Modification of Current Transmitted From Apical Dendrite to Soma by Blockade of Voltage- and Ca2+-Dependent Conductances in Rat Neocortical Pyramidal Neurons

Peter C. Schwindt and Wayne E. Crill

Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington 98195-7290

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Schwindt, Peter C. and Wayne E. Crill. Modification of current transmitted from apical dendrite to soma by blockade of voltage- and Ca2+-dependent conductances in rat neocortical pyramidal neurons. J. Neurophysiol. 78: 187-198, 1997. The axial current transmitted to the soma during the long-lasting iontophoresis of glutamate at a distal site on the apical dendrite was measured by somatic voltage clamp of rat neocortical pyramidal neurons. Evidence for voltage- and Ca2+-gated channels in the apical dendrite was sought by examining the modification of this transmitted current resulting from the alteration of membrane potential and the application of channel-blocking agents. After N-methyl-D-aspartate receptor blockade, iontophoresis of glutamate on the soma evoked a current whose amplitude decreased linearly with depolarization to an extrapolated reversal potential near 0 mV. Under the same conditions, glutamate iontophoresis on the apical dendrite 241-537 µm from the soma resulted in a transmitted axial current that increased with depolarization over the same range of membrane potential (about -90 to -40 mV). Current transmitted from dendrite to soma was thus amplified during depolarization from resting potential (about -70 mV) and attenuated during hyperpolarization. After Ca2+ influx was blocked to eliminate Ca2+-dependent K+ currents, application of 10 mM tetraethylammonium chloride (TEA) altered the amplitude and voltage dependence of the transmitted current in a manner consistent with the reduction of dendritic voltage-gated K+ current. We conclude that dendritic, TEA-sensitive, voltage-gated K+ channels can be activated by tonic dendritic depolarization. The most prominent effects of blocking Ca2+ influx resembled those elicited by TEA application, suggesting that these effects were caused predominantly by blockade of a dendritic Ca2+-dependent K+ current. When cells were impaled with microelectrodes containing ethylene glycol-bis(beta -amino-ethyl ether)-N,N',N'-tetraacetic acid to prevent a rise in intracellular Ca2+ concentration, blockade of Ca2+ influx altered the tonic transmitted current in different manner consistent with the blockade of a inward dendritic current carried by high-threshold-activated Ca2+ channels. We conclude that the primary effect of Ca2+ influx during tonic dendritic depolarization is the activation of a dendritic Ca2+-dependent K+ current. The hyperpolarizing attenuation of transmitted current was unaffected by blocking all known voltage-gated inward currents except the hyperpolarization-activated cation current (Ih). Extracellular Cs+ (3 mM) reversibly abolished both the hyperpolarizing attenuation of transmitted current and Ih measured at the soma. We conclude that activation of Ih by hyperpolarization of the proximal apical dendrite would cause less axial current to arrive at the soma from a distal site than in a passive dendrite. Several functional implications of dendritic K+ and Ih channels are discussed.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

In previous studies employing long-lasting iontophoresis of glutamate at a distal site on the apical dendrite, we observed two types of responses in the soma: spike responses during the onset of the iontophoresis, and a subsequent graded, tonic current (Schwindt and Crill 1995-1997). We found that the amplitude of the tonic axial current transmitted from dendrite to soma increased as the soma was depolarized. Part of this amplification was caused by activation of a tetrodotoxin (TTX)-sensitive, noninactivating Na+ current (INaP) in the dendrites, and part was caused by the voltage dependence of the current flowing through N-methyl-D-aspartate (NMDA)-preferring glutamate channels at theiontophoretic site (Schwindt and Crill 1995, 1996). The iontophoretically evoked spikes were local Ca2+ spikes that normally were restricted from active propagation along the dendrite by a tetraethylammonium chloride (TEA)-sensitive K+ current (Schwindt and Crill 1997). Because adequate dendritic depolarization can cause Ca2+ influx through voltage-gated Ca2+ channels in much or all of the dendritic tree (Markram et al. 1995; Schiller et al. 1995; Stuart and Sakmann 1994; Yuste et al. 1994), we hypothesized that, in addition to their involvement in dendritic spikes, the dendritic Ca2+ and K+ channels also may influence the amplitude of the tonic component of the axial current transmitted to the soma.

To investigate this question, we used the same method employed in previous investigations. We used the iontophoresis of glutamate to depolarize a region of the apical dendrite at a known, fixed distance from the soma. The glutamate iontophoresis allowed us to evoke a graded, long-lasting dendritic depolarization in the presence of pharmacological agents that would block or alter synaptic transmission. If voltage-gated channels are present on the apical dendrite, they may be opened (or closed) by the glutamate-evoked depolarization. During the glutamate-evoked dendritic depolarization we voltage clamped the soma to isolate and measure directly the axial current that was transmitted from the dendritic site to the soma. By keeping soma membrane potential constant (by voltage clamp) during the glutamate-evoked dendritic depolarization we ensured that the dendritic depolarization caused no change in the baseline activity of voltage-gated channels in the soma and axon. We examined the behavior of the axial current transmitted from dendrite to soma during two experimental manipulations: alteration of somatic membrane potential and application of specific channel-blocking agents. Voltage is well controlled (clamped) only at the soma, but an imposed change in soma potential alters membrane potential along a portion of the apical dendrite (Rall and Segev 1985; Spruston et al. 1993). We have presented evidence that we can alter dendritic membrane potential out to >= 300-400 µm from the soma by altering somatic membrane potential (Schwindt and Crill 1995). Thus by voltage clamping the soma to different DC potentials we indirectly change membrane potential in a portion of the apical dendrite. A change of dendritic membrane potential during the same dendritic glutamate iontophoresis may alter 1) the gating of dendritic voltage-gated channels and 2) the net dendritic membrane current, and therefore 3) the axial current flowing from dendrite to soma. Voltage-dependent changes of transmitted current can thus provide evidence for dendritic voltage-gated channels, and the channel type may then be identified with the use of specific channel-blocking agents.

Our rationale for detecting dendritic voltage-gated channels with the use of this indirect method is as follows: although direct recording of dendritic channel activity proves that voltage-gated channels exist in a region, it is difficult to judge whether the channels are dense enough or distributed widely enough to significantly influence dendritic electrical signals. Our indirect approach reveals whether voltage-gated dendritic channels do in fact alter the signal delivered to the soma and how they alter it.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Methods were similar to those described previously (Schwindt and Crill 1995-1997). Sprague-Dawley rats of either sex (21-35 days postnatal) were anesthetized with ketamine (150 mg/kg) and xylazine (10 mg/kg) and killed by carotid section. A coronal section of cortex 0-3 mm posterior to bregma was isolated, and slices 350 µm thick were prepared and maintained as described. Recorded cells in this study lay 0.89-1.30 mm below the pial surface (mode: 1.18 mm) and 2.04-3.18 mm from midline (mode: 2.78 mm), corresponding to layer 5 of areas HL and FL of sensorimotor cortex (Zilles and Wree 1985). Three cells in this study were recovered after being injected with biocytin (0.5% in 2.7 M KCl) and visualized after standard histological processing. Each had a pyramidal-shaped soma in deep layer 5 and an apical dendrite extending to the pial surface with a terminal tuft and with the first major branch point 500-600 µm from the soma (see Fig. 1A of Schwindt and Crill 1997).


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FIG. 1. Comparison of responses to glutamate iontophoresis on soma and apical dendrite of same cell. A1: currents recorded in soma (top) during voltage clamp at 2 holding potentials (middle) in response to 1-s iontophoresis (bottom) applied to soma. Delta I: amplitude of current through glutamate-sensitive channels at end of 1-s iontophoresis. Holding potential records show membrane potential actually measured during voltage clamp, not just command potential. A2: plot of glutamate current amplitude (Delta I) measured at different somatic holding potentials for cell of A1. Best-fit line (least-squares method) was fitted to data points. B1: traces from same cell (arranged as in A1) when iontophoresis was applied to apical dendrite 307 µm from soma. Current scale is half that of A1. In this and following figures Delta I measures amplitude of axial current transmitted from iontophoretic site to soma at end of 1-s iontophoresis. B2: plot of transmitted current amplitude (Delta I) obtained at different somatic holding potentials for conditions of B1. Dashed lines and associated labels: values used to quantify amplification of transmitted current during depolarization and effective slope of relation between transmitted current amplitude and soma potential (Delta I-V relation, see text). In this and following figures, solid curves were fit to data points with the use of 1st- or 2nd-order least-squares fits as visual guide to trend of data. They have no theoretical significance. Bathing solution in both A and B contained 100 µM D-2-amino-5-phosphonopentoic acid (APV).

Recordings were made in a chamber with the slice submerged and maintained at 33-34°C. Slices were perfused with a physiological saline consisting of (in mM) 130 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 dextrose saturated with 95% O2-5% CO2, pH 7.4. D-2-amino-5-phosphonopentoic acid (APV, 100 µM) or 10 µM MK801 or 3 mM CsCl was added to this perfusate. TEA (10 mM) was substituted for 10 mM NaCl. To block voltage-gated Ca2+ channels, one of the following ion substitutions was employed in a particular experiment: 1 or 2 mM MnCl2 or 2 mM NiCl2 or 200 µM CdCl2 was substituted for CaCl2 in an equimolar amount and NaH2PO4 was omitted to avoid precipitation.

Cells were impaled with sharp microelectrodes made from standard borosilicate tubing (1.0 mm OD). In most experiments the microelectrodes contained 2.7 M KCl (DC resistance 30-40 MOmega ). In some experiments they were filled with 2.5 M KCL and 100 mM ethylene glycol-bis(beta -amino-ethyl ether)-N,N',N'-tetraacetic acid (EGTA) and buffered to pH 7.2 with 30 mM 3-(N-morpholino)propanesulfonic acid.

During iontophoresis, somatic membrane potential was maintained constant with the use of an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) in single-electrode voltage-clamp mode with a switching rate of 2.5-5.5 kHz (30% duty cycle). Somatic membrane potential was maintained at a DC level for1-2 s before the iontophoresis was applied.

The second current-clamp amplifier and headstage of the same Axoclamp-2A was used to pass a constant current through the iontophoretic microelectrode. Iontophoretic microelectrodes were broken to a tip diameter of ~2 µm and filled with 0.5 M sodium glutamate adjusted to pH 7.4 with NaOH. Negative iontophoretic currents of 15-70 nA (mode: -50 nA) were employed from a +5-nA holding current. Positive iontophoretic currents were tested but never produced a postsynaptic response. The iontophoretic electrode was positioned with the use of a separate micromanipulator, and an effective site was found near a line extending from the recording electrode normal to the pial surface. Vertical electrode movements of ~10 µm caused response amplitude to vary from zero to maximum. Postsynaptic responses, evoked by an iontophoresis 1 s in duration repeated each 15-20 s for the duration of the experiment, were stable and reproducible (cf. Hu and Hvalby 1992). The distance between recording and iontophoretic electrodes was measured at the slice surface with the use of a calibrated eyepiece on a dissecting microscope.

Membrane potential, membrane current, and iontophoretic current were monitored, amplified, and recorded on a multichannel video cassette recorder with pulse code modulation (Neuro-Data, New York, NY). Membrane potential and injected current were filtered at 2-10 kHz. Membrane current evoked during voltage clamp was filtered at 100 Hz. Resting potential was taken as the difference between the intracellular and extracellular potentials recorded on a chart recorder. Recorded data were played back into a storage oscilloscope for photography or digitized for further analysis by computer.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Cell properties

Cell impalements >= 1 h in duration were required because of the time needed first to find an effective dendritic site and then to collect control data and apply a series of blocking agents. Cells were accepted for analysis only if they exhibited a stationary resting potentials and responses to iontophoresis both during the control period and after the application of each drug. Data were obtained from 36 cells meeting these criteria. In these 36 cells resting potential ranged from -67 to -80 mV (mode: -70 mV) and was little affected by most blocking agents employed. All cells displayed a sag of membrane potential back toward resting potential during the application of a 1-s hyperpolarizing current pulse, but rarely during depolarization (see Fig. 1B of Schwindt and Crill 1997). Plots of membrane potential versus injected current were well fit by two lines intersecting near resting potential (see Fig. 1C of Schwindt and Crill 1997). The slopes of these lines gives input resistance, which, at the end of the 1-s pulse, averaged 27.6 MOmega for depolarization (range: 13.2-90.1 MOmega ) and 15.8 MOmega for hyperpolarization (range: 6.5-38.3 MOmega ). On average, steady-state input resistance during depolarization was 1.7 times greater than during hyperpolarization. A sufficiently large injected current pulse 1 s in duration evoked tonic repetitive firing in all cells. Thirty-five percent of the cells displayed an initial burst of action potentials at the onset of the pulse.

Different responses to somatic versus dendritic glutamate iontophoresis

We would expect the activation of voltage-independent (non-NMDA) glutamate channels by the iontophoresis of glutamate on an isopotential region of the neuron, such as the soma, to evoke a current that decreases as the voltage-clamped region is depolarized toward reversal potential. In contrast, activation of non-NMDA channels at a distal dendritic site should result in a current arriving at the soma whose variation with membrane potential depends on the voltage-dependent properties of the region between the iontophoretic site and the soma. To confirm this interpretation, we iontophoresed glutamate on the somata (i.e., on a membrane under good voltage-clamp control) of four cells. In practice, the tip of the iontophoretic electrode was positioned within 20 µm of the recording electrode on the slice surface and lowered until a glutamate response was obtained when it was near the same depth as the recording microelectrode. Figure 1A illustrates our results. The current caused by opening of glutamate-sensitive channels was taken as the difference between the steady holding current at each potential and the current at the end of a 1-s glutamate iontophoresis (Delta I in Fig. 1A1). In these four experiments 100 µM APV was present to block NMDA receptors (which would otherwise cause the glutamate current to exhibit voltage dependence in the presence of extracellular Mg2+) (cf. Mayer et al. 1984). Under these conditions glutamate current amplitude decreased with depolarization (Fig. 1A2). A line was fit to the data points, as in Fig. 1A2, and extrapolated to zero current to estimate reversal potential. This extrapolated reversal potential was within 4 mV of zero in each of the four cells, which is similar to the reversal potential of the current evoked either by glutamate iontophoresis or by electrically evoked excitatory postsynaptic potentials (EPSPs) in hippocampal pyramidal neurons in the research of Hablitz and Langmoen (1982).

In the cell of Fig. 1 we were able to locate an effective site on the apical dendrite after performing the somatic iontophoresis (Fig. 1B). Again, Delta I was measured as the difference between baseline holding current and the current at the end of the 1-s iontophoresis (Fig. 1B1), but in this situation Delta I is not a direct measure of the glutamate current at the iontophoretic site. Rather, Delta I is the axial current arriving at the soma after traveling along the imperfectly clamped dendrite. Delta I will have this meaning in all subsequent figures, and is referred to as "transmitted current." At a given holding potential Delta I amplitude cannot be influenced by voltage-gated channels in soma or axon because somatic voltage is held constant during the iontophoresis. Any contribution of somatic or axonic voltage-gated channels to the baseline holding current is subtracted from our measurement of Delta I.

To alter the activity of dendritic voltage-gated channels, we alter dendrite membrane potential indirectly (in an uncontrolled manner) by changing soma membrane potential to a different DC potential via voltage clamp. We then repeat the iontophoresis to measure the amplitude of the transmitted current at the new potential. We will refer to the relation between transmitted current amplitude and soma potential as the "Delta I-V relation." In the cell of Fig. 1, with APV still present, transmitted current from the dendrite increased as the soma (thus the dendrite) was depolarized (Fig. 1B2). This increase of dendritic transmitted current with somatic depolarization (which we call "amplification") is inconsistent with a passive dendrite and must be due to activation of voltage-gated dendritic channels.

The amplification of transmitted current was seen in every cell examined. Amplification was quantified as the increase in transmitted current at firing level (the somatic potential at which a spike was initiated during a long injected current pulse) above the transmitted current at resting potential expressed as a percent of the transmitted current at resting potential [(Delta IFL - Delta IRP)/Delta IRP: see dashed lines in Fig. 1B2], where RP refers to resting potential and FL to firing level. Typically, firing level was ~20 mV positive to resting potential. When measured in physiological saline (see METHODS), amplification in individual cells ranged from 28% to 267% (mean: 99%), which is similar to values obtained previously (Schwindt and Crill 1995, 1996). In our experiments voltage-clamp depolarizations were confined to values less than or equal to firing level because larger depolarizations evoked fast, large, TTX-sensitive, inward action currents that the discontinous single-electrode voltage clamp was unable to fully control. Our inability to control these action currents may result both from their initiation in the axon initial segment (a region not expected to be under voltage control) and the temporal limitations of the single-electrode voltage clamp.

In most cells, we examined the response to glutamate iontophoresis at a single dendritic site and usually to a single iontophoretic current strength. The iontophoretic sites ranged from 241 to 537 µm from the soma (mode: 370 µm), and most were 330-400 µm from the soma. Because the first major branch point of the apical dendritic occurred 500-600 µm from the soma in biocytin-stained cells (see METHODS), it is likely that all of our iontophoretic sites were on the proximal half of the apical dendrite. We never obtained a robust response when exploring more distal regions, perhaps because the erratic path of the dendrites after the branch point and their small diameter prevented us from finding an effective site within a reasonable search time.

In the present experiments we wanted to depolarize the dendritic site sufficiently that dendritic voltage-gated currents having a high activation threshold would be evoked if present. Thus we chose an iontophoretic current large enough to evoke low-rate repetitive firing in most (70%) of the cells (and a just-subthreshold response in the remainder) when recorded in current clamp. The amplitude of the transmitted current measured during voltage clamp at resting potential averaged 380 pA (range: 120-780 pA).

Blockade of TEA-sensitive, voltage-gated K+ channels

We have provided evidence that localized dendritic Ca2+ spikes actively propagate along the apical dendrite after dendritic K+ currents were reduced by application of 10 mM TEA (Schwindt and Crill 1997). Neocortical neurons exhibit several voltage-gated K+ conductances that are sensitive to TEA (Foehring and Surmeier 1993; Spain et al. 1991). These TEA-sensitive K+ channels are first activated near -50 mV, so the dendritic membrane need not be excessively depolarized to activate these channels. We tested the idea that dendritic voltage-gated K+ channels may be activated tonically during dendritic depolarization by examining the effect of 10 mM TEA on the amplitude of the tonic transmitted current. To ensure that the TEA affected only voltage-gated K+ channels, we first blocked voltage-gated Ca2+ channels, and thus any Ca2+-dependent K+ current [IK(Ca)] by partial or full substitution of other divalent cations for Ca2+ (see METHODS). Adequate Ca2+ channel blockade by the divalent cation substitution was confirmed by our inability to evoke Ca2+ spikes by somatic depolarization or by iontophoresis in the presence of 10 mM TEA and 1 µM TTX after the divalent cation substitution, whereas Ca2+ spikes always could be evoked in the presence of TEA and TTX if the Ca2+ channel-blocking cations were not present (cf. Reuveni et al. 1993; Schwindt and Crill 1997; Stafstrom et al. 1985; Yuste et al. 1994). When these cells were depolarized adequately (by current injection or iontophoresis) after the addition of TEA and blockade of Ca2+ channels, an initial action potential was followed by a prolonged, TTX-sensitive plateau depolarization (data not shown) as described previously by Stafstrom et al. (1985). The cells' steady-state current-voltage relations (obtained from DC voltage clamp of the soma in the absence of iontophoresis) exhibited greater inward rectification as membrane potential approached firing level after TEA application (data not shown). Nevertheless, the triggering of plateau depolarizations or corresponding uncontrolled action currents was prevented if membrane potential was kept negative to firing level.

After Ca2+ channel blockade, TEA application caused a major alteration in the Delta I-V relation in each of five cells tested. Figure 2 shows two examples that represent opposite ends of the spectrum of Delta I-V modifications that we observed. After TEA application in the cell of Fig. 2A, transmitted current amplitude became larger than in control solution at more negative potentials. In this cell the slope of the Delta I-V relation changed from negative to positive. That is, transmitted current amplitude decreased with depolarization, similar to the response observed when glutamate was iontophoresed on the soma (cf. Fig. 1A2). Three other cells tested with TEA exhibited similar behavior. The Delta I-V slope changed from negative to positive in one, from negative to zero in another, and to a less negative value in the last. In the four cells in which changes of Delta I-V relations were similar to those in Fig. 2A, the average reduction of Delta I-V slope by TEA was 107% (i.e., the average slope changed from negative to a small positive value). In addition, transmitted current amplitude became larger than control at more negative potentials in each of these cells (averaging 31% larger than control at resting potential). These effects of TEA were similar whether 100 µM APV was present (2 cells) or absent (2 cells) in both Cd2+-containing control and test solutions. TTX (1 µM) was added to the test solution in two of these cells, and the slope of the Delta I-V relation changed from negative or zero to positive (data not shown), indicating that activation of a persistent inward Na+ current (INaP) countered the slope-reducing effect of TEA. TEA altered the Delta I-Vrelation in the cell of Fig. 2B in a different way from that in the other four cells. Delta I became greater than control at less negative potentials and was unchanged from control at more negative potentials. This TEA-enhanced amplification of transmitted current was essentially eliminated by the subsequent addition of TTX. The iontophoretic distance for this cell (307 µm from the soma) was in the same range as for the other four cells tested (241-407 µm; mean: 313 µm).


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FIG. 2. Effect of 10 mM tetraethylammonium chloride (TEA) on Delta I. Plots in A and B are from 2 different cells and illustrate different effects of TEA on transmitted current. Response in A was typical of most cells in that Delta I in TEA was greater than control at more negative potentials. In both experiments all solutions contained 100 µM APV, and divalent cations were partially or fully substituted for Ca2+ (200 µM Cd2+ in A; 2 mM Ni2+ in B) to block Ca2+ influx and Ca2+-dependent K+ currents. In B, 1 µM tetrodotoxin (TTX) was also added in last solution change. Iontophoretic sites were 277 and 307 µm from soma for cells of A and B, respectively.

The fact that the Delta I-V relations were significantly altered by TEA after the blockade of Ca2+ channels allows us to conclude that TEA-sensitive, voltage-gated K+ channels in the apical dendrite influence the transmission of tonic axial current to the soma. A separate question is how the reduction of dendritic K+ currents could result in the specific alterations of the Delta I-V relations that we observed. In the DISCUSSION we take up this question and argue that the alterations of the Delta I-V relations shown in Fig. 2 are consistent with the reduction of a voltage-gated K+ current in a dendritic membrane that also can generate a voltage-gated inward current.

Blockade of Ca2+ influx through voltage-gated Ca2+ channels

Evidence from calcium imaging studies suggests that voltage-gated Ca2+ channels exist on the apical dendrite, and that these channels may be activated even by single subthreshold EPSPs (Magee and Johnston 1995; Magee et al. 1995; Markram and Sakmann 1994). In addition, dendritic depolarization can evoke dendritic Ca2+ spikes (Schwindt and Crill 1997). We tested the hypothesis that Ca2+ influx through dendritic voltage-gated Ca2+ channels also may influence the transmission of tonic axial current to the soma by examining the effects of Ca2+ channel blockade on the Delta I-V relation. The voltage-gated Ca2+ channels were blocked by partial or full substitution of other divalent cations for Ca2+ (see METHODS).

Twenty-three trials were performed on 18 cells. Multiple trials in the same cell involved returning to control solution after the divalent cation substitution and observing the effect of TTX, perhaps followed by a second cation substitution in the presence of TTX, or by comparing effects of more than one iontophoretic current strength in each solution to see whether the effect of Ca2+ blockade varied with the degree of dendritic depolarization. We found that the blockade of Ca2+ influx often had little or no effect on the Delta I-Vrelation. Because of the variable effectiveness of Ca2+ blockade, in 17 of the 23 trials we compared the effect of blocking Ca2+ influx with a much more consistent effect, the blockade of INaP with TTX.

Both TTX application and divalent cation substitution (when it was effective) decreased the slope of Delta I-V relation (Fig. 3, B-D). To quantify this effect, we calculated an effective slope of the Delta I-V relation as (Delta IFL - Delta IRP)/(VFL - VRP) (see dashed lines in Fig. 1B2). This slope was measured in each solution (e.g., control, TTX, Ca2+ blockade), and the percent reduction in effective slope was calculated with respect to the immediately preceding solution.The histograms of Fig. 4A show that the magnitude of Delta I-Vslope reduction depended on which type of voltage-gated channel (Na+ or Ca2+) was blocked first. If TTX was applied first, subsequent Ca2+ blockade usually caused little or no reduction of Delta I-V slope, whereas Delta I-V slope usually was reduced if Ca2+ influx was blocked first. In about half of the trials 100 µM APV was present in both control and test solutions, but its presence or absence made no difference in these results. Because it represents an average, the left histogram in Fig. 3A does not adequately express the variability of Ca2+ blockade in reducing Delta I-V slope. In the seven trials where TTX was applied first, it accounted for essentially all the slope reduction (as suggested by the right histogram in Fig. 3A), but it was equally effective in 4 of 10 trials where Ca2+ influx was blocked first. Among these 10 trials, blockade of Ca2+ influx caused a slope reduction greater than TTX in only 3 of the trials and a reduction equivalent to TTX in 3 other trials. When the 17 trials where Ca2+ and Na+ influx were blocked sequentially were separated into groups according to whether TTX was more effective, whether Ca2+ blockade was more effective or whether both blocking agents produced equivalent slope reductions, there was no significant difference among these groups in iontophoretic distance, amplification in control solution, or transmitted current measured at resting potential in control solution (1-way analysis of variance, P > 0.1).


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FIG. 3. Effects of divalent cation substitution on Delta I. A: histogram summarizing average reduction of Delta I-V relation slope observed when voltage-gated Na+ and Ca2+ channels were blocked serially by 1 µM TTX and by full or partial substitution of divalent cation for Ca2+ (Ca block), respectively. Effectiveness of Ca2+ blockade depended on whether it occurred before Na+ blockade. Error bars: means ± SE. B-D: examples of Delta I-V relations obtained by substituting 1 mM Mn2+ for 1 mM Ca2+ (Mn) on Delta I-V relations in 2 different cells. Records in D were obtained from cell of C after return to normal physiological saline. Iontophoretic sites were 307 and 333 µm from soma for cells of B, C, and D, respectively.


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FIG. 4. Effect of divalent cation substitution on Delta I after intracellular injection of ethylene glycol-bis(beta -amino-ethyl ether)-N,N',N'-tetraacetic acid (EGTA). A-C are from 3 different cells impaled for approx 0.5 h with recording microelectrode containing 100 mM EGTA before records were taken. A: superimposed records of current (bottom) measured during 1-s glutamate iontophoresis (-20 nA, not shown) at 2 different somatic holding potentials (top). Vertical arrow: time before earliest current spike where Delta I was measured in both control and test solutions to construct Delta I-V relations (see text). After substitution of 2 mM Mn2+ for 2 mM Ca2+ (Mn), transmitted current was reduced below control value (saline) and current spike was abolished at -58 mV, but transmitted current was unaffected at -72 mV. B: Delta I-V relations from another cell showing that effects of substituting Mn2+ for Ca2+ were reversible, and Delta I at most negative potentials was unaffected by Mn2+ substitution. C: Delta I-V relations from another cell where 100 µM APV was present in all solutions and 200 µM Cd2+ was substituted for equimolar Ca2+. Final addition of 1 µM TTX caused transmitted current to decrease with somatic depolarization. Iontophoretic sites were 370, 315, and 370 µm from soma for A-C, respectively.

Possible reasons for the greater effectiveness of TTX in reducing Delta I-V slope when it was applied first and the variability in results from cell to cell will be taken up in the DISCUSSION. Here we note that the highly nonlinear effect of sequential application of these blocking agents eliminates the possibility of making a quantitative assessment of the contribution of the blocked channel type to the Delta I-V relation. If the Delta I-V relation was significantly altered after application of a blocking agent, the corresponding channel type must have played a role in shaping the control Delta I-V relation, but the percent change in Delta I-V slope need not be directly related to the contribution of the blocked channel to the control Delta I-V relation.

The slope of the Delta I-V relation was reduced (by 50.1% on average) by divalent cation substitution in 14 of the 23 trials. (The effects of TTX were not tested in 6 of these 14 trials.) The plots of Fig. 3, B-D, illustrate the effects of divalent cation substitution seen in the majority (10 of 14) of these trials in which Ca2+ blockade reduced Delta I-V slope. In these 10 trials Ca2+ blockade not only decreased Delta I-V slope but also caused Delta I to become larger than control at more negative potentials, similar to the most common effect of TEA (cf. Fig. 2A). In the cell of Fig. 3B, for example, the Delta I-V slope became shallower and Delta I amplitude became greater than control at more negative potentials. Ca2+ blockade also caused Delta I to become smaller than control at less negative potentials; i.e., the control and test Delta I-V relations crossed. Figure 3C illustrates this effect and shows that it was reversible. After Ca2+ blockade in these 10 trials Delta I averaged 21% larger than control at resting potential and 17% smaller than control at firing level (which was ~20 mV positive to resting potential). Figure 3D shows the results of serial TTX application and Mn2+ substitution in the cell of Fig. 3C after its return to control solution. Both TTX application and Mn2+ substitution decreased the slope of the Delta I-V relation, and the residual amplification of transmitted current was abolished by addition of MK801. The Mn2+ substitution caused Delta I to become larger than control at more negative potentials even when TTX was present. Because the effects of Ca2+ blockade were similar to the most common effects of TEA, we hypothesized that the increase of Delta I at more negative potentials was caused by blockade of a K+ current, a Ca2+-dependent K+ current [IK(Ca)] in this case.

To test the idea that the alteration of the Delta I-V curve by blockade of Ca2+ influx was caused primarily by blockade of dendritic IK(Ca), we impaled five cells with microelectrodes containing 100 mM EGTA (see METHODS) to prevent the rise in intracellular Ca2+ concentration that activates IK(Ca). Shortly after impalement with the EGTA-containing electrodes, all five cells exhibited abnormal excitability consisting of repetitive bursts of action potential in response to constant current injection (cf. Friedman and Gutnick 1989). To be effective, however, the EGTA needed to diffuse from the soma to the site of iontophoresis, and 0.5 h was allowed for this diffusion before iontophoretic data were obtained.

In four of the five EGTA-injected cells, divalent cation substitution reduced Delta I-V slope (by 58% on average) without changing Delta I amplitude at the most negative potentials examined. Examples are shown in Fig. 4. In these four cells a slow "current spike" was evoked during iontophoresis in control solution when the somatic membrane potential was held positive to resting potential, and this spike was abolished reversibly by divalent cation substitution (Fig. 4A). The appearance of this Ca2+ spike in the EGTA-injected cells was probably related to blockade of IK(Ca). Its appearance precluded our usual measurement of Delta I at the end of the 1-s iontophoresis. Thus Delta I-V plots were constructed with the use of Delta I amplitude at a fixed time (in both control and test solutions) before the occurrence of the earliest Ca2+ spike in the test solution (as indicated, e.g., by the vertical arrow in Fig. 4A). Figure 4B shows that the alteration of the Delta I-V curve by divalent cation substitution was reversible and that there was no change in Delta I at the most negative potentials (see also Fig. 4A, bottom current traces). Figure 4C makes the same point in another cell and shows that, with APV present in all solutions, the residual amplification was abolished by the addition of TTX, and the Delta I-V relation then resembled that seen when glutamate was iontophoresed on the soma (cf. Figs. 1A2 and 2A). In the fifth cell injected with EGTA (not shown), divalent cation substitution caused a small increase of transmitted current at negative potentials, similar to the effect of Mn2+ relative to TTX in Fig. 3D. It is possible that insufficient EGTA diffused into the dendrite of this cell.

Contribution of high-threshold Ca2+ current

The alteration of the Delta I-V relation observed after blockade of Ca2+ influx might have been due entirely to blockade of IK(Ca). Although the alteration of the Delta I-V relation shows that Ca2+ entered the dendrite during depolarization, it possible that the inward Ca2+ current was too small to play any direct role in the amplification of transmitted current. Rather, Ca2+ influx may have served only to activate IK(Ca), which by itself would tend to reduce the amplification of transmittedcurrent. In contrast, the effect of Ca2+ blockade on the Delta I-Vrelation of cells injected with EGTA was similar to the effect of adding TTX to physiological saline: Delta I amplitude was smaller than control during depolarization and identical to control at more negative potentials. This result is consistent with the blockade of a depolarization-activated inward dendritic current. Results from the EGTA-injected cells suggest, therefore, that Ca2+ current can contribute to the amplification of tonic transmitted curent. The contribution of the Ca2+ current to amplification may be exaggerated in the EGTA-injected cells, however, because a normally present IK(Ca) was blocked.

We obtained evidence that dendritic Ca2+ current can contribute to the amplification of transmitted current from four cells not injected with EGTA. Examples of responses to Ca2+ blockade in two of these cells are shown in Fig. 5. Our conclusion that Ca2+ current contributes to amplification in these cells rests on the fact that their responses to divalent cation substitution resembled those of the EGTA-injected cells. Delta I amplitude was smaller than control during depolarization and identical to control at more negative potentials. The Ca2+ influx in these cells probably activated less IK(Ca) than in most cells tested for unknown reasons. The cell of Fig. 5B was tested with the use of two iontophoretic currents. When the smaller current was used, Cd2+ substitution had no effect on the Delta I-V relation. The larger iontophoretic current resulted in a larger amplification in control solution, and the Cd2+ substitution caused Delta I amplitude to become smaller than the control during depolarization. Similarly, Mn2+ substitution in the cell of Fig. 5A caused Delta I to become smaller than control only during depolarization, whereas TTX application caused Delta I to become smaller than control at all potentials except the most negative. The data from both cells are consistent with the idea that the Ca2+ current contributing to amplification of steady-state transmitted current flows through high-threshold-activated (HVA) Ca2+ channels. For example, if the affected dendritic region was ~20 mV more depolarized than the soma in the cell of Fig. 5A, TTX would begin to affect transmitted current when the local dendritic potential was about -60 mV, and Mn2+ would start to have its effect when the local dendritic potential was about -45 mV. These correspond to potentials where INaP and HVA Ca2+ current first appear when measured at the soma (Brown et al. 1993, 1994; Sayer et al. 1990; Stafstrom et al. 1985).


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FIG. 5. High-voltage-activated Ca2+ channels may contribute to amplification of Delta I. A: Delta I-V relations from cell in which addition of 1 µM TTX and substitution of 1 mM Mn2+ for 1 mM Ca2+ were performed serially. TTX reduced transmitted current over full range of potentials examined. Further reduction of Delta I by Mn2+ substitution (with TTX still present) occurred only positive to -70 mV. B: Delta I-V relations from another cell in which 1 µM TTX and 100 µM APV were present in all solutions. Substitution of 200 µM Cd2+ for equimolar Ca2+ had no effect on transmitted current resulting from smaller iontophoretic current (-50 nA). Larger iontophoretic current (-60 nA), presumably causing larger dendritic depolarization, enhanced transmitted current positive to -65 mV and this enhancement was abolished by Cd2+ substitution. Iontophoretic sites were 307 and 389 µm from soma for A and B, respectively.

Blockade of hyperpolarization-activated cation current

In most cells examined in this and previous studies (Schwindt and Crill 1995, 1996), we observed that the transmitted current decreased (was attenuated) when the soma was hyperpolarized, even after the depolarizing amplification was greatly reduced or eliminated by blocking agents (Fig. 6A). We hypothesized that dendritic hyperpolarization-activated cation current (Ih) channels opened during hyperpolarization, increased dendritic slope conductance, and thereby allowed some of the transmitted current to leak out of the dendritic membrane before it reached the soma. Ih was present in all recorded cells judging by the membrane potential "sag" observed during hyperpolarizing current pulses (see Fig. 1B of Schwindt and Crill 1997) (Fig. 6C, inset, top trace 1). Because extracellular Cs+ is known to block Ih (Mayer and Westbrook 1983; Spain et al. 1987), we examined the effect of 3 mM extracellular Cs+ on the hyperpolarizing attenuation after first blocking all other known voltage-gated inward currents: Na+ currents by TTX, Ca2+ currents by substituting Mn2+ for Ca2+, and NMDA current by MK801 or APV. As seen in Fig. 6B, the hyperpolarizing attenuation of transmitted current survived this combination of blocking agents but was abolished (reversibly) by extracellular Cs+. Figure 6C shows the effect of the Cs+ application on somatic responses of the same cell. Hyperpolarizing rectification (measured from the steady-state somatic holding current at each potential) was decreased negative to -60 mV; resting potential hyperpolarized (Fig. 6C, inset, traces 2 vs. traces 1); membrane potential sag was abolished, and input resistance increased. All these effects signify that Cs+ abolished Ih at the soma. Presumably, Cs+ eliminated hyperpolarizing attenuation of transmitted current (Fig. 6B) by the same mechanism, namely, by the blockade of dendritic Ih. A similar elimination of hyperpolarizing attenuation by Cs+ was observed in each of six cells tested.


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FIG. 6. Effect of extracellular Cs+ on attenuation of Delta I during hyperpolarization. A: attenuation of transmitted current at potentials negative to -70 mV was unchanged after TTX abolished amplification of transmitted current at less negative potentials. B: different cell in which hyperpolarizing attenuation of transmitted current was unaffected by presence of several blocking agents (1 µM TTX, 10 µM MK801, and substitution of 2 mM Mn2+ for 2 mM Ca2+). Subsequent addition of 3 mM extracellular Cs+ abolished attenuation. C: data from cell of B under same experimental conditions. Holding current [I(soma)] recorded at each holding potential was reduced at potentials negative to -60 mV after Cs+ application. Inset: resting potentials and voltage responses (top) evoked by hyperpolarizing injected current pulses (bottom) before (traces 1) and after (traces 2) Cs+ application. Iontophoretic sites were 278 and 307 µm from soma for cell of A and cell of B and C, respectively.

These data suggest that Ih channels are present in the apical dendrite, but they shed no light on whether Ih channels extend as far as the iontophoretic site. The Ih channels close when membrane potential is positive to about -60 mV (Mayer and Westbrook 1983; Spain et al. 1987). Because the membrane at the iontophoretic site is significantly depolarized by glutamate, it is likely that Ih channels, if present at that site, would always be closed during the iontophoresis and thus unaffected by Cs+. Dendritic Ih channels nearest the soma probably are responsible for the attenuation of transmitted current during somatic hyperpolarization.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Our results confirm the idea that the amplification of the current transmitted to the soma from a site on the apical dendrite is a consistent feature of neocortical pyramidal cells. Previously, we found that the amplification of transmitted current depends partly on activation of dendritic INaP and partly on current flowing through NMDA channels at the iontophoretic site (Schwindt and Crill 1995). In the present study we have identified four other types of dendritic channels that can modify the tonic axial current transmitted to the soma, namely, TEA-sensitive voltage-gated K+ channels, Ca2+-dependent K+ channels, voltage-gated Ca2+ channels, and Ih channels.

Our detection of these dendritic channels depends not on the direct measurement of channel activity but on the measurement of transmitted current, its variation with somatic membrane potential, and the effects of channel-blocking agents. Despite this indirect measurement, it is hard to imagine how the transmitted current could exhibit voltage dependence and be altered in different, specific, voltage-dependent ways by the various channel-blocking agents by a mechanism other than altered activity of dendritic ion channels. Using glutamate iontophoresis on the soma, we have found only the expected linear decrease of current amplitude with depolarization, indicating that an unusual glutamate response in the dendritic is unlikely to be responsible for voltage-dependent effects except insofar as NMDA receptors may be stimulated at that site, an effect that was eliminated by NMDA receptor blockade. To identify the channels responsible for modification of the transmitted current, we relied on the blocking agents employed to affect specific voltage-gated channels and not, e.g., glutamate-sensitive channels. We have evidence that the blocking agents affect the appropriate channels during the experiment. For example, TTX abolished the steady-state inward rectification that was measured from the steady somatic current (see Fig. 1E of Schwindt and Crill 1995), and this rectification is known to be due to INaP activation (Stafstrom et al. 1985). Divalent cation substitution reduced evoked spike afterhyperpolarizations, known to be at least partly Ca2+ dependent (e.g., Greene et al. 1994; Schwindt et al. 1988), and increased excitability; it also blocked presumed dendritic Ca2+ spikes (Fig. 4A). Extracellular Cs+ blocked Ih-dependent effects measured at the soma in voltage clamp and current clamp. That TEA acted appropriately on dendritic membrane was documented previously (Schwindt and Crill 1997). On the basis of these considerations it seems reasonable to conclude that if a given blocking agent significantly altered the Delta I-V relation, the corresponding channel type had influenced the Delta I-V relation in the control solution. Just how the blockade of a certain type of channel leads to a specific alteration of the Delta I-V relation requires some interpretation, however, as we now discuss with respect to the blockade of K+ channels.

Theoretical modeling studies have focused on how neuronal cable properties would be altered if K+ channels were added to a passive cable or dendrite. A general conclusion of these studies is that activation of K+ channels in a region of the cable causes more axial current to leak out of that region than in a passive cable because of the increased membrane conductance caused by K+ channel activation. For a given synaptic current, less axial current is available to travel down the cable and depolarize a distal site (Jack et al. 1975; Wilson 1995). On the basis of this theoretical effect of adding K+ channels, we would expect the blockade of dendritic K+ channels to cause the opposite effect, namely, the leakage of axial current through the dendritic membrane would be reduced. Consequently, more axial current would reach the soma, and Delta I would became larger than control, as was in fact observed at more negative potentials after TEA application and Ca2+ blockade (Figs. 2A and 3, B-D).

We also observed that the negative slope of the Delta I-V relation was reduced (or converted to a positive slope) after K+ channel blockade. How can the blockade of K+ channels account for these observations? As explained above, K+ channel blockade is expected to result in less leakage of axial current through the dendritic membrane. Consider the extreme case where no axial current leaks out of the dendrite after K+ channel blockade. Dendrite and soma then become isopotential, and there is no transmitted axial current. Delta I would now reflect only the properties of the receptor-operated channels, as if the iontophoresis were directly on the soma, and we would expect Delta I to decrease with depolarization as membrane potential approaches glutamate reversal potential (Fig. 1A2). If the control Delta I-V curve had a negative slope, it would now have a positive slope (Fig. 2A). This change of the Delta I-V curve would occur even when the dendrites also can generate an inward current. The inward dendritic current can amplify the axial current transmitted to the soma only if there is an axial current. If blockade of the K+ channels makes the cell isopotential, membrane potential at the iontophoretic site and the soma become identical and there would be no axial current, as explained above. The inward current generated by the dendrite would contribute only to the baseline current that is subtracted out of the measurement of Delta I.

In reality, dendrite and soma are not expected to become perfectly isopotential after K+ channel blockade, but they may become more isopotential. Delta I would become larger than control at negative potentials and the slope of the Delta I-V relation would become less negative than the control or even positive. In many cells tested after Ca2+ blockade, Delta I became smaller than the control values during depolarization (Fig. 3, B-D). Whether the control and test Delta I-V curves cross in this manner would depend partly on how nearly isopotential the cell becomes after K+ channel blockade (i.e., how much the Delta I-V slope shifts toward a positive value).

The inward dendritic current clearly continues to influence the slope of the Delta I-V relation after reduction of K+ currents because its subsequent blockade (e.g., by TTX) caused the Delta I-V slope to become less negative (Figs. 2B and 3D) or even positive (Fig. 4C). Alteration of the Delta I-V curve by blockade of inward current is one indication that the cell was not perfectly isopotential after K+ channel blockade. Furthermore, it is possible that voltage control of the dendritic site may not improve significantly after K+ channel blockade. This would depend on the spatial extent and the magnitude of the dendritic K+ conductance and the degree to which the conductance is reduced by the blocking agent. For example, K+ channels could exist around the iontophoretic site but be sparse or nonexistent for much of the region between that site and the soma. In this case, the net inward dendritic current at the iontophoretic site may become larger after K+ channel blockade both because it is unopposed by outward current and because the local membrane potential is not under good control. This enhanced inward current at the dendritic site would then cause the axial current transmitted to the soma to become larger than control, but only at the depolarized potentials where the inward dendritic current is activated. Amplification of transmitted current would increase in this situation. We presume this is the explanation for the behavior of the cell of Fig. 2B, namely, that K+ channel blockade resulted in a greater net inward current at the iontophoretic site but did not result in improved voltage control of the site.

Because our observations are consistent with the presence of dendritic K+ channels, additional predictions of theoretical studies are of interest, such as the prediction that K+ channel activation will limit the amplitude of synaptic depolarization in the region where the channels are activated (Wilson 1995). This limitation may provide a "safety valve" to prevent deleterious effects of excessive depolarization, such as excessive Ca2+ influx. This limitation would also prevent local membrane potential from approaching reversal potential of the glutamate current. Saturation of the glutamate current would be prevented, and a wider range of dendritic membrane potential would be available over which increased synaptic input results in increased synaptic current, an effect sometimes called "linearization" (Bernander et al. 1994; Wilson 1995). Another predicted effect of K+ channel activation is the electrotonic isolation of the depolarized region from adjacent regions (Wilson 1995). Evidence that the dendritic K+ channels support this function was presented previously (Schwindt and Crill 1997).

Blockade of Ca2+ influx produced less consistent and often smaller effects on the tonic transmitted current among different cells than TTX application. This variability may derive simply from the extent to which the dendrites were depolarized in a given experiment, particularly if we assume that HVA Ca2+ channels cause most of the Ca2+ influx during tonic dendritic depolarization. INaP is first activated at about -60 mV (Brown et al. 1994; Stafstrom et al. 1985), but HVA Ca2+ currents are not activated until about -45 mV (Brown et al. 1993; Sayer et al. 1990). It is conceivable that activation of INaP is required for dendritic membrane potential to depolarize to the level where HVA Ca2+ channels are activated. Thus blocking INaP would preclude adequate depolarization for HVA Ca2+ current activation, and no effect of subsequent Ca2+ channel blockade would be apparent even though Ca2+ influx occurred in the control response. This idea would explain our finding that Ca2+ blockade usually had little or no effect on the Delta I-V relation when TTX was applied first. According to the above idea, these negative results have no bearing on whether dendritic Ca2+ influx occurred in normal saline. This question can be answered only by first blocking Ca2+ influx. In most cells where this was done, an effect on the Delta I-V curve was apparent. The remaining variability in the effects of Ca2+ blockade in this set of cells may result from differences among pyramidal neurons or dendrites in the density or distribution of the Ca2+ or K+ channels.

A role of the inward Ca2+ current in shaping the Delta I-V relation was difficult to detect, but this difficulty was not entirely unexpected. A minor role for dendritic Ca2+ current during long-lasting depolarization seems reasonable considering the properties of Ca2+ currents observed during voltage clamp. LVA Ca2+ channels would inactivate completely during long-lasting depolarizations positive to about -70 mV (Sayer et al. 1990). In contrast, a tonic HVA Ca2+ current is maintained for >= 10 s in neocortical neurons during long-lasting voltage steps, but after only 1 s of depolarization the Ca2+ current becomes much smaller than the peak current at the onset of the depolarization (Brown et al. 1993). Thus only HVA channels would be active during long-lasting depolarization, and the steady Ca2+ current would be relatively small. We found evidence consistent with a role of HVA Ca2+ currents in aiding depolarizing amplification in EGTA-injected cells where IK(Ca) was reduced and in some normal cells where IK(Ca) may have been small. Evidence that a relatively large dendritic depolarization was required to activate this Ca2+ current was obtained in cells where the effect of Ca2+ blockade was apparent only at larger iontophoretic strength (i.e., dendritic depolarization). In most cells the principal role of Ca2+ influx in modifying transmitted current during tonic dendritic depolarization appeared to be activation of IK(Ca), which would reduce amplification. Evidence for dendritic IK(Ca) activation also has been obtained from intradendritic recordings in hippocampal pyramidal cells (Andreasen and Lambert 1995).

Because voltage-gated channels require a minimum depolarization to activate significantly, a minimum amplitude and duration of dendritic depolarization may be required for their effect on transmission of axial current from dendrite to soma to become significant, i.e., they may not significantly affect the transmission of synaptic current resulting from a single, small EPSP. This would be particularly true for Ca2+ and K+ channels that have slower onset kinetics and a higher activation voltage than, e.g., INaP. We would expect their influence on transmitted current to be most important during large, tonic dendritic depolarization of the type we employed. Increased activity of most of the dendritic channels identified in this study would tend to reduce transmission of current to the soma. Activation of both the Ih conductance (Gh) by hyperpolarization and the K+ conductance by depolarization would result in a shunt of axial current through the dendritic membrane before it reached the soma. It seems remarkable, therefore, that amplification of the steady-state transmitted current is normally observed over the subthreshold range of somatic membrane potentials. This may result from the fact that INaP is relatively unopposed by steady Ca2+ [thus IK(Ca)] or K+ currents over this range of membrane potentials. These latter currents are activated more strongly during larger depolarizations and may serve to attenuate the transmission of current from dendrite to soma during large dendritic depolarizations. We were unable to test this idea in the present study. We indirectly depolarized the dendrite by depolarizing the soma, and the range of somatic depolarization was limited by the inability of the single-electrode voltage clamp to control action currents evoked by large somatic depolarizations.

We found that activation of Gh is responsible for the hyperpolarizing attenuation of transmitted current. A small shunting action of Gh also may occur at resting potential because Ih is partially activated at resting potential, as evidenced e.g., by the membrane potential hyperpolarization observed when Cs+ was added to block Ih (Fig. 8C, inset) (Spain et al. 1987). Spike afterhyperpolarizations or inhibitory postsynaptic potentials reduce excitability in part by moving membrane potential away from spike threshold. This inhibitory effect of hyperpolarization may be reinforced by activating Ih in the proximal dendrite and thereby shunting excitatory current flowing from the distal dendrite. Our method is incapable of determining whether Gh exists at more distal dendritic sites. If it does (see Spruston et al. 1995), modulation of Gh by noradrenalin or serotonin would depolarize the neuron (McCormick and Pape 1990), allow dendritic depolarization to activate dendritic INap more easily, and thereby more readily amplify excitatory currents flowing into the soma. In fact, each type of dendritic current identified in this study (Ih, HVA Ca2+ current, and various voltage- and Ca2+-dependent K+ currents) has been shown to be subject to modulation by certain neurotransmitters. Ca2+ currents are decreased by most neuromodulators, but different K+ currents may increase or decrease (e.g., Anwyl 1991; Nicoll 1988). In light of the role that dendritic ion channels can play in altering the current transmitted from dendrite to soma, it is possible that the modulation of these channels could have a profound effect. In particular, the efficacy of transmission from remote dendrites might be controlled by this means. The effects of neuromodulation on transmitted current remain to be discovered.

    ACKNOWLEDGEMENTS

  We thank G. Hinz and P. Newman for excellent technical assistance.

  This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-16792 and the Keck Foundation.

    FOOTNOTES

  Address for reprint requests: P. C. Schwindt, Dept. of Physiology and Biophysics, University of Washington School of Medicine, Box 357290, Seattle, WA 98195-7290

  

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society