Role of GABAA and GABAC receptors in the biphasic GABA responses in neurons of the rat major pelvic ganglia.

The role of gamma-aminobutyric acid-A (GABAA) and GABAC receptors in the GABA-induced biphasic response in neurons of the rat major pelvic ganglia (MPG) were examined in vitro. Application of GABA (100 microM) to MPG neurons produced a biphasic response, an initial depolarization (GABAd) followed by a hyperpolarization (GABAh). The input resistance of the MPG neurons was decreased during the GABAd, whereas it was increased during the GABAh. The GABAd could be further separated into the early component (early GABAd) with a duration of 27 +/- 5 s (mean +/- SE; n = 11) and the late component (late GABAd) with a duration of 109 +/- 11 s (n = 11). The duration of the GABAh was 516 +/- 64 s (n = 11). The effects of GABA (5-500 microM) in producing the depolarization and the hyperpolarization were concentration-dependent. GABA (5-30 microM) induced only late depolarizations. The early component of the depolarization appeared when the concentration of GABA was >50 microM. Muscimol produced only early depolarizing responses. Baclofen (100 microM) had no effect on the membrane potential and input resistance of MPG neurons. Bicuculline (60 microM) blocked the early GABAd but not the late GABAd and the GABAh. Application of picrotoxin (100 microM) with bicuculline (60 microM) blocked both the late GABAd and the GABAh. CGP55845A (3 microM), a selective GABAB receptor antagonist, did not affect the GABA-induced responses. cis-4-Aminocrotonic acid (CACA, 1 mM) and trans-4-aminocrotonic acid (TACA, 1 mM), selective GABAC receptor agonists, produced late biphasic responses in the MPG neurons. The duration of the CACA responses was almost the same as those of the late GABAd and GABAh obtained in the presence of bicuculline. Imidazole-4-acetic acid (I4AA, 100 microM), a GABAC receptor antagonist, depressed the late GABAd and the GABAh but not the early GABAd. I4AA (100 microM) and picrotoxin (100 microM) also suppressed the biphasic response to CACA. The early GABAd and the late GABAd were reversed in polarity at -32 +/- 3 mV (n = 7) and -38 +/- 2 mV (n = 4), respectively, in the Krebs solution. The reversal potential of the GABAh was -34 +/- 2 mV (n = 4) in the Krebs solution. The reversal potentials of the late GABAd and the GABAh shifted to -20 +/- 3 mV (n = 5) and -22 +/- 3 mV (n = 5), respectively, in 85 mM Cl- solution. These results indicate that the late GABA(d) and the GABAh are mediated predominantly by bicuculline-insensitive, picrotoxin-sensitive GABA receptors, GABAC (or GABAAOr) receptors, in neurons of the rat MPG.     

Potassium distribution and membrane potential of sensory neurons in the leech nervous system

The intracellular K activity (aKi) and membrane potential of sensory neurons in the leech central nervous system were measured in normal and altered external K+ concentrations, [K+]o, using double-barreled, liquid ion-exchanger microelectrodes. In control experiments membrane potential measurements were made using potassium chloride-filled single-barreled microelectrodes. All values are means +/- SD. At the normal [K+]o (4 mM) the mean aKi of all cells tested was 72.6 +/- 10.6 mM (n = 40) and the average membrane potential was -47.3 +/- 5.2 mM (n = 40). When measured with single-barreled microelectrodes, the membrane potential averaged -45.3 +/- 2.9 mV (n = 12). Assuming an intracellular K+ activity coefficient of 0.75, the intracellular K+ concentration of sensory neurons would be 96.8 +/- 14.1 mM). With an extracellular K+ concentration of 5.8 mM in the intact ganglion compared to the K+ concentration of 4 mM in the bath, the K+ equilibrium potential was -71.5 mV. When the ganglion capsule was opened, the extracellular K+ concentrations in the ganglion were similar to that of the bathing medium and the calculated K+ equilibrium potential was -81 mV. The membrane of sensory neurons depolarized following the changes to elevated [K+]o (greater than or equal to 10-100 mM), whereas aKi changed only little or not at all. At very low [K+]o (0.2, 0 mM) aKi and membrane potential showed little short-term (less than 3 min) effect but began to change after longer exposure (greater than 3 min). Reduction of [K+]o from 4 to 0.2 mM (or 0 mM) produced first a slow, and then a more rapid decrease of aKi and membrane resistance, accompanied by a slow membrane hyperpolarization. Following readdition of normal [K+]o, the membrane first depolarized and then transiently hyperpolarized, eventually returning slowly to the normal membrane potential.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bullfrog dorsal root ganglion cells having tetrodotoxin-resistant spikes are endowed with nicotinic receptors

1. Intracellular recordings were made from bullfrog dorsal root ganglion (DRG) neurons in vitro. They were divided into three types, As, Ar, and C, according to their conduction velocity and their sensitivity to tetrodotoxin [TTX (less than or equal to 1 microM)]; an As neuron had a fast conduction velocity (13-50 m/s, mean = 31 m/s, n = 73) and TTX-sensitive sodium soma spikes: an Ar neuron showed a fast conduction velocity (4-28 m/s, mean = 14 m/s, n = 52) and TTX-resistant sodium soma spikes; and a C neuron had a slow conduction velocity (0.16-0.8 m/s, mean = 0.4 m/s, n = 49) and TTX-resistant sodium-calcium soma spikes. 2. Superfusion of acetylcholine [ACh (0.3 microM-1 mM)] produced a fast depolarization in 70% of Ar and in 50% of C neurons. No As neuron showed a fast depolarization in response to ACh. The ACh-induced fast response persisted in calcium-free or TTX-containing solutions. 3. The response in both Ar and C neurons was similar except in time course; the response was always more rapid in C than in Ar neurons. The response was always associated with a decreased membrane resistance and reversed in polarity at about -30 mV. The reversal potential varied with both sodium and potassium concentrations of the superfusing solutions. 4. Nicotine, (+)-tubocurarine [(+)-TC], and hexamethonium reversibly blocked the ACh fast response.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-Hydroxytryptamine effects on the somata of bullfrog primary afferent neurons

Intracellular recordings were made from neurons in the isolated dorsal root ganglia of bullfrogs. 5-Hydroxytryptamine was applied by superfusion and by ionophoresis. The most common response to 5-hydroxytryptamine in C neurons was a membrane hyperpolarization and this was observed in 80% of cells. This was due to an increase in membrane potassium conductance because it reversed its polarity at about -90 mV. It was blocked by removal of calcium or addition of calcium blockers. (+)-Tubocurarine, methysergide, ketanserin, quipazine, picrotoxin, caffeine and ouabain blocked this response. The next most common response in C neurons was a fast depolarization, particularly readily observed when 5-hydroxytryptamine was applied by ionophoresis. Since this response reversed its polarity at about -10 mV and was blocked by removal of sodium, this was due to an increase in membrane conductance to both sodium and potassium ions. This response was reduced by superfusion of acetylcholine and gamma-aminobutyric acid. (+)-Tubocurarine, quipazine, picrotoxin and caffeine blocked the response. A small proportion of C neurons (16%) responded to superfusion of 5-hydroxytryptamine with a slow depolarization accompanied by an increase in input resistance. This response reversed its polarity at about -90 mV and, therefore, is presumed to result from potassium inactivation. It was blocked by methysergide and ketanserin but not by (+)-tubocurarine or quipazine. A few type A neurons (8%) caused a fast and transient depolarization like the fast depolarization of C neurons. About half of the A neurons showed a slow depolarization associated with a fall in input resistance. This slow response was assumed to be due to an increase in membrane conductance to both potassium and calcium ions because the response reversed its polarity at about -65 mV and was sensitive to change in external concentrations of those ions. This slow response was blocked by (+)-tubocurarine, methysergide, ketanserin, picrotoxin, caffeine and ouabain but not by quipazine. The effects of 5-hydroxytryptamine are discussed in relation to the similar actions described on a variety of other vertebrate and invertebrate nerve cells. The findings imply that dorsal root ganglion cells of bullfrogs are sensitive to 5-hydroxytryptamine and causes multiple types of 5-hydroxytryptamine responses.     

Sulfur dioxide derivatives modulation of high-threshold calcium currents in rat dorsal root ganglion neurons

This study addressed the effect of sulfur dioxide (SO(2)) derivatives on high-voltage-activated calcium currents (HVA-I(Ca)) in somatic membrane of freshly isolated rat dorsal root ganglion (DRG) neurons by using the whole-cell configuration of patch-clamp technique. High-threshold Ca(2+) channels are highly expressed in small dorsal root ganglion neurons. SO(2) derivatives increased the amplitudes of calcium currents in a concentration-dependent and voltage-dependent manner. The 50% enhancement concentrations (EC(50)) of SO(2) derivatives on HVA-I(Ca) was about 0.4 microM. In addition, SO(2) derivatives significantly shifted the activation and inactivation curve in the depolarizing direction. Parameters for the fit of a Boltzmann equation to mean values for the activation were V(1/2)=-17.9+/-1.3 mV before and -12.5+/-1.1 mV after application 0.5 microM SO(2) derivatives 2 min (P<0.05). The half inactivation of HVA-I(Ca) was shifted 9.7 mV to positive direction (P<0.05). Furthermore, SO(2) derivatives significantly prolonged the slow constant of inactivation, slowed the fast recovery but markedly accelerated the slow recovery of HVA-I(Ca) from inactivation. From HP of -60 mV 0.5 microM SO(2) derivatives increased the amplitude of HVA-I(Ca) with a depolarizing voltage step to -10 mV about 54.0% in small DRG neurons but 33.3% in large DRG neurons. These results indicated a possible correlation between the change of calcium channels and SO(2) inhalation toxicity, which might cause periphery neurons abnormal regulation of nociceptive transmission via calcium channels.     

The effects of ouabain on resting membrane potential and hyperpolarization-activated current in neonatal rat nodose ganglion neurons

To determine whether the responses of resting membrane potential (RMP) and hyperpolarization-activated current (IH) are altered by the application of ouabain, one of the Na+-K+ pump inhibitors, in neonatal rat small-diameter (<30microm) nodose ganglion (NG) neurons, we examined the effects of 1microM ouabain on those responses using perforated patch-clamp techniques. In current-clamp mode, the RMP was 40.2+/-1.6mV (n=31). Twenty of 31 cells tested were depolarized by ouabain application, and these responses were associated with an increase in the cell input resistance. In the remaining 11 cells studied, 3 showed hyperpolarization in response to ouabain and 8 showed no effect on RMP. In voltage-clamp mode, 1muM ouabain application enhanced the IH in all of 10 neurons examined. These results suggest that ouabain application at 1microM is capable of setting both the RMP level and the neuronal excitability in small-diameter NG neurons.     

Transient outward currents in cochlear ganglion neurons of the chick embryo.

Cochlear ganglion neurons were isolated from chick embryos and membrane currents recorded using the patch-clamp technique. Depolarizing voltage steps elicited transient outward currents whose inactivation was best fitted by a double-exponential function with time constants < 30 ms and > 100 ms. The fast inactivating transient outward current (Ito,f) had a threshold for activation of -61 +/- 5.5 mV; steady-state inactivation was voltage-dependent between -90 and -60 mV, with half-inactivation near -75 mV. The slowly inactivating outward current (Ito,s) showed an activation threshold of 34 +/- 4 mV. Half-inactivation was at -67 +/- 3 mV. Ito,f was blocked by 4-aminopyridine which did not affect Ito,s. The effect was concentration- and voltage-dependent. Tetraethylammonium had no effect on either fast or slow transient currents but reduced the amplitude of the non-inactivating outward current in a dose-dependent manner. Ito,f was strongly inhibited by removing Ca2+ from the extracellular bathing solution. Cobalt ions inhibited Ito,f in a dose-dependent manner between 2 and 20 mM. The inhibitory effect of Co2+ was voltage-dependent, displaying a bell-shaped inhibition curve as a function of membrane voltage, maximal inhibition occurring between -20 and 0 mV. Ca2+ removal did not affect Ito,s and partially reduced the amplitude of the steady-state current. These results provide kinetic and pharmacological evidence for the presence of two distinct transient outward currents in cochlear neurons. These currents may play a role in the first synaptic relay of sound transmission.     

Electrophysiological properties and chemosensitivity of acutely dissociated trigeminal somata innervating the cornea

Adult rat sensory trigeminal ganglion neurons innervating the cornea (cTGNs) were isolated and identified following retrograde dye labeling with FM1-43. Using standard whole-cell patch clamp recording techniques, cTGNs could be subdivided by their action potential (AP) duration. Fast cTGNs had AP durations <1 ms (40%) while slow cTGNs had AP durations >1 ms and an inflection on the repolarization phase of the AP. With the exception of membrane input resistance, the passive membrane properties of fast cTGNs were different from those of slow cTGNs (capacitance: 61+/-4.5 pF vs. 42+/-2.6 pF, resting membrane potential: -59+/-0.7 mV vs. -53+/-0.9 mV, for fast and slow cTGNs respectively). Active membrane properties also differed between fast and slow cTGNs. Slow cTGNs had a higher AP threshold (-25+/-1.6 mV vs. -38+/-0.8 mV), a larger rheobase (14+/-1.9 pA/pF vs. 6.8+/-1.0 pA/pF), and a smaller AP undershoot (-56+/-1.7 mV vs. -67+/-2.5 mV). The AP overshoot, however was similar between the two types of neurons (46+/-3.1 mV vs. 48+/-4 mV). Slow cTGNs were depolarized by capsaicin (1 microM, 80%) and 60% of their APs were blocked by tetrodotoxin (TTX) (100 nM). Fast cTGNs were unaffected by capsaicin and 100% of their APs were blocked by TTX. Similarly, cTGNs were also heterogeneous with respect to their responses to exogenous ATP and 5-HT. The current work shows that cTGNs have distinctive electrophysiological properties and chemosensitivity profiles. These characteristics may mirror the distinct properties of corneal sensory nerve terminals. The availability of isolated identified cTGNs constitutes a tractable model system to investigate the biophysical and pharmacological properties of corneal sensory nerve terminals.     

Glycine binding site of the synaptic NMDA receptor in subpostremal NTS neurons

The nucleus of the tractus solitarius (NTS) plays an important role in the control of several autonomic reflex functions and has glutamate and GABA as main neurotransmitters. In this work, we used patch-clamp recordings in transverse slice preparations from rats to study whether the glycine binding site of the N-methyl-D-aspartate (NMDA) receptor is saturated or not in neurons of the subpostremal NTS. Except at hyperpolarized voltages and close to the reversal potential, glycine potentiated the NMDA responses in a concentration-dependent manner. The total charge transferred by glutamatergic currents was enhanced by glycine (500 microM; from 28 +/- 13 to 42 +/- 18 pC at +50 mV, n = 7, P < 0.05). Glycine increased the conductance of the postsynaptic membrane, without altering its reversal potential, both in the presence (from 2.4 +/- 0.06 to 3.4 +/- 0.09 nS; n = 7) and absence (from 3.1 +/- 0.06 to 4.4 +/- 0.10 nS; n = 8) of Mg2+ in the bathing solution. d-serine, in the presence of strychnine, also increased the amplitude of the NMDA component (by 68 +/- 19%, P < 0.05, n = 5). The membrane potential was hyperpolarized (16 +/- 6 mV, n = 8) by glycine, suggesting the presence of inhibitory glycinergic receptors. Our results indicate that the glycine site of the NMDA receptor in neurons of the subpostremal NTS is not saturated and that glycine may act as a modulator of the NMDA transmission in this nucleus.     

Nitric oxide is an autocrine regulator of Na(+) currents in axotomized C-type DRG neurons

In this study, we examined whether nitric oxide synthase (NOS) is upregulated in small dorsal root ganglion (DRG) neurons after axotomy and, if so, whether the upregulation of NOS modulates Na(+) currents in these cells. We identified axotomized C-type DRG neurons using a fluorescent label, hydroxystilbamine methanesulfonate and found that sciatic nerve transection upregulates NOS activity in 60% of these neurons. Fast-inactivating tetrodotoxin-sensitive (TTX-S) Na(+) ("fast") current and slowly inactivating tetrodotoxin-resistant (TTX-R) Na(+) ("slow") current were present in control noninjured neurons with current densities of 1.08 +/- 0. 09 nA/pF and 1.03 +/- 0.10 nA/pF, respectively (means +/- SE). In some control neurons, a persistent TTX-R Na(+) current was observed with current amplitude as much as approximately 50% of the TTX-S Na(+) current amplitude and 100% of the TTX-R Na(+) current amplitude. Seven to 10 days after axotomy, current density of the fast and slow Na(+) currents was reduced to 0.58 +/- 0.05 nA/pF (P < 0.01) and 0.2 +/- 0.05 nA/pF (P < 0.001), respectively. Persistent TTX-R Na(+) current was not observed in axotomized neurons. Nitric oxide (NO) produced by the upregulation of NOS can block Na(+) currents. To examine the role of NOS upregulation on the reduction of the three types of Na(+) currents in axotomized neurons, axotomized DRG neurons were incubated with 1 mM N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. The current density of fast and slow Na(+) channels in these neurons increased to 0.82 +/- 0.08 nA/pF (P < 0.01) and 0.34 +/- 0.04 nA/pF (P < 0.05), respectively. However, we did not observe any persistent TTX-R current in axotomized neurons incubated with L-NAME. These results demonstrate that endogenous NO/NO-related species block both fast and slow Na(+) current in DRG neurons and suggest that NO functions as an autocrine regulator of Na(+) currents in injured DRG neurons.     

Excitatory effects of human immunodeficiency virus 1 Tat on cultured rat cerebral cortical neurons

Human immunodeficiency virus 1 (HIV-1) Tat protein is one of the neurotoxins involved in the pathogenesis of HIV-1-associated neuronal disorders. Combined electrophysiological and optical imaging experiments were undertaken to investigate whether HIV-1 Tat30-86, herein referred to as Tat30-86, acted directly or indirectly via the release of glutamate or both and to test its effect on the properties of spontaneous quantal events in cultured cortical neurons. Whole-cell patch recordings were made from cultured rat cortical neurons in either current- or voltage-clamp mode. Tat30-86 (50-1000 nM) induced in a population of cortical neurons a long-lasting depolarization, which was accompanied by a decrease of membrane resistance and persisted in a Krebs solution containing tetrodotoxin (TTX, 0.5 microM). Depolarizations were slightly reduced by pretreatment with glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM) and d-2-amino-5-phosphonovaleric acid (AP-5) (50 microM), and were markedly reduced in a Ca(2+)-free Krebs solution; the differences were statistically significant. Tat30-86-induced inward currents had a reversal potential between -30 and 0 mV. While not causing a noticeable depolarization, lower concentrations of Tat30-86 (10 nM) increased membrane excitability, as indicated by increased numbers of neuronal discharge in response to a step depolarizing pulse. Tat30-86 (10 nM) increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), while not significantly affecting their amplitude. Tat30-86 (10 nM) moderately increased the frequency as well as the amplitude of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Ratiometric Ca(2+) imaging studies showed that Tat30-86 produced three types of Ca(2+) responses: 1) a fast and transitory increase, 2) Ca(2+) oscillations, and 3) a fast increase followed by a plateau; the glutamate receptor antagonists eliminated the late component of Ca(2+) response. The result suggests that Tat30-86 is an active fragment and that it excites cortical neurons directly and indirectly via releasing glutamate from adjacent neurons.     

Calcium regulates L-Type Ca2+ channel expression in rat skeletal muscle cells

Primary skeletal muscle cells were cultured in a normal- (1.8 mM) or high- (4.8 mM) Ca2+ culture medium to determine whether Ca2+ modulates the number of L-type Ca2+ channels. Skeletal myoballs cultured in a normal medium showed, when exposed to a high extracellular [Ca2+], ([Ca2+]e) a transient increase in intracellular [Ca2+] ([Ca2+]i) from a resting concentration of 60 to 160 nM. By day 3, however, when the experiments were made, [Ca2+]i no longer differed from control (pre-exposure to high Ca2+). The maximum charge movements in myoballs incubated in 1.8 and 4.8 mM were 16.4+/-1.05 (n=56) and 24.1+/-1.18 nC/microF (n=58; P<0.01), respectively, and peak Ca2+ currents at 20 mV were -10.8+/-1.09 (n=46) and -12.8+/-0.75 nA/microF (n=82), respectively (P>0.05). The tail current amplitudes in 1.8 and 4.8 mM Ca2+-treated cells were -9.3+/-1.23 and -14.2+/-1.37 nA/microF (P<0.05), respectively, at 10 mV and -15.3+/-1.76 and -23.6+/-2.02 nA/microF (P<0.05), respectively at 60 mV. The maximum binding of [3H]PN200-110 (a radioligand specific for L-type Ca2+ channel alpha1 subunits) in myoballs cultured in 1.8 and 4.8 mM [Ca2+]e was 1.34+/-0.23 and 3.2+/-0.63 pmol/mg protein (n=8; P<0.02), respectively. The increase in [Ca2+]i associated with the increases in charge movements, tail currents and the number of L-type Ca2+ channel alpha1 subunits in skeletal muscle cells cultured in high [Ca2+]e support the concept that extracellular Ca2+ influx modulates the expression of L-type Ca2+ channels in skeletal muscle cells.     

Voltage-dependent, slowly activating K+ current (I Ks) and its augmentation by carbachol in rat pancreatic acini

Acetylcholine-stimulated exocrine secretion of Cl- and water requires the concomitant activation of K+ channels. However, there has not been much investigation of the carbachol- (CCH-) activated K+ channel of rodent pancreatic acini. Here, in a study of rat pancreatic acini, we characterize a voltage-dependent, slowly activating outward current (I(Ks)) that is augmented by CCH. Intact acini were obtained by enzymatic digestion and fast-whole-cell patch-clamp was applied. With symmetrical [Cl-] (32 mmol/l) in the pipette and bath solution, acinar cells had resting membrane voltages of -45+/-0.8 mV (n=97) under current-clamp conditions. CCH (10 micromol/l), which is known to activate Cl- channels via a Ca2+-mediated pathway, sharply depolarized the membrane to -4+/-0.5 mV, which was more negative than E(Cl) (0 mV), and reversed it to -41+/-0.9 mV (n=83) by washout. A clamp voltage of 0 mV activated I(Ks) under control conditions (91+/-8.6 pA, n=83). During CCH application an increase of outward current was observed at 0 mV, and at -50 mV a marked increase of inward Cl current occurred. In the presence of CCH the slow activation of I(Ks) was rarely distinguishable because of interference by the huge Cl- conductance. During CCH washout and decrease of inward current, a persistent augmentation of I(Ks) was revealed (486+/-36.3 pA, n=83). I(Ks) and its augmentation were abolished by substituting K+ in the pipette solution with Cs+. Augmentation of I(Ks) was mimicked by applying ionomycin (0.1 micromol/l), a Ca2+ ionophore. Pharmacological blockers were tested. The chromanol 293B and clotrimazole blocked I(Ks) at micromolar concentrations (IC50=3 micromol/l and 9 micromol/l, respectively) and Ba2+ was a poor blocker (IC50=3 mmol/l). In the presence of CCH (0.2 micromol/l), the membrane was depolarized to around -20 mV and the addition of 293B (10 micromol/l) further depolarized the membrane by 11+/-3 mV (n=5). These data suggest the presence of I(Ks) channels in rat pancreatic acini and their muscarinic activation.     

The nicotinic activation of the denervated sympathetic neuron of the rat

Nicotinic responses to endogenous acetylcholine and to exogenously applied agonists have been studied in the intact or denervated rat sympathetic neuron in vitro, by using the two-microelectrode voltage-clamp technique. Preganglionic denervation resulted in progressive decrease of the synaptic current (excitatory postsynaptic current, EPSC) amplitude, which disappeared within 24 h. These effects were accompanied by changes in ion selectivity of the nicotinic channel (nAChR). The extrapolated EPSC null potential (equilibrium potential for acetylcholine action, E(Syn)) shifted from a mean value of -15.9+/-0.7 mV, in control, to -7.4+/-1.6 mV, in denervated neurons, indicating a decrease of the permeability ratio for the main components of the synaptic current (P(K)/P(Na)) from 1.56 to 1.07. The overall properties of AChRs were investigated by applying dimethylphenylpiperazinium or cytisine and by examining the effects of endogenous ACh, diffusing within the ganglion after preganglionic tetanization in the presence of neostigmine. The null potentials of these macrocurrents (equilibrium potential for dimethylphenylpiperazinium action, E(DMPP); and equilibrium potential for diffusing acetylcholine, E(ACh), respectively) were evaluated by applying voltage ramps and from current-voltage plots. In normal neurons, E(Syn) (-15.9+/-0.7 mV) was significantly different from E(DMPP) (-26.1+/-1.0) and E(ACh) (-31.1+/-3.3); following denervation, nerve-evoked currents displayed marked shifts in their null potentials (E(Syn)=-7.4+/-1.6 mV), whereas the amplitude and null potential of the agonist-evoked macrocurrents were unaffected by denervation and its duration (E(DMPP)=-26.6+/-1.2 mV). It is suggested that two populations of nicotinic receptors, synaptic and extrasynaptic, are present on the neuron surface, and that only the synaptic type displays sensitivity to denervation.     

Activation of N-methyl-D-aspartate receptors evokes calcium spikes in the dendrites of rat hypothalamic paraventricular nucleus neurons

Activation of dendritic voltage-dependent calcium (Ca2+) conductances in neuroendocrine cells of the hypothalamus may underlie previously documented Ca2+ spikes in these cells. The present study, in which whole-cell recordings were obtained from paraventricular nucleus neurons in a hypothalamic slice preparation, addresses this issue by directly activating dendritic N-methyl-D-aspartate receptors in the presence of tetrodotoxin. Application of tetrodotoxin abolished spontaneous action potentials in all paraventricular nucleus neurons tested (n = 27). Following tetrodotoxin, spikes were evoked by depolarizing current pulses, in an all-or-none fashion in the majority of cells (n = 20). Removal of extracellular Ca2+ (n = 6) or addition of 500 microM CdCl2 (n = 4) abolished the spikes in response to pulses. Repetitive spiking activity (in tetrodotoxin) was also observed following N-methyl-D-aspartate agonist application in 75% of the cells tested (n = 15). The spikes, underscored by a slow membrane depolarization, were abolished by the administration of CdCl2 (n = 4). N-Methyl-D-aspartate agonist elicited a slow inward current in cells voltage-clamped at -60 mV (n = 5). Additionally, larger amplitude, transient inward currents were observed near the onset of the response. The activation threshold to elicit spikes following N-methyl-D-aspartate agonist application was significantly more negative (-54.6+/-3.6 mV) than the potential at which spikes were initiated as a result of depolarizing current injection (-32.3+/-1.8 mV; Student's t-test: P < 0.0001). In contrast to this, Na+ spikes in control solution had an invariable threshold (-49.6+/-0.7 mV vs -51.5+/-1.2 mV; P > 0.05), regardless of the stimulus used to initiate the spikes. These observations suggest that direct activation of N-methyl-D-aspartate receptors located on the dendrites of paraventricular nucleus neurons triggers Ca2+ spikes. Although the precise function of these spikes is unclear, previous data reporting dendritic neuropeptide release in the paraventricular nucleus raise the possibility that dendritically initiated spikes may serve as a local signal to trigger such release.     

Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons

C-type dorsal root ganglion (DRG) neurons can generate tetrodotoxin-resistant (TTX-R) sodium-dependent action potentials. However, multiple sodium channels are expressed in these neurons, and the molecular identity of the TTX-R sodium channels that contribute to action potential production in these neurons has not been established. In this study, we used current-clamp recordings to compare action potential electrogenesis in Na(v)1.8 (+/+) and (-/-) small DRG neurons maintained for 2-8 h in vitro to examine the role of sodium channel Na(v)1.8 (alpha-SNS) in action potential electrogenesis. Although there was no significant difference in resting membrane potential, input resistance, current threshold, or voltage threshold in Na(v)1.8 (+/+) and (-/-) DRG neurons, there were significant differences in action potential electrogenesis. Most Na(v)1.8 (+/+) neurons generate all-or-none action potentials, whereas most of Na(v)1.8 (-/-) neurons produce smaller graded responses. The peak of the response was significantly reduced in Na(v)1.8 (-/-) neurons [31.5 +/- 2.2 (SE) mV] compared with Na(v)1.8 (+/+) neurons (55.0 +/- 4.3 mV). The maximum rise slope was 84.7 +/- 11.2 mV/ms in Na(v)1.8 (+/+) neurons, significantly faster than in Na(v)1.8 (-/-) neurons where it was 47.2 +/- 1.3 mV/ms. Calculations based on the action potential overshoot in Na(v)1.8 (+/+) and (-/-) neurons, following blockade of Ca(2+) currents, indicate that Na(v)1.8 contributes a substantial fraction (80-90%) of the inward membrane current that flows during the rising phase of the action potential. We found that fast TTX-sensitive Na(+) channels can produce all-or-none action potentials in some Na(v)1.8 (-/-) neurons but, presumably as a result of steady-state inactivation of these channels, electrogenesis in Na(v)1.8 (-/-) neurons is more sensitive to membrane depolarization than in Na(v)1.8 (+/+) neurons, and, in the absence of Na(v)1.8, is attenuated with even modest depolarization. These observations indicate that Na(v)1.8 contributes substantially to action potential electrogenesis in C-type DRG neurons.     

APV-sensitive dorsal root afferent transmission to neonate rat sympathetic preganglionic neurons in vitro

1. Intracellular recordings were made from antidromically identified sympathetic preganglionic neurons (SPNs) in transverse thoracolumbar spinal cord slices from neonate (12- to 22-day-old) rats. 2. Electrical stimulation of dorsal roots or dorsal root entry zone elicited in SPNs an excitatory postsynaptic potential (EPSP) or multiple EPSPs of varying latencies. The EPSP could be graded by varying the stimulus intensity and, on reaching the threshold, discharged an action potential. 3. The dorsal root-evoked EPSPs had a mean synaptic latency of 2.6 ms (range: 1.2-11 ms), suggesting a polysynaptic pathway. The EPSPs were characteristically slow in onset with a mean rise time and half-decay time of 8.3 and 23 ms, respectively. 4. At the resting membrane potential of -50 to -60 mV, the amplitude of EPSPs recorded in normal (1.3 mM Mg2+) Krebs solution was reduced by membrane hyperpolarization or depolarization. In Mg2(+)-free solution, EPSPs were potentiated and reached threshold for spike discharge. 5. The EPSPs were suppressed by the nonselective glutamate receptor antagonist kynurenic acid (0.1-0.5 mM) and by the N-methyl-D-aspartate (NMDA) receptor antagonists D-2-amino-5-phosphonovaleric acid (APV; 1-10 microM) and ketamine (5-10 microM), but not by the quisqualate (QA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 1-10 microM). The latter depressed the EPSPs elicited by stimulation of lateral funiculus in the same SPNs. 6. NMDA applied by pressure elicited a depolarization in the SPNs. In normal Krebs solution the response was voltage dependent with the peak amplitude occurring around -60 mV; conditioning depolarization or hyperpolarization diminished the response.(ABSTRACT TRUNCATED AT 250 WORDS)     

The dependence of the transmembrane salivary secretory potential on the external potassium and sodium concentration

1. The submandibular gland of the cat was perfused with equi-osmolar Locke solutions of different cationic composition. The increase in acinar membrane potential (secretory potential), measured with a glass capillary micro-electrode, was recorded after intra-arterial injection of acetylcholine.2. When nearly all Na in the perfusion fluid was replaced by tetraethylammonium (TEA), the size of the secretory potentials was significantly (P < 0.001) greater than of those recorded during perfusion with normal Locke solution.3. When the K concentration in the TEA Locke solution was augmented, the size of the secretory potentials was greatly diminished. Thus at a [K](o) of 4 m-equiv/l. the size of the secretory potentials was 43.3 mV +/- 2.5 (n = 19) but at a [K](o) of 10 m-equiv/l. the size was only 24.7 mV +/- 2.0 (n = 13) and at a [K](o) of 20 m-equiv/l. the size of the secretory potentials was 12.0 mV +/- 1.0 (n = 21). When the submandibular gland was perfused with a K-free TEA Locke solution the size of the secretory potentials was 49.7 mV +/- 2.2 (n = 17).4. The size of the secretory potentials recorded during perfusion with normal (Na) Locke solution increased significantly (P < 0.001) when K was omitted from the perfusion fluid. The decrease of the size with increasing [K](o) was considerably less marked than that found during perfusion with TEA Locke solution. Increasing [K](o) by a factor of 5 diminished the size of the secretory potentials recorded in Na Locke solution by a factor of 2, whereas the size of the secretory potentials recorded in TEA Locke solution, with the same changes in [K](o), diminished by a factor of 3.5.5. The results seem to indicate that the increase in acinar membrane potential obtained after stimulation of the gland is due to an enhanced permeability of the basal acinar cell membrane to K. However, it seems that the permeability to Na is also enhanced so that the outwardly directed K current is partly short-circuited by an inward Na current.     

Effect of intracellular dialysis of ATP on the hyperpolarization-activated cation current in rat dorsal root ganglion neurons

The mechanism of the effect of intracellular ATP on the hyperpolarization-activated non-selective cation current (Ih) in rat dorsal root ganglion neurons was investigated using a whole cell voltage-clamp technique. Under voltage-clamp conditions, Ih was activated by hyperpolarizing pulses raised to a voltage of between -70 and -130 mV. The activation curve of Ih in rat dorsal root ganglion (DRG) neurons shifted by about 15 mV in the positive direction with an intracellular solution containing 1 mM cAMP. When ATP (2 mM) was applied intracellularly, the half-maximal activation voltage (Vhalf) of Ih shifted from -97.4 +/- 1.9 to -86.8 +/- 1.6 mV, resulting in an increase in the current amplitude of Ih by the pulse to between -80 and -90 mV. In the presence of an adenylate cyclase inhibitor, SQ-22536 (100 microM), the intracellular dialysis of ATP also produced a shift in the voltage-dependence of Ih in rat DRG neurons, indicating that the effect of ATP was not caused by cAMP converted by adenylate cyclase. Intracellular dialysis of a nonhydrolysable ATP analog, AMP-PNP or ATP-gamma-S, also produced a positive shift in the voltage-dependence of Ih activation, suggesting that the effect of ATP results from its direct action on the channel protein. These results indicate that cytosolic ATP directly regulates the voltage dependence of Ih activation as an intracellular modulating factor.