Natalia Kulesskaya

Fear Erasure in Mice Requires Synergy Between Antidepressant Drugs and Extinction Training

Long-term loss of fearful memories can be achieved through a combination of antidepressant drugs and exposure therapy. Antidepressant drugs and psychotherapy combined are more effective in treating mood disorders than either treatment alone, but the neurobiological basis of this interaction is unknown. To investigate how antidepressants influence the response of mood-related systems to behavioral experience, we used a fear-conditioning and extinction paradigm in mice. Combining extinction training with chronic fluoxetine, but neither treatment alone, induced an enduring loss of conditioned fear memory in adult animals. Fluoxetine treatment increased synaptic plasticity, converted the fear memory circuitry to a more immature state, and acted through local brain-derived neurotrophic factor. Fluoxetine-induced plasticity may allow fear erasure by extinction-guided remodeling of the memory circuitry. Thus, the pharmacological effects of antidepressants need to be combined with psychological rehabilitation to reorganize networks rendered more plastic by the drug treatment.

Assessment of mouse anxiety-like behavior in the light-dark box and open-field arena: role of equipment and procedure.

Light-dark box and open field are conventional tests for assessment of anxiety-like behavior in the laboratory mice, based on approach-avoidance conflict. However, except the basic principles, variations in the equipment and procedures are very common. Therefore, contribution of certain methodological issues in different settings was investigated. Three inbred strains (C57BL/6, 129/Sv, DBA/2) and one outbred stock (ICR) of mice were used in the experiments. An effect of initial placement of mice either in the light or dark compartment was studied in the light-dark test. Moreover, two tracking systems were applied - position of the animals was detected either by infrared sensors in square box (1/2 dark) or by videotracking in rectangular box (1/3 dark). Both approaches revealed robust and consistent strain differences in the exploratory behavior. In general, C57BL/6 and ICR mice showed reduced anxiety-like behavior as compared to 129/Sv and DBA/2 strains. However, the latter two strains differed markedly in their behavior. DBA/2 mice displayed high avoidance of the light compartment accompanied by thigmotaxis, whereas the hypoactive 129 mice spent a significant proportion of time in risk-assessment behavior at the opening between two compartments. Starting from the light side increased the time spent in the light compartment and reduced the latency to the first transition. In the open field arena, black floor promoted exploratory behavior - increased time and distance in the center and increased rearing compared to white floor. In conclusion, modifications of the apparatus and procedure had significant effects on approach-avoidance behavior in general whereas the strain rankings remained unaffected.

Evaluation of Social and Physical Enrichment in Modulation of Behavioural Phenotype in C57BL/6J Female Mice

Housing conditions represent an important environmental variable playing a critical role in the assessment of mouse behaviour. In the present study the effects of isolation and nesting material on the behaviour of female C57BL/6J mice were evaluated. The mice were subjected to different rearing conditions from weaning (at the age of 3 weeks). The study groups were group- and single-housed mice, divided further into groups with or without nesting material (species-specific enrichment). After 8 weeks spent in respective conditions the behavioural testing began. Both factors (social conditions and nesting material) appeared to have a significant impact on the behavioural phenotype. However, it is important to stress that the interaction between the factors was virtually absent. We established that isolation increased locomotor activity and reduced anxiety-like behaviour in several tests of exploration. In contrast, absence of nesting material increased anxiety-like behaviour. Neither factor affected rota-rod performance, nociception and prepulse inhibition. Contextual fear memory was significantly reduced in single-housed mice, and interestingly, in mice with nesting material. Cued fear memory was reduced by single-housing, but not affected by enrichment. Mice from enriched cages displayed faster and better learning and spatial search strategy in the water maze. In contrast, isolation caused significant impairment in the water maze. In conclusion, both isolation and species-specific enrichment have profound effects on mouse behaviour and should be considered in design of the experiments and in assessment of animal welfare issues.

Effect of S-COMT deficiency on behavior and extracellular brain dopamine concentrations in mice.

IntroductionCatechol-O-methyltransferase (COMT) has soluble (S-COMT) and membrane bound (MB-COMT) isoforms. Our aims were to assess the behavioral phenotype of S-COMT mutant mice and to clarify the role of MB-COMT in dopamine metabolism in different brain areas.MethodsBehavioral phenotype of the S-COMT mutant mice was assessed using a test battery designed to describe anxiety phenotype, spontaneous locomotor activity, sensorymotor gating, social behavior, and pain sensitivity. Microdialysis was used to explore the effect of S-COMT deficiency on extracellular dopamine under an L-dopa load (carbidopa /L-dopa 30/10 mg/kg i.p.).ResultsIn behavioral tests, mature adult S-COMT mutants that only possessed MB-COMT exhibited enhanced acoustic startle without alterations in sensorimotor gating. They also showed barbering of vibrissae and nonaggressive social dominance, suggesting a change in their social interactions. In addition, S-COMT deficiency slightly and sex-dependently affected spinal pain reflex and the effect of morphine on hot-plate latency. In microdialysis studies under L-dopa load, S-COMT mutants of both sexes had higher accumbal dopamine levels, but male S-COMT mutant mice showed paradoxically lower prefrontal cortical dopamine concentrations than wild-type animals. S-COMT deficiency induced the accumulation of 3,4-dihydroxyphenylacetic acid in all brain areas, which was accentuated after L-dopa loading. The lack of S-COMT decreased extracellular homovanillic acid levels. However, after L-dopa loading, homovanillic acid concentrations in the prefrontal cortex of S-COMT mutants were similar to those of wild-type mice.ConclusionA lack of S-COMT has a notable, albeit small, brain-area and sex-dependent effect on the O-methylation of dopamine and 3,4-dihydroxyphenylacetic acid in the mouse brain. It also induces subtle changes in mouse social interaction behaviors and nociception.

CD73 Is a Major Regulator of Adenosinergic Signalling in Mouse Brain

CD73 (ecto-5’-nucleotidase) is a cell surface enzyme that regulates purinergic signalling by desphosphorylating extracellular AMP to adenosine. 5′-nucleotidases are known to be expressed in brain, but the expression of CD73 and its putative physiological functions at this location remain elusive. Here we found, using immunohistochemistry of wild-type and CD73 deficient mice, that CD73 is prominently expressed in the basal ganglia core comprised of striatum (caudate nucleus and putamen) and globus pallidus. Furthermore, meninges and the olfactory tubercle were found to specifically express CD73. Analysis of wild type (wt) and CD73 deficient mice revealed that CD73 confers the majority of 5’-nucleotidase activity in several areas of the brain. In a battery of behavioural tests and in IntelliCage studies, the CD73 deficient mice demonstrated significantly enhanced exploratory locomotor activity, which probably reflects the prominent expression of CD73 in striatum and globus pallidus that are known to control locomotion. Furthermore, the CD73 deficient mice displayed altered social behaviour. Overall, our data provide a novel mechanistic insight into adenosinergic signalling in brain, which is implicated in the regulation of normal and pathological behaviour.

KCC2-deficient mice show reduced sensitivity to diazepam, but normal alcohol-induced motor impairment, gaboxadol-induced sedation, and neurosteroid-induced hypnosis.

GABAA receptors mediate both fast phasic inhibitory postsynaptic potentials and slower tonic extrasynaptic inhibition. Hyperpolarizing phasic GABAergic inhibition requires the activity of neuron-specific chloride-extruding potassium–chloride cotransporter KCC2 in adult CNS. However, the possible role of KCC2 in tonic GABAergic inhibition and the associated behaviors is unknown. Here, we have examined the role of KCC2 in phasic vs tonic GABA inhibition by measuring the behavioral effects of pharmacological agents that presumably enhance phasic vs tonic inhibition in mice that retain 15–20% of normal KCC2 protein levels. These KCC2-deficient mice show decreased sensitivity to diazepam-induced sedation and motor impairment consistent with the reported role for KCC2 in fast hyperpolarizing inhibition. In contrast, the mice exhibit normal responses to low-dose alcohol-induced motor impairment, gaboxadol-induced sedation, and neurosteroid-induced hypnosis; behaviors thought to be associated with tonic GABAergic inhibition. Electrophysiological recordings show that the tonic conductance is not affected. The results suggest that KCC2 activity is more critical for behaviors dependent on phasic than tonic GABAergic inhibition.

LRRTM1-deficient mice show a rare phenotype of avoiding small enclosures--a tentative mouse model for claustrophobia-like behaviour.

The LRRTM family proteins have been shown to act as synaptogenic cell adhesion molecules via interaction with presynaptic neurexins and are associated with neuropsychiatric disorders. LRRTM1-knockout mice have subtle morphological deficits in excitatory hippocampal synapses and were suggested to have impaired cognitive function. Here we report that LRRTM1-knockout mice exhibit an extraordinary phenotype of avoiding small enclosures. In the light-dark box, the knockout mice escape to dark through a standard opening as quickly as wild-type littermates but avoid escaping through a small doorway. While all wild-type mice spontaneously enter a small tube, most knockout mice do not. This apparent aversion to enter narrow space may explain other abnormalities such as increased time in open arms in the elevated plus maze and less visits through a tunnel in the IntelliCage. Moreover, LRRTM1-knockout mice show increased social interaction, reduced nest building and MK801-induced locomotion, and slower swim speed but normal water maze learning. Since LRRTM1 is predominantly expressed in thalamus, hippocampus and limbic cortex, specific synaptic defects in those areas presumably cause these behavioural abnormalities.

JNK1 controls dendritic field size in L2/3 and L5 of the motor cortex, constrains soma size, and influences fine motor coordination

Genetic anomalies on the JNK pathway confer susceptibility to autism spectrum disorders, schizophrenia, and intellectual disability. The mechanism whereby a gain or loss of function in JNK signaling predisposes to these prevalent dendrite disorders, with associated motor dysfunction, remains unclear. Here we find that JNK1 regulates the dendritic field of L2/3 and L5 pyramidal neurons of the mouse motor cortex (M1), the main excitatory pathway controlling voluntary movement. In Jnk1-/- mice, basal dendrite branching of L5 pyramidal neurons is increased in M1, as is cell soma size, whereas in L2/3, dendritic arborization is decreased. We show that JNK1 phosphorylates rat HMW-MAP2 on T1619, T1622, and T1625 (Uniprot P15146) corresponding to mouse T1617, T1620, T1623, to create a binding motif, that is critical for MAP2 interaction with and stabilization of microtubules, and dendrite growth control. Targeted expression in M1 of GFP-HMW-MAP2 that is pseudo-phosphorylated on T1619, T1622, and T1625 increases dendrite complexity in L2/3 indicating that JNK1 phosphorylation of HMW-MAP2 regulates the dendritic field. Consistent with the morphological changes observed in L2/3 and L5, Jnk1-/- mice exhibit deficits in limb placement and motor coordination, while stride length is reduced in older animals. In summary, JNK1 phosphorylates HMW-MAP2 to increase its stabilization of microtubules while at the same time controlling dendritic fields in the main excitatory pathway of M1. Moreover, JNK1 contributes to normal functioning of fine motor coordination. We report for the first time, a quantitative Sholl analysis of dendrite architecture, and of motor behavior in Jnk1-/- mice. Our results illustrate the molecular and behavioral consequences of interrupted JNK1 signaling and provide new ground for mechanistic understanding of those prevalent neuropyschiatric disorders where genetic disruption of the JNK pathway is central.

Mixed housing with DBA/2 mice induces stress in C57BL/6 mice: implications for interventions based on social enrichment

Several behavioral interventions, based on social enrichment and observational learning are applied in treatment of neuropsychiatric disorders. However, the mechanism of such modulatory effect and the safety of applied methods on individuals involved in social support need further investigation. We took advantage of known differences between inbred mouse strains to reveal the effect of social enrichment on behavior and neurobiology of animals with different behavioral phenotypes. C57BL/6 and DBA/2 female mice displaying multiple differences in cognitive, social, and emotional behavior were group-housed either in same-strain or in mixed-strain conditions. Comprehensive behavioral phenotyping and analysis of expression of several plasticity- and stress-related genes were done to measure the reciprocal effects of social interaction between the strains. Contrary to our expectation, mixed housing did not change the behavior of DBA/2 mice. Nevertheless, the level of serum corticosterone and the expression of glucocorticoid receptor Nr3c1 in the brain were increased in mixed housed DBA/2 as compared with those of separately housed DBA/2 mice. In contrast, socially active C57BL/6 animals were more sensitive to the mixed housing, displaying several signs of stress: alterations in learning, social, and anxiety-like behavior and anhedonia. These behavioral impairments were accompanied by the elevated serum corticosterone and the reduced expression of Nr3c1, as well as the elevated Bdnf levels in the cortex and hippocampus. Our results demonstrate the importance of social factors in modulation of both behavior and the underlying neurobiological mechanisms in stress response, and draw attention to the potential negative impact of social interventions for individuals involved in social support.

Deficiency of prolyl oligopeptidase in mice disturbs synaptic plasticity and reduces anxiety-like behaviour, body weight, and brain volume.

Prolyl oligopeptidase (PREP) has been implicated in neurodegeneration and neuroinflammation and has been considered a drug target to enhance memory in dementia. However, the true physiological role of PREP is not yet understood. In this paper, we report the phenotyping of a mouse line where the PREP gene has been knocked out. This work indicates that the lack of PREP in mice causes reduced anxiety but also hyperactivity. The cortical volumes of PREP knockout mice were smaller than those of wild type littermates. Additionally, we found increased expression of diazepam binding inhibitor protein in the cortex and of the somatostatin receptor-2 in the hippocampus of PREP knockout mice. Furthermore, immunohistochemistry and tail suspension test revealed lack of response of PREP knockout mice to lipopolysaccharide insult. Further analysis revealed significantly increased levels of polysialylated-neural cell adhesion molecule in PREP deficient mice. These findings might be explained as possible alteration in brain plasticity caused by PREP deficiency, which in turn affect behaviour and brain development.