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Roles of calcium- and voltage-sensitive potassium currents in the generation of neuromagnetic signals and field potentials in a CA3 longitudinal slice of the guinea-pig
Clinical Neurophysiology 111 (2000) 150±160 www.elsevier.com/locate/clinph
Roles of calcium- and voltage-sensitive potassium currents in the generation of neuromagnetic signals and ®eld potentials in a CA3 longitudinal slice of the guinea-pig Jie Wu a, b, Yoshio C. Okada a, b,* a
Department of Neurology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
b
Accepted 29 July 1999
Abstract Objectives: Roles of calcium- and voltage-sensitive potassium currents in generation of neuromagnetic signals and ®eld potentials were evaluated using the longitudinal CA3 slice preparation of the guinea-pig. Methods: Their roles were evaluated by using selective channel blockers (tetraethyl-ammonium (TEA) and 4-aminopyridine (4AP)) and measuring their effects on the two types of signals and intracellular potentials. Fast g-aminobutyric acid type A inhibition was blocked with picrotoxin. Results: Stimulation of the apical dendrites with an array of extracellular bipolar electrodes produced triphasic evoked magnetic ®elds with a spike and a slow wave typical of the slices. The evoked potentials in the apical and basal areas of the pyramidal cells closely resembled the magnetic ®eld waveforms. Blockade of the potassium currents with TEA and 4AP had only subtle effects on the initial spike, but dramatically altered the slow wave. They also induced long-lasting spontaneous burst discharges synchronized across the slice. The results could be interpreted in terms of their known pre- and postsynaptic effects. Their post-synaptic effects were con®rmed with intracellular recordings. Conclusion: Our results are consistent with a hypothesis that the calcium- and voltage-sensitive potassium currents, especially the A and C currents, play important roles in shaping the slow wave of the neuromagnetic and ®eld potential signals produced by the mammalian hippocampus. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Biomagnetism; Magnetoencephalography; Electroencephalography; Evoked potential; Potassium conductance; CA3; Hippocampus; Tetraethylammonium; 4-aminopyridine; Epilepsy
1. Introduction Discoveries of numerous active conductances over the past 20 years (Hille, 1992) have motivated us to initiate a systematic re-examination of the genesis of magnetoencephalography (MEG) and electroencephalography (EEG) signals in light of the new concepts of dendritic and somatic electrophysiology of central nervous system (CNS) neurons (Kyuhou and Okada, 1993; Okada et al., 1997; Wu and Okada, 1998, 1999). The presence of voltage- and calcium-sensitive intrinsic conductances in the dendrites and soma of CNS neurons renders the post-synaptic currents more complex than the classic picture which painted the * Corresponding author. Tel.: 11-505-256-2874; fax: 11-505-2600165. E-mail address: [email protected] (Y.C. Okada)
currents in the dendrites and soma to have been passively (electrotonically) generated by neurotransmitter inputs. Yet, the relationships between MEG and EEG signals on one hand and the intracellular currents produced by opening and closing of these active conductances on the other are still poorly understood. This issue was examined in the longitudinal CA3 slice preparation of the guinea-pig since its cellular organization is relatively simple and its single-cell electrophysiology is well-understood for relating the MEG and EEG signals produced by this preparation to intracellular events in the principal neurons called the pyramidal cells. A variety of intrinsic ionic conductances are thought to be important in controlling neuronal activity of the pyramidal cells. They include a sodium conductance (gNa), a calcium conductance (gCa), voltage-sensitive potassium conductances of delayed recti®er type (gK(DR)), A type (gK(A)) and D type (gK(D)), a
1388-2457/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S13 88-2457(99)0020 7-2
CLINPH 98141
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Fig. 1. (A) A hippocampus was placed on an agar block and two 400-mm thick longitudinal slices were cut with a Vibratome just above the ®mbria. (B) A rectangular longitudinal CA3 slice was prepared by separating CA3 from CA1 with a razor blade. The slice was placed in a recording chamber (cf. Okada et al., 1997). Six pairs of bipolar stimulating electrodes (125 mm diameter Ag±AgCl wires) were placed from below the slice along the pyramidal cell layer with negative electrodes in stratum radiatum to stimulate the apical dendrites. Three Ag±AgCl (125 mm diameter) wires were used to record ®eld potentials from the basal, soma and apical regions of the pyramidal cells. Neuromagnetic ®elds were recorded with a 4 channel superconducting magnetic ®eld detector called a microSQUID. Its detection coils were 4 mm in diameter, separated by 8.4 mm diagonally and placed 2 mm above the slice. (C) An example of the apical stimulation-evoked magnetic ®elds and ®eld potentials recorded by the 4 microSQUID sensors and 3 electrodes. Note that the magnetic signals were strong and mirror-images of each other at SQ-2 and -4, whereas they were close to the baseline at SQ-1 and -3, indicating that the underlying currents were along the longitudinal axis of the pyramidal cells (perpendicular to the cell layer). The magnetic ®eld waveforms were most closely related with the ®eld potentials in the basal and apical areas and less so in the soma area. Thus, the magnetic data are compared with apical and basal ®eld potentials in the subsequent ®gures. Outward ®eld is up in this and all the ®gures. Stimulus artifacts not removed in this and subsequent ®gures. 30 epochs/ave.
calcium- and voltage-sensitive potassium conductance of C type (gK(C)), and a calcium-sensitive potassium conductance of afterhyperpolarization (AHP) type (gK(AHP)) (Storm, 1990). These active conductances have their own characteristic non-linear activation and inactivation kinetics (Traub and Miles, 1991; Traub et al., 1994). Therefore, the temporal waveform and spatial distribution of the currents along the longitudinal axis of the pyramidal neurons depend profoundly on the interplay of these active channels as well as on the network properties of the hippocampus. Consequently, the temporal waveform of MEG and EEG signals may be profoundly affected by these active conductances. In order to evaluate the roles of a number of potassium active conductances, we chose the strategy of using selective channel blockers to block some of these conductances and study the consequences on the MEG and EEG signals
produced by the remaining conductances. In the preceding report (Wu and Okada, 1999), we have described the role of gK(AHP) in modulating MEG and EEG signals. Here, we used tetraethylammonium (TEA) and 4-aminopyridine (4AP) to manipulate other principal potassium conductances. We carefully selected the concentrations of TEA and 4AP in order to be able to infer the role of gK(DR), gK(A), gK(D), and gK(C) in generating MEG and EEG signals from the hippocampus. Also, we recorded ®eld potentials simultaneously with intracellular potentials from the pyramidal cells during the manipulations with selective channel blockers in order to clarify the roles of the active conductances. Eventually, it will be necessary to interpret the effects of these experimental manipulations with the help of a mathematical model (e.g. Traub and Miles, 1991; Traub et al., 1991, 1994, 1995) in order to arrive at a clearer understanding of roles
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of the active potassium currents in generating MEG and EEG signals. Some of the results have been presented in abstract form (Wu and Okada, 1996). 2. Methods 2.1. Slice preparation and solution The preparation of CA3 longitudinal slices was described in detail in a recent paper (Okada et al., 1997). The procedure will be thus only brie¯y described. Readers are referred to earlier studies by Miles, Wong and others for electrophysiology of this preparation (Miles and Wong, 1987b; Miles et al., 1988). Twenty-six animals were used in this study. Each animal was anesthetized with sodium pentobarbital (60 mg/kg i.p.) and decapitated. Each hippocampus was placed on an agar block and two 400-mm thick slices were cut parallel to the agar surface just above the ®mbria using a Vibratome (Technical Products International) (Fig. 1A). Then, the CA3 was separated from CA1 for each slice with a razor blade and was placed in chilled oxygenated Ringer solution (concentration in mM: NaCl, 124; KCl, 5; KH2PO4, 1.2; MgCl2, 1.3; CaCl2, 2.5; NaHCO3, 26 and glucose, 10) and incubated in the medium for 1 h or more until the medium attained room temperature. Its pH was maintained at 7.4 with 95% O2/5% CO2. One slice was then placed in a recording chamber in which the slice was immersed in a bath containing 36 ^ 0.58C oxygenated Ringer solution. TEA was used in experiments with 15 animals. 4AP was used on 15 animals, sometimes together with TEA. Picrotoxin (PTX, 0.1 mM) was used to produce a high degree of synchronization of the neuronal activity across the slice. This was needed for relating the MEG signals, which are due to a weighted sum of currents by all active cells in the slice, and the EEG signals, which are due to population activity within the vicinity of the electrode, and the intracellular potential signals from individual pyramidal neurons. (See Okada et al., 1997, for more on this issue.) 2.2. Stimulation and recording procedures Fig. 1B shows the stimulation and recording arrangements. The slice was stimulated with 6 pairs of bipolar electrodes (Ag±AgCl 125 mm diameter wires), separated by 0.8±1.0 mm between pairs and by about 0.4 mm within pairs. The cathode of each pair was placed against the stratum radiatum and the anode against the basal side of the pyramidal cell layer from below the slice. This mode of stimulation will be designated as `apical stimulation'. The slice was stimulated with constant current pulses (0.7±1.0 mA, 50 ms, 0.1±0.3 Hz). Evoked magnetic ®elds were measured with a 4 channel microSQUID (Okada et al., 1994). The detection coils were placed directly over the slice at a distance of about 2 mm above the slice. The spatial pattern of the magnetic signal was initially determined by
moving the microSQUID over the slice. Once the center of the activity was found, the detection coils were placed around this point at the orientation shown in Fig. 1B. Extracellular ®eld potentials were recorded simultaneously with the magnetic signal using an array of three electrodes (Ag± AgCl 125 mm diameter wires) placed in the tissue from below. The electrodes were placed in the stratum oriens, cell layer and stratum radiatum to record the ®eld potentials in the area of the basal dendrites (`basal'), soma (`soma') and apical dendrites (`apical') (see Fig. 1B). Both magnetic and electrical signals were passed through FET preampli®ers, ampli®ed and ®ltered with Butterworth-type bandpass ®lters (roll off 24 dB/octave, 0.1±1000 Hz), and then recorded at a 10 kHz sampling rate. Conventional intracellular recording methods were used to record activity from 18 slices placed in the same chamber as for the magnetic recordings. Twenty-three pyramidal cells were activated either by stimulation with the same array of electrodes used for the magnetic recording or by directly passing depolarizing current into the pyramidal cells with the recording electrode. Intracellular potentials (Vin) were measured in the soma of the pyramidal cells with a glass micropipette ®lled with potassium acetate (4 M; 50±90 MV). Only cells with resting membrane potentials of at least 255 mV were used. All intracellular recordings were performed simultaneously with a ®eld potential recording (Vex). Intracellular potentials were recorded with a bandwidth of DC to 10 kHz using Axoprobe 1A (Axon Instrument).
3. Results 3.1. Basic features of evoked magnetic and electrical signals in the presence of PTX In the presence of 0.1 mM PTX, the apical stimulation evoked a typical triphasic neuromagnetic ®eld with mirrorimage waveforms recorded at SQ-2 and SQ-4 and nearbaseline signals at SQ-1 and SQ-3 (Fig. 1C). This spatial pattern is consistent with a more complete mapping of the ®eld distribution over the bath (Okada et al., 1997; Wu and Okada, 1998), indicating that the currents responsible for the neuromagnetic signals were directed along the longitudinal axis of the pyramidal cells which is perpendicular to the cell layer. The waveform consisted of an initial `spike', a return to the baseline or a slight overshoot past the baseline, and a slow wave. The ®eld polarity indicates that the underlying intracellular current was directed from the apical to basal side for both the initial spike and slow wave. This direction is consistent with the current underlying the ®eld potentials recorded along the longitudinal axis of the pyramidal cells (Fig. 1C). The potential waveform consisted of a train of synchronized population spikes, followed by a slow wave. In the apical area, a negative spike (labeled a) was followed by a positive spike (labeled
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Fig. 2. Effects of TEA in presence of 0.1 mM PTX on apical stimulation-evoked magnetic ®elds measured at SQ-2 and SQ-4, ®eld potentials in the basal and apical areas, and on intracellular potential in a pyramidal cell (Vin) and simultaneously measured ®eld potential in the basal area (Vex-unaveraged). Magnetic ®elds and ®eld potentials (top 4 traces) were recorded simultaneously from the same slice. Vin and Vex were recorded simultaneously from another one. Arrows indicate the stimulus onset; stimulus artifacts not removed. 30 epochs/ave for the top 4 traces. Single-trial data for Vin and Vex.
b). These spikes corresponded in latency to the initial and last part of the spike peak in the magnetic ®eld, as indicated by two vertical lines next to the magnetic ®eld peaks at SQ-2 and SQ-4. Spikes a and b in the apical area matched in latency with a positive spike (`a') and a negative spike (`b'), respectively, in the ®eld potential in the soma area. Spike b in the apical area also matched in latency with a positive spike (`b') in the basal dendrites area. The polarities and relative amplitudes of these spikes are consistent with directions of the underlying intracellular currents inferred from the magnetic data. Spike a appears to have been generated by a current sink in the apical dendrites with the intracellular current directed toward the soma since the extracellular potential was negative in the apical area and positive in the cell layer. Spike b generated by a current sink near the soma, as revealed by the extracellular negativity in the cell layer, was directed toward the basal dendrites, since spike b was more positive in the basal than in the apical area. Synchronization becomes less across the slice for the later spikes in the spike train (Okada et al., 1997). Consequently, they are much weaker or absent in the magnetic signals. The slow wave in the soma area always peaked earlier
than the slow wave of the magnetic ®eld. On the other hand, the slow waves in the apical and basal areas were quite similar to that of the magnetic ®eld in peak latency and duration. The polarity was negative in the apical region and positive in the basal region, indicating that the underlying intracellular current was directed from the apical to basal side as was inferred from the magnetic ®eld data. In sum there is a high degree of correspondence in waveform, as corroborated in the subsequent ®gures of this paper, between the magnetic ®elds recorded on two sides of the slice and the ®eld potentials recorded in the apical and basal areas of the pyramidal cells. In presenting the results, we show in the subsequent ®gures only the magnetic data from SQ-2 and SQ-4 since the ®elds at SQ-1 and SQ-3 were close to zero. In comparing the magnetic ®eld data with the other types of signals, we present the ®eld potentials from the apical and basal areas since their waveforms best corresponded to the magnetic ®elds. 3.2. Effects of manipulating gK(C) by TEA on evoked magnetic and electrical signals The role of gK(C) in shaping neuromagnetic signals was
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Fig. 3. (A) Effects of TEA on evoked magnetic ®eld (SQ-2), ®eld potential (Basal) and intracellular potential (Vin). Magnetic signal and ®eld potential were recorded simultaneously from one slice. Vin was recorded from another slice. (B) Concentration-ef®ciency curve of TEA for the slow wave of the magnetic signal. MEF, magnetic evoked ®eld. All responses were normalized to the magnetic signal in Ringer with 0.1 mM PTX (see symbol marked with star). Each point is an average of 4 cases and the vertical bars show ^ 1 SEM. (C) Broadening of the initial intracellular spike in the records shown in (A) at two levels of TEA concentration.
studied with TEA in the presence of PTX. TEA at concentrations of 0.5±2 mM selectively blocks gK(C) of the hippocampal pyramidal cells (Lancaster and Adams, 1986; Lancaster and Nicoll, 1987; Storm, 1987). At this level of concentration, TEA also enhances release of excitatory neurotransmitters (Rutecki et al., 1990). At higher concentrations (25±30 mM) it also completely blocks gK(DR) (Storm, 1990). As shown in Figs. 2 and 3 mM TEA in the bath had only subtle effects on the initial component of the evoked magnetic ®elds, but dramatically enhanced the slow wave. The arrows indicate the time of stimulus onset; the stimulus artifacts were not removed in this and all the subsequent ®gures. The amplitude of the initial spike relative to the control condition was 1:07 ^ 0:12 (mean ^ 1 SEM (standard error of mean), n 4). The slow wave amplitude was 3:30 ^ 0:49 times the control amplitude (t 4:69, n 4, P , 0:01). Its peak latency was 1:61 ^ 0:08 (n 4) times longer than the control. The ®eld potentials measured in the basal and apical areas were very close in waveform to the simultaneously measured magnetic ®elds. Accordingly, their initial component consisting of a train of synchronized population spikes was not changed clearly, but the subse-
quent slow wave was enhanced and its peak latency was lengthened by TEA. These effects were reversible (see Washout). In a separate set of experiments, the intracellular potential was measured from the pyramidal cells simultaneously with ®eld potentials in order to obtain insight at the cellular level for the observed effects on the population responses. In the example shown in Fig. 2 (bottom two traces), the slow wave of the unaveraged ®eld potential (Vex) was enhanced by 3 mM TEA replicating the results for the above experiment. This concentration of TEA slightly broadened the initial sodium spike of the intracellular potential, but did not affect its peak amplitude. It, however, produced a clear calcium spike (initial broad spike) detectable in the somatic recording and broadened the following spikes which are probably dominated by calcium currents as well. The paroxysmal depolarizing shift (PDS) terminated abruptly at about 80 ms post-stimulus. All these TEA effects were reversible (Fig. 2, Washout). There was no component in the intracellular record that corresponded directly to the slow wave of the magnetic ®eld because our intracellular measurements were all made in the soma rather than in the apical dendrites. However, previous intradendritic recordings have shown that the hippocampal pyramidal cells are capable of locally producing bursts mediated by calcium currents (e.g. Wong and Stewart, 1992). Fig. 3 shows the apical-stimulation evoked magnetic and electrical population responses in the presence of various concentrations of TEA. The synchronized population spikes in the ®eld potential were slightly larger and broader in width at a TEA concentration of 3 mM in comparison to the control. This effect is consistent with the known action of blocking the repolarizing gK(C). Fig. 3C shows the initial spike of the intracellular recording (Vin) presented at the bottom of Fig. 3A. The repolarizing portion of the initial spike clearly became longer in duration when TEA in the bath was increased to 3 mM. Increasing TEA to 10 mM reduced the amplitudes of the initial component of the magnetic signal and the initial spike of the ®eld potential. This reduction may be due to the further prolongation of the action potentials by blocking gK(DR) since the initial spike of the intracellular potential shown in Fig. 3C is broadened even more. Such a block should lead to reduced repetitive ®ring. The increase did not further enhance the slow wave
Fig. 4. Effects of TEA on the intracellular potential elicited by injection of constant current into the recorded pyramidal cell under a current clamp mode. Bath concentration of 10 mM TEA broadened the sharp spike, increased spike frequency and activated a calcium spike.
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Fig. 5. Increased spontaneous activity of the longitudinal CA3 slice in the presence of 10 mM TEA in the bath. (A) Magnetic ®eld at two locations and ®eld potential near the soma simultaneously recorded from a slice in control medium (Ringer 1 0.1 mM PTX), in the Ringer with PTX 1 TEA, and in control medium during the Washout. (B) Intracellular potential (Vin) and simultaneously recorded ®eld potential near the soma (Vex) from another slice in the 3 conditions.
amplitude, but broadened its waveform and delayed the peak latency. Fig. 3B shows that the effect of TEA on slow wave amplitude peaked at a TEA concentration of about 3 mM. Since TEA may not only potentiate post-synaptic potentials, but also increase transmitter release from pre-synaptic terminals, we directly examined post-synaptic effects of TEA on the pyramidal cells, by injecting dc current from the recording pipette. As shown in Fig. 4, 10 mM TEA clearly broadened the spikes in a manner similar to the effects of TEA on the sodium and calcium spikes evoked by the apical stimulation (Fig. 3A), namely, it broadened the sodium spikes and produced a clear calcium spike. Also the spike accommodation was reduced, consistent with a known action of TEA. These results indicate that the effects of TEA observed in the intracellular recording above are at least in part due to its post-synaptic effects. 3.3. Effects of manipulating gK(C) by TEA on spontaneous magnetic and electrical signals TEA in the bath at a concentration of 10 mM heightens excitability of the longitudinal CA3 slice as shown by the simultaneously recorded single-epoch magnetic and extracellular ®eld potential signals (Fig. 5A) and by the simultaneously recorded intracellular potential in a pyramidal cell
and ®eld potential near the soma (Fig. 5B). In the presence of 0.1 mM PTX the slice exhibited spontaneous single burst discharges that were synchronized suf®ciently across the slice to be seen as a population response in the magnetic signal. The magnetic ®eld during the burst was outward at location SQ-2 and inward at SQ-4, indicating that its underlying current was directed from the apical to basal side just as for the slow wave of the evoked response. TEA reversibly produced a train of burst discharges lasting 1±2 s. The direction of the steady shift of the magnetic ®eld during each train of bursts was downward at SQ-2 and upward at SQ4, indicating that its underlying current was directed from the basal/soma toward apical side. Intracellular records in the control condition showed a mixture of simple sodium spikes which were not seen in the simultaneously recorded ®eld potential and calcium bursts which were synchronized suf®ciently to be seen by the ®eld potential recording electrode (Fig. 5B, bottom). In bath with 10 mM TEA, the intracellular record showed a mixture of single spikes, which were again not seen in the ®eld potential recording, and strong, long-lasting repetitive bursts. These long-lasting bursts corresponded to the multiple spikes in the ®eld potential. During the washout phase, the duration of individual bursts became shorter, eventually returning to the activity pattern of the control condition. The increased excitability in presence of TEA is consistent with
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Fig. 6. Effects of 4AP on apical stimulation-evoked magnetic ®elds at locations SQ-2 and SQ-4, ®eld potentials in the basal and apical areas recorded simultaneously from one slice (30 epochs/ave), and also on intracellular potential (Vin) and simultaneously recorded ®eld potential in the soma area (Vex) from another slice.
®ndings from transverse slice preparations (e.g. Fueta and Avoli, 1993). 3.4. Effects of manipulating gK(A) and gK(D) with 4AP on evoked population responses The gK(A) was ®rst described in CA3 cells of hippocampal slices from adult guinea-pigs by Gustafsson et al. (1982) and byZbica and Weight (1985) This class of conductance is voltage-sensitive, but calcium-independent. The gK(A) in the hippocampus is blocked by extracellular 4AP concentrations of 0.1±0.5 mM (Storm, 1990). 4AP blocks not only gK(A), but also gK(D). The gK(D) is similar to gK(A) in that both are calciumindependent and 4AP sensitive, but it inactivates more slowly and is more sensitive to 4AP than gK(A) in that it is completely blocked by approximately 30 mM (Storm, 1990). Since there are two distinguishable types of 4AP sensitive transient potassium currents in the CA3 cells, we evaluated the relative contributions of these two types of conductances to the magnetic and electrical population responses. Fig. 6 shows that 4AP at a concentration of 0.1 mM slightly increased the initial `spike' in the triphasic magnetic
and electrical population responses. The mean amplitude of the spike in the magnetic signal was 1:36 ^ 0:14 times the control (t 2:57, n 4, P , 0:05). As for the slow wave, 4AP also dramatically enhanced its amplitude by 3:74 ^ 0:58 times the control (t 4:72, n 4, P , 0:01), shortened its peak latency (81 ^ 7%) and reduced duration (73 ^ 7%) relative to the control. In intracellular recordings, 4AP clearly widened the spikes and enhanced the PDS. The ®eld potential (Vex-soma) was recorded on the basal side of the cell layer and thus the population activity was not seen as strongly as, for example, in the somal recording of Fig. 1, but was closer to the basal recording in the same ®gure. The observed effects of 4AP may be due to a combined effect of gK(A) and gK(D) since they should be both blocked by 0.1 mM 4AP. We thus studied the effects of 4AP at a concentration (0.01 mM) where it should selectively block gK(D) and at a higher concentration (0.1 mM) where gK(A) should be blocked by 4AP in addition to gK(D). As shown in Fig. 7A, 4AP enhanced the amplitude and shortened the peak latency of the slow wave of both the neuromagnetic ®eld and ®eld potential even at a concentration of 0.01 mM, indicating that blocking gK(D) may enhance the slow wave. However, the slow wave was further enhanced by 0.1 mM
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Fig. 7. (A) 4AP effects on evoked magnetic ®eld (SQ-2), ®eld potential (basal) and intracellular potential (Vin) at 0.01 mM which selectively blocks D currents mediated by gK(D) and at 0.1 mM which blocks gK(A) and gK(D). Magnetic signal and ®eld potential were recorded simultaneously from one slice. Vin was recorded from another slice. (B) Concentration-ef®ciency curve of 4AP for the slow wave of the magnetic signal. All responses were normalized to the magnetic signal in Ringer with 0.1 mM PTX (see symbol marked with a star). Each point shows the average of 4 cases and the vertical bars show ^ 1 SEM. (C) Broadening of the initial intracellular spike in the records shown in (A) at two levels of 4AP concentration.
4AP. The concentration-ef®ciency curve in Fig. 7B indicates that the effect of blocking gK(D) was fairly small compared to that of blocking the A current, since the enhancement effect was maximal at the concentration where gK(A) is expected to be blocked. Supporting this conclusion, the initial spike of the intracellular response shown in Fig. 7A was modi®ed only slightly at the concentration of 0.01 mM, but was clearly broader in width at a concentration of 0.1 mM 4AP (Fig. 7C). This result indicates that blocking gK(D) did not have a large effect on the action potential, but blocking gK(A) did. As in the case of TEA, 4AP facilitates synaptically mediated responses by pre- and post-synaptic mechanisms. We assessed the post-synaptic effects of 4AP on the pyramidal cells in our preparation at a concentration of 0.1 mM by directly injecting current into a pyramidal cell under a current clamp condition and measuring the voltage response of the cell. Fig. 8 shows that 0.1 mM 4AP increased the number of spikes and increased the height of each spike. This result demonstrates that 4AP had some post-synaptic effects. 3.5. Effects of 4AP on spontaneous magnetic and electrical responses As in the case of TEA, 4AP enhanced the spontaneous
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activity of cells in the longitudinal CA3 slice. In the control condition with 0.1 mM PTX in the bath (Fig. 9A), there were spontaneously occurring single bursts detectable with the magnetic and electrical techniques. Just as in the control condition of Fig. 5, the current producing the burst was directed from the apical to basal side. 4AP at a concentration of 0.1 mM that should be suf®ciently high to block gK(A) increased the spontaneous activity with more numerous trains of bursts. The currents during each burst was again directed from the apical to basal side. The direction of this current is consistent with the laminar ®eld potential pro®le of this component produced by 4AP in CA1 (Voskuyl and Albus, 1985). The steady shift during each train of bursts was directed from the basal to apical side just as in the case of TEA (Fig. 5). Intracellular recordings from the pyramidal cells were consistent with the population responses. In the example shown in Fig. 9B, the spontaneous activity consisted of a mixture of single spikes and bursts. Only the bursts were suf®ciently synchronized locally to produce detectable ®eld potentials. 4AP changed the spontaneous activity to a mixture of single bursts and long-lasting trains of bursts that could be seen as multiple bursts in the simultaneously recorded extracellular potentials (Vex). The increased spontaneous activity in the longitudinal CA3 slices in the presence of 4AP is consistent with the earlier results for transverse hippocampal slice preparations (e.g. Perreault and Avoli, 1991).
4. Discussion The main ®nding in this study is that bath application of potassium channel blockers, TEA and 4AP, had only small effects on the initial component of the triphasic magnetic signal and the corresponding component of the evoked potential, but strongly enhanced their slow wave elicited by the apical stimulation in CA3 longitudinal slices. TEA and 4AP have a number of effects on the behavior of neurons in the hippocampus both pre- and post-synaptically. Therefore, it is necessary to carefully consider possible effects of these channel blockers in order to infer roles of the calcium- and voltage-sensitive potassium conductances in the genesis of neuromagnetic signals and ®eld potentials.
Fig. 8. Effects of 4AP on the intracellular potential elicited by injection of constant current into the recorded pyramidal cell under a current clamp mode.
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Fig. 9. Increased spontaneous activity of the longitudinal CA3 slice in presence of 0.1 mM 4AP in the bath. (A) Magnetic ®eld at two locations and ®eld potential near the soma simultaneously recorded from a slice in control medium (Ringer 1 0.1 mM PTX), in the Ringer with PTX 1 4AP, and in control medium during the Washout. (B) Intracellular potential (Vin) and simultaneously recorded ®eld potential near the soma (Vex) from another slice in the 3 conditions.
4.1. Roles of the potassium currents in generating the spike Our hypothesis is that the initial spike in the magnetic signal was produced by sodium spikes in the soma and trunk of the apical dendrites. According to our view, the apical stimulation depolarizes the soma and dendritic trunk. The densities of gNa and gK(DR) are thought to be high in the proximal trunk and soma of the pyramidal cells (Traub et al., 1994). Thus, both of these regions are capable of generating sodium spikes. Our results indicate that a spike was ®rst generated in the apical trunk followed by a spike in the soma area. Spike a of the ®eld potential was negative in the apical area and positive in the soma area, suggesting that the spike was generated in the apical area close to the site of stimulation. The polarities in the apical and soma areas indicate that the underlying intracellular current was directed from the apical dendrites to the soma. Spike b appears to have been generated in the soma. We inferred from the polarities of spike b that its underlying intracellular current was directed from the soma toward the basal dendrites. The current generated in the soma should be directed toward the basal dendrites because the apical dendrites should be strongly depolarized by the initial apical spike and thus the potential gradient inside the cell forces the current to be directed toward the basal dendrites. The latencies of these spikes matched the broad peak of the magnetic ®eld
in latency. Thus, the spike of the magnetic ®eld appears to have been produced by currents generated in the apical trunk and soma. The intracellular recordings have shown that both TEA and 4AP did have post-synaptic effects on the spikes generated by pyramidal cells in our longitudinal CA3 slices. The response to injected current under a current clamp mode showed broader spikes, a calcium spike and less accommodation, all indicating post-synaptic effects of TEA. The intracellular potential showed larger and broader spikes with less accommodation in the current clamp mode in presence of 0.1 mM 4AP. Thus, TEA and 4AP should have had some post-synaptic effects on the spikes in the ®eld potential and magnetic ®eld generated by the apical stimulation. The bath concentration of TEA at 3 mM broadened the spikes of the evoked intracellular potentials. Raising the concentration to 10 mM broadened the spikes further. The corresponding spikes of the magnetic ®eld and ®eld potential were broader, but were not signi®cantly enhanced in presence of 3 mM TEA compared to the control. Amplitude of the spike in the neuromagnetic signals was slightly, but signi®cantly elevated by 0.1 mM 4AP. Although the broadening of the spikes under TEA and the enhancement in the presence of 4AP must be in part due to their post-synaptic effects, it is not possible to separate the effects from possible pre-synaptic increase in neurotrans-
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mitter release that may be caused by both TEA and 4AP. They are both thought to increase neurotransmitter release and enhance synaptic transmission in both excitatory and inhibitory neurons at the concentrations used in our study (0.01±0.1 mM for 4AP and 1±10 mM for TEA) (Buckle and Haas, 1982; Avoli and Perreault, 1987; Rutecki et al., 1987, 1990; Perreault and Avoli, 1989, 1991; Traub et al., 1995; Wheeler et al., 1996). In our study the effects of TEA and 4AP on GABAA inhibitory neurotransmitter release can be ignored since 0.1 mM PTX used in all the experiments is strong enough to block the synaptic transmission in hippocampal slices (Miles and Wong, 1987). GABAB type inhibition should be functional in our preparation, but this slow inhibition probably does not signi®cantly affect the slow wave which has a relatively short latency. However, we may have to take into account possible increases in neurotransmitter release at the recurrent excitatory synapses that connect the pyramidal cells to each other (Miles and Wong, 1986, 1987a) and possible release of neurotransmitters from the mossy ®ber terminals that are cut but may be functional in order to more fully understand the effects of TEA and 4AP of the spike. These issues will have to be clari®ed in future studies. 4.2. Roles of the potassium currents in generating the slow wave The effects of TEA and 4AP on the slow wave of the magnetic ®eld and ®eld potential were quite robust. The slow wave increased in amplitude and its peak latency became shorter in presence of 4AP. The concentration-ef®ciency curve for 4AP indicated that the enhancement of the slow wave was relatively small at a concentration of 4AP where it is expected to block gK(D), but became greater with stronger concentrations of 4AP, peaking at a concentration of 0.1 mM where it is expected to block gK(A). Thus, we infer that the A current, rather than the D current, plays a dominant role in generating the slow wave. According to this hypothesis, blocking gK(A) with 4AP should reduce the repolarization of the spikes mediated by Na currents which in turn should help activate the high-threshold gCa (Wong and Stewart, 1992). This hypothesis, however, requires further validation, since it would be necessary to show that the range of concentrations of 4AP used in this study selectively blocks gK(A) and gK(D) and that selective elimination of these channels produces the changes in the kinetics of gCa opening and closing that are presumably responsible for the enhancement of the slow wave. As for the effects of TEA, the slow wave amplitude and peak latency increased in presence of TEA. The effect of TEA on slow wave amplitude peaked at a concentration of 3 mM. This suggests that the amplitude increased at concentrations of TEA where gK(C) is fully blocked, but not gK(DR). The lack of further enhancement of the slow wave amplitude in presence of 10 mM TEA may be because of reduced inputs onto the apical dendrites via the recurrent excitatory
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synapses due to reduced population spikes. Blocking of gK(C) by TEA should delay the repolarization of the spikes mediated by gCa. Thus, the manipulations with 4AP and TEA should both lead to an increase the apical dendritic currents mediated by gCa. The greater depolarization of the apical dendrites should increase the opening of NMDA (Nmethyl-d-aspartate) channels by excitatory inputs in the recurrent excitatory synapses of the pyramidal cells. The slow wave thus in our view is due to a mixture of currents produced by the intrinsic gCa and synaptic currents carried by the NMDA channels. We are not able to assess their contributions separately based on the present set of data. The increase in intracellular calcium concentration should enhance the activation of gK(AHP) since it is not affected by TEA or 4AP. The activation should help terminate the depolarization earlier (Traub et al., 1993) and hasten the return of the slow wave to the baseline as was seen clearly in Fig. 2. 4.3. Roles of TEA and 4AP in producing spontaneous activity In the presence of TEA or 4AP, the spontaneous activity that was present in Ringer with PTX became more synchronized across the entire slice. Single spikes transformed to bursts. The polarities of the magnetic ®eld and ®eld potentials during these spontaneous bursts were the same as for the evoked potentials. The analysis of enhanced spontaneous activity and temporal waveform of each spontaneous burst would follow the above framework for evoked responses. However, there are some additional complications in the analysis. For example, 4AP produces spontaneously occurring ectopic axonal spikes which can propagate anti- and orthodromically to help initiate synchronized population spikes via orthodromic recurrent excitatory synapses (Traub et al., 1995). In the case of evoked responses, this ectopic spike can be ignored since the population of neurons are synchronously stimulated externally. Detailed analysis is, however, beyond the scope of this paper. 4.4. Signi®cance It should be emphasized again that the above interpretation of effects of TEA and 4AP on the magnetic ®eld and ®eld potential should be viewed as a hypothesis since this study is still in the state of infancy. It is dif®cult to cleanly block only one conductance of interest without any other complicating effect using any so-called selective channel blocker. Furthermore, even if it is possible, blocking one conductance has ramifying consequences on the rest of the channels and the functioning of the entire neuronal circuitry. Thus, we believe that further advances in our understanding of the genesis of MEG and EEG signals are possible only through a careful study combining a mathematical model of the CNS tissue of interest with experimen-
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tal analyses at the level of neuronal ensemble, single cells and channels. Nevertheless, we believe that this study has demonstrated important roles of the potassium currents in determining the temporal waveform of magnetic ®elds and extracellular potentials. Since the kinetics of these channels are highly non-linear, the consequences of altering any one channel on the waveform of the magnetic and electrical signals may be profound as seen in the present study. This implies that abnormality of these potassium conductance can profoundly in¯uence the temporal waveform of MEG and EEG signals in general. Acknowledgements The present study was performed at the VA Medical Center, Albuquerque, NM 87108, USA. The authors thank the staff of the Research Service of the VA for providing animal care and our research assistant, Ms. Anna Barrios. This study was supported by NIH grant R01-NS21149 to Y.C.O. References Avoli M, Perreault P. A GABAergic depolarizing potential in the hippocampus disclosed by the convulsant 4-aminopyridine. Brain Res 1987;400:191±195. Buckle PJ, Haas HL. Enhancement of synaptic transmission by 4-aminopyridine in hippocampal slices of the rat. J Physiol 1982;326:109±122. Fueta Y, Avoli M. tetraethylammonium-induced epileptiform activity in young and adult rat hippocampus. Dev Brain Res 1993;72:51±58. Gustafsson B, Galvan M, Grafe P, Wigstrom HA. Transient outward current in mammalian central neuron blocked by 4-aminopyridine. Nature 1982;299:252±254. Hille B. Ionic channels of excitable membranes, Sunderland, MA: Sinauer, 1992. Kyuhou SI, Okada YC. Detection of magnetic evoked ®elds associated with synchronous population activities in the transverse CA1 slice of the guinea pig. J Neurophysiol 1993;70:2665±2668. Lancaster B, Adams PR. Calcium-dependent current generating the after hyperpolarization of hippocampal neurons. J Neurophysiol 1986;55:1268±1282. Lancaster B, Nicoll RA. Properties of two calcium-activated hyperpolarizations in rat hippocampal neurons. J Physiol 1987;389:187±203. Miles R, Wong RKS. Excitatory synaptic interactions between CA3 neurones in the guinea-pig hippocampus. J Physiol 1986;373:397± 418. Miles R, Wong RKS. Inhibitory control of local excitatory circuits in the guinea-pig hippocampus. J Physiol 1987a;388:611±629. Miles R, Wong RKS. Latent synaptic pathways revealed after tetanic stimulation in the hippocampus. Nature 1987b;329:724±726. Miles R, Traub RD, Wong RKS. Spread of synchronous ®ring in longitudinal slices from the CA3 region of the hippocampus. J Neurophysiol 1988;60:1481±1496.
Okada YC, Shah B, Huang J-C. Ferromagnetic high-permeability alloy alone can provide suf®cient low-frequency and eddy-current shieldings for biomagnetic measurements. IEEE Trans Biomed Eng 1994;41:688± 697. Okada YC, Wu J. Kyuhou. S.I. Genesis of neuromagnetic signals in a mammalian CNS structure. Electroenceph clin Neurophysiol 1997;103:474±485. Perreault P, Avoli M. Effects of low concentrations of 4-aminopyridine of CA1 pyramidal cells of the hippocampus. J Neurophysiol 1989;61:953± 970. Perreault P, Avoli M. Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices. J Neurophysiol 1991;65:771±785. Rutecki PA, Lebeda FJ, Johnston D. 4-aminopyridine produces epileptiform activity in hippocampus and enhances synaptic excitation and inhibition. J Neurophysiol 1987;57:1911±1924. Rutecki PA, Lebeda FJ, Johnston D. Epileptiform activity in the hippocampus produced by tetraethylammonium. J Neurophysiol 1990;64:1077± 1088. Storm JF. Action potential repolarization and a fast after-hyperpolarization in rat hippocampal pyramidal cells. J Physiol 1987;385:733±759. Storm JF. Potassium currents in hippocampal pyramidal cells. Prog Brain Res 1990;83:161±185. Traub RD, Miles R. Neuronal networks of the hippocampus, Cambridge, MA: Cambridge University Press, 1991. Traub RD, Wong RKS, Miles R, Michelson H. A model of a CA3 hippocampal pyramidal neuron incorporating voltage-clamp data on intrinsic conductances. J Neurophysiol 1991;66:635±650. Traub RD, Miles R, Jefferys JGR. Synaptic and intrinsic conductances shape picrotoxin-induced synchronized after-discharges in the guineapig hippocampal slice. J Physiol 1993;461:525±547. Traub RD, Jefferys JGR, Miles R, Whittington MA, ToÂth K. A branching dendritic model of a rodent CA3 pyramidal neurone. J Physiol 1994;481:79±95. Traub RD, Colling SB, Jefferys GR. Cellular mechanisms of 4-aminopyridine-induced synchronized after-discharges in the rat hippocampal slice. J Physiol 1995;489:127±140. Voskuyl RA, Albus H. Spontaneous epileptiform discharges in hippocampal slices induced by 4-aminpyridine. Brain Res 1985;342:54± 66. Wheeler DB, Randall A, Tsien RW. Changes in action potential duration alter reliance of excitatory synaptic transmission on multiple types of Ca 21 channels in rat hippocampus. J Neurosci 1996;16:2226±2237. Wong RKS, Stewart M. Different ®ring patterns generated in dendrites and somata of CA1 pyramidal neurones in guinea-pig hippocampus. J Physiol 1992;457:675±687. Wu J, Okada CY. Voltage- and Ca 21-dependent K 1 conductances contribute to the shape and size of biomagnetic signals in guinea pig hippocampal CA3 longitudinal slice. Presented at the 10th 1996;16±21. Wu J, Okada CY. Physiological bases of the synchronized population spikes and slow wave of the magnetic ®eld generated by a guinea-pig longitudinal CA3 slice preparation. Electroenceph clin Neurophysiol 1998;107:361±373. Wu J, Okada CY. Roles of a potassium afterhyperpolarization current in generating neuromagnetic ®elds in longitudinal CA3 slices of the guinea-pig. Electroenceph clin Neurophysiol submitted 1999;in press. Zbica KL, Weight EF. Transient voltage and calcium-dependent outward currents in hippocampal neurons. J. Neurophysiol. 1985;53:1038±1058.
Roles of calcium- and voltage-sensitive potassium currents in the generation of neuromagnetic signals and ®eld potentials in a CA3 longitudinal slice of the guinea-pig Jie Wu a, b, Yoshio C. Okada a, b,* a
Department of Neurology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
b
Accepted 29 July 1999
Abstract Objectives: Roles of calcium- and voltage-sensitive potassium currents in generation of neuromagnetic signals and ®eld potentials were evaluated using the longitudinal CA3 slice preparation of the guinea-pig. Methods: Their roles were evaluated by using selective channel blockers (tetraethyl-ammonium (TEA) and 4-aminopyridine (4AP)) and measuring their effects on the two types of signals and intracellular potentials. Fast g-aminobutyric acid type A inhibition was blocked with picrotoxin. Results: Stimulation of the apical dendrites with an array of extracellular bipolar electrodes produced triphasic evoked magnetic ®elds with a spike and a slow wave typical of the slices. The evoked potentials in the apical and basal areas of the pyramidal cells closely resembled the magnetic ®eld waveforms. Blockade of the potassium currents with TEA and 4AP had only subtle effects on the initial spike, but dramatically altered the slow wave. They also induced long-lasting spontaneous burst discharges synchronized across the slice. The results could be interpreted in terms of their known pre- and postsynaptic effects. Their post-synaptic effects were con®rmed with intracellular recordings. Conclusion: Our results are consistent with a hypothesis that the calcium- and voltage-sensitive potassium currents, especially the A and C currents, play important roles in shaping the slow wave of the neuromagnetic and ®eld potential signals produced by the mammalian hippocampus. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Biomagnetism; Magnetoencephalography; Electroencephalography; Evoked potential; Potassium conductance; CA3; Hippocampus; Tetraethylammonium; 4-aminopyridine; Epilepsy
1. Introduction Discoveries of numerous active conductances over the past 20 years (Hille, 1992) have motivated us to initiate a systematic re-examination of the genesis of magnetoencephalography (MEG) and electroencephalography (EEG) signals in light of the new concepts of dendritic and somatic electrophysiology of central nervous system (CNS) neurons (Kyuhou and Okada, 1993; Okada et al., 1997; Wu and Okada, 1998, 1999). The presence of voltage- and calcium-sensitive intrinsic conductances in the dendrites and soma of CNS neurons renders the post-synaptic currents more complex than the classic picture which painted the * Corresponding author. Tel.: 11-505-256-2874; fax: 11-505-2600165. E-mail address: [email protected] (Y.C. Okada)
currents in the dendrites and soma to have been passively (electrotonically) generated by neurotransmitter inputs. Yet, the relationships between MEG and EEG signals on one hand and the intracellular currents produced by opening and closing of these active conductances on the other are still poorly understood. This issue was examined in the longitudinal CA3 slice preparation of the guinea-pig since its cellular organization is relatively simple and its single-cell electrophysiology is well-understood for relating the MEG and EEG signals produced by this preparation to intracellular events in the principal neurons called the pyramidal cells. A variety of intrinsic ionic conductances are thought to be important in controlling neuronal activity of the pyramidal cells. They include a sodium conductance (gNa), a calcium conductance (gCa), voltage-sensitive potassium conductances of delayed recti®er type (gK(DR)), A type (gK(A)) and D type (gK(D)), a
1388-2457/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S13 88-2457(99)0020 7-2
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Fig. 1. (A) A hippocampus was placed on an agar block and two 400-mm thick longitudinal slices were cut with a Vibratome just above the ®mbria. (B) A rectangular longitudinal CA3 slice was prepared by separating CA3 from CA1 with a razor blade. The slice was placed in a recording chamber (cf. Okada et al., 1997). Six pairs of bipolar stimulating electrodes (125 mm diameter Ag±AgCl wires) were placed from below the slice along the pyramidal cell layer with negative electrodes in stratum radiatum to stimulate the apical dendrites. Three Ag±AgCl (125 mm diameter) wires were used to record ®eld potentials from the basal, soma and apical regions of the pyramidal cells. Neuromagnetic ®elds were recorded with a 4 channel superconducting magnetic ®eld detector called a microSQUID. Its detection coils were 4 mm in diameter, separated by 8.4 mm diagonally and placed 2 mm above the slice. (C) An example of the apical stimulation-evoked magnetic ®elds and ®eld potentials recorded by the 4 microSQUID sensors and 3 electrodes. Note that the magnetic signals were strong and mirror-images of each other at SQ-2 and -4, whereas they were close to the baseline at SQ-1 and -3, indicating that the underlying currents were along the longitudinal axis of the pyramidal cells (perpendicular to the cell layer). The magnetic ®eld waveforms were most closely related with the ®eld potentials in the basal and apical areas and less so in the soma area. Thus, the magnetic data are compared with apical and basal ®eld potentials in the subsequent ®gures. Outward ®eld is up in this and all the ®gures. Stimulus artifacts not removed in this and subsequent ®gures. 30 epochs/ave.
calcium- and voltage-sensitive potassium conductance of C type (gK(C)), and a calcium-sensitive potassium conductance of afterhyperpolarization (AHP) type (gK(AHP)) (Storm, 1990). These active conductances have their own characteristic non-linear activation and inactivation kinetics (Traub and Miles, 1991; Traub et al., 1994). Therefore, the temporal waveform and spatial distribution of the currents along the longitudinal axis of the pyramidal neurons depend profoundly on the interplay of these active channels as well as on the network properties of the hippocampus. Consequently, the temporal waveform of MEG and EEG signals may be profoundly affected by these active conductances. In order to evaluate the roles of a number of potassium active conductances, we chose the strategy of using selective channel blockers to block some of these conductances and study the consequences on the MEG and EEG signals
produced by the remaining conductances. In the preceding report (Wu and Okada, 1999), we have described the role of gK(AHP) in modulating MEG and EEG signals. Here, we used tetraethylammonium (TEA) and 4-aminopyridine (4AP) to manipulate other principal potassium conductances. We carefully selected the concentrations of TEA and 4AP in order to be able to infer the role of gK(DR), gK(A), gK(D), and gK(C) in generating MEG and EEG signals from the hippocampus. Also, we recorded ®eld potentials simultaneously with intracellular potentials from the pyramidal cells during the manipulations with selective channel blockers in order to clarify the roles of the active conductances. Eventually, it will be necessary to interpret the effects of these experimental manipulations with the help of a mathematical model (e.g. Traub and Miles, 1991; Traub et al., 1991, 1994, 1995) in order to arrive at a clearer understanding of roles
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of the active potassium currents in generating MEG and EEG signals. Some of the results have been presented in abstract form (Wu and Okada, 1996). 2. Methods 2.1. Slice preparation and solution The preparation of CA3 longitudinal slices was described in detail in a recent paper (Okada et al., 1997). The procedure will be thus only brie¯y described. Readers are referred to earlier studies by Miles, Wong and others for electrophysiology of this preparation (Miles and Wong, 1987b; Miles et al., 1988). Twenty-six animals were used in this study. Each animal was anesthetized with sodium pentobarbital (60 mg/kg i.p.) and decapitated. Each hippocampus was placed on an agar block and two 400-mm thick slices were cut parallel to the agar surface just above the ®mbria using a Vibratome (Technical Products International) (Fig. 1A). Then, the CA3 was separated from CA1 for each slice with a razor blade and was placed in chilled oxygenated Ringer solution (concentration in mM: NaCl, 124; KCl, 5; KH2PO4, 1.2; MgCl2, 1.3; CaCl2, 2.5; NaHCO3, 26 and glucose, 10) and incubated in the medium for 1 h or more until the medium attained room temperature. Its pH was maintained at 7.4 with 95% O2/5% CO2. One slice was then placed in a recording chamber in which the slice was immersed in a bath containing 36 ^ 0.58C oxygenated Ringer solution. TEA was used in experiments with 15 animals. 4AP was used on 15 animals, sometimes together with TEA. Picrotoxin (PTX, 0.1 mM) was used to produce a high degree of synchronization of the neuronal activity across the slice. This was needed for relating the MEG signals, which are due to a weighted sum of currents by all active cells in the slice, and the EEG signals, which are due to population activity within the vicinity of the electrode, and the intracellular potential signals from individual pyramidal neurons. (See Okada et al., 1997, for more on this issue.) 2.2. Stimulation and recording procedures Fig. 1B shows the stimulation and recording arrangements. The slice was stimulated with 6 pairs of bipolar electrodes (Ag±AgCl 125 mm diameter wires), separated by 0.8±1.0 mm between pairs and by about 0.4 mm within pairs. The cathode of each pair was placed against the stratum radiatum and the anode against the basal side of the pyramidal cell layer from below the slice. This mode of stimulation will be designated as `apical stimulation'. The slice was stimulated with constant current pulses (0.7±1.0 mA, 50 ms, 0.1±0.3 Hz). Evoked magnetic ®elds were measured with a 4 channel microSQUID (Okada et al., 1994). The detection coils were placed directly over the slice at a distance of about 2 mm above the slice. The spatial pattern of the magnetic signal was initially determined by
moving the microSQUID over the slice. Once the center of the activity was found, the detection coils were placed around this point at the orientation shown in Fig. 1B. Extracellular ®eld potentials were recorded simultaneously with the magnetic signal using an array of three electrodes (Ag± AgCl 125 mm diameter wires) placed in the tissue from below. The electrodes were placed in the stratum oriens, cell layer and stratum radiatum to record the ®eld potentials in the area of the basal dendrites (`basal'), soma (`soma') and apical dendrites (`apical') (see Fig. 1B). Both magnetic and electrical signals were passed through FET preampli®ers, ampli®ed and ®ltered with Butterworth-type bandpass ®lters (roll off 24 dB/octave, 0.1±1000 Hz), and then recorded at a 10 kHz sampling rate. Conventional intracellular recording methods were used to record activity from 18 slices placed in the same chamber as for the magnetic recordings. Twenty-three pyramidal cells were activated either by stimulation with the same array of electrodes used for the magnetic recording or by directly passing depolarizing current into the pyramidal cells with the recording electrode. Intracellular potentials (Vin) were measured in the soma of the pyramidal cells with a glass micropipette ®lled with potassium acetate (4 M; 50±90 MV). Only cells with resting membrane potentials of at least 255 mV were used. All intracellular recordings were performed simultaneously with a ®eld potential recording (Vex). Intracellular potentials were recorded with a bandwidth of DC to 10 kHz using Axoprobe 1A (Axon Instrument).
3. Results 3.1. Basic features of evoked magnetic and electrical signals in the presence of PTX In the presence of 0.1 mM PTX, the apical stimulation evoked a typical triphasic neuromagnetic ®eld with mirrorimage waveforms recorded at SQ-2 and SQ-4 and nearbaseline signals at SQ-1 and SQ-3 (Fig. 1C). This spatial pattern is consistent with a more complete mapping of the ®eld distribution over the bath (Okada et al., 1997; Wu and Okada, 1998), indicating that the currents responsible for the neuromagnetic signals were directed along the longitudinal axis of the pyramidal cells which is perpendicular to the cell layer. The waveform consisted of an initial `spike', a return to the baseline or a slight overshoot past the baseline, and a slow wave. The ®eld polarity indicates that the underlying intracellular current was directed from the apical to basal side for both the initial spike and slow wave. This direction is consistent with the current underlying the ®eld potentials recorded along the longitudinal axis of the pyramidal cells (Fig. 1C). The potential waveform consisted of a train of synchronized population spikes, followed by a slow wave. In the apical area, a negative spike (labeled a) was followed by a positive spike (labeled
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Fig. 2. Effects of TEA in presence of 0.1 mM PTX on apical stimulation-evoked magnetic ®elds measured at SQ-2 and SQ-4, ®eld potentials in the basal and apical areas, and on intracellular potential in a pyramidal cell (Vin) and simultaneously measured ®eld potential in the basal area (Vex-unaveraged). Magnetic ®elds and ®eld potentials (top 4 traces) were recorded simultaneously from the same slice. Vin and Vex were recorded simultaneously from another one. Arrows indicate the stimulus onset; stimulus artifacts not removed. 30 epochs/ave for the top 4 traces. Single-trial data for Vin and Vex.
b). These spikes corresponded in latency to the initial and last part of the spike peak in the magnetic ®eld, as indicated by two vertical lines next to the magnetic ®eld peaks at SQ-2 and SQ-4. Spikes a and b in the apical area matched in latency with a positive spike (`a') and a negative spike (`b'), respectively, in the ®eld potential in the soma area. Spike b in the apical area also matched in latency with a positive spike (`b') in the basal dendrites area. The polarities and relative amplitudes of these spikes are consistent with directions of the underlying intracellular currents inferred from the magnetic data. Spike a appears to have been generated by a current sink in the apical dendrites with the intracellular current directed toward the soma since the extracellular potential was negative in the apical area and positive in the cell layer. Spike b generated by a current sink near the soma, as revealed by the extracellular negativity in the cell layer, was directed toward the basal dendrites, since spike b was more positive in the basal than in the apical area. Synchronization becomes less across the slice for the later spikes in the spike train (Okada et al., 1997). Consequently, they are much weaker or absent in the magnetic signals. The slow wave in the soma area always peaked earlier
than the slow wave of the magnetic ®eld. On the other hand, the slow waves in the apical and basal areas were quite similar to that of the magnetic ®eld in peak latency and duration. The polarity was negative in the apical region and positive in the basal region, indicating that the underlying intracellular current was directed from the apical to basal side as was inferred from the magnetic ®eld data. In sum there is a high degree of correspondence in waveform, as corroborated in the subsequent ®gures of this paper, between the magnetic ®elds recorded on two sides of the slice and the ®eld potentials recorded in the apical and basal areas of the pyramidal cells. In presenting the results, we show in the subsequent ®gures only the magnetic data from SQ-2 and SQ-4 since the ®elds at SQ-1 and SQ-3 were close to zero. In comparing the magnetic ®eld data with the other types of signals, we present the ®eld potentials from the apical and basal areas since their waveforms best corresponded to the magnetic ®elds. 3.2. Effects of manipulating gK(C) by TEA on evoked magnetic and electrical signals The role of gK(C) in shaping neuromagnetic signals was
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Fig. 3. (A) Effects of TEA on evoked magnetic ®eld (SQ-2), ®eld potential (Basal) and intracellular potential (Vin). Magnetic signal and ®eld potential were recorded simultaneously from one slice. Vin was recorded from another slice. (B) Concentration-ef®ciency curve of TEA for the slow wave of the magnetic signal. MEF, magnetic evoked ®eld. All responses were normalized to the magnetic signal in Ringer with 0.1 mM PTX (see symbol marked with star). Each point is an average of 4 cases and the vertical bars show ^ 1 SEM. (C) Broadening of the initial intracellular spike in the records shown in (A) at two levels of TEA concentration.
studied with TEA in the presence of PTX. TEA at concentrations of 0.5±2 mM selectively blocks gK(C) of the hippocampal pyramidal cells (Lancaster and Adams, 1986; Lancaster and Nicoll, 1987; Storm, 1987). At this level of concentration, TEA also enhances release of excitatory neurotransmitters (Rutecki et al., 1990). At higher concentrations (25±30 mM) it also completely blocks gK(DR) (Storm, 1990). As shown in Figs. 2 and 3 mM TEA in the bath had only subtle effects on the initial component of the evoked magnetic ®elds, but dramatically enhanced the slow wave. The arrows indicate the time of stimulus onset; the stimulus artifacts were not removed in this and all the subsequent ®gures. The amplitude of the initial spike relative to the control condition was 1:07 ^ 0:12 (mean ^ 1 SEM (standard error of mean), n 4). The slow wave amplitude was 3:30 ^ 0:49 times the control amplitude (t 4:69, n 4, P , 0:01). Its peak latency was 1:61 ^ 0:08 (n 4) times longer than the control. The ®eld potentials measured in the basal and apical areas were very close in waveform to the simultaneously measured magnetic ®elds. Accordingly, their initial component consisting of a train of synchronized population spikes was not changed clearly, but the subse-
quent slow wave was enhanced and its peak latency was lengthened by TEA. These effects were reversible (see Washout). In a separate set of experiments, the intracellular potential was measured from the pyramidal cells simultaneously with ®eld potentials in order to obtain insight at the cellular level for the observed effects on the population responses. In the example shown in Fig. 2 (bottom two traces), the slow wave of the unaveraged ®eld potential (Vex) was enhanced by 3 mM TEA replicating the results for the above experiment. This concentration of TEA slightly broadened the initial sodium spike of the intracellular potential, but did not affect its peak amplitude. It, however, produced a clear calcium spike (initial broad spike) detectable in the somatic recording and broadened the following spikes which are probably dominated by calcium currents as well. The paroxysmal depolarizing shift (PDS) terminated abruptly at about 80 ms post-stimulus. All these TEA effects were reversible (Fig. 2, Washout). There was no component in the intracellular record that corresponded directly to the slow wave of the magnetic ®eld because our intracellular measurements were all made in the soma rather than in the apical dendrites. However, previous intradendritic recordings have shown that the hippocampal pyramidal cells are capable of locally producing bursts mediated by calcium currents (e.g. Wong and Stewart, 1992). Fig. 3 shows the apical-stimulation evoked magnetic and electrical population responses in the presence of various concentrations of TEA. The synchronized population spikes in the ®eld potential were slightly larger and broader in width at a TEA concentration of 3 mM in comparison to the control. This effect is consistent with the known action of blocking the repolarizing gK(C). Fig. 3C shows the initial spike of the intracellular recording (Vin) presented at the bottom of Fig. 3A. The repolarizing portion of the initial spike clearly became longer in duration when TEA in the bath was increased to 3 mM. Increasing TEA to 10 mM reduced the amplitudes of the initial component of the magnetic signal and the initial spike of the ®eld potential. This reduction may be due to the further prolongation of the action potentials by blocking gK(DR) since the initial spike of the intracellular potential shown in Fig. 3C is broadened even more. Such a block should lead to reduced repetitive ®ring. The increase did not further enhance the slow wave
Fig. 4. Effects of TEA on the intracellular potential elicited by injection of constant current into the recorded pyramidal cell under a current clamp mode. Bath concentration of 10 mM TEA broadened the sharp spike, increased spike frequency and activated a calcium spike.
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Fig. 5. Increased spontaneous activity of the longitudinal CA3 slice in the presence of 10 mM TEA in the bath. (A) Magnetic ®eld at two locations and ®eld potential near the soma simultaneously recorded from a slice in control medium (Ringer 1 0.1 mM PTX), in the Ringer with PTX 1 TEA, and in control medium during the Washout. (B) Intracellular potential (Vin) and simultaneously recorded ®eld potential near the soma (Vex) from another slice in the 3 conditions.
amplitude, but broadened its waveform and delayed the peak latency. Fig. 3B shows that the effect of TEA on slow wave amplitude peaked at a TEA concentration of about 3 mM. Since TEA may not only potentiate post-synaptic potentials, but also increase transmitter release from pre-synaptic terminals, we directly examined post-synaptic effects of TEA on the pyramidal cells, by injecting dc current from the recording pipette. As shown in Fig. 4, 10 mM TEA clearly broadened the spikes in a manner similar to the effects of TEA on the sodium and calcium spikes evoked by the apical stimulation (Fig. 3A), namely, it broadened the sodium spikes and produced a clear calcium spike. Also the spike accommodation was reduced, consistent with a known action of TEA. These results indicate that the effects of TEA observed in the intracellular recording above are at least in part due to its post-synaptic effects. 3.3. Effects of manipulating gK(C) by TEA on spontaneous magnetic and electrical signals TEA in the bath at a concentration of 10 mM heightens excitability of the longitudinal CA3 slice as shown by the simultaneously recorded single-epoch magnetic and extracellular ®eld potential signals (Fig. 5A) and by the simultaneously recorded intracellular potential in a pyramidal cell
and ®eld potential near the soma (Fig. 5B). In the presence of 0.1 mM PTX the slice exhibited spontaneous single burst discharges that were synchronized suf®ciently across the slice to be seen as a population response in the magnetic signal. The magnetic ®eld during the burst was outward at location SQ-2 and inward at SQ-4, indicating that its underlying current was directed from the apical to basal side just as for the slow wave of the evoked response. TEA reversibly produced a train of burst discharges lasting 1±2 s. The direction of the steady shift of the magnetic ®eld during each train of bursts was downward at SQ-2 and upward at SQ4, indicating that its underlying current was directed from the basal/soma toward apical side. Intracellular records in the control condition showed a mixture of simple sodium spikes which were not seen in the simultaneously recorded ®eld potential and calcium bursts which were synchronized suf®ciently to be seen by the ®eld potential recording electrode (Fig. 5B, bottom). In bath with 10 mM TEA, the intracellular record showed a mixture of single spikes, which were again not seen in the ®eld potential recording, and strong, long-lasting repetitive bursts. These long-lasting bursts corresponded to the multiple spikes in the ®eld potential. During the washout phase, the duration of individual bursts became shorter, eventually returning to the activity pattern of the control condition. The increased excitability in presence of TEA is consistent with
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Fig. 6. Effects of 4AP on apical stimulation-evoked magnetic ®elds at locations SQ-2 and SQ-4, ®eld potentials in the basal and apical areas recorded simultaneously from one slice (30 epochs/ave), and also on intracellular potential (Vin) and simultaneously recorded ®eld potential in the soma area (Vex) from another slice.
®ndings from transverse slice preparations (e.g. Fueta and Avoli, 1993). 3.4. Effects of manipulating gK(A) and gK(D) with 4AP on evoked population responses The gK(A) was ®rst described in CA3 cells of hippocampal slices from adult guinea-pigs by Gustafsson et al. (1982) and byZbica and Weight (1985) This class of conductance is voltage-sensitive, but calcium-independent. The gK(A) in the hippocampus is blocked by extracellular 4AP concentrations of 0.1±0.5 mM (Storm, 1990). 4AP blocks not only gK(A), but also gK(D). The gK(D) is similar to gK(A) in that both are calciumindependent and 4AP sensitive, but it inactivates more slowly and is more sensitive to 4AP than gK(A) in that it is completely blocked by approximately 30 mM (Storm, 1990). Since there are two distinguishable types of 4AP sensitive transient potassium currents in the CA3 cells, we evaluated the relative contributions of these two types of conductances to the magnetic and electrical population responses. Fig. 6 shows that 4AP at a concentration of 0.1 mM slightly increased the initial `spike' in the triphasic magnetic
and electrical population responses. The mean amplitude of the spike in the magnetic signal was 1:36 ^ 0:14 times the control (t 2:57, n 4, P , 0:05). As for the slow wave, 4AP also dramatically enhanced its amplitude by 3:74 ^ 0:58 times the control (t 4:72, n 4, P , 0:01), shortened its peak latency (81 ^ 7%) and reduced duration (73 ^ 7%) relative to the control. In intracellular recordings, 4AP clearly widened the spikes and enhanced the PDS. The ®eld potential (Vex-soma) was recorded on the basal side of the cell layer and thus the population activity was not seen as strongly as, for example, in the somal recording of Fig. 1, but was closer to the basal recording in the same ®gure. The observed effects of 4AP may be due to a combined effect of gK(A) and gK(D) since they should be both blocked by 0.1 mM 4AP. We thus studied the effects of 4AP at a concentration (0.01 mM) where it should selectively block gK(D) and at a higher concentration (0.1 mM) where gK(A) should be blocked by 4AP in addition to gK(D). As shown in Fig. 7A, 4AP enhanced the amplitude and shortened the peak latency of the slow wave of both the neuromagnetic ®eld and ®eld potential even at a concentration of 0.01 mM, indicating that blocking gK(D) may enhance the slow wave. However, the slow wave was further enhanced by 0.1 mM
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Fig. 7. (A) 4AP effects on evoked magnetic ®eld (SQ-2), ®eld potential (basal) and intracellular potential (Vin) at 0.01 mM which selectively blocks D currents mediated by gK(D) and at 0.1 mM which blocks gK(A) and gK(D). Magnetic signal and ®eld potential were recorded simultaneously from one slice. Vin was recorded from another slice. (B) Concentration-ef®ciency curve of 4AP for the slow wave of the magnetic signal. All responses were normalized to the magnetic signal in Ringer with 0.1 mM PTX (see symbol marked with a star). Each point shows the average of 4 cases and the vertical bars show ^ 1 SEM. (C) Broadening of the initial intracellular spike in the records shown in (A) at two levels of 4AP concentration.
4AP. The concentration-ef®ciency curve in Fig. 7B indicates that the effect of blocking gK(D) was fairly small compared to that of blocking the A current, since the enhancement effect was maximal at the concentration where gK(A) is expected to be blocked. Supporting this conclusion, the initial spike of the intracellular response shown in Fig. 7A was modi®ed only slightly at the concentration of 0.01 mM, but was clearly broader in width at a concentration of 0.1 mM 4AP (Fig. 7C). This result indicates that blocking gK(D) did not have a large effect on the action potential, but blocking gK(A) did. As in the case of TEA, 4AP facilitates synaptically mediated responses by pre- and post-synaptic mechanisms. We assessed the post-synaptic effects of 4AP on the pyramidal cells in our preparation at a concentration of 0.1 mM by directly injecting current into a pyramidal cell under a current clamp condition and measuring the voltage response of the cell. Fig. 8 shows that 0.1 mM 4AP increased the number of spikes and increased the height of each spike. This result demonstrates that 4AP had some post-synaptic effects. 3.5. Effects of 4AP on spontaneous magnetic and electrical responses As in the case of TEA, 4AP enhanced the spontaneous
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activity of cells in the longitudinal CA3 slice. In the control condition with 0.1 mM PTX in the bath (Fig. 9A), there were spontaneously occurring single bursts detectable with the magnetic and electrical techniques. Just as in the control condition of Fig. 5, the current producing the burst was directed from the apical to basal side. 4AP at a concentration of 0.1 mM that should be suf®ciently high to block gK(A) increased the spontaneous activity with more numerous trains of bursts. The currents during each burst was again directed from the apical to basal side. The direction of this current is consistent with the laminar ®eld potential pro®le of this component produced by 4AP in CA1 (Voskuyl and Albus, 1985). The steady shift during each train of bursts was directed from the basal to apical side just as in the case of TEA (Fig. 5). Intracellular recordings from the pyramidal cells were consistent with the population responses. In the example shown in Fig. 9B, the spontaneous activity consisted of a mixture of single spikes and bursts. Only the bursts were suf®ciently synchronized locally to produce detectable ®eld potentials. 4AP changed the spontaneous activity to a mixture of single bursts and long-lasting trains of bursts that could be seen as multiple bursts in the simultaneously recorded extracellular potentials (Vex). The increased spontaneous activity in the longitudinal CA3 slices in the presence of 4AP is consistent with the earlier results for transverse hippocampal slice preparations (e.g. Perreault and Avoli, 1991).
4. Discussion The main ®nding in this study is that bath application of potassium channel blockers, TEA and 4AP, had only small effects on the initial component of the triphasic magnetic signal and the corresponding component of the evoked potential, but strongly enhanced their slow wave elicited by the apical stimulation in CA3 longitudinal slices. TEA and 4AP have a number of effects on the behavior of neurons in the hippocampus both pre- and post-synaptically. Therefore, it is necessary to carefully consider possible effects of these channel blockers in order to infer roles of the calcium- and voltage-sensitive potassium conductances in the genesis of neuromagnetic signals and ®eld potentials.
Fig. 8. Effects of 4AP on the intracellular potential elicited by injection of constant current into the recorded pyramidal cell under a current clamp mode.
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Fig. 9. Increased spontaneous activity of the longitudinal CA3 slice in presence of 0.1 mM 4AP in the bath. (A) Magnetic ®eld at two locations and ®eld potential near the soma simultaneously recorded from a slice in control medium (Ringer 1 0.1 mM PTX), in the Ringer with PTX 1 4AP, and in control medium during the Washout. (B) Intracellular potential (Vin) and simultaneously recorded ®eld potential near the soma (Vex) from another slice in the 3 conditions.
4.1. Roles of the potassium currents in generating the spike Our hypothesis is that the initial spike in the magnetic signal was produced by sodium spikes in the soma and trunk of the apical dendrites. According to our view, the apical stimulation depolarizes the soma and dendritic trunk. The densities of gNa and gK(DR) are thought to be high in the proximal trunk and soma of the pyramidal cells (Traub et al., 1994). Thus, both of these regions are capable of generating sodium spikes. Our results indicate that a spike was ®rst generated in the apical trunk followed by a spike in the soma area. Spike a of the ®eld potential was negative in the apical area and positive in the soma area, suggesting that the spike was generated in the apical area close to the site of stimulation. The polarities in the apical and soma areas indicate that the underlying intracellular current was directed from the apical dendrites to the soma. Spike b appears to have been generated in the soma. We inferred from the polarities of spike b that its underlying intracellular current was directed from the soma toward the basal dendrites. The current generated in the soma should be directed toward the basal dendrites because the apical dendrites should be strongly depolarized by the initial apical spike and thus the potential gradient inside the cell forces the current to be directed toward the basal dendrites. The latencies of these spikes matched the broad peak of the magnetic ®eld
in latency. Thus, the spike of the magnetic ®eld appears to have been produced by currents generated in the apical trunk and soma. The intracellular recordings have shown that both TEA and 4AP did have post-synaptic effects on the spikes generated by pyramidal cells in our longitudinal CA3 slices. The response to injected current under a current clamp mode showed broader spikes, a calcium spike and less accommodation, all indicating post-synaptic effects of TEA. The intracellular potential showed larger and broader spikes with less accommodation in the current clamp mode in presence of 0.1 mM 4AP. Thus, TEA and 4AP should have had some post-synaptic effects on the spikes in the ®eld potential and magnetic ®eld generated by the apical stimulation. The bath concentration of TEA at 3 mM broadened the spikes of the evoked intracellular potentials. Raising the concentration to 10 mM broadened the spikes further. The corresponding spikes of the magnetic ®eld and ®eld potential were broader, but were not signi®cantly enhanced in presence of 3 mM TEA compared to the control. Amplitude of the spike in the neuromagnetic signals was slightly, but signi®cantly elevated by 0.1 mM 4AP. Although the broadening of the spikes under TEA and the enhancement in the presence of 4AP must be in part due to their post-synaptic effects, it is not possible to separate the effects from possible pre-synaptic increase in neurotrans-
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mitter release that may be caused by both TEA and 4AP. They are both thought to increase neurotransmitter release and enhance synaptic transmission in both excitatory and inhibitory neurons at the concentrations used in our study (0.01±0.1 mM for 4AP and 1±10 mM for TEA) (Buckle and Haas, 1982; Avoli and Perreault, 1987; Rutecki et al., 1987, 1990; Perreault and Avoli, 1989, 1991; Traub et al., 1995; Wheeler et al., 1996). In our study the effects of TEA and 4AP on GABAA inhibitory neurotransmitter release can be ignored since 0.1 mM PTX used in all the experiments is strong enough to block the synaptic transmission in hippocampal slices (Miles and Wong, 1987). GABAB type inhibition should be functional in our preparation, but this slow inhibition probably does not signi®cantly affect the slow wave which has a relatively short latency. However, we may have to take into account possible increases in neurotransmitter release at the recurrent excitatory synapses that connect the pyramidal cells to each other (Miles and Wong, 1986, 1987a) and possible release of neurotransmitters from the mossy ®ber terminals that are cut but may be functional in order to more fully understand the effects of TEA and 4AP of the spike. These issues will have to be clari®ed in future studies. 4.2. Roles of the potassium currents in generating the slow wave The effects of TEA and 4AP on the slow wave of the magnetic ®eld and ®eld potential were quite robust. The slow wave increased in amplitude and its peak latency became shorter in presence of 4AP. The concentration-ef®ciency curve for 4AP indicated that the enhancement of the slow wave was relatively small at a concentration of 4AP where it is expected to block gK(D), but became greater with stronger concentrations of 4AP, peaking at a concentration of 0.1 mM where it is expected to block gK(A). Thus, we infer that the A current, rather than the D current, plays a dominant role in generating the slow wave. According to this hypothesis, blocking gK(A) with 4AP should reduce the repolarization of the spikes mediated by Na currents which in turn should help activate the high-threshold gCa (Wong and Stewart, 1992). This hypothesis, however, requires further validation, since it would be necessary to show that the range of concentrations of 4AP used in this study selectively blocks gK(A) and gK(D) and that selective elimination of these channels produces the changes in the kinetics of gCa opening and closing that are presumably responsible for the enhancement of the slow wave. As for the effects of TEA, the slow wave amplitude and peak latency increased in presence of TEA. The effect of TEA on slow wave amplitude peaked at a concentration of 3 mM. This suggests that the amplitude increased at concentrations of TEA where gK(C) is fully blocked, but not gK(DR). The lack of further enhancement of the slow wave amplitude in presence of 10 mM TEA may be because of reduced inputs onto the apical dendrites via the recurrent excitatory
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synapses due to reduced population spikes. Blocking of gK(C) by TEA should delay the repolarization of the spikes mediated by gCa. Thus, the manipulations with 4AP and TEA should both lead to an increase the apical dendritic currents mediated by gCa. The greater depolarization of the apical dendrites should increase the opening of NMDA (Nmethyl-d-aspartate) channels by excitatory inputs in the recurrent excitatory synapses of the pyramidal cells. The slow wave thus in our view is due to a mixture of currents produced by the intrinsic gCa and synaptic currents carried by the NMDA channels. We are not able to assess their contributions separately based on the present set of data. The increase in intracellular calcium concentration should enhance the activation of gK(AHP) since it is not affected by TEA or 4AP. The activation should help terminate the depolarization earlier (Traub et al., 1993) and hasten the return of the slow wave to the baseline as was seen clearly in Fig. 2. 4.3. Roles of TEA and 4AP in producing spontaneous activity In the presence of TEA or 4AP, the spontaneous activity that was present in Ringer with PTX became more synchronized across the entire slice. Single spikes transformed to bursts. The polarities of the magnetic ®eld and ®eld potentials during these spontaneous bursts were the same as for the evoked potentials. The analysis of enhanced spontaneous activity and temporal waveform of each spontaneous burst would follow the above framework for evoked responses. However, there are some additional complications in the analysis. For example, 4AP produces spontaneously occurring ectopic axonal spikes which can propagate anti- and orthodromically to help initiate synchronized population spikes via orthodromic recurrent excitatory synapses (Traub et al., 1995). In the case of evoked responses, this ectopic spike can be ignored since the population of neurons are synchronously stimulated externally. Detailed analysis is, however, beyond the scope of this paper. 4.4. Signi®cance It should be emphasized again that the above interpretation of effects of TEA and 4AP on the magnetic ®eld and ®eld potential should be viewed as a hypothesis since this study is still in the state of infancy. It is dif®cult to cleanly block only one conductance of interest without any other complicating effect using any so-called selective channel blocker. Furthermore, even if it is possible, blocking one conductance has ramifying consequences on the rest of the channels and the functioning of the entire neuronal circuitry. Thus, we believe that further advances in our understanding of the genesis of MEG and EEG signals are possible only through a careful study combining a mathematical model of the CNS tissue of interest with experimen-
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tal analyses at the level of neuronal ensemble, single cells and channels. Nevertheless, we believe that this study has demonstrated important roles of the potassium currents in determining the temporal waveform of magnetic ®elds and extracellular potentials. Since the kinetics of these channels are highly non-linear, the consequences of altering any one channel on the waveform of the magnetic and electrical signals may be profound as seen in the present study. This implies that abnormality of these potassium conductance can profoundly in¯uence the temporal waveform of MEG and EEG signals in general. Acknowledgements The present study was performed at the VA Medical Center, Albuquerque, NM 87108, USA. The authors thank the staff of the Research Service of the VA for providing animal care and our research assistant, Ms. Anna Barrios. This study was supported by NIH grant R01-NS21149 to Y.C.O. References Avoli M, Perreault P. A GABAergic depolarizing potential in the hippocampus disclosed by the convulsant 4-aminopyridine. Brain Res 1987;400:191±195. Buckle PJ, Haas HL. Enhancement of synaptic transmission by 4-aminopyridine in hippocampal slices of the rat. J Physiol 1982;326:109±122. Fueta Y, Avoli M. tetraethylammonium-induced epileptiform activity in young and adult rat hippocampus. Dev Brain Res 1993;72:51±58. Gustafsson B, Galvan M, Grafe P, Wigstrom HA. Transient outward current in mammalian central neuron blocked by 4-aminopyridine. Nature 1982;299:252±254. Hille B. Ionic channels of excitable membranes, Sunderland, MA: Sinauer, 1992. Kyuhou SI, Okada YC. Detection of magnetic evoked ®elds associated with synchronous population activities in the transverse CA1 slice of the guinea pig. J Neurophysiol 1993;70:2665±2668. Lancaster B, Adams PR. Calcium-dependent current generating the after hyperpolarization of hippocampal neurons. J Neurophysiol 1986;55:1268±1282. Lancaster B, Nicoll RA. Properties of two calcium-activated hyperpolarizations in rat hippocampal neurons. J Physiol 1987;389:187±203. Miles R, Wong RKS. Excitatory synaptic interactions between CA3 neurones in the guinea-pig hippocampus. J Physiol 1986;373:397± 418. Miles R, Wong RKS. Inhibitory control of local excitatory circuits in the guinea-pig hippocampus. J Physiol 1987a;388:611±629. Miles R, Wong RKS. Latent synaptic pathways revealed after tetanic stimulation in the hippocampus. Nature 1987b;329:724±726. Miles R, Traub RD, Wong RKS. Spread of synchronous ®ring in longitudinal slices from the CA3 region of the hippocampus. J Neurophysiol 1988;60:1481±1496.
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