Tone Responses Research Articles

Transient Receptor Potential Channels in Vascular Mechanotransduction.

Transmural pressure and shear stress are mechanical forces that profoundly affect the smooth muscle cells (SMCs) comprising the vascular wall and the endothelial cells (ECs) lining the lumen. Pressure and flow are detected by mechanosensors in these cells and translated into appropriate responses to regulate blood pressure and flow. This review focuses on the role of the transient receptor potential (TRP) superfamily of cation channels in this process. We discuss how specific members of the TRP superfamily (TRPC6, TRPM4, TRPV1, TRPV4, and TRPP1) regulate the resting membrane and intracellular Ca2+ levels in SMCs and ECs to promote changes in vascular tone in response to intraluminal pressure and shear stress. Although TRP channels participate in vascular mechanotransduction, little evidence supports their intrinsic mechanosensitivity. Therefore, we also examine the evidence exploring the force-sensitive signal transduction pathways acting upstream of vascular TRP channels. Understanding the interplay between mechanosensors, force-induced signaling cascades, and TRP channels holds promise for the development of targeted therapies for diseases caused by vascular dysfunction.

Developmental encoding of natural sounds in the mouse auditory cortex.

Mice communicate through high-frequency ultrasonic vocalizations, which are crucial for social interactions such as courtship and aggression. Although ultrasonic vocalization representation has been found in adult brain areas along the auditory pathway, including the auditory cortex, no evidence is available on the neuronal representation of ultrasonic vocalizations early in life. Using in vivo two-photon calcium imaging, we analyzed auditory cortex layer 2/3 neuronal responses to USVs, pure tones (4 to 90kHz), and high-frequency modulated sweeps from postnatal day 12 (P12) to P21. We found that ACx neurons are tuned to respond to ultrasonic vocalization syllables as early as P12 to P13, with an increasing number of responsive cells as the mouse age. By P14, while pure tone responses showed a frequency preference, no syllable preference was observed. Additionally, at P14, USVs, pure tones, and modulated sweeps activate clusters of largely nonoverlapping responsive neurons. Finally, we show that while cell correlation decreases with increasing processing of peripheral auditory stimuli, neurons responding to the same stimulus maintain highly correlated spontaneous activity after circuits have attained mature organization, forming neuronal subnetworks sharing similar functional properties.

The combined effects of bilingualism and musicianship on listeners’ perception of non-native lexical tones

Non-native lexical tone perception can be affected by listeners’ musical or linguistic experience, but it remains unclear of whether there will be combined effects and how these impacts will be modulated by different types of non-native tones. This study adopted an orthogonal design with four participant groups, namely, Mandarin-L1 monolinguals and Mandarin-L1 and Cantonese-L2 bilinguals with or without musical training, to investigate effects of bilingualism and musicianship on perception of non-native lexical tones. The closely matched four groups, each encompassing an equal number of 20 participants, attended a modified ABX discrimination task of lexical tones of Teochew, which was unknown to all participants and consists of multiple tone types of level tones, contour tones, and checked tones. The tone perceptual sensitivity index of d’ values and response times were calculated and compared using linear mixed-effects models. Results on tone sensitivity and response time revealed that all groups were more sensitive to contour tones than level tones, indicating the effect of native phonology of Mandarin tones on non-native tone perception. Besides, as compared to monolinguals, bilinguals obtained a higher d’ value when discriminating non-native tones, and musically trained bilinguals responded faster than their non-musician peers. It indicates that bilinguals enjoy a perceptual advantage in non-native tone perception, with musicianship further enhancing this advantage. This extends prior studies by showing that an L2 with a more intricate tone inventory than L1 could facilitate listeners’ non-native tone perception. The pedagogical implications were discussed.

8705 Autoimmune Polyglandular Syndrome Type 2 Presenting As Recurrent Cardiac Tamponade

Abstract Disclosure: E.M. Niedzialkowska: None. D. Shelden: None. Introduction: Autoimmune polyglandular syndrome type 2 (APS2) is a rare endocrinopathy with co-occurrence of autoimmune adrenal insufficiency, autoimmune thyroiditis and/or type 1 diabetes. In rare cases, APS2 can predispose patients to recurrent pericardial effusion and tamponade. Case presentation: 32 y.o male with medical history of hypothyroidism on replacement therapy diagnosed 2 weeks prior to admission presented with progressive dyspnea and hypotension. The patient was found to have pericardial effusion with tamponade requiring pressor support. Initial lab tests showed TSH 148.7 uIU/ml (N 0.4-4.5), free t4 0.7 ng/dl(N 0.7-1.5 ), free t3 2 pg/ml (N 1.7-3.5), Hb a1c 4.7% (N <5.6%), thyroglobulin antibody 851 IU/ml (N <20), TPO antibody >1 000 IU/ml (N<=35), TSI 0.1 IU/l (<0.1), sodium 127 mmol/l (N 135-145), and a negative adrenal antibody panel. The patient was started on IV levothyroxine (400 mcg daily) and stress dose steroids in the setting of shock requiring 4 pressors. Shock resolved after pericardiocentesis and the patient was weaned off pressor support. Pericardial fluid showed neutrophil predominance and was negative for an infectious process, lab tests showed negative ANA, ANCA and GBM. The day following admission lab tests showed TSH of 23.86 uIU/ml, ft4 1.3 ng/dl, the patient was transitioned to oral levothyroxine 125 mcg and the steroid regimen was tapered and discontinued. Follow up tests showed normal TSH and resolution of pericardial effusion. Four weeks after initial admission the patient was readmitted due to hypotension. Laboratory tests showed random cortisol <1.0 ug/dl (N 2.9-19.4), ACTH 17 pg/ml (N 6-48), adrenal antibody titers 1:20 (N < 1:10), cosyntropin stimulation test was positive for adrenal insufficiency and the patient was started on steroid regimen with symptomatic improvement. He was diagnosed with APS type 2 based on his constellation of endocrinopathies. Repeat echocardiogram was negative for pericardial effusion. Two months later, the patient presented with chest pain. Imaging was positive for recurrent pericardial effusion requiring pressor support and pericardiocentesis. The patient was subsequently started on colchicine. Infectious and rheumatologic workup continued to be negative. The patient did not exhibit symptoms of connective tissue disease. Seven months after the initial presentation the patient was readmitted with COVID-19 infection complicated by hypotension and a small pericardial effusion. Symptoms improved after initiation of stress dose steroids. Conclusion: Cardiac tamponade is a rare presentation of APS2 with 9 cases reported in the literature up to date. Increased incidence of pericardial effusion is presumed to be a result of autoimmune inflammation leading to fluid accumulation. The patients are prone to tamponade due to intravascular volume depletion, decreased vascular tone and impaired stress response. Presentation: 6/3/2024

Low frequency ultrasound elicits broad cortical responses inhibited by ketamine in mice

The neuromodulatory effects of >250 kHz ultrasound have been well-demonstrated, but the impact of lower-frequency ultrasound, which can transmit better through air and the skull, on the brain is unclear. This study investigates the biological impact of 40 kHz pulsed ultrasound on the brain using calcium imaging and electrophysiology in mice. Our findings reveal burst duration-dependent neural responses in somatosensory and auditory cortices, resembling responses to 12 kHz audible tone, in vivo. In vitro brain slice experiments show no neural responses to 300 kPa 40 kHz ultrasound, implying indirect network effects. Ketamine fully blocks neural responses to ultrasound in both cortices but only partially affects 12 kHz audible tone responses in the somatosensory cortex and has no impact on auditory cortex 12 kHz responses. This suggests that low-frequency ultrasound’s cortical effects rely heavily on NMDA receptors and may involve mechanisms beyond indirect auditory cortex activation. This research uncovers potential low-frequency ultrasound effects and mechanisms in the brain, offering a path for future neuromodulation.

The Role of Endothelial Cell Mitophagy in Age-Related Cardiovascular Diseases.

Aging is a major risk factor for cardiovascular diseases (CVD), and mitochondrial autophagy impairment is considered a significant physiological change associated with aging. Endothelial cells play a crucial role in maintaining vascular homeostasis and function, participating in various physiological processes such as regulating vascular tone, coagulation, angiogenesis, and inflammatory responses. As aging progresses, mitochondrial autophagy impairment in endothelial cells worsens, leading to the development of numerous cardiovascular diseases. Therefore, regulating mitochondrial autophagy in endothelial cells is vital for preventing and treating age-related cardiovascular diseases. However, there is currently a lack of systematic reviews in this area. To address this gap, we have written this review to provide new research and therapeutic strategies for managing aging and age-related cardiovascular diseases.

Bile Acids as Emerging Players at the Intersection of Steatotic Liver Disease and Cardiovascular Diseases.

Affecting approximately 25% of the global population, steatotic liver disease (SLD) poses a significant health concern. SLD ranges from simple steatosis to metabolic dysfunction-associated steatohepatitis and fibrosis with a risk of severe liver complications such as cirrhosis and hepatocellular carcinoma. SLD is associated with obesity, atherogenic dyslipidaemia, and insulin resistance, increasing cardiovascular risks. As such, identifying SLD is vital for cardiovascular disease (CVD) prevention and treatment. Bile acids (BAs) have critical roles in lipid digestion and are signalling molecules regulating glucose and lipid metabolism and influencing gut microbiota balance. BAs have been identified as critical mediators in cardiovascular health, influencing vascular tone, cholesterol homeostasis, and inflammatory responses. The cardio-protective or harmful effects of BAs depend on their concentration and composition in circulation. The effects of certain BAs occur through the activation of a group of receptors, which reduce atherosclerosis and modulate cardiac functions. Thus, manipulating BA receptors could offer new avenues for treating not only liver diseases but also CVDs linked to metabolic dysfunctions. In conclusion, this review discusses the intricate interplay between BAs, metabolic pathways, and hepatic and extrahepatic diseases. We also highlight the necessity for further research to improve our understanding of how modifying BA characteristics affects or ameliorates disease.

Patients with uncontrolled hypertension subjected to cardiopulmonary bypass have altered coronary vasomotor responses to serotonin

BackgroundWe previously found that cardioplegic arrest and cardiopulmonary bypass are associated with altered coronary arteriolar response to serotonin in patients undergoing cardiac surgery. In this study, we investigated the effects of hypertension on coronary microvascular vasomotor tone in response to serotonin and alterations in serotonin receptor protein expression in the setting of cardioplegic arrest and cardiopulmonary bypass. MethodsCoronary arterioles were dissected from harvested pre– and post–cardioplegic arrest and cardiopulmonary bypass right atrial tissue samples of patients undergoing cardiac surgery with normotension, well-controlled hypertension, and uncontrolled hypertension. Vasomotor tone was assessed by video-myography, and protein expression was measured with immunoblotting. ResultsPre–cardioplegic arrest and cardiopulmonary bypass, serotonin induced moderate relaxation responses of coronary arterioles in normotension and well-controlled hypertension patients, whereas serotonin caused moderate contractile responses in uncontrolled hypertension patients. Post–cardioplegic arrest and cardiopulmonary bypass, serotonin caused contractile responses of coronary arterioles in all 3 groups. The post–cardioplegic arrest and cardiopulmonary bypass contractile response to serotonin was significantly higher in the uncontrolled hypertension group compared with the normotension or well-controlled hypertension groups (P < .05). Pre–cardioplegic arrest and cardiopulmonary bypass, expression of the serotonin 1A receptor was significantly lower in the uncontrolled hypertension group compared with the well-controlled hypertension and normotension groups (P = .01 and P < .001). Serotonin 1B receptor expression was higher in the uncontrolled hypertension group compared with the normotension or well-controlled hypertension groups post–cardioplegic arrest and cardiopulmonary bypass (P = .03 and P = .046). ConclusionUncontrolled hypertension is associated with an increased coronary contractile response of coronary microvessels to serotonin and altered serotonin receptor protein expression after cardioplegic arrest and cardiopulmonary bypass. These findings may contribute to a worse postoperative coronary spasm and worsened recovery of coronary perfusion in patients with uncontrolled hypertension after cardioplegic arrest and cardiopulmonary bypass and cardiac surgery.

A Magnetic Particle Imaging Approach for Minimally Invasive Imaging and Sensing With Implantable Bioelectronic Circuits.

Minimally-invasive and biocompatible implantable bioelectronic circuits are used for long-term monitoring of physiological processes in the body. However, there is a lack of methods that can cheaply and conveniently image the device within the body while simultaneously extracting sensor information. Magnetic Particle Imaging (MPI) with zero background signal, high contrast, and high sensitivity with quantitative images is ideal for this challenge because the magnetic signal is not absorbed with increasing tissue depth and incurs no radiation dose. We show how to easily modify common implantable devices to be imaged by MPI by encapsulating and magnetically-coupling magnetic nanoparticles (SPIOs) to the device circuit. These modified implantable devices not only provide spatial information via MPI, but also couple to our handheld MPI reader to transmit sensor information by modulating harmonic signals from magnetic nanoparticles via switching or frequency-shifting with resistive or capacitive sensors. This paper provides proof-of-concept of an optimized MPI imaging technique for implantable devices to extract spatial information as well as other information transmitted by the implanted circuit (such as biosensing) via encoding in the magnetic particle spectrum. The 4D images present 3D position and a changing color tone in response to a variable biometric. Biophysical sensing via bioelectronic circuits that take advantage of the unique imaging properties of MPI may enable a wide range of minimally invasive applications in biomedicine and diagnosis.

First Real-Life Data on the Diving Response in Healthy Children.

Swimming and diving are popular recreational activities, representing an effective option in maintaining and improving cardiovascular fitness in healthy people. To date, only little is known about the cardiovascular adaption to submersion in children. This study was conducted to improve an understanding thereof. We used a stepwise apnea protocol with apnea at rest, apnea with facial immersion, and at last apnea during whole body submersion. Continuous measurement of heart rate, oxygen saturation, and peripheral resistance index was done. Physiologic data and analysis of influencing factors on heart rate, oxygen saturation, and peripheral vascular tone response are reported. The current study presents the first data of physiologic diving response in children. Data showed that facial or whole body submersion leads to a major drop in heart rate, and increase of peripheral resistance, while the oxygen saturation seems to be unaffected by static apnea in most children, with apnea times of up to 75s without change in oxygen saturation.

Effects of Rhythm and Accent Patterns on Tempo-Keeping Property of Finger Tapping

Tempo of music performance is often accelerated irrespective of players’ intention. Though the characteristics of the tempo deviation phenomenon have been investigated using a finger-tapping task, most such studies dealt with tapping with a fixed interval and intensity; few studies considered the effects of rhythm and accent, both important factors of music performance. Here, we asked how different rhythm and accent patterns affected the tempo-keeping property using a synchronization-continuation task paradigm: Participants were asked to keep tapping with reproducing the given rhythm/accent patterns designated by the target tones. Tapping tempo was significantly deviated depending on rhythm/accent patterns, but their magnitudes were only several percent in 150 s, much smaller than those in real music performance. We also ran experiments in the conditions that participants need not reproduce the accent patterns but only response tones were accented. These auditory modulations affected the tempo deviation, implying that perceptual processes induced by the auditory feedback also influence the tempo maintenance. Inter-tap intervals (ITIs) were modulated by the accent pattern, but no clear relationship was observed between ITI fluctuations and long-term tempo deviation, suggesting that different mechanisms are involved. In sum, the present finding shows that non-uniform motor actions required for music articulation can disturb the long-term tempo maintenance but their effect is rather small. We also reported the tempo deviation when the response tone was modulated by loudness (crescendo vs. diminuendo) or pitch (ascending vs. descending).

Breathing Against the Conducting Gesture? The Effect of Congruent and Incongruent Gesture-Task Combinations on Breathing Behavior and Resulting Loudness of Choral Singers

Previous research suggests that the conductor's choice of preparatory gesture affects choral singers’ breathing behavior as well as the corresponding loudness and sound quality. As these gestures are in choral practice often combined with instructions on the desired breathing technique, the aim of this study was to explore whether congruent and incongruent combinations of preparatory gestures with breathing instructions affect the breathing behavior and resulting loudness in choral singers. In our within-subjects study design, 18 healthy choral singers were asked to sing a tone in response to 4 different video stimuli, consisting of two congruent and two incongruent gesture-instruction combinations. We recorded chest wall kinematics via three-dimensional motion capture and voice samples. The results show that the used preparatory gesture has an influence on the predetermined inhalation type. Particularly, the most common inward-upward gesture combined with an abdominal inhalation results in a significantly reduced chest wall expansion and loudness of the resulting tone. Furthermore, the moment of maximum inhalation and onset are delayed after inward-upward gestures. Consequently, in choral practice, it is essential to generally consider that the selected preparatory gesture has significant influence on the resulting sound and should therefore be chosen according to the desired type of inhalation.

Anesthetic efficacy of dexmedetomidine-ketamine in eastern box turtles (Terrapene carolina carolina) is enhanced with the addition of midazolam and when administered in the forelimb versus the hindlimb.

To compare dexmedetomidine-ketamine (DK; 0.1 and 10 mg/kg, respectively) with midazolam (M; 1.0 mg/kg) or 0.9% sodium chloride (S; 0.2 mL/kg) administered IM in the forelimb (F) or hindlimb (H) in eastern box turtles (Terrapene carolina carolina). 20 clinically healthy, captive adult eastern box turtles. In a randomized, blinded, complete crossover study with 1-week washout periods, turtles were administered each of 3 treatments: F-DKS, F-DKM, or H-DKM. Palpebral reflex, muscle tone, and withdrawal responses were serially assessed and used to calculate cumulative sedation scores at each 5-minute time point. The ability to intubate was evaluated. At 60 minutes, atipamezole (1.0 mg/kg) and either flumazenil (F-DKM, H-DKM; 0.05 mg/kg) or 0.9% sodium chloride (F-DKS; 0.5 mL/kg) were administered IM. All treatments resulted in clinically relevant anesthetic effects. F-DKM produced significantly higher sedation scores than H-DKM or F-DKS at all time points between 10 and 60 minutes (P < .05). Sedation score variability was observed with all treatments with significantly higher variability for H-DKM (P < .05). Intubation was successful in 32, 89, and 11% of turtles in F-DKS, F-DKM, and H-DKM, respectively. Median (range) recovery time was 10 (5-22), 16 (7-45), and 12 (4-28) minutes for F-DKS, F-DKM, and H-DKM, respectively. In eastern box turtles, forelimb dexmedetomidine-ketamine resulted in clinically relevant anesthetic effects that were heightened with the addition of midazolam. Hindlimb administration of midazolam-dexmedetomidine-ketamine resulted in reduced and more variable anesthetic effects compared to forelimb administration, supporting a hepatic first-pass effect.

Change detection in the primate auditory cortex through feedback of prediction error signals

Although cortical feedback signals are essential for modulating feedforward processing, no feedback error signal across hierarchical cortical areas has been reported. Here, we observed such a signal in the auditory cortex of awake common marmoset during an oddball paradigm to induce auditory duration mismatch negativity. Prediction errors to a deviant tone presentation were generated as offset calcium responses of layer 2/3 neurons in the rostral parabelt (RPB) of higher-order auditory cortex, while responses to non-deviant tones were strongly suppressed. Within several hundred milliseconds, the error signals propagated broadly into layer 1 of the primary auditory cortex (A1) and accumulated locally on top of incoming auditory signals. Blockade of RPB activity prevented deviance detection in A1. Optogenetic activation of RPB following tone presentation nonlinearly enhanced A1 tone response. Thus, the feedback error signal is critical for automatic detection of unpredicted stimuli in physiological auditory processing and may serve as backpropagation-like learning.

Salt-sensitive trait of normotensive individuals is associated with altered autonomous cardiac regulation: a randomized controlled intervention study.

Blood pressure (BP) responses to sodium intake show great variation, discriminating salt-sensitive (SS) from salt-resistant (SR) individuals. The pathophysiology behind salt sensitivity is still not fully elucidated. We aimed to investigate salt-induced effects on body fluid, vascular tone, and autonomic cardiac response with regard to BP change in healthy normotensive individuals. We performed a randomized crossover study in 51 normotensive individuals with normal body mass index and estimated glomerular filtration rate. Subjects followed both a low-Na+ diet (LSD, <50 mmol/day) and a high-Na+ diet (HSD, >200 mmol/day). Cardiac output, systemic vascular resistance (SVR), and cardiac autonomous activity, through heart rate variability and cross-correlation baroreflex sensitivity (xBRS), were assessed with noninvasive continuous finger BP measurements. In a subset, extracellular volume (ECV) was assessed by iohexol measurements. Subjects were characterized as SS if mean arterial pressure (MAP) increased ≥3 mmHg after HSD. After HSD, SS subjects (25%) showed a 6.1-mmHg (SD 1.9) increase in MAP. No differences between SS and SR in body weight, cardiac output, or ECV were found. SVR was positively correlated with Delta BP (r = 0.31, P = 0.03). xBRS and heart rate variability were significantly higher in SS participants compared to SR participants after both HSD and LSD. Sodium loading did not alter heart rate variability within groups. Salt sensitivity in normotensive individuals is associated with an inability to decrease SVR upon high salt intake that is accompanied by alterations in autonomous cardiac regulation, as reflected by decreased xBRS and heart rate variability. No discriminatory changes upon high salt were observed among salt-sensitive individuals in body weight and ECV.NEW & NOTEWORTHY Extracellular fluid expansion in normotensive individuals after salt loading is present in both salt-sensitive and salt-resistant individuals and is not discriminatory to the blood pressure response to sodium loading in a steady-state measurement. In normotensive subjects, the ability to sufficiently vasodilate seems to play a pivotal role in salt sensitivity. In a normotensive cohort, differences in sympathovagal balance are also present in low-salt conditions rather than being affected by salt loading. Whereas treatment and prevention of salt-sensitive blood pressure increase are mostly focused on renal sodium handling and extracellular volume regulation, our study suggests that an inability to adequately vasodilate and altered autonomous cardiac functioning are additional key players in the pathophysiology of salt-sensitive blood pressure increase.

Pulmonary Arterial Hypertension Attenuates Vasoconstrictor Responses Caused by Activation of Alpha-1-Adrenoreceptors in the Systemic Circulation

Pulmonary arterial hypertension (PAH) accompanied by an arterial pressure increase in the pulmonary circulation, remodeling of pulmonary arteries and a change in its sensitivity to regulatory factors; PAH is accompanied by activation of the sympathetic nervous system and renin-angiotensin-aldosterone system and increased production of atrial natriuretic peptide. The change in the sensitivity of the vessels of the systemic circulation (SC) to regulatory influences in PAH has not been investigated. Vasoconstrictor reactions in SC with monocrotaline (MCT) were studied in the work models of PAH in rats (Wistar, 350 ± 50 g, 4 months). Mean arterial pressure (MAP) was recorded against the background of a double autonomous blockade with the administration of the α1-adrenergic receptor agonist (α1-AR) phenylephrine (PE) to conscious rats at the start of experiment, then 2 and 4 weeks after the induction of PAH with MCT or saline injection for control animals. Registration of MAP under the action of PE was also performed during angiotensin-II (ATII) infusion. The maximal amplitude (Amax) of the change in MAP and the longest half-return time of MAP (T∆MAP1/2) to the baseline level in rats in response to the Phe injection were estimated. It was found that in response to PE, Amah did not change in rats with PAH, whereas in control animals it significantly increased. In rats with PAH 2 (n = 6) and 4 weeks after the induction of PAH with MCT, T∆MAP1/2 is significantly less than in control rats. ATII leads to delayed changes in T∆MAP1/2 in both control rats and rats with PAH. In rats with MAP, the potentiation with angiotensin T∆MAP1/2 is significantly less than in control rats. Thus, in animals with PAH, the ability of the resistive arteries of the systemic circulation to maintain tone in response to the activation of α1-AR decreases. In addition, PAH suppresses the ability of ATII to stimulate sympathetic responses in the SC. Firstly, in vivo, it has been demonstrated remodeling and changing the functional state of the pulmonary circulation leads to changes in the regulation of vascular tone of the systemic circulation.

ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

Pulmonary arterial (PA) smooth muscle cells (PASMC) generate vascular tone in response to agonists coupled to Gq-protein receptor signaling. Such agonists stimulate oscillating calcium waves, the frequency of which drives the strength of contraction. These Ca2+ events are modulated by a variety of ion channels including voltage-gated calcium channels (CaV1.2), the Tmem16a or Anoctamin-1 (ANO1)-encoded calcium-activated chloride (CaCC) channel, and Ca2+ release from the sarcoplasmic reticulum through inositol-trisphosphate receptors (IP3R). Although these calcium events have been characterized, it is unclear how these calcium oscillations underly a sustained contraction in these muscle cells. We used smooth muscle-specific ablation of ANO1 and pharmacological tools to establish the role of ANO1, CaV1.2, and IP3R in the contractile and intracellular Ca2+ signaling properties of mouse PA smooth muscle expressing the Ca2+ biosensor GCaMP3 or GCaMP6. Pharmacological block or genetic ablation of ANO1 or inhibition of CaV1.2 or IP3R, or Ca2+ store depletion equally inhibited 5-HT-induced tone and intracellular Ca2+ waves. Coimmunoprecipitation experiments showed that an anti-ANO1 antibody was able to pull down both CaV1.2 and IP3R. Confocal and superresolution nanomicroscopy showed that ANO1 coassembles with both CaV1.2 and IP3R at or near the plasma membrane of PASMC from wild-type mice. We conclude that the stable 5-HT-induced PA contraction results from the integration of stochastic and localized Ca2+ events supported by a microenvironment comprising ANO1, CaV1.2, and IP3R. In this model, ANO1 and CaV1.2 would indirectly support cyclical Ca2+ release events from IP3R and propagation of intracellular Ca2+ waves.

Abstract P235: The Absence Of Inhibitory G Protein Regulation By Rgs2 Increases Basal Phosphodiesterase Activity To Decrease Forskolin-stimulated Camp Generation And Enhances Myogenic Response In Uterine Arteries Of Non-pregnant Mice

G protein regulation by regulators of G protein signaling (RGS) proteins is crucial to maintaining normal vascular tone for promoting uterine vascular bed perfusion. However, the specific regulatory mechanisms involved are incompletely understood. Previously, we showed that loss of regulation of Gi/o and Gq/11 by RGS2 contributes to enhanced myogenic tone and decreased uterine blood flow in non-pregnant mice. Similarly, RGS5, closely related to RGS2, was found to fine-tune G protein signaling and modulate GPCR-evoked vascular reactivity. RGS2 and RGS5 are co-expressed in the uterine vascular bed. However, it is unclear whether their regulatory activities are coordinated in fine-tuning G protein signaling to modulate vascular tone. Here, we tested whether the combined activity of RGS2 and 5 reduces myogenic tone by negatively regulating Gi/o signaling to promote cAMP-dependent uterine artery relaxation. We used commercially available kits to measure forskolin-stimulated cAMP generation and basal phosphodiesterase (PDE) activity in freshly isolated uterine arteries from non-pregnant wild type, Rgs5 KO and Rgs2 KO mice. Basal tone and myogenic response to increasing intraluminal pressure were assessed using ex vivo pressure myography. We found that the absence of RGS2 but not RGS5 decreased forskolin-stimulated cAMP generation. Conversely, basal PDE activity was augmented in both Rgs2 KO and Rgs5 KO arteries, though the increase was less robust in Rgs5 KO arteries. Basal tone and myogenic response were reduced in Rgs5 KO arteries compared to WT or Rgs2 KO arteries in the absence or presence of nitric oxide synthase blockade with L-NAME. Activating Gi/o with dopamine increased myogenic constriction in all genotypes but less robustly in Rgs5 KO arteries, while pre-incubation of the vessels with exogenous cAMP or PDE inhibition with IBMX normalized maximal myogenic response to similar levels in all groups. Collectively, these findings indicate that altered Gi/o regulation due to RGS2 absence enhances myogenic tone by promoting PDE-mediated inhibition of cAMP-dependent vasodilation. While both RGS2 and RGS5 negatively regulate this novel Gi/o-PDE-cAMP signaling pathway, RGS5 dampens the inhibitory efficacy of RGS2 towards Gi/o in uterine arteries.

Statistical Information Affects Spoken Word Recognition of Tone Languages in Stutterers: Evidence From an Auditory-Perceptual Gating Study.

Previous studies have shown that individuals who stutter exhibit abnormal speech perception in addition to disfluent production as compared with their nonstuttering peers. This study investigated whether adult Chinese-speaking stutterers are still able to use knowledge of statistical regularities embedded in their native language to recognize spoken words and, if so, how much acoustic information is needed to trigger this information. Seventeen stutterers and 20 typical, nonstuttering controls participated in a gating experiment. All participants listened to monosyllabic words that consisted of syllables and lexical tones and were segmented into eight successive gates. These words differed in syllable token frequency and syllable-tone co-occurrence probability in line with a Chinese spoken word corpus. The correct syllable-only, correct tone-only, correct syllable-tone word, and correct syllable-incorrect tone responses were analyzed between the two groups using mixed-effects models. Stutterers were less accurate overall than controls, with fewer correct syllables, tones, and their combination as words. However, stutterers showed consistent and reliable perceptual patterns triggered by statistical information of speech, as reflected by more accurate responses to high-frequency syllables, high-probability tones, and tone errors all in manners similar to those of nonstuttering controls. Stutterers' atypical speech perception is not due to a lack of statistical learning. Stutterers were able to perceive spoken words with phonological tones based on statistical regularities embedded in their native speech. This finding echoes previous production studies of stuttering and lends some support for a link between perception and production. Implications of pathological, diagnostic, and therapeutic conditions of stuttering are discussed.

Abstract P1001: Comprehensive “Tool Box” For Delineating Adrenergic Receptor Function

Sympathetic regulation of the cardiovascular system is required to maintain cardiac output and vascular tone in response to varied physiological stressors. This includes the release norepinepherine and epinephrine. Nor/epinephrine acts on nine, evolutionarily conserved, adrenergic, G-protein coupled receptors. Three alpha 1 ( Adra1 ), 3 alpha 2 ( Adra2 ), and 3 beta ( Adrb ) receptors. Understanding the distribution and cell-specific function of each of these receptors may lead to new targeted therapies for cardiovascular disease. To delineate the relative roles of each of the 9 adrenergic receptors, we are taking a genetic approach to study these receptors in mice; specifically, by using the Cre-loxP system to target the varied receptors in a tissue-specific manner. Unfortunately, there is a dearth of such tools to study these receptors. We therefore are in the process of generating three sets of tools for each of the 9 adrenergic receptors using the CRISPR-Cas9 method. 1. Adrenergic receptor floxed mice. 2. Adrenergic receptor reactivation (transcriptional blocking) mice and 3. Adrenergic receptor Cre mice. Thus far, we have generated Adra1a , Adra1b , Adra1d , Adra2a , Adrb2 , and Adrb3 floxed mice by inserting loxP sites flanking at least one exon near the 5’ end of the gene. The floxed mice were crossed to a germline-Cre (CMV-Cre), generating mice with whole-body loss of function alleles. Complementing the conditional loss of function alleles, we are also generating reactivation alleles by inserting a loxP-flanked transcriptional blocking cassette (TB) in each of the 9 endogenous adrenergic receptor genes. These TB alleles will allow us to reactivate endogenous expression of each adrenergic receptor in a Cre-dependent manner in an otherwise global knockout background. This model will allow us to identify tissues which are sufficient for the function of each adrenergic receptor. Finally, the ires-Cre models will allow for targeting of each adrenergic receptor-expressing cells with genetic reporters, and physiological studies using chemo and/or optogenetics. These new tools will assist in identifying the site of action and physiological function of various receptor subtypes and provide mechanistic insights into adrenergic receptor signaling.