An examination of frog myelinated axons using intracellular microelectrode recording: the role of voltage-dependent and leak conductances on the steady-state electrical properties

1. Intracellular microelectrode current-clamp technique was used to study the steady-state membrane properties of single intact large primary afferent axons (conduction velocity > 10 m/s) attached to isolated hemisected frog spinal cord. 2. Hyperpolarizing electrotonic potentials (ETPs) had a slow complex multiphasic charging. This complex charging could be approximated by two time constants: one in the range of 70–210 ms, the other of < 20 ms. 3. Two regions of outward and inward rectification hyperpolarized to the resting membrane potential were observed, in addition to the previously characterized outward rectification active at potentials depolarized to resting membrane potential. The peak and steady-state input resistance of these axons in tetrodotoxin Ringer solution was on average 65.6 +/- 21.1 and 31.1 +/- 10.8 M omega, mean +/- SE, respectively. 4. Application of external tetraethylammonium (10–20 mM) significantly depolarized the axon and decreased the outward rectification just hyperpolarized to the resting membrane potential. This outward rectification could also be blocked by external barium ions (2–10 mM). 5. Activation of an inward or anomalous rectification in these axons was observed 300–600 ms after the start of a current pulse. In addition, a depolarizing afterpotential (DAP) (1–3 mV in amplitude, 500 ms-10 s in duration) was evident after a current pulse in which inward rectification had been activated. This DAP most likely reflected the slow inactivation of the inwardly rectifying conductance. 6. Inward rectification was blocked by external application of cesium ions (1–3 mM) but it was insensitive to external application of barium ions (2–10 mM). The blockade of the voltage attenuation was accompanied by a disappearance of the DAP and an increase in the charging time constant of the axon. This blockade resulted in a single linear voltage-current (V-I) relationship. Axons now had, on average, an input resistance of 114 +/- 19.1 M omega. 7. Reducing the concentration of external potassium ions increased both the peak and steady-state slope resistance. Reducing the external sodium concentration altered the ETPs and the V-I relationship little but it consistently reduced the magnitude and length of the DAP. These results are compatible with the hypothesis that anomalous rectification is a mixed ionic conductance dependent on potassium and sodium ions in the external media. 8. Overall, the V-I relationship of these intact axons had both linear and nonlinear regions reflecting the activity of numerous slowly activating and inactivating conductances. (ABSTRACT TRUNCATED AT 400 WORDS)

Hyperpolarization-activated currents in neurons of the rat basolateral amygdala

1. A single microelectrode was used to obtain current-clamp or voltage-clamp recordings from two neuronal cell types (pyramidal and late-firing neurons) in the basolateral nucleus of the amygdala (BLA) in slices of the rat ventral forebrain. Conductances activated by hyperpolarizing voltage steps from a holding potential of -70 mV were identified and their sensitivity to muscarinic modulation was determined using bath superfusion of carbachol. 2. Unclamped pyramidal neurons exhibited anomalous rectification, seen as a slowly developing depolarizing sag in the electronic potential in response to a hyperpolarizing current pulse. 3. Stepping voltage-clamped pyramidal neurons to command potentials of between -70 and -100 mV activated a slowly developing inward current (ISlow) that followed a single exponential time course. Larger hyperpolarizing voltage steps evoked a rapidly developing inward current (IFast) that preceded the development of ISlow. 4. The ISlow component reversed at a level positive to the -70 mV holding potential. Its rate of activation accelerated as the hyperpolarizing voltage step was made more negative. The threshold for activation of the conductance underlying ISlow was approximately -60 mV, with half-activation occurring at -90 mV. 5. Extracellular Cs+ (2 mM) blocked ISlow and eliminated anomalous rectification in unclamped pyramidal neurons. The inhibition of ISlow by Cs+ was also associated with membrane hyperpolarization and reduction of the medium afterhyperpolarization. ISlow was unaffected by extracellular Ba2+ (100 microM). The properties of this current appeared similar to that of the mixed cationic H-current previously identified in other neurons. 6. In comparison with pyramidal cells, unclamped late-firing neurons displayed a lesser but more rapidly developing anomalous rectification in response to large hyperpolarizations from rest. In voltage clamp, hyperpolarizing steps to command potentials more negative than -100 mV elicited IFast. Late-firing neurons expressed little or no ISlow. 7. The properties of IFast were identical in both pyramidal and late-firing neurons. This current reversed at a potential negative to -70 mV. Its rate of current activation increased with the magnitude of the hyperpolarizing voltage step. This rate was approximately sevenfold faster than ISlow activation recorded at the same membrane potential. IFast was blocked by 2 mM extracellular Cs+ and reduced by 100 microM extracellular Ba2+. The threshold for activation of the underlying conductance was approximately -85 mV, with half-activation occurring at -112 mV. The properties of IFast were similar to those of the inward rectifier current previously identified in other central neurons. 8. Carbachol (40 microM) largely blocked IFast without affecting its rate of activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane properties of guinea pig cingulate cortical neurons in vitro

1. The passive and active membrane properties of guinea pig cingulate cortical neurons were studied in vitro using the slice preparation. Results were reported for intracellular recordings made from neurons that were penetrated in layers V/VI of the anterior cingulate cortex areas 1 and 3. 2. The neurons had an average resting potential of -71 mV, an input resistance of 71 M omega, a spike amplitude of 93 mV, and a spike duration of 1.6 ms. The firing occurred regularly at an average rate of 13 spikes/s at the membrane potential of -55 mV, suggesting that they are probably regular spiking pyramidal cells. 3. The voltage decay following a hyperpolarizing current pulse could always be fitted by two exponentials in most cells. The slope of the charging function was analyzed to estimate the two cable theory parameters of the neurons, based on a simple Rall model: the electrotonic length (LN) of the equivalent dendritic cylinder and the conductance ratio (rho) of the dendrites to that of the soma. There were no significant differences in the LN (0.9-1.1) and the rho (2.8-3.0) of neurons in normal media and solutions containing tetrodotoxin (TTX), Cs+ and low Ca2+, indicating that the neurons may be electrically compact. 4. In most cells the steady-state current-voltage (I-V) relationship revealed three distinct types of rectification: an anomalous inward rectification in the hyperpolarizing direction, a subthreshold inward rectification, and a delayed outward rectification in the depolarizing direction. 5. The anomalous rectification was increased in high K+ solutions and was decreased in low K+ solutions. Analysis of the Ba2+ and Cs+ sensitivity confirmed that the anterior cingulate neurons had two distinct types of anomalous rectification, one that was time dependent and Ba2+ insensitive and the other that was fast and Ba2+ sensitive. Ionic analyses indicated that the time-dependent anomalous rectification was due to an increased permeability to both Na+ and K+, whereas the fast, Ba(2+)-sensitive rectification was probably only K+ dependent. 6. The subthreshold inward rectification was depressed by TTX, lidocaine, or Co2+, as well as the reduction of extracellular Na+, whereas it was augmented by extracellular Ba2+. This persistent Na(+)-Ca2+ conductance triggered the generation of Na(+)-dependent action potentials. 7. The delayed outward rectification was recorded in the potential range between -65 and -20 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane properties of rat substantia gelatinosa neurons in vitro

1. The membrane properties of substantia gelatinosa (SG) neurons in an in vitro adult rat transverse spinal cord slice preparation with attached dorsal root have been examined. Intracellular recordings were obtained from identified SG neurons. 2. Seventy-six percent of SG neurons exhibited a time-dependent anomalous rectification (AR) when the membrane was hyperpolarized from the resting potential. The time-dependent AR was blocked by cesium (Cs+, 2 mM) but not by barium (Ba2+, 2 mM). Application of Cs+ itself caused membrane hyperpolarization in those SG neurons that expressed the time-dependent AR. The activation of the time-dependent AR was maximal at potentials 5-10 mV below the resting membrane potential. 3. In a few SG neurons, the current-voltage relationship revealed a marked inward rectification, even though there was no detectable time-dependent anomalous rectification during hyperpolarization. Analysis of the Ba2+- and Cs+-sensitivity of these neurons confirmed that SG neurons expressed two distinct ARs, one of which is fast and Ba2+-sensitive and the other of which is time-dependent and Ba2+-insensitive. 4. Fifty-one percent of SG neurons exhibited a transient outward rectification when hyperpolarizing current pulses were applied from potentials more positive than -60 mV or when depolarizing pulses were applied from potentials more negative than -65 mV. The transient outward rectification persisted for 0.3-2 s when hyperpolarizing pulses were applied at -55 mV. 5. The transient outward rectification was associated with a decrease in membrane resistance and was enhanced in low K+ solutions. 4-aminopyridine (4-AP, 2 mM) reversibly blocked the transient outward rectification. 6. The time-dependent anomalous and transient outward rectifying currents exerted opposite effects on the firing properties of SG neurons. Activation of the time-dependent AR increased neuronal excitability. In neurons that exhibited the time-dependent AR, membrane depolarization caused the appearance of a rebound depolarization that resulted in the generation of spikes with only a short delay after application of the depolarizing pulse. In contrast, the transient outward rectifying current markedly delayed spike firing in response to depolarizing pulses. This delay was blocked by application of 4-AP. 7. The diversity in response properties of subpopulations of SG neurons may result in part from this heterogeneity in membrane properties.

Anomalous rectification in neurons from cat sensorimotor cortex in vitro

The ionic mechanisms underlying anomalous rectification in large neurons from layer V of cat sensorimotor cortex were studied in an in vitro brain slice. The anomalous rectification was apparent as an increase of slope conductance during membrane hyperpolarization, and the development of anomalous rectification during a hyperpolarizing current pulse was signaled by a depolarizing sag of membrane potential toward resting potential (RP). Voltage-clamp analysis revealed the time- and voltage-dependent inward current (IAR) that produced anomalous rectification. IAR reversal potential (EAR) was estimated to be approximately -50 mV from extrapolation of linear, instantaneous, current-voltage relations. The conductance underlying IAR (GAR) had a sigmoidal steady-state activation characteristic. GAR increased with hyperpolarization from -55 to -105 mV with half-activation at approximately -82 mV. The time course of both GAR and IAR during a voltage step was described by two exponentials. The faster exponential had a time constant (tau F) of approximately 40 ms; the slow time constant (tau S) was approximately 300 ms. Neither tau F nor tau S changed with voltage in the range -60 mV to -110 mV. The fast component constituted approximately 80% of IAR at each potential. Both IAR and GAR increased in raised extracellular potassium [( K+]o) and EAR shifted positive, but the GAR activation curve did not shift along the voltage axis. Solutions containing an impermeable Na+ substitute caused an initial transient decrease in IAR followed by a slower increase of IAR. Brain slices bathed in Na+-substituted solution developed a gradual increase in [K+]o as measured with K+-sensitive microelectrodes. We conclude that GAR is permeable to both Na+ and K+, but the full contribution of Na+ was masked by the slow increase of [K+]o that occurred in Na+ substituted solutions. Chloride did not appear to contribute significantly to IAR since estimates of EAR were similar in neurons impaled with microelectrodes filled with potassium chloride or methylsulfate, whereas, ECl (estimated from reversal of a GABA-induced ionic current) was approximately 30 mV more positive with the KCl-filled microelectrodes. Extracellular Cs+ caused a reversible dose- and voltage-dependent reduction of GAR, whereas intracellular Cs+ was ineffective. The parameters measured during voltage clamp were used to formulate a quantitative empirical model of IAR.(ABSTRACT TRUNCATED AT 400 WORDS)