Modulation of Dendritic Action Currents Decreases the Reliability of Population Spikes

L. López-Aguado, J. M. Ibarz, and O. Herreras

Departmento de Investigación, Hospital Ramón y Cajal, 28034 Madrid, Spain


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

López-Aguado, L., J. M. Ibarz, and O. Herreras. Modulation of Dendritic Action Currents Decreases the Reliability of Population Spikes. J. Neurophysiol. 83: 1108-1114, 2000. During synchronous action potential (AP) firing of CA1 pyramidal cells, a population spike (PS) is recorded in the extracellular space, the amplitude of which is considered a reliable quantitative index of the population output. Because the AP can be actively conducted and differentially modulated along the soma and dendrites, the extracellular part of the dendritic inward currents variably contributes to the somatic PS by spreading in the volume conductor to adjacent strata. This contribution has been studied by current-source density analysis and intracellular recordings in vivo during repetitive backpropagation that induces their selective fading. Both the PS and the ensemble action currents declined during high-frequency activation, although at different rates and timings. The decline was much stronger in dendrites than in the somatic region. At specific frequencies and for a short number of impulses the decrease of the somatic PS was neither due to fewer firing cells nor to decreased somatic action currents but to the blockade of dendritic action currents. The dendritic contribution to the peak of the somatic antidromic PS was estimated at ~30-40% and up to 100% at later times in the positive-going limb. The blockade of AP dendritic invasion was in part due to a decreased transfer of current from the soma that underwent a cumulative increase of conductance and slow depolarization during the train that eventually extended into the axon. The possibility of differential modulation of soma and dendritic action currents during APs should be checked when using the PS as a quantitative parameter.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

During synchronous action potential (AP) firing of CA1 pyramidal cells the action currents from individual cells sum in the extracellular space raising a sharp negative field potential (FP) known as the population spike (PS). Its amplitude increases linearly with the number of firing neurons (Andersen et al. 1971) and is considered a reliable quantitative index of the population output. Variations of the PS may, however, be due to factors other than the cell number or the firing synchronism, such as changes in the amount of current contributed by the firing cells to the extracellular space. In the laminated CA1, the PS is typically recorded at the stratum pyramidale, which is dominated by cell somata, but may also be contributed by currents arising from activated neuronal elements nearby, such as dendrites. In fact, the PS negativity spans through the basal and the proximal apical region, a result that is taken as an indication for active dendritic AP generation (Fujita and Sakata 1962; Sperti et al. 1966). Subsequent current-source density (CSD) analysis and intradendritic recordings have definitely demonstrated that CA1 pyramidal cell dendrites can actively conduct and even initiate the AP (Herreras 1990; Leung 1979b; Turner et al. 1991; Wong et al. 1979). Because soma and dendrites can be differentially modulated (e.g., Callaway and Ross 1995; Herreras and Somjen 1993; Mackenzie and Murphy 1998; Spruston et al. 1995), we investigated the extent of the relative dendritic current contribution to the PS in the s. pyramidale.

For this purpose we calculated the average local currents of somata and dendrites by CSD and compared its amplitude and waveform with that of the PS. CSD provides the net local current generated within a small volume of tissue (Nicholson and Freeman 1975), and in the strongly stratified CA1 region reflects the sum of transmembrane currents from analogous specific neuronal subregions (e.g., Herreras 1990; Richardson et al. 1987). Therefore, discrepancies in the temporal evolution of FPs and CSDs can be used to disclose the contribution from neuronal generators located in different strata. We have unmasked the dendritic contribution using the selective gradual fading of backpropagated APs during high-frequency antidromic activation (Lorente de Nó 1947; Spruston et al. 1995). A unitary intracellular study was also made to assess whether individual cells fired during the protocol.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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REFERENCES

Female Sprague-Dawley rats weighing 200-250 g were anesthetized with urethane (1.2-1.5 g/kg ip). Surgical and stereotaxic procedures were as previously described (Herreras 1990). Antidromic activation of the CA1 was achieved by alvear stimuli (0.07-0.1 ms, 0.1-0.5 mA) delivered in short (150 ms) tetani of <= 300 Hz.

Extracellular recordings and CSD

Two micropipettes (filled with 150 mM NaCl; 3-6 MOmega ) connected to DC-coupled field-effect transistor (FET) input stages were used. One remained stationary in the CA1 s. pyramidale to test the constancy of the evoked PS amplitude, and another was used to explore dorsoventral trajectories in 50-µm steps. After filtering (1 Hz to 5 kHz band-pass) and amplification, signals were recorded on VCR, stored in a computer (20-40 kHz acquisition rate, Digidata 1200, Axon Instruments, Burlingame, CA), processed by Axotape software (Axon), and then further analyzed by the Axum program (Trimetrix, Seattle, WA). Depth profiles of evoked potentials were used for CSD analysis (Nicholson and Freeman 1975). At each recording station six to eight trains of stimuli were delivered 1 min apart. Although in most cases trains contained 30 impulses, only the first 15 were used for this study, because the constancy of responses was less reliable in the second half of the train. Trains were rejected for averaging when some of their PSs at the stationary somatic electrode differed more than 10% of those from a grand average made through the entire profile. Averages at each station had at least four valid trials. A detailed account of technical and theoretical considerations for the calculation of CSD in vivo has been presented elsewhere (Herreras 1990). We used the customary unidimensional approach for the calculation of the extracellular currents (Freeman and Nicholson 1975). In preliminary experiments we checked that relative tissue resistivity between strata was unchanged during the stimulating protocol, so that CSD values are given in proportional units. Smoothing procedures intended to decrease high spatial noise were not used, because they introduce substantial perturbations in the relative amplitude and spatial distribution of high-frequency components, i.e., action currents (Herreras 1990).

The PS in the s. pyramidale was measured as the amplitude to the peak of its negative-going limb compared with the precedent baseline. On some occasions, when the artifact partially overlapped the evoked potential, the most positive value between them was used instead. In the stratum radiatum it was measured as the voltage from the negative peak (when discernible) to an imaginary line linking the precedent positive crest and the following inflection point in the rising phase. The precedent positivity is clearly discernible in all but the s. pyramidale, where the negative-going limb departs from baseline. Current sinks were similarly measured. In this case, a sink was present when the inflection point was at a negative value, otherwise a pure source was assumed and the value taken as zero. All values are given as the percentage of control (mean ± SE). Mean values were obtained with six valid FP profiles from different animals.

Intracellular study

Micropipettes (1.5 mm OD) were backfilled with 2-4 M potassium acetate (60-120 MOmega ). Signals were amplified using a bridge circuit amplifier, filtered at 10 kHz, and stored on VCR for later analysis by using pClamp and Axotape computer programs (20-40 kHz acquisition rate). Recordings were made from electrophysiologically identified pyramidal cells located within the s. pyramidale of the CA1, identified by the characteristic PS. Cells that did not fire a single antidromic AP after alvear stimulation were rejected. We considered healthy cells to be those with resting membrane potential more negative than -60 mV (-66.3 ± 2.7 mV; n = 7) and overshooting APs. The average apparent input resistance calculated from the linear range of current voltage (I-V) plots was 30.2 ± 4.2 MOmega (range, 22-41 MOmega ). Once stabilized, all cells fired spontaneous APs at a very low rate (<1 Hz).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Extracellular potentials and subcellular current generators

Antidromic activation raised a sharp negative PS (24.96 ± 0.51 mV) followed by a slower (10-15 ms), small, positive wave at the level of the s. pyramidale. The PS became a positive-negative diphasic spike for ~200-250 µm within the proximal s. radiatum (Fig. 1A, FP, see first pulse), and a pure positive spike at more distal positions, as the negative component gradually faded off. Within the stratum oriens, the PS unfolded in a two-spike sequence toward the alveus. The first remained stationary, whereas the second increased in latency and declined faster. In the CSD analysis (Fig. 1A, right), the earliest current was a sink (black areas) at the level of the s. pyramidale, corresponding primarily to the population inward currents during synchronous somatic APs. This somatic sink propagated actively into the apical and basal dendritic trees for ~225 and 125 µm, respectively, and was always preceded by a passive source (small arrows in pulse 1, Fig. 1, A and B) corresponding to the leading outward passive currents depolarizing the membrane in front of the AP. The peak amplitude at each of the three successive stages below the s. pyramidale (taken as reference) was 71 ± 4%, 49 ± 4%, and 61 ± 5%, respectively, decreasing thereafter to extinction (data obtained from n = 19 animals in another series of experiments). The enlarged tracings in Fig. 1B (left) show the short duration of the sinks at different strata and their temporal relation to the PS waveform recorded at the s. pyramidale, whose duration spans all sinks. A decreasing source was obtained in the region of distal dendrites in the stratum moleculare, as expected where the AP propagation became passive. Negative PSs were recorded over a wider dendritic band than current sinks, because these give rise to negative potentials that spread in the extracellular space beyond the active membrane region (Lorente de Nó 1947; Rall 1962).



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Fig. 1. Fading of dendritic action currents during repetitive antidromic activation. A: field potential (FP) recordings at different positions along the somatodendritic axis (left) in 50-µm steps during a train at 200 Hz and the calculated current-source density (CSD; right) for sample pulses indicated by the small arrows on the FPs. Net sinks of current are filled in black for clarity. Note the progressive decrease of the negative population spike (PS), more pronounced in dendritic regions, and the gradual fading of the current sinks, showing decreased action potential (AP) invasion of dendrites. The approximate position of recording is illustrated by the scheme on the left. Small arrows in pulse 1 mark the leading passive sources (outward currents) in front of the active sink, characteristic of dendritic positions. Artifacts in FP recordings have been deleted (gaps). Inset (top): plots of the baseline potential value before each of 30 impulses recorded at the stratum pyramidale (st. pyr., ) and stratum radiatum (st. r., ap100, open circle ). Zero potential is marked by the horizontal dashed line. B: enlargement of currents in the control (1) and in the last (15) pulses of a train superimposed on the PS waveform recorded at the s. pyramidale. Note the temporal relation of sinks and somatic PS negativity. Vertical dashed lines are for temporal reference. Small arrows in pulse 15 indicate the complementary time course of the late phase at the somatic sink and the ap50 sink, denoting reciprocal cancellation. C: average evolution of the PS in the s. pyramidale and s. radiatum (ap50-200) given as the percentage value of the first pulse (mean ± SE, n = 7 animals). b, basal; ap, apical; numbers in labels of position, distance in micrometers from the s. pyramidale.

Dendritic action currents contribute to the PS at the s. pyramidale

During repetitive activation, a negative potential envelope of 2-4 mV developed in the s. pyramidale and s. oriens in parallel with a smaller positive potential in the apical region, except during the initial impulses that were positive throughout (see inset in Fig. 1A). This slow envelope outlasted the stimulating period, returning to baseline within 1 s. To simplify the comparison with the subsequent intracellular study we will refer mainly to the initial 15 pulses of a train (see METHODS). As a whole, both the PS and the ensemble action currents declined during high-frequency alvear stimulation, although somatic and dendritic regions declined at different rates and timings (Fig. 1, A and C). The rate of decline was faster the higher the stimulus frequency and was stronger the farther away from the cell soma layer. It was also stronger in the basal than in the apical dendritic tree. Note the selective blockade of the second negative peak in the uppermost row of the FPs in the s. oriens, whereas the first peak, attributed to axon initial segment currents (Sperti et al. 1966), remained unchanged. As an example in apical dendrites, the PS at 100 µm below the s. pyramidale (ap100) had decreased by the 15th pulse to 57.6 ± 5.5% and 40.9 ± 4.1% at 100 and 200 Hz, respectively (see Fig. 1C, upward triangles). As it concerns the ensemble currents, the basal sink (in the s. oriens) was extremely labile, fading out after a few pulses (Fig. 1A, right, 5). At 100 Hz, the apical sink at a location of 100 µm below the s. pyramidale had decreased to 37.8 ± 22% by the 15th pulse, whereas at 200 Hz it had disappeared entirely below 50 µm of the apical region, remaining only within a narrow band dominated by the thick apical shafts (Fig. 1A, CSD, and Fig. 1B, pulse 15). Except in this region, the source-sink sequences were gradually transformed into pure sources (top and two bottom rows in Fig. 1A, CSD, and enlargements in Fig. 1B, pulse 15), verifying that shorter distances were progressively invaded by a regenerative AP, whose spread became passive. It is notable that negative PSs could be recorded during the train over a somatopetally shifting dendritic band, whereas the corresponding currents at the border were already pure sources. This was the expected finding slightly beyond the farthest activated region and demonstrates that negative PSs do not necessarily denote active events at the recording position. The negativity on this boundary zone is caused by the spread of currents in the extracellular space from the nearest active region, the so-called volume propagation (see following description and schematic in Fig. 2D).



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Fig. 2. Divergent evolution of somatic PS and CSD unmasks the contribution of dendritic action currents to the s. pyramidale. A and B: typical experiment at 200 Hz. Whereas the PS continuously decreased (FP), the corresponding CSD first increased and then began to decrease (compare first and last pulse in the thick traces in B). A (small arrow): source that follows the active somatic sink, corresponding to the passive currents of the delayed active dendritic sinks. The fading of the dendritic sink and its matching source in the s. pyramidale unmasked the late portion of the somatic sink (curved arrow in B). C: duration of the somatic sink (thin traces) was much shorter than the negative phase of the PS (thick traces) in a control pulse (1st). For most of the PS rising limb there was no net sink (shaded area), and the negative value of the potential must be entirely caused by volume-propagated currents from dendrites. At earlier times, both somata and dendrites contributed variably. When dendritic currents faded out (15th pulse) the PS and CSD followed a similar time course, as expected. Artifacts have been deleted in A-C and marked by dots in B and C. D: some prominent features of intra- and extracellular currents during AP in control (1) and after block of dendritic AP invasion (2). Gray zone indicates the membrane surface actively invaded by the AP. Inward and outward currents are depicted in the right and left sides of the schemes, respectively. Dashed arrow inside marks the AP direction. Extracellular sinks of current (represented by dots) occurred at any extracellular region where inward currents occurred through the membranes actively invaded by the AP. These returned to the extracellular space as passive outward currents at the front of the propagating AP (b, arrows), depolarizing the membrane and causing the extracellular leading source in dendrites (see Fig. 1). A fraction of the return currents from the dendritic sink left the cell through the soma membrane (a, arrows) causing the cancellation of the still active somatic sink in its late phase. In the extracellular space, the somatic and dendritic sinks created negative fields (radiating arrows) that extended beyond the active membrane, generating volume-propagated currents and contributing to each other's negativity. When the dendritic sink was blocked, the extracellular somatic sink was the only current to build up the extracellular negative field, and both matched.

In the s. pyramidale, the PS and the averaged somatic currents evolved differently (an example is illustrated in Fig. 2A). By the 15th pulse, the PS was always smaller than control (66.4 ± 8%; see evolution in Fig. 2B, FP, and  in Fig. 1C). The inward currents first increased and then decreased but at lower rate than the PS, remaining at 100.3 ± 7.4% (measured at the peak) by the 15th pulse (compare 1st and 15th pulses in Fig. 2B, CSD). In parallel, the duration of the somatic sink increased because of the gradual fading of a delayed current source at the positive-going limb of the PS (marked by the small arrows in Fig. 2, A and C, and the curved arrow in Fig. 2B). This fading source corresponded to the outward passive currents from the delayed active dendritic sinks (see matching currents in Fig. 1B). If the somatic currents were measured at the time of the PS peak instead of their own, they were even larger (122.5 ± 6.3% by the 15th pulse), increasing further the divergence with the decreased PS peak. For longer trains (more than 15 impulses), both the PS and the ensemble somatic currents decreased still further, but once dendritic invasion was minimized, they decreased at similar rates (not shown).

The decrease of the PS at the s. pyramidale was time related to the missing current contribution from the extracellular dendritic sinks nearby as they faded out. If these events are causally related, the dendritic contribution to the PS recorded at the s. pyramidale should be smaller at the peak than at the rising phase, because dendritic inward currents are slightly delayed (see Fig. 1, A and B). In fact, when the temporal course of the population currents and FPs were compared, it was evident that the PS peaked later than the current sink, and most important, the PS negativity lasted much longer (Fig. 2C, 1st pulse). From the instant when the somatic sink ended (i.e., when the CSD value becames positive; see arrowhead in Fig. 2C), the negativity of the PS (shaded area) could be entirely attributed to extracellular current spread from dendritic generators (see temporal correlation in Fig. 1B). On the contrary, in the 15th pulse, the time course of currents and potentials in the s. pyramidale was almost identical (Fig. 2C, 15th pulse), indicating that the delayed contributors to the PS negativity (dendritic sinks) had disappeared (Fig. 1, A and B). A moderate contribution from the still-invaded proximal-most apical shafts is very likely. That is, the PS at the s. pyramidale faithfully reflected the evolution of the local (somatic) currents only when it was not contributed by distant generators.

The PS decrease at the cell body layer is not caused by fewer firing cells

The PS decrease in the s. pyramidale in spite of the maintained or increased population somatic inward currents could also be due to fewer firing cells, individually contributing more current. To check this possibility, we made intracellular recordings from seven pyramidal cells located within the s. pyramidale. Single alvear pulses triggered an AP followed by a fast inhibitory postsynaptic potential (IPSP) lasting ~150 ms. During repetitive antidromic activation (Fig. 3, A and B, bottom) the AP initially decreased in amplitude and half-width, and then, increased much more in half-width, became delayed, and decreased the rate of rise, revealing an AB break (small arrow in Fig. 3B). Eventually, it failed to invade. Occasional AP failures occurred in some of the initial pulses, coinciding with the highest rate of conductance increase during the IPSP (Fig. 3B). These changes were more marked and required fewer pulses the higher the frequency. During the course of the train, a gradual depolarization with increased conductance developed (arrow in Fig. 3A, top) that outlasted the stimulating period (double-headed arrow). Its amplitude varied considerably between cells (<= 30 mV) and was not totally inactivated, because often some spontaneous APs appeared at the top, beyond the stimuli (not shown). The duration was ~1 s and roughly matched the extracellular sustained negative potential recorded in the s. pyramidale (see inset in Fig. 1A) Overall, pyramidal cells followed longer trains the lower the frequency. Regarding the specific windows used for the CSD study (first 15 impulses), all neurons followed activation at 100 Hz for at least 300 ms (i.e., 30 pulses; see an example of 45 pulses in Fig. 3A). At 200 Hz, they could follow trains up to 150 ms long (30 pulses at 5-ms intervals, Fig. 3B), except two (of 7) that began to fail in the second half of the train (beyond pulse 15). All seven cells showed AP failures for longer train duration, i.e., outside the window where a dendritic contribution to the somatic PS has been shown. Small all-or-nothing spikes (~25 and ~10 mV, see arrows 1 and 2 in Fig. 3C at 300 Hz) attributed to the axon initial segment and first node were visible in most neurons after the AP failed to invade the cell soma. Eventually, the AP block progressed into axonic regions as these spikes gradually disappeared as well. Regarding the relevant first 15 pulses at 200 Hz, when discrepancies between the PS and CSD amplitudes were found, all seven cells followed regularly. Therefore, the decrease of the PS amplitude was not caused by fewer firing cells.



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Fig. 3. Pyramidal cells can follow high-frequency antidromic activation. A, B, and C: intracellular somatic recordings from three different neurons activated at 100, 200, and 300 Hz, respectively. A slow depolarization developed (small arrow, A) that outlasted the stimulation period (double-headed arrow). Increased conductance is denoted by the lower voltage drop to hyperpolarizing pulses (*). A and B (bottom): detail of the evolution of the AP waveform during the train. B (dashed line): initial resting membrane potential; (small arrow) the AB break, unmasked before the AP failed to invade the soma. Afterwards, small-amplitude all-or-nothing spikes were usually recorded in two discrete amplitudes, as those marked 1 and 2 in C, which most likely correspond to the electrotonic spread from the axon initial segment and first node APs, respectively. These are similarly modulated and blocked at later times, indicating a gradual blockade of the antidromic AP at successively more distal positions in the axon.


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The most relevant result of this study is that the PS recorded in the s. pyramidale was significantly attributable to dendritic action currents during backpropagation of APs. Because of the obligatory delay of dendritic action currents during antidromic activation, this contribution was larger at later times---i.e., during the positive-going limb of the PS waveform. In this phase it could reach up to 100%, because the net somatic sink terminated well before the negativity of the PS. From a practical point of view, it is the dendritic contribution at the time of the PS peak that may be of more interest. A higher limit could be set at ~30-40%, which constituted the maximum divergence between the evolution of the ensemble somatic currents and the PS peak. Once dendritic regenerative AP invasion was blocked, the somatic PS reflected only the evolution of somatic action currents, with some contribution of the initial portion of the apical shafts. After the chosen window of 15 pulses, a further PS decrease may be accounted for by a decreased number of firing cells and/or a decreased unitary current contribution, making a unitary study mandatory. Therefore, the possible modulation of active dendritic properties should be taken into account when interpreting changes of the PS measured at a single point in the s. pyramidale.

An interesting observation is that at least part of the longer duration of the somatic sink was caused by the fading of a delayed current source. These outward currents corresponded to the return currents matching the active dendritic sinks (Fig. 2D, arrows marked a), because both disappeared in parallel. Those more delayed were responsible for the net source marked by an arrow in Fig. 2C (the subsequent longer source corresponded to IPSP currents), whereas those corresponding to more proximal locations overlapped in time with the late phase of the somatic sink and caused its partial cancellation and shortening. A fraction of the return outward current constituted the depolarizing front of propagating APs (arrows at b, Fig. 2D), but obviously, a major fraction left the membrane locally and through the adjacent active sites of greatly increased conductance (arrows at a, Fig 2D). This observation illustrates the strong interaction between adjacent membranes during active AP propagation, acting as reciprocal shunts. An example of this reciprocal cancellation can be appreciated in the currents of somata and proximal apical shafts shown in Fig. 1B (pulse 15). Note how in spite of the increased duration of the somatic sink due to the disappearance of delayed sources a positive hump leaving reduced amplitude is observed that matches the time course of the remaining lower ap50 sink (small arrows). In fact, it can be easily demonstrated using computational methods that the somatic transmembrane current is much larger and longer in passive-dendrite neurons (Varona et al. 1998). Although this was barely reflected in the intracellular somatic AP waveform, it had a strong impact on the extracellular fields, as shown in this study on an experimental basis.

We have shown that individual pyramidal cells can follow short periods of activation at high frequencies. Even higher rates have been reported using extracellular recordings (Leung 1979a) in the same in vivo preparation. The lower efficiency in our experiments may have been due to the mechanical stress and somatic shunt caused by the intracellular electrode.

As previously reported, the blockade of dendritic AP backpropagation during repetitive activity is in part due to prolonged sodium channel inactivation (e.g., Colbert et al. 1997; Jung et al. 1997). Some of the present data point to a major role of somatic mechanisms at higher frequencies. The interstimulus interval fell well within the AP partial refractory period, so that cumulative sodium channel inactivation accounts for the gradual AP change before its eventual failure. These changes were stronger and faster than those obtained at the lower frequencies used to study the attenuation of backpropagating spikes. The slowing in the rising phase of the AP was most likely caused by a decreased inward current (e.g., see Fig. 1 in Jung et al. 1997), which should cause the decrease of the somatic sink. In fact, this was the overall trend (note the slower rate of increase in Fig. 2B), although the uncovering of late phases (see earlier description), and the coincidence of an initial distinct increment for the first three to eight impulses, slowed the rate and timing of the decrease.

The AP changes are time related to the extracellularly observed dendritic AP block and to the somatic cumulative depolarization with increased conductance (Fig. 3A). The nature of this somatic depolarization was not investigated; reversed IPSP, extracellular potassium accumulation or overlapping depolarizing afterpotentials are among the possible causes. However, it matched the extracellular slow potential that was negative in the somatic-basal region and positive in the apical tree, a dipole configuration typical of active inward somatic currents. A similar result has long been known to occur during tetanic antidromic activation of some brain regions and is taken as evidence for cumulative depolarization of somata but not of dendrites (e.g., Lorente de Nó 1947), the first of which was shown in these experiments.

The present results are compatible with a gradually shorter dendritic AP invasion due to reduction of depolarizing currents entering dendrites from the soma, rather than a putative smaller excitability of the apical dendrites, although this also contributed. Such a gating function of the soma may result from the cumulative inactivation of sodium channels and the increased shunt during the slow depolarization. In fact, our results show that the AP invasion block progressed into the axon initial segment, probably as the somatic depolarization extended into this region. In short, the increasing evidence of local modulation of somatodendritic properties not only enhances our knowledge of the repertoire of integrative properties, but it also demands a continuous reevaluation and refinement of the methods used to explore them.


    ACKNOWLEDGMENTS

We thank M. J. Yagüe for technical assistance.

This work was supported by grants 8.5/15/98 from the Comunidad Autónoma de Madrid and PB97/1448 from the Spanish Dirección General de Investigación Científica y Técnica.


    FOOTNOTES

Address for reprint requests: O. Herreras, Dept. Investigación, Hospital Ramón y Cajal, Ctra. Colmenar km 9, 28034 Madrid, Spain.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 July 1999; accepted in final form 11 October 1999.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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