Neurochemically distinct populations of the bed nucleus of stria terminalis modulate innate fear response to weak threat evoked by predator odor stimuli

Anxiety and trauma-related disorders are characterized by significant alterations in threat detection, resulting in inadequate fear responses evoked by weak threats or safety stimuli. Recent research pointed out the important role of the bed nucleus of stria terminalis (BNST) in threat anticipation and fear modulation under ambiguous threats, hence, exaggerated fear may be traced back to altered BNST function. To test this hypothesis, we chemogenetically inhibited specific BNST neuronal populations (corticotropin-releasing hormone - BNSTCRH and somatostatin - BNSTSST expressing neurons) in a predator odor-evoked innate fear paradigm. The rationale for this paradigm was threefold: (1) predatory cues are particularly strong danger signals for all vertebrate species evoking defensive responses on the flight-avoidance-freezing dimension (conservative mechanisms), (2) predator odor can be presented in a scalable manner (from weak to strong), and (3) higher-order processing of olfactory information including predatory odor stimuli is integrated by the BNST. Accordingly, we exposed adult male mice to low and high predatory threats presented by means of cat urine, or low- and high-dose of 2-methyl-2-thiazoline (2MT), a synthetic derivate of a fox anogenital product, which evoked low and high fear response, respectively. Then, we tested the impact of chemogenetic inhibition of BNSTCRH and BNSTSST neurons on innate fear responses using crh- and sst-ires-cre mouse lines. We observed that BNSTSST inhibition was effective only under low threat conditions, resulting in reduced avoidance and increased exploration of the odor source. In contrast, BNSTCRH inhibition had no impact on 2MT-evoked responses, but enhanced fear responses to cat odor, representing an even weaker threat stimulus. These findings support the notion that BNST is recruited by uncertain or remote, potential threats, and CRH and SST neurons orchestrate innate fear responses in complementary ways

Béta-amiloid peptidek aggregációja és kölcsönhatása fehérjékkel; új neuroprotektív vegyületek alkalmazása az Alzheimer-kór megelőzésére = Beta-amyloid aggregation and interaction with proteins; novel neuroprotective compounds for prevention of Alzheimer's disease

Új, standardizálható módszert dolgoztunk ki toxikus �béta-amiloid (Abéta) 1-42 peptid oligomerek előállítására, a preparált oligomereket fiziko-kémiai módszerekkel jellemeztük. Két új neuroprotektív peptidmimetikum vegyületcsaládot találtunk, ezek az anyagok megvédik a neuronokat az Alzheimer-kór (AK) állatmodelljében az Abéta neurotoxikus hatásától. Mindkét vegyületcsoportot szabadalmilag védjük, mint az AK potenciális gyógyszerjelölt vegyületeit. Új ex vivo módszert dolgoztunk ki az Abéta peptidek toxicitásának mérésére (patkány hippocampus szelet, MTT-teszt), a módszer alkalmas az új neuroprotektív vegyületeink aktivitásmérésére is. Az ex vivo hippocampus szeleteket sikerrel alkalmaztuk a neuronális plaszticitás (LTP) mérésére, az Abéta-toxicitás meghatározására, multielektród array (MEA) technikával. In vivo, egysejt-elvezetéses elektrofiziológiai mérésekkel bizonyítottuk az új peptidmimetikumaink neuroprotektív hatását. Proteomikai módszerekkel azonosítottuk az Abéta peptidekkel kölcsönhatásba lépő fehérjéket, ezek elsősorban plazmamembrán, ill. intraneuronális fehérjék (mitokondrium, endoplazmás reticulum, mikrotubuláris rendszer). Az intraneuronális fehérjék és az Abéta peptidek kölcsönhatásai kulcsszerepet játszhatnak az AK patogenezisében. Igazoltuk, hogy a Zn2+ ionok toxikus Abéta-aggregátumok képződését indukálják. Az AK transzgén állatmodelljén bizonyítottuk, hogy a Zn-kelátorok (pl. Perindopril) neuroprotektív hatásúak. Új AK-állatmodellt dolgoztunk ki az Abéta oligomerek icv bevitelével. | A new method was introduced for the preparation of toxic beta-amyloid (Abeta) 1-42 oligomers, these assemblies were characterized with physicochemical methods. Two families of novel neuroprotective peptidomimetics were found, these substances protect neurons against the toxic effect of Abeta in tg mouse models of Alzheimer’s disease (AD). Both groups of the novel substances will be patented as putative drug candidates for AD treatment. A new ex vivo method was introduced for toxicity measurement of Abeta peptides (rat hippocampal slices, MTT-assay); this method proved to be suitable for activity measurement of the novel neuroprotective substances. Hippocampal slices were successfully used for measurement of neuronal plasticity (LTP) for demonstrating neurotoxicity of Abeta aggregates, applying multielectrode array (MEA) technique. The neuroprotective effect of our novel peptidomimetics was demonstrated also in vivo, using one-cell electrophysiology. Proteomic methods were used for identification of proteins interacting with Abeta peptides; these are mainly plasma membrane and intraneuronal (mitochondrial, endoplasmatic reticular and microtubular) proteins. Interaction of intracellular proteins with Abeta may play key role in AD pathogenesis. The role of Zn2+ ions in formation of toxic Abeta-aggregates was demonstrated. Zn2+-chelators (e.g. Perindopril) were neuroprotective in a tg-mouse model of AD. A new AD rat model was introduced using icv administration of synthetic Abeta oligomers

Modelling posttraumatic stress disorders in animals

Animal models of posttraumatic stress disorder are useful tools to reveal the neurobiological basis of the vulnerability to traumatic events, and to develop new treatment strategies, as well as predicting treatment response contributing to personalized medicine approach. Different models have different construct, face and predictive validity and they model different symptoms of the disease. The most prevalent models are the single prolonged stress, electric foot-shock and predator odor. Freezing as 're-experiencing' in cluster B and startle as 'arousal' in cluster E according to DSM-5 are the most frequently studied parameters; however, several other symptoms related to mood, cognitive and social skills are part of the examinations. Beside behavioral characteristics, symptoms of exaggerated sympathetic activity and hypothalamic-pituitary-adrenocortical axis as well as signs of sleep disturbances are also warranted. Test battery rather than a single test is required to describe a model properly and the results should be interpreted in a comprehensive way, e.g. creating a z-score. Research is shifting to study larger populations and identifying the features of the resilient and vulnerable individuals, which cannot be easily done in humans. Incorporation of the "three hit theory" in animal models may lead to a better animal model of vulnerability and resilience. As women are twice as vulnerable as men, more emphasize should be taken to include female animals. Moreover, hypothesis free testing and big data analysis may help to identify an array of biomarkers instead of a single variable for identification of vulnerability and for the purpose of personalized medicine

Vasopressin and post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric condition with a wide range of behavioral disturbances and serious consequences for both patient and society. One of the main reasons for unsuccessful therapies is insufficient knowledge about its underlying pathomechanism. In the search for centrally signaling molecules that might be relevant to the development of PTSD we focus here on arginine vasopressin (AVP). So far AVP has not been strongly implicated in PTSD, but different lines of evidence suggest a possible impact of its signaling in all clusters of PTSD symptomatology. More specifically, in laboratory rodents, AVP agonists affect behavior in a PTSD-like manner, while significant reduction of AVP signaling in the brain e.g. in AVP-deficient Brattleboro rats, ameliorated defined behavioral parameters that can be linked to PTSD symptoms. Different animal models of PTSD also show alterations in the AVP signaling in distinct brain areas. However, pharmacological treatment targeting central AVP receptors via systemic routes is hampered by possible side effects that are linked to the peripheral action of AVP as a hormone. Indeed, the V1a receptor, the most common receptor subtype in the brain, is implicated in vasoconstriction. Thus, systemic treatment with V1a receptor antagonists would be implicated in hypotonia. This implies that novel treatment concepts are needed to target AVP receptors not only at brain level but also in distinct brain areas, to offer alternative treatments for PTSD

Median raphe region GABAergic neurons contribute to social interest in mouse

Gamma-aminobutyric acid (GABA) is a well-known inhibitory neurotransmitter implicated in numerous physiological and pathological behaviors including social interest. Dysregulation of the median raphe region (MRR), a main serotoninergic nucleus, is also characterized by increased social problems. As the majority of MRR cells are GABAergic, we aimed to reveal the social role of these cells. Chemogenetic techniques were used in vesicular GABA transporter Cre mice and with the help of adeno-associated virus vectors artificial receptors (DREADDs, stimulatory, inhibitory or control, containing only a fluorophore) were expressed in MRR GABAergic cells confirmed by immunohistochemistry. Four weeks after viral injection a behavioral test battery (sociability; social interaction; resident-intruder) was conducted. The artificial ligand (clozapine-N-oxide, 1 mg/10 ml/kg) was administrated 30 min before the tests. As possible confounding factors, locomotion (open field/OF), anxiety-like behavior (elevated plus maze/EPM), and short-term memory (Y-maze) were also evaluated. Stimulation of the GABAergic cells in MRR had no effect on locomotion or working and social memory; however, it increased social interest during sociability and social interaction but not in resident-intruder tests. Accordingly, c-Fos elevation in MRR-GABAergic cells was detected after sociability, but not resident-intruder tests. In the EPM test, the inhibitory group entered into the open arms later, suggesting an anxiogenic-like tendency. We confirmed the role of MRR-GABAergic cells in promoting social interest. However, different subpopulations (e.g. long vs short projecting, various neuropeptide containing) might have divergent roles, which might remain hidden and requires further studies

Searching for glycomic biomarkers for predicting resilience and vulnerability in a rat model of posttraumatic stress disorder

Posttraumatic stress disorder (PTSD) is triggered by traumatic events in 10-20% of exposed subjects. N-linked glycosylation, by modifying protein functions, may provide an important environmental link predicting vulnerability.Our goals were (1) to find alterations in plasma N-glycome predicting stress-vulnerability; (2) to investigate how trauma affects N-glycome in the plasma (PGP) and in three PTSD-related brain regions (prefrontal cortex, hippocampus and amygdala; BGP), hence uncover specific targets for PTSD treatment. We examined male (1) controls, (2) traumatized vulnerable and (3) traumatized resilient rats both before and several weeks after electric footshock. Vulnerable and resilient groups were separated by z-score analysis of behavior.Higher freezing behavior and decreased social interest were detected in vulnerable groups compared to control and resilient rats. Innate anxiety did not predict vulnerability, but pretrauma levels of PGP10(FA1G1Ga1), PGP11(FA2G2) and PGP15(FA3G2) correlated positively with it, the last one being the most sensitive. Traumatic stress induced a shift from large, elaborate N-glycans towards simpler neutral structures in the plasma of all traumatized animals and specifically in the prefrontal cortex of vulnerable rats. In plasma trauma increased PGP17(A2G2S) level in vulnerable animals. In all three brain regions BGP11(F(6)A2B) was more abundant in vulnerable rats, while most behavioral correlations occurred in the prefrontal cortex.In conclusion, we found N-glycans (especially PGP15(FA3G2)) in plasma as possible biomarkers of vulnerability to trauma that warrants further investigation. Posttrauma PGP17(A2G2S1) increase showed overlap with human results highlighting the utility and relevance of this animal model. Prefrontal cortex is a key site of trauma-induced glycosylation changes that could modulate the behavioral outcome

Somatostatin neurons of the bed nucleus of stria terminalis enhance associative fear memory consolidation in mice

Excessive fear learning and generalized, extinction-resistant fear memories are core symptoms of anxiety and trauma-related disorders. Despite significant evidence from clinical studies reporting hyperactivity of the bed nucleus of stria terminalis (BNST) under these conditions, the role of BNST in fear learning and expression is still not clarified. Here, we tested how BNST modulates fear learning in male mice using a chemogenetic approach. Activation of GABAergic neurons of BNST during fear conditioning or memory consolidation resulted in enhanced cue-related fear recall. Importantly, BNST activation had no acute impact on fear expression during conditioning or recalls, but it enhanced cue-related fear recall subsequently, potentially via altered activity of downstream regions. Enhanced fear memory consolidation could be replicated by selectively activating somatostatin (SOM), but not corticotropin- releasing factor (CRF) neurons of the BNST, which was accompanied by increased fear generalization. Our findings suggest the significant modulation of fear memory strength by specific circuits of the BNST.SIGNIFICANCE STATEMENT:The bed nucleus of stria terminalis mediates different defensive behaviors and its connections implicate its integrative modulatory role in fear memory formation, however, the involvement of BNST in fear learning has yet to be elucidated in detail. Our data highlight that BNST stimulation enhances fear memory formation without direct effects on fear expression. Our study identified somatostatin cells within the extended amygdala as specific neurons promoting fear memory formation. These data underline the importance of anxiety circuits in maladaptive fear memory formation, indicating elevated BNST activity as a potential vulnerability factor to anxiety and trauma-related disorders

Investigation of Anxiety- and Depressive-like Symptoms in 4- and 8-Month-Old Male Triple Transgenic Mouse Models of Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. Approximately 50% of AD patients show anxiety and depressive symptoms, which may contribute to cognitive decline. We aimed to investigate whether the triple-transgenic mouse (3xTg-AD) is a good preclinical model of this co-morbidity. The characteristic histological hallmarks are known to appear around 6-month; thus, 4- and 8-month-old male mice were compared with age-matched controls. A behavioral test battery was used to examine anxiety- (open field (OF), elevated plus maze, light-dark box, novelty suppressed feeding, and social interaction (SI) tests), and depression-like symptoms (forced swim test, tail suspension test, sucrose preference test, splash test, and learned helplessness) as well as the cognitive decline (Morris water maze (MWM) and social discrimination (SD) tests). Acetylcholinesterase histochemistry visualized cholinergic fibers in the cortex. Dexamethasone-test evaluated the glucocorticoid non-suppression. In the MWM, the 3xTg-AD mice found the platform later than controls in the 8-month-old cohort. The SD abilities of the 3xTg-AD mice were missing at both ages. In OF, both age groups of 3xTg-AD mice moved significantly less than the controls. During SI, 8-month-old 3xTg-AD animals spent less time with friendly social behavior than the controls. In the splash test, 3xTg-AD mice groomed themselves significantly less than controls of both ages. Cortical fiber density was lower in 8-month-old 3xTg-AD mice compared to the control. Dexamethasone non-suppression was detectable in the 4-month-old group. All in all, some anxiety- and depressive-like symptoms were present in 3xTg-AD mice. Although this strain was not generally more anxious or depressed, some aspects of comorbidity might be studied in selected tests, which may help to develop new possible treatments