Endocannabinoids mediate acute fear adaptation via glutamatergic neurons independently of corticotropin-releasing hormone signaling.

Recent evidence showed that the endocannabinoid system plays an important role in the behavioral adaptation of stress and fear responses. In this study, we chose a behavioral paradigm that includes criteria of both fear and stress responses to assess whether the involvement of endocannabinoids in these two processes rely on common mechanisms. To this end, we delivered a footshock and measured the fear response to a subsequently presented novel tone stimulus. First, we exposed different groups of cannabinoid receptor type 1 (CB(1))-deficient mice (CB(1) (-/-)) and their wild-type littermates (CB(1) (+/+)) to footshocks of different intensities. Only application of an intense footshock resulted in a sustained fear response to the tone in CB(1) (-/-). Using the intense protocol, we next investigated whether endocannabinoids mediate their effects via an interplay with corticotropin-releasing hormone (CRH) signaling. Pharmacological blockade of CB(1) receptors by rimonabant in mice deficient for the CRH receptor type 1 (CRHR1(-/-)) or type 2 (CRHR2(-/-)), and in respective wild-type littermates, resulted in a sustained fear response in all genotypes. This suggests that CRH is not involved in the fear-alleviating effects of CB(1). As CRHR1(-/-) are known to be severely impaired in stress-induced corticosterone secretion, our observation also implicates that corticosterone is dispensable for CB(1)-mediated acute fear adaptation. Instead, conditional mutants with a specific deletion of CB(1) in principal neurons of the forebrain (CaMK-CB(1) (-/-)), or in cortical glutamatergic neurons (Glu-CB(1) (-/-)), showed a similar phenotype as CB(1) (-/-), thus indicating that endocannabinoid-controlled glutamatergic transmission plays an essential role in acute fear adaptation

Supplementary Material for: NextGen Brain Microdialysis: Applying Modern Metabolomics Technology to the Analysis of Extracellular Fluid in the Central Nervous System

Microdialysis is a powerful method for in vivo neurochemical analyses. It allows fluid sampling in a dynamic manner in specific brain regions over an extended period of time. A particular focus has been the neurochemical analysis of extracellular fluids to explore central nervous system functions. Brain microdialysis recovers neurotransmitters, low-molecular-weight neuromodulators and neuropeptides of special interest when studying behavior and drug effects. Other small molecules, such as central metabolites, are typically not assessed despite their potential to yield important information related to brain metabolism and activity in selected brain regions. We have implemented a liquid chromatography online mass spectrometry metabolomics platform for an expanded analysis of mouse brain microdialysates. The method is sensitive and delivers information for a far greater number of analytes than commonly used electrochemical and fluorescent detection or biochemical assays. The metabolomics platform was applied to the analysis of microdialysates in a foot shock-induced mouse model of posttraumatic stress disorder (PTSD). The rich metabolite data information was then used to delineate affected prefrontal molecular pathways that reflect individual susceptibility for developing PTSD-like symptoms. We demonstrate that hypothesis-free metabolomics can be adapted to the analysis of microdialysates for the discovery of small molecules with functional significance

Conditional Ablation of the Neural Cell Adhesion Molecule Reduces Precision of Spatial Learning, Long-Term Potentiation, and Depression in the CA1 Subfield of Mouse Hippocampus

10.1523/JNEUROSCI.3298-03.2004Journal of Neuroscience2471565-1577JNRS

A robust and reliable non-invasive test for stress responsivity in mice.

Stress and an altered stress response have been associated with many multifactorial diseases, such as psychiatric disorders or neurodegenerative diseases. As currently mouse mutants for each single gene are generated and phenotyped in a large-scale manner, it seems advisable also to test these mutants for alterations in their stress responses. Here we present the determinants of a robust and reliable non-invasive test for stress-responsivity in mice. Stress is applied through restraining the mice in tubes and recording behavior in the Open Field 20 min after cessation of the stress. Two hours, but not 15 or 50 min of restraint lead to a robust and reproducible increase in distance traveled and number of rearings during the first 5 min in the Open Field in C57BL/6 mice. This behavioral response is blocked by the corticosterone synthesis inhibitor metyrapone, but not by RU486 treatment, indicating that it depends on corticosteroid secretion, but is not mediated via the glucocorticoid receptor type II. We assumed that with a stress duration of 15 min one could detect hyper-responsivity, and with a stress duration of 2 h hypo-responsivity in mutant mouse lines. This was validated with two mutant lines known to show opposing effects on corticosterone secretion after stress exposure, corticotropin-releasing hormone (CRH) over-expressing mice and CRH receptor 1 knockout (KO) mice. Both lines showed the expected phenotype, i.e., increased stress responsivity in the CRH over-expressing mouse line (after 15 min restraint stress) and decreased stress responsivity in the CRHR1-KO mouse line (after 2 h of restraint stress). It is possible to repeat the acute stress test several times without the stressed animal adapting to it, and the behavioral response can be robustly evoked at different ages, in both sexes and in different mouse strains. Thus, locomotor and rearing behavior in the Open Field after an acute stress challenge can be used as reliable, non-invasive indicators of stress responsivity and corticosterone secretion in mice

Context and trade-offs characterize real-world threat detection systems: A review and comprehensive framework to improve research practice and resolve the translational crisis

Contains fulltext : 219339.pdf (Publisher’s version ) (Closed access)A better understanding of context in decision-making - that is, the internal and external conditions that modulate decisions - is required to help bridge the gap between natural behaviors that evolved by natural selection and more arbitrary laboratory models of anxiety and fear. Because anxiety and fear are mechanisms evolved to manage threats from predators and other exigencies, the large behavioral, ecological and evolutionary literature on predation risk is useful for re-framing experimental research on human anxiety-related disorders. We review the trade-offs that are commonly made during antipredator decision-making in wild animals along with the context under which the behavior is performed and measured, and highlight their relevance for focused laboratory models of fear and anxiety. We then develop an integrative mechanistic model of decision-making under risk which, when applied to laboratory and field settings, should improve studies of the biological basis of normal and pathological anxiety and may therefore improve translational outcomes.9 p

Supplementary Material for: Fluoxetine Treatment Rescues Energy Metabolism Pathway Alterations in a Posttraumatic Stress Disorder Mouse Model

<br>Posttraumatic stress disorder (PTSD) is a prevalent psychiatric disorder. Several studies have attempted to characterize molecular alterations associated with PTSD, but most findings were limited to the investigation of specific cellular markers in the periphery or defined brain regions. In the current study, we aimed to unravel affected molecular pathways/mechanisms in the fear circuitry associated with PTSD. We interrogated a foot shock-induced PTSD mouse model by integrating proteomics and metabolomics profiling data. Alterations at the proteome level were analyzed using in vivo ¹⁵N metabolic labeling combined with mass spectrometry in the prelimbic cortex (PrL), anterior cingulate cortex (ACC), basolateral amygdala, central nucleus of the amygdala and CA1 of the hippocampus between shocked and nonshocked (control) mice, with and without fluoxetine treatment. In silico pathway analyses revealed an upregulation of the citric acid cycle pathway in PrL, and downregulation in ACC and nucleus accumbens (NAc). Chronic fluoxetine treatment prevented decreased citric acid cycle activity in NAc and ACC and ameliorated conditioned fear response in shocked mice. Our results shed light on the role of energy metabolism in PTSD pathogenesis and suggest potential therapy through mitochondrial targeting

Beware of your Cre-Ation: lacZ expression impairs neuronal integrity and hippocampus-dependent memory.

Expression of the lacZ-sequence is a widely used reporter-tool to assess the transgenic and/or transfection efficacy of a target gene in mice. Once activated, lacZ is permanently expressed. However, protein accumulation is one of the hallmarks of neurodegenerative diseases. Furthermore, the protein product of the bacterial lacZ gene is &szlig;-galactosidase, an analog to the mammalian senescence-associated &szlig;-galactosidase, a molecular marker for aging. Therefore we studied the behavioral, structural and molecular consequences of lacZ expression in distinct neuronal sub-populations. lacZ expression in cortical glutamatergic neurons resulted in severe impairments in hippocampus-dependent memory accompanied by marked structural alterations throughout the CNS. In contrast, GFP expression or the expression of the ChR2/YFP fusion product in the same cell populations did not result in either cognitive or structural deficits. GABAergic lacZ expression caused significantly decreased hyper-arousal and mild cognitive deficits. Attenuated structural and behavioral consequences of lacZ expression could also be induced in adulthood, and lacZ transfection in neuronal cell cultures significantly decreased their viability. Our findings provide a strong caveat against the use of lacZ reporter mice for phenotyping studies and point to a particular sensitivity of the hippocampus formation to detrimental consequences of lacZ expression

Neddylation inhibition impairs spine development, destabilizes synapses and deteriorates cognition.

Neddylation is a ubiquitylation-like pathway that controls cell cycle and proliferation by covalently conjugating Nedd8 to specific targets. However, its role in neurons, nonreplicating postmitotic cells, remains unexplored. Here we report that Nedd8 conjugation increased during postnatal brain development and is active in mature synapses, where many proteins are neddylated. We show that neddylation controls spine development during neuronal maturation and spine stability in mature neurons. We found that neddylated PSD-95 was present in spines and that neddylation on Lys202 of PSD-95 is required for the proactive role of the scaffolding protein in spine maturation and synaptic transmission. Finally, we developed Nae1(CamKII&alpha;-CreERT2) mice, in which neddylation is conditionally ablated in adult excitatory forebrain neurons. These mice showed synaptic loss, impaired neurotransmission and severe cognitive deficits. In summary, our results establish neddylation as an active post-translational modification in the synapse regulating the maturation, stability and function of dendritic spines