 |
INTRODUCTION |
The effects of neuromodulators on CNS neurons were generally evaluated with recordings from the cell body. From this position the most interesting results concern changes in firing rate induced by changes in conductance close to the soma. For example, serotonin (5-HT) applied to hippocampal pyramidal neurons causes changes in several K+-conductances (Andrade and Nicoll 1987
; Colino and Halliwell 1987
; reviewed in Nicoll 1988
; Nicoll et al. 1990
), usually leading to a suppression of tonic firing rates.
Control of spike propagation and associated [Ca2+]i changes may be additional targets for neuromodulatory effects. In pyramidal neurons, action potentials usually are initiated in the axon hillock region. From this position they propagate orthodromically down the axon and backward into the dendritic tree (Colbert and Johnston 1996
; Spruston et al. 1995
; Turner et al. 1991
). Propagation in the apical arbor is sustained by voltage-dependent Na+ and Ca2+ channels that are distributed all over the dendrites (Magee and Johnston 1995
). Not all spikes actively spread to the tips of the dendrites (Andreasen and Lambert 1995
; Callaway and Ross 1995
; Spruston et al. 1995
), suggesting that propagation in this region is labile. Therefore, mechanisms that change dendritic conductances and potentials could affect the extent of active propagation. This kind of regulation could be important because backpropagating action potentials were implicated in the induction of some forms of long-term potentiation (Magee and Johnston 1997
; Markram et al. 1997
) and may modulate dendritic conductances (e.g., Cook and Johnston 1997
).
Previously, it was shown that synaptic inhibition could block backpropagating action potentials (Buzsaki et al. 1996
; Tsubokawa and Ross 1996
) and that cholinergic agonists could enhance backpropagation (Tsubokawa and Ross 1997
). Inhibition also decreased dramatically the associated [Ca2+]i changes in the dendrites, whereas carbachol (CCh) enhanced the [Ca2+]i changes. These results suggest that other modulatory mechanisms might affect dendritic propagation. We examine the effects of bath-applied 5-HT. We report that 5-HT exerts a different effect on backpropagating spikes. It reduces the peak spike potential in the dendrites but not in the soma. Interestingly, the spike-evoked [Ca2+]i increase was reduced by 5-HT at all locations. The reduction in the [Ca2+]i change at the soma, where the peak spike potential did not change significantly, indicates that 5-HT also reduces Ca2+ entry by directly modulating the Ca2+ channels that are opened by the action potentials. Some of these results were reported in abstract form (Sandler and Ross 1997
).
 |
METHODS |
Transverse hippocampal slices (250-300 µm thick) were prepared from 3- to 5-wk-old Sprague Dawley rats as previously described (Tsubokawa and Ross 1997
). Slices were kept at room temperature and then transferred to a submerged recording chamber maintained at 30-32°C. The normal incubation solution [artificial cerebrospinal fluid (ACSF)] was composed of (in mM) 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose, bubbled with a mixture of 95% O2-5% CO2, making the final pH 7.4.
Experiments were made in two different recording setups. In one (Callaway et al. 1995
), the chamber was set on the stage of an Olympus IMT-2F inverted microscope. Bipolar electrodes were placed either on the alveus for stimulating antidromic spikes or on the stratum oriens (SO) for generating excitatory postsynaptic potentials (EPSPs). Recordings were made with patch pipettes pulled from 1.5-mm OD diam thick-walled glass tubing (No. 1511-M, Friderick and Dimmock, Millville, NJ). The electrode was positioned with a dissecting microscope mounted over the preparation, but the dendrites were approached blindly (Blanton et al. 1989
). Tight seals were made with pipettes containing (in mM) 130 K-gluconate, 10 Na-gluconate, 4 NaCl, 2 Mg-ATP, 0.3 Na-GTP, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH-adjusted to 7.2 with KOH. In some test experiments, K-gluconate was replaced with K-methylsulfate. For simultaneous electrical and [Ca2+]i measurements, 100 µM bis-fura-2 was added to this solution. This concentration of indicator (also a Ca2+ buffer) appears to be well below the level that modulates the effect of some transmitters in other preparations (Beech et al. 1991
). No additional Ca2+ buffers were used. Similar spike potentials and responses to 5-HT were recorded with electrodes containing 100-200 µM bis-fura-2 or no indicator. Open resistance of the pipettes was 5-7 M
for somatic recording and 6-10 M
for dendritic recording. After breaking into the cell the holding current was always <0.05 nA and usually zero. Uncompensated series resistance was usually <20 M
. No correction was made for the junction potential between the bath and the pipette. Full correction for this potential would make the resting potentials ~11 mV more negative than indicated (Neher 1992
).
Some [Ca2+]i measurements on pyramidal neurons were made after loading the cells with the cell-permeant form of fura-2 (Grynkiewicz et al. 1985
). For these experiments, the indicator was prepared by dissolving fura-2-AM in dimethyl sulfoxide (DMSO; 3.3 mM), diluting to 15 µM with normal ACSF, and sonicating. Slices from 10- to 17-day-old rats were incubated with this solution for 13-15 min at 36°C. They were then washed with normal ACSF at 32°C for
30 min before any measurements were made. Fresh solutions were prepared for each experiment. Control experiments established that 0.5% DMSO by itself had no measurable effect on the pyramidal neurons (n = 3) (see also Markram et al. 1995
).
Calcium concentration measurements and some electrical measurements were made in a different chamber mounted on an upright Olympus BX50WI microscope. For combined measurements on individual neurons, tight seals were made on the cell bodies with video-enhanced differential interference contrast (DIC) optics (Stuart and Sakmann 1994
) with a ×40 water-immersion lens (Olympus). After allowing the indicator to diffuse into the cell for
15 min, we recorded high-speed image sequences (25- to 30-ms frame intervals) with a cooled charge-coupled device camera (Lasser-Ross et al. 1991
). Changes in [Ca2+]i are presented as the spatial average of
F/F (percent), in which F is the fluorescence intensity at resting membrane potential and
F is the time-dependent change in fluorescence corrected for bleaching. Bis-fura-2 fluorescence was measured with excitation of 382 ± 6 nm and emission >455 nm. Similar procedures were followed when making measurements of fura-2-AM-loaded cells except that no electrical recordings were made. Because the purpose of these experiments was to compare the [Ca2+]i changes under different pharmacological conditions no corrections were made for background fluorescence in either the patch-loaded or AM-loaded experiments. This procedure will not affect the percentage change in
F/F as long as the resting fluorescence (F) is not affected by the solution change.
Most chemicals were obtained from Sigma (St. Louis, MO). K-methylsulfate was purchased from ICN Biomedicals (Aurora, OH); DL-2-amino-5-phosphovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and spiperone hydrochloride were obtained from RBI (Natick, MA). Fura-2-AM and bis-fura-2 were obtained from Molecular Probes (Eugene, OR). Errors were calculated and presented as means + SE.
 |
RESULTS |
When 10 µM 5-HT was added to the bath the resting potential decreased 8-10 mV. In both somatic recordings (data not shown) and in dendritic recordings (n = 7; Fig. 1A) the hyperpolarization was accompanied by an increase in conductance. If 5-HT remained in the bath, the membrane potential relaxed to the original resting potential and sometimes depolarized the cell. If depolarizing current was injected into the dendrites to restore the potential to resting level, the conductance increase remained (Fig. 1B). This demonstrates that the conductance increase was not strongly voltage dependent. 5-HT did reduce the sag in the voltage response (Fig. 1C). The hyperpolarization was prevented by 10 µM spiperone added to the bath 10 min before the addition of 5-HT (data not shown). These results from dendritic recordings are similar to those previously reported from somatic recordings (e.g., Andrade and Nicoll 1987
; McCormick and Pape 1990
), suggesting that 5HT1A receptors are the dominant mediator of this effect. Dendritic hyperpolarization was also observed in response to focal pressure-applied 5-HT on the distal apical dendrites (not shown), suggesting that 5-HT receptors and coupled channels are found in this region. This result is consistent with the immunocytochemical localization of 5-HT1A receptors on hippocampal pyramidal cell dendrites (Kia et al. 1996
).

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| FIG. 1.
Serotonin (5-HT) hyperpolarizes the dendrites of pyramidal neurons and increases the membrane conductance. A: constant current hyperpolarizing pulses (500-ms duration) were given every 30 s through the recording electrode as 10 µM 5-HT was washing into the bath. Each vertical line is a compressed version of the response. Individual responses are shown in C. At the recording site (215 µm from the soma), the cell hyperpolarized 8 mV. The amplitude of the voltage responses diminished, indicating that the conductance increased. After returning to control solution the membrane potential and conductance slowly recovered. B: similar experiment except that at the peak of the response additional depolarizing current in step increments was injected through the recording electrode. The potential was restored to resting level, but the conductance increase remained. C: selected responses from this experiment on an expanded time scale.
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To test the effects of 5-HT on spike amplitude we evoked action potentials antidromically with stimulating pulses in the alveus in the presence of 10 µM CNQX and 50 µM APV to block fast excitatory transmission. Under these conditions, single action potentials of almost constant amplitude were recorded with patch electrodes in the dendrites (200-300 µm from the soma). The amplitude of the first action potential in the dendrites was smaller than the 100-mV amplitude usually recorded in the soma with patch electrodes. (Amplitude was measured as the difference between the peak potential and the potential just before evoking the spike.) When spikes were evoked at 20 Hz their amplitudes declined during the train (Fig. 2A, control) as previously reported (Callaway and Ross 1995
; Spruston et al. 1995
). 5-HT (10 µM) added to the bath hyperpolarized the cell and decreased the peak amplitude (absolute potential) of all the spikes in the train reversibly (Fig. 2A). On average, the peak potentials of the earlier spikes were reduced by 14 mV and of the later spikes by 10 mV (Fig. 2B). Because the membrane hyperpolarized only ~8 mV the absolute amplitude decreased slightly (4-6 mV). Figure 2, C and D, summarizes the effect of 5-HT on the peak potential and absolute amplitude of the action potentials in a train (n = 9).

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| FIG. 2.
5-HT reduces the peak potential and the absolute amplitude of backpropagating action potentials recorded in the apical dendrites. A: train of 10 spikes evoked at 50-ms intervals recorded 270 µm from the soma in normal artificial cerebrospinal fluid (ACSF). Spike amplitudes decrement in an activity-dependent manner; 10 µM 5-HT hyperpolarized the cell and reduced the peak potential of the action potentials. Washing out the 5-HT restored the control recording. B: summary of the effect of 5-HT on the changes in peak potential and absolute amplitude. Nine cells were analyzed; all were recorded 200-300 µm from the soma. The early spikes in the train were reduced by ~15 mV; later spikes were reduced 8-10 mV. The absolute amplitude was reduced by a few millivolts or stayed the same for all spikes in the train. C: summary of the peak potentials of the 10 spikes in normal and 5-HT containing ACSF. D: summary of the absolute amplitudes of the action potentials under the same conditions. The error bars in C and D are greater than in B because of the variation in amplitudes from cell to cell caused by recording conditions or distance from the soma.
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This weak effect of 5-HT on dendritically recorded action potentials resembles the effect of membrane hyperpolarization evoked by current injection into the dendrites (Tsubokawa and Ross 1996
). To directly compare the two effects we combined the application of 5-HT and current injection in different combinations in the same cell. Figure 3 (typical of experiments on 3 cells) shows that
0.08 nA mimicked the effect of 10 µM 5-HT, and +0.08 nA in the presence of 5-HT restored the spike profile to the control response. This result differs from that observed with CCh (Tsubokawa and Ross 1997
) where current injection could not reproduce or reverse the response.

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| FIG. 3.
Current injection into the dendrites mimics the effect of 5-HT on dendritic action potentials. Control recordings at 270 µm from the soma demonstrate the typical activity-dependent amplitude profile. Injection of 0.08 nA steady current lowers the resting potential and reduces the peak spike potential. The first, larger amplitude spike is more affected. Removal of the current and application of 10 µM 5-HT has a similar effect. Injection of +0.08 nA in the presence of 5-HT restores the spike profile to the control response. (One spike failed in this trial.) Finally, removal of current and washout of 5-HT restores the control response.
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Previously (Tsubokawa and Ross 1996
), we found that membrane hyperpolarization caused by current injection had little effect on the peak amplitude recorded at the soma. Therefore we repeated the 5-HT experiments with somatic recordings to test whether the 5-HT-induced hyperpolarization also affected the spikes in the two regions differently. Figure 4A shows that when the action potentials were evoked antidromically the peak potential of the spikes did not change when the soma hyperpolarized 8 mV, increasing the absolute amplitude by 8 mV. Note that in both normal conditions and in 5-HT all the somatic spikes in the train have approximately the same amplitude (Callaway and Ross 1995
; Spruston et al. 1995
). Figure 4C shows that on average the peak potentials of all the somatic spikes in the train decreased by ~3 mV, and the absolute amplitude increased by ~4 mV. Figure 4, D and E, summarizes the effects on the peak potential and absolute amplitude in the soma (n = 14).

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| FIG. 4.
5-HT increases the absolute amplitude of action potentials in the soma with little affect on the peak potential. A: train of 10 antidromic spikes evoked at 50-ms intervals. All spikes have approximately the same amplitude. In 10 µM 5-HT the cell hyperpolarized, but the peak potential was unchanged. B: overlay of the first spikes in control and 5-HT containing ACSF. Only the amplitude was affected by 5-HT. C: summary of the effect of 5-HT on the changes in peak potential and absolute amplitude in 14 recordings from the soma. The absolute amplitude of all spikes increased by ~4 mV. The peak potential was reduced by 2-3 mV. D: summary of the peak potential in the soma of the 10 spikes in normal and 5-HT containing ACSF. E: summary of the absolute amplitudes of the spikes under the same conditions in the same cells. The larger error bars in D and E compared with C reflect cell to cell variation.
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One important consequence of spike backpropagation into the dendrites is that these action potentials cause large changes in [Ca2+]i in this region (e.g., Jaffe et al. 1992
). Mechanisms that alter backpropagation change the magnitude and spatial extent of these changes (Tsubokawa and Ross 1996
, 1997
). Because 5-HT reduced spike amplitude in the dendrites, we reasoned that this reduction would also affect the associated [Ca2+]i change. The experiments shown in Fig. 5 confirm this idea. In normal ACSF backpropagating action potentials caused [Ca2+]i increases at all locations (Callaway and Ross 1995
; Jaffe et al. 1992
; Regehr and Tank 1992
). In 10 µM 5-HT the [Ca2+]i increase evoked by a train of 10 action potentials was reversibly reduced in the dendrites by ~20% (Fig. 5B). In addition, 5-HT reduced the somatic
F/F change by 30%, although the peak potential was not significantly reduced in this region. The resting fluorescence level (F) did not change in 5-HT (not shown), demonstrating that there was no change in resting [Ca2+]i. Figure 5C summarizes the results of five experiments. In all regions of the cell, the reduction in
F/F was >20%. The reductions were all statistically significant (P < 0.002).

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| FIG. 5.
5-HT reduces spike-evoked [Ca2+]i changes in all parts of patch-loaded pyramidal neurons. A: fluorescence image of bis-fura-2-loaded pyramidal neuron. Rectangles indicate regions from which time-dependent traces are taken. Scale 20 µm. B: fluorescence changes in 3 regions in response to a train of 10 antidromic action potentials evoked at 50-ms intervals. Control recordings made just before changing to ACSF containing 10 µM 5-HT and 32 min after breaking into cell. 5-HT recording 4 min after changing solution. Wash recording 9 min after returning to normal ACSF. 5-HT was in the bath for 3 min. Three trials averaged for each condition. Voltage traces show the first of the 3 responses. Spike amplitude did not completely return to control values, but the fluorescence change recovered. C: summary of changes in 3 regions for 5 cells. Control values were normalized to 100%. The middle (M) and distal (D) boxes were typically 100 and 200 µm from the soma (S).
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A difficulty in these experiments was that the concentration of bis-fura-2 in the dendrites rarely reached a stable value in <30 min. The indicator slowly diffused to these distal sites from the site of injection in the soma. As the indicator concentration increased, buffering of the [Ca2+]i change increased, reducing
F/F for constant levels of Ca2+ entry (Helmchen et al. 1996
). In addition, higher values of indicator fluorescence altered the significance of background fluorescence. To minimize these effects, we tried to wait as long as possible after breaking into the cell before making the measurements. Typically, we waited
15 min before making the first control measurement and 30 min before making the first measurement in 5-HT containing ACSF. We also bracketed all measurements with control observations after returning to normal ACSF.
In another series of experiments, we tried to avoid this problem by loading pyramidal neurons in the slice with the acetoxymethylester form of fura-2 (Grynkiewicz et al. 1985
). By using this procedure, all parts of the neuron were loaded simultaneously, and diffusion was not a problem. The final concentration of fura-2 in the cells was unknown. However, the one-half recovery time for single spike-evoked fluorescence transients typically was <200 ms in the soma and <150 ms in the dendrites (Fig. 6A). If the indicator in these experiments responded similarly to indicator loaded from patch pipettes, these fast recovery times would indicate that the final concentration was <100 µM (Helmchen et al. 1996
). The protocol for these experiments was similar to the whole cell experiments except that there was no electrode in the cell. Although this procedure was not able to directly monitor the potentials in the neurons, it had the advantage of avoiding any washout of important intracellular constituents. We confirmed that the pyramidal cells fired antidromic action potentials by observing the all-or-none fluorescence response as the stimulus intensity in the alveus was increased. These fluorescence changes were blocked by adding 1 µM tetrodotoxin to the bath, showing that they resulted from propagating Na+ spikes (n = 3; not shown). Figure 6B shows results from a typical experiment. Fluorescence transients were evoked with a train of 10 backpropagating action potentials. 5-HT (10 µM) clearly caused a reversible reduction in spike-evoked
F/F in the soma of ~35%. Transients in the dendrites, 200 µm from the soma, were too noisy to measure accurately with this technique. Analysis of 19 cells (Fig. 6C) shows that 10 µM 5-HT on average reduced the [Ca2+]i change in the soma by 27% (P < 0.0005), similar to the level observed in whole cell experiments (Fig. 5C).

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| FIG. 6.
5-HT reduces spike-evoked [Ca2+]i changes in the cell bodies of fura-2-AM-loaded pyramidal neurons. A: fluorescence image of a hippocampal slice loaded with fura-2-AM. The cell body and proximal dendritic region of one neuron are indicated. Scale 50 µm. The traces below the image show the fluorescence change in the 2 regions in response to a single stimulus to the alveus. No averaging was required. Note the fast recovery times of the transients. B: effect of 5-HT on the amplitude of the spike-evoked fluorescence changes. Each point represents the average of 5 trials of 10 antidromically activated spikes. The lines are arbitrary smooth curves fit to the points. Selected traces are shown below the time points. C: summary of results from 19 cells in 4 slices.
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Because 5-HT hyperpolarized the resting potential it is possible that this reduction was responsible for the reduced [Ca2+]i change, even when the peak spike potential was unchanged. To test this possibility directly we hyperpolarized the soma with current and compared the [Ca2+]i increase with that recorded without injected current. Figure 7 shows that the [Ca2+]i increase was unchanged when the soma was hyperpolarized by 10 mV. In four measurements from the soma of this cell the ratio of
F/F in hyperpolarized and normal conditions was 0.98 ± 0.06. Similar unchanging results were found in five other cells.

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| FIG. 7.
Membrane hyperpolarization, by itself, does not reduce spike evoked [Ca2+]i increases. A: fluorescence increases in the soma and proximal dendrites evoked by 10 antidromic action potentials. The image shows the 2 selected regions and the recording electrode in the soma. Scale 50 µm. B: fluorescence increases from the same regions when the soma was hyperpolarized by 10 mV with current through the recording electrode. The fluorescence changes are approximately the same as in A.
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In the experiments presented so far, the action potentials were evoked by intrasomatic depolarization or antidromic stimulation. To test whether 5-HT could affect dendritic spike amplitudes under more physiological protocols we stimulated the pyramidal cells synaptically while recording in the dendrites. For these experiments, APV and CNQX were not included in the bathing solution. When the stimulating electrode was in the stratum radiatum the peak spike potentials were not significantly reduced in 5-HT (data not shown). These unchanged spike amplitudes, even when the cell hyperpolarized, probably were due to the large EPSP supporting the action potential at the recording site, counteracting the hyperpolarization caused by 5-HT. To avoid this problem we placed the stimulating electrode in the SO, evoking EPSPs primarily in the basal dendrites. EPSPs that were at threshold in the somatic region were electrotonically reduced to lower amplitudes at the recording site in the apical dendrites. Under these conditions, 10 µM 5-HT reversibly reduced the peak potential of the dendritically recorded action potentials (Fig. 8, top). This reduction was greater for earlier spikes in the train than for later ones. This differential effect on earlier spikes is similar to the effect of direct hyperpolarization on distal dendrites (Fig. 3) (see also Tsubokawa and Ross 1996
) Thus in some protocols 5-HT modulates synaptically activated backpropagating action potentials.

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| FIG. 8.
5-HT reduces synaptically activated action potentials in the dendrites when stimulated in the stratum oriens. Left: response to 10 stimuli in the stratum oriens at 50 ms-intervals in normal ACSF. Recording 250 µm from the soma. The first response was below threshold. Later responses facilitated and evoked spikes. The first 2 activated responses (time window indicated by solid line) are shown in the expanded trace below. Center: responses in 10 µM 5-HT. The cell-hyperpolarized and spike potentials were reduced. The expanded traces below (time window indicated by dotted line) show that the amplitudes measured from threshold (arrows) also were reduced. Right: return to normal ACSF restored the control responses.
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 |
DISCUSSION |
These experiments show that 5-HT has at least two effects on dendritic properties. First, it hyperpolarized the resting potential in this region and increased the membrane conductance. This hyperpolarization matched the change measured in the soma and probably reflects the same 5-HT1A receptor-mediated increase in K+ conductance determined previously (Andrade and Nicoll 1987
; Colino and Halliwell 1987
). We do not know the spatial distribution of the conductance increase in these cells, but the high-density of 5-HT1A receptors on the dendrites (Kia et al. 1996
) suggests that some of the increase occurs in this part of the cell.
Dendritic recordings also revealed a reduction in the peak potential of backpropagating action potentials. The reduction usually was greater than the reduction of the resting potential, decreasing slightly the amplitude of the earlier spikes in the train. In contrast, 5-HT lowered the resting potential in the soma without affecting the peak potential. Consequently, the amplitude increased at that location. These effects are similar to the effects of injecting current to hyperpolarize the cell (Tsubokawa and Ross 1996
).
The almost equal reduction in peak dendritic spike potential for all the action potentials in the train, without a significant change in amplitude, differs from the effects of CCh on backpropagating spikes (Tsubokawa and Ross 1997
). In those experiments, CCh reversed the activity-dependent amplitude reduction in a train, making all spikes have almost equal amplitude when recorded in the dendrites. Both the peak potential and amplitude of later spikes were enhanced by CCh. There was little effect on the first spike in the train. This difference means that in the hippocampus 5-HT will affect almost all spikes equally, whether they are isolated or occur in bursts. In contrast, cholinergic modulation will have more dramatic effects on closely spaced action potentials than on isolated spikes.
This conclusion may not apply to the most distal dendrites (>300 µm from the soma). At this distance, CCh was ineffective in reversing the activity-dependent reduction in spike amplitude (Tsubokawa and Ross 1997
). In addition, hyperpolarization of the distal dendrites by current injection was particularly effective in reducing the amplitude of the first (and sometimes second) propagating action potential. Later spikes in the train were not reduced because they already failed at a more proximal location. Peak potentials of all spikes were lowered (Tsubokawa and Ross 1996
). 5-HT acts similarly to hyperpolarization at this location.
Because the peak spike potential is largely due to the balance of conductances at that time, the different effects in the soma and dendrites are probably a result of a different proportion of Na+ and K+ conductances in these two regions. If the balance in the soma is dominated by the Na+ conductance, a small increase in K+ conductance and hyperpolarization mediated by 5-HT would not lower the peak potential significantly. However, if the balance in the dendrites is more equal, an increase in K+ conductance and hyperpolarization would lower the peak potential in that region. One possibility is that there is a high-density of Na+ channels near the soma, perhaps in the axon hillock as traditionally assumed. However, Colbert and Johnston (1996)
found that the density of Na+ channels was not significantly different in the axon hillock than in other regions of hippocampal pyramidal neurons. A second possibility is that the density of K+ channels is higher in the dendrites. Recently, Hoffman et al. (1997)
found that the conductance underlying the A-current increased with distance away from the soma in pyramidal neurons. Because the A-current activates rapidly during the upstroke of the action potential, the higher conductance in the dendrites could be responsible for the lower spike amplitude in this region. Other K+ conductances, including those active at resting potential, also could contribute to the balance of currents in the dendrites.
The second effect of 5-HT was the reduction in the spike-mediated [Ca2+]i increase in both the soma and dendrites. Some of this reduction probably was due to the reduction in peak spike potential in the dendrites. The lower potential would open fewer Ca2+ channels causing less Ca2+ entry. A similar reduction in spike-evoked [Ca2+]i increase by 5-HT was recently observed by Chen and Lambert (1997)
in hippocampal neurons loaded with fura-2-AM. They used dendritic field potential recordings to support the idea that spike peak reduction is responsible for the reduction in the [Ca2+]i change. One important difference between their experiments and ours is that we observed a consistent 5-HT-mediated reduction in the spike-evoked [Ca2+]i change in the soma. They observed a much smaller effect of 5-HT in this region. A possible explanation for the difference is that they used adenosine as an agonist in most of their experiments. Although adenosine and 5-HT are thought to act through similar pathways (Hille 1994
), it is possible that the distribution of receptors on pyramidal neurons is different. A second possibility is that they detected the [Ca2+]i changes from all the alveus-stimulated neurons in a field of view. These may have included some interneurons. In contrast, in both our experiments with whole cell-loaded and fura-2-AM-loaded cells we recorded from single identified pyramidal neurons.
The reduction of the spike-evoked [Ca2+]i change in the soma, where the action potential peak potential was not reduced and the amplitude increased, suggests that 5-HT also affects Ca2+ channels directly. This conclusion is supported by the fact that the action potential shape was not altered by 5-HT (Fig. 4B) and by the observation that membrane hyperpolarization by itself did not affect the [Ca2+]i change (Fig. 7). Previous work suggests that 5-HT modulates several different kinds of Ca2+ channels in CNS neurons, although not to the same extent (Anwyl 1991
; Hille 1994
). In particular, N- and P-type channels appear to be more susceptible than L- or T-type channels (Bayliss et al. 1995
; Foehring 1996
). These modulations generally occur through G-protein, membrane-delimited mechanisms. Imaging (Christie et al. 1995
), electrophysiological (Magee and Johnston 1995
), and immunocytochemical (Westenbroek et al. 1990
) studies indicate that several Ca2+ channels types are expressed in pyramidal cells. All channel types are found in all regions of the cell. However, L-type channels appear to be more concentrated near the soma, and T-type channels appear to be more concentrated in the dendrites. The distribution of other high-threshold channels, including N-type, is more uniform (Magee and Johnston 1995
). Our experiments made no attempt to distinguish which channel type was affected by 5-HT. Assuming that some Ca2+ channels were affected in all regions of the cell, our results suggest that in the dendrites the reduction in the [Ca2+]i change was caused by both a reduction in peak spike amplitude and direct modulation of Ca2+ channels. However, the relative contribution of each effect was not determined in these experiments. Modulation of dendritic Ca2+ channels is consistent with the demonstration of G-protein-mediated inhibition of voltage-gated Ca2+ currents in isolated dendritic segments (Kavalali et al. 1997
).
Previously, we demonstrated that spike propagation and dendritic [Ca2+]i changes could be affected by synaptic inhibition and muscarinic modulation. The dramatic reduction in spike-evoked [Ca2+]i in the distal dendrites caused by synaptic inhibition (Tsubokawa and Ross 1996
) clearly was due to a reduction in spike amplitude. No change in Ca2+ channel properties would be expected from
-aminobutyric acid-A (GABAA)-mediated inhibition. GABAB-mediated changes in channel properties are possible (Anwyl 1991
) but are inconsistent with the narrow time window for the inhibitory effect in those experiments. The CCh-mediated increase in [Ca2+]i change in the dendrites resulting from a train of action potentials (Tsubokawa and Ross 1997
) was predominantly due to the enhanced backpropagation of later spikes in the train. Modulation of Ca2+ channel properties was not a major contributor to this effect. One piece of evidence for this conclusion is that neither the amplitude of the first spike nor the [Ca2+]i increase evoked by this spike was significantly altered by CCh. However, we did not examine in detail the effect of CCh on dendritic Ca2+ channels.
Because inhibition and cholinergic modulation affected [Ca2+]i changes via changes in spike amplitude, the changes in [Ca2+]i were restricted to the dendrites where the amplitude change was greatest. The largest changes were in the distal processes. Spike amplitudes and associated [Ca2+]i changes in the soma were unaffected. 5-HT, in contrast, affected spike-evoked [Ca2+]i changes in all parts of the cell, although spike potentials in the soma were not significantly reduced.
The functional consequences of the reduction in action potential amplitude in the dendrites and the associated [Ca2+]i change are unknown. A role for backpropagating action potentials in synaptic plasticity was demonstrated (Magee and Johnston 1997
; Markram et al. 1997
). Either the action potentials themselves or the spike-evoked [Ca2+]i changes might contribute to this effect. 5-HT has been shown to inhibit the induction of long-term potentiation in the hippocampus in some experiments (Corradetti et al. 1992
; Sakai and Tanaka 1993
; Villani and Johnston 1993
). However, it is not known if the effects on spike backpropagation described in this paper contribute to this inhibition. The magnitude of the changes we observed was not large. Therefore it is possible that the effects of 5-HT on dendrites are not relevant to changes in synaptic plasticity.
A second possible role for backpropagating spikes is the control of membrane conductances in different parts of the cell, possibly by activating Ca2+-sensitive K+ conductances (Storm 1993
). These conductances could shape dendritic EPSPs and inhibitory postsynaptic potentials IPSPs, influencing synaptic integration and spike accommodation. Related to this possibility, Torres et al. (1996)
recently have shown that 5-HT reduces the slow Ca2+-dependent afterhyperpolarization in CA1 pyramidal neurons.