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 of 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 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 of the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35-45 % reduction of 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

Chronic stress and impaired glutamate function elicit a depressive-like phenotype and common changes in gene expression in the mouse frontal cortex

Major depression might originate from both environmental and genetic risk factors. The environmental chronic mild stress (CMS) model mimics some environmental factors contributing to human depression and induces anhedonia and helplessness. Mice heterozygous for the synaptic vesicle protein (SVP) vesicular glutamate transporter 1 (VGLUT1) have been proposed as a genetic model of deficient glutamate function linked to depressive-like behaviour. Here, we aimed to identify, in these two experimental models, gene expression changes in the frontal cortex, common to stress and impaired glutamate function. Both VGLUT1+/- and CMS mice showed helpless and anhedonic-like behavior. Microarray studies in VGLUT1+/- mice revealed regulation of genes involved in apoptosis, neurogenesis, synaptic transmission, protein metabolic process or learning and memory. In addition, RT-PCR studies confirmed gene expression changes in several glutamate, GABA, dopamine and serotonin neurotransmitter receptors. On the other hand, CMS affected the regulation of 147 transcripts, some of them involved in response to stress and oxidoreductase activity. Interestingly, 52 genes were similarly regulated in both models. Specifically, a dowregulation in genes that promote cell proliferation (Anapc7), cell growth (CsnK1g1), cell survival (Hdac3), inhibition of apoptosis (Dido1) was observed. Genes linked to cytoskeleton (Hspg2, Invs), psychiatric disorders (Grin1, MapK12) or an antioxidant enzyme (Gpx2) were also downregulated. Moreover, genes that inhibit the MAPK pathways (Dusp14), stimulate oxidative metabolism (Eif4a2) and enhance glutamate transmission (Rab8b) were upregulated. We suggest that these genes could form part of the altered “molecular context” underlying depressive-like behaviour in animal models. The clinical relevance of these findings is discussed

Interactions between age, stress and insulin on cognition: implications for Alzheimer's disease

There is much interest in understanding the mechanisms responsible for interactions among stress, aging, memory and Alzheimer's disease. Glucocorticoid secretion associated with early life stress may contribute to the variability of the aging process and to the development of neuro- and psychopathologies. Maternal separation (MS), a model of early life stress in which rats experience 3 h of daily separation from the dam during the first 3 weeks of life, was used to study the interactions between stress and aging. Young (3 months) MS rats showed an altered hypothalamic-pituitary-adrenal (HPA) axis reactivity, depressive-like behavior in the Porsolt swimming test and cognitive impairments in the Morris water maze and new object recognition test that persisted in aged (18 months) rats. Levels of insulin receptor, phosphorylated insulin receptor and markers of downstream signaling pathways (pAkt, pGSK3 beta, pTau, and pERK1 levels) were significantly decreased in aged rats. There was a significant decrease in pERK2 and in the plasticity marker ARC in MS aged rats compared with single MS or aged rats. It is interesting to note that there was a significant increase in the C99 : C83 ratio, A beta levels, and BACE1 levels the hippocampus of MS aged rats, suggesting that in aged rats subjected to early life stress, there was an increase in the amyloidogenic processing of amyloid precursor protein (APP). These results are integrated in a tentative mechanism through which aging interplay with stress to influence cognition as the basis of Alzheimer disease (AD). The present results may provide the proof-of-concept for the use of glucocorticoid-/insulin-related drugs in the treatment of AD

Increased vulnerability to depressive-like behaviour of mice with decreased expression of VGLUT1

Background: Many studies have linked depression to an increase in the excitatory-inhibitory ratio in the forebrain. Presynaptic alterations in a shared pathway of the glutamate/GABA cycle may account for this imbalance. Recent evidence suggests that decreased vesicular glutamate transporter 1 (VGLUT1) levels in the forebrain affects the glutamate/GABA cycle and induces helpless behaviour. Here we studied decreased VGLUT1 as a potencial factor enhancing a depressive-like phenotype in an animal model. Methods: Glutamate and GABA synthesis as well as oxidative metabolism were studied in heterozygous mice for the vesicular glutamate transporter 1 (VGLUT1+/-) and WT. Subsequently, the regulation of neurotransmitter levels, proteins involved in the glutamate/GABA cycle and behaviour by both genotype and chronic mild stress (CMS) was studied. Finally, the effect of chronic imipramine on VGLUT1 control and CMS mice was also studied. Results: VGLUT1+/- mice showed increased neuronal synthesis of glutamate, decreased cortical and hippocampal GABA, VGLUT1 and EAAT1, as well as helplessness and anhedonia. CMS induced an increase of glutamate and a decrease of GABA, VGAT and GAD65 in both areas and led to upregulation EAAT1 in the hippocampus. Moreover, CMS induced anhedonia, helplessness, anxiety and impaired recognition memory. VGLUT1+/- CMS mice showed a combined phenotype (genotype plus stress) and specific alterations, such as an upregulation of VGLUT2 and hyperlocomotion. Moreover, an increased vulnerability to anhedonia and helplessness reversible by chronic imipramine was shown. Conclusions: These studies highlight a crucial role for decreased VGLUT1 in the forebrain as a biological mediator of increased vulnerability to chronic mild stress

Regulation of markers of synaptic function in mouse models of depression: chronic mild stress and decreased expression of VGLUT1

Depression has been linked to failure in synaptic plasticity originating from environmental and/or genetic risk factors. The chronic mild stress (CMS) model regulates the expression of synaptic markers of neurotransmitter function and associated depressive-like behaviour. Moreover, mice heterozygous for the synaptic vesicle protein (SVP) vesicular glutamate transporter 1 (VGLUT1), have been proposed as a genetic model of deficient glutamate function linked to depressive-like behaviour. Here, we aimed to identify, in these two experimental models, mechanisms of failure in synaptic plasticity, common to stress and impaired glutamate function. First, we show that CMS induced a transient decrease of different plasticity markers (VGLUT1, synapsin 1, sinaptophysin, rab3A and activity regulated cytoskeletal protein Arc) but a long-lasting decrease of the brain derived neurotrophic factor (BDNF) as well as depressive-like behaviour. The immediate early gene (IEG) Arc was also downregulated in VGLUT1+/- heterozygous mice. In contrast, an opposite regulation of synapsin 1 was observed. Finally, both models showed a marked increase of cortical Arc response to novelty. Increased Arc response to novelty could be suggested as a molecular mechanism underlying failure to adapt to environmental changes, common to chronic stress and altered glutamate function. Further studies should investigate whether these changes are associated to depressive-like behaviour both in animal models and in depressed patients

Estudio conductual y neurobiológico del estrés crónico social en ratón

Major depression is a mental disorder often preceded by exposure to chronic stress or stressful life events. Recently, animal models based on social conflict such as chronic social defeat stress (CSDS) are proposed to be more relevant to stress-induced human psychopathology compared to environmental models like the chronic mild stress (CMS). Although CSDS has been proposed as a model of depression, most CSDS studies rely only on analysis of stress-induced social avoidance. Moreover, the predictive validity of the model has been poorly analyzed let alone the possible alterations in dendritic and synaptic structures of brain regions involved in emotional processing. Therefore, the aims of this work were to study comparatively social versus environmental stress using the CMS and CSDS models. Secondly we have fully characterized the validity of the CSDS model as a possible model of depression. Comparing both stress models we observed that CMS induced a clear depressive-like profile including anhedonia, helplessness and memory impairment. CSDS induced anhedonia, hyperactivity, anxiety, social avoidance and freezing, signs also common to anxiety and posttraumatic stress disorders. Both models disrupted the excitatory inhibitory balance in the prefrontal cortex. Moreover, CSDS decreased dopamine in the prefrontal cortex and brainstem. We suggests that while depressive-like behaviours might be associated to altered aminoacid neurotransmission in cortical areas, CSDS induced anxiety behaviours might be linked to specific alteration of dopaminergic pathways involved in rewarding processes. We next explored the predictive validity of the CSDS model using different classes of antidepressants and we observed that this validity was limited. Firstly, repeated venlafaxine showed antidepressant-like activity and both venlafaxine and tianeptine behaved as effective anxiolytics. However CSDS-induced social avoidance was only partially reverted by tianeptine. On the other hand, fluoxetine failed to revert most of the behavioral alterations. In addition, defeated mice showed a downregulation of VGLUT1 mRNA in amygdale and VGLUT1+/- mice showed an enhanced vulnerability to stress-induced social avoidance. However, unlike the environmental model of CMS, these mice did not show enhanced vulnerability to depressive behaviour when exposed to CSDS. Moreover, CSDS induced an opposite regulation of the spine density and different synaptic plasticity markers in the prefrontal cortex and the amygdale. Specifically while in the prefrontal cortex spine density, GluR1 AMPA receptor subunit, PSD95 and Synaptophysin were increased, a decrease was found in the amygdale. In addition, in the amygdale a downregulation of NR1 NMDA subunit, mGlur4, brain derived neurotrophic factor (BDNF) and CREB was also found. In addition, stressed mice exposed to the social interaction test with a dominant mouse experienced marked freezing and showed increased in pCREB/CREB ratio, proBDNF and mature BDNF in the amygdale. Taken together these results suggest that CSDS is not a pure model of depression. Indeed it addresses relevant aspects of anxiety-related disorders. Firstly, CSDS induced anhedonia and social avoidance is not associated in this model. Moreover, CSDS might be affecting brain areas mainly involved in the processing of social behaviour, such as the amygdale, where glutamatergic mechanism could play a key role. In addition, these results suggest a persistent maladaptive function of the prefrontal cortex and amygdale of socially defeated mice. Moreover, this would be the first evidence that late reencounter with an aversive social stimuli can triggers fear conditioning in socially defeated mice and support the validity of CSDS to model clinical aspects of post-traumatic stress disorders

Role of HDAC5 and SIRT2 in depression and clinical efficacy of antidepressants

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Role of HDAC5 and SIRT2 in depression and clinical efficacy of antidepressants

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Estudio conductual y neurobiológico del estrés crónico social en ratón

Major depression is a mental disorder often preceded by exposure to chronic stress or stressful life events. Recently, animal models based on social conflict such as chronic social defeat stress (CSDS) are proposed to be more relevant to stress-induced human psychopathology compared to environmental models like the chronic mild stress (CMS). Although CSDS has been proposed as a model of depression, most CSDS studies rely only on analysis of stress-induced social avoidance. Moreover, the predictive validity of the model has been poorly analyzed let alone the possible alterations in dendritic and synaptic structures of brain regions involved in emotional processing. Therefore, the aims of this work were to study comparatively social versus environmental stress using the CMS and CSDS models. Secondly we have fully characterized the validity of the CSDS model as a possible model of depression. Comparing both stress models we observed that CMS induced a clear depressive-like profile including anhedonia, helplessness and memory impairment. CSDS induced anhedonia, hyperactivity, anxiety, social avoidance and freezing, signs also common to anxiety and posttraumatic stress disorders. Both models disrupted the excitatory inhibitory balance in the prefrontal cortex. Moreover, CSDS decreased dopamine in the prefrontal cortex and brainstem. We suggests that while depressive-like behaviours might be associated to altered aminoacid neurotransmission in cortical areas, CSDS induced anxiety behaviours might be linked to specific alteration of dopaminergic pathways involved in rewarding processes. We next explored the predictive validity of the CSDS model using different classes of antidepressants and we observed that this validity was limited. Firstly, repeated venlafaxine showed antidepressant-like activity and both venlafaxine and tianeptine behaved as effective anxiolytics. However CSDS-induced social avoidance was only partially reverted by tianeptine. On the other hand, fluoxetine failed to revert most of the behavioral alterations. In addition, defeated mice showed a downregulation of VGLUT1 mRNA in amygdale and VGLUT1+/- mice showed an enhanced vulnerability to stress-induced social avoidance. However, unlike the environmental model of CMS, these mice did not show enhanced vulnerability to depressive behaviour when exposed to CSDS. Moreover, CSDS induced an opposite regulation of the spine density and different synaptic plasticity markers in the prefrontal cortex and the amygdale. Specifically while in the prefrontal cortex spine density, GluR1 AMPA receptor subunit, PSD95 and Synaptophysin were increased, a decrease was found in the amygdale. In addition, in the amygdale a downregulation of NR1 NMDA subunit, mGlur4, brain derived neurotrophic factor (BDNF) and CREB was also found. In addition, stressed mice exposed to the social interaction test with a dominant mouse experienced marked freezing and showed increased in pCREB/CREB ratio, proBDNF and mature BDNF in the amygdale. Taken together these results suggest that CSDS is not a pure model of depression. Indeed it addresses relevant aspects of anxiety-related disorders. Firstly, CSDS induced anhedonia and social avoidance is not associated in this model. Moreover, CSDS might be affecting brain areas mainly involved in the processing of social behaviour, such as the amygdale, where glutamatergic mechanism could play a key role. In addition, these results suggest a persistent maladaptive function of the prefrontal cortex and amygdale of socially defeated mice. Moreover, this would be the first evidence that late reencounter with an aversive social stimuli can triggers fear conditioning in socially defeated mice and support the validity of CSDS to model clinical aspects of post-traumatic stress disorders

The role of HDAC5, SIRT2 and VGLUT1 in antidepressant action

La depresión sigue siendo una de las enfermedades crónicas con mayor repercusión a nivel mundial, principalmente en los países desarrollados. Las terapias actuales se basan en el tratamiento con antidepresivos que aumentan los niveles de monoaminas en el cerebro, concretamente, serotonina y/o noradrenalina. La acción antidepresiva se ha relacionado con el aumento de la plasticidad sináptica en la corteza prefrontal (PFC) en la que diversos mecanismos epigenéticos podrían estar implicados. Mediante estudios previos en nuestro laboratorio, hemos identificado dos dianas epigenéticas, las enzimas histona desacetilasas (HDAC) HDAC5 y SIRT2, cuya expresión se regula opuestamente por el estrés crónico y el tratamiento antidepresivo. HDAC5 pertenece a la clase IIa de la superfamilia de HDAC y tiene la capacidad de migrar desde el núcleo hasta el citoplasma a través de un mecanismo de fosforilación. La otra enzima, SIRT2, pertenece a la clase III y es dependiente de NAD+. Este trabajo consta de tres objetivos principales. En el primero de ellos se utilizó el modelo in vitro SH-SY5Y, una línea celular proveniente de neuroblastoma humano, para estudiar el efecto de los antidepresivos imipramina, fluoxetina y reboxetina sobre la expresión de las dianas epigenéticas HDAC5 y SIRT2. También se estudió cómo se ve afectada la acetilación de las histonas 3 y 4 (AcH3 y AcH4), así como el factor neurotrófico derivado del cerebro (BDNF) y el trasportador vesicular de glutamato 1 (VGLUT1), considerados marcadores de plasticidad sináptica. Nuestro estudio mostró que la administración de imipramina y reboxetina aumentó la forma fosforilada de HDAC5 (P-HDAC5) favoreciendo la traslocación al citoplasma de esta enzima. Sin embargo, la expresión de SIRT2 se vio disminuida por todos los antidepresivos. Además, los antidepresivos aumentaron la expresión de BDNF, VGLUT1, AcH3 y AcH4. Finalmente, la inhibición selectiva de HDAC5 y SIRT2 con los compuestos MC3822 y 33i, respectivamente, aumentó los marcadores de neuroplasticidad. Paralelamente se realizaron estudios en ratones a los que se administró de forma crónica estos antidepresivos y se midió la expresión de todas estas proteínas en la PFC obteniendo resultados similares a los ensayos in vitro. En conjunto, se sugiere que la traslocación nucleocitoplasmática de HDAC5 y la disminución de SIRT2 son mecanismos epigenéticos mediante los cuales estos antidepresivos estimulan la neuroplasticidad. En el siguiente objetivo, estudiamos el potencial terapéutico de SIRT2 para el tratamiento de la depresión. Concretamente, el inhibidor selectivo de SIRT2 33i revirtió la anhedonia en ratones heterocigotos para VGLUT1 (VGLUT1+/-), considerado un modelo genético de depresión. Sin embargo, este modelo no mostró cambios en la expresión de los marcadores epigenéticos seleccionados (HDACs y acetilación de histonas) ni en los marcadores de la plasticidad sináptica. Este resultado evidencia que la inhibición de SIRT2 es capaz de revertir un fenotipo depresivo también cuando éste no está asociado a alteraciones epigenéticas. Paralelamente, se llevó a cabo un tercer objetivo. Se sabe que la actividad serotonérgica en los núcleos del rafe está regulada por neuronas glutamatérgicas provenientes de la PFC. Utilizando el modelo VGLUT1+/-, se examinó el papel de VGLUT1 en la acción antidepresiva. Se demostró que la inyección estereotáxica del virus adenoasociado diseñado en este trabajo, para expresar el gen Vglut1, dio como resultado un aumento de VGLUT1 tanto en el soma neuronal como en fibras en la PFC, así como en fibras en el núcleo dorsal del rafe. Además, la expresión inducida de esta proteína en la PFC provocó una clara acción antianhedónica. En conjunto, esta es la primera evidencia que relaciona directamente VGLUT1 con la acción antidepresiva. Las enzimas histona desacetilasa HDAC5 y SIRT2 así como VGLUT1 podrían proponerse como dianas farmacológicas implicadas en la acción antidepresiva