Na+ action potentials propagate into the dendrites of pyramidal neurons driving an influx of Ca2+ that seems to be important for associative synaptic plasticity. During repetitive (10–50 Hz) firing, dendritic action potentials display a marked and prolonged voltage-dependent decrease in amplitude. Such a decrease is not apparent in somatic action potentials. We investigated the mechanisms of the different activity dependence of somatic and dendritic action potentials in CA1 pyramidal neurons of adult rats using whole-cell and cell-attached patch-clamp methods. There were three main findings. First, dendritic Na+ currents decreased in amplitude when repeatedly activated by brief (2 msec) depolarizations. Recovery was slow and voltage-dependent. Second, Na+ currents decreased much less in somatic than in dendritic patches. Third, although K+ currents remained constant during trains, K+ currents were necessary for dendritic action potential amplitude to decrease in whole-cell experiments. These results suggest that regional differences in Na+ and K+ channels determine the differences in the activity dependence of somatic and dendritic action potential amplitudes.
Pyramidal neurons often initiate action potentials in the axosomatic region (Turner et al., 1991; Stuart and Sakmann, 1994; Spruston et al., 1995; Colbert and Johnston, 1996b). Once the action potential fires, it propagates not only along the axon but also “backward” throughout much of the dendritic arbor (Turner et al., 1991; Spruston et al., 1995). These backpropagating dendritic action potentials are primarily mediated by Na+ ions but drive an influx of Ca2+ ions that may provide a sufficient postsynaptic signal for associative synaptic plasticity (Jaffe et al., 1992; Markram and Tsodyks, 1996; Magee and Johnston, 1997). Unlike somatic action potentials, which remain relatively constant in amplitude, dendritic action potentials display an activity-dependent decrease in amplitude that is voltage-dependent and slow in its recovery (Callaway and Ross, 1995; Spruston et al., 1995;Tsubokawa and Ross, 1996a). These decreases in amplitude are accompanied by reduced Ca2+ influx (Callaway and Ross, 1995; Spruston et al., 1995) and are, therefore, likely to determine some of the time- and activity-dependent properties of synaptic plasticity. The mechanism of the decrease in dendritic action potential amplitude, however, is not known. A number of candidate mechanisms have been considered in the theoretical literature (Migliore, 1996).
The present study was aimed at identifying properties of voltage-gated ion channels that might underlie the activity-dependent decrease in dendritic action potential amplitude. We hypothesized that repetitive activation of voltage-gated channels, as would occur during a train of dendritic action potentials, might alter channel properties. Using cell-attached patch-clamp techniques, we observed isolated currents in patches from the soma and dendrites of hippocampal pyramidal neurons under conditions of repetitive activation. Dendritic K+ currents showed no activity-dependent change in amplitude during trains of brief depolarizations. Dendritic Na+ currents, however, displayed a striking decrease in amplitude during such trains. The recovery from this attenuation was slow and voltage-dependent. Somatic Na+ currents also decreased during trains but to a much lesser degree. These results represent the first electrophysiological evidence of regional differences in somatic and dendritic Na+ channel properties in pyramidal neurons (cf. Stuart and Sakmann, 1994; Magee and Johnston, 1995).
The finding that somatic Na+ currents decrease during trains, although to a lesser degree than dendritic Na+ currents, seemed inconsistent with the constant somatic action potential amplitude reported previously (Callaway and Ross, 1995; Spruston et al., 1995; Tsubokawa and Ross, 1996a). Thus, we tested whether factors other than the decrease in Na+ current might contribute to the decrease in dendritic action potential amplitude. We found that the relationship between Na+ current and action potential amplitude became stronger as the Na+/K+permeability ratio decreased. Thus, in the dendrites, in which the density of A-type K+ channels is high compared with the density in the soma (Hoffman et al., 1997), the effect of losing Na+ current during a train is more pronounced. Thus, regional differences in both density and electrophysiological properties of voltage-gated channels provide the basis for the different activity dependence of somatic and dendritic action potential amplitude.
Portions of this work have appeared in abstract form (Colbert and Johnston, 1996a).
MATERIALS AND METHODS
Preparation and solutions. The present study used 4–10-week-old male Sprague Dawley rats. Animals were anesthetized with a lethal dose of a combination of ketamine, xylazine, and acepromazine. Once deeply anesthetized, they were perfused through the heart with cold, modified artificial CSF (ACSF) containing (in mm): 110 choline-Cl, 2.5 KCl, 1.2 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7.0 MgCl2, and 20 dextrose. After removal of the brain, 400-μm-thick slices were cut using a Vibratome (Lancer), incubated submerged in a holding chamber for 30 min at 32°C, and stored submerged at room temperature.
During the slicing procedure, the slices were maintained in the same ACSF used for the perfusion. The external recording solution contained (in mm): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2.0 CaCl2, 1.0 MgCl2, and 25 dextrose. Slices were maintained in a submerged holding chamber in normal external recording solution. All external solutions were bubbled continuously with 95%O2/5%CO2. The internal pipette solution used for whole-cell recordings contained (in mm): 120 potassium gluconate, 20 KCl, 10 HEPES, 0.4 EGTA, 4.0 NaCl, 4.0 Mg2ATP, 0.3 Mg2GTP, and 14 phosphocreatine. pH was adjusted to 7.25 with KOH. The pipette solution for cell-attached patch recordings of Na+ currents contained (in mm): 120 NaCl, 30 tetraethylammonium (TEA) chloride, 10 HEPES, 2 CaCl2, 3 KCl, 1 MgCl2, and 5 4-aminopyridine (4-AP). pH was adjusted to 7.4 with NaOH. For recording K+ currents, the pipette contained (in mm): 125 NaCl, 10 HEPES, 2 CaCl2, 1 MgCl2, and 2.5 KCl, plus 1 μm tetrodotoxin (TTX). pH was adjusted to 7.4 with NaOH.
Recording techniques. Recordings were made from somata and dendrites of hippocampal CA1 pyramidal neurons. Neurons were visualized using infrared-illuminated, differential interference contrast (DIC) optics (Axioskop; Ziess) according to standard techniques (Stuart et al., 1993). Whole-cell patch recordings were made using microelectrode amplifiers (Axoclamp 2A, Axon Instruments; IX2-700 Dagan Instruments) in bridge mode. Cell-attached patch recordings were made using a patch-clamp amplifier with a capacitive headstage (Axopatch 200A or 1D; Axon Instruments). Pipettes (3–5 MΩ for whole-cell and 7–12 MΩ for cell-attached) were made from borosilicate glass and pulled using a P-87 Flaming-Brown pipette puller (Sutter Instruments). Except as noted, cell-attached patch recordings were made submerged at room temperature (∼25°C). Membrane potentials were determined by rupturing the patch after the channel data were recorded. Whole-cell recordings were made submerged at ∼35°C. Whole-cell series resistance was 6–20 MΩ for somatic recordings and 15–50 MΩ for dendritic recordings. Whole-cell recordings were low-pass filtered at 3 kHz (6 dB/octave) and digitized at 10 kHz. Cell-attached patch recordings were filtered at 2 kHz (eight-pole Bessel filter) and sampled at 10 kHz. Data were digitized at 16-bit resolution (ADC488/16; IOTech) and stored by computer for off-line analysis (Next Computer). Antidromic action potentials were stimulated by constant current pulses (Neurolog; Digitimer Ltd. or WPI Instruments) through tungsten electrodes (AM Systems) placed in the alveus. In some experiments, trains of backpropagating action potentials were evoked by brief depolarizing current pulses through the somatic electrode. Significance was determined by t test with p < 0.05 considered significant. Data are reported as mean ± SEM.
Dendritic action potential amplitudes decrease during trains
Simultaneous whole-cell recordings from the soma (Fig.1 A) and dendrites (Fig.1 B) of CA1 pyramidal neurons demonstrated a number of differences between somatic and dendritic action potentials. First, single action potentials were larger in the soma (88 ± 1.4 mV;n = 8) than in the apical dendrites at ∼200μm from the soma (50 ± 4.5 mV; n = 8). Second, evoked at rates of 5–50 Hz, action potentials maintained a relatively constant amplitude in the soma (Vm , Fig.1 A) but decreased with successive action potentials in the dendrites (V m, Fig.1 B). The amplitude ratio of the 10th to the first action potential was 97 ± 1% (n = 8) in the soma and 59 ± 2% in the dendrites. The maximum rate of rise of the somatic action potential was more sensitive than its amplitude, revealing that the somatic action potential changed throughout the train (dVm /dt, Fig.1 A,C). The dV m/dt ratio of the 10th to the first action potential was 86 ± 2%. In contrast, the rate of rise of the dendritic action potential (dV m/dt, Fig.1 B) decreased similarly to its amplitude (Fig.1 C). The dV m/dtratio of the 10th to the first dendritic action potential was 50 ± 2%. Thus, action potentials displayed quite different activity dependencies in the soma and dendrites of CA1 pyramidal neurons.
Na+ channel current decreases during trains of simulated action potentials
To identify the underlying changes in voltage-gated currents that determine the frequency dependence of action potentials during trains, we took the approach of observing pharmacologically isolated currents in cell-attached patches of somatic and dendritic membranes. Patches typically had 7–20 channels, estimated from peak currents and unitary current amplitudes (Magee and Johnston, 1995; Hoffman et al., 1997). We simulated trains of action potentials in the cell-attached patches by trains (20 or 50 Hz) of brief depolarizations (2 msec; Fig.2 A). Ensemble averages were constructed from 20–40 individual sweeps. The change in the amplitudes of the ensemble currents was quantified as the relative amplitude of the current evoked by the 10th step in the train compared with the amplitude of the ensemble current evoked by the first step.
The first channel type that we explored was the Na+channel. As described in our previous studies (Magee and Johnston, 1995; Colbert and Johnston, 1996b), we isolated Na+currents by including 4-AP and TEA in the patch pipette. Because of the fast kinetics of Na+ channels, the channels activated and substantially inactivated during the 2 msec command potential (Fig. 2 B). From a holding potential equal to the resting potential of the cell (approximately −67 mV), ensemble Na+ currents decreased in amplitude during trains. Consistent with the change in dV m/dt from the whole-cell recordings (compare Fig. 1), the decrease was modest in somatic patches but quite striking in dendritic patches (Soma and Dendrite, Fig.2 A,B). This difference was apparent at both 20 and 50 Hz frequencies. In the dendrites, the ratio of the 10th current amplitude to the first was 38 ± 3% (n = 16) during the 50 Hz train and 37 ± 3% (n = 11) during the 20 Hz train (Fig. 2 C). Na+ currents evoked in somatic patches decreased to 63 ± 5% (n = 11) at 50 Hz and 73 ± 4% (n = 12) at 20 Hz.
To determine whether the decrease in Na+ current during trains was dependent on holding potential, we held the patch at 20–30 mV hyperpolarized to its rest potential. With the hyperpolarized holding potential, the Na+ current decreased significantly less than when held at rest (Fig. 2 C). In the soma the current decreased by the 10th step to 85 ± 3% (n = 4) at 50 Hz and to 84 ± 3% (n = 10) at 20 Hz. In the dendrites the current decreased to 49 ± 6% (n = 7) at 50 Hz and to 47 ± 4% (n = 5) at 20 Hz. Thus, the decrease in both somatic and dendritic Na+ channels was voltage-dependent. The difference in the magnitude of the decrease in the soma and dendrites, however, could not be explained by differences in resting potential. Even when held at 20 mV hyperpolarized to rest, the dendritic Na+ currents decreased more than the somatic Na+ currents held at rest.
Spruston et al. (1995) reported that the recovery of action potential amplitude in the dendrites after a train of action potentials was slow and voltage-dependent. If the loss of Na+ current during trains was responsible for the loss of action potential amplitude in the dendrites, then the recovery of the Na+ current amplitude after a train should be slow as well. To test this hypothesis, we observed the recovery of Na+ current amplitude after a 20 Hz train of brief depolarizations. After giving a train, we waited a variable length of time (100, 400, 800, or 1600 msec) and then gave a final brief test depolarization (Fig. 3 A). The minimum duration before a new train was given was 5 sec. To quantify the time course of recovery, we first normalized the amplitude of each test current in the train to the amplitude of the first current. We then scaled the amplitude of each test current (Fig. 3 B) between the amplitude of the 10th current in the 20 Hz train (i.e., 0% recovery) and the amplitude of the first current in the 20 Hz train (i.e., 100% recovery). Finally, these normalized values were fit with single exponentials to determine an approximate time constant of recovery. Dendritic Na+ currents recovered at rest potential with a single-exponential time constant of 5.6 sec (Fig.3 C, Dendrite). Somatic Na+ currents recovered with a single-exponential time constant of 4.1 sec (Soma, Fig. 3 C). That the time constant of recovery for the Na+ current was measured in seconds suggests that the decrease in Na+ current was more consistent with reported slow inactivations of the Na+ channel than with accumulation of fast inactivation (see Discussion).
To test for voltage dependence of recovery, we repeated the recovery paradigm using a holding potential 20–30 mV hyperpolarized from rest. At this holding potential, the rate of recovery increased in both somatic and dendritic patches (Fig. 3 C). The single-exponential time constant of recovery was 0.79 sec for somatic patches and 2.2 sec for dendritic patches. As seen with the magnitude of the decrease in Na+ current, the voltage dependence of recovery of Na+ current seemed to differ between somatic and dendritic patches.
K+ currents remain constant during trains
After identifying the activity dependence of Na+ channels in cell-attached patches, we turned to the observation of K+ channels. From a negative holding potential, a long step to +40 mV evoked an outward current with two major components: a fast transient A-type current and a smaller sustained delayed-rectifier-type (DR-type) component (Fig.4 A). We have characterized these currents previously and found them to be the dominant outward currents in dendritic patches from CA1 pyramidal cells (Hoffman et al., 1997).
As described for the Na+ channels, we tested the activity dependence of the K+ channels by simulating trains of action potentials with trains of brief depolarizations of dendritic cell-attached patches. Trains of 8–10 brief depolarizations from rest to +40 mV resulted in essentially no change in the evoked current between the first and the last depolarization (0.98 ± 0.02). To provide a better comparison with the Na+current experiments and to simulate a dendritic action potential, we repeated the trains in some patches from rest to −10 mV (Fig.4 B,C). We also tried a number of different combinations of frequency and duration of the steps (up to 30 msec; data not shown). In all cases there was no change in the outward current during the train. The outward current deactivates very rapidly at the end of the 2 msec depolarization, and there does not seem to be any residual current in the waveform between the depolarizing steps (Fig. 4 B,C). These experiments additionally provide a useful technical control for the Na+ channel experiments; there is no recording artifact that results in an apparent alteration of currents during a train.
Hyperpolarization between action potentials reduces the decrease in action potential amplitude during trains
The results of the cell-attached patch experiments suggested that trains of action potentials in CA1 pyramidal neurons lead to a slowly recovering, voltage-dependent decrease in available Na+ current but no long-lasting change in K+ currents. To test the role of this decrease in Na+ current on action potential amplitude during trains, we gave transient hyperpolarizing current injections, via the dendritic pipette, between each individual action potential in a train. Such membrane hyperpolarization should reduce Na+channel inactivation during the train, allowing action potential amplitude and maximum rate of rise to be maintained. Hyperpolarizations of 20–25 mV (Fig. 5 A, arrow S) produced a graded effect on the attenuation of action potential amplitude, whereas even larger hyperpolarizations of 35–45 mV (Fig.5 A, arrow L) completely eliminated the decrease in action potential amplitude (3 ± 2% decrease; n = 5) and rate of rise (11 ± 7% decrease; n = 5) that normally occurred during trains (Fig. 5 A, arrow C). We repeated a similar paradigm at the same temperature as the whole-cell experiments using the cell-attached patches. Forty msec, 40 mV hyperpolarizing steps were given during the period between test commands to −10 mV in a train as described in Figure2 A. Without the hyperpolarizing steps, the attenuation was 55 ± 3% (n = 3). With the hyperpolarizing steps, the attenuation was reduced to 20 ± 1% (n = 3). The agreement between the channel data and the maximum rate of rise is consistent with the notion that the Na+ channels underlie the activity-dependent decrease in action potential amplitude.
We have found previously that in CA1 pyramidal neurons the dendritic A-type K+ channel density increases linearly with distance from the soma to reach a value fivefold higher than that found in the soma (Hoffman et al., 1997). Thus, we wanted to investigate the role of the relatively high density of dendritic A-type K+ channels in the decrease of action potential amplitude during trains. The elevated A-channel density determines that dendritic membrane has a Na+/K+permeability ratio that is significantly lower than that of somatic membrane. As a result, similar reductions in Na+current could potentially result in greater attenuation of action potential amplitude in the dendrites. We bath applied 4-AP (3–8 mm) to reduce the density of A-type K+channels, diminishing the somatodendritic gradient of Na+/K+ permeability. Cd2+ at 200 μm was also included to reduce Ca2+ spikes. After application of 4-AP, the decrease in action potential amplitude was reduced from 41 ± 3% to 15 ± 2% (n = 5; Fig.6 A,B), whereas the decrease in maximum rate of rise remained unchanged at 50 ± 3% (n = 5) for control versus 51 ± 4% (n = 5) with 4-AP application. These data suggest that without the high density of dendritic K+current, the decrease in Na+ current during a train of spikes [which was unchanged by 4-AP application (see dV m/dt plot in Fig.4 B)] is less effective in reducing action potential amplitude. Thus, along with the Na+ channel inactivation observed in the dendrites, the elevated dendritic A-channel density enhances the decrease in action potential amplitude during trains.
To test this idea further, we lowered the relatively high Na+/K+ permeability ratio of the somatic membrane by applying a low concentration of TTX (100 nm) to the bath (Fig. 6 C,D). TTX increased the drop-off in somatic action potential amplitude during a train from 4 ± 1% to 18 ± 3% (n = 5). Furthermore, there was a greater decrease in the maximum dVm /dt: 21 ± 2% (n = 5) for control versus 33 ± 4% (n = 5) with TTX application. Thus, lowering the Na+ channel density of somatic membrane allowed the ∼20% decrease in Na+ current occurring during a train to have a more pronounced affect on somatic action potential amplitude. However, even in the presence of TTX, the decrease in somatic action potential amplitude was never as great as that recorded in the dendritic regions under control conditions (21% in the soma vs 41% in the dendrites).
In the present study, we have identified properties of ion channels that taken together predict the different activity dependence of action potential amplitude in the soma and apical dendrites of CA1 pyramidal neurons. First, repetitive activation of Na+ channels leads to a loss of available Na+ current that has a slow and voltage-dependent rate of recovery. The magnitude of this loss of current seems to be considerably greater in the dendrites than in the soma. Second, the degree to which action potential amplitude depends on the magnitude of Na+ current is a function of the Na+/K+ permeability ratio. Therefore, the relatively high density of A-type K+channels and the relatively greater decrease in Na+current in the dendrites determine that backpropagation of action potentials into the dendritic arborization will be severely reduced during repetitive firing.
Slow Na+ channel inactivation
Slowly accumulating inactivation and slow recovery from inactivation have been described in Na+ channels from a number of preparations including skeletal muscle (Ruff et al., 1988), squid giant axon (Moore et al., 1964; Rudy, 1978), suprachiasmatic neurons (Huang, 1993), and most recently neocortical neurons (Fleidervish et al., 1996). Importantly, slow inactivation of Na+ channels has traditionally been associated with long-sustained depolarizations. Because of this association, its role as a physiological or even pathophysiological process has been questioned (Cannon, 1996). In the present study, however, the slow inactivation state is rapidly entered (i.e., within a single train) and, thus, is likely to have a physiological role (see below).
Fleidervish et al. (1996) described previously a slow inactivation of Na+ channels in somata of neocortical pyramidal neurons. Using somatic recordings, they found that multiple long depolarizations (>500 msec) decreased somatic firing frequency. Using cell-attached patches on the soma, they found that a single depolarization to approximately −10 mV required 2 sec duration to inactivate 50% of the current. Recovery from this inactivation had a rate constant similar to that seen in the present study and, thus, may represent the same process. Fleidervish et al. suggested that such slow inactivation may explain the activity dependence of dendritic action potential amplitude (Callaway and Ross, 1995; Spruston et al., 1995). The present findings make this suggestion more plausible. First, we showed that a single train of brief depolarizations, rather than sustained depolarization, greatly decreases Na+current in dendritic patches. Second, we demonstrated that the relatively low K+ channel density in the soma together with the more modest decrease in somatic Na+ current predicts the modest activity dependence of somatic action potential amplitude. Importantly, this scheme reconciles the experimentally observed uniform density of Na+ channels throughout the dendrites, soma, and initial segment (Magee and Johnston, 1995; Colbert and Johnston, 1996b) with the notion of different Na+/K+ permeability ratios in these regions. Traditionally, electrophysiological properties of pyramidal neurons consistent with a high Na+/K+ permeability ratio have been attributed to an extremely high density of Na+channels in the axon.
Differences in Na+ inactivation between soma and dendrites
The observed differences in the attenuation of Na+ current in the soma and dendrites imply regional differences in either the type of channels or their modulation. In previous electrophysiological studies of Na+channels in pyramidal neurons (Stuart and Sakmann, 1994; Magee and Johnston, 1995), no regional differences were noted in the steady-state or kinetic properties of activation or fast inactivation to suggest different channel types. Although there is evidence of segregation of Na+ channel α-subunits within the neuron [i.e., a relatively high density of type I in the somatic region and a relatively high density of type II or IIA in fiber tracts (Westenbroek et al., 1989)], type IIA apparently accounts for ∼80% of the total in all regions. Furthermore, because it is difficult to distinguish the presynaptic or postynaptic location of the subunits in the dendritic fields, it is not clear whether the distribution of α-subunits can account for the regional differences in electrophysiological properties of the Na+ channels reported here.
A number of modulations of the voltage dependence of activation and fast inactivation have been reported (reviewed in Catterall, 1992), including phosphorylation by protein kinase C (Renganathan et al., 1995; Cantrell et al., 1996) and by cAMP-dependent pathways (Li et al., 1992). Effects of these various pathways have been studied primarily in somata or in expression systems; thus differences in regional activity of the various modulators are not known. Regional differences in the activity of the various modulatory systems would provide an explanation for the differences in the magnitude of Na+ current attenuation seen here without the need to invoke regional differences in Na+ channel subtypes.
The decrease in dendritic action potential amplitude represents a time- and activity-dependent process. It is important to note that the rates of action potential generation that decrease dendritic action potential amplitude include not only high frequency burst firing but also sustained firing that is not much above background rates (Tsubokawa and Ross, 1997). Spikes that occur at 5–10 Hz will cause significant reductions in dendritic Na+ current. Furthermore, these reductions will impact not only action potential amplitude but any process that depends on Na+current, such as boosting of EPSPs or setting thresholds for action potential initiation. Although we demonstrated that a significant block of A-type K+ channels greatly minimized the effect of Na+ current attenuation on spike amplitude, significant activity-dependent attenuation of dendritic spikes still occurs under less extreme conditions in which inactivation of A-type channels boosts the amplitude of the early spikes in the train (Andreasen and Lambert, 1995; Spruston et al., 1995).
A decrease in the influx of Ca2+ ions accompanies any decrease in dendritic action potential amplitude (Jaffe et al., 1992; Callaway and Ross, 1995; Spruston et al., 1995; Tsubokawa and Ross, 1996b). Therefore, any process that depends on the influx of Ca2+ will be affected by changes in dendritic action potential amplitude. Although the relationships between Ca2+ influx and processes such as long-term potentiation are not fully understood, the backpropagating dendritic action potential has been demonstrated to be an important postynaptic signal for the induction of such processes (Magee and Johnston, 1997;Markram et al., 1997). Thus, it is probable that regulation of dendritic spike amplitude determines some of the activity dependence of the induction of various synaptic plasticities. The present result that both Na+ and K+ channels contribute to the expression of the decrease in dendritic action potential amplitude suggests two independent targets for modulation of dendritic action potential amplitude by second-messenger systems (Tsubokawa and Ross, 1997).
This work was supported by NS11535, MH44754, MH48432, and Human Frontiers in Science Program to D.J.
Correspondence should be addressed to Dr. Costa M. Colbert, Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
Dr. Magee’s present address: Neuroscience Center, Louisiana State University, Medical Center, 2020 Gravier, New Orleans, LA 70112.