Translational studies of estradiol and progesterone in fear and PTSD.

Translational models of fear have greatly informed our understanding of PTSD and its underlying fear circuitry. One of the most replicated findings in the field is the two-fold higher PTSD incidence in females compared to males. While sociocultural factors play a role, the most robust biological influencers to date are gonadal hormones, such as estradiol and progesterone, which fluctuate across the menstrual cycle. Among studies that account for these hormones, most do so in isolation or collect both and only report one. Variation in study findings suggests that the ratio between these two hormones (the P/E ratio) may be an important and missing variable to further understand gonadal hormone influences on fear. Here we review cross-species examinations of fear and PTSD, within the contexts of estradiol and progesterone as well as P/E ratios that were calculated based on extant literature. We then provide recommendations for best practices in assay methods and reporting to improve research on the P/E ratio in fear and PTSD. Ultimately, greater understanding of this important variable will advance efforts to characterize gonadal hormone influences on fear learning processes in humans and animals.

Los modelos traslacionales del miedo han enriquecido nuestro conocimiento sobre el TEPT y el circuito del miedo subyacente. Uno de los hallazgos más replicados en el campo es la incidencia dos veces mayor de TEPT en mujeres en comparación a los hombres. Aunque los factores socioculturales juegan un rol, las influencias biológicas más robustas a la fecha son las hormonas gonadales, tales como el estradiol y la progesterona, que fluctúan durante el ciclo menstrual. Entre los estudios que dan cuenta de estas hormonas, la mayoría las estudian de forma aislada u obtienen ambas y solo reportan una. La variación en los hallazgos de los estudios sugiere que la razón entre estas dos hormonas (la razón P/E) puede ser una variable poco estudiada e importante para mejorar la comprensión de la influencia de las hormonas gonadales en el miedo. En este trabajo hacemos una revisión de hallazgos trans-especie sobre el miedo y el TEPT, dentro del contexto del estradiol y la progresterona así como de la razón P/E que fue calculada basándose en la literatura existente Posteriormente proveemos recomendaciones para mejorar las prácticas en los métodos de ensayo y reportes para mejorar la investigación sobre la razón P/E en miedo y TEPT. En última instancia, mayor comprensión de esta importante variable hará que progresen los esfuerzos para caracterizar la influencia de las hormonas gonadales en procesos de aprendizaje del miedo en humanos y animales.

An mGlu5-Positive Allosteric Modulator Rescues the Neuroplasticity Deficits in a Genetic Model of NMDA Receptor Hypofunction in Schizophrenia.

There is substantial evidence that NMDA receptor (NMDAR) hypofunction contributes to the pathophysiology of schizophrenia (SCZ). A recent large-scale genome-wide association study identified serine racemase (SR), the enzyme that produces the NMDAR co-agonist D-serine, as a risk gene for SCZ. Serine racemase knockout (SR-/-) mice, which lack D-serine, exhibit many of the neurochemical and behavioral abnormalities observed in SCZ. Metabotropic glutamate receptor 5 (mGlu5)-positive allosteric modulators (PAMs) are currently being developed to treat cognitive dysfunction. We used in vitro electrophysiology to determine whether the mGlu5 PAM VU0409551 directly enhances NMDAR function in hippocampal slices from adult male SR-/- mice. We administered VU0409551 systemically for 5 days to adult male wild-type C57BL/6 animals to determine the optimal dose to test in SR-/- mice. We used western blot analyses and trace-fear conditioning to determine whether 5 days of VU0409551 treatment could reverse the neuroplasticity and learning deficits, respectively, in SR-/- mice. We show that VU0409551 enhances NMDAR function and rescues long-term potentiation in hippocampal slices obtained from SR-/- mice. Systemic treatment with VU0409551 (10 and 30 mg/kg) to wild-type mice causes a dose-dependent increase in the Akt/GS3Kα/ß signaling pathway, which is reduced in SR-/- mice and in SCZ. Furthermore, the administration of VU0409551 to SR-/- mice reverses their deficits in several neuroplasticity signaling pathways and improves their contextual fear memory. These results support positive allosteric modulation of mGlu5, particularly with VU0409551, as a viable mechanism to reverse the deficits in NMDAR function, synaptic plasticity, and memory that are known to be impaired in SCZ.

Stress-related disorders, pituitary adenylate cyclase-activating peptide (PACAP)ergic system, and sex differences.

Trauma-related disorders, such as posttraumatic stress disorder (PTSD) are remarkably common and debilitating, and are often characterized by dysregulated threat responses. Across numerous epidemiological studies, females have been found to have an approximately twofold increased risk for PTSD and other stress-related disorders. Understanding the biological mechanisms of this differential risk is of critical importance. Recent data suggest that the pituitary adenylate cyclase-activating polypeptide (PACAP) pathway is a critical regulator of the stress response across species. Moreover, increasing evidence suggests that this pathway is regulated by both stress and estrogen modulation and may provide an important window into understanding mechanisms of sex differences in the stress response. We have recently shown that PACAP and its receptor (PAC1R) are critical mediators of abnormal processes after psychological trauma. Notably, in heavily traumatized human subjects, there appears to be a robust sex-specific association of PACAP blood levels and PAC1R gene variants with fear physiology, PTSD diagnosis, and symptoms, specifically in females. The sex-specific association occurs within a single-nucleotide polymorphism (rs2267735) that resides in a putative estrogen response element involved in PAC1R gene regulation. Complementing these human data, the PAC1R messenger RNA is induced with fear conditioning or estrogen replacement in rodent models. These data suggest that perturbations in the PACAP-PAC1R pathway are regulated by estrogen and are involved in abnormal fear responses underlying PTSD.

Los trastornos relacionados con el trauma, como el trastorno por estrés postraumático (TEPT) en humanos, son extraordinariamente comunes y desgastadores, y a menudo están caracterizados por respuestas desreguladas a la amenaza. En numerosos estudios epidemiológicos se ha encontrado que las mujeres tienen un riesgo aumentado al doble para el TEPT y otros trastornos relacionados con el estrés. La comprensión de los mecanismos biológicos de este riesgo diferencial es de gran importancia. Hay datos recientes que sugieren que, a través de las especies, la vía del polipéptido activador de la adenilato ciclasa hipofisiaria (PAACH) tiene una regulación central en la respuesta de estrés. Sin embargo, hay evidencia creciente que sugiere que esta vía está regulada por el estrés y la modulación estrogénica y puede aportar una ventana importante para la comprensión de los mecanismos de las diferencias por sexo en la respuesta de estrés. Nosotros hemos mostrado recientemente que el PAACH y su receptor (R1PAC) son importantes mediadores de los procesos anormales después del trauma psicológico. Es notable que, específicamente en mujeres víctimas de grandes traumas, al parecer hay una potente asociación específica entre los niveles sanguíneos de PAACH y variantes del gen R1PAC con la fisiología del miedo, y síntomas y diagnóstico de TEPT. La asociación específica con el sexo ocurre con el polimorfismo del nucleótido único (rs2267735) que se encuentra en un elemento de la respuesta putativa de estrógeno involucrada en la regulación del gen R1PAC. Como complemento a estos datos humanos, en modelos de roedores el ARN mensajero del R1PAC está inducido por el condicionamiento al miedo o por el reemplazo de estrógenos. Estos datos sugieren que las perturbaciones en la vía del PACCH-R1PAC están reguladas por estrógenos y participan en las respuestas anormales al miedo del TEPT.

Les troubles liés aux traumatismes, comme les troubles du stress post-traumatique (TSPT) chez les humains, sont extrêmement courants et invalidants et souvent caractérisés par une dérégulation des réponses à la menace. De nombreuses études épidémiologiques indiquent que les femmes ont environ deux fois plus de risque de TSPT et d'autres troubles liés au stress. La compréhension des mécanismes biologiques de ce risque différentiel est d'une importance essentielle. Selon des données récentes, la voie du PACAP (pituitary adenylate cyclase-activating polypeptide) est un régulateur capital de la réponse au stress commun à l'ensemble des espèces. De plus, il existe des preuves croissantes de la régulation de ces voies par la modulation à la fois du stress et des estrogènes et de leur apport d'une ouverture importante dans la compréhension du mécanisme des différences selon le sexe dans la réponse au stress. Nous avons récemment montré que la PACAP et son récepteur (PAC1R) sont des médiateurs essentiels des processus anormaux après un traumatisme psychologique. Il semble y avoir, en particulier chez les humains fortement traumatisés, une importante association des concentrations sanguines de PACAP et des variants du gène PAS1R spécifiques du sexe avec la physiologie de la peur, le diagnostic de TSPT et des symptômes, en particulier chez les femmes. L'association spécifique au sexe est liée à un polymorphisme nucléotidique (rs2267735) localisé dans une séquence d'ADN supposée être un élément de réponse à l'estrogène impliqué dans la régulation du gène PAC1R. Pour compléter ces données humaines, l'ARN messager du PAC1R est induit avec le conditionnement à la peur ou le remplacement des estrogènes dans des modèles murins. Ces données suggèrent que les perturbations dans la voie du PACAP-PAC1R sont régulées par les estrogènes et impliquées dans les réponses anormales à la peur sous-tendant le TSPT.

Fluoxetine Facilitates Fear Extinction Through Amygdala Endocannabinoids.

Pharmacologically elevating brain endocannabinoids (eCBs) share anxiolytic and fear extinction-facilitating properties with classical therapeutics, including the selective serotonin reuptake inhibitor, fluoxetine. There are also known functional interactions between the eCB and serotonin systems and preliminary evidence that antidepressants cause alterations in brain eCBs. However, the potential role of eCBs in mediating the facilitatory effects of fluoxetine on fear extinction has not been established. Here, to test for a possible mechanistic contribution of eCBs to fluoxetine's proextinction effects, we integrated biochemical, electrophysiological, pharmacological, and behavioral techniques, using the extinction-impaired 129S1/Sv1mJ mouse strain. Chronic fluoxetine treatment produced a significant and selective increase in levels of anandamide in the BLA, and an associated decrease in activity of the anandamide-catabolizing enzyme, fatty acid amide hydrolase. Slice electrophysiological recordings showed that fluoxetine-induced increases in anandamide were associated with the amplification of eCB-mediated tonic constraint of inhibitory, but not excitatory, transmission in the BLA. Behaviorally, chronic fluoxetine facilitated extinction retrieval in a manner that was prevented by systemic or BLA-specific blockade of CB1 receptors. In contrast to fluoxetine, citalopram treatment did not increase BLA eCBs or facilitate extinction. Taken together, these findings reveal a novel, obligatory role for amygdala eCBs in the proextinction effects of a major pharmacotherapy for trauma- and stressor-related disorders and anxiety disorders.

Genetic disruption of 2-arachidonoylglycerol synthesis reveals a key role for endocannabinoid signaling in anxiety modulation.

Endocannabinoid (eCB) signaling has been heavily implicated in the modulation of anxiety and depressive behaviors and emotional learning. However, the role of the most-abundant endocannabinoid 2-arachidonoylglycerol (2-AG) in the physiological regulation of affective behaviors is not well understood. Here, we show that genetic deletion of the 2-AG synthetic enzyme diacylglycerol lipase α (DAGLα) in mice reduces brain, but not circulating, 2-AG levels. DAGLα deletion also results in anxiety-like and sex-specific anhedonic phenotypes associated with impaired activity-dependent eCB retrograde signaling at amygdala glutamatergic synapses. Importantly, acute pharmacological normalization of 2-AG levels reverses both phenotypes of DAGLα-deficient mice. These data suggest 2-AG deficiency could contribute to the pathogenesis of affective disorders and that pharmacological normalization of 2-AG signaling could represent an approach for the treatment of mood and anxiety disorders.

Multiple mechanistically distinct modes of endocannabinoid mobilization at central amygdala glutamatergic synapses.

The central amygdala (CeA) is a key structure at the limbic-motor interface regulating stress responses and emotional learning. Endocannabinoid (eCB) signaling is heavily implicated in the regulation of stress-response physiology and emotional learning processes; however, the role of eCBs in the modulation of synaptic efficacy in the CeA is not well understood. Here we describe the subcellular localization of CB1 cannabinoid receptors and eCB synthetic machinery at glutamatergic synapses in the CeA and find that CeA neurons exhibit multiple mechanistically and temporally distinct modes of postsynaptic eCB mobilization. These data identify a prominent role for eCBs in the modulation of excitatory drive to CeA neurons and provide insight into the mechanisms by which eCB signaling and exogenous cannabinoids could regulate stress responses and emotional learning.

CaMKII regulates diacylglycerol lipase-α and striatal endocannabinoid signaling.

The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates activity-dependent depression of excitatory neurotransmission at central synapses, but the molecular regulation of 2-AG synthesis is not well understood. Here we identify a functional interaction between the 2-AG synthetic enzyme diacylglycerol lipase-α (DGLα) and calcium/calmodulin dependent protein kinase II (CaMKII). Activated CaMKII interacted with the C-terminal domain of DGLα, phosphorylated two serine residues and inhibited DGLα activity. Consistent with an inhibitory role for CaMKII in 2-AG synthesis, in vivo genetic inhibition of CaMKII increased striatal DGL activity and basal levels of 2-AG, and CaMKII inhibition augmented short-term retrograde endocannabinoid signaling at striatal glutamatergic synapses. Lastly, blockade of 2-AG breakdown using concentrations of JZL-184 that have no effect in wild-type mice produced a hypolocomotor response in mice with reduced CaMKII activity. These findings provide mechanistic insights into the molecular regulation of striatal endocannabinoid signaling with implications for physiological control of motor function.

Reversible gating of endocannabinoid plasticity in the amygdala by chronic stress: a potential role for monoacylglycerol lipase inhibition in the prevention of stress-induced behavioral adaptation.

Chronic stress is the primary environmental risk factor for the development and exacerbation of affective disorders, thus understanding the neuroadaptations that occur in response to stress is a critical step in the development of novel therapeutics for depressive and anxiety disorders. Brain endocannabinoid (eCB) signaling is known to modulate emotional behavior and stress responses, and levels of the eCB 2-arachidonoylglycerol (2-AG) are elevated in response to chronic homotypic stress exposure. However, the role of 2-AG in the synaptic and behavioral adaptations to chronic stress is poorly understood. Here, we show that stress-induced development of anxiety-like behavior is paralleled by a transient appearance of low-frequency stimulation-induced, 2-AG-mediated long-term depression at GABAergic synapses in the basolateral amygdala, a key region involved in motivation, affective regulation, and emotional learning. This enhancement of 2-AG signaling is mediated, in part, via downregulation of the primary 2-AG-degrading enzyme monoacylglycerol lipase (MAGL). Acute in vivo inhibition of MAGL had little effect on anxiety-related behaviors. However, chronic stress-induced anxiety-like behavior and emergence of long-term depression of GABAergic transmission was prevented by chronic MAGL inhibition, likely via an occlusive mechanism. These data indicate that chronic stress reversibly gates eCB synaptic plasticity at inhibitory synapses in the amygdala, and in vivo augmentation of 2-AG levels prevents both behavioral and synaptic adaptations to chronic stress.