A Reversible Low Frequency Alternating Current Nerve Conduction Block Applied to Mammalian Autonomic Nerves.

Electrical stimulation can be used to modulate activity within the nervous system in one of two modes: (1) Activation, where activity is added to the neural signalling pathways, or (2) Block, where activity in the nerve is reduced or eliminated. In principle, electrical nerve conduction block has many attractive properties compared to pharmaceutical or surgical interventions. These include reversibility, localization, and tunability for nerve caliber and type. However, methods to effect electrical nerve block are relatively new. Some methods can have associated drawbacks, such as the need for large currents, the production of irreversible chemical byproducts, and onset responses. These can lead to irreversible nerve damage or undesirable neural responses. In the present study we describe a novel low frequency alternating current blocking waveform (LFACb) and measure its efficacy to reversibly block the bradycardic effect elicited by vagal stimulation in anaesthetised rat model. The waveform is a sinusoidal, zero mean(charge balanced), current waveform presented at 1 Hz to bipolar electrodes. Standard pulse stimulation was delivered through Pt-Black coated PtIr bipolar hook electrodes to evoke bradycardia. The conditioning LFAC waveform was presented either through a set of CorTec® bipolar cuff electrodes with Amplicoat® coated Pt contacts, or a second set of Pt Black coated PtIr hook electrodes. The conditioning electrodes were placed caudal to the pulse stimulation hook electrodes. Block of bradycardic effect was assessed by quantifying changes in heart rate during the stimulation stages of LFAC alone, LFAC-and-vagal, and vagal alone. The LFAC achieved 86.2±11.1% and 84.3±4.6% block using hook (N = 7) and cuff (N = 5) electrodes, respectively, at current levels less than 110 µAp (current to peak). The potential across the LFAC delivering electrodes were continuously monitored to verify that the blocking effect was immediately reversed upon discontinuing the LFAC. Thus, LFACb produced a high degree of nerve block at current levels comparable to pulse stimulation amplitudes to activate nerves, resulting in a measurable functional change of a biomarker in the mammalian nervous system.

An afferent explanation for sexual dimorphism in the aortic baroreflex of rat.

Sex differences in baroreflex (BRx) function are well documented. Hormones likely contribute to this dimorphism, but many functional aspects remain unresolved. Our lab has been investigating a subset of vagal sensory neurons that constitute nearly 50% of the total population of myelinated aortic baroreceptors (BR) in female rats but less than 2% in male rats. Termed "Ah," this unique phenotype has many of the nonoverlapping electrophysiological properties and chemical sensitivities of both myelinated A-type and unmyelinated C-type BR afferents. In this study, we utilize three distinct experimental protocols to determine if Ah-type barosensory afferents underlie, at least in part, the sex-related differences in BRx function. Electron microscopy of the aortic depressor nerve (ADN) revealed that female rats have less myelin (P < 0.03) and a smaller fiber cross-sectional area (P < 0.05) per BR fiber than male rats. Electrical stimulation of the ADN evoked compound action potentials and nerve conduction profiles that were markedly different (P < 0.01, n = 7 females and n = 9 males). Selective activation of ADN myelinated fibers evoked a BRx-mediated depressor response that was 3-7 times greater in female (n = 16) than in male (n = 17) rats. Interestingly, the most striking hemodynamic difference was functionally dependent upon the rate of myelinated barosensory fiber activation. Only 5-10 Hz of stimulation evoked a rapid, 20- to 30-mmHg reduction in arterial pressure of female rats, whereas rates of 50 Hz or higher were required to elicit a comparable depressor response from male rats. Collectively, our experimental results are suggestive of an alternative myelinated baroreceptor afferent pathway in females that may account for, at least in part, the noted sex-related differences in autonomic control of cardiovascular function.

Differential distribution of voltage-gated channels in myelinated and unmyelinated baroreceptor afferents.

Voltage gated ion channels (VGC) make possible the frequency coding of arterial pressure and the neurotransmission of this information along myelinated and unmyelinated fiber pathways. Although many of the same VGC isoforms are expressed in both fiber types, it is the relative expression of each that defines the unique discharge properties of myelinated A-type and unmyelinated C-type baroreceptors. For example, the fast inward Na⁺ current is a major determinant of the action potential threshold and the regenerative transmembrane current needed to sustain repetitive discharge. In A-type baroreceptors the TTX-sensitive Na(v)1.7 VGC contributes to the whole cell Na⁺ current. Na(v)1.7 is expressed at a lower density in C-type neurons and in conjunction with TTX-insensitive Na(v)1.8 and Na(v)1.9 VGC. As a result, action potentials of A-type neurons have firing thresholds that are 15-20 mV more negative and upstroke velocities that are 5-10 times faster than unmyelinated C-type neurons. A more depolarized threshold in conjunction with a broader complement of non-inactivating K(V) VGC subtypes produces C-type action potentials that are 3-4 times longer in duration than A-type neurons and at markedly lower levels of cell excitability. Unmyelinated baroreceptors also express KCa1.1 which provides approximately 25% of the total outward K⁺ current. KCa1.1 plays a critically important role in shaping the action potential profile of C-type neurons and strongly impacts neuronal excitability. A-type neurons do not functionally express the KCa1.1 channel despite having a whole cell Ca(V) current quite similar to that of C-type neurons. As a result, A-type neurons do not have the frequency-dependent braking forces of KCa1.1. Lack of a KCa current and only a limited complement of non-inactivating K(V) VGC in addition to a hyperpolarization activated HCN1 current that is nearly 10 times larger than in C-type neurons leads to elevated levels of discharge in A-type neurons, a hallmark of myelinated baroreceptors. Interestingly, HCN2 and HCN4 expression levels are comparable in both fiber types. Collectively, such apportion of VGC constrains the neural coding of myelinated A-type baroreceptors to low threshold, high frequency, high fidelity discharge but with a limited capacity for neuromodulation of afferent bandwidth. Unmyelinated C-type baroreceptors require greater depolarizing forces for spike initiation and have a low frequency discharge profile that is often poorly correlated with the physiological stimulus. But the complement of VGC in C-type neurons provides far greater capacity for neuromodulation of cell excitability than can be obtained from A-type baroreceptors.

FKBP12 is a critical regulator of the heart rhythm and the cardiac voltage-gated sodium current in mice.

RATIONALE: FK506 binding protein (FKBP)12 is a known cis-trans peptidyl prolyl isomerase and highly expressed in the heart. Its role in regulating postnatal cardiac function remains largely unknown. METHODS AND RESULTS: We generated FKBP12 overexpressing transgenic (αMyHC-FKBP12) mice and cardiomyocyte-restricted FKBP12 conditional knockout (FKBP12(f/f)/αMyHC-Cre) mice and analyzed their cardiac electrophysiology in vivo and in vitro. A high incidence (38%) of sudden death was found in αMyHC-FKBP12 mice. Surface and ambulatory ECGs documented cardiac conduction defects, which were further confirmed by electric measurements and optical mapping in Langendorff-perfused hearts. αMyHC-FKBP12 hearts had slower action potential upstrokes and longer action potential durations. Whole-cell patch-clamp analyses demonstrated an ≈ 80% reduction in peak density of the tetrodotoxin-resistant, voltage-gated sodium current I(Na) in αMyHC-FKBP12 ventricular cardiomyocytes, a slower recovery of I(Na) from inactivation, shifts of steady-state activation and inactivation curves of I(Na) to more depolarized potentials, and augmentation of late I(Na), suggesting that the arrhythmogenic phenotype of αMyHC-FKBP12 mice is attributable to abnormal I(Na). Ventricular cardiomyocytes isolated from FKBP12(f/f)/αMyHC-Cre hearts showed faster action potential upstrokes and a more than 2-fold increase in peak I(Na) density. Dialysis of exogenous recombinant FKBP12 protein into FKBP12-deficient cardiomyocytes promptly recapitulated alterations in I(Na) seen in αMyHC-FKBP12 myocytes. CONCLUSIONS: FKBP12 is a critical regulator of I(Na) and is important for cardiac arrhythmogenic physiology. FKPB12-mediated dysregulation of I(Na) may underlie clinical arrhythmias associated with FK506 administration.

KCa1.1 channel contributes to cell excitability in unmyelinated but not myelinated rat vagal afferents.

High conductance calcium-activated potassium (BK(Ca)) channels can modulate cell excitability and neurotransmitter release at synaptic and afferent terminals. BK(Ca) channels are present in primary afferents of most, if not, all internal organs and are an intriguing target for pharmacological manipulation of visceral sensation. Our laboratory has a long-standing interest in the neurophysiological differences between myelinated and unmyelinated visceral afferent function. Here, we seek to determine whether there is a differential distribution of BK(Ca) channels in myelinated and unmyelinated vagal afferents. Immunocytochemistry studies with double staining for the BK-type K(Ca)1.1 channel protein and isolectin B4 (IB4), a reliable marker of unmyelinated peripheral afferents, reveal a pattern of IB4 labeling that strongly correlates with the expression of the K(Ca)1.1 channel protein. Measures of cell size and immunostaining intensity for K(Ca)1.1 and IB4 cluster into two statistically distinct (P < 0.05) populations of cells. Smaller diameter neurons most often presented with strong IB4 labeling and are presumed to be unmyelinated (n = 1,390) vagal afferents. Larger diameter neurons most often lacked or exhibited a very weak IB4 labeling and are presumed to be myelinated (n = 58) vagal afferents. Complimentary electrophysiological studies reveal that the BK(Ca) channel blockers charybdotoxin (ChTX) and iberiotoxin (IbTX) bring about a comparable elevation in excitability and action potential widening in unmyelinated neurons but had no effect on the excitability of myelinated vagal afferents. This study is the first to demonstrate using combined immunohistochemical and electrophysiological techniques that K(Ca)1.1 channels are uniquely expressed in unmyelinated C-type vagal afferents and do not contribute to the dynamic discharge characteristics of myelinated A-type vagal afferents. This unique functional distribution of BK-type K(Ca) channels may provide an opportunity for afferent selective pharmacological intervention across a wide range of visceral pathophysiologies, particularly those with a reflexogenic etiology and pain.

Functional impact of the hyperpolarization-activated current on the excitability of myelinated A-type vagal afferent neurons in the rat.

1. The hyperpolarization-induced, cation-selective current I(h) is widely observed in peripheral sensory neurons of the vagal and dorsal root ganglia, but the peak magnitude and voltage- and time-dependent properties of this current vary widely across afferent fibre type. 2. Using patch clamp investigations of rat isolated vagal ganglion neurons (VGN) identified as myelinated A-type afferents, we established a compendium of functional correlates between changes in membrane potential and the dynamic discharge properties of these sensory neurons as a result of the controlled recruitment of I(h) using the current clamp technique. 3. Two robust responses were observed in response to hyperpolarizing step currents: (i) upon initiation of the negative step current, there was a rapid hyperpolarization of membrane potential followed by a depolarizing voltage sag (DVS) towards a plateau in membrane potential as a result of steady state recruitment of I(h); and (ii) upon termination of the negative step current, there was a rapid return to the pretest resting membrane potential that often led to spontaneous action potential discharge. These data were strongly correlated (r(2) > 0.9) with a broad compendium of dynamic discharge characteristics in these A-type VGN. 4. In response to depolarizing step currents of increasing magnitude, the discharge frequency of the A-type VGN responded with increases in the rate of sustained repetitive discharge. Upon termination of the depolarizing step current, there was a post-excitatory membrane hyperpolarization of a magnitude that was strongly correlated with action potential discharge rate (r(2) > 0.9). 5. Application of the selective hyperpolarization-activated cyclic nucleotide gated (HCN) channel blockers ZD7288 (10 micromol/L) or CsCl (1.0 mmol/L) abolished I(h) and all of the aforementioned functional correlates. In addition to reducing the excitability of the A-type VGN to step depolarizing currents. 6. Because there is increasing evidence that the HCN channel current may represent a valid target for pharmacological intervention, the quantitative relationships described in the present study could potentially help guide the molecular and/or chemical modification of HCN channel gating properties to effect a particular outcome in VGN discharge properties, ideally well beyond merely selective blockade of a particular HCN channel subtype.

17Beta-estradiol restores excitability of a sexually dimorphic subset of myelinated vagal afferents in ovariectomized rats.

We recently identified a myelinated vagal afferent subpopulation (Ah type) far more prevalent in female than male rats and showed that this difference extends to functionally specific visceral sensory afferents, baroreceptors of the aortic arch. Excitability of myelinated Ah-type afferents is markedly reduced after ovariectomy (OVX). Here we tested the hypothesis that 17beta-estradiol can selectively restore excitability of these sex-specific vagal afferents. Acutely isolated vagal afferent neurons (VGN) from intact and OVX adult female rats were used with patch-clamp technique and current-clamp protocols to assess the effect of acute application of 17beta-estradiol on neuronal excitability. At over physiologically relevant 17beta-estradiol concentrations for rat (1-10 nM) excitability of myelinated Ah-type vagal afferents is restored to discharge frequencies comparable to those in intact females, albeit with some interesting differences related to burst and sustained patterns of neuronal discharge. Restoration of excitability occurs within 3 min of hormone application and is stereo specific, because 1,000 nM 17alpha-estradiol fails to alter excitability. Furthermore, activation of G protein-coupled estrogen receptor GPR30 with highly selective agonist G-1 similarly restores excitability of Ah-type afferents. The effectiveness of 17beta-estradiol and G-1 is completely eliminated by application of high-affinity estrogen receptor ligand ICI-182,780. 17beta-Estradiol conjugated with BSA is approximately 70% as effective as 17beta-estradiol alone in restoring Ah-type VGN excitability. These data support our conclusions that the cellular mechanisms leading to rapid restoration of neuronal excitability of myelinated Ah-type VGN after OVX occur, at least in part, via membrane-bound estrogen receptors. We contend that recovery of high-frequency discharge at physiologically relevant 17beta-estradiol concentrations implies that this unique subtype of low-threshold myelinated vagal afferent may account for some of the sex-related differences in visceral organ system function. Sex differences in cardiovascular and gastrointestinal function and the potential role of GPR30 in modulation of sex-specific myelinated Ah-type vagal afferents are discussed.

Characterization of persistent TTX-R Na+ currents in physiological concentration of sodium in rat visceral afferents.

Persistent tetrodotoxin-resistant (TTX-R) Na(+) (Na(v)1.9/SCN11A) currents are not normally recorded in vagal afferent neurons (VANs) with 50 mM of extracellular Na(+) although the functional expression of this current was observed in the presence of PGE(2) or forskolin. However, it is uncertain whether this current can be seen under physiological condition (150 mM Na(+)). Using the whole-cell patch-clamp technique, we showed that persistent TTX-R Na(+) currents were expressed in 9 out of 38 VANs bathed in 150 mM Na(+). The current density, but not the whole-cell capacitance, was significantly enhanced in the VANs expressing Nav1.9. Persistent TTX-R Na(+) channels were activated at a more hyperpolarized membrane potential near -60 mV, compared with TTX-sensitive (TTX-S at -40 mV) and TTX-R Na(+) channels (at -20 mV). This indicates that persistent TTX-R Na(+) channels provide a wider activation window than TTX-S and TTX-R Na channels to up-regulate neuronal excitability. These results suggest that the persistent TTX-R Na(+) currents may be involved in the neuronal excitability by setting a lower pressure-discharge threshold and higher discharge frequency of VANs, especially the unique subset and gender-specific distribution of myelinated Ah-type VANs, including Ah-type aortic baroreceptor neurons, identified in our previous study.

Electrophysiological and neuroanatomical evidence of sexual dimorphism in aortic baroreceptor and vagal afferents in rat.

Evidence for sexual dimorphism in autonomic control of cardiovascular function is both compelling and confounding. Across healthy and disease populations sex-associated differences in neurocirculatory hemodynamics are far too complex to be entirely related to sex hormones. As an initial step toward identifying additional physiological mechanisms, we investigated whether there is a sex bias in the relative expression of low-threshold-myelinated and high-threshold-unmyelinated aortic baroreceptor afferents in rats. These two types of afferent fibers have markedly different reflexogenic effects upon heart rate and blood pressure and thus the potential impact upon baroreflex dynamics could be substantial. Our results, using a combination of a patch-clamp study of fluorescently identified aortic baroreceptor neurons (ABN) and morphometric analysis of aortic baroreceptor nerve fibers, demonstrate that females exhibit a greater percentage of myelinated baroreceptor fibers (24.8% vs. 18.7% of total baroreceptor fiber population, P < 0.01) and express a functional subtype of myelinated ABN rarely found in age-matched males (11% vs. 2.3%, n = 107, P < 0.01). Interestingly, this neuronal phenotype is more prevalent in the general population of female vagal afferent neurons (17.7% vs. 3.8%, n = 169, P < 0.01), and ovariectomy does not alter its expression but does lessen neuronal excitability. These data suggest there are fundamental neuroanatomical and electrophysiological differences between aortic baroreceptor afferents of female and male rats. Possible explanations are presented as to how such a greater prevalence of low-threshold myelinated afferents could be a contributing factor to the altered baroreflex sensitivity and vagal tone of females compared with males.

The KCNQ/M-current modulates arterial baroreceptor function at the sensory terminal in rats.

The ion channels responsible for the pattern and frequency of discharge in arterial baroreceptor terminals are, with few exceptions, unknown. In this study we examined the contribution of KCNQ potassium channels that underlie the M-current to the function of the arterial baroreceptors. Labelled aortic baroreceptor neurons, immunohistochemistry and an isolated aortic arch preparation were used to demonstrate the presence and function of KCNQ2, KCNQ3 and KCNQ5 channels in aortic baroreceptors. An activator (retigabine) and an inhibitor (XE991) of the M-current were used to establish a role for these channels in setting the resting membrane potential and in regulating the response to ramp increases in arterial pressure. Retigabine raised the threshold for activation of arterial baroreceptors and shifted the pressure-response curve to higher aortic pressures. XE991, on the other hand, produced an increase in excitability as shown by an increase in discharge at elevated pressures as compared to control. We propose that KCNQ2, KCNQ3 and KCNQ5 channels provide a hyperpolarizing influence to offset the previously described depolarizing influence of the HCN channels in baroreceptor neurons and their terminals.

Theoretical and electrophysiological evidence for axial loading about aortic baroreceptor nerve terminals in rats.

Arterial baroreceptors are essential for neurocirculatory control, providing rapid hemodynamic feedback to the central nervous system. The pressure-dependent discharge of carotid and aortic baroreceptor afferents has been extensively studied. A common assumption has been that circumferential deformation of the arterial wall is the predominant mechanical force affecting baroreceptor discharge. However, in vivo the arterial tree is under significant longitudinal tension, leading to the hypothesis that axially directed forces may contribute to baroreceptor function. To test this hypothesis, we utilized a combination of finite element modeling methods and an in vitro rat aortic arch preparation. Model formulation utilized traditional analytic constructs available in the literature followed by refinement of model material parameters through direct comparison of computationally and experimentally generated pressure-diameter curves. The numerical simulations strongly indicated a functional role for axial loading within the region of the baroreceptive nerve terminal. This prediction was confirmed through single-fiber recording of baroreceptor nerve discharge under conditions with and without longitudinal tension in the vessel preparation. The recordings (n = 5) demonstrated that longitudinal tension significantly (P < 0.02) lowered both the pressure threshold (P(th), mmHg) for baroreceptor discharge and sensitivity (S(th), Hz/mmHg). The effect was nearly instantaneous and sustained; i.e., under longitudinal tension average P(th) was 84 +/- 3 mmHg and S(th) was 0.71 +/- 0.15 Hz/mmHg, which immediately increased to a P(th) of 94 +/- 4 mmHg and a S(th) of 1.20 +/- 0.32 Hz/mmHg with loss of axial tension. Possible explanations of how an abrupt change in axial loading could result in a synchronized increase in afferent drive of the baroreceptor reflex, and the potentiating effect this could have on neurogenically mediated orthostatic intolerance are discussed.

Unmyelinated visceral afferents exhibit frequency dependent action potential broadening while myelinated visceral afferents do not.

Sensory information arising from visceral organ systems is encoded into action potential trains that propagate along afferent fibers to target nuclei in the central nervous system. These information streams range from tight patterns of action potentials that are well synchronized with the sensory transduction event to irregular, patternless discharge with no clear correlation to the sensory input. In general terms these afferent pathways can be divided into unmyelinated and myelinated fiber types. Our laboratory has a long standing interest in the functional differences between these two types of afferents in terms of the preprocessing of sensory information into action potential trains (synchrony, frequency, duration, etc.), the reflexogenic consequences of this sensory input to the central nervous system and the ionic channels that give rise to the electrophysiological properties of these unique cell types. The aim of this study was to determine whether there were any functional differences in the somatic action potential characteristics of unmyelinated and myelinated vagal afferents in response to different rates of sensory nerve stimulation. Our results showed that activity and frequency-dependent widening of the somatic action potential was quite prominent in unmyelinated but not myelinated vagal afferents. Spike broadening often leads to increased influx of Ca(2+) ions that has been associated with a diverse range of modulatory mechanisms both at the cell body and central synaptic terminations (e.g. increased neurotransmitter release.) We conclude that our observations are indicative of fundamentally different mechanisms for neural integration of sensory information arising from unmyelinated and myelinated vagal afferents.

Electrophysiological and pharmacological validation of vagal afferent fiber type of neurons enzymatically isolated from rat nodose ganglia.

An unavoidable consequence of enzymatic dispersion of sensory neurons from intact ganglia is loss of the axon and thus the ability to classify afferent fiber type based upon conduction velocity (CV). An intact rat nodose ganglion preparation was used to randomly sample neurons (n=76) using the patch clamp technique. Reliable electrophysiological and chemophysiological correlates of afferent fiber type were established for use with isolated neuron preparations. Myelinated afferents (approximately 25%) formed two groups exhibiting strikingly different functional profiles. One group (n=10) exhibited CVs in excess of 10 m/s and narrow (<1 ms) action potentials (APs) while the other (n=9) had CVs as low as 4m/s and broad (>2 ms) APs that closely approximated those identified as unmyelinated afferents (n=57) with CVs less than 1m/s. A cluster analysis of select measures from the AP waveforms strongly correlated with CV, producing three statistically unique populations (p<0.05). These groupings aligned with our earlier hypothesis (Jin et al., 2004) that a differential sensitivity to the selective purinergic and vanilloid receptor agonists can be used as reliable pharmacological indicators of vagal afferent fiber type. These metrics were further validated using an even larger population of isolated (n=240) nodose neurons. Collectively, these indicators of afferent fiber type can be used to provide valuable insight concerning the relavence of isolated cellular observations to integrated afferent function of visceral organ systems.

Persistent tetrodotoxin-resistant Na+ currents are activated by prostaglandin E2 via cyclic AMP-dependent pathway in C-type nodose neurons of adult rats.

It has been documented that nodose neurons express TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na(+) channels. However, wheteher nodose neurons functionally express persistent TTX-R Na(+) currents has not been reported. The present study first demonstrated persistent TTX-R Na(+) channel activities in 7/19 C-type nodose neurons in the presence of PGE(2) using whole-cell patch. Voltage-dependent property showed that persistent TTX-R Na(+) currents were activated at near -60mV and channels were maintained open. The average peak was approximately 300-500pA. The mid-point of activation exhibited a greater shift to a more hyperpolarized potential in the neurons co-expressing TTX-R and persistent TTX-R Na(+) currents than those expressing TTX-R only. This effect of PGE(2) was also mimicked by Forskolin. The fact that persistent TTX-R Na(+) currents were only activated by PGE(2) suggested that the modulatory effects of PGE(2) on persistent TTX-R Na(+) currents are crucial in PGE(2)-mediated neuronal excitability, and may have a great impact on specifically physiological significance.

Purinergic and vanilloid receptor activation releases glutamate from separate cranial afferent terminals in nucleus tractus solitarius.

Vanilloid (VR1) and purinergic (P2X) receptors are found in cranial afferent neurons in nodose ganglia and their central terminations within the solitary tract nucleus (NTS), but little is known about their function. We mechanically dissociated dorsomedial NTS neurons to preserve attached native synapses and tested for VR1 and P2X function primarily in spindle-shaped neurons resembling intact second-order neurons. All neurons (n = 95) exhibited spontaneous glutamate (EPSCs) and GABA (IPSCs)-mediated synaptic currents. VR1 agonist capsaicin (CAP; 100 nm) reversibly increased EPSC frequency, effects blocked by capsazepine. ATP (100 microm) increased EPSC frequency, actions blocked by P2X antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS; 20 microm). In all CAP-resistant neurons, P2X agonist alphabeta-methylene-ATP (alphabeta-m-ATP) increased EPSC frequency. Neither CAP nor alphabeta-m-ATP altered EPSC amplitudes, kinetics, or holding currents. Thus, activation of VR1 and P2X receptors selectively facilitated presynaptic glutamate release on different NTS neurons. PPADS and 2',3'-O-(2,4,6-trinitrophenyl)-ATP blocked alphabeta-m-ATP responses, but P2X1-selective antagonist NF023 (8,8'-[carbonylbis (imino-3,1-phenylene carbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid) did not. The pharmacological profile and transient kinetics of ATP responses are consistent with P2X3 homomeric receptors. TTX and Cd(2+) did not eliminate agonist-evoked EPSC frequency increases, suggesting that voltage-gated sodium and calcium channels are not required. In nodose ganglia, CAP but not alphabeta-m-ATP evoked inward currents in slow conducting neurons and the converse pattern in myelinated, rapidly conducting neurons (n = 14). Together, results are consistent with segregation of glutamatergic terminals into either P2X sensitive or VR1 sensitive that correspondingly identify myelinated and unmyelinated afferent pathways at the NTS.

Synaptic and membrane properties of neurons in the dorsomedial hypothalamus.

Studies in intact rats have shown that the dorsomedial hypothalamus (DMH) plays a key role in generating stress-induced physiologic changes, including activation of the hypothalamic-pituitary-adrenal axis through direct projections to paraventricular hypothalamic nucleus (PVN). However, little is known about the cellular properties of DMH neurons. We employed whole-cell patch-clamp recording techniques to characterize membrane properties and spontaneous post-synaptic currents (PSCs) in DMH neurons, including those projecting to PVN (identified by prior injection of DiI into PVN), in rat hypothalamic slices. DMH neurons (n=86 total) had uniform membrane properties. However, PVN-projecting neurons (n=32) had higher action potential (AP) thresholds, and fired fewer APs in response to current injection. Spontaneous PVN-projecting neurons (n=20) also fired APs at lower rates (4.8+/-0.6 Hz) than spontaneous neurons of unknown projection (n=38; 7.3+/-1.1 Hz). Spontaneous PSCs were observed in all neurons: One population expressed rapid decay characteristics (1.5-2.0 ms) and was blocked by non-NMDA ionotropic glutamate receptor antagonists NBQX or CNQX. Remaining PSCs reversed near E(Cl), were blocked by the GABA(A) receptor antagonists picrotoxin or bicuculline methiodide (BMI), and had longer decay time constants (4.5-6.0 ms) that were modulated by pentobarbital. Tetrodotoxin markedly reduced the frequency of PSCs sensitive to NBQX but not to BMI. Thus, DMH is made up of electrophysiologically similar neurons and PVN-projecting neurons are less excitable than neurons of unknown projection. Furthermore, as suggested by studies in intact rats, neurons in the DMH, including those projecting to the PVN, are regulated by tonic GABA(A) and non-NMDA glutamate receptor-mediated synaptic transmission.

Ketamine differentially blocks sensory afferent synaptic transmission in medial nucleus tractus solitarius (mNTS).

BACKGROUND: Ketamine increases blood pressure and heart rate by unknown mechanisms, but studies suggest that an intact central nervous system and arterial baroreceptors are required. In the brain stem, medial nucleus tractus solitarius receives afferents from nodose neurons that initiate cardiovascular autonomic reflexes. Here, the authors assessed ketamine actions on afferent medial nucleus tractus solitarius synaptic transmission. METHODS: Ketamine was applied to horizontally sliced brain stems. Solitary tract (ST) stimulation evoked excitatory postsynaptic currents (eEPSCs) in medial nucleus tractus solitarius neurons. Capsaicin (200 nm) block of ST eEPSCs sorted neurons into sensitive (n = 19) and resistant (n = 23). In nodose ganglion slices, shocks to the peripheral vagal trunk activated afferent action potentials in sensory neurons classified by conduction velocities and capsaicin. RESULTS: Ketamine potently (10-100 mciro m) blocked small, ST-evoked -methyl-d-aspartate synaptic currents found only in a subset of capsaicin-resistant neurons (6 of 12). Surprisingly, ketamine reversibly inhibited ST eEPSC amplitudes and induced synaptic failure at lower concentrations in capsaicin-sensitive than in capsaicin-resistant neurons (P < 0.005; n = 11 and 11). Spontaneous EPSCs using non- -methyl-d-aspartate receptors were insensitive even to 1-3 mm ketamine, suggesting that ST responses were blocked presynaptically. Similarly, ketamine blocked C-type action potential conduction at lower concentrations than A-type nodose sensory neurons. CONCLUSION: The authors conclude that ketamine inhibits postsynaptic -methyl-d-aspartate receptors and presynaptic afferent processes in medial nucleus tractus solitarius. Unexpectedly, capsaicin-sensitive (C-type), unmyelinated afferents are significantly more susceptible to block than capsaicin-resistant (A-type), myelinated afferents. This differentiation may be related to tetrodotoxin-resistant sodium currents. Since C-type afferents mediate powerful arterial baroreflexes effects, these differential actions may contribute to ketamine-induced cardiovascular dysfunction.

Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons.

Voltage-gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0-3) and in 85-90 % of the neurons from older animals (postnatal days 21-60). In voltage clamp studies, alpha-dendrotoxin (alpha-DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K(+) currents of the sensory neurons. alpha-DTX blocked current in both A- and C-type neurons. The current had characteristics of a delayed rectifier with activation positive to -50 mV and little inactivation during 250 ms pulses. In current-clamp experiments alpha-DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A- and C-type neurons. However, there were prominent effects on excitability. alpha-DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after-hyperpolarization and increased the frequency/pattern of discharge of A- and C-type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K(+) currents function in response to the loss of the alpha-DTX-sensitive current to effect these changes in action potential wave shape and discharge.

Comparisons of somatic action potentials from dispersed and intact rat nodose ganglia using patch-clamp technique.

AIM: To differentiate the electrophysiological characteristics of somatic action potentials (AP) from isolated Neo and Juv nodose sensory neurons (NSN) and those from slices of intact Juv and adult rat nodose ganglia. METHODS: For isolated cell recordings nodose ganglia from 3-8 d old Neo and 4 weeks old Juv rats were dissociated using trypsin and collagenase, respectively. Nodose ganglia slices with attached vagus were prepared using a sequential treatment of collagenase and trypsin for both Juv and adult rats. Conduction velocity (CV) was collected by vagal stimulation. Whole-cell patch was applied for somatic AP recordings. RESULTS: (1) 281 NSN from both isolated cells and nodose slices were studied. Across all age groups, there was no difference observed among either C- or A-types. The difference between C- and A-type was significant. (2) Neurons exhibiting AP with prominent repolarization hump, broader APD50 (>2.0 ms), upstroke velocity at the point of APD50 (UV(APD50)) and downstroke velocity at the point of APD50 (DV(APD50)) below 100 V . s-1 and 50 V . s-1 were classified as C-type (n = 222). Those without a hump, brief APD50 (<1.0 ms), and UV(APD50) and DV(APD50) greater than 200 V . s-1 and 100 V . s-1 were classified as A-type (n = 59). (3) With slices, except for hump, APD, UV, and DV, significant differences were also observed (C- vs A-type) in CV from both Juv (0.56 +/- 0.15 vs 15.6 m.s-1) and adult (0.9 +/- 0.4 vs 14.5 +/- 1.0 m . s-1) nodose slices, discharge rate (single or burst vs repetitive), and frequency follow (<20 Hz vs >100 Hz). (4) Phase analysis showed that C-type had higher AP firing threshold, and lower total in- and outward peak currents than A-type. CONCLUSION: C- and A-type AP from isolated NSN of Neo or Juv rats exhibited electrophysiological characteristics that closely followed those of AP recorded in nodose slices. Therefore, isolated NSN can effectively serve as an experimental surrogate for electrophysiological studies of NSN requiring prior identification of afferent fiber type. Features of the AP wave-form such as the repolarization hump, APD50), and UV are strongly correlated with CV and are therefore reliable measures for classifying isolated NSN as either C- or A-type. It is most important that the nodose slice enables us to study the AP and ion channel characteristics on identified NSN by afferent fiber CV with adult animals.