ACUTE KETAMINE RESCUES CELLULAR AND MOLECULAR CHANGES INDUCED BY CHRONIC MILD STRESS IN VULNERABLE RATS

Numerosi studi hanno associato la disfunzione del sistema glutammatergico con la fisiopatologia dei disturbi neuropsichiatrici correlati allo stress, compreso il disturbo depressivo maggiore. Studi clinici su pazienti depressi hanno mostrato variazioni volumetriche e funzionali in aree cerebrali dove prevale la trasmissione glutammatergica, come l'ippocampo (HPC) e la corteccia prefrontale e frontale (PFC/FC). In parallelo, studi preclinici su modelli animali di depressione basati sullo stress hanno osservato alterazioni della neurotrasmissione glutammatergica e dell'arborizzazione dendritica nelle stesse aree cerebrali coinvolte nei pazienti. Studi recenti hanno mostrato che una singola dose sub-anestetica di ketamina (KET), un antagonista del recettore NMDA, induce un rapido e sostenuto effetto antidepressivo, sia nei pazienti depressi, che nei modelli animali di depressione. Tuttavia, i meccanismi molecolari che sottendono l'effetto terapeutico di KET rimangono al momento poco conosciuti. Utilizzando il chronic mild stress (CMS), un modello animale di depressione, abbiamo studiato i cambiamenti funzionali, morfologici e molecolari associati all'azione antidepressiva rapida di KET. I ratti sono stati sottoposti a CMS per 5 settimane. Attraverso il test di preferenza per il saccarosio siamo riusciti a distinguere i ratti resilienti (CMS-R) da quelli vulnerabili (CMS-V) allo stress. KET (10 mg/kg), somministrata in acuto ai CMS-V 24 ore prima del sacrificio, \ue8 stata in grado di invertire il comportamento anedonico. Il CMS ha indotto importanti cambiamenti fenotipici in tutti i ratti stressati, anche se la diminuzione nell\u2019incremento ponderale, l'aumento dei livelli serici di corticosterone e l'aumento del peso delle ghiandole surrenali sono risultati pi\uf9 elevati nei CMS-V. Una riduzione del rilascio di glutammato, sia basale, che evocato da depolarizzazione, \ue8 stata osservata selettivamente in sinaptosomi in superfusione da HPC di CMS-V e nei sinaptosomi da PFC/FC di CMS-R. KET \ue8 stata in grado di ripristinare il rilascio di glutammato basale, ma non quello depolarizzato, nei CMS-V. Mediante Western Blot, sono state misurate alterazioni area e frazione cellulare specifiche nei livelli di espressione e di fosforilazione di determinate proteine coinvolte nel rilascio di glutammato e nella risposta dello stress, in omogenato totale, sinaptosomi e membrane sinaptiche da HPC e PFC/FC. Tramite qPCR abbiamo rivelato una significativa riduzione dei trascritti totali di BDNF e delle sue varianti di splicing in HPC e PFC/FC di tutti i ratti stressati, inclusi quelli trattati con KET. Tuttavia, studi di ibridazione in situ hanno riscontrato un ridotto traffico dendritico di mRNA totale di BDNF e BDNF-2/BDNF-6 in CA1 e CA3 di CMS-V, mentre il trattamento con KET, pur non ripristinando i cambiamenti nei livelli di mRNA totale di BDNF, \ue8 stato in grado di ristabilire il traffico dendritico in CA3 di CMS-V. L'analisi morfologica dei neuroni piramidali in CA3, in sezioni marcate con il metodo di Golgi-Cox, ha mostrato una riduzione della lunghezza totale e della ramificazione dei dendriti apicali, ma non di quelli basali. KET ha ripristinato tali modifiche ai livelli di controllo. Nel complesso, i nostri risultati mostrano che l'esposizione allo stress cronico induce alterazioni funzionali e morfologiche in HPC e PFC/FC di ratti vulnerabili. \uc8 interessante notare che una singola amministrazione di KET \ue8 in grado di recuperare la maggior parte di questi deficit.Increasing evidence has associated dysfunction of the glutamate system with the pathophysiology of stress-related neuropsychiatric disorders, including major depressive disorder. Clinical studies on depressed patients have shown consistent volumetric and functional changes in brain areas where glutamatergic transmission is predominant, such as hippocampus (HPC) and prefrontal and frontal cortex (PFC/FC). In parallel, preclinical studies on stress-based animal models of depression reported impaired glutamate neurotransmission and dendritic arborisation in the same brain regions affected in patients. Intriguingly, consistent evidence reported that a single sub-anaesthetic dose of the NMDA receptor antagonist ketamine (KET) induces rapid and sustained antidepressant effect, both in patients and in rodent models of depression. However, molecular mechanisms underlying KET therapeutic effect remain poorly understood. Using a chronic mild stress (CMS) rat model of depression, we aimed at studying the functional, morphological and molecular changes associated with fast antidepressant action of KET. Rats were subjected to CMS for 5 weeks. Sucrose preference test allowed to distinguish stress resilient (CMS-R) from vulnerable (CMS-V) rats. 10 mg/kg KET, acutely administered to CMS-V 24 hours before sacrifice, reversed anhedonic behavior. CMS induced significant phenotypic changes in all CMS rats, although the decrease in body weight gain and the increase of circulating corticosterone levels and adrenal glands weight were higher in CMS-V. A decrease in basal and depolarization-evoked glutamate release was selectively measured in HPC synaptosomes in superfusion from CMS-V, and in PFC/FC synaptosomes from CMS-R. Intriguingly, KET restored basal, but not depolarization-evoked, glutamate release in CMS-V. Area- and subcellular fraction-specific modifications in the expression and phosphorylation levels of selected proteins involved in glutamate release and stress response were measured by Western Blot in total homogenate, synaptosomes and synaptic membranes from HPC and PFC/FC. qPCR revealed a significant reduction in total-BDNF and BDNF splice variants transcripts in HPC and PFC/FC of all CMS rats, including those treated with KET. However, in situ hybridization studies found reduced dendritic trafficking of total-BDNF, and BDNF-2/BDNF-6 variants mRNAs in CA1 and CA3 of CMS-V, while KET treatment, although not reversing changes in BDNF mRNA levels, completely rescued dendritic trafficking in CA3 of CMS-V. Morphological analysis of CA3 pyramidal neurons in Golgi-Cox stained sections showed a reduction in total length and branching of apical, but not basal, dendrites. KET restored these changes to control levels. Overall, our results show that chronic exposure to mild stress induces functional and morphological alterations in HPC and PFC/FC of vulnerable rats. Interestingly, a single administration of KET was able to reverse most of these deficits

Sun protection habits and behaviors of a group of outdoor workers and students from the agricultural and construction sectors in north-Italy

Background: Despite the relevant frequency of ultraviolet induced adverse health effects in workers, solar ultraviolet radiation (UVR) exposure is an occupational risk not adequately minimized in Italy. Objective: To assess the characteristics and prevalence of sun exposure habits and behaviors in a group of students and outdoor workers (OW) from the agricultural and construction sectors of a north-Italian region. Methods: Based on a previously developed standardized questionnaire, we collected full information on individual sun exposure habits at work and during leisure activities. Results: In 2018, 380 high school students and OW from the agricultural and construction sectors participated in a sun-safety campaign. More than a third (39.0%) of OW reported never using sunglasses, 52.8% never applied sunscreens at work, and a quarter never wear a UV protective hat. Considering leisure-time, students reported more frequent sunburns compared to OW: 25.0% vs. 13.8%; half (51.6%) of students and a third (36.4%) of OW reported never wearing a UV protective hat. A third (30.1%) of students and 37.2% of OW never or only seldom applied sunscreens on holidays. Discussion: The majority of OW in our study reported poor protective solar exposure habits. Young students of the construction and agricultural sectors indicated even worse sun-protective behaviors, both during apprenticeship and leisure activities. Our study highlights the low health literacy related to solar UVR in OW and apprentices. Further educational initiatives are required in Italy to improve the adoption of protective behaviors during outdoor activities

The effects of ecolabels on environmentally- and health-friendly cars : an online survey and two experimental studies

Purpose: Given the increasing importance of political decision-making to reduce emission targets, the main purpose of the current paper is to identify and test the considerations that would nudge consumers towards an environmentally and health-friendly motor vehicle. Methods: An online survey was conducted to assess public responses and the role of public authorities to a voluntary emission standard for passenger cars. In addition, two online experiments were conducted to test incentives in the design of ecolabels (e.g. price, safety, performance) for optimization. A random sample of 6400 individuals was drawn from eight countries: Germany, Ireland, Italy, the Netherlands, Spain, UK, Czech Republic and Lithuania. An online survey was conducted among 3200 respondents, 400 in each of the 8 countries, and 2 online experiments with 3200 subjects, 400 in each of the 8 countries, allowing for 200 respondents for each experiment in each country. Results and discussion: The survey shows that Europeans are aware of the health and environmental impact of cars. The findings also confirm the gap between self-reported attitudes/intentions and actual behaviours. In influencing car purchase decisions, health and environmental concerns are less important than other attributes such as price, safety and performance. The experiments show that all these attributes have a significant effect on consumers\u2019 choices. However, message content was found to have the strongest effect. Respondents are more likely to choose European Union Low Emitting carS (EULES)-friendly cars when the label shows information on lower costs or lower taxes and less likely to be influenced by health-related benefits, convenient parking or access fees. Finally, combinations of one message with other elements\u2014EULES logo, CO2 logo or both\u2014within the same label have a small but positive effect on respondents\u2019 choices. Conclusions: The findings of this study assist governmental decision-making processes by identifying those issues that have the greatest impact on consumers\u2019 car purchasing decisions. Furthermore, the results will help to guide environmentally conscious customers towards the purchase of vehicles with clean emission profiles

What acute stress protocols can tell us about PTSD and stress-related neuropsychiatric disorders

Posttraumatic stress disorder (PTSD), the fifth most prevalent mental disorder in the United States, is a chronic, debilitating mental illness with as yet limited options for treatment. Hallmark symptoms of PTSD include intrusive memory of trauma, avoidance of reminders of the event, hyperarousal and hypervigilance, emotional numbing, and anhedonia. PTSD is often triggered by exposure to a single traumatic experience, such as a traffic accident, a natural catastrophe, or an episode of violence. This suggests that stressful events have a primary role in the pathogenesis of the disorder, although genetic background and previous life events are likely involved. However, pathophysiology of this mental disorder, as for major depression and anxiety disorders, is still poorly understood. In particular, it is unknown how can a single traumatic, stressful event induce a disease that can last for years or decades. A major shift in the conceptual framework investigating neuropsychiatric disorders has occurred in recent years, from a monoamine-oriented hypothesis (which dominated pharmacological research for over half a century) to a neuroplasticity hypothesis, which posits that structural and functional changes in brain circuitry (largely in the glutamate system) mediate psychopathology and also therapeutic action. Rodent stress models are very useful to understand pathophysiology of PTSD. Recent studies with acute or subacute stress models have shown that exposure to short-time stressors (from several minutes to a few hours) can induce not only rapid, but also sustained changes in synaptic function (glutamate release, synaptic transmission/plasticity), neuroarchitecture (dendritic morphology, synaptic spines), and behavior (cognitive functions). Some of these changes, e.g., stress-induced increased glutamate release and dendrite retraction, are likely connected and occur more rapidly than previously thought. We propose here to use a modified version of a simple and validated protocol of footshock stress to explore different trajectories in the individual response to acute stress. This new conceptual framework may enable us to identify determinants of resilient versus vulnerable response as well as new targets for treatment, in particular for rapid-acting antidepressants. It will be interesting to investigate the putative prophylactic action of ketamine toward the maladaptive effects of acute stress in this new protocol

What acute stress protocols can tell us about PTSD and stress-related neuropsychiatric disorders

Posttraumatic stress disorder (PTSD), the fifth most prevalent mental disorder in the United States, is a chronic, debilitating mental illness with as yet limited options for treatment. Hallmark symptoms of PTSD include intrusive memory of trauma, avoidance of reminders of the event, hyperarousal and hypervigilance, emotional numbing, and anhedonia. PTSD is often triggered by exposure to a single traumatic experience, such as a traffic accident, a natural catastrophe, or an episode of violence. This suggests that stressful events have a primary role in the pathogenesis of the disorder, although genetic background and previous life events are likely involved. However, pathophysiology of this mental disorder, as for major depression and anxiety disorders, is still poorly understood. In particular, it is unknown how can a single traumatic, stressful event induce a disease that can last for years or decades. A major shift in the conceptual framework investigating neuropsychiatric disorders has occurred in recent years, from a monoamine-oriented hypothesis (which dominated pharmacological research for over half a century) to a neuroplasticity hypothesis, which posits that structural and functional changes in brain circuitry (largely in the glutamate system) mediate psychopathology and also therapeutic action. Rodent stress models are very useful to understand pathophysiology of PTSD. Recent studies with acute or subacute stress models have shown that exposure to short-time stressors (from several minutes to a few hours) can induce not only rapid, but also sustained changes in synaptic function (glutamate release, synaptic transmission/plasticity), neuroarchitecture (dendritic morphology, synaptic spines), and behavior (cognitive functions). Some of these changes, e.g., stress-induced increased glutamate release and dendrite retraction, are likely connected and occur more rapidly than previously thought. We propose here to use a modified version of a simple and validated protocol of footshock stress to explore different trajectories in the individual response to acute stress. This new conceptual framework may enable us to identify determinants of resilient versus vulnerable response as well as new targets for treatment, in particular for rapid-acting antidepressants. It will be interesting to investigate the putative prophylactic action of ketamine toward the maladaptive effects of acute stress in this new protocol

Modulation by chronic stress and ketamine of ionotropic AMPA/NMDA and metabotropic glutamate receptors in the rat hippocampus

Converging clinical and preclinical evidence has shown that dysfunction of the glutamate system is a core feature of major depressive disorder. In this context, the N-methyl-d-aspartate (NMDA) receptor antagonist ketamine has raised growing interest as fast acting antidepressant. Using the chronic mild stress (CMS) rat model of depression, performed in male rats, we aimed at analyzing whether hippocampal specific changes in subunit expression and regulation of \u3b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or NMDA ionotropic receptors and in metabotropic glutamate receptors could be associated with behavioral vulnerability/resilience to CMS. We also assessed whether acute ketamine (10 mg/kg) was able to dampen the alterations in CMS vulnerable animals. Although chronic stress and ketamine had no effect on ionotropic glutamate receptors mRNAs (expression, RNA editing and splicing), we found selective modulations in their protein expression, phosphorylation and localization at synaptic membranes. AMPA GluA2 expression at synaptic membranes was significantly increased only in CMS resilient rats (although a trend was found also in vulnerable animals), while its phosphorylation at Ser880 was higher in both CMS resilient and vulnerable rats, a change partially dampened by ketamine. In the hippocampus from all stressed groups, despite NMDA receptor expression levels were reduced in total extract, the levels of GluN2B-containing NMDA receptors were remarkably increased in synaptic membranes. Finally, mGlu2 underwent a selective downregulation in stress vulnerable animals, which was completely restored by acute ketamine. Overall, these results are in line with a hypofunction of activity-dependent glutamatergic synaptic transmission induced by chronic stress exposure in all the animals, as suggested by the alterations of ionotropic glutamate receptors expression and localization at synaptic level. At the same time, the selective modulation of mGlu2 receptor, confirms its previously hypothesized functional role in regulating stress vulnerability and, for the first time here, suggests a mGlu2 involvement in the fast antidepressant effect of ketamine

Time-dependent changes induced by acute stress in function and architecture of excitatory synapses in prefrontal and frontal cortex

Stressful life events impact on brain and bodily function and represent major risk factors for stress-related neuropsychiatric disorders. The response to stressful events can promote adaptive plasticity and improved cognition, when the physiological stress response is efficiently activated and inactivated in due time, or maladaptive and harmful effects, when the response is overused or dysregulated. In turn, the outcome of a maladaptive stress response can be associated with the triggering of brain, systemic and metabolic disorders. Chronic stress has been shown to induce reduction of density of synapses and dendrites in prefrontal and frontal cortex (PFC/FC), with concomitant impairments in neuronal activity and cognitive functions. Instead, the early and rapid effects of acute stress on synaptic function and plasticity are often opposite, with enhancement of glutamate release/transmission, increased number of spines and synapses, enhancement of synaptic strength. However, the delayed effects of acute stress have not been investigated, although this could give crucial information on the time- dependent changes in the brain stress response. We have previously characterized the synaptic effects of acute footshock (FS)-stress, which induces enhancement of glutamate release/transmission in PFC/FC, due to the increase of the readily releasable pool (RRP), in turn mediated by rapid non-genomic corticosterone action at synapses (Mol. Psy., 19:433-443, 2014). Here we have analyzed the effects of acute FS-stress in the PFC/FC of rats at different times after completion of the stress protocol. We found that acute stress induced early and sustained increase of RRP over time in excitatory perforated synapses, while the number of non-perforated and axo-spinous synapses was increased (without changes in vesicle pools). The total number of synaptic spines was increased up to 24 h, while apical dendrites showed decreased density 2 weeks after acute stress (with no significant changes at earlier times). In behavioral tests for working memory, FS-stress improved performance 2 h after stress and impaired it after 24 h. Changes in glutamate release, RRP, number of synapses and spines are blocked or attenuated by prior chronic treatment with the antidepressant desipramine. The different glutamatergic modifications in functional and morphological plasticity suggest a bi-phasic process, during which the stress response in PFC/FC may turn from early increased excitatory activation into its opposite. The identification of these points and the players involved in the switch are crucial for the understanding of the dynamics of stress-related pathology

Acute Ketamine Facilitates Fear Memory Extinction in a Rat Model of PTSD Along With Restoring Glutamatergic Alterations and Dendritic Atrophy in the Prefrontal Cortex

Stress represents a major risk factor for psychiatric disorders, including post-traumatic stress disorder (PTSD). Recently, we dissected the destabilizing effects of acute stress on the excitatory glutamate system in the prefrontal cortex (PFC). Here, we assessed the effects of single subanesthetic administration of ketamine (10 mg/kg) on glutamate transmission and dendritic arborization in the PFC of footshock (FS)-stressed rats, along with changes in depressive, anxious, and fear extinction behaviors. We found that ketamine, while inducing a mild increase of glutamate release in the PFC of na\uefve rats, blocked the acute stress-induced enhancement of glutamate release when administered 24 or 72 h before or 6 h after FS. Accordingly, the treatment with ketamine 6 h after FS also reduced the stress-dependent increase of spontaneous excitatory postsynaptic current (sEPSC) amplitude in prelimbic (PL)-PFC. At the same time, ketamine injection 6 h after FS was found to rescue apical dendritic retraction of pyramidal neurons induced by acute stress in PL-PFC and facilitated contextual fear extinction. These results show rapid effects of ketamine in animals subjected to acute FS, in line with previous studies suggesting a therapeutic action of the drug in PTSD models. Our data are consistent with a mechanism of ketamine involving re-establishment of synaptic homeostasis, through restoration of glutamate release, and structural remodeling of dendrites

Elevated blood pressure, cardiometabolic risk and target organ damage in youth with overweight and obesity

Background and aim: To compare cardiometabolic risk profile and preclinical signs of target organ damage in youth with normal and elevated blood pressure (BP), according to the American Academy of Pediatrics (AAP) guidelines. Methods and results: This cross-sectional multicenter study included 2739 youth (5-17 year-old; 170 normal-weight, 610 overweight and 1959 with obesity) defined non hypertensive by the AAP guidelines. Anthropometric, biochemical and liver ultrasound data were available in the whole population; carotid artery ultrasound and echocardiographic assessments were available respectively in 427 and 264 youth. Elevated BP was defined as BP 65 90th to <95th percentile for age, gender and height in children or BP 65 120/80 to <130/80 in adolescents. The overall prevalence of elevated BP was 18.3%, and significantly increased from normal-weight to obese youth. Young people with elevated BP showed higher levels of body mass index (BMI), insulin resistance and a higher prevalence of liver steatosis (45% vs 36%, p < 0.0001) than normotensive youth, whilst they did not differ for the other cardiometabolic risk factors, neither for carotid intima media thickness or left ventricular mass. Compared with normotensive youth, individuals with elevated BP had an odds ratio (95%Cl) of 3.60 (2.00\u20136.46) for overweight/obesity, 1.46 (1.19\u20131.78) for insulin-resistance and 1.45 (1.19\u20131.77) for liver steatosis, controlling for centers, age and prepubertal stage. The odds for insulin resistance and liver steatosis persisted elevated after correction for BMI-SDS. Conclusion: Compared to normotensive youth, elevated BP is associated with increased BMI, insulin resistance and liver steatosis, without significant target organ damage