavioral phenotypes of transgenic and gene knockout mice is a powerful means to better understand the pathophysiology of neuropsychiatric disorders and ultimately improve their treatment. This paper provides an overview of the methods and findings of studies that have tested for anxiety-related behavioral phenotypes in gene mutant mice. In the context of improving the side effect burden of benzodiazepines, gene targeting has been valuable for dissociating the functional roles (i.e., anxiolytic, sedative, amnestic) of individual GABA(A) receptor subunits. Supporting the link between abnormalities in CRH function and anxiety, CRH overexpressing transgenic mice and CRH-R2 receptor knockout mutants have displayed significantly increased anxiety-like behavior, while CRH-R1 receptor knockout mice have shown an anxiolytic-like phenotype. Consistent with an important role for the serotonergic system in anxiety, 5-HT1(A) receptor deficient mice have consistently exhibited heightened anxiety-like behavior, while the evidence from 5-HT1(B) and 5-HT2(C) deficient mice remains somewhat equivocal. Mutant mice lacking either of the monoamine degrading enzymes, MAOA or COMT, have shown a number of behavioral and neurological effects, including alterations in anxiety-like behavior. With enhanced spatial and temporal control over gene mutations, in combination with an improved battery of behavioral tests, gene mutant mice will provide an increasingly valuable tool for understanding the neural substrates of anxiety.     

Expression pattern of the cannabinoid receptor genes in the frontal cortex of mood disorder patients and mice selectively bred for high and low fear

Although the endocannabinoid system (ECS) has been implicated in brain development and various psychiatric disorders, precise mechanisms of the ECS on mood and anxiety disorders remain unclear. Here, we have investigated developmental and disease-related expression pattern of the cannabinoid receptor 1 (CB1) and the cannabinoid receptor 2 (CB2) genes in the dorsolateral prefrontal cortex (PFC) of humans. Using mice selectively bred for high and low fear, we further investigated potential association between fear memory and the cannabinoid receptor expression in the brain. The CB1, not the CB2, mRNA levels in the PFC gradually decrease during postnatal development ranging in age from birth to 50 years (r2 > 0.6 & adj. p < 0.05). The CB1 levels in the PFC of major depression patients were higher when compared to the age-matched controls (adj. p < 0.05). In mice, the CB1, not the CB2, levels in the PFC were positively correlated with freezing behavior in classical fear conditioning (p < 0.05). These results suggest that the CB1 in the PFC may play a significant role in regulating mood and anxiety symptoms. Our study demonstrates the advantage of utilizing data from postmortem brain tissue and a mouse model of fear to enhance our understanding of the role of the cannabinoid receptors in mood and anxiety disorders.     

Social, Communication, and Cortical Structural Impairments in Epac2-Deficient Mice

Deficits in social and communication behaviors are common features of a number of neurodevelopmental disorders. However, the molecular and cellular substrates of these higher order brain functions are not well understood. Here we report that specific alterations in social and communication behaviors in mice occur as a result of loss of the EPAC2 gene, which encodes a protein kinase A-independent cAMP target. Epac2-deficient mice exhibited robust deficits in social interactions and ultrasonic vocalizations, but displayed normal olfaction, working and reference memory, motor abilities, anxiety, and repetitive behaviors. Epac2-deficient mice displayed abnormal columnar organization in the anterior cingulate cortex, a region implicated in social behavior in humans, but not in somatosensory cortex. In vivo two-photon imaging revealed reduced dendritic spine motility and density on cortical neurons in Epac2-deficient mice, indicating deficits at the synaptic level. Together, these findings provide novel insight into the molecular and cellular substrates of social and communication behavior.     

Motor neurons are rich in non-phosphorylated neurofilaments: cross-species comparison and alterations in ALS

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is believed to play an essential role for GABA synthesis in the central nervous system. Using mice with targeted disruption of the GAD65 gene (GAD65^−/− mice) we investigated the contribution of GAD65 to GABA synthesis in different brain areas during postnatal development and in adulthood. In the amygdala, hypothalamus and parietal cortex of GAD65^+/+ mice an increase of GABA levels was observed during postnatal development, most prominently between the first and second month after birth. This increase appeared to be dependent on GAD65, as it was delayed by 2 months in GAD65^+/− mice and was not observed in GAD65^−/− mice. Likely as a consequence of their GABA deficit, adult GAD65^−/− mice showed a largely abnormal neural activity with frequent paroxysmal discharges and spontaneous seizures. They furthermore displayed increased anxiety-like behaviour in a light/dark avoidance test and reduced intermale aggression, as well as a reduced forced-swimming-induced immobility indicative of an antidepressant-like behavioural change. Adult GAD65^+/− mice did not show behavioural disturbances except for a reduced aggressive behaviour that was comparable to that in GAD65^−/− mice. We conclude that GAD65-mediated GABA synthesis may be crucially involved in control of emotional behaviour and indispensable for a tonic inhibition that prevents the development of hyperexcitability in the maturating central nervous system. Aggressive, and possibly other social behaviour may be especially prone to regulation through GAD65-mediated GABA synthesis.     

Cannabinoid receptor type 1 receptors on GABAergic vs. glutamatergic neurons differentially gate sex‐dependent social interest in mice

Abnormalities in social behavior are found in almost all psychiatric disorders, such as anxiety, depression, autism, and schizophrenia. Thus, comprehension of the neurobiological basis of social interaction is important for a better understanding of numerous pathologies and improved treatments. Several findings have suggested that an alteration of cannabinoid receptor type 1 (CB1) receptor function could be involved in the pathophysiology of such disorders. However, the role of CB1 receptors is still unclear, and their localisation on different neuronal subpopulations may produce distinct outcomes. To dissect the role of CB1 receptors in different neuronal populations, we used male knockout mice and their respective control littermates [total deletion (CB1^?/?); specific deletion on cortical glutamatergic neurons (Glu‐CB1^?/?) or on GABAergic interneurons (GABA‐CB1^?/?), and wild‐type (WT) mice treated with the CB1 antagonist/inverse agonist SR141716A (3 mg/kg). Mice were required to perform different social tasks – direct social interaction and social investigation. Direct interaction of two male mice was not modified in any group; however, when they were paired with females, Glu‐CB1^?/? mice showed reduced interaction. Also, exploration of the male stimulus subject in the three‐chamber social investigation test was almost unaffected. The situation was completely different when a female was used as the stimulus subject. In this case, Glu‐CB1^?/? mice showed reduced interest in the social stimulus, mimicking the phenotype of CB1^?/? or WT mice treated with SR141716A. GABA‐CB1^?/? mice showed the opposite phenotype, by spending more time investigating the social stimulus. In conclusion, we provide evidence that CB1 receptors specifically modulate the social investigation of female mice in a neuronal subtype‐specific manner.     

Genetic dissection of the role of cannabinoid type-1 receptors in the emotional consequences of repeated social stress in mice

The endocannabinoid system (ECS) tightly controls emotional responses to acute aversive stimuli. Repeated stress alters ECS activity but the role played by the ECS in the emotional consequences of repeated stress has not been investigated in detail. This study used social defeat stress, together with pharmacology and genetics to examine the role of cannabinoid type-1 (CB(1)) receptors on repeated stress-induced emotional alterations. Seven daily social defeat sessions increased water (but not food) intake, sucrose preference, anxiety, cued fear expression, and adrenal weight in C57BL/6N mice. The first and the last social stress sessions triggered immediate brain region-dependent changes in the concentrations of the principal endocannabinoids anandamide and 2-arachidonoylglycerol. Pretreatment before each of the seven stress sessions with the CB(1) receptor antagonist rimonabant prolonged freezing responses of stressed mice during cued fear recall tests. Repeated social stress abolished the increased fear expression displayed by constitutive CB(1) receptor-deficient mice. The use of mutant mice lacking CB(1) receptors from cortical glutamatergic neurons or from GABAergic neurons indicated that it is the absence of the former CB(1) receptor population that is responsible for the fear responses in socially stressed CB(1) mutant mice. In addition, stress-induced hypolocomotor reactivity was amplified by the absence of CB(1) receptors from GABAergic neurons. Mutant mice lacking CB(1) receptors from serotonergic neurons displayed a higher anxiety but decreased cued fear expression than their wild-type controls. These mutant mice failed to show social stress-elicited increased sucrose preference. This study shows that (i) release of endocannabinoids during stress exposure impedes stress-elicited amplification of cued fear behavior, (ii) social stress opposes the increased fear expression and delayed between-session extinction because of the absence of CB(1) receptors from cortical glutamatergic neurons, and (iii) CB(1) receptors on central serotonergic neurons are involved in the sweet consumption response to repeated stress.     

TNFalpha signaling in depression and anxiety: behavioral consequences of individual receptor targeting.

BACKGROUND: Increased serum levels of TNFalpha and other pro-inflammatory cytokines have been found in patients with major depression and several other psychiatric conditions. In rodents, these cytokines produce symptoms commonly referred to as "sickness behavior." Some of these, including reduced feeding and decreased social and exploratory behavior, are reminiscent of those seen in depressed patients. Interpretation of these effects is complicated by the malaise caused by acute injections of pro-inflammatory cytokines, however. Thus, it is unclear whether cytokines are involved in the etiology of depressive symptoms. METHODS: We used a panel of behavioral assays to assess TNFR1(-/-) and TNFR2(-/-) mice for anxiety and depression-like behaviors. RESULTS: We show that deletion of either TNFR1 or TNFR2 leads to an antidepressant-like response in the forced swim test and that mice lacking TNFR2 demonstrate a hedonic response in a sucrose drinking test compared with wildtype littermates. In addition, deletion of TNFR1 leads to decreased fear conditioning. There were no differences in behavior in anxiety tests for either null mutant. CONCLUSIONS: These results are consistent with the hypothesis that TNFalpha can induce depression-like symptoms even in the absence of malaise and demonstrate that both receptor subtypes can be involved in this response.     

The latent structure of anxiety symptoms in anxiety disorders.

OBJECTIVE: Research in the psychopathology of panic and anxiety disorders, particularly agoraphobia, suggests that fear of fear may be the basis of these conditions. However, there is little empirical research on the definition and validity of the concept of fear of fear in a clinical study group. The authors' aims are 1) to determine empirically if particular associations between symptoms and beliefs exist in a group of patients with anxiety disorders and what underlying dimensions of perceived threat they represent and 2) to assess the relative importance of these associations in agoraphobia with panic attacks, panic disorder, social phobia, and generalized anxiety disorder. METHOD: In an anxiety disorders treatment unit, 390 outpatients with anxiety disorders diagnosed according to DSM-III criteria completed the Anxiety Symptoms and Beliefs Scale. RESULTS: A principal components analysis of the patients' ratings on the Anxiety Symptoms and Beliefs Scale produced a four-factor solution in which specific sets of anxiety symptoms loaded with specific beliefs. These four factors were interpreted as respiratory symptoms, vestibular symptoms, autonomic arousal, and psychological threat. Respiratory and vestibular symptoms were more associated with panic disorder diagnoses than with social phobia and generalized anxiety disorder diagnoses. CONCLUSIONS: These findings support a conception of fear of fear in anxiety disorders as fearful beliefs concerning the experience of anxiety symptoms. Associations between symptoms and fear of fear are present across anxiety disorders but are most pronounced in agoraphobia with panic attacks.     

Reduced anxiety and improved stress coping ability in mice lacking NPY-Y2 receptors

Neuropeptide Y (NPY) has been implicated in the pathophysiology of certain mood disorders, including depression and anxiety. It is, however, not known which of the five cloned NPY receptors mediate these functions. We investigated the effect of Y2 receptor deletion on anxiety and stress-related behaviours. In the elevated plus maze, Y2 knock out (Y2(-/-)) mice showed a 2.7-fold higher frequency of entering into, and spent 3.8 times more time within, the open arms compared to controls, while entries into the closed arms did not differ. Similarly Y2(-/-) mice entered the central area of the open field 1.7 times more frequently and also spent 1.8 times more time there. In the light/dark test Y2(-/-) mice had a 4.8-fold lower latency to enter the lit area but stayed there 2.6 times longer than control mice. Y2(-/-) mice displayed 3.2-fold less immobility in the forced swim test, indicating improved stress coping ability. Y2 receptors are predominantly located presynaptically where they mediate feedback inhibition of neurotransmitter release. Deletion of these receptors may result in enhanced release of NPY, GABA and/or glutamate in brain areas linked to the manifestation of anxiety, and stress-related behaviour such as the amygdala. Taken together, deletion of the Y2 receptor has revealed an important role of Y2 receptors in the generation of anxiety-related and stress-related behaviours in mice.     

Targeting the NMDA receptor for fear-related disorders

The gap between basic neuroscience and clinical psychiatry in the treatment of anxiety disorders has been steadily diminishing over recent years. Among the leading advances in this field of translation research is the application of knowledge on the neurobiology of fear memory to the treatment of anxiety disorders in humans. Animal studies have identified N-methyl-D-aspartate (NMDA) receptors as crucial in fear memory acquisition and consolidation, as well as in its extinction and reconsolidation. Based on this, the NMDA receptor partial agonist D-cycloserine, which facilitates fear extinction in rodents, has been shown to increase the effect of exposure therapy in psychiatric patients for conditions such as phobias, social anxiety and obsessive-compulsive disorder. In this article, we review current strategies for targeting the NMDA receptor in the treatment of fear-related disorders, analyzing experimental results, clinical data, and recent patents in the field, while also addressing potential new approaches to explore this field of drug discovery.     

Current status of virtual reality exposure therapy in anxiety disorders: editorial review

PURPOSE OF REVIEW: In recent years, a considerable number of publications have appeared on different aspects of virtual reality exposure therapy in the treatment of anxiety disorders. The purpose of the present article is to review the evidence that has emerged to support the potential of this therapy in the treatment of anxiety disorders. RECENT FINDINGS: Case histories and open comparative studies, as well as randomized controlled studies, have been published on the use of virtual reality exposure therapy in the treatment of anxiety disorders. Most studies in the field have been on specific phobias, in particular on fear of flying, acrophobia, fear of driving, claustrophobia and fear of spiders. In addition, several studies have been published on the use of virtual reality exposure therapy for social anxiety disorders, posttraumatic stress disorder and panic disorder with or without agoraphobia. SUMMARY: Recent studies provide evidence that virtual reality exposure therapy is a promising tool for the treatment of several anxiety disorders.     

The circadian gene Nr1d1 in the mouse nucleus accumbens modulates sociability and anxiety‐related behaviour

Nuclear receptor subfamily 1, group D, member 1 (Nr1d1) (also known as Rev‐erb alpha) has been linked to circadian rhythm regulation, mood‐related behaviour and disorders associated with social deficits. Recent work from our laboratory found striking decreases in Nr1d1 in the nucleus accumbens (NAc) in the maternal condition and indirect evidence that Nr1d1 was interacting with numerous addiction and reward‐related genes to modulate social reward. In this study, we applied our insights from the maternal state to nonparental adult mice to determine whether decreases in Nr1d1 expression in the NAc via adeno‐associated viral (AAV) vectors and short hairpin RNA (shRNA)‐mediated gene knockdown were sufficient to modulate social behaviours and mood‐related behaviours. Knockdown of Nr1d1 in the NAc enhanced sociability and reduced anxiety, but did not affect depressive‐like traits in female mice. In male mice, Nr1d1 knockdown had no significant behavioural effects. Microarray analysis of Nr1d1 knockdown in females identified changes in circadian rhythm and histone deacetylase genes and suggested possible drugs, including histone deacetylase inhibitors, that could mimic actions of Nr1d1 knockdown. Quantitative real‐time PCR (qPCR) analysis confirmed expression upregulation of gene period circadian clock 1 (Per1) and period circadian clock 2 (Per2) with Nr1d1 knockdown. The evidence for roles for opioid‐related genes opioid receptor, delta 1 (Oprd1) and preproenkephalin (Penk) was also found. Together, these results suggest that Nr1d1 in the NAc modulates sociability and anxiety‐related behaviour in a sex‐specific manner, and circadian, histone deacetylase and opioid‐related genes may be involved in the expression of these behavioural phenotypes.