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Meredith Hay - One of the best experts on this subject based on the ideXlab platform.
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cellular mechanisms regulating synaptic vesicle exocytosis and endocytosis in Aortic Baroreceptor neurons
Annals of the New York Academy of Sciences, 2006Co-Authors: Meredith Hay, Caroline J Hoang, Jaya PamidimukkalaAbstract:: The purpose of this chapter is to review some of the recent progress in the understanding of the cellular and biophysical mechanisms that are involved in the regulation of arterial Baroreceptor neurotransmssion. Synaptic depression or fatigue following repeated neuronal stimulation has been shown at central Baroreceptor synapses in vivo and in vitro. As most of the central neurons have a limited number of vesicles, vesicle retrieval or endocytosis following exocytosis is thought to play a major role in preserving synaptic transmission. We have hypothesized that central Baroreceptor terminals may inhibit their own synaptic transmission via feedback activation of presynaptic metabotropic glutamate receptors (mGluRs). We have analyzed the effects of mGluR autoreceptors (group III mGluRs) on voltage-gated calcium channels using standard patch-clamp techniques and on the process of exocytosis and endocytosis in Aortic Baroreceptor neurons using the quantitative imaging dye FM1–43 and FM2–10. Usng the whole-cell patch-clamp technique, we have found that activation of group III mGluRs with L-AP4 inhibits peak calcium channel current. Furthermore, activation of group III mGluRs with L-AP4 markedly decreases stimulation-induced exocytosis in Aortic Baroreceptor neurons, as measured with FM1–43, and inhibits synapsin I phosphorylation. These results suggest that activation of group III mGluRs may inhibit synaptic transmission by (1) inhibiting calcium influx, (2) decreasing synaptic vesicle exocytosis, and (3) modulating the mechanisms governing synaptic vesicle recovery and endocytosis. These effects of mGluRs on Baroreceptor synaptic vesicles may contribute to the Baroreceptor/nucleus tractus solitarius synaptic depression observed in vivo.
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frequency dependence of synaptic vesicle exocytosis in Aortic Baroreceptor neurons and the role of group iii mglurs
Brain Research, 2004Co-Authors: Jaya Pamidimukkala, Meredith HayAbstract:Abstract Synaptic transmission between Baroreceptor afferents and the nucleus tractus solitarius (NTS) is essential for reflex regulation of blood pressure. High frequency stimulation of the afferents in vivo leads to a decrease in synaptic strength and is generally attributed to reduction in presynaptic neurotransmitter release. It has been hypothesized that during high frequency stimulation glutamate a major neurotransmitter at the Baroreceptor afferent terminals inhibits its own release via presynaptic group III metabotropic glutamate receptors (mGluRs). A key player in modulation of presynaptic release is vesicle exocytosis. The present study utilized cultured Aortic Baroreceptor neurons and the styryl dye FM2–10 to characterize (1) the dependence of exocytosis at these afferent nerve terminals on the frequency of neuronal activation, (2) the effect of duration of stimulation on the rate of exocytosis and (3) the role of mGluRs in the frequency-dependent modulation of exocytosis. Destaining in the FM2–10 loaded boutons during 3 min of stimulation, a measure of exocytosis, progressively decreased with increasing frequency (0.5, 1.0 and 10 Hz). Blockade of group III mGluRs with 300 μM (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG) facilitated exocytosis evoked by 10 Hz stimulation but not at 0.5 Hz. The data suggest that Aortic Baroreceptor terminals exhibit frequency-dependent depression of exocytosis and support a role for group III mGluRs in the frequency-dependent modulation of exocytosis.
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frequency dependence of endocytosis in Aortic Baroreceptor neurons and role of group iii mglurs
American Journal of Physiology-heart and Circulatory Physiology, 2001Co-Authors: Jaya Pamidimukkala, Meredith HayAbstract:Synaptic transmission between Baroreceptor afferents and the nucleus tractus solitarius (NTS) is known to exhibit frequency-dependent depression. Reductions in neurotransmitter release and alterations in mechanisms regulating synaptic transmission are hypothesized to be involved in the activity-dependent depression observed in Baroreceptor afferent neurons. The present study utilized cultured Aortic Baroreceptor neurons and the fluorescent dyes FM1-43 and FM2-10 to characterize the process of endocytosis or vesicle retrieval and its dependence on 1) frequency of neuronal activation, 2) metabotropic glutamate receptor (mGluR) activation, and 3) calcium concentrations inside and outside the cell. Endocytosis per spike, measured in fluorescence units after a 10-s stimulus applied at frequencies of 0.5 (53 +/- 4), 1.0 (23 +/- 1), and 10.0 Hz (2.7 +/- 0.2), was significantly depressed at higher frequencies. Blockade of group III mGluRs with (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG) facilitated endocytosis at all frequencies, suggesting that this receptor subtype may be involved in the inhibition of endocytosis. Manipulating the extracellular and intracellular calcium concentrations subsequent to exocytosis had no effect on endocytosis. These results suggest that frequency-dependent depression of endocytosis observed in vitro could contribute to the frequency-dependent depression of Baroreceptor afferent neurotransmission and that group III mGluRs inhibit endocytosis.
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measurement of synaptic vesicle exocytosis in Aortic Baroreceptor neurons
American Journal of Physiology-heart and Circulatory Physiology, 1998Co-Authors: Meredith Hay, Eileen M HasserAbstract:The purpose of this study was to evaluate the use of the fluorescent membrane label FM1–43 as a measure of synaptic terminal exocytosis during stimulation of labeled Aortic Baroreceptor and unlabel...
Robert L Muelleman - One of the best experts on this subject based on the ideXlab platform.
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altered enac is associated with Aortic Baroreceptor dysfunction in chronic heart failure
American Journal of Hypertension, 2016Co-Authors: Dongze Zhang, Robert L MuellemanAbstract:Background Abnormal Baroreceptor function contributes to attenuated arterial baroreflex sensitivity in chronic heart failure (CHF). As a mechanosensor in mammalian nonepithelium, the epithelial sodium channel (ENaC) is an amiloride-sensitive and voltage-independent ion channel. The ENaC is thought to be a component of Baroreceptor mechanosensitive ion channels in Aortic Baroreceptor cell bodies and nerve terminals. In this study, therefore, we measured the expression and activation of the ENaC in nodose neuronal cell bodies and Aortic Baroreceptor nerve terminals in sham and CHF rats. Methods and results CHF was induced by surgical ligation of left coronary artery. The development of CHF was confirmed by hemodynamic and morphological characteristics. The Aortic Baroreceptor sensitivity was blunted in anesthetized CHF rats, compared with that in sham rats. The data from immunostaining and western blot analysis showed that the protein of β- and γ-ENaC subunits was expressed in nodose neuronal cell bodies and Aortic Baroreceptor nerve terminals, whereas the protein of α-ENaC subunit was undetectable. CHF reduced protein expression of β- and γ-ENaC subunits in nodose neuronal cell bodies and Aortic Baroreceptor nerve terminals. Additionally, the data recorded by the whole cell patch-clamp technique demonstrated that ENaC currents in Aortic Baroreceptor neurons were lower in CHF rats than that in sham rats. Conclusion These results suggest that reduced protein expression of the ENaC decreases the ENaC activation, which could be involved in attenuation of the Aortic Baroreceptor sensitivity in the CHF state. Baroreceptors should be a potential therapeutic target for reducing mortality in CHF.
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effect of angiotensin ii on voltage gated sodium currents in Aortic Baroreceptor neurons and arterial baroreflex sensitivity in heart failure rats
Journal of Hypertension, 2015Co-Authors: Dongze Zhang, Hong Zheng, Jinxu Liu, Robert L MuellemanAbstract:Background Impairment of arterial baroreflex sensitivity is associated with mortality in patients with chronic heart failure (CHF). Elevation of plasma angiotension II (Ang II) contributes to arterial baroreflex dysfunction in CHF. A reduced number of voltage-gated sodium (Nav) channels in Aortic Baroreceptor neurons are involved in CHF-blunted arterial baroreflex. Method In this study, we investigated acute effect of Ang II on Nav currents in the Aortic Baroreceptor neuron and on arterial baroreflex in sham and coronary artery ligation-induced CHF rats. Results Using Ang II I radioimmunoassay, real-time reverse transcription-PCR and western blot, we found that Ang II levels, and mRNA and protein expression of angiotension II type 1 receptor in nodose ganglia from CHF rats were higher than that from sham rats. Local microinjection of Ang II (0.2 nmol) into the nodose ganglia decreased the arterial baroreflex sensitivity in sham rats, whereas losartan (1 nmol, an angiotension II type 1 receptor antagonist) improved the arterial baroreflex sensitivity in CHF rats. Data from patch-clamp recording showed that Ang II (100 nmol/l) acutely inhibited Nav currents in the Aortic Baroreceptor neurons from sham and CHF rats. In particular, inhibitory effect of Ang II on Nav currents in the Aortic Baroreceptor neurons was larger in CHF rats than that in sham rats. Losartan (1 μmol/l) totally abolished the inhibitory effect of Ang II on Nav currents in sham and CHF Aortic Baroreceptor neurons. Conclusion These results suggest that elevation of endogenous Ang II in the nodose ganglia contributes to impairment of the arterial baroreflex function in CHF rats through inhibiting Nav channels.
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Angiotensin II-superoxide-NFκB Signaling and Aortic Baroreceptor Dysfunction in Chronic Heart Failure
Frontiers Media S.A., 2015Co-Authors: Dongze Ezhang, Robert L Muelleman, Yulong EliAbstract:Chronic heart failure (CHF) affects approximately 5.7 million people in the United States. Increasing evidence from both clinical and experimental studies indicates that the sensitivity of arterial baroreflex is blunted in the CHF state, which is a predictive risk factor for sudden cardiac death. Normally, the arterial baroreflex regulates blood pressure and heart rate through sensing mechanical alteration of arterial vascular walls by Baroreceptor terminals in the Aortic arch and carotid sinus. There are Aortic Baroreceptor neurons in the nodose ganglion (NG), which serve as the main afferent component of the arterial baroreflex. Functional changes of Baroreceptor neurons are involved in the arterial baroreflex dysfunction in CHF. In the CHF state, circulating angiotensin II (Ang II) and local Ang II concentration in the NG are elevated, and AT1R mRNA and protein are overexpressed in the NG. Additionally, Ang II-superoxide-NFκB signaling pathway regulates the neuronal excitability of Aortic Baroreceptors through influencing the expression and activation of Nav channels in Aortic Baroreceptors, and subsequently causes the impairment of the arterial baroreflex in CHF. These new findings provide a basis for potential pharmacological interventions for the improvement of the arterial baroreflex sensitivity in the CHF state. This review summarizes the mechanisms responsible for the arterial baroreflex dysfunction in CHF
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in vivo transfection of manganese superoxide dismutase gene or nuclear factor κb shrna in nodose ganglia improves Aortic Baroreceptor function in heart failure rats
Hypertension, 2014Co-Authors: Dongze Zhang, Robert L Muelleman, Jinxu Liu, Kurtis G CornishAbstract:Arterial baroreflex sensitivity is attenuated in chronic heart failure (CHF) state, which is associated with cardiac arrhythmias and sudden cardiac death in patients with CHF. Our previous study showed that CHF-induced sodium channel dysfunction in the Baroreceptor neurons was involved in the blunted baroreflex sensitivity in CHF rats. Mitochondria-derived superoxide overproduction decreased expression and activation of the sodium channels in the Baroreceptor neurons from CHF rats. However, the molecular mechanisms responsible for the sodium channel dysfunction in the Baroreceptor neurons from CHF rats remain unknown. We tested the involvement of nuclear factor κB (NFκB) in the sodium channel dysfunction and evaluated the effects of in vivo transfection of manganese superoxide dismutase gene and NFκB shRNA on the baroreflex function in CHF rats. CHF was developed at 6 to 8 weeks after left coronary artery ligation in adult rats. Western blot and chromatin immunoprecipitation data showed that phosphorylated NFκB p65 and ability of NFκB p65 binding to the sodium channel promoter were increased in the nodose ganglia from CHF rats. In vivo transfection of adenoviral manganese superoxide dismutase gene or lentiviral NFκB p65 shRNA into the nodose ganglia partially reversed CHF-reduced sodium channel expression and cell excitability in the Baroreceptor neurons and improved CHF-blunted arterial baroreflex sensitivity. Additionally, transfection of adenoviral manganese superoxide dismutase also inhibited the augmentation of phosphorylated NFκB p65 in the nodose neurons from CHF rats. The present study suggests that superoxide-NFκB signaling contributes to CHF-induced Baroreceptor dysfunction and resultant impairment of baroreflex function.
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blunted excitability of Aortic Baroreceptor neurons in diabetic rats involvement of hyperpolarization activated channel
Cardiovascular Research, 2008Co-Authors: Thai P Tran, Robert L Muelleman, Harold D SchultzAbstract:Aims Although dysfunction of arterial baroreflex occurs in human and animal models of type-1 diabetes (T1D), the mechanisms involved in the impairment of the baroreflex still remain unclear. The nodose ganglion (NG) contains the cell bodies of the Aortic Baroreceptor (AB) neurons. Hyperpolarizationactivated cyclic nucleotide-gated (HCN) channels are expressed in AB neurons and play an important role in regulating the cell excitability. We investigated whether the excitability of AB neurons is depressed in streptozotocin (STZ)-induced T1D rats and whether HCN channels are involved in this depression. Methods and results Using the whole-cell patch clamp technique, we found that AB neuron excitability (action potential frequency at 50 pA current stimulation) in the T1D rats was lower than that in the sham rats (0.4+ 0.5 vs. 4.8+ 0.6 spikes/s, P , 0.05; AB neurons were identified by DiI staining). In addition, HCN current density in AB neurons from the T1D rats was bigger than that from the sham rats (60.2+ 6.1 vs. 30.7+ 4.9 pA/pF at test pulse 2140 from holding potential 240 mV, P , 0.05). Furthermore, HCN channel blockers (5 mM cesium chloride and 100 mM ZD7288) significantly reduced HCN currents and increased action potential frequency of the AB neurons in sham and T1D rats. Immunofluorescent and western blot analyses demonstrated that the expression of HCN1 and HCN2 channel protein in the NG from the T1D rats was higher than that from the sham rats. Conclusion These results indicate that the HCN channels influence the excitability of AB neurons, and more importantly, contribute to the decreased excitability of AB neurons in T1D rats.
Francois M Abboud - One of the best experts on this subject based on the ideXlab platform.
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mechanosensitive ion channels in putative Aortic Baroreceptor neurons
American Journal of Physiology-heart and Circulatory Physiology, 1998Co-Authors: Ruth E Wachtel, Francois M Abboud, Thomas J Cunningham, Shane Kraske, G Hajduczok, Mark W ChapleauAbstract:Cell-attached patch-clamp experiments were performed on dissociated neurons from nodose ganglia of adult rats. Putative Aortic Baroreceptor neurons were identified by labeling nerve endings in the adventitia of the Aortic arch with the carbocyanine dye DiI. Whereas previous experiments demonstrated the presence of mechanosensitive (MS) whole cell currents, these experiments studied single MS ion channels and examined the influence of culture conditions on their expression. Single MS channels were activated by applying negative pressure through the recording pipette. Channel openings became more frequent as the negative pressure was increased, with open probability increasing significantly above 30 mmHg. MS channels had a slope conductance of 114 pS and a reversal potential of ∼0 mV, consistent with a nonspecific cation conductance. Channels were not affected by antagonists of voltage-gated conductances but were blocked by 20 μM gadolinium, a known blocker of MS ion channels. When nodose neurons were cocultured with Aortic endothelial cells, but not Aortic smooth muscle cells, the percentage of patches exhibiting MS ion channels increased significantly, suggesting that Aortic endothelial cells secrete a diffusible factor that increases channel expression.
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the prostacyclin analogue carbacyclin inhibits ca2 activated k current in Aortic Baroreceptor neurones of rats
The Journal of Physiology, 1997Co-Authors: Hon Chi Lee, Mark W Chapleau, Klaus Bielefeldt, Francois M AbboudAbstract:1. Previous studies indicate that prostacyclin (PGI2) increases the activity of Baroreceptor afferent fibres. The purpose of this study was to test the hypothesis that PGI2 inhibits Ca(2+)-activated K+ current (IK(Ca))in isolated Baroreceptor neurones in culture. 2. Rat Aortic Baroreceptor neurones in the nodose ganglia were labelled in vivo by applying a fluorescent dye (DiI) to the Aortic arch 1-2 weeks before dissociation of the neurones. Outward K+ currents in Baroreceptor neurones evoked by depolarizing voltage steps from a holding potential of -40 mV were recorded using the whole-cell patch-clamp technique. 3. Exposure of Baroreceptor neurones to the stable PGI2 analogue carbacyclin significantly inhibited the steady-state K+ current in a dose-dependent and reversible manner. The inhibition of K+ current was not caused indirectly by changes in cytosolic Ca2+ concentration. The Ca(2+)-activated K+ channel blocker charybdotoxin (ChTX, 10(-7) M) also inhibited the K+ current. In the presence of ChTX or in the absence of Ca2+, carbacyclin failed to inhibit the residual K+ current. Furthermore, in the presence of high concentrations of carbacyclin, ChTX did not cause further reduction of K+ current. 4. Carbacyclin-induced inhibition of IK(Ca) was mimicked by 8-bromo-cAMP and by activation of G-protein with GTP gamma S. The inhibitory effect of carbacyclin on IK(Ca) was abolished by GDP beta S, which blocks G-protein activation, and by a selective inhibitor of cAMP-dependent protein kinase, PKI5-24. 5. The results demonstrate that carbacyclin inhibits ChTX-sensitive IK(Ca) in isolated Aortic Baroreceptor neurones by a G-protein-coupled activation of cAMP-dependent protein kinase. This mechanism may contribute to the PGI2-induced increase in Baroreceptor activity demonstrated previously.
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non voltage gated ca2 influx through mechanosensitive ion channels in Aortic Baroreceptor neurons
Circulation Research, 1997Co-Authors: Margaret J Sullivan, Ruth E Wachtel, Mark W Chapleau, Ram V Sharma, Laurie J Waite, Ramesh C Bhalla, Francois M AbboudAbstract:Abstract The mechanisms underlying mechanotransduction in Baroreceptor neurons (BRNs) are undefined. In this study, we specifically identified Aortic Baroreceptor neurons in primary neuronal cell cultures from nodose ganglia of rats. Aortic Baroreceptor neurons were identified by labeling their soma with the fluorescent dye 1,1′-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) applied to the Aortic arch. Using Ca 2+ imaging with fura 2, we examined these BRNs for evidence of Ca 2+ influx and determined its mechanosensitivity and voltage dependence. Mechanical stimuli were produced by ejecting buffer from a micropipette onto the cell surface with a pneumatic picopump, producing a shift in the center of mass of the cell that was related to intensity of stimulation. Ninety-three percent of DiI-labeled neurons responded to mechanical stimulation with an increase in [Ca 2+ ] i . The magnitude of the increases in [Ca 2+ ] i was directly related to the intensity of the stimulus and required the presence of external Ca 2+ . The trivalent cations Gd 3+ or La 3+ in equimolar concentrations (20 μmol/L) eliminated the K + -induced rises in [Ca 2+ ] i , demonstrating that both trivalent cations are equally effective at blocking voltage-gated Ca 2+ channels in these Baroreceptor neurons. In contrast, the mechanically induced increases in [Ca 2+ ] i were blocked by Gd 3+ (20 μmol/L) only and not by La 3+ (20 μmol/L). Stretch-activated channels (SACs) have been shown in other preparations to be blocked by Gd 3+ specifically. Our data demonstrate that (1) BRNs, specifically identified as projecting to the Aortic arch, have ion channels that are sensitive to mechanical stimuli; (2) mechanically induced Ca 2+ influx in these cells is mediated by a Gd 3+ -sensitive ion channel and not by voltage-gated Ca 2+ channels; (3) the magnitude of the Ca 2+ influx is dependent on the intensity of the stimulus and the degree and duration of deformation; and (4) repeated stimuli of the same intensity result in comparable increases in [Ca 2+ ] i . We conclude that mechanical stimulation increases Ca 2+ influx into Aortic BRNs independent of voltage-gated Ca 2+ channels. The results suggest that Gd 3+ -sensitive SACs are the mechanoelectrical transducers in Baroreceptors.
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mechanical stimulation of neurites generates an inward current in putative Aortic Baroreceptor neurons in vitro
Brain Research, 1997Co-Authors: Thomas J Cunningham, Ruth E Wachtel, Francois M AbboudAbstract:Abstract We investigated the responses of putative Aortic Baroreceptor neurons to mechanical stimulation of their processes. Putative Aortic Baroreceptor neurons were identified by applying the carbocyanine dye DiI to the adventitia of the Aortic arch of anesthetized rats. After at least 1 week, the nodose ganglia were removed and the neurons were cultured. Within 2–3 days, neurite outgrowth was evident on many neurons. The soma was voltage-clamped using whole cell patch clamp techniques while the neurites were deformed with pneumatic ejection of bath solution at 5–15 psi using a glass pipette (7–15 μ m) positioned at least 50 μ m from the neurite. Mechanical stimulation induced an inward current in 15 out of 17 putative Aortic Baroreceptor neurons. The magnitude of the current was related to the intensity of stimulation. The current was blocked by 20 μ M gadolinium ( n =11), a reported blocker of mechanically sensitive ion channels, or by incubating the cells overnight in 10 μ M phalloidin, which binds to actin filaments ( n =5). We conclude that mechanical deformation of neurites of putative Baroreceptor neurons activates a mechanosensitive inward current in the soma and that the cytoskeletal actin filaments are involved in the generation of this current.
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mechanosensitive currents in putative Aortic Baroreceptor neurons in vitro
Journal of Neurophysiology, 1995Co-Authors: Joseph Thomas Cunningham, Ruth E Wachtel, Francois M AbboudAbstract:1. Whole cell patch-clamp experiments were conducted to determine whether rat Aortic Baroreceptor neurons contain mechano-sensitive conductances. 2. Putative Aortic Baroreceptor neurons in the nodose ganglia were identified by injecting DiI onto the adventitia of the Aortic arch. Nodose ganglia neurons were dissociated after > or = 1 wk. A fluorescein-conjugated tetanus toxin fragment was used to confirm that the cells labeled with DiI in culture were neurons. 3. Hypoosmotic stretch significantly increased the conductance of DiI-labeled neurons (n = 19). The reversal potential of the response was -11 +/- 1 (SE) mV. 4. In experiments on unlabeled neurons, only 7 of 13 cells showed increases in conductance. BC3H1 cells, a mouse tumor cell line, showed no changes in conductance. 5. Gadolinium (20 microM), a putative blocker of mechanosensitive channels, prevented the increase in conductance produced by hypoosmolality in seven of seven labeled cells. Equimolar concentrations of lanthanum (n = 6) and omega-conotoxin GVIA (1 microM, n = 4), which block voltage-gated calcium channels, failed to significantly affect the inward current.
Ruth E Wachtel - One of the best experts on this subject based on the ideXlab platform.
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mechanosensitive ion channels in putative Aortic Baroreceptor neurons
American Journal of Physiology-heart and Circulatory Physiology, 1998Co-Authors: Ruth E Wachtel, Francois M Abboud, Thomas J Cunningham, Shane Kraske, G Hajduczok, Mark W ChapleauAbstract:Cell-attached patch-clamp experiments were performed on dissociated neurons from nodose ganglia of adult rats. Putative Aortic Baroreceptor neurons were identified by labeling nerve endings in the adventitia of the Aortic arch with the carbocyanine dye DiI. Whereas previous experiments demonstrated the presence of mechanosensitive (MS) whole cell currents, these experiments studied single MS ion channels and examined the influence of culture conditions on their expression. Single MS channels were activated by applying negative pressure through the recording pipette. Channel openings became more frequent as the negative pressure was increased, with open probability increasing significantly above 30 mmHg. MS channels had a slope conductance of 114 pS and a reversal potential of ∼0 mV, consistent with a nonspecific cation conductance. Channels were not affected by antagonists of voltage-gated conductances but were blocked by 20 μM gadolinium, a known blocker of MS ion channels. When nodose neurons were cocultured with Aortic endothelial cells, but not Aortic smooth muscle cells, the percentage of patches exhibiting MS ion channels increased significantly, suggesting that Aortic endothelial cells secrete a diffusible factor that increases channel expression.
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non voltage gated ca2 influx through mechanosensitive ion channels in Aortic Baroreceptor neurons
Circulation Research, 1997Co-Authors: Margaret J Sullivan, Ruth E Wachtel, Mark W Chapleau, Ram V Sharma, Laurie J Waite, Ramesh C Bhalla, Francois M AbboudAbstract:Abstract The mechanisms underlying mechanotransduction in Baroreceptor neurons (BRNs) are undefined. In this study, we specifically identified Aortic Baroreceptor neurons in primary neuronal cell cultures from nodose ganglia of rats. Aortic Baroreceptor neurons were identified by labeling their soma with the fluorescent dye 1,1′-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) applied to the Aortic arch. Using Ca 2+ imaging with fura 2, we examined these BRNs for evidence of Ca 2+ influx and determined its mechanosensitivity and voltage dependence. Mechanical stimuli were produced by ejecting buffer from a micropipette onto the cell surface with a pneumatic picopump, producing a shift in the center of mass of the cell that was related to intensity of stimulation. Ninety-three percent of DiI-labeled neurons responded to mechanical stimulation with an increase in [Ca 2+ ] i . The magnitude of the increases in [Ca 2+ ] i was directly related to the intensity of the stimulus and required the presence of external Ca 2+ . The trivalent cations Gd 3+ or La 3+ in equimolar concentrations (20 μmol/L) eliminated the K + -induced rises in [Ca 2+ ] i , demonstrating that both trivalent cations are equally effective at blocking voltage-gated Ca 2+ channels in these Baroreceptor neurons. In contrast, the mechanically induced increases in [Ca 2+ ] i were blocked by Gd 3+ (20 μmol/L) only and not by La 3+ (20 μmol/L). Stretch-activated channels (SACs) have been shown in other preparations to be blocked by Gd 3+ specifically. Our data demonstrate that (1) BRNs, specifically identified as projecting to the Aortic arch, have ion channels that are sensitive to mechanical stimuli; (2) mechanically induced Ca 2+ influx in these cells is mediated by a Gd 3+ -sensitive ion channel and not by voltage-gated Ca 2+ channels; (3) the magnitude of the Ca 2+ influx is dependent on the intensity of the stimulus and the degree and duration of deformation; and (4) repeated stimuli of the same intensity result in comparable increases in [Ca 2+ ] i . We conclude that mechanical stimulation increases Ca 2+ influx into Aortic BRNs independent of voltage-gated Ca 2+ channels. The results suggest that Gd 3+ -sensitive SACs are the mechanoelectrical transducers in Baroreceptors.
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mechanical stimulation of neurites generates an inward current in putative Aortic Baroreceptor neurons in vitro
Brain Research, 1997Co-Authors: Thomas J Cunningham, Ruth E Wachtel, Francois M AbboudAbstract:Abstract We investigated the responses of putative Aortic Baroreceptor neurons to mechanical stimulation of their processes. Putative Aortic Baroreceptor neurons were identified by applying the carbocyanine dye DiI to the adventitia of the Aortic arch of anesthetized rats. After at least 1 week, the nodose ganglia were removed and the neurons were cultured. Within 2–3 days, neurite outgrowth was evident on many neurons. The soma was voltage-clamped using whole cell patch clamp techniques while the neurites were deformed with pneumatic ejection of bath solution at 5–15 psi using a glass pipette (7–15 μ m) positioned at least 50 μ m from the neurite. Mechanical stimulation induced an inward current in 15 out of 17 putative Aortic Baroreceptor neurons. The magnitude of the current was related to the intensity of stimulation. The current was blocked by 20 μ M gadolinium ( n =11), a reported blocker of mechanically sensitive ion channels, or by incubating the cells overnight in 10 μ M phalloidin, which binds to actin filaments ( n =5). We conclude that mechanical deformation of neurites of putative Baroreceptor neurons activates a mechanosensitive inward current in the soma and that the cytoskeletal actin filaments are involved in the generation of this current.
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mechanosensitive currents in putative Aortic Baroreceptor neurons in vitro
Journal of Neurophysiology, 1995Co-Authors: Joseph Thomas Cunningham, Ruth E Wachtel, Francois M AbboudAbstract:1. Whole cell patch-clamp experiments were conducted to determine whether rat Aortic Baroreceptor neurons contain mechano-sensitive conductances. 2. Putative Aortic Baroreceptor neurons in the nodose ganglia were identified by injecting DiI onto the adventitia of the Aortic arch. Nodose ganglia neurons were dissociated after > or = 1 wk. A fluorescein-conjugated tetanus toxin fragment was used to confirm that the cells labeled with DiI in culture were neurons. 3. Hypoosmotic stretch significantly increased the conductance of DiI-labeled neurons (n = 19). The reversal potential of the response was -11 +/- 1 (SE) mV. 4. In experiments on unlabeled neurons, only 7 of 13 cells showed increases in conductance. BC3H1 cells, a mouse tumor cell line, showed no changes in conductance. 5. Gadolinium (20 microM), a putative blocker of mechanosensitive channels, prevented the increase in conductance produced by hypoosmolality in seven of seven labeled cells. Equimolar concentrations of lanthanum (n = 6) and omega-conotoxin GVIA (1 microM, n = 4), which block voltage-gated calcium channels, failed to significantly affect the inward current.
Jaya Pamidimukkala - One of the best experts on this subject based on the ideXlab platform.
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cellular mechanisms regulating synaptic vesicle exocytosis and endocytosis in Aortic Baroreceptor neurons
Annals of the New York Academy of Sciences, 2006Co-Authors: Meredith Hay, Caroline J Hoang, Jaya PamidimukkalaAbstract:: The purpose of this chapter is to review some of the recent progress in the understanding of the cellular and biophysical mechanisms that are involved in the regulation of arterial Baroreceptor neurotransmssion. Synaptic depression or fatigue following repeated neuronal stimulation has been shown at central Baroreceptor synapses in vivo and in vitro. As most of the central neurons have a limited number of vesicles, vesicle retrieval or endocytosis following exocytosis is thought to play a major role in preserving synaptic transmission. We have hypothesized that central Baroreceptor terminals may inhibit their own synaptic transmission via feedback activation of presynaptic metabotropic glutamate receptors (mGluRs). We have analyzed the effects of mGluR autoreceptors (group III mGluRs) on voltage-gated calcium channels using standard patch-clamp techniques and on the process of exocytosis and endocytosis in Aortic Baroreceptor neurons using the quantitative imaging dye FM1–43 and FM2–10. Usng the whole-cell patch-clamp technique, we have found that activation of group III mGluRs with L-AP4 inhibits peak calcium channel current. Furthermore, activation of group III mGluRs with L-AP4 markedly decreases stimulation-induced exocytosis in Aortic Baroreceptor neurons, as measured with FM1–43, and inhibits synapsin I phosphorylation. These results suggest that activation of group III mGluRs may inhibit synaptic transmission by (1) inhibiting calcium influx, (2) decreasing synaptic vesicle exocytosis, and (3) modulating the mechanisms governing synaptic vesicle recovery and endocytosis. These effects of mGluRs on Baroreceptor synaptic vesicles may contribute to the Baroreceptor/nucleus tractus solitarius synaptic depression observed in vivo.
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frequency dependence of synaptic vesicle exocytosis in Aortic Baroreceptor neurons and the role of group iii mglurs
Brain Research, 2004Co-Authors: Jaya Pamidimukkala, Meredith HayAbstract:Abstract Synaptic transmission between Baroreceptor afferents and the nucleus tractus solitarius (NTS) is essential for reflex regulation of blood pressure. High frequency stimulation of the afferents in vivo leads to a decrease in synaptic strength and is generally attributed to reduction in presynaptic neurotransmitter release. It has been hypothesized that during high frequency stimulation glutamate a major neurotransmitter at the Baroreceptor afferent terminals inhibits its own release via presynaptic group III metabotropic glutamate receptors (mGluRs). A key player in modulation of presynaptic release is vesicle exocytosis. The present study utilized cultured Aortic Baroreceptor neurons and the styryl dye FM2–10 to characterize (1) the dependence of exocytosis at these afferent nerve terminals on the frequency of neuronal activation, (2) the effect of duration of stimulation on the rate of exocytosis and (3) the role of mGluRs in the frequency-dependent modulation of exocytosis. Destaining in the FM2–10 loaded boutons during 3 min of stimulation, a measure of exocytosis, progressively decreased with increasing frequency (0.5, 1.0 and 10 Hz). Blockade of group III mGluRs with 300 μM (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG) facilitated exocytosis evoked by 10 Hz stimulation but not at 0.5 Hz. The data suggest that Aortic Baroreceptor terminals exhibit frequency-dependent depression of exocytosis and support a role for group III mGluRs in the frequency-dependent modulation of exocytosis.
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frequency dependence of endocytosis in Aortic Baroreceptor neurons and role of group iii mglurs
American Journal of Physiology-heart and Circulatory Physiology, 2001Co-Authors: Jaya Pamidimukkala, Meredith HayAbstract:Synaptic transmission between Baroreceptor afferents and the nucleus tractus solitarius (NTS) is known to exhibit frequency-dependent depression. Reductions in neurotransmitter release and alterations in mechanisms regulating synaptic transmission are hypothesized to be involved in the activity-dependent depression observed in Baroreceptor afferent neurons. The present study utilized cultured Aortic Baroreceptor neurons and the fluorescent dyes FM1-43 and FM2-10 to characterize the process of endocytosis or vesicle retrieval and its dependence on 1) frequency of neuronal activation, 2) metabotropic glutamate receptor (mGluR) activation, and 3) calcium concentrations inside and outside the cell. Endocytosis per spike, measured in fluorescence units after a 10-s stimulus applied at frequencies of 0.5 (53 +/- 4), 1.0 (23 +/- 1), and 10.0 Hz (2.7 +/- 0.2), was significantly depressed at higher frequencies. Blockade of group III mGluRs with (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG) facilitated endocytosis at all frequencies, suggesting that this receptor subtype may be involved in the inhibition of endocytosis. Manipulating the extracellular and intracellular calcium concentrations subsequent to exocytosis had no effect on endocytosis. These results suggest that frequency-dependent depression of endocytosis observed in vitro could contribute to the frequency-dependent depression of Baroreceptor afferent neurotransmission and that group III mGluRs inhibit endocytosis.