Noradrenaline Efflux Research Articles

Tyrosine hydroxylase-positive neurons in the rostral ventrolateral medulla mediate sympathetic activation in sepsis

AimSepsis results in high mortality and is associated with organ dysfunction caused by infection. The present study aimed to elucidate whether early-stage sympathetic activation is associated with the prognosis of sepsis and its possible mechanisms. MethodsPatients with sepsis and healthy controls were included. Sepsis in rats was induced by lipopolysaccharide. Dexmedetomidine, a α2-adrenergic receptor agonist, was used in patients and rats with sepsis to evaluate the role of the sympathetic nervous system in sepsis. Holter monitoring was used to detect heart rate variability, while plasma samples were obtained to measure levels of norepinephrine and inflammatory markers. Mean arterial pressure, heart rate, and renal sympathetic nerve activity were recorded. Immunofluorescence was used to detect the activation of neurons in the rostral ventrolateral medulla (RVLM). ResultsIn patients with sepsis, plasma levels of norepinephrine and interleukin-1β were higher compared with those in controls and positively correlated with acute physiology and chronic health evaluation (APACHEII). SDNN and SDANN were significantly reduced as well as negatively correlated with APACHEII. Meanwhile, rats with sepsis showed increased of sympathetic outflow and plasma levels of norepinephrine, with increased c-fos levels in the RVLM. Treatment with dexmedetomidine could improve prognosis. Lesion of tyrosine hydroxylase-positive neurons in the RVLM attenuated sympathetic activation and target organs damage in septic rats as well as improved survival. ConclusionThe results suggest that tyrosine hydroxylase-positive neurons in the RVLM might contribute to the prognosis of sepsis via activation of the sympathetic nervous system, while dexmedetomidine could ameliorate sepsis via inhibiting sympathetic activation.

Potentiation of prefrontal cortex dopamine function by the novel cognitive enhancer d-govadine

Cognitive impairment is a debilitating feature of psychiatric disorders including schizophrenia, mood disorders and substance use disorders for which there is a substantial lack of effective therapies. d-Govadine (d-GOV) is a tetrahydroprotoberberine recently shown to significantly enhance working memory and behavioural flexibility in several prefrontal cortex (PFC)-dependent rodent tasks. d-GOV potentiates dopamine (DA) efflux in the mPFC and not the nucleus accumbens, a unique pharmacology that sets it apart from many dopaminergic drugs and likely contributes to its effects on cognitive function. However, specific mechanisms involved in the preferential effects of d-GOV on mPFC DA function remain to be determined. The present study employs brain dialysis in male rats to deliver d-GOV into the mPFC or ventral tegmental area (VTA), while simultaneously sampling DA and norepinephrine (NE) efflux in the mPFC. Intra-PFC delivery or systemic administration of d-GOV preferentially potentiated medial prefrontal DA vs NE efflux. This differential effect of d-GOV on the primary catecholamines known to affect mPFC function further underscores its specificity for the mPFC DA system. Importantly, the potentiating effect of d-GOV on mPFC DA was disrupted when glutamatergic transmission was blocked in either the mPFC or the VTA. We hypothesize that d-GOV acts in the mPFC to engage the mesocortical feedback loop through which prefrontal glutamatergic projections activate a population of VTA DA neurons that specifically project back to the PFC. The activation of a PFC-VTA feedback loop to elevate PFC DA efflux without affecting mesolimbic DA release represents a novel approach to developing pro-cognitive drugs.

Intermittent hypoxia in neonatal rodents affects facial bone growth.

Preterm human infants often show periodic breathing (PB) or apnea of prematurity (AOP), breathing patterns which are accompanied by intermittent hypoxia (IH). We examined cause-effect relationships between transient IH and reduced facial bone growth using a rat model. Neonatal pups from 14 timed pregnant Sprague-Dawley rats were randomly assigned to an IH condition, with oxygen altering between 10% and 21% every 4 min for 1 h immediately after birth, or to a litter-matched control group. The IH pups were compared with their age- and sex-matched control groups in body weight (WT), size of facial bones and nor-epinephrine (NE) levels in blood at 3, 4, and 5-weeks. Markedly increased activity of osteoclasts in sub-condylar regions of 3-week-old IH-treated animals appeared, as well as increased numbers of sympathetic nerve endings in the same region of tissue sections. Male IH-pups showed significantly higher levels of NE levels in sera at 3, 4 as well as 5-week-old time points. NE levels in 4- and-5-week-old female pups did not differ significantly. Intercondylar Width, Mandible Length and Intermolar Width measures consistently declined after IH insults in 3- and 4-week-old male as well as female animals. Three-week-old male IH-pups only showed a significantly reduced (p < 0.05) body weight compared to those of 3-week controls. However, female IH-pups were heavier than age-matched controls at all 3 time-points. Trabecular bone configuration, size of facial bones, and metabolism are disturbed after an IH challenge 1 h immediately after birth. The findings raise the possibility that IH, introduced by breathing patterns such as PB or AOP, induce significantly impaired bone development and metabolic changes in human newborns. The enhanced NE outflow from IH exposure may serve a major role in deficient bone growth, and may affect bone and other tissue influenced by that elevation.

Alpha-2A but not 2B/C noradrenergic receptors in ventral tegmental area regulate phasic dopamine release in nucleus accumbens core

Adrenergic receptors (AR) in the ventral tegmental area (VTA) modulate local neuronal activity and, as a consequence, dopamine (DA) release in the mesolimbic forebrain. Such modulation has functional significance: intra-VTA blockade of α1-AR attenuates behavioral responses to salient environmental stimuli in rat models of drug seeking and conditioned fear as well as phasic DA release in the nucleus accumbens (NAc). In contrast, α2-AR in the VTA has been suggested to act primarily as autoreceptors, limiting local noradrenergic input. The regulation of noradrenaline efflux by α2-AR could be of clinical interest, as α2-AR agonists are proposed as promising pharmacological tools in the treatment of PTSD and substance use disorder. Thus, the aim of our study was to determine the subtype-specificity of α2-ARs in the VTA capable of modulating phasic DA release. We used fast scan cyclic voltammetry (FSCV) in anaesthetized male rats to measure DA release in the NAc after combined electrical stimulation and infusion of selected α2-AR antagonists into the VTA. Intra-VTA microinfusion of idazoxan – a non-subtype-specific α2-AR antagonist, as well as BRL-44408 – a selective α2A-AR antagonist, attenuated electrically-evoked DA in the NAc. In contrast, local administration of JP-1302 or imiloxan (α2B- and α2C-AR antagonists, respectively) had no effect. The effect of BRL-44408 on DA release was attenuated by intra-VTA DA D2 antagonist (raclopride) pre-administration. Finally, we confirmed the presence of α2A-AR protein in the VTA using western blotting. In conclusion, these data specify α2A-, but not α2B- or α2C-AR as the receptor subtype controlling NA release in the VTA.

Consequences of Acute or Chronic Methylphenidate Exposure Using Ex Vivo Neurochemistry and In Vivo Electrophysiology in the Prefrontal Cortex and Striatum of Rats.

Methylphenidate (MPH) is among the main drugs prescribed to treat patients with attention-deficit and hyperactivity disease (ADHD). MPH blocks both the norepinephrine and dopamine reuptake transporters (NET and DAT, respectively). Our study was aimed at further understanding the mechanisms by which MPH could modulate neurotransmitter efflux, using ex vivo radiolabelled neurotransmitter assays isolated from rats. Here, we observed significant dopamine and norepinephrine efflux from the prefrontal cortex (PFC) after MPH (100 µM) exposure. Efflux was mediated by both dopamine and norepinephrine terminals. In the striatum, MPH (100 µM) triggered dopamine efflux through both sodium- and vesicular-dependent mechanisms. Chronic MPH exposure (4 mg/kg/day/animal, voluntary oral intake) for 15 days, followed by a 28-day washout period, increased the firing rate of PFC pyramidal neurons, assessed by in vivo extracellular single-cell electrophysiological recordings, without altering the responses to locally applied NMDA, via micro-iontophoresis. Furthermore, chronic MPH treatment resulted in decreased efficiency of extracellular dopamine to modulate NMDA-induced firing activities of medium spiny neurons in the striatum, together with lower MPH-induced (100 µM) dopamine outflow, suggesting desensitization to both dopamine and MPH in striatal regions. These results indicate that MPH can modulate neurotransmitter efflux in brain regions enriched with dopamine and/or norepinephrine terminals. Further, long-lasting alterations of striatal and prefrontal neurotransmission were observed, even after extensive washout periods. Further studies will be needed to understand the clinical implications of these findings.

Repeated administration of rapastinel produces exceptionally prolonged rescue of memory deficits in phencyclidine-treated mice

Rapastinel, a positive N-methyl-D-aspartate receptor (NMDAR) modulator with rapid-acting antidepressant properties, rescues memory deficits in rodents. We have previously reported that a single intravenous dose of rapastinel, significantly, but only transiently, prevented and rescued deficits in the novel object recognition (NOR) test, a measure of episodic memory, produced by acute or subchronic administration of the NMDAR antagonists, phencyclidine (PCP) and ketamine. Here, we tested the ability of single and multiple subcutaneous doses per day of rapastinel to restore NOR and operant reversal learning (ORL) deficits in subchronic PCP-treated mice. Rapastinel, 1 or 3 mg/kg, administered subcutaneously, 30 min before NOR or ORL testing, respectively, transiently rescued both deficits in subchronic PCP mice. This effect of rapastinel on NOR and ORL was mammalian target of rapamycin (mTOR)-dependent. Most importantly, 1 mg/kg rapastinel given twice daily for 3 or 5 days, but not 1 day, restored NOR for at least 9 and 10 weeks, respectively, which is an indication of neuroplastic effects on learning and memory. Both rapastinel (3 mg/kg) and ketamine (30 mg/kg), moderately increased the efflux of dopamine, norepinephrine, and serotonin in medial prefrontal cortex; however, only ketamine increased cortical glutamate efflux. This observation was likely the basis for the contrasting effects of the two drugs on cognition.

Molecular Signaling Mechanisms for the Antidepressant Effects of NLX-101, a Selective Cortical 5-HT1A Receptor Biased Agonist

Depression is the most prevalent of the mental illnesses and serotonin (5-hydroxytryptamine, 5-HT) is considered to be the major neurotransmitter involved in its etiology and treatment. In this context, 5-HT1A receptors have attracted interest as targets for therapeutic intervention. Notably the activation of presynaptic 5-HT1A autoreceptors delays antidepressant effects whereas the stimulation of postsynaptic 5-HT1A heteroreceptors is needed for an antidepressant action. NLX-101 (also known as F15599) is a selective biased agonist which exhibits preferred activation of cortical over brain stem 5-HT1A receptors. Here, we used behavioral, neurochemical and molecular methods to examine the antidepressant-like effects in rats of a single dose of NLX-101 (0.16 mg/kg, i.p.). NLX-101 reduced immobility in the forced swim test when measured 30 min but not 24 h after drug administration. NLX-101 increased extracellular concentrations of glutamate and dopamine in the medial prefrontal cortex, but no changes were detected in the efflux of noradrenaline or 5-HT. NLX-101 also produced an increase in the activation of pmTOR, pERK1/2 and pAkt, and the expression of PSD95 and GluA1, which may contribute to its rapid antidepressant action.

Splenic Denervation Attenuates Repeated Social Defeat Stress-Induced T Lymphocyte Inflammation

BackgroundPosttraumatic stress disorder (PTSD) is a devastating psychological disorder. Patients with PTSD canonically demonstrate an increased risk for inflammatory diseases as well as increased sympathetic tone and norepinephrine outflow. Yet, the exact etiology and causal nature of these physiologic changes remain unclear. Previously, we demonstrated that exogenous norepinephrine alters mitochondrial superoxide in T lymphocytes to produce a proinflammatory T helper 17 phenotype and observed similar T helper 17 polarization in a preclinical model of PTSD. Therefore, we hypothesized sympathetic-driven neuroimmune interactions could mediate psychological trauma–induced T lymphocyte inflammation. MethodsRepeated social defeat stress (RSDS) is a preclinical murine model that recapitulates the behavioral, autonomic, and inflammatory aspects of PTSD. Targeted splenic denervation was performed to deduce the contribution of splenic sympathetic nerves to RSDS-induced inflammation. Denervation or sham operation was performed in 85 C57BL/6J mice, followed by RSDS or control paradigms. Animals were assessed for behavioral, autonomic, inflammatory, and redox profiles. ResultsDenervation did not alter the antisocial or anxiety-like behavior induced by RSDS. In circulation, denervation/RSDS animals exhibited diminished levels of T lymphocyte–specific cytokines (interleukin 2 [IL-2], IL-17A, and IL-22) compared with intact animals, whereas other nonspecific inflammatory cytokines (e.g., IL-6, tumor necrosis factor α, and IL-10) were unaffected by denervation. Importantly, denervation specifically ameliorated the increases in RSDS-induced T lymphocyte mitochondrial superoxide, T helper 17 polarization, and proinflammatory gene expression with minimal impact to non–T lymphocyte immune populations. ConclusionsOverall, our data suggest that sympathetic nerves regulate RSDS-induced splenic T lymphocyte inflammation but play less of a role in the behavioral and non–T lymphocyte inflammatory phenotypes induced by this psychological trauma paradigm.

Impact of Cell Membrane Cholesterol Alterations on Function, Oligomerization, and Binding Affinity of Monoamine Transporters

Objective and hypothesis Cholesterol is the major lipid constituent of mammalian cell membranes and contributes to membrane fluidity and permeability. Additionally, cholesterol is important for membrane trafficking, lipid and protein sorting, and the formation of lipid nanodomains and lipid rafts. Changes in membrane cholesterol concentration may affect the function and regulation of the neurotransmitter transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET). Therefore, alterations of cholesterol levels may potentially contribute to the development and progression of various psychiatric diseases. Methods Cell membranes of SERT, DAT, and NET-expressing human embryonic kidney (HEK) 293 cells were depleted of or replenished with cholesterol using methyl-β-cyclodextrin (MBCD) or cholesterol-saturated MBCD, respectively. In these modified cells, uptake and efflux function was first quantified by liquid scintillation counting of tritiated neurotransmitters. In a next step, the oligomerization of SERT, DAT, and NET at the plasma membrane was assessed with Förster resonance energy transfer (FRET). Additionally, the binding affinity of specific radioactively labeled inhibitors at the neurotransmitter transporters was determined by liquid scintillation counting. Results Depletion of membrane cholesterol reduced monoamine uptake for all tested transporters in a concentration-dependent manner. Cholesterol replenishment slightly increased the uptake kinetics of SERT but had no effect on DAT and NET function. However, cholesterol depletion significantly reduced the stimulant-induced dopamine and norepinephrine efflux, whereas cholesterol replenishment had no effect. In contrast, cholesterol depletion and replenishment significantly increased and decreased the stimulant-induced serotonin efflux, respectively. Furthermore, the membrane surface oligomerization of SERT, but not of DAT and NET, was significantly increased after cholesterol depletion. Cholesterol replenishment had no effect on either transporter. After cholesterol depletion, the binding affinity of specific inhibitors at SERT, DAT, and NET was significantly diminished after cholesterol depletion. Cholesterol replenishment resulted in increased binding affinity at DAT and NET, but not at SERT. Conclusion Membrane cholesterol alterations similarly affected uptake function and binding affinities at SERT, DAT, and NET. However, drug-induced serotonin efflux and SERT oligomerization was increased due to cholesterol depletion, which was not observed for DAT and NET. This indicates the possibility that SERT but not DAT and NET-mediated efflux is dependent on the oligomeric state of the transporters. Furthermore, these findings suggest different effects of cholesterol alterations in psychiatric diseases, depending on the monoaminergic systems involved.

Sympathetic Nerves Control Splenic T-Lymphocyte Inflammation and the Mitochondrial Redox Environment During Repeated Social Defeat Stress

Post-traumatic stress disorder (PTSD) is a devastating psychological disorder that increases the risk for autoimmune disease by >3 fold, with the exact etiology still unclear. A known hallmark of PTSD is increased sympathetic tone and norepinephrine (NE) outflow. We have previously demonstrated that exogenous NE induces a pro-inflammatory T-helper 17 (TH17) phenotype in T-lymphocytes through a mitochondrial superoxide-dependent manner. Therefore, we hypothesized sympathetic-driven neuroimmune interactions could mediate psychological trauma-induced T-lymphocyte inflammation.

Antidepressant-Like Effects of CX717, a Positive Allosteric Modulator of AMPA Receptors.

Conventional antidepressant drugs elevate the availability of monoamine neurotransmitters. However, these pharmacological therapies have limited efficacy and a slow onset of action as main limitations. New glutamatergic drugs such as ketamine have shown promise as a rapid-acting antidepressant drugs although with adverse effects. The mechanism of action of ketamine is hypothesized to involve a dis-inhibition of cortical pyramidal neurons produced by an stimulation of AMPA receptors by glutamate. In this context, low-impact positive allosteric modulators of the AMPA receptors (a.k.a. ampakines) have been regarded as potential antidepressant drugs. Here, we have examined the behavioral, biochemical, and molecular effects of a low-impact ampakine, CX717. Our results show that CX717 has a rapid (30min) but short-lasting (up to 24h) antidepressant-like effect in the forced swim test. Intra-cortical infusion of CX717 increases the efflux of noradrenaline, dopamine, and serotonin, but not glutamate. However, systemic CX717 does not alter these neurotransmitters. CX717 also produced a rapid (up to 1h) increase of brain-derived neurotrophic factor (BDNF) and a more sustained (up to 6h) increase of p11. Overall, CX717 appears to possess a rapid but not sustained antidepressant action possibly caused by rapid increases of BDNF and p11.

Behavioral Effects of d‐Methamphetamine and sin vivo microdialysis in rats

The present studies were conducted to examine the relationship between locomotor stimulant effects of d‐methamphetamine (d‐MA) and its ability to increase extracellular concentrations of d‐MA, its active metabolite d‐amphetamine (d‐AMP), dopamine (DA), serotonin (5‐HT), norepinephrine (NE), γ‐aminobutyric acid (GABA), and glutamate (GLU) in dialysate samples obtained from the rat nucleus accumbens (nAcc) shell. First, the effects of cumulatively administered d‐MA (0.3–5.6 mg/kg) on locomotor activity were determined in rats. In other studies, in vivo microdialysis and LC/MS analysis of dialysate samples were used to determine the effects of cumulatively administered d‐MA (0.3–5.6 mg/kg) on extracellular levels of d‐MA, d‐AMP, DA, 5‐HT, NE, GABA, and GLU in the nAcc shell. d‐MA produced dose‐related effects on locomotor activity, characterized by an inverted‐U dose‐response curve; 0.3–3 mg/kg increased activity, whereas 5.6 mg/kg suppressed activity (induced stereotypy). Results from microdialysis studies show that levels of d‐MA (0.3–5.6 mg/kg i.p.) in the nAcc shell increased in a dose‐ and time‐dependent manner with concentrations ranging from 12–319 nM, respectively; peak levels (334 nM) detected 20 min after 5.6 mg/kg slowly declined over the next hour. An increase in d‐AMP was also observed with concentrations ranging from 4.5–130 nM after 0.3–5.6 mg/kg d‐MA; peak levels of d‐AMP were detected 20 min (164 nM) after 5.6 mg/kg d‐MA and remained at this level for more than the next hour 100 min. d‐MA produced dose‐ and time‐dependent increases in DA, 5‐HT, and NE efflux in the nAcc shell to maxima of approximately 642% (17 nM), 582% (1.2 nM), and 422% (0.7 nM) of control values, respectively. In contrast, d‐MA did not produce significant changes in GABA or GLU levels. Time course and dose‐response analysis show that increases in d‐MA levels after 0.3–3.0 mg/kg are concordant with increases in DA, 5‐HT, and NE efflux. However, levels of d‐MA after 5.6 mg/kg appear to reflect increases in DA and 5‐HT but not NE. Correlation analysis revealed an inverse relationship between d‐MA‐induced increases in locomotor activity and in vivo extracellular levels of 5‐HT and d‐AMP. Although not statistically significant, a trend towards an inverse relationship was also observed between d‐MA‐induced increases in locomotor activity and extracellular levels of d‐MA and other neurotransmitters in the nAcc shell. Taken together, the ability to detect and correlate the presence and clearance of in vivo brain levels of d‐MA, and its active metabolite d‐AMP in the nAcc shell of rats to neurochemical and behavioral effects can provide insight into the neuropharmacological effects of d‐MA.

Effects of NBI-98782, a selective vesicular monoamine transporter 2 (VMAT2) inhibitor, on neurotransmitter efflux and phencyclidine-induced locomotor activity: Relevance to tardive dyskinesia and antipsychotic action

Valbenazine, a vesicular monoamine transporter 2 (VMAT2, SLC18A2) inhibitor, is a newly approved treatment for tardive dyskinesia. VMAT2 is present in the membrane of secretory vesicles and transports dopamine (DA), norepinephrine (NE), serotonin (5-HT), histamine, glutamate (Glu), and GABA into vesicles for presynaptic release. We utilized microdialysis in awake, freely moving mice to determine the effect of NBI-98782, the active metabolite of valbenazine, alone, or in combination with several antipsychotic drugs (APDs), to influence neurotransmitter efflux in the medial prefrontal cortex (mPFC), dorsal striatum (dSTR), hippocampus and nucleus accumbens (NAC); we also compared it with tetrabenazine, the prototypical VMAT2 inhibitor. Acute NBI-98782 and tetrabenazine decreased mPFC, dSTR, hippocampus, and NAC DA, 5-HT, and NE efflux, while increasing that of DOPAC, HVA, and 5-HIAA. Sub-chronic NBI-98782 (7 days) decreased baseline DA and 5-HT efflux in both mPFC and dSTR. NBI-98782 elicited similar effects on neurotransmitter efflux in sub-chronic NBI-98782-treated mice but also enhanced ACh and GABA; the decrease in DA efflux in mPFC and dSTR was not significant in the sc-treated animals. NBI-98782 suppressed clozapine-, olanzapine- and risperidone-induced DA efflux in both mPFC and dSTR, and ACh efflux in mPFC. NBI-98782 suppressed the haloperidol-induced DA efflux in dSTR, with minimal effect on GABA efflux. NBI-98782 attenuated PCP-induced DA, 5-HT, NE and Glu efflux, and AMPH-induced DA and NE efflux, in both mPFC and dSTR, as well as PCP- and AMPH-induced hyperlocomotion, suggesting possible beneficial antipsychotic effects.

Role of Serotonin and Noradrenaline in the Rapid Antidepressant Action of Ketamine.

Depression is a chronic and debilitating illness that interferes severely with many human behaviors, and is the leading cause of disability in the world. There is data suggesting that deficits in serotonin neurotransmission can contribute to the development of depression. Indeed, >90% of prescribed antidepressant drugs act by increasing serotonergic transmission at the synapse. However, this increase is offset by a negative feedback operating at the level of the cell body of the serotonin neurons in the raphe nuclei. In the present work, we demonstrate: first, the intracortical infusion of ketamine induced an antidepressant-like effect in the forced swim test, comparable to that produced by systemic ketamine; second, systemic and intracortical ketamine increased serotonin and noradrenaline efflux in the prefrontal cortex, but not in the dorsal raphe nucleus; third, systemic and intracortical administration of ketamine increased the efflux of glutamate in the prefrontal cortex and dorsal raphe nucleus; fourth, systemic ketamine did not alter the functionality of 5-HT1A receptors in the dorsal raphe nucleus. Taken together, these findings suggest that the antidepressant-like effects of ketamine are caused by the stimulation of the prefrontal projection to the dorsal raphe nucleus and locus coeruleus caused by an elevated glutamate in the medial prefrontal cortex, which would stimulate release of serotonin and noradrenaline in the same area. The impact of both monoamines in the antidepressant response to ketamine seems to have different time frames.

S21. Identification of a Novel T-Lymphocyte Inflammatory Protein in Psychological Stress-Induced Hypertension

Post-Traumatic Stress Disorder (PTSD) dysregulates physiological systems leading to elevated sympathetic tone and norepinephrine (NE) outflow, pronounced inflammation, and a >50% increased risk in developing comorbid cardiovascular diseases like hypertension (HTN). We hypothesized that NE increases T-lymphocyte-driven inflammation leading to HTN after trauma exposure.

Identification of a Novel T‐lymphocyte Inflammatory Protein in Psychological Stress‐ Induced Hypertension

Post‐traumatic stress disorder (PTSD) is a psychiatric condition characterized by hyperarousal, anxiety, anhedonia, and depression. While the behavioral manifestations are often the focus of this disease, PTSD also alters physiological processes leading to elevated sympathetic tone and norepinephrine (NE) outflow, pronounced inflammation, and a &gt;50% increased risk in developing co‐morbid cardiovascular diseases like hypertension. Our laboratory previously reported that exposure to NE can modulate levels of T‐lymphocyte pro‐inflammatory cytokines, and understanding that hypertension is regulated in part due to inflammation, we posited a connection between the autonomic nervous, immune, and cardiovascular systems in PTSD patients. We hypothesized that psychological stress‐induced sympathoexcitation leads to increased pro‐inflammatory cytokine production from T‐lymphocytes, which impacts the development of hypertension. Utilizing a mouse‐model of PTSD‐like psychological trauma, lymphatic sympathoexcitation was confirmed by a 2.5 fold increase in NE and 4.1 fold increase in tyrosine hydroxylase content in spleens from stressed animals (p&lt;0.001). This increased sympathetic tone correlated with elevated pro‐inflammatory cytokines IL‐17A, IL‐6, and IL‐2 (+4.4, +3.1, and +3.0 fold, respectively, p&lt;0.01) both in circulation and also specifically within splenic T‐lymphocytes. Examination of hemodynamics in the stressed mice elucidated a stable increase in resting mean arterial pressure (+19.5 mmHg, p&lt;0.05) over the stress‐induction period with an even greater (+39.8 mmHg, p&lt;0.01) rise after stress context re‐exposure compared to control animals. To investigate the causal contribution of T‐lymphocytes in driving this hypertensive response, recombination activating gene 2 (Rag2) knock‐out animals, devoid of mature lymphocytes, were utilized. Rag2 knock‐out mice showed no changes in pro‐inflammatory cytokines, a 75% decrease in splenic TH levels (p&lt;0.01), and a blunted blood pressure response upon context re‐exposure (+8.8 mmHg, p&lt;0.01). Single cell RNAseq analysis of T‐lymphocytes from stressed wild‐type animals demonstrated a significant increase (p=8.6×10−19) in calprotectin (S100a8 and S100a9) mRNA levels compared to control. Calprotectin is both an intracellular and extracellular protein involved in many inflammatory processes, but has been thought to be granulocyte‐specific making its detection in T‐lymphocytes highly unexpected. We confirmed 40 and 30‐fold increases in S100a8 and S100a9 mRNA, respectively, in purified T‐lymphocytes from stressed animals by qRT‐PCR (p&lt;0.01). Additionally, a 3‐fold increase (p&lt;0.001) in levels of calprotectin protein was observed in circulation of stressed animals. Last, we identified an approximate 2‐fold increase (p&lt;0.05) in calprotectin protein levels in circulation from human patients with PTSD with comorbid hypertension compared to PTSD alone. Taken together, our data suggest a new role for T‐lymphocytes in the regulation of stress‐induced inflammation and hypertension, and present an undescribed protein target for future investigation and potential therapeutic intervention.Support or Funding InformationNIH R00HL123471This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

37 - Psychological stress-mediated hypertension is regulated via redox-signaling in T-lymphocytes

Post-traumatic stress disorder (PTSD) is a debilitating psychological disorder that increases sympathoexcitation, norepinephrine (NE) outflow, and the risk of comorbid diseases such as hypertension by >50%, but the mechanisms remain unclear. Hypertension is regulated in part due to inflammation, and our laboratory previously reported that exposure to NE can modulate levels of T-lymphocyte pro-inflammatory cytokines by redox signaling mechanisms. Therefore, we hypothesized that psychological stress-induced sympathoexcitation leads to a redox-regulated increase in pro-inflammatory cytokine production from T-lymphocytes, which impacts the development of hypertension. Utilizing a mouse-model of PTSD-like psychological stress, increased sympathoexcitation was confirmed by a 2.5 fold increase in splenic NE and 4.1 fold increase in tyrosine hydroxylase content (TH; p

Sex differences in the effect of cannabinoid type 1 receptor deletion on locus coeruleus-norepinephrine neurons and corticotropin releasing factor-mediated responses.

Cannabinoids are capable of modulating mood, arousal, cognition and behavior, in part via their effects on the noradrenergic nucleus locus coeruleus (LC). Dysregulation of LC signaling and norepinephrine (NE) efflux in the medial prefrontal cortex (mPFC) can lead to the development of psychiatric disorders, and CB1r deletion results in alterations of α2- and β1-adrenoceptors in the mPFC, suggestive of increased LC activity. To determine how CB1r deletion alters LC signaling, whole-cell patch-clamp electrophysiology was conducted in LC-NE neurons of male and female wild type (WT) and CB1r-knock out (KO) mice. CB1r deletion caused a significant increase in LC-NE excitability and input resistance in male but not female mice when compared to WT. CB1r deletion also caused adaptations in several indices of noradrenergic function. CB1r/CB2r-KO male mice had a significant increase in cortical NE levels and tyrosine hydroxylase and CRF levels in the LC compared to WT males. CB1r/CB2r-KO female mice showed a significant increase in LC α2-AR levels compared to WT females. To further probe actions of the endocannabinoid system as an anti-stress neuromediator, the effect of CB1r deletion on CRF-induced responses in the LC was investigated. The increase in LC-NE excitability observed in male and female WT mice following CRF (300nM) bath application was not observed in CB1r-KO mice. These results indicate that cellular adaptations following CB1r deletion cause a disruption in LC-NE signaling in males but not females, suggesting underlying sex differences in compensatory mechanisms in KO mice as well as basal endocannabinoid regulation of LC-NE activity.

Abstract 023: Redox-Driven T-Lymphocyte Inflammation Sensitizes Mice to Psychological Stress-Mediated Hypertension

Post-traumatic stress disorder (PTSD) is a debilitating psychological disease that increases sympathoexcitation, norepinephrine (NE) outflow, and the risk of cardiovascular diseases like hypertension by &gt;50%, but the mechanisms remain unclear. Hypertension is regulated in part due to immune system-driven inflammation, and our laboratory has previously reported that exposure to NE can modulate levels of T-lymphocyte pro-inflammatory cytokines by redox signaling mechanisms. Therefore, we hypothesized that psychological stress-induced sympathoexcitation leads to a redox-regulated increase in pro-inflammatory cytokine production from T-lymphocytes, which may impact the development of hypertension. Utilizing a mouse-model of PTSD-like psychological stress ( i.e. social defeat), increased sympathoexcitation was confirmed by a 2.5 fold increase in splenic NE content and 4.1 fold increase in tyrosine hydroxylase (TH; p&lt;0.05). Splenic and peripheral T-lymphocytes demonstrated increased reactive oxygen species (ROS) levels as evidenced by enhanced dihydroethidium and MitoSOX Red oxidation (p&lt;0.05). Increases in ROS were correlated with elevated pro-inflammatory cytokines IL-17A, IL-6, and IL-2 in circulation as well as specifically within T-lymphocytes (p&lt;0.05). Examination of hemodynamics elucidated a progressive increase in mean arterial pressure (15 mmHg, p&lt;0.05) over the stress-induction period, correlating with the elevation of T-lymphocyte driven inflammation. This hypertension was not associated with an increase in circulating renin levels. To investigate the causal contribution of T-lymphocytes in driving this hypertension, recombination activating gene 2 (Rag2) knock-out animals, which are devoid of mature lymphocytes, were utilized. Rag2 -/- mice showed no changes in pro-inflammatory cytokines, a 75% decrease in splenic TH levels (p&lt;0.05), and decreased blood pressure after stress, suggesting T-lymphocytes may regulate the stress-induced hypertension. Overall, our data suggest the potential for a new paradigm involving redox control of T-lymphocytes in the regulation of psychological stress-driven hypertension.

TPA-023 attenuates subchronic phencyclidine-induced declarative and reversal learning deficits via GABAA receptor agonist mechanism: possible therapeutic target for cognitive deficit in schizophrenia

GABAergic drugs are of interest for the treatment of anxiety, depression, bipolar disorder, pain, cognitive impairment associated with schizophrenia (CIAS), and other neuropsychiatric disorders. Some evidence suggests that TPA-023, (7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b] pyridazine), a GABAA α2,3 subtype-selective GABAA partial agonist and α1/5 antagonist, and the neurosteroid, pregnenolone sulfate, a GABAA antagonist, may improve CIAS in pilot clinical trials. The goal of this study was to investigate the effect of TPA-023 in mice after acute or subchronic (sc) treatment with the N-methyl-d-aspartate receptor (NMDAR) antagonist, phencyclidine (PCP), on novel object recognition (NOR), reversal learning (RL), and locomotor activity (LMA) in rodents. Acute TPA-023 significantly reversed scPCP-induced NOR and RL deficits. Co-administration of sub-effective dose (SED) TPA-023 with SEDs of the atypical antipsychotic drug, lurasidone, significantly potentiated the effect of TPA-023 in reversing the scPCP-induced NOR deficit. Further, scTPA-023 co-administration significantly prevented scPCP-induced NOR deficit for 5 weeks. Also, administration of TPA-023 for 7 days following scPCP reversed the NOR deficit for 1 week. However, TPA-023 did not blunt acute PCP-induced hyperactivity, suggesting lack of efficacy as a treatment for psychosis. Systemic TPA-023 significantly blocked lurasidone-induced increases in cortical acetylcholine, dopamine, and glutamate without affecting increases in norepinephrine and with minimal effect on basal release of these neurotransmitters. TPA-023 significantly inhibited PCP-induced cortical and striatal dopamine, serotonin, norepinephrine, and glutamate efflux. These results suggest that TPA-023 and other GABAA agonists may be of benefit to treat CIAS.