The association between serotonin transporter availability and the neural correlates of fear bradycardia.

Susceptibility to stress-related psychopathology is associated with reduced expression of the serotonin transporter (5-HTT), particularly in combination with stress exposure. Aberrant physiological and neuronal responses to threat may underlie this increased vulnerability. Here, implementing a cross-species approach, we investigated the association between 5-HTT expression and the neural correlates of fear bradycardia, a defensive response linked to vigilance and action preparation. We tested this during threat anticipation induced by a well-established fear conditioning paradigm applied in both humans and rodents. In humans, we studied the effect of the common 5-HTT-linked polymorphic region (5-HTTLPR) on bradycardia and neural responses to anticipatory threat during functional magnetic resonance imaging scanning in healthy volunteers (n = 104). Compared with homozygous long-allele carriers, the 5-HTTLPR short-allele carriers displayed an exaggerated bradycardic response to threat, overall reduced activation of the medial prefrontal cortex (mPFC), and increased threat-induced connectivity between the amygdala and periaqueductal gray (PAG), which statistically mediated the effect of the 5-HTTLPR genotype on bradycardia. In parallel, 5-HTT knockout (KO) rats also showed exaggerated threat-related bradycardia and behavioral freezing. Immunohistochemistry indicated overall reduced activity of glutamatergic neurons in the mPFC of KO rats and increased activity of central amygdala somatostatin-positive neurons, putatively projecting to the PAG, which-similarly to the human population-mediated the 5-HTT genotype's effect on freezing. Moreover, the ventrolateral PAG of KO rats displayed elevated overall activity and increased relative activation of CaMKII-expressing projection neurons. Our results provide a mechanistic explanation for previously reported associations between 5-HTT gene variance and a stress-sensitive phenotype.

Using Yoda-1 to mimic laminar flow in vitro: A tool to simplify drug testing.

The endothelium is an attractive drug target and an important site of adverse drug reactions. Endothelial dysfunction is strongly associated with inflammation and contributes to drug-induced cardiovascular toxicity. Endothelial cells in the circulation are exposed to haemodynamic forces including shear stress. Including shear stress may improve future endothelial cell drug discovery or toxicity screening. Piezo-1 is required for endothelial cells to respond to shear stress. In this study, we investigated whether a small molecule activator of Piezo-1, Yoda-1, can mimic the effect of laminar flow-induced shear stress on endothelial cell inflammation, and endothelial cytotoxicity in response to the chemotherapy agent, doxorubicin. First, we tested whether Yoda-1 could mimic the effects of shear stress of expression of the endothelial adhesion molecules, ICAM-1 and VCAM-1. Human umbilical vein endothelial cells (HUVEC) were cultured in static conditions (with or without Yoda-1) or under laminar flow-induced shear stress (5 dyn/cm2). Yoda-1 and laminar flow had similar anti-inflammatory effects, reducing the ability of TNF-α to induce ICAM-1 and VCAM-1 expression. We then tested whether Yoda-1 could mimic the effect of shear stress on doxorubicin-induced cytotoxicity. Both laminar flow and Yoda-1 treatment of static cultures increased the cytotoxicity of doxorubicin. These findings show that Piezo-1 activation with Yoda-1 in static culture leads to an endothelial cell phenotype that mimics endothelial cells under laminar flow. Pharmacological activation of Piezo-1 may be a useful approach to mimic constant shear stress in static cultures, which may improve endothelial drug discovery and toxicity testing.

Acute inescapable stress alleviates fear extinction recall deficits caused by serotonin transporter abolishment.

Life stress increases risk for developing post-traumatic stress disorder (PTSD), and more prominently so in short-allele carriers of the serotonin transporter linked polymorphic region (5-HTTLPR). Serotonin transporter knockout (5-HTT-/-) rats show compromised extinction (recall) of conditioned fear, which might mediate the increased risk for PTSD and reduce the therapeutic efficacy of exposure therapy. Here, we assessed whether acute inescapable stress (IS) differentially affects fear extinction and extinction recall in 5-HTT-/- rats and wildtype controls. Surprisingly, IS experience improved fear extinction recall in 5-HTT-/- rats to the level of wildtype animals, while wildtypes were unaffected by this IS. Thus, whereas 5-HTT-/- rats evidently were more responsive to the stressor, the behavioral consequences presented themselves as adaptive.

Ketamine accelerates fear extinction via mTORC1 signaling.

Impaired fear extinction contributes to the persistence of post-traumatic stress disorder (PTSD), and can be utilized for the study of novel therapeutic agents. Glutamate plays an important role in the formation of traumatic memories, and in the pathophysiology and treatment of PTSD, highlighting several possible drug targets. Recent clinical studies demonstrate that infusion of ketamine, a glutamate NMDA receptor antagonist, rapidly and significantly reduces symptom severity in PTSD patients. In the present study, we examine the mechanisms underlying the actions of ketamine in a rodent model of fear conditioning, extinction, and renewal. Rats received ketamine or saline 24h after fear conditioning and were then subjected to extinction-training on each of the following three days. Ketamine administration enhanced extinction on the second day of training (i.e., reduced freezing behavior to cue) and produced a long-lasting reduction in freezing on exposure to cue plus context 8days later. Additionally, ketamine and extinction exposure increased levels of mTORC1 in the medial prefrontal cortex (mPFC), a region involved in the acquisition and retrieval of extinction, and infusion of the selective mTORC1 inhibitor rapamycin into the mPFC blocked the effects of ketamine on extinction. Ketamine plus extinction also increased cFos in the mPFC and administration of a glutamate-AMPA receptor antagonist blocked the effects of ketamine. These results support the hypothesis that ketamine produces long-lasting mTORC1/protein synthesis and activity dependent effects on neuronal circuits that enhance the expression of extinction and could represent a novel approach for the treatment of PTSD.

GLYX-13 Produces Rapid Antidepressant Responses with Key Synaptic and Behavioral Effects Distinct from Ketamine.

GLYX-13 is a putative NMDA receptor modulator with glycine-site partial agonist properties that produces rapid antidepressant effects, but without the psychotomimetic side effects of ketamine. Studies were conducted to examine the molecular, cellular, and behavioral actions of GLYX-13 to further characterize the mechanisms underlying the antidepressant actions of this agent. The results demonstrate that a single dose of GLYX-13 rapidly activates the mTORC1 pathway in the prefrontal cortex (PFC), and that infusion of the selective mTORC1 inhibitor rapamycin into the medial PFC (mPFC) blocks the antidepressant behavioral actions of GLYX-13, indicating a requirement for mTORC1 similar to ketamine. The results also demonstrate that GLYX-13 rapidly increases the number and function of spine synapses in the apical dendritic tuft of layer V pyramidal neurons in the mPFC. Notably, GLYX-13 significantly increased the synaptic responses to hypocretin, a measure of thalamocortical synapses, compared with its effects on 5-HT responses, a measure of cortical-cortical responses mediated by the 5-HT2A receptor. Behavioral studies further demonstrate that GLYX-13 does not influence 5-HT2 receptor induced head twitch response or impulsivity in a serial reaction time task (SRTT), whereas ketamine increased responses in both tests. In contrast, both GLYX-13 and ketamine increased attention in the SRTT task, which is linked to hypocretin-thalamocortical responses. The differences in the 5-HT2 receptor synaptic and behavioral responses may be related to the lack of psychotomimetic side effects of GLYX-13 compared with ketamine, whereas regulation of the hypocretin responses may contribute to the therapeutic benefits of both rapid acting antidepressants.

Neural circuits and mechanisms involved in fear generalization: Implications for the pathophysiology and treatment of posttraumatic stress disorder.

Compelling evidence suggests that fear generalization (i.e. the transfer of fear from a particular stimulus to another one sharing similarities with the original stimulus) may contribute to the development of posttraumatic stress disorder (PTSD), for which current treatments are ineffective. Deficits in hippocampus-mediated pattern separation, the process by which memories are stored as unique representations that are resistant to confusion, have been solely proposed as a putative underlying marker of generalization. We delineate instead an enlarged scenario, wherein conditioned and generalized fear memories share a common neurocircuitry, with the hippocampus being the nub of contextual fear, and the prefrontal cortex of both cued and contextual fear. The potential contribution of the amygdala and insula will be highlighted as well. Finally, we will consider vulnerability factors that may contribute to the development of PTSD, and suggest avenues for novel therapeutics. A better understanding of the mechanisms behind fear generalization is fundamental to provide further insight into treatment of debilitating conditions such as PTSD.

Improved Stress Control in Serotonin Transporter Knockout Rats: Involvement of the Prefrontal Cortex and Dorsal Raphe Nucleus.

Variations in serotonin transporter (5-HTT) expression have been associated with altered sensitivity to stress. Since controllability is known to alter the impact of a stressor through differential activation of the medial prefrontal cortex (mPFC) and dorsal raphe nucleus (DRN), and that these regions are functionally affected by genetic 5-HTT down-regulation, we hypothesized that 5-HTT expression modulates the effect of controllability on stressor impact and coping. Here, we investigated the effects of a signaled stress controllability task or a yoked uncontrollable stressor on behavioral responding and mPFC and DRN activation. 5-HTT(-/-) rats proved better capable of acquiring the active avoidance task than 5-HTT(+/+) animals. Controllability determined DRN activation in 5-HTT(+/+), but not 5-HTT(-/-), rats, whereas controllability-related activation of the mPFC was independent of genotype. These findings suggest that serotonergic activation in the DRN is involved in stress coping in a 5-HTT expression dependent manner, whereas mPFC activation seems to be implicated in control over stress independently of 5-HTT expression. We speculate that alterations in serotonergic feedback in the DRN might be a potential mechanism driving this differential stress coping.