Limbic Neuropeptidergic Modulators of Emotion and Their Therapeutic Potential for Anxiety and Post-Traumatic Stress Disorder.

Post-traumatic stress disorder (PTSD) is characterized by hypervigilance, increased reactivity to unpredictable versus predictable threat signals, deficits in fear extinction, and an inability to discriminate between threat and safety. First-line pharmacotherapies for psychiatric disorders have limited therapeutic efficacy in PTSD. However, recent studies have advanced our understanding of the roles of several limbic neuropeptides in the regulation of defensive behaviors and in the neural processes that are disrupted in PTSD. For example, preclinical studies have shown that blockers of tachykinin pathways, such as the Tac2 pathway, attenuate fear memory consolidation in mice and thus might have unique potential as early post-trauma interventions to prevent PTSD development. Targeting this pathway might also be beneficial in regulating other symptoms of PTSD, including trauma-induced aggressive behavior. In addition, preclinical and clinical studies have shown the important role of angiotensin receptors in fear extinction and the promise of using angiotensin II receptor blockade to reduce PTSD symptom severity. Additional preclinical studies have demonstrated that the oxytocin receptors foster accurate fear discrimination by facilitating fear responses to predictable versus unpredictable threats. Complementary human imaging studies demonstrate unique neural targets of intranasal oxytocin and compare its efficacy with well-established anxiolytic treatments. Finally, promising data from human subjects have demonstrated that a selective vasopressin 1A receptor antagonist reduces anxiety induced by unpredictable threats. This review highlights these novel promising targets for the treatment of unique core elements of PTSD pathophysiology.

Microanalysis of Brain Angiotensin Peptides Using Ultrasensitive Capillary Electrophoresis Trapped Ion Mobility Mass Spectrometry.

While the role of the renin-angiotensin system (RAS) in peripheral circulation is well characterized, we still lack an in-depth understanding of its role within the brain. This knowledge gap is sustained by lacking technologies for trace-level angiotensin detection throughout tissues, such as the brain. To provide a bridging solution, we enhanced capillary electrophoresis (CE) nanoflow electrospray ionization (ESI) with large-volume sample stacking and employed trapped ion mobility time-of-flight (timsTOF) tandem HRMS detection. A dynamic pH junction helped stack approximately 10 times more of the sample than optimal using the field-amplified reference. In conjunction, the efficiency of ion generation was maximized by a cone-jet nanospray on a low sheath-flow tapered-tip nano-electrospray emitter. The platform provided additional peptide-dependent information, the collision cross section, to filter chemical noise and improve sequence identification and detection limits. The lower limit of detection reached sub-picomolar or ∼30 zmol (∼18,000 copies) level. All nine targeted angiotensin peptides in mouse tissue samples were detectable and quantifiable from the paraventricular nucleus (PVN) of the hypothalamus even after removal of circulatory blood components (perfusion). We anticipate CE-ESI with timsTOF HRMS to be broadly applicable for the ultrasensitive detection of brain peptidomes in pursuit of a better understanding of the brain.

Involvement of the brain-heart axis in the link between PTSD and cardiovascular disease.

Posttraumatic stress disorder (PTSD) has long been associated with a heightened risk of cardiovascular disease (CVD). A number of mechanisms have been implicated to underlie this brain-heart axis relationship, such as altered functioning of the autonomic nervous system and increased systemic inflammation. While neural alterations have repeatedly been observed in PTSD, they are rarely considered in the PTSD-CVD link. The brain-heart axis is a pathway connecting frontal and limbic brain regions to the brainstem and periphery via the autonomic nervous system and it may be a promising model for understanding CVD risk in PTSD given its overlap with PTSD neural deficits. We first provide a summary of the primary mechanisms implicated in the association between PTSD and CVD. We then review the brain-heart axis and its relevance to PTSD, as well as findings from PTSD trials demonstrating that a number of PTSD treatments have effects on areas of the brain-heart axis. Finally, we discuss sex considerations in the PTSD-CVD link. A critical next step in this study is to determine if PTSD treatments that affect the brain-heart axis (e.g., brain stimulation that improves autonomic function) also reduce the risk of CVD.

Symptom severity impacts sympathetic dysregulation and inflammation in post-traumatic stress disorder (PTSD).

Post-traumatic stress disorder (PTSD) is associated with a greater risk of incident hypertension and cardiovascular disease. Inflammation, impaired baroreflex sensitivity (BRS) decreased parasympathetic nervous system (PNS) and overactive sympathetic nervous system (SNS) activity are suggested as contributing mechanisms. Increasing severity of PTSD symptoms has been linked to greater cardiovascular risk; however, the impact of PTSD symptom severity on inflammation and autonomic control of blood pressure has not yet been explored. We hypothesized that increasing PTSD symptom severity is linked to higher inflammation, greater SNS activity, lower PNS reactivity and impaired BRS. Seventy Veterans participated in this study: 28 with severe PTSD ((Clinical Administered PTSD Scale (CAPS) > 60; S-PTSD), 16 with moderate PTSD (CAPS ≥ 45 ≤ 60; M-PTSD) and 26 Controls (CAPS < 45; NO-PTSD). We recorded continuous blood pressure (BP), heart rate (HR) via EKG, heart rate variability (HRV) markers reflecting PNS and muscle sympathetic nerve activity (MSNA) at rest, during arterial baroreflex sensitivity (BRS) testing via the modified Oxford technique, and during 3 min of mental stress via mental arithmetic. Blood samples were analyzed for 12 biomarkers of systemic and vascular inflammation. While BP was comparable between severity groups, HR tended to be higher (p = 0.055) in S-PTSD (76 ±â€¯2 beats/min) than in Controls (67 ±â€¯2 beats/min) but comparable to M-PTSD (70 ±â€¯3 beats/min). There were no differences in resting HRV and MSNA between groups; however, cardiovagal BRS was blunted (p = 0.021) in S-PTSD (10 ±â€¯1 ms/mmHg) compared to controls (16 ±â€¯3 ms/mmHg) but comparable to M-PTSD (12 ±â€¯2 ms/mmHg). Veterans in the S-PTSD group had a higher (p < 0.001) combined inflammatory score compared to both M-PTSD and NO-PTSD. Likewise, while mental stress induced similar SNS and cardiovascular responses between the groups, there was a greater reduction in HRV in S-PTSD compared to both M-PTSD and NO-PTSD. In summary, individuals with severe PTSD symptoms have higher inflammation, greater impairment of BRS, a trend towards higher resting HR and exaggerated PNS withdrawal at the onset of mental stress that may contribute to cardiovascular risk in severe PTSD.

A microanalytical capillary electrophoresis mass spectrometry assay for quantifying angiotensin peptides in the brain.

The renin-angiotensin system (RAS) of the brain produces a series of biologically active angiotensinogen-derived peptides involved in physiological homeostasis and pathophysiology of disease. Despite significant research efforts to date, a comprehensive understanding of brain RAS physiology is lacking. A significant challenge has been the limited set of bioanalytical assays capable of detecting angiotensin (Ang) peptides at physiologically low concentrations (2-15 fmol/g of wet tissue) and sufficient chemical specificity for unambiguous molecular identifications. Additionally, a complex brain anatomy calls for microanalysis of specific tissue regions, thus further taxing sensitivity requirements for identification and quantification in studies of the RAS. To fill this technology gap, we here developed a microanalytical assay by coupling a laboratory-built capillary electrophoresis (CE) nano-electrospray ionization (nano-ESI) platform to a high-resolution mass spectrometer (HRMS). Using parallel reaction monitoring, we demonstrated that this technology achieved confident identification and quantification of the Ang peptides at approx. 5 amol to 300 zmol sensitivity. This microanalytical assay revealed differential Ang peptide profiles between tissues that were micro-sampled from the subfornical organ and the paraventricular nucleus of the hypothalamus, important brain regions involved in thirst and water homeostasis and neuroendocrine regulation to stress. Microanalytical CE-nano-ESI-HRMS extends the analytical toolbox of neuroscience to help better understand the RAS.

Acute effects of device-guided slow breathing on sympathetic nerve activity and baroreflex sensitivity in posttraumatic stress disorder.

Patients with posttraumatic stress disorder (PTSD) have elevated sympathetic nervous system reactivity and impaired sympathetic and cardiovagal baroreflex sensitivity (BRS). Device-guided slow breathing (DGB) has been shown to lower blood pressure (BP) and sympathetic activity in other patient populations. We hypothesized that DGB acutely lowers BP, heart rate (HR), and improves BRS in PTSD. In 23 prehypertensive veterans with PTSD, we measured continuous BP, ECG, and muscle sympathetic nerve activity (MSNA) at rest and during 15 min of DGB at 5 breaths/min ( n = 13) or identical sham device breathing at normal rates of 14 breaths/min (sham; n = 10). Sympathetic and cardiovagal BRS was quantified using pharmacological manipulation of BP via the modified Oxford technique at baseline and during the last 5 min of DGB or sham. There was a significant reduction in systolic BP (by -9 ± 2 mmHg, P < 0.001), diastolic BP (by -3 ± 1 mmHg, P = 0.019), mean arterial pressure (by -4 ± 1 mmHg, P = 0.002), and MSNA burst frequency (by -7.8 ± 2.1 bursts/min, P = 0.004) with DGB but no significant change in HR ( P > 0.05). Within the sham group, there was no significant change in diastolic BP, mean arterial pressure, HR, or MSNA burst frequency, but there was a small but significant decrease in systolic BP ( P = 0.034) and MSNA burst incidence ( P = 0.033). Sympathetic BRS increased significantly in the DGB group (-1.08 ± 0.25 to -2.29 ± 0.24 bursts·100 heart beats-1·mmHg-1, P = 0.014) but decreased in the sham group (-1.58 ± 0.34 to -0.82 ± 0.28 bursts·100 heart beats-1·mmHg-1, P = 0.025) (time × device, P = 0.001). There was no significant difference in the change in cardiovagal BRS between the groups (time × device, P = 0.496). DGB acutely lowers BP and MSNA and improves sympathetic but not cardiovagal BRS in prehypertensive veterans with PTSD. NEW & NOTEWORTHY Posttraumatic stress disorder is characterized by augmented sympathetic reactivity, impaired baroreflex sensitivity, and an increased risk for developing hypertension and cardiovascular disease. This is the first study to examine the potential beneficial effects of device-guided slow breathing on hemodynamics, sympathetic activity, and arterial baroreflex sensitivity in prehypertensive veterans with posttraumatic stress disorder.

Elevated resting blood pressure augments autonomic imbalance in posttraumatic stress disorder.

Posttraumatic stress disorder (PTSD) is characterized by increased sympathetic nervous system (SNS) activity, blunted parasympathetic nervous system (PNS) activity, and impaired baroreflex sensitivity (BRS), which contribute to accelerated cardiovascular disease. Patients with PTSD also have chronic stress-related elevations in resting blood pressure (BP), often in the prehypertensive range; yet, it is unclear if elevated resting blood pressure (ERBP) augments these autonomic derangements in PTSD. We hypothesized that compared with normotensive PTSD (N-PTSD), those with ERBP (E-PTSD) have further increased SNS, decreased PNS activity, and impaired BRS at rest and exaggerated SNS reactivity, PNS withdrawal, and pressor responses during stress. In 16 E-PTSD and 17 matched N-PTSD, we measured continuous BP, ECG, muscle sympathetic nerve activity (MSNA), and heart rate variability (HRV) markers reflecting cardiac PNS activity [standard deviation of R-R intervals (SDNN), root mean square of differences in successive R-R intervals (RMSSD), and high frequency power (HF)] during 5 min of rest and 3 min of mental arithmetic. Resting MSNA ( P = 0.943), sympathetic BRS ( P = 0.189), and cardiovagal BRS ( P = 0.332) were similar between groups. However, baseline SDNN (56 ± 6 vs. 78 ± 8 ms, P = 0.019), RMSSD (39 ± 6 vs. 63 ± 9 ms, P = 0.018), and HF (378 ± 103 vs. 693 ± 92 ms2, P = 0.015) were lower in E-PTSD versus N-PTSD. During mental stress, the systolic blood pressure response ( P = 0.011) was augmented in E-PTSD. Although MSNA reactivity was not different ( P > 0.05), the E-PTSD group had an exaggerated reduction in HRV during mental stress ( P < 0.05). PTSD with ERBP have attenuated resting cardiac PNS activity, coupled with exaggerated BP reactivity and PNS withdrawal during stress.

Baroreflex dysfunction and augmented sympathetic nerve responses during mental stress in veterans with post-traumatic stress disorder.

KEY POINTS: Patients with post-traumatic stress disorder (PTSD) are at a significantly higher risk of developing hypertension and cardiovascular disease. The mechanisms underlying this increased risk are not known. Studies have suggested that PTSD patients have an overactive sympathetic nervous system (SNS) that could contribute to cardiovascular risk; however, sympathetic function has not previously been rigorously evaluated in PTSD patients. Using direct measurements of sympathetic nerve activity and pharmacological manipulation of blood pressure, we show that veterans with PTSD have augmented SNS and haemodynamic reactivity during both combat-related and non-combat related mental stress, impaired sympathetic and cardiovagal baroreflex sensitivity, and increased inflammation. Identifying the mechanisms contributing to increased cardiovascular (CV) risk in PTSD will pave the way for developing interventions to improve sympathetic function and reduce CV risk in these patients. ABSTRACT: Post-traumatic stress disorder (PTSD) is associated with increased cardiovascular (CV) risk. We tested the hypothesis that PTSD patients have augmented sympathetic nervous system (SNS) and haemodynamic reactivity during mental stress, as well as impaired arterial baroreflex sensitivity (BRS). Fourteen otherwise healthy Veterans with combat-related PTSD were compared with 14 matched Controls without PTSD.  Muscle sympathetic nerve activity (MSNA), continuous blood pressure (BP) and electrocardiography were measured at baseline, as well as during two types of mental stress:  combat-related mental stress using virtual reality combat exposure (VRCE) and non-combat related stress using mental arithmetic (MA). A cold pressor test (CPT) was administered for comparison. BRS was tested using pharmacological manipulation of BP via the Modified Oxford technique at rest and during VRCE. Blood samples were analysed for inflammatory biomarkers. Baseline characteristics, MSNA and haemodynamics were similar between the groups. In PTSD vs. Controls, MSNA (+8.2 ± 1.0 vs. +1.2 ± 1.3 bursts min-1 , P < 0.001) and heart rate responses (+3.2 ± 1.1 vs. -2.3 ± 1.0 beats min-1 , P = 0.003) were significantly augmented during VRCE.  Similarly, in PTSD vs. Controls, MSNA (+21.0 ± 2.6 vs. +6.7 ± 1.5 bursts min-1 , P < 0.001) and diastolic BP responses (+6.3 ± 1.0 vs. +3.5 ± 1.0 mmHg, P = 0.011) were significantly augmented during MA but not during CPT (P = not significant). In the PTSD group, sympathetic BRS (-1.2 ± 0.2 vs. -2.0 ± 0.3 burst incidence mmHg-1 , P = 0.026) and cardiovagal BRS (9.5 ± 1.4 vs. 23.6 ± 4.3 ms mmHg-1 , P = 0.008) were significantly blunted at rest. PTSD patients had significantly higher highly sensitive-C-reactive protein levels compared to Controls (2.1 ± 0.4 vs. 1.0 ± 0.3 mg L-1 , P = 0.047). Augmented SNS and haemodynamic responses to mental stress, blunted BRS and inflammation may contribute to an increased CV risk in PTSD.

Plastics and cardiovascular health: phthalates may disrupt heart rate variability and cardiovascular reactivity.

Plastics have revolutionized medical device technology, transformed hematological care, and facilitated modern cardiology procedures. Despite these advances, studies have shown that phthalate chemicals migrate out of plastic products and that these chemicals are bioactive. Recent epidemiological and research studies have suggested that phthalate exposure adversely affects cardiovascular function. Our objective was to assess the safety and biocompatibility of phthalate chemicals and resolve the impact on cardiovascular and autonomic physiology. Adult mice were implanted with radiofrequency transmitters to monitor heart rate variability, blood pressure, and autonomic regulation in response to di-2-ethylhexyl-phthalate (DEHP) exposure. DEHP-treated animals displayed a decrease in heart rate variability (-17% SD of normal beat-to-beat intervals and -36% high-frequency power) and an exaggerated mean arterial pressure response to ganglionic blockade (31.5% via chlorisondamine). In response to a conditioned stressor, DEHP-treated animals displayed enhanced cardiovascular reactivity (-56% SD major axis Poincarè plot) and prolonged blood pressure recovery. Alterations in cardiac gene expression of endothelin-1, angiotensin-converting enzyme, and nitric oxide synthase may partly explain these cardiovascular alterations. This is the first study to show an association between phthalate chemicals that are used in medical devices with alterations in autonomic regulation, heart rate variability, and cardiovascular reactivity. Because changes in autonomic balance often precede clinical manifestations of hypertension, atherosclerosis, and conduction abnormalities, future studies are warranted to assess the downstream impact of plastic chemical exposure on end-organ function in sensitive patient populations. This study also highlights the importance of adopting safer biomaterials, chemicals, and/or surface coatings for use in medical devices.NEW & NOTEWORTHY Phthalates are widely used in the manufacturing of consumer and medical products. In the present study, di-2-ethylhexyl-phthalate exposure was associated with alterations in heart rate variability and cardiovascular reactivity. This highlights the importance of investigating the impact of phthalates on health and identifying suitable alternatives for medical device manufacturing.

Role of chemokine RANTES in the regulation of perivascular inflammation, T-cell accumulation, and vascular dysfunction in hypertension.

Recent studies have emphasized the role of perivascular inflammation in cardiovascular disease. We studied mechanisms of perivascular leukocyte infiltration in angiotensin II (Ang II)-induced hypertension and their links to vascular dysfunction. Chronic Ang II infusion in mice increased immune cell content of T cells (255 ± 130 to 1664 ± 349 cells/mg; P < 0.01), M1 and M2 macrophages, and dendritic cells in perivascular adipose tissue. In particular, the content of T lymphocytes bearing CC chemokine receptor (CCR) 1, CCR3, and CCR5 receptors for RANTES chemokine was increased by Ang II (CCR1, 15.6 ± 1.5% vs. 31 ± 5%; P < 0.01). Hypertension was associated with an increase in perivascular adipose tissue expression of the chemokine RANTES (relative quantification, 1.2 ± 0.2 vs. 3.5 ± 1.1; P < 0.05), which induced T-cell chemotaxis and vascular accumulation of T cells expressing the chemokine receptors CCR1, CCR3, and CCR5. Mechanistically, RANTES(-/-) knockout protected against vascular leukocyte, and in particular T lymphocyte infiltration (26 ± 5% in wild type Ang II vs. 15 ± 4% in RANTES(-/-)), which was associated with protection from endothelial dysfunction induced by Ang II. This effect was linked with diminished infiltration of IFN-γ-producing CD8(+) and double-negative CD3(+)CD4(-)CD8(-) T cells in perivascular space and reduced vascular oxidative stress while FoxP3(+) T-regulatory cells were unaltered. IFN-γ ex vivo caused significant endothelial dysfunction, which was reduced by superoxide anion scavenging. In a human cohort, a significant inverse correlation was observed between circulating RANTES levels as a biomarker and vascular function measured as flow-mediated dilatation (R = -0.3, P < 0.01) or endothelial injury marker von Willebrand factor (R = +0.3; P < 0.01). Thus, chemokine RANTES is important in the regulation of vascular dysfunction through modulation of perivascular inflammation.-Mikolajczyk, T. P., Nosalski, R., Szczepaniak, P., Budzyn, K., Osmenda, G., Skiba, D., Sagan, A., Wu, J., Vinh, A., Marvar, P. J., Guzik, B., Podolec, J., Drummond, G., Lob, H. E., Harrison, D. G., Guzik, T. J. Role of chemokine RANTES in the regulation of perivascular inflammation, T-cell accumulation, and vascular dysfunction in hypertension.

Systemic leukotriene B4 receptor antagonism lowers arterial blood pressure and improves autonomic function in the spontaneously hypertensive rat.

KEY POINTS: Evidence indicates an association between hypertension and chronic systemic inflammation in both human hypertension and experimental animal models. Previous studies in the spontaneously hypertensive rat (SHR) support a role for leukotriene B4 (LTB4 ), a potent chemoattractant involved in the inflammatory response, but its mode of action is poorly understood. In the SHR, we observed an increase in T cells and macrophages in the brainstem; in addition, gene expression profiling data showed that LTB4 production, degradation and downstream signalling in the brainstem of the SHR are dynamically regulated during hypertension. When LTB4 receptor 1 (BLT1) receptors were blocked with CP-105,696, arterial pressure was reduced in the SHR compared to the normotensive control and this reduction was associated with a significant decrease in systolic blood pressure (BP) indicators. These data provide new evidence for the role of LTB4 as an important neuro-immune pathway in the development of hypertension and therefore may serve as a novel therapeutic target for the treatment of neurogenic hypertension. ABSTRACT: Accumulating evidence indicates an association between hypertension and chronic systemic inflammation in both human hypertension and experimental animal models. Previous studies in the spontaneously hypertensive rat (SHR) support a role for leukotriene B4 (LTB4 ), a potent chemoattractant involved in the inflammatory response. However, the mechanism for LTB4 -mediated inflammation in hypertension is poorly understood. Here we report in the SHR, increased brainstem infiltration of T cells and macrophages plus gene expression profiling data showing that LTB4 production, degradation and downstream signalling in the brainstem of the SHR are dynamically regulated during hypertension. Chronic blockade of the LTB4 receptor 1 (BLT1) receptor with CP-105,696, reduced arterial pressure in the SHR compared to the normotensive control and this reduction was associated with a significant decrease in low and high frequency spectra of systolic blood pressure, and an increase in spontaneous baroreceptor reflex gain (sBRG). These data provide new evidence for the role of LTB4 as an important neuro-immune pathway in the development of hypertension and therefore may serve as a novel therapeutic target for the treatment of neurogenic hypertension.

Carotid sinus denervation ameliorates renovascular hypertension in adult Wistar rats.

KEY POINTS: Peripheral chemoreflex sensitization is a feature of renovascular hypertension. Carotid sinus nerve denervation (CSD) has recently been shown to relieve hypertension and reduce sympathetic activity in other rat models of hypertension. We show that CSD in renovascular hypertension halts further increases in blood pressure. Possible mechanisms include improvements in baroreceptor reflex sensitivity and renal function, restoration of cardiac calcium signalling towards control levels, and reduced neural inflammation. Our data suggest that the peripheral chemoreflex may be a viable therapeutic target for renovascular hypertension. ABSTRACT: The peripheral chemoreflex is known to be hyper-responsive in both spontaneously hypertensive (SHR) and Goldblatt hypertensive (two kidney one clip; 2K1C) rats. We have previously shown that carotid sinus nerve denervation (CSD) reduces arterial blood pressure (ABP) in SHR. In the present study, we show that CSD ameliorates 2K1C hypertension and reveal the potential underlying mechanisms. Adult Wistar rats were instrumented to record ABP via telemetry, and then underwent CSD (n = 9) or sham CSD (n = 9) 5 weeks after renal artery clipping, in comparison with normal Wistar rats (n = 5). After 21 days, renal function was assessed, and tissue was collected to assess sympathetic postganglionic intracellular calcium transients ([Ca2+ ]i ) and immune cell infiltrates. Hypertensive 2K1C rats showed a profound elevation in ABP (Wistar: 98 ± 4 mmHg vs. 2K1C: 147 ± 8 mmHg; P < 0.001), coupled with impairments in renal function and baroreflex sensitivity, increased neuroinflammatory markers and enhanced [Ca2+ ]I in stellate neurons (P < 0.05). CSD reduced ABP in 2K1C+CSD rats and prevented the further progressive increase in ABP seen in 2K1C+sham CSD rats, with a between-group difference of 14 ± 2 mmHg by week 3 (P < 0.01), which was accompanied by improvements in both baroreflex control and spectral indicators of cardiac sympatho-vagal balance. Furthermore, CSD improved protein and albuminuria, decreased [Ca2+ ]i evoked responses from stellate neurons, and also reduced indicators of brainstem inflammation. In summary, CSD in 2K1C rats reduces the hypertensive burden and improves renal function. This may be mediated by improvements in autonomic balance, functional remodelling of post-ganglionic neurons and reduced inflammation. Our results suggest that the peripheral chemoreflex may be considered as a potential therapeutic target for controlling renovascular hypertension.

The Physiology of Fear: Reconceptualizing the Role of the Central Amygdala in Fear Learning.

The historically understood role of the central amygdala (CeA) in fear learning is to serve as a passive output station for processing and plasticity that occurs elsewhere in the brain. However, recent research has suggested that the CeA may play a more dynamic role in fear learning. In particular, there is growing evidence that the CeA is a site of plasticity and memory formation, and that its activity is subject to tight regulation. The following review examines the evidence for these three main roles of the CeA as they relate to fear learning. The classical role of the CeA as a routing station to fear effector brain structures like the periaqueductal gray, the lateral hypothalamus, and paraventricular nucleus of the hypothalamus will be briefly reviewed, but specific emphasis is placed on recent literature suggesting that the CeA 1) has an important role in the plasticity underlying fear learning, 2) is involved in regulation of other amygdala subnuclei, and 3) is itself regulated by intra- and extra-amygdalar input. Finally, we discuss the parallels of human and mouse CeA involvement in fear disorders and fear conditioning, respectively.

Autonomic and inflammatory consequences of posttraumatic stress disorder and the link to cardiovascular disease.

Stress- and anxiety-related disorders are on the rise in both military and general populations. Over the next decade, it is predicted that treatment of these conditions, in particular, posttraumatic stress disorder (PTSD), along with its associated long-term comorbidities, will challenge the health care system. Multiple organ systems are adversely affected by PTSD, and PTSD is linked to cancer, arthritis, digestive disease, and cardiovascular disease. Evidence for a strong link between PTSD and cardiovascular disease is compelling, and this review describes current clinical data linking PTSD to cardiovascular disease, via inflammation, autonomic dysfunction, and the renin-angiotensin system. Recent clinical and preclinical evidence regarding the role of the renin-angiotensin system in the extinction of fear memory and relevance in PTSD-related immune and autonomic dysfunction is also addressed.

The Impact of Chronic Phthalate Exposure on Rodent Anxiety and Cognition.

Background: There is a growing importance for environmental contributions to psychiatric disorders and understanding the impact of the exposome (i.e., pollutants and toxins). For example, increased biomonitoring and epidemiological studies suggest that daily phthalate chemical exposure contributes to neurological and behavioral abnormalities; however, these mechanisms remain poorly understood. Therefore, the current study was aimed at examining the effects of chronic phthalate exposure on rodent anxiety behaviors and cognition and the impact on hypothalamic-pituitary-adrenal axis function. Methods: Adult male mice (C57BL6/J) were administered MEHP via drinking water (1 mg/mL), and anxiety-like behavior and cognition combined with hypothalamic-pituitary-adrenal axis and inflammatory assays were assessed after 3 weeks of MEHP exposure. Results: MEHP-treated mice exhibited enhanced generalized anxiety-like behaviors, as demonstrated by reduced time spent in the open-arm of the elevated plus maze and center exploration in the open field. Tests of spatial memory and cognition were unchanged. Following MEHP administration, circulating levels of corticosterone and proinflammatory cytokines were significantly increased, while at the tissue level, there were MEHP-dependent reductions in glucocorticoid metabolism genes Hsd11b1 and Hsd11b2. Conclusions: These data suggest that chronic MEHP exposure leads to enhanced generalized anxiety behaviors independent of rodent measures of cognition and memory, which may be driven by MEHP-dependent effects on hypothalamic-pituitary-adrenal axis and peripheral glucocorticoid metabolism function.

Tyrosine metabolism during interferon-alpha administration: association with fatigue and CSF dopamine concentrations.

Chronic exposure to interferon (IFN)-alpha, an innate immune cytokine, produces high rates of behavioral disturbances, including depression and fatigue. These effects may be mediated by the actions of IFN-alpha on dopamine (DA) metabolism in the basal ganglia. Diminished conversion of phenylalanine (Phen) to tyrosine (Tyr), the primary amino acid precursor of DA, has been associated with inflammation, and may reflect decreased activity of the enzyme phenylalanine-hydroxylase (PAH). This study investigated the peripheral Phen/Tyr ratio in relation to cerebrospinal fluid (CSF) concentrations of DA and its metabolites in subjects treated with IFN-alpha plus ribavirin for hepatitis C and controls awaiting IFN-alpha therapy. Plasma Phen/Tyr ratios were significantly increased in IFN-alpha-treated subjects (n=25) compared to controls (n=9), and were negatively correlated with CSF DA (r=-0.59, df=15, p<0.05) and its metabolite, homovanillic acid (r=-0.67, df=15, p<0.01), and positively correlated with fatigue (r=0.44, df=23, p<.05) in IFN-alpha-treated patients but not controls. Given the role of tetrahydrobiopterin (BH4) in the PAH conversion of Phen to Tyr, CSF concentrations of BH4 and its inactive oxidized form, dihydrobiopterin (BH2), were examined along with CSF interleukin (IL)-6 in a subset of patients. BH2 concentrations were significantly increased in IFN-alpha-treated patients (n=12) compared to controls (n=7), and decreased CSF BH4 concentrations correlated with increased CSF IL-6 (r=-0.57, df=12, p<0.05). These results indicate that IFN-alpha is associated with decreased peripheral conversion of Phen to Tyr, which in turn is associated with reduced DA in the brain as well as fatigue. These alterations may be related to oxidation of BH4 secondary to IFN-alpha-induced activation of a CNS inflammatory response.

Central amygdala angiotensin type 1 receptor (Agtr1) expressing neurons contribute to fear extinction.

The renin-angiotensin system (RAS) has been linked to the pathophysiology of posttraumatic stress disorder (PTSD) however, the underlying neurobiological mechanism(s) remain elusive. Here we utilized angiotensin II receptor type 1 (AT1R) transgenic mice combined with neuroanatomical, behavioral, and electrophysiological approaches, to examine the role of the central amygdala (CeA) expressing AT1R neurons in fear and anxiety-related behavior. Within the major amygdala subdivisions, AT1R+ neurons were localized to gamma-aminobutyric acid (GABA) expressing neurons in the lateral division of the central amygdala (CeL), and the majority of them were identified as protein kinase C-δ positive (PKCδ+) neurons. Following CeA-AT1R deletion using cre-expressing lentiviral delivery in AT1R-Flox mice, generalized anxiety and locomotor activity as well as the acquisition of conditioned fear were unaltered while the acquisition of extinction learning, as measured by percent freezing behavior, was significantly enhanced. During electrophysiological recordings of CeL-AT1R+ neurons, the application of angiotensin II (1 µm) increased the amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) and decreased the excitability of CeL-AT1R+ neurons. Overall, these findings demonstrate that CeL-AT1R-expressing neurons play a role in fear extinction, potentially through facilitated CeL-AT1R+ GABAergic inhibition. These results provide new evidence for mechanisms of angiotensinergic neuromodulation of the CeL and its role in fear extinction and may aid in further advancing targeted novel therapies for improving maladaptive fear learning processes associated with PTSD.

Sex-dependent effects of angiotensin type 2 receptor expressing medial prefrontal cortex (mPFC) interneurons in fear extinction learning.

Background: The renin-angiotensin system (RAS) has been identified as a potential therapeutic target for PTSD, though its mechanisms are not well understood. Brain angiotensin type 2 receptors (AT2Rs) are a subtype of angiotensin II receptors located in stress and anxiety-related regions, including the medial prefrontal cortex (mPFC), but their function and mechanism in the mPFC remain unexplored. We therefore used a combination of imaging, cre/lox, and behavioral methods to investigate mPFC-AT2R-expressing neuron involvement in fear learning. Methods: To characterize mPFC-AT2R-expressing neurons in the mPFC, AT2R-Cre/td-Tomato male and female mice were used for immunohistochemistry (IHC). mPFC brain sections were stained with glutamatergic or interneuron markers, and density of AT2R+ cells and colocalization with each marker was quantified. To assess fear-related behaviors in AT2R-flox mice, we selectively deleted AT2R from mPFC neurons using an AAV-Cre virus. Mice then underwent Pavlovian auditory fear conditioning, approach/avoidance, and locomotion testing. Results: IHC results revealed that AT2R is densely expressed in the mPFC. Furthermore, AT2R is primarily expressed in somatostatin interneurons in females but not males. Following fear conditioning, mPFC-AT2R deletion impaired extinction in female but not male mice. Locomotion was unaltered by mPFC-AT2R deletion in males or females, while AT2R-deleted females had increased exploratory behavior. Conclusion: These results lend support for mPFC-AT2R+ neurons as a novel subgroup of somatostatin interneurons that influence fear extinction in a sex-dependent manner. This furthers underscores the role of mPFC in top-down regulation and a unique role for peptidergic (ie., angiotensin) mPFC regulation of fear and sex differences.

The Impact of Chronic Phthalate Exposure on Rodent Anxiety and Cognition.

There is a growing importance for environmental contributions to psychiatric disorders and understanding the impact of the exposome (i.e., pollutants and toxins). Increased biomonitoring and epidemiological studies, for example, suggest that daily phthalate chemical exposure contribute to neurological and behavioral abnormalities, however these mechanisms remain poorly understood. The current study therefore aimed to examine the effects of chronic phthalate exposure on rodent anxiety behaviors, cognition, and the impact on hypothalamic-pituitary- adrenal (HPA)-axis function. Adult male mice (C57BL6/J) were administered mono-2-ethylhexyl phthalate (MEHP) via drinking water (1 mg/ml), and anxiety-like behavior, cognition combined with HPA- axis and inflammatory assays were assessed after 3 weeks of MEHP exposure. MEHP-treated mice exhibited enhanced generalized anxiety-like behaviors, as demonstrated by reduced time spent in the open-arm of the elevated plus maze (EPM) and center exploration in the open field (OF). Tests of spatial, cognition and memory function were unchanged. Following MEHP administration, circulating levels of corticosterone and pro- inflammatory cytokines were significantly increased, while at the tissue level, MEHP-dependent reductions in glucocorticoid metabolism genes 11ß-hydroxysteroid dehydrogenase (11ß-HSD) 1 and 2. These data suggest that chronic MEHP exposure leads to enhanced generalized-anxiety behaviors independent of rodent measures of cognition and memory, which maybe driven by MEHP-dependent effects on HPA-axis and peripheral glucocorticoid metabolism function.