Vladislav Snitsarev

Imaging Synaptic Activity in Intact Brain and Slices with FM1-43 in C. elegans, Lamprey, and Rat

The fluorescent probe FM1-43 has been used extensively for imaging vesicle recycling; however, high nonspecific adsorption resulting in elevated background levels has precluded its use in certain tissues, notably brain slices. We have found that a sulfobutylated derivative of beta-cyclodextrin (ADVASEP-7) has a higher affinity for FM1-43 than the plasma membrane. ADVASEP-7 was used as a carrier to remove FM1-43 nonspecifically bound to the outer leaflet of the plasma membrane or extracellular molecules, significantly reducing background staining. This has enabled us to visualize synaptic vesicle recycling in the nematode C. elegans, intact lamprey spinal cord, and rat brain slices.

The Ion Channel ASIC2 Is Required for Baroreceptor and Autonomic Control of the Circulation

Arterial baroreceptors provide a neural sensory input that reflexly regulates the autonomic drive of circulation. Our goal was to test the hypothesis that a member of the acid-sensing ion channel (ASIC) subfamily of the DEG/ENaC superfamily is an important determinant of the arterial baroreceptor reflex. We found that aortic baroreceptor neurons in the nodose ganglia and their terminals express ASIC2. Conscious ASIC2 null mice developed hypertension, had exaggerated sympathetic and depressed parasympathetic control of the circulation, and a decreased gain of the baroreflex, all indicative of an impaired baroreceptor reflex. Multiple measures of baroreceptor activity each suggest that mechanosensitivity is diminished in ASIC2 null mice. The results define ASIC2 as an important determinant of autonomic circulatory control and of baroreceptor sensitivity. The genetic disruption of ASIC2 recapitulates the pathological dysautonomia seen in heart failure and hypertension and defines a molecular defect that may be relevant to its development.

Fluorescent Detection of Zn2+-Rich Vesicles with Zinquin: Mechanism of Action in Lipid Environments

High concentrations of free Zn2+ ions are found in certain glutamatergic synaptic vesicles in the mammalian brain. These terminals can be visualized histochemically with quinoline sulfonamide compounds that form fluorescent complexes with Zn2+. The present study was undertaken to examine the interaction of the water-soluble quinoline sulfonamide probe, Zinquin (2-methyl-8-(toluene-p-sulfonamido)-6-quinolyloxyacetic acid) with the complex heterogeneous cellular environment. Experiments on rat hippocampal and neocortical slices gave indications that Zinquin in its free acid form was able to diffuse across the plasma and synaptic vesicle membranes. Further experiments were undertaken on unilamellar liposomes to study the interaction of Zinquin and its metal complexes in membranes. These experiments confirmed that Zinquin is able to diffuse across lipid bilayers. Steady-state and time-resolved fluorimetric studies showed that Zinquin in aqueous solution mainly forms a 1:2 (metal:ligand) complex with small amounts of a 1:1 complex. Formation of the 1:1 complex was favored by the presence of lipid, suggesting that it partitions into membranes. Evidence is presented that Zinquin can act as a Zn(2+)-ionophore, exchanging Zn2+ for two protons. The presence of a pH gradient across vesicles traps the Zn(2+)-probe complex within the vesicles. Zinquin is useful as a qualitative probe for detecting the presence of vesicular Zn2+; however, its tendency to partition into membranes and to serve as an ionophore should be borne in mind.

Mechanosensory transduction of vagal and baroreceptor afferents revealed by study of isolated nodose neurons in culture

Changes in arterial pressure and blood volume are sensed by baroreceptor and vagal afferent nerves innervating aorta and heart with soma in nodose ganglia. The inability to measure membrane potential at the nerve terminals has limited our understanding of mechanosensory transduction. Goals of the present study were to: (1) Characterize membrane potential and action potential responses to mechanical stimulation of isolated nodose sensory neurons in culture; and (2) Determine whether the degenerin/epithelial sodium channel (DEG/ENaC) blocker amiloride selectively blocks mechanically induced depolarization without suppressing membrane excitability. Membrane potential of isolated rat nodose neurons was measured with sharp microelectrodes. Mechanical stimulation with buffer ejected from a micropipette (5, 10, 20 psi) depolarized 6 of 10 nodose neurons (60%) in an intensity-dependent manner. The depolarization evoked action potentials in 4 of the 6 neurons. Amiloride (1 microM) essentially abolished mechanically induced depolarization (15 +/- 4 mV during control vs. 1 +/- 2 mV during amiloride with 20-psi stimulation, n = 6) and action potential discharge. In contrast, amiloride did not inhibit the frequency of action potential discharge in response to depolarizing current injection (n = 6). In summary, mechanical stimulation depolarizes and triggers action potentials in a subpopulation of nodose sensory neurons in culture. The DEG/ENaC blocker amiloride at a concentration of 1 microM inhibits responses to mechanical stimulation without suppressing membrane excitability. The results support the hypothesis that DEG/ENaC subunits are components of mechanosensitive ion channels on vagal afferent and baroreceptor neurons.

Acid-sensing ion channels interact with and inhibit BK K+ channels

Acid-sensing ion channels (ASICs) are neuronal non-voltage-gated cation channels that are activated when extracellular pH falls. They contribute to sensory function and nociception in the peripheral nervous system, and in the brain they contribute to synaptic plasticity and fear responses. Some of the physiologic consequences of disrupting ASIC genes in mice suggested that ASIC channels might modulate neuronal function by mechanisms in addition to their H+-evoked opening. Within ASIC channels large extracellular domain, we identified sequence resembling that in scorpion toxins that inhibit K+ channels. Therefore, we tested the hypothesis that ASIC channels might inhibit K+ channel function by coexpressing ASIC1a and the high-conductance Ca2+- and voltage-activated K+ (BK) channel. We found that ASIC1a associated with BK channels and inhibited their current. Reducing extracellular pH disrupted the association and relieved the inhibition. BK channels, in turn, altered the kinetics of ASIC1a current. In addition to BK, ASIC1a inhibited voltage-gated Kv1.3 channels. Other ASIC channels also inhibited BK, although acidosis-dependent relief of inhibition varied. These results reveal a mechanism of ion channel interaction and reciprocal regulation. Finding that a reduced pH activated ASIC1a and relieved BK inhibition suggests that extracellular protons may enhance the activity of channels with opposing effects on membrane voltage. The wide and varied expression patterns of ASICs, BK, and related K+ channels suggest broad opportunities for this signaling system to alter neuronal function.

Mechano-and chemosensitivity of rat nodose neurones - : selective excitatory effects of prostacyclin

Nodose ganglion sensory neurones exert a significant reflex autonomic influence. We contrasted their mechanosensitivity, excitability and chemosensitivity in response to the stable prostacyclin (PGI2) analogue carbacyclin (cPGI) in culture. Under current clamp conditions we measured changes in membrane potential (ΔmV) and action potential (AP) responses to mechanically induced depolarizations and depolarizing current injections before and after superfusion of cPGI (1 μm and 10 μm). Chemosensitivity was indicated by augmentation of AP firing frequency and increased maximum gain of AP frequency (max. dAP/dΔmV), during superfusion with cPGI. Results indicate that two groups of neurones, A and B, are mechanosensitive (MS) and one group, C, is mechanoinsensitive (MI). Group A shows modest depolarization without AP generation during mechanical stimulation, and no increase in max. dAP/dΔmV, despite a marked increase in electrical depolarization with cPGI. Group B shows pronounced mechanical depolarization accompanied by enhanced AP discharge with cPGI, and an increase in max. dAP/dΔmV. Group C remains MI after cPGI but is more excitable and markedly chemosensitive (CS) with a pronounced enhancement of max. dAP/dΔmV with cPGI. The effect of cPGI on ionic conductances indicates that it does not sensitize the mechanically gated depolarizing degenerin/epithelial Na+ channels (DEG/ENaC), but it inhibits two voltage‐gated K+ currents, Maxi‐K and M‐current, causing enhanced AP firing frequency and depolarization, respectively. We conclude that MS nodose neurones may be unimodal MS or bimodal MS/CS, and that MI neurones are unimodal CS, and much more CS to cPGI than MS/CS neurones. We suggest that the known excitatory effect of PGI2 on baroreceptor and vagal afferent fibres is mediated by inhibition of voltage‐gated K+ channels (Maxi‐K and M‐current) and not by an effect on mechanically gated DEG/ENaC channels.

Neuronal Prostacyclin Is an Autocrine Regulator of Arterial Baroreceptor Activity

We tested the hypothesis that neuronal prostacyclin is an autocrine regulator of arterial baroreceptor neuronal activity. In isolated rat aortic nodose baroreceptor neurons, mechanical stimulation depolarized 12 neurons by 13.1±3.4 mV and triggered action potentials in 5 of them, averaging 18.2±9.5 spikes. Current injections depolarized 21 neurons by 29.9±8.0 mV and triggered action potentials averaging 17.0±2.4 spikes. After a period of prolonged neuronal activation with pulses of 1 nA at 20 Hz for 1 minute, the action potential responses to mechanical stimulation and to current injections were first markedly suppressed (0.2±0.2 and 2.1±0.7 spikes, respectively) and then enhanced, reaching levels above control (29.0±8.0 and 21.7±2.6 spikes, respectively) over the subsequent 15 minutes. In contrast, there was no inhibition of the depolarizations caused by mechanical stimulation or current injections. The recovery and enhancement of action potentials, which reached 150±5.4% of control values at 15 minutes (n=13), were abrogated by 10 &mgr;mol/L of indomethacin and replaced by sustained inhibition for over 15 minutes. Carbacyclin (10 &mgr;mol/L) reversed the inhibition and restored action potential responses. Prostacyclin production by cultured nodose neurons was enhanced by arachidonic acid and electrical field stimulation and inhibited by indomethacin. We conclude that prostacyclin provides an autocrine feedback that restores and enhances the responsiveness of arterial baroreceptor neurons after their inhibition from excessive neuronal activation. We speculate that reduced synthesis of neuronal prostacyclin contributes to the resetting phenomenon and the suppressed activity of arterial baroreceptors in hypertension.

Dual mechanisms of angiotensin‐induced activation of mouse sympathetic neurones

Ang II directly activates neurones in sympathetic ganglia. Our goal was to define the electrophysiological basis of this activation. Neurones from mouse aortic–renal and coeliac ganglia were identified as either ‘tonic’ or ‘phasic’. With injections of depolarizing currents, action potentials (APs) were abundant and sustained in tonic neurones (TNs) and scarce or absent in phasic neurones (PNs). Resting membrane potentials were equivalent in TNs (−48 ± 2 mV, n= 18) and PNs (−48 ± 1 mV, n= 23) while membrane resistance was significantly higher in TNs. Ang II depolarized and increased membrane resistance equally in both TNs (n= 8) and PNs (n= 8) but it induced APs only in TNs, and enhanced current‐evoked APs much more markedly in TNs (P < 0.05). The AT1 receptor antagonist losartan (2 μm, n= 6) abolished all responses to Ang II, whereas the AT2 receptor blocker PD123,319 had no effect. The transient K+ current (IA), which was more than twice as large in TNs as in PNs, was significantly inhibited by Ang II in TNs only whereas the delayed sustained K+ current (IK), which was comparable in both TNs and PNs, was not inhibited. M currents were more prominent in PNs and were inhibited by Ang II. The IA channel blocker 4‐aminopyridine triggered AP generation in TNs and prevented the Ang II‐induced APs but not the depolarization. Blockade of M currents by oxotremorine M or linopirdine prevented the depolarizing action of Ang II. The protein kinase C (PKC) inhibitor H7 (10 μm, n= 9) also prevented the Ang II‐induced inhibition of IA and the generation APs but not the depolarization nor the inhibition of M currents. Conversely, the PKC agonist phorbol 12‐myristate 13‐acetate mimicked the Ang II effects by triggering APs. The results indicate that Ang II may increase AP generation in sympathetic neurones by inducing a PKC‐dependent inhibition of IA currents, and a PKC‐independent depolarization through inhibition of M currents. The differential expression of various K+ channels and their sensitivity to phosphorylation by PKC may determine the degree of activation of sympathetic neurones and hence may influence the severity of the hypertensive response.