Journal of Neurophysiology

Wang, X.; Treistman, S. N.; Lemos, J. R.

doi: N/Apmid: 8283218

Abstract 1. Ca2+ currents through single channels in acutely dissociated nerve terminals from rat neurohypophyses were recorded using cell-attached patch recordings with 110 mM Ba2+ as the charge carrier. 2. One type (Nt, where the t denotes terminal) of single Ca2+ channel current was evoked only by depolarizing steps from holding potentials less negative than -50 mV. Because this channel opened primarily at the beginning of a 180-ms-long voltage pulse, the averaged ensemble current decayed rapidly (approximately 30 ms). Infrequently, the channel opened throughout such a long pulse, resulting in a long-lasting averaged ensemble current. The averaged channel open time constant (tau) was 0.34 ms and the two averaged closed time constants were 1.78 (tau 1) and 86.57 (tau 2) ms. The mean unitary slope conductance for this channel was 11 pS and its threshold for activation was approximately -10 mV. 3. The other type (L) of single Ca2+ channel current could be evoked in isolation by depolarizations from holding potentials more positive than or equal to -50 mV. This channel opened throughout an entire 180-ms-long voltage pulse. The averaged ensemble current, therefore, showed little inactivation. The averaged channel open-time constant was 0.49 ms and the two average closed time constants were 2.02 (tau 1) and 79.91 (tau 2) ms. The mean unitary slope conductance for this channel was 25 pS. 4. Bay K 8644 (5 microM), a dihydropyridine (DHP) Ca2+ channel agonist, increased the open probability of the larger-conductance L-type Ca2+ channel by prolonging the average duration (to 2.79 ms) of channel openings, but did not alter the single channel slope conductance. In contrast, the same concentration of Bay K 8644 did not affect the smaller-conductance Nt-type Ca2+ channel. The DHP Ca2+ channel antagonist nicardipine (5 microM), but not nifedipine (5 microM), reduced the open probability of the large-conductance L-type Ca2+ channel by shortening the duration (to 0.36 ms) of channel openings. 5. The voltage- and time-dependent properties of these two types of single Ca2+ channel currents are in close agreement with those of the two components of macroscopic Ca2+ currents previously reported using the "whole-terminal" recording method. Therefore these two types of single channels appear to underlie the macroscopic current. 6. Our studies suggest that the terminal Nt-type Ca2+ channel differs from the conventional somatic N- and T-type Ca2+ channels in some respects, and that the terminal L-type Ca2+ channel is similar to the conventional somatic L-type Ca2+ channel.(ABSTRACT TRUNCATED AT 400 WORDS) Copyright © 1993 the American Physiological Society

Nabekura, J.; Ebihara, S.; Akaike, N.

doi: N/Apmid: 8283213

Abstract 1. The effects of acetylcholine (ACh) on granule cells freshly dissociated from rat dentate gyrus (DG) were studied using the nystatin perforated patch technique. This method allowed us to study ACh-induced currents (IACh) under voltage clamp without "run-down" of the ACh response. In some experiments, we used the conventional whole-cell method for intracellular application of drugs not permeable to cell membrane. 2. At a holding potential of -40 mV, ACh induced an outward current. The amplitude of IACh increased in a sigmoidal fashion with increasing ACh concentration. The half-maximal response and the Hill coefficient determined from the relation between ACh concentration and response were 4.98 x 10(-7) M and 1.70, respectively. 3. The reversal potential of IACh was close to the K+ equilibrium potential. The IACh was accompanied by an enhancement of the K+ current. 4. Muscarine and McN-A-343 mimicked the ACh response, whereas oxotremorine induced no response. 5. Muscarinic antagonists reversibly suppressed the IACh (10(-5) M) in a concentration-dependent manner, where the values of half-inhibition concentration (IC50) were 1.03 x 10(-6) M for pirenzepine and 2.21 x 10(-5) M for AF-DX-116. 6. Intracellular perfusion with GDP-beta S suppressed the IACh greatly. The IACh persisted in the neurons pretreated with an external solution containing pertussis toxin (IAP) for 18 h. 7. In the neurons perfused with Ca(2+)-free external solution containing 2 mM ethylene glycol-O,O'-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 10 mM Mg2+, the first application of ACh induced the IACh with an amplitude similar to that in the standard solution.(ABSTRACT TRUNCATED AT 250 WORDS) Copyright © 1993 the American Physiological Society

Fieber, L. A.; McCleskey, E. W.

doi: N/Apmid: 7506754

Abstract 1. Whole-cell and cell-attached patch-clamp recordings were made from enzymatically isolated type I cells from the carotid body of adult rats. Voltage-dependent K+ and Ca2+ channels were observed, but there was no detectable Na+ current. In this respect, rat carotid body cells are unlike those from rabbit, which have Na+ currents and Na(+)-dependent action potentials. 2. The observed Ca2+ channels had the following properties: 1) activation requires voltage steps above -20 mV; 2) little inactivation occurred with holding voltages below -40 mV; 3) one single-channel conductance of 21 pS was found with 90 or 110 mM Ba2+ in the cell-attached pipette and this was the only conductance observed; 4) open probability was increased by the dihydropyridine Ca2+ channel agonist Bay K 8644 and was decreased by the antagonist nifedipine; and 5) omega-conotoxin had little or no effect on the channels. These are properties expected of L-type Ca2+ channels. 3. To investigate whether these voltage-dependent channels would be available for opening on membrane depolarization, we measured the type I cell resting membrane potential noninvasively using unitary openings of the L-type Ca2+ channel with Bay K 8644 in the cell-attached pipette. Resting potentials ranged from -62 to -13 mV, with a mean of -32 mV in 12 cells. 4. Judging from single-channel conductance and pharmacology, the Ca2+ current is mostly, if not solely, carried by L channels. Thus it should be possible to use modulators of L channel activity to determine the role of Ca2+ channels in stimulus-secretion coupling in the rat carotid body. Copyright © 1993 the American Physiological Society

Carp, J. S.

doi: N/Apmid: 8283216

Abstract 1. Homonymous and heteronymous monosynaptic composite excitatory postsynaptic potentials (EPSPs) were evaluated by intracellular recordings from 89 motoneurons innervating triceps surae (n = 59) and more distal (n = 30) muscles in 14 pentobarbital-anesthetized monkeys (Macaca nemestrina). 2. Homonymous EPSPs were found in all motoneurons tested. The mean values +/- SD for maximum EPSP amplitude of triceps surae motoneurons were 2.5 +/- 1.3, 1.8 +/- 1.3 and 4.5 +/- 2.0 mV for medial gastrocnemius, lateral gastrocnemius, and soleus motoneurons, respectively. Heteronymous EPSPs were almost always smaller than their corresponding homonymous EPSPs. 3. Triceps surae EPSP amplitude was larger in motoneurons with higher input resistance. However, this relationship was weak, suggesting that factors related to input resistance play a limited role in determining the magnitude of the EPSP. 4. The mean ratio +/- SD of the amplitude of the EPSP elicited by combined stimulation of all triceps surae nerves to the amplitude of the algebraic sum of the three individual EPSPs was 0.95 +/- 0.05. This ratio was greater in motoneurons with lower rheobase. 5. Some patterns of synaptic connectivity in the macaque are consistent with previously reported differences between primates and cat (e.g., heteronymous EPSPs elicited by medial gastrocnemius nerve stimulation in soleus motoneurons are small in macaque and other primates but large in cat). However, no overall pattern emerges from a comparison of the similarities and differences in EPSPs among species in which they have been studied (i.e., macaque, baboon, and cat). That is, there are no two species in which EPSP properties are consistently similar to each other, but different from those of the third species.(ABSTRACT TRUNCATED AT 250 WORDS) Copyright © 1993 the American Physiological Society

Thoreson, W. B.; Miller, R. F.

doi: N/Apmid: 7506752

Abstract 1. Whole-cell patch-clamp recordings were obtained from ON bipolar cells in a retinal slice preparation of the mudpuppy, Necturus maculosus. The effects of excitatory amino acid (EAA) agonists applied in the presence of cobalt (2-5 mM) were examined. 2. At the holding potential of -50 mV, L-2-amino-4-phosphonobutanoic acid (L-AP4, 5-10 microM) evoked an outward current accompanied by a conductance decrease. The zero current potential of the L-AP4-evoked current was near 0 mV independent of whether the intracellular Ringer solution contained CsCl or CsCH3SO4. The currents evoked by light were also accompanied by a conductance decrease and reversed near 0 mV. Replacing external sodium with choline or N-methyl-D-glucamine generated an outward current and suppressed the response to L-AP4. The response to L-AP4 was enhanced by removing extracellular calcium and suppressed by increasing extracellular calcium. These results indicate that L-AP4 closes nonspecific cation channels that are blocked by extracellular calcium. 3. In 2 mM cobalt, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA, 50-100 microM) evoked membrane currents that were accompanied by a conductance increase. AMPA-evoked currents exhibited a significant chloride dependence and were suppressed by gamma-aminobutyric acid-A (GABAA) antagonists bicuculline and picrotoxin; a GABA uptake blocker, nipecotic acid; and a glycine antagonist, strychnine. AMPA-induced currents were virtually absent in the presence of 5 mM cobalt and nominally 0 mM extracellular calcium. These results indicate that the conductance increase induced by AMPA in the presence of 2 mM cobalt is largely the result of calcium-dependent synaptic inputs onto GABAA and glycine receptors of ON bipolar cells. 4. N-methyl-D-aspartic acid (250 microM) was ineffective when applied in the presence of 100 microM cadmium or 2 mM cobalt. 5. 1S,3R/1R,3S-1-aminocyclopentane-1,3-dicarboxylic acid (100-200 microM) evoked an outward current accompanied by a conductance decrease and appears to be an agonist at the L-AP4 receptor. 6. The findings of this study suggest that the only type of EAA receptor in mudpuppy ON bipolar cells is the L-AP4 receptor and that L-AP4 receptor activation results in the closing of nonspecific cation channels that are blocked by extracellular calcium. Copyright © 1993 the American Physiological Society

Gallemore, R. P.; Hernandez, E.; Tayyanipour, R.; Fujii, S.; Steinberg, R. H.

doi: N/Apmid: 8283221

Abstract 1. We characterized the basolateral membrane Cl- and K+ conductances of the dark-adapted chick neural retina-retinal pigment epithelium (RPE)-choroid preparation. Conventional microelectrodes were used to measure apical (V(ap)) and basolateral (Vba) membrane voltage, and double-barreled Cl- and K+ selective microelectrodes were used to follow the time course and magnitude of ion concentration changes outside the basolateral (basal) membrane. 2. In response to a fivefold decrease in basal Cl-o, Vba rapidly depolarized by 6.4 +/- 0.7 (SE) mV, and the apparent resistance of the basolateral membrane (Rba) increased. The Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) suppressed the Vba depolarization by 40% and blocked the Rba increase. Estimates of the relative Cl- conductance (transference number, TCl) from the DIDS-sensitive component of the Cl- diffusion potential gave an average value for TCl of 0.22 +/- 0.03. 3. Further evidence for a Cl- conductance was obtained by measuring changes in intracellular Cl- activity (aCli) induced by transtissue current. Depolarizing Vba elevated aiCl, whereas hyperpolarizing Vba had the opposite effect, consistent with conductive Cl- movement across the basal membrane. TCl estimated from these data averaged 0.23 +/- 0.02. 4. In response to a sixfold increase in basal K+o, Vba depolarized 6.1 +/- 0.8 mV. The amplitude of this K+ diffusion potential was inhibited 44 and 67% by 5 and 10 mM Ba2+, respectively. TK was estimated to be 0.61 +/- 0.05. 5. The rapid c-wave membrane hyperpolarizations in response to the light-evoked decrease in subretinal K+o were used to calculate the equivalent resistances of the apical membrane (R(ap)), basolateral membrane (Rba), and the paracellular shunt pathway (Rs). They were 152 +/- 10, 615 +/- 38, and 138 +/- 7 omega.cm2 (n = 11 tissues), respectively. From these data the equivalent electromotive force for the basal (Eba) and apical (Eap) membranes were estimated to be -45 +/- 2 and -77 +/- 1 mV, respectively. This estimate of Eba is in the range of that predicted from our estimates of TCl and TK, indicating that, in the dark-adapted chick retina, the resting conductance of the basal membrane can largely be accounted for by the Cl- and K+ conductances described here. Copyright © 1993 the American Physiological Society

Vogt, M. B.; Smith, D. V.

doi: N/Apmid: 8283203

Abstract 1. Although taste experience generally arises from a mixture of gustatory stimuli, most neurophysiological studies of the mammalian central gustatory system have focused on responses to single chemical stimuli. Recently, in a study of single third-order neurons in the hamster parabrachial nucleus (PbN), we reported that mixture suppression occurs in the responses to binary mixtures of sucrose and QHCl presented to the anterior tongue. Mixture suppression was reflected both in reduced response frequencies and in an altered pattern of responses across neurons. In the current report we extend our investigation of CNS neuron responses to binary mixtures of heterogeneous stimuli to include sucrose+citric acid mixtures and NaCl+citric acid mixtures. The response to each mixture was compared with the response to the more effective component (MEC) presented alone, and those that differed by more than a selected criterion (based on response variability) were identified. 2. For all mixture responses recorded, 29% (79/256) involved mixture suppression (mixture response < MEC response), only 6% (18/276) were greater than the response to MEC, and 65% (179/276) did not differ from the response to the MEC. 3. In Experiments 1 and 2, neurons were tested with four concentrations of sucrose or citric acid each presented alone and in binary mixtures with a single strong concentration of the other stimulus. Sucrose suppression (mixture response < sucrose response) occurred in 24% of mixture responses and was exhibited almost exclusively by sucrose-best neurons, primarily to the mixtures that contained the stronger sucrose and citric acid concentrations. Sucrose suppression involved a 40% reduction of mixture response frequencies compared with responses to the sucrose component alone. 4. In Experiments 3 and 4, neurons were tested with four concentrations of NaCl or citric acid each presented alone and in binary mixtures with a single strong concentration of the other stimulus. NaCl suppression (mixture response < NaCl response) occurred in 21% of mixture responses and was displayed by both sucrose-best and NaCl-best neurons. NaCl suppression involved a 28% reduction in mixture response frequencies compared with responses to the NaCl component alone. In all experiments citric acid suppression (mixture response < citric acid response) was observed in only 6% of mixture responses and was relatively small in magnitude. 5. The across-neuron patterns (ANPs) of taste responses, which are correlated with behavioral measures of taste similarity, were compared for mixtures and components.(ABSTRACT TRUNCATED AT 400 WORDS) Copyright © 1993 the American Physiological Society

Lin, X.; Hume, R. I.; Nuttall, A. L.

doi: N/Apmid: 7506758

Abstract 1. The effects of externally applied ATP and neomycin on whole cell currents of isolated guinea pig cochlear outer hair cells (OHCs) were studied using the whole cell voltage-clamp technique. In OCHs held at -70 mV, ATP activated a large inward current. In the presence of neomycin, the ATP-induced whole cell current activated along a relatively unaltered time course, but the current then decreased to a reduced steady level. The neomycin inhibition of the ATP-induced current was dose dependent. The half-inhibitory concentration (IC50) of neomycin measured at steady state was estimated to be 90 microM. 2. Neomycin inhibition of the ATP response could not be reversed by increasing the concentration of ATP, indicating that the effect was noncompetitive. The inhibition was voltage dependent and was greatly reduced when OHCs were held at positive potentials. 3. Cells treated with 100 microM ATP gave maximal current responses. Addition of neomycin substantially increased membrane current noise of the 100 microM ATP responses. When neomycin concentration was varied from 10 to 500 microM, the current noise level peaked between 50 and 100 microM. The noise increase was observed at negative holding potentials but not at positive potentials. 4. The neomycin-induced whole cell current noise was used to estimate the size of the underlying elementary current. The ATP-induced single channel current of OHCs at -70 mV was estimated to be approximately 0.3 pA. The number of ATP-activated channels in a single OHC was estimated to be in the range of a few thousand. 5. The characteristics of the neomycin inhibition of ATP-induced currents were consistent with an open channel blocking mechanism. Analysis of the voltage dependence of the steady state neomycin inhibition suggested a neomycin binding site at an electrical distance of 0.3 from the extracellular side. Copyright © 1993 the American Physiological Society

Huang, R. C.

doi: N/Apmid: 7904302

Abstract 1. Neurons were acutely dissociated from the suprachiasmatic nucleus (SCN) of adult rats and studied with whole-cell and perforated-patch recordings at room temperature. 2. Acutely dissociated SCN neurons had spherical cell bodies of 12 microns in average diameter. The recorded cells were randomly selected and had either no process (38%), one (41%), two (19%), or three processes (2%). They had a resting potential of about -60 mV, an input resistance of approximately 5 G omega, and a cell capacitance of approximately 7 pF. 3. The dissociated neurons had variable spontaneous firing rates, typically (76%) < 1 Hz. 4. Under current clamp, continuous current injection elicited repetitive action potentials. 1 microM tetrodotoxin (TTX) reduced the amplitudes of the action potentials as well as the firing rate, whereas 200 microM Cd2+ stopped repetitive firing altogether. Action potentials were completely eliminated with Cd2+ and TTX present. These results suggest that both Na+ and Ca2+ contribute to the action potential in these cells. 5. With 200 microM Cd2+ present to block calcium currents, a train of brief depolarizing pulses could still elicit repetitive sodium action potentials, but these became attenuated at stimulating frequencies as low as 1 Hz. 6. Under voltage clamp, the sodium current was activated at about -40 mV and peaked at about -10 mV. It inactivated with a time constant of approximately 0.5 ms at 0 mV, and in steady state the current was half-inactivated at about -60 mV. Recovery of the current from inactivation showed two very different phases with time constants of approximately 30 and 600 ms at -60 mV. The slow phase was probably responsible for the very low firing rate of the sodium action responsible for the very low firing rate of the sodium action potential. 7. In the absence of external sodium, depolarization-activated calcium action potentials were preferentially blocked by 20 microM Cd2+, whereas a posthyperpolarizing depolarizing (or anode break) was preferentially reduced by 100 microM Ni2+. These differential effects hinted at the presence of both low-threshold and high-threshold calcium currents in these cells. 8. Voltage-clamp experiments confirmed the presence of a low-threshold, transient calcium current that was activated by depolarizations above -70 mV. It inactivated with a time constant of approximately 25 ms between -50 and -30 mV. Steady-state inactivation was half-complete at about -90 mV and complete at about -70 mV.(ABSTRACT TRUNCATED AT 400 WORDS) Copyright © 1993 the American Physiological Society

Manabe, T.; Wyllie, D. J.; Perkel, D. J.; Nicoll, R. A.

doi: N/Apmid: 7904300

Abstract 1. Whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs) were made from guinea pig hippocampal CA1 pyramidal cells. The sensitivity of paired pulse facilitation (PPF) and EPSC variance to changes in synaptic transmission was investigated and the results were compared with the changes in these parameters evoked by long-term potentiation (LTP). 2. Presynaptic manipulations, such as activation of presynaptic gamma-aminobutyric acid-B receptors by baclofen, blockade of presynaptic adenosine receptors by theophylline, blockade of presynaptic potassium channels by cesium, and increasing the Ca(2+)-Mg2+ ratio in the external recording solution, each reliably changed PPF in a fashion reciprocal to the change in the EPSC amplitude. However, recruitment of additional synaptic release sites by increasing stimulus strength and antagonism of non-N-methyl-D-aspartate (NMDA) glutamate receptors by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) failed to alter PPF. 3. Presynaptic manipulations including increased stimulus strength gave the predicted changes in the value of mean 2/variance (M2/sigma 2). Moreover, postsynaptic manipulations that altered EPSC amplitude, including blockade of non-NMDA receptors by CNQX, or changing the holding potential of the postsynaptic cell, gave little change in M2/sigma 2, as would be predicted for manipulations resulting in a uniform postsynaptic change. 4. LTP caused no change in PPF, whereas the presynaptic manipulations, which caused a similar amount of potentiation to that induced by LTP, significantly decreased PPF. On the other hand, LTP did increase M2/sigma 2, although the increase was less than that predicted for a purely presynaptic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS) Copyright © 1993 the American Physiological Society