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Physiological bases of the synchronized population spikes and slow wave of the magnetic field generated by a guinea-pig longitudinal CA3 slice preparation
Electroencephalography and clinical Neurophysiology 107 (1998) 361–373
Physiological bases of the synchronized population spikes and slow wave of the magnetic field generated by a guinea-pig longitudinal CA3 slice preparation Jie Wu, Yoshio C. Okada* Departments of Neurology and Neurosciences, University of New Mexico School of Medicine, Albuquerque, New Mexico, 87131 USA Accepted for publication: 1 July 1998
Abstract Objective: The physiological bases of evoked magnetic fields were examined in a guinea-pig hippocampal slice preparation, motivated by new concepts in central nervous system (CNS) electrophysiology brought about by discoveries of active conductances in the dendrites and soma of neurons. Methods: Their origins were elucidated by comparing them with intracellular and extracellular field potentials. Results: With excitatory synaptic transmissions blocked, the magnetic signal elicited by an electrical stimulus applied to the pyramidal cell layer consisted of a spike and a depolarizing afterpotential-like waveform. With the excitatory synaptic transmissions intact, but with inhibitory synaptic transmissions blocked, the magnetic signal was bi- or triphasic depending on whether the cell layer or the apical dendrite area of the pyramidal cells was, respectively, depolarized. In both cases the signal consisted of a train of synchronized population spikes superimposed on a brief wave followed by a longer, slow wave. The spike train was correlated with synaptically mediated intracellular spikes. The underlying currents for the slow wave were directed from the apical to the basal side for both types of stimulation. It was most likely generated by depolarization of the apical dendrites, caused by recurrent excitatory synaptic activation. Conclusions: This analysis illustrates how synaptic connections and intrinsic conductances in a disinhibited mammalian CNS structure can generate spikes and waves of the magnetic field and electrical potential. 1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: Biomagnetism; Electroencephalography; Magnetoencephalography; Evoked potential; Hippocampus; Voltage-sensitive conductances; Ligand-gated channels; a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; N-Methyl-d-aspartate
1. Introduction Concepts of dendritic and somatic physiology have recently undergone dramatic changes due to discoveries of many types of ligand- and voltage-sensitive conductances in central nervous system (CNS) neurons (Llina´s, 1984, 1988; Baxter and Byrne, 1991; Hille, 1992). These discoveries call for an examination of the genesis of magnetoencephalography (MEG) signals within this new framework. Likewise, the genesis of electroencephalographic (EEG) signals which was thoroughly studied about 30 years ago (Pollen, 1969;
* Corresponding author. Department of Neurology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. Tel.: +1 505 2562874; fax: +1 505 2600165; e-mail: [email protected]
Creutzfeldt et al., 1966a,b; Humphrey, 1968a,b) should be re-examined. Our current understanding of the genesis of the evoked potential is still largely based on studies carried out when the dendrites were viewed as passive, electrotonic core conductor cables having excitatory and inhibitory neurotransmitter receptors whose opening produced excitatory and inhibitory post-synaptic potentials (EPSPs and IPSPs, respectively) and produced action potentials in the axon hillock by electrotonic conduction of the EPSPs. One approach for elucidating the origin of MEG signals is to analyze and compare such signals produced in a relatively simple CNS preparation. We chose the hippocampus of the guinea-pig for this purpose. The anatomy and electrophysiology of this structure are quite well understood to the point where it is possible to construct a mathematical model of CA1 and CA3 incorporating various intrinsic pas-
0013-4694/98/$ - see front matter 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0013-4694 (98 )0 0098-4
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sive and active membrane conductances and network properties that have been actually experimentally determined (Traub and Miles, 1991; Traub et al., 1991, 1993, 1994). Thus, physiological mechanisms for the evoked magnetic fields may be understood by comparing them with the evoked extracellular field potentials simultaneously measured with the magnetic signals and with the intracellular potentials. We have used a simplified preparation in order to make it feasible to interpret the MEG data in terms of underlying cellular events (Okada et al., 1997). The present study was carried out on a longitudinal CA3 slice in which the longitudinal axes of the pyramidal cells are parallel to each other along the direction perpendicular to the cell layer. Thus, the slice is effectively a two dimensional structure with the principal core conductors from a single subfield of the hippocampus arranged along the main axes of a rectangular coordinate system. This preparation is even simpler than the conventional transverse slice which contains many subfields and related areas of the hippocampus. The slice contains excitatory pyramidal neurons which are connected to each other via recurrent excitatory connections (MacVicar and Dudek, 1980; Miles and Wong, 1986), and inhibitory neurons which receive excitatory inputs from the pyramidal neurons and provide g-aminobutyric acid A (GABAA) and GABAB type inhibitory connections to each other and to the pyramidal cells (Newberry and Nicoll, 1984, 1985; Miles and Wong, 1987; Miles, 1990). The recurrent excitatory connections among the pyramidal cells are mediated by glutamatergic synapses (Johnston and Brown, 1981), having N-methyl-d-aspartate (NMDA) receptors (Jefferys, 1989; Neumann et al., 1989) and a-amino-3-hydroxy-5-methyl4-isoxazole propionic acid (AMPA) receptors, since NMDA and AMPA channels are co-localized (Bekkers and Stevens, 1989). The connections are sufficiently strong to produce synchronous population activities (Miles and Wong, 1983, 1986) that can be measured with MEG (Kyuhou and Okada, 1993; Okada and Xu, 1996). The degree of synchronization can be sufficiently high across the entire slice (Okada et al., 1997) so that the magnetic field outside the slice can be analyzed in terms of events at a single cell level. In the first paper of this series (Okada et al., 1997), we have presented some basic characteristics of MEG signals in CA1 and CA3 slice preparations. Here, we describe the physiological bases of spikes and waves of the evoked magnetic fields generated in the longitudinal CA3 slices. Some of this work was previously presented in abstract form (Wu and Okada, 1996).
2. Methods 2.1. Slice preparation The longitudinal slice of CA3 was prepared with a
method described previously in detail (Okada et al., 1997). Briefly, adult guinea-pigs (Hartley, 200–400 g, 80 animals) were anesthetized with sodium pentobarbital (60 mg/kg i.p.) and decapitated with a guillotine. The brain was quickly removed and submerged in Ringer solution at 2–4°C for 1–2 min. Both hippocampi were removed from the brain, the dentate surface of a hippocampus was glued flat to a 3% agar block and the assembly was placed horizontally in a well of Vibratome filled with cold Ringer solution (Fig. 1A). The hippocampus was cut parallel to the agar surface just above the fimbria to obtain two 400 mm-thick slices. A rectangular longitudinal CA3 slice (Fig. 1C) was obtained by cutting CA1 away from the slice and trimming both edges. All slices were incubated in the Ringer solution bubbled with 95% O2 –5% CO2 gas at room temperature for 1 h or more. After the incubation, one slice was transferred to an experimental chamber (Fig. 1B) that contained 36°C oxygenated Ringer solution. The slice was placed between a pair of nylon nets on top of a cylindrical platform in the chamber. Warm oxygenated Ringer continuously flowed out of the platform from below to superfuse the slice. The slice was kept in this chamber for at least one half hour before starting measurements. 2.2. Solutions and chemicals The Ringer solution in the recording chamber had the following composition (in mM): NaCl 124, KCl 5, KH2 PO4 1.2, MgCl2 1.3, CaCl2 2.5, NaHCO3 26 and glucose 10. The pH was kept at 7.4 with 95% O2 –5% CO2 gas mixture. Intracellular potentials in the pyramidal cells were measured with a glass micropipette filled with potassium acetate (4 M; 50–90 MQ). The channel blockers used in the present experiments were picrotoxin (PTX) which blocks GABAA receptors, tetrodotoxin (TTX) which blocks the sodium conductance (gNa), d-2 amino-5-phosphonovaleric acid (APV) dissolved in normal Ringer solution which blocks NMDA receptors and 6-cyano-2,3-dihydroxy-7nitroquinoxaline (CNQX), dissolved in dimethyl sulphoxide (DMSO, concentration ,0.01%; Tocris Neuramin), which blocks AMPA receptors. 2.3. Stimulation The longitudinal CA3 slice was stimulated with 6–10 pairs of electrodes (Ag-AgCl wire, 125 mm diameter) placed along the pyramidal cell layer from below the slice (Fig. 1C). Here an arrangement with 6 pairs of electrodes is illustrated. One row of electrodes was placed near the cell layer on the side of the stratum oriens and the second row on the stratum lucidum or radiatum. The electrodes were negative in the cell layer and positive in the stratum lucidum or radiatum for ‘somatic’ stimulations and opposite in polarity for ‘apical’ stimulations. The stimulating parameters were: 0.7–1.0 mA, 50–100 ms, 0.1–0.3 Hz.
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Fig. 1. Schematic of experimental arrangement. (A) Orientation of the hippocampus for cutting longitudinal CA3 slices. Two 400 mm-thick slices parallel to the pyramidal cells were obtained just above the fimbria. (B) Experimental chamber. A slice was sandwiched between a pair of nylon nets. Warm (36°C) oxygenated Ringer superfused the slice from below, flowing out of the chamber. Magnetic field detectors were placed 2 mm above the slice. Stimulating and recording electrodes were placed from below the slice. (C) Arrangement of the stimulating electrodes (six pairs of bipolar Ag-AgCl electrodes about 0.8 mm apart shown as black squares) along the pyramidal cell layer, 3 field potential recording electrodes (chlorided 125 mm diameter silver wires) along the longitudinal axis of pyramidal cells (shown enlarged in the inset) in the region of the basal dendrites in the stratum oriens (s.o.), the soma in the stratum pyramidale (s. p.), and apical dendrites in the stratum lucidum (s. l.) and radiatum (s. r.) and 4 channels of the m-SQUID (Superconducting Quantum Interference Device) sensor (SQ1–4). All in correct relative dimensions. Stimulating electrodes were negative in the cell layer and positive in the stratum lucidum or radiatum for ‘somatic’ stimulations and opposite in polarity for ‘apical’ stimulations.
2.4. Recording procedures The magnetic field recording was carried out using a superconducting magnetic field sensor called a mSQUID (Buchanan et al., 1989). It was equipped with four channels of a first-order, asymmetric, axial gradiometer with a 4.0 mm diameter bottom detection coil, an 8.5 mm diagonal separation between channels, a 16 mm baseline, a 1.2 mm separation between the detection coils and the outside surface of the cryogenic container of the mSQUID and a field sensitivity of 50 fT/ÎHz (fT = femtotesla; Okada et al., 1994). The detection coils were placed directly over the slice at a distance of about 2 mm above the slice. The SQUID outputs were fed to a 4-channel amplifier with a 24 dB/octave Butterworth filter. All the magnetic field data were collected with a bandpass filter having a highand a low-pass cut-off frequencies of 0.1 Hz and 2 kHz, respectively. The magnetic field recordings were carried out in a magnetically shielded room (Okada et al., 1994). Field potentials were recorded simultaneously with the magnetic fields using 125 mm-diameter Ag-AgCl electrodes placed along the longitudinal axis of the pyramidal cells. Three such electrodes labeled ‘basal’, ‘soma’ and ‘apical’ were placed in the region of the basal dendrites, soma and apical dendrites. The potentials were fed to an FET preamplifier and then to an amplifier/filter equipped with a 24 dB/octave Butterworth filter with a bandpass of 0.1 Hz to 2kHz. Intracellular potentials were recorded from 14 slices for a total of 33 pyramidal cells using an Axoclamp 1A with a 10 kHz lowpass filter. Only cells with resting membrane poten-
tials of at least −55 mV were used. In the presence of 0.1 mM PTX, the cellular resting potential, amplitude of action potential and input resistance were 64.9 ± 1.5 mV (SEM), 79.7 ± 2.7 mV and 26.0 ± 2.2 MQ, respectively. All intracellular recordings were performed simultaneously with extracellular field potential recordings.
3. Results 3.1. Characteristics of the evoked magnetic fields Fig. 2 shows three types of signals. The first two rows show the magnetic fields recorded at positions SQ2 and SQ4 (see Fig. 1C). The next three rows show the field potentials recorded along the longitudinal axis of the pyramidal cells. These records were obtained simultaneously. The bottom two rows show a field potential simultaneously measured with an intracellular potential in the soma of a pyramidal cell in another slice. The records on the right side were obtained by stimulating the apical area about 400 mm from the soma with a negative electrode; those on the left were obtained by stimulating the soma area. In the presence of 0.1 mM PTX, the magnetic fields were biphasic for the ‘somatic’ stimulation and triphasic for the ‘apical’ stimulation, replicating our earlier results (Okada et al., 1997). The magnetic fields detected at locations SQ2 and SQ4 were mirror images of each other in both cases with a near baseline level of signal at locations SQ1 and SQ3 (not shown). This spatial distribution is consistent with a dipolar field pattern seen in extensive maps (Okada et al., 1997),
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indicating that the underlying currents were directed along the longitudinal axis of the pyramidal cells. The somatic stimulation produced 1–3 spikes in the magnetic field (peak latency of the first spike, mean = 2.2 ms with a range of 1.4–4.3 ms, n = 20) riding on a brief wave (this complex is labeled ‘1’), followed by a later slow wave labeled ‘2’ lasting about 70 ms. Applying the right-hand rule of electromagnetism, one can deduce from directions of the magnetic fields that the initial spike and the brief, early wave were both due to intracellular currents directed from the basal to the apical side of the pyramidal cells, whereas the following slow wave was due to oppositely directed currents. These conclusions are consistent with the polarities of the field potentials. The large spike in the
field potentials corresponding in latency to the first spike of magnetic field was negative in the basal dendrite area and soma, and positive in the apical area, indicating that the corresponding intracellular currents were directed from the basal to the apical side. The field potential during the slow wave of the magnetic field was negative on the apical side and positive on the soma and basal side, indicating that the underlying intracellular currents were directed from the apical to the basal side. The apical stimulation produced a train of synchronized population spikes in the magnetic field (peak latency of the first spike, mean = 3.0 ms with a range of 1.7–5.2 ms, n = 23) riding on a brief wave (labeled as ‘1’), followed by a return to the baseline (‘2’), and then a slow wave
Fig. 2. Magnetic fields, field potentials and intracellular potentials produced by longitudinal CA3 slices. Left: somatic stimulation; right: apical stimulation. Top two rows: magnetic fields; middle 3 rows: field potentials. These five traces were recorded simultaneously (30 epochs/ave). Bottom two rows: field potential in the soma area and simultaneously recorded intracellular potential from the soma of a pyramidal cell. Note: time scale is the same for all three types of signal. pT, picoTesla.
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(‘3’). Interestingly, the currents underlying both the initial spike and the early wave were opposite in direction to those seen with the somatic stimulation. They were directed from the apical toward the basal side. Consistent with this direction, the early wave of the field potential was negative on the apical and soma side and positive on the basal dendrite side of the pyramidal cells. In contrast, the currents for the slow wave, according to the magnetic field, were directed from the apical to the basal side just as for the somatic stimulation. Accordingly, the field potential was negative on the apical side and positive on the soma and basal sides. 3.2. Relationships between synchronized population spikes of the magnetic field and field potential The relationships between the magnetic field and field potential of the synchronized population spikes seen in Fig. 2 are more clearly illustrated in Fig. 3. The latencies of the spikes were highly correlated between the magnetic and electrical signals. In the case of somatic stimulation (Fig. 3A), the field potentials recorded near the soma and basal dendrites
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showed an initial negativity overlapping with the stimulus artifact, followed by a small positivity (‘a’), then a strong negativity (‘b’) with additional several spikes. It is difficult to distinguish the initial negativity from a stimulus artifact in this record, but it most likely reflects a depolarization of the basal and soma areas since a negative stimulus was applied with six pairs of bipolar electrodes to these regions (about 100 mm from the cell layer on the basal side). This conclusion is consistent with the results presented in Figs. 4 and 5. The initial spike was not seen in the magnetic field, masked by the stimulus artifact. The spike ‘a’, however, was seen in the magnetic records. It is probably due to activation of fast conductances in the proximal apical dendrites since the field potential was negative in the apical area. The underlying current for the magnetic field of spike ‘a’ was directed from the apical to the basal side consistent with this interpretation. The spikes labeled ‘b’, ‘c’ and ‘d’ were seen slightly earlier (0.5 ms, n = 8) in the basal area compared to those in the soma and apical areas. This difference in peak latency probably reflects a difference in activating a spike in the axon hillock and a spike in the soma area. The spikes in the magnetic signals matched in latency with the corre-
Fig. 3. Comparison of the spikes of simultaneously recorded magnetic fields and field potentials. (A) Somatic stimulation (−electrode near the cell layer and +electrode in apical dendrite area). (B) Apical stimulation (+electrode near the cell layer and −electrode in apical dendrite area; 30 epochs/ave). (a–d) Synchronized population spike components. See text for details.
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sponding spikes in the field potentials recorded in the soma and apical areas, suggesting that the currents associated with these somatic and apical spikes contained a stronger dipolar component than the currents associated with an action potential in the axon hillock. The magnetic field polarity indicated the underlying currents to have been directed from the soma toward the apical area consistent with the polarities of the field potentials. In the case of apical stimulation (Fig. 3B) there was an initial negativity (spike ‘a’) at 3.5 ms followed by a positivity at 5 ms (‘b’) in the apical area. The potential was positive in the soma and basal area and negative in the apical area for spike ‘a’. We hypothesize that spike ‘a’ was produced by rapidly changing currents originating in the apical area which produced passive extracellular current sources in the soma and basal areas. The evoked magnetic fields consisted of a small deflection at the latency of spike ‘a’ riding on a larger spike labeled ‘b’. The direction of the underlying currents for spike ‘a’ was from the apical to the basal side, consistent with the above hypothesis. The polarities of both the field potentials and magnetic fields for spike ‘b’ and the subsequent spikes ‘c’ and ‘d’ were the same. The polarity of the field potential was negative in the soma area and positive in the apical and basal areas for spike ‘b’, suggesting the presence of currents from the soma toward the basal and apical directions. The underlying currents for these spikes, according to the magnetic signals, were directed from the apical to the basal side. Thus, the currents toward the basal direction were evidently stronger than the currents toward the apical direction.
highly correlated with the first two negative spikes seen in the field potential. The latency of the first spike depends on exact locations of the stimulating electrodes and stimulus amplitude. Thus, it was seen more clearly here than in Fig. 3. The latencies were not perfectly matched because the field potential electrode was far away from the intracellular recording electrode. The synchronization of populations spikes across the slice was high, but not perfect as one would expect in actual experimental preparations. Thus, the degree of synchronization degrades as the distance between two recording electrodes becomes larger. The same stimulus did not produce the short 1 ms-latency spike in a cell away from the stimulating electrodes as shown in Fig. 4B. This cell, instead, generated a train of spikes with the first spike occurring at the same time as the second spike in the field potential recorded close to the cell (see the inset). In some cases such as the one shown here the subsequent spikes of the intracellular and field potentials matched in latency. In other cases the latency match was not exact, but the latencies were closely correlated. The intracellular recordings in Fig. 4 thus indicate that pyramidal cells close to each pair of stimulating electrodes generated a short-latency spike, whereas those relatively far away did not. In contrast all the neurons appear to have fired at a latency of about 3–4 ms. The number of cells involved and a high degree of synchrony explain the large amplitude
3.3. Origins of the synchronized population spikes in the magnetic field We inferred from Fig. 3 that a somatic stimulation produces two types of population spikes, one that is directly activated by membrane depolarization due to the stimulus and the other that is elicited either by propagation of action potentials within the pyramidal cells or by synaptic activation via recurrent excitatory connections among the pyramidal cells. In the above experiments, electrical stimuli were applied with 125 mm-diameter Ag-AgCl electrodes spaced about 800 mm along the stratum pyramidale. Thus, they may elicit directly-activated spikes in the cells close to these electrodes, but the cells between the electrode pairs may not be activated in this manner, but only through a recurrent excitatory activation. This possibility was confirmed by intracellular recordings. In Fig. 4A, the soma area was stimulated with 6 pairs of bipolar electrodes with the polarities of the electrodes as shown in the inset and the intracellular potential was recorded from a pyramidal cell close to the stimulating electrodes simultaneously with the field potential. The cell generated a somatic spike with a peak latency of 1 ms, followed by a train of spikes which may have been produced either intrinsically or synaptically. The first two spikes were
Fig. 4. Comparison of the spikes of simultaneously recorded field potential and intracellular potential. Intracellular recording was obtained from a pyramidal cell away from (A) and close (B) to the stimulating electrodes. Field potential recording at the same location (labeled Rec. elec.) in two conditions. The arrows indicate the onset of a 50 ms-long stimulus.
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Fig. 5. Magnetic fields and field potentials measured in normal Ringer with [Ca2+]o = 2.5 mM and 0.1 mM PTX in the Ringer with elevated [Ca2+]o (5.0 mM; 20 min after the solution change), in the presence of added CNQX and finally in presence of added TTX. (A) Simultaneously recorded magnetic field and field potentials. (B) Left two columns: comparisons of the magnetic fields at SQ2 and field potentials in the soma area before and after raising [Ca2+]o. Right two columns: comparisons of the magnetic fields at SQ2 and field potentials in the soma area before and after adding CNQX (100 epochs/ave).
of the first, clearly visible spike in the magnetic field. The next section shows the waveform of the magnetic field associated with the non-synaptic, stimulus-activated spike. Also, it shows that the spike in the magnetic field with a latency of 3–4 ms was synaptically mediated. 3.4. Origins of the early and slow waves of the magnetic field The possible origins of the early wave and the later slow wave of the magnetic field were studied by manipulations that affect recurrent excitatory synaptic interactions among the pyramidal cells. High concentrations of Ca2+ in the bathing medium ([Ca2+]o) reduce recurrent synaptic interactions by increasing firing thresholds of the neurons via screening or binding of the membrane negative charge (Wong and Traub, 1983; Miles and Wong, 1987). As shown in Fig. 5,
increasing [Ca2+]o from 2.5 to 5.0 mM slightly reduced the initial spike and either reduced the amplitude or delayed the latencies of the subsequent spikes of the magnetic field and field potential (Fig. 5B, left). Also, the amplitude of the brief, early wave of the magnetic field was reduced. These results suggest that an elevated level of [Ca2+]o reduced the amplitude or probability of spikes generated in the pyramidal cells, leading to less depolarization in the cells. Elevated [Ca2+]o had the largest effect on the late slow wave. The reduced recurrent excitation, due to less firing in the pyramidal cell axons, probably resulted in reduced depolarization of the apical dendrites, leading to a diminution of the late slow wave. Bath application of CNQX (10 mM) abolished the synchronized spikes and the early wave as well as the late slow wave, revealing a response consisting of an initial spike followed by a slow wave resembling the depolarizing after-
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potential (DAP) commonly seen in intracellular recordings (see, for example, the intracellular record in Fig. 7 obtained with a 10 Hz stimulation rate). The right two traces in Fig. 5B compare the magnetic fields and field potentials obtained before and after adding CNQX to the bath. The spike in the magnetic signal was directed so that its underlying current was oriented from the soma toward the apical dendrites, just as for the large spike seen before adding CNQX. However, the peak latency of the spike (1 ms) was faster than the main spike seen in the elevated calcium medium. The spike amplitude was reliably larger in presence of CNQX, but its cause is unknown. The current for the subsequent slow wave was also directed from the soma toward the apical dendrites. Bath application of 0.5 mM TTX to the preceding solution abolished the responses seen in the presence of CNQX. The signals seen in the TTX medium reflect a discharge through the membrane capacitance at the end of stimulus application and the currents produced by activation of various voltagesensitive conductances other than gNa which was blocked
by TTX. These results indicate that the first large spike, as well as the superimposed brief wave and the late slow wave, seen with excitatory synaptic transmission intact were all synaptically mediated. The magnetic field seen in the presence of CNQX must be due to a direct, stimulus activation of gNa and fast potassium conductances. Voltage-sensitive sodium and potassium channels are numerous near the soma (Traub and Miles, 1991; Traub et al., 1991). Thus, opening of these channels can produce the intracellular currents directed from the soma toward the apical dendrites responsible for the spike and subsequent wave in the magnetic signal. The sensitivity of the late slow wave to an elevated level of [Ca2+]o and the apical-to-basal direction of the underlying currents indicate that this component was produced by depolarization in the apical dendrites produced by recurrent excitatory synaptic inputs. This notion is supported by the effect of an NMDA antagonist, d-APV (0.1 mM), on the slow wave shown in Fig. 6. As illustrated at the bottom of Fig. 6, APV did not strongly affect the spikes in the intracellular response
Fig. 6. Effects of NMDA receptor antagonist, APV. Simultaneously recorded magnetic fields and field potentials in normal Ringer with 0.1 mM PTX (control), after applying 0.1 mM APV and 20 min after washout of APV (30 epochs/ave). Intracellular potentials at the bottom were recorded from another slice under the same conditions.
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Fig. 7. Effects of stimulation frequency on three types of signal. Top five records: magnetic fields and field potentials were simultaneously recorded from a slice (30 epochs/ave). Bottom two records: simultaneously recorded field potential from the soma area and intracellular potential from another slice under the three conditions. Note: the time scale is the same for all records.
unlike CNQX, but it shortened the depolarization shift compared to the control (Ringer with 0.1 mM PTX). Consistent with this intracellular effect, the slow wave of the magnetic field and the corresponding wave of the field potential were both reduced in duration in the presence of APV. Evidently, blocking synaptic currents through NMDA channels, which appear to be located in the apical dendrites (Traub et al., 1993), shortened the duration of the apical depolarization, resulting in shortening of the duration and reduction of the amplitude of the late slow wave. As shown in Fig. 7, increasing stimulation frequency from 0.1 to 1 Hz reduced the amplitude of the magnetic field starting with the slower components. The slow wave of the magnetic field first showed reduction in its duration and gradually in its amplitude as well, this component disappearing at the 10 Hz stimulation rate. The spikes were also gradually reduced in amplitude and number. At 10 Hz, the early component partially remained. These changes are similar to the changes seen in the field potential and intracellular recordings. Just as for the magnetic field, the slow wave of the field potential was first reduced in duration, then
in amplitude with an increase in stimulation frequency. The spikes were reduced in number and amplitude by the increase in frequency. The depolarizing shift in the intracellular potential was also reduced in duration and amplitude with an increase in stimulation frequency. The spikes were reduced in number. A single spike followed by a DAP remained at 10 Hz. The reduction in response amplitude of these signals with stimulus frequency may be in part due to an increase in tonic inhibition mediated by GABAB (Traub et al., 1993), since the resting membrane became hyperpolarized with an increase in stimulus frequency even in presence of PTX which blocks GABAA inhibition (Fig. 7).
4. Discussion 4.1. Experimental analyses of the relationships among the magnetic field, field potential and intracellular potential Our aim was to elucidate the origins of spikes and waves
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in the evoked magnetic fields produced by a hippocampal slice preparation. An experimental analysis of this issue is feasible only if measured magnetic fields can be related to cellular events. Thus, we chose a preparation with a fairly simple cellular arrangement and contrived an experimental arrangement capable of generating highly synchronized activity. As in our previous study (Okada et al., 1997), the population activity was highly synchronized across the entire slice since the population spikes of the magnetic field were highly correlated with the field potentials and intracellular potentials measured in the pyramidal cells. This enabled us to interpret the physiological bases of the evoked magnetic fields at a single-cell level. Even in this type of simplified preparation, however, experimental analyses are not straightforward since the temporal waveform of the magnetic field is determined by a combination of intrinsic neuronal properties such as those of the active membrane conductances and network properties such as distribution and type of excitatory and inhibitory synaptic connections. Therefore, experimental analyses must be eventually combined with mathematical analyses for obtaining deeper and more definite insights into the physiological bases of MEG and EEG signals. We consider that an experimental analysis such as the one presented here is a necessary pre-requisite to such a more advanced analysis. It seems helpful to start with a brief theoretical account of the relationships between the magnetic field, field potential and intracellular potential before discussing the results presented above (cf. Swinney and Wikswo, 1980; Plonsey, 1981; Roth and Wikswo, 1985; Malmivuo and Plonsey, 1995). All three types of signals originate from transmembrane currents which flow in neurons as a result of changes in the conductance of either ligand- or voltage-gated channels. They in turn produce intra- and extracellular currents. Importantly, MEG and EEG signals measured far away from the active neurons are due to these intra- and extracellular currents along the inner and outer walls of the neuronal membrane. The transmembrane currents do not produce the signals because the neuronal membranes are thin and the currents are radial. Thus, the intra- and extracellular currents along the membrane surfaces are labeled as primary currents. The extracellular currents along any conductivity boundary surface other than the active neuronal membranes are labeled as secondary currents. The intracellular currents appear to account for about 75% of the magnetic signal in a CNS structure (turtle cerebellum) (Okada, 1989). Thus, we consider that both MEG and EEG signals are produced by intracellular currents (Okada et al., 1997). Both signals are linearly related to intracellular currents (Swinney and Wikswo, 1980; Roth and Wikswo, 1985; Barth and Sutherling, 1988; Barth and Di, 1991; Okada, 1989). The comparisons are, however, not necessarily straightforward for the data obtained from a slice. The magnetic field should be mostly due to the dipolar component of the
intracellular currents since it is measured far away from the cells (Swinney and Wikswo, 1980). The field potential measured in an extracellular space at some point along the longitudinal axis of the pyramidal cells within the slice is related to more local currents. Transmembrane currents as well as the intra- and extracellular currents should contribute to the field potential measured within an active tissue. The current distribution at any point in time along the axis may be complex, containing not only a dipolar, but also multipolar moments. This means that the magnetic field and field potential may not be always highly correlated if there are strong quadrupolar or higher moments in the distribution. A laminar field potential profile indicated that the magnetic field of a population spike may be quite weak even if there is a strong spike in the field potential because of a weak dipolar component (single current sink-source pair) and a strong quadrupolar component (two current sinksource pairs) along the longitudinal axis of the pyramidal cells (Okada et al., 1997). The latency of a component of a field potential may not always match with the corresponding component of the magnetic field (cf. Fig. 3A). This can occur, for example, when a spike is generated first in the axon hillock and then propagates to the soma as we suppose happened under the somatic stimulation (Fig. 3A). The currents associated with the spike in the axon hillock may have a weak dipolar component and a strong quadrupolar component, whereas the spike in the soma may have a stronger dipolar component. This can lead to a latency difference between the spike of the field potential measured in the basal area and the spike of the magnetic field. Similarly, the latency of a spike in the magnetic field may not match that of a spike of an intracellular potential because the potential is measured in one part of a cell, usually in the soma, and whereas the spike that gives rise to the magnetic field may be generated in another part of the cell. Nevertheless, our results here indicate that it seems possible to experimentally analyze the physiological bases of evoked magnetic fields from our slice preparation as long as one is cognizant of these possible complications. 4.2. Physiological bases of synchronized population spikes in the evoked magnetic field The initial spike in the intracelllular record from a pyramidal cell close to the stimulating electrodes was produced non-synaptically by a direct stimulus activation, since its peak latency was about 1 ms. The spike in the magnetic field had a peak latency of 1 ms, similar to that seen intracellularly. This spike was revealed in an experiment where the artifact was relatively small and the synaptic transmissions mediated by AMPA receptors were blocked by CNQX. The current giving rise to the spike in the magnetic field was oriented from the soma toward the apical dendrites and thus it was consistent with a notion that the spike was due to currents produced by opening of gNa in the soma. The spike was followed by a slow variation of the magnetic
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field which resembled the DAP of the intracellular potential. The mechanism for this DAP-like waveform must be elucidated in the future. The second spike in cells close to a pair of stimulating electrodes and the first spike in cells relatively far away from the stimulating electrodes were close together in their latencies. They were also highly time-locked to the first spike of the field potential and magnetic field clearly visible in a bath without synaptic transmission blockers. This component disappeared in a bath with CNQX (Fig. 5). Thus, it was produced synaptically. Judging from its latency, it was produced monosynaptically. The high degree of correlation with intracellular spikes suggests that it was produced by gNa and associated fast potassium conductances. The direction of the large spike in the magnetic field was opposite for the somatic and apical stimulations. In describing this result, we noted that the field potential was negative in the soma area for both types of stimulation. We concluded that the currents for spike ‘b’ was directed from the soma toward the apical dendrites in the case of the somatic stimulation and from the soma toward the basal dendrites in the case of the apical stimulation. How is it that the currents were directed toward the opposite directions even though the field potential was negative in the soma area? The field potentials along the longitudinal axis of the pyramidal cells (Fig. 3) had different profiles for these two types of stimulation. They were negative in both the basal and soma areas and positive in the apical area for the somatic stimulation, whereas they were positive on the basal and apical areas and negative in the soma area for the apical stimulation. Thus, the current sink was most likely closer to the apical side for the apical stimulation compared to the somatic stimulation. It is also possible that the currents may be directed oppositely even if the current sinks are the same. This can happen depending on the history of cellular activity just before a spike is initiated. For example, the basal dendrites may be more strongly depolarized than the apical dendrites when a spike is initiated in the axon hillock. Then, the current could flow toward the apical dendrites. Conversely, the currents could flow toward the basal dendrites if the apical dendrites were more strongly depolarized than the basal side, depending on the magnitude of the spatial gradient of the transmembrane potential along the longitudinal axis. Regardless of the exact locations of the current sink, the spikes of the magnetic field were opposite in polarity for the two types of stimulation. This was one of the most robust findings. Thus, it must be reconciled with earlier reports demonstrating the back-propagation of action spikes in pyramidal cells of the hippocampus (Traub et al., 1994) and the neocortex (Stuart and Sakmann, 1994). These reports have shown that an orthodromic excitation of the apical dendrites produces an action potential first in the soma followed by action potentials which propagate backward up the dendrites, even though the dendrites are depolarized initially. We hypothesize that the pyramidal cells can generate, depending on the exact stimulation parameters, either the
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back-propagation of this type or an action potential in the proximal apical dendritic trunk that is presumed to be the basis for the large spike in the magnetic field evoked by the apical stimulation. In our study, the depolarization in the apical dendritic trunk due to the electrical stimulation and the activation of gNa in this area must have been sufficiently high to maintain the trunk depolarized until the arrival of the monosynaptic recurrent synaptic inputs in the apical area. This can put the membrane potential in the trunk area near the threshold for firing, leading to an action potential in this area before an action potential in the axon hillock. 4.3. Physiological bases of the early wave and the slow wave of the magnetic field The early wave was eliminated by an application of CNQX. Thus, it was synaptically mediated. The currents underlying the brief early wave were directed from the basal dendrites and soma side toward the apical dendrites when the soma area was stimulated with a depolarizing negative potential extracellularly. It was directed toward the opposite direction when an apical area was depolarized with the extracellular electrodes. Furthermore, the direction of this current coincided with the direction of the spikes (Fig. 3). This suggests that the cellular events responsible for the early wave were related to the mechanism responsible for the spikes. They may reflect a depolarization in the soma in the case of the somatic stimulation and in the apical trunk in the case of the apical stimulation. The later slow wave was also synaptically mediated. It appears to be produced by currents originating in the apical dendrites since the magnetic field polarity indicated the underlying currents to have been directed from the apical to the basal side. The underlying currents were most probably produced by depolarization of the apical membrane due to both excitatory post-synaptic currents (EPSCs) at recurrent excitatory synapses and voltage-sensitive calcium conductances. This hypothesis was successfully used to account for the intracellular potentials observed in hippocampal slices bathed in PTX (Traub et al., 1993). Our study has obtained some independent evidence for this hypothesis. The elevated [Ca2+]o abolished the slow wave. This effect is probably due to reduced recurrent synaptic inputs, since a high level of [Ca2+]o raises firing threshold (Berry and Pentreath, 1976; Wong and Traub, 1983; Miles and Wong, 1987). APV reduced the duration of the late slow wave. This reduction correlated with the shortening of duration of the depolarizing shift in the intracellular record. This indicates that the slow wave was maintained in part by NMDA channels which remain open for 100–1000 ms (Traub et al., 1993). NMDA channels are localized in the recurrent excitatory synapses (Jefferys, 1989; Neumann et al., 1989). Thus, the slow wave appears to have been produced in part by the EPSCs carried by the NMDA channels. Swann et al. (1986) found a large extracellular current observed in both the apical and basal dendrite areas during
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a depolarizing shift in epileptiform bursts (Swann et al., 1986). Our results indicate that the current sink in the apical area was stronger than the sink in the basal area. The effects of stimulation frequency were also consistent with the above hypothesis. Increasing the stimulation frequency initially shortened the duration and finally suppressed the slow wave. This correlated with the shortening of the depolarizing shift in the intracellular potential seen with an increase in stimulation frequency. Increasing the stimulation rate can hyperpolarize the membrane by activating GABAB channels and thus shunt the membrane potential, reducing the depolarization caused by the recurrent synaptic inputs. In conclusion, the detection of the magnetic field associated with the non-synaptic, stimulus-activated spike in the intracellular potential indicates that it is feasible in future to study the way various intrinsic membrane conductances generate the magnetic field in a simplified situation without contributions from the network properties of the slice. The synchronized populations spikes seen clearly in the magnetic signals from slices with excitatory synaptic connections intact were synaptically mediated. The short duration of the spikes and a high degree of correlation with intracellular spikes indicate that they were produced by voltage-sensitive conductances with fast kinetics such as gNa and potassium conductances of the delayed rectifier type and A-type (Traub et al., 1991, 1994). It is necessary to verify whether the early wave reflects a relatively slow depolarization of the soma or apical trunk produced by activation of these fast conductances. The late slow wave of the magnetic field appears to be due to currents generated in the apical dendrites, probably by a combination of excitatory post-synaptic currents carried by NMDA and AMPA channels and calcium-mediated currents. It is necessary to verify this hypothesis and separate their relative contributions. These currents are evidently capable of producing magnetic fields with a slow onset and a long duration in the absence of GABAA inhibition, suggesting that the evoked magnetic fields produced by a single focal area of a CNS structure can be quite complex in temporal waveform. Acknowledgements This research was carried out at Veterans Affairs Medical Center, Albuquerque, NM 87108. The authors thank Professor Roger Traub for his encouragement and the VAMC for providing the laboratory space. We also thank Ms. AnaLorena Barrios for her assistance. This work was generously supported by US DHHS grant RO1-NS21149. References Barth, D.S. and Di, S. The electrophysiological basis of epileptiform magnetic fields in neocortex: spontaneous ictal phenomena. Brain Res., 1991, 557: 95–102.
Barth, D.S. and Sutherling, W. Current source-density and neuromagnetic analysis of the direct cortical response in rat cortex. Brain Res., 1988, 450: 280–294. Baxter, D.A. and Byrne, J.H. Ionic conductance mechanisms contributing to the electrophysiological properties of neurons. Curr. Opin. Neurobiol., 1991, 1: 105–112. Bekkers, J.M. and Stevens, C.F. NMDA and non-NMDA receptors are colocalized at individual excitatory synapses in cultured rat hippocampus. Nature, 1989, 341: 230–233. Berry, M.S. and Pentreath, V.W. Criteria for distinguishing between monosynaptic and polysynaptic transmission. Brain Res., 1976, 105: 1–20. Buchanan, D.S., Crum, D.B., Cox, D. and Wikswo, J.P., Jr. MicroSQUID: a close-spaced four channel magnetometer. In: S.J. Williamson, M. Hoke, G. Stroink and M. Kotani (Eds.), Advances in Biomagnetism. Plenum, New York, 1989, pp. 677–679. Creutzfeldt, O.D., Watanable, S. and Lux, H.D. Relations between EEG phenomena and potentials of single cortical cells. I. Evoked responses after thalamic and epicortical stimulation. Electroenceph. clin. Neurophysiol., 1966a, 20: 1–18. Creutzfeldt, O.D., Watanable, S. and Lux, H.D. Relations between EEG phenomena and potentials of single cortical cells. ii. spontaneous and convulse activity. Electroenceph. Clin. Neurophysiol., 1966b, 20: 19– 37. Hille, B. Ionic Channels of Excitable Membranes. Sinauer, Sunderland, MA, 1992. Humphrey, D.R. Re-analysis of the antidromic cortical response. I. Potentials evoked by stimulation of the isolated pyramidal trace. Electroenceph. clin. Neurophysiol., 1968a, 24: 116–129. Humphrey, D.R. Re-analysis of the antidromic cortical response. II. On the contribution of cell discharge and PSPS to the evoked potentials. Electroenceph. clin. Neurophysiol., 1968b, 24: 421–442. Jefferys, J.G.R. Chronic epileptic foci in vitro in hippocampal slices from rats with tetanus toxin epileptic syndrome. J. Neurophysiol., 1989, 62: 458–468. Johnston, D. and Brown, T.H. Giant synaptic potential hypothesis for epileptiform activity. Science, 1981, 211: 294–297. Kyuhou, S.I. and Okada, Y.C. Detection of magnetic evoked fields associated with synchronous population activities in the transverse CA1 slice of the guinea-pig. J. Neurophysiol., 1993, 70: 2665–2668. Llina´s, R.R. Comparative electrobiology of mammalian central neurons. In: R. Dingledine (Ed.), Brain Slices. Plenum, New York, 1984, pp. 7– 24. Llina´s, R.R. The intrinsic electrophysiological properties of mammalian neurons: insights into central nervous system function. Science, 1988, 242: 1654–1664. MacVicar, B.A. and Dudek, F.E. Local synaptic circuits in rat hippocampus: interactions between pyramidal cells. Brain Res., 1980, 184: 220– 223. Malmivuo, J. and Plonsey, R. Bioelectromagnetism. Oxford University Press, New York, 1995. Miles, R. Synaptic excitation of inhibitory cells by single CA3 hippocampal pyramidal cells of the guinea-pig in vitro. J. Physiol., 1990, 428: 61– 77. Miles, R. and Wong, R.K.S. Single neurones can initiate synchronized population discharge in the hippocampus. Nature, 1983, 306: 371– 373. Miles, R. and Wong, R.K.S. Excitatory synaptic interactions between CA3 neurones in the guinea-pig hippocampus. J. Physiol., 1986, 373: 397– 418. Miles, R. and Wong, R.K.S. Inhibitory control of local excitatory circuits in the guinea-pig hippocampus. J. Physiol., 1987, 388: 611–629. Neumann, R.S., Cherubini, E. and Ben-Ari, Y. Endogenous and network bursts induced by N-methyl-d-aspartate and magnesium-free medium in the CA3 region of the hippocampal slice. Neuroscience, 1989, 28: 393– 399. Newberry, N.R. and Nicoll, R.A. A bicuculline-resistant inhibitory post-
J. Wu, Y.C. Okada / Electroencephalography and clinical Neurophysiology 107 (1998) 361–373 synaptic potential in rat hippocampal pyramidal cells in vitro. J. Physiol., 1984, 348: 239–254. Newberry, N.R. and Nicoll, R.A. Comparison of the action of baclofen with g-aminobutyric acid on rat hippocampal cells in vitro. J. Physiol., 1985, 360: 161–185. Okada, Y.C. Recent developments on the physiological basis of magnetoencephalography (MEG). In: S.J. Williamson, M. Hoke, G. Stroink and M. Kotani (Eds.), Advances in Biomagnetism. Plenum, New York, 1989, pp. 273–278. Okada, Y.C., Shah, B. and Huang, J.-C. Ferromagnetic high-permeability alloy alone can provide sufficient low-frequency and eddy-current shieldings for biomagnetic measurements. IEEE Trans. BME, 1994, 41: 688–697. Okada, Y.C., Wu, J. and Kyuhou, S.I. Genesis of magnetic signals in a mammalian CNS structure. Electroenceph. clin. Neurophysiol., 1997, 103: 474–485. Okada, Y.C. and Xu, C. Single-epoch neuromagnetic signals during epileptiform activities in guinea-pig longitudinal CA3 slices. Neurosci. Lett., 1996, 211: 155–158. Plonsey, R. Generation of magnetic fields by the human body. In: S.N. Erne´, H.D. Hahlbohm and H. Lu¨bbig (Eds.), Biomagnetism. de Gruyter, Berlin, 1981, pp. 177–205. Pollen, D.A. On the generation of neocortical potentials. In: H.H. Jasper, A.A. Ward and A. Pope (Eds.), Basic Mechanisms of the Epilepsies. Little Brown. Boston, MA, 1969, pp. 411–420. Roth, B.J. and Wikswo, J.P. Jr. The magnetic field of a single axon: a comparison of theory and experiment. Biophys. J., 1985, 48: 93–109.
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Physiological bases of the synchronized population spikes and slow wave of the magnetic field generated by a guinea-pig longitudinal CA3 slice preparation Jie Wu, Yoshio C. Okada* Departments of Neurology and Neurosciences, University of New Mexico School of Medicine, Albuquerque, New Mexico, 87131 USA Accepted for publication: 1 July 1998
Abstract Objective: The physiological bases of evoked magnetic fields were examined in a guinea-pig hippocampal slice preparation, motivated by new concepts in central nervous system (CNS) electrophysiology brought about by discoveries of active conductances in the dendrites and soma of neurons. Methods: Their origins were elucidated by comparing them with intracellular and extracellular field potentials. Results: With excitatory synaptic transmissions blocked, the magnetic signal elicited by an electrical stimulus applied to the pyramidal cell layer consisted of a spike and a depolarizing afterpotential-like waveform. With the excitatory synaptic transmissions intact, but with inhibitory synaptic transmissions blocked, the magnetic signal was bi- or triphasic depending on whether the cell layer or the apical dendrite area of the pyramidal cells was, respectively, depolarized. In both cases the signal consisted of a train of synchronized population spikes superimposed on a brief wave followed by a longer, slow wave. The spike train was correlated with synaptically mediated intracellular spikes. The underlying currents for the slow wave were directed from the apical to the basal side for both types of stimulation. It was most likely generated by depolarization of the apical dendrites, caused by recurrent excitatory synaptic activation. Conclusions: This analysis illustrates how synaptic connections and intrinsic conductances in a disinhibited mammalian CNS structure can generate spikes and waves of the magnetic field and electrical potential. 1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: Biomagnetism; Electroencephalography; Magnetoencephalography; Evoked potential; Hippocampus; Voltage-sensitive conductances; Ligand-gated channels; a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; N-Methyl-d-aspartate
1. Introduction Concepts of dendritic and somatic physiology have recently undergone dramatic changes due to discoveries of many types of ligand- and voltage-sensitive conductances in central nervous system (CNS) neurons (Llina´s, 1984, 1988; Baxter and Byrne, 1991; Hille, 1992). These discoveries call for an examination of the genesis of magnetoencephalography (MEG) signals within this new framework. Likewise, the genesis of electroencephalographic (EEG) signals which was thoroughly studied about 30 years ago (Pollen, 1969;
* Corresponding author. Department of Neurology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. Tel.: +1 505 2562874; fax: +1 505 2600165; e-mail: [email protected]
Creutzfeldt et al., 1966a,b; Humphrey, 1968a,b) should be re-examined. Our current understanding of the genesis of the evoked potential is still largely based on studies carried out when the dendrites were viewed as passive, electrotonic core conductor cables having excitatory and inhibitory neurotransmitter receptors whose opening produced excitatory and inhibitory post-synaptic potentials (EPSPs and IPSPs, respectively) and produced action potentials in the axon hillock by electrotonic conduction of the EPSPs. One approach for elucidating the origin of MEG signals is to analyze and compare such signals produced in a relatively simple CNS preparation. We chose the hippocampus of the guinea-pig for this purpose. The anatomy and electrophysiology of this structure are quite well understood to the point where it is possible to construct a mathematical model of CA1 and CA3 incorporating various intrinsic pas-
0013-4694/98/$ - see front matter 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0013-4694 (98 )0 0098-4
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sive and active membrane conductances and network properties that have been actually experimentally determined (Traub and Miles, 1991; Traub et al., 1991, 1993, 1994). Thus, physiological mechanisms for the evoked magnetic fields may be understood by comparing them with the evoked extracellular field potentials simultaneously measured with the magnetic signals and with the intracellular potentials. We have used a simplified preparation in order to make it feasible to interpret the MEG data in terms of underlying cellular events (Okada et al., 1997). The present study was carried out on a longitudinal CA3 slice in which the longitudinal axes of the pyramidal cells are parallel to each other along the direction perpendicular to the cell layer. Thus, the slice is effectively a two dimensional structure with the principal core conductors from a single subfield of the hippocampus arranged along the main axes of a rectangular coordinate system. This preparation is even simpler than the conventional transverse slice which contains many subfields and related areas of the hippocampus. The slice contains excitatory pyramidal neurons which are connected to each other via recurrent excitatory connections (MacVicar and Dudek, 1980; Miles and Wong, 1986), and inhibitory neurons which receive excitatory inputs from the pyramidal neurons and provide g-aminobutyric acid A (GABAA) and GABAB type inhibitory connections to each other and to the pyramidal cells (Newberry and Nicoll, 1984, 1985; Miles and Wong, 1987; Miles, 1990). The recurrent excitatory connections among the pyramidal cells are mediated by glutamatergic synapses (Johnston and Brown, 1981), having N-methyl-d-aspartate (NMDA) receptors (Jefferys, 1989; Neumann et al., 1989) and a-amino-3-hydroxy-5-methyl4-isoxazole propionic acid (AMPA) receptors, since NMDA and AMPA channels are co-localized (Bekkers and Stevens, 1989). The connections are sufficiently strong to produce synchronous population activities (Miles and Wong, 1983, 1986) that can be measured with MEG (Kyuhou and Okada, 1993; Okada and Xu, 1996). The degree of synchronization can be sufficiently high across the entire slice (Okada et al., 1997) so that the magnetic field outside the slice can be analyzed in terms of events at a single cell level. In the first paper of this series (Okada et al., 1997), we have presented some basic characteristics of MEG signals in CA1 and CA3 slice preparations. Here, we describe the physiological bases of spikes and waves of the evoked magnetic fields generated in the longitudinal CA3 slices. Some of this work was previously presented in abstract form (Wu and Okada, 1996).
2. Methods 2.1. Slice preparation The longitudinal slice of CA3 was prepared with a
method described previously in detail (Okada et al., 1997). Briefly, adult guinea-pigs (Hartley, 200–400 g, 80 animals) were anesthetized with sodium pentobarbital (60 mg/kg i.p.) and decapitated with a guillotine. The brain was quickly removed and submerged in Ringer solution at 2–4°C for 1–2 min. Both hippocampi were removed from the brain, the dentate surface of a hippocampus was glued flat to a 3% agar block and the assembly was placed horizontally in a well of Vibratome filled with cold Ringer solution (Fig. 1A). The hippocampus was cut parallel to the agar surface just above the fimbria to obtain two 400 mm-thick slices. A rectangular longitudinal CA3 slice (Fig. 1C) was obtained by cutting CA1 away from the slice and trimming both edges. All slices were incubated in the Ringer solution bubbled with 95% O2 –5% CO2 gas at room temperature for 1 h or more. After the incubation, one slice was transferred to an experimental chamber (Fig. 1B) that contained 36°C oxygenated Ringer solution. The slice was placed between a pair of nylon nets on top of a cylindrical platform in the chamber. Warm oxygenated Ringer continuously flowed out of the platform from below to superfuse the slice. The slice was kept in this chamber for at least one half hour before starting measurements. 2.2. Solutions and chemicals The Ringer solution in the recording chamber had the following composition (in mM): NaCl 124, KCl 5, KH2 PO4 1.2, MgCl2 1.3, CaCl2 2.5, NaHCO3 26 and glucose 10. The pH was kept at 7.4 with 95% O2 –5% CO2 gas mixture. Intracellular potentials in the pyramidal cells were measured with a glass micropipette filled with potassium acetate (4 M; 50–90 MQ). The channel blockers used in the present experiments were picrotoxin (PTX) which blocks GABAA receptors, tetrodotoxin (TTX) which blocks the sodium conductance (gNa), d-2 amino-5-phosphonovaleric acid (APV) dissolved in normal Ringer solution which blocks NMDA receptors and 6-cyano-2,3-dihydroxy-7nitroquinoxaline (CNQX), dissolved in dimethyl sulphoxide (DMSO, concentration ,0.01%; Tocris Neuramin), which blocks AMPA receptors. 2.3. Stimulation The longitudinal CA3 slice was stimulated with 6–10 pairs of electrodes (Ag-AgCl wire, 125 mm diameter) placed along the pyramidal cell layer from below the slice (Fig. 1C). Here an arrangement with 6 pairs of electrodes is illustrated. One row of electrodes was placed near the cell layer on the side of the stratum oriens and the second row on the stratum lucidum or radiatum. The electrodes were negative in the cell layer and positive in the stratum lucidum or radiatum for ‘somatic’ stimulations and opposite in polarity for ‘apical’ stimulations. The stimulating parameters were: 0.7–1.0 mA, 50–100 ms, 0.1–0.3 Hz.
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Fig. 1. Schematic of experimental arrangement. (A) Orientation of the hippocampus for cutting longitudinal CA3 slices. Two 400 mm-thick slices parallel to the pyramidal cells were obtained just above the fimbria. (B) Experimental chamber. A slice was sandwiched between a pair of nylon nets. Warm (36°C) oxygenated Ringer superfused the slice from below, flowing out of the chamber. Magnetic field detectors were placed 2 mm above the slice. Stimulating and recording electrodes were placed from below the slice. (C) Arrangement of the stimulating electrodes (six pairs of bipolar Ag-AgCl electrodes about 0.8 mm apart shown as black squares) along the pyramidal cell layer, 3 field potential recording electrodes (chlorided 125 mm diameter silver wires) along the longitudinal axis of pyramidal cells (shown enlarged in the inset) in the region of the basal dendrites in the stratum oriens (s.o.), the soma in the stratum pyramidale (s. p.), and apical dendrites in the stratum lucidum (s. l.) and radiatum (s. r.) and 4 channels of the m-SQUID (Superconducting Quantum Interference Device) sensor (SQ1–4). All in correct relative dimensions. Stimulating electrodes were negative in the cell layer and positive in the stratum lucidum or radiatum for ‘somatic’ stimulations and opposite in polarity for ‘apical’ stimulations.
2.4. Recording procedures The magnetic field recording was carried out using a superconducting magnetic field sensor called a mSQUID (Buchanan et al., 1989). It was equipped with four channels of a first-order, asymmetric, axial gradiometer with a 4.0 mm diameter bottom detection coil, an 8.5 mm diagonal separation between channels, a 16 mm baseline, a 1.2 mm separation between the detection coils and the outside surface of the cryogenic container of the mSQUID and a field sensitivity of 50 fT/ÎHz (fT = femtotesla; Okada et al., 1994). The detection coils were placed directly over the slice at a distance of about 2 mm above the slice. The SQUID outputs were fed to a 4-channel amplifier with a 24 dB/octave Butterworth filter. All the magnetic field data were collected with a bandpass filter having a highand a low-pass cut-off frequencies of 0.1 Hz and 2 kHz, respectively. The magnetic field recordings were carried out in a magnetically shielded room (Okada et al., 1994). Field potentials were recorded simultaneously with the magnetic fields using 125 mm-diameter Ag-AgCl electrodes placed along the longitudinal axis of the pyramidal cells. Three such electrodes labeled ‘basal’, ‘soma’ and ‘apical’ were placed in the region of the basal dendrites, soma and apical dendrites. The potentials were fed to an FET preamplifier and then to an amplifier/filter equipped with a 24 dB/octave Butterworth filter with a bandpass of 0.1 Hz to 2kHz. Intracellular potentials were recorded from 14 slices for a total of 33 pyramidal cells using an Axoclamp 1A with a 10 kHz lowpass filter. Only cells with resting membrane poten-
tials of at least −55 mV were used. In the presence of 0.1 mM PTX, the cellular resting potential, amplitude of action potential and input resistance were 64.9 ± 1.5 mV (SEM), 79.7 ± 2.7 mV and 26.0 ± 2.2 MQ, respectively. All intracellular recordings were performed simultaneously with extracellular field potential recordings.
3. Results 3.1. Characteristics of the evoked magnetic fields Fig. 2 shows three types of signals. The first two rows show the magnetic fields recorded at positions SQ2 and SQ4 (see Fig. 1C). The next three rows show the field potentials recorded along the longitudinal axis of the pyramidal cells. These records were obtained simultaneously. The bottom two rows show a field potential simultaneously measured with an intracellular potential in the soma of a pyramidal cell in another slice. The records on the right side were obtained by stimulating the apical area about 400 mm from the soma with a negative electrode; those on the left were obtained by stimulating the soma area. In the presence of 0.1 mM PTX, the magnetic fields were biphasic for the ‘somatic’ stimulation and triphasic for the ‘apical’ stimulation, replicating our earlier results (Okada et al., 1997). The magnetic fields detected at locations SQ2 and SQ4 were mirror images of each other in both cases with a near baseline level of signal at locations SQ1 and SQ3 (not shown). This spatial distribution is consistent with a dipolar field pattern seen in extensive maps (Okada et al., 1997),
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indicating that the underlying currents were directed along the longitudinal axis of the pyramidal cells. The somatic stimulation produced 1–3 spikes in the magnetic field (peak latency of the first spike, mean = 2.2 ms with a range of 1.4–4.3 ms, n = 20) riding on a brief wave (this complex is labeled ‘1’), followed by a later slow wave labeled ‘2’ lasting about 70 ms. Applying the right-hand rule of electromagnetism, one can deduce from directions of the magnetic fields that the initial spike and the brief, early wave were both due to intracellular currents directed from the basal to the apical side of the pyramidal cells, whereas the following slow wave was due to oppositely directed currents. These conclusions are consistent with the polarities of the field potentials. The large spike in the
field potentials corresponding in latency to the first spike of magnetic field was negative in the basal dendrite area and soma, and positive in the apical area, indicating that the corresponding intracellular currents were directed from the basal to the apical side. The field potential during the slow wave of the magnetic field was negative on the apical side and positive on the soma and basal side, indicating that the underlying intracellular currents were directed from the apical to the basal side. The apical stimulation produced a train of synchronized population spikes in the magnetic field (peak latency of the first spike, mean = 3.0 ms with a range of 1.7–5.2 ms, n = 23) riding on a brief wave (labeled as ‘1’), followed by a return to the baseline (‘2’), and then a slow wave
Fig. 2. Magnetic fields, field potentials and intracellular potentials produced by longitudinal CA3 slices. Left: somatic stimulation; right: apical stimulation. Top two rows: magnetic fields; middle 3 rows: field potentials. These five traces were recorded simultaneously (30 epochs/ave). Bottom two rows: field potential in the soma area and simultaneously recorded intracellular potential from the soma of a pyramidal cell. Note: time scale is the same for all three types of signal. pT, picoTesla.
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(‘3’). Interestingly, the currents underlying both the initial spike and the early wave were opposite in direction to those seen with the somatic stimulation. They were directed from the apical toward the basal side. Consistent with this direction, the early wave of the field potential was negative on the apical and soma side and positive on the basal dendrite side of the pyramidal cells. In contrast, the currents for the slow wave, according to the magnetic field, were directed from the apical to the basal side just as for the somatic stimulation. Accordingly, the field potential was negative on the apical side and positive on the soma and basal sides. 3.2. Relationships between synchronized population spikes of the magnetic field and field potential The relationships between the magnetic field and field potential of the synchronized population spikes seen in Fig. 2 are more clearly illustrated in Fig. 3. The latencies of the spikes were highly correlated between the magnetic and electrical signals. In the case of somatic stimulation (Fig. 3A), the field potentials recorded near the soma and basal dendrites
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showed an initial negativity overlapping with the stimulus artifact, followed by a small positivity (‘a’), then a strong negativity (‘b’) with additional several spikes. It is difficult to distinguish the initial negativity from a stimulus artifact in this record, but it most likely reflects a depolarization of the basal and soma areas since a negative stimulus was applied with six pairs of bipolar electrodes to these regions (about 100 mm from the cell layer on the basal side). This conclusion is consistent with the results presented in Figs. 4 and 5. The initial spike was not seen in the magnetic field, masked by the stimulus artifact. The spike ‘a’, however, was seen in the magnetic records. It is probably due to activation of fast conductances in the proximal apical dendrites since the field potential was negative in the apical area. The underlying current for the magnetic field of spike ‘a’ was directed from the apical to the basal side consistent with this interpretation. The spikes labeled ‘b’, ‘c’ and ‘d’ were seen slightly earlier (0.5 ms, n = 8) in the basal area compared to those in the soma and apical areas. This difference in peak latency probably reflects a difference in activating a spike in the axon hillock and a spike in the soma area. The spikes in the magnetic signals matched in latency with the corre-
Fig. 3. Comparison of the spikes of simultaneously recorded magnetic fields and field potentials. (A) Somatic stimulation (−electrode near the cell layer and +electrode in apical dendrite area). (B) Apical stimulation (+electrode near the cell layer and −electrode in apical dendrite area; 30 epochs/ave). (a–d) Synchronized population spike components. See text for details.
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sponding spikes in the field potentials recorded in the soma and apical areas, suggesting that the currents associated with these somatic and apical spikes contained a stronger dipolar component than the currents associated with an action potential in the axon hillock. The magnetic field polarity indicated the underlying currents to have been directed from the soma toward the apical area consistent with the polarities of the field potentials. In the case of apical stimulation (Fig. 3B) there was an initial negativity (spike ‘a’) at 3.5 ms followed by a positivity at 5 ms (‘b’) in the apical area. The potential was positive in the soma and basal area and negative in the apical area for spike ‘a’. We hypothesize that spike ‘a’ was produced by rapidly changing currents originating in the apical area which produced passive extracellular current sources in the soma and basal areas. The evoked magnetic fields consisted of a small deflection at the latency of spike ‘a’ riding on a larger spike labeled ‘b’. The direction of the underlying currents for spike ‘a’ was from the apical to the basal side, consistent with the above hypothesis. The polarities of both the field potentials and magnetic fields for spike ‘b’ and the subsequent spikes ‘c’ and ‘d’ were the same. The polarity of the field potential was negative in the soma area and positive in the apical and basal areas for spike ‘b’, suggesting the presence of currents from the soma toward the basal and apical directions. The underlying currents for these spikes, according to the magnetic signals, were directed from the apical to the basal side. Thus, the currents toward the basal direction were evidently stronger than the currents toward the apical direction.
highly correlated with the first two negative spikes seen in the field potential. The latency of the first spike depends on exact locations of the stimulating electrodes and stimulus amplitude. Thus, it was seen more clearly here than in Fig. 3. The latencies were not perfectly matched because the field potential electrode was far away from the intracellular recording electrode. The synchronization of populations spikes across the slice was high, but not perfect as one would expect in actual experimental preparations. Thus, the degree of synchronization degrades as the distance between two recording electrodes becomes larger. The same stimulus did not produce the short 1 ms-latency spike in a cell away from the stimulating electrodes as shown in Fig. 4B. This cell, instead, generated a train of spikes with the first spike occurring at the same time as the second spike in the field potential recorded close to the cell (see the inset). In some cases such as the one shown here the subsequent spikes of the intracellular and field potentials matched in latency. In other cases the latency match was not exact, but the latencies were closely correlated. The intracellular recordings in Fig. 4 thus indicate that pyramidal cells close to each pair of stimulating electrodes generated a short-latency spike, whereas those relatively far away did not. In contrast all the neurons appear to have fired at a latency of about 3–4 ms. The number of cells involved and a high degree of synchrony explain the large amplitude
3.3. Origins of the synchronized population spikes in the magnetic field We inferred from Fig. 3 that a somatic stimulation produces two types of population spikes, one that is directly activated by membrane depolarization due to the stimulus and the other that is elicited either by propagation of action potentials within the pyramidal cells or by synaptic activation via recurrent excitatory connections among the pyramidal cells. In the above experiments, electrical stimuli were applied with 125 mm-diameter Ag-AgCl electrodes spaced about 800 mm along the stratum pyramidale. Thus, they may elicit directly-activated spikes in the cells close to these electrodes, but the cells between the electrode pairs may not be activated in this manner, but only through a recurrent excitatory activation. This possibility was confirmed by intracellular recordings. In Fig. 4A, the soma area was stimulated with 6 pairs of bipolar electrodes with the polarities of the electrodes as shown in the inset and the intracellular potential was recorded from a pyramidal cell close to the stimulating electrodes simultaneously with the field potential. The cell generated a somatic spike with a peak latency of 1 ms, followed by a train of spikes which may have been produced either intrinsically or synaptically. The first two spikes were
Fig. 4. Comparison of the spikes of simultaneously recorded field potential and intracellular potential. Intracellular recording was obtained from a pyramidal cell away from (A) and close (B) to the stimulating electrodes. Field potential recording at the same location (labeled Rec. elec.) in two conditions. The arrows indicate the onset of a 50 ms-long stimulus.
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Fig. 5. Magnetic fields and field potentials measured in normal Ringer with [Ca2+]o = 2.5 mM and 0.1 mM PTX in the Ringer with elevated [Ca2+]o (5.0 mM; 20 min after the solution change), in the presence of added CNQX and finally in presence of added TTX. (A) Simultaneously recorded magnetic field and field potentials. (B) Left two columns: comparisons of the magnetic fields at SQ2 and field potentials in the soma area before and after raising [Ca2+]o. Right two columns: comparisons of the magnetic fields at SQ2 and field potentials in the soma area before and after adding CNQX (100 epochs/ave).
of the first, clearly visible spike in the magnetic field. The next section shows the waveform of the magnetic field associated with the non-synaptic, stimulus-activated spike. Also, it shows that the spike in the magnetic field with a latency of 3–4 ms was synaptically mediated. 3.4. Origins of the early and slow waves of the magnetic field The possible origins of the early wave and the later slow wave of the magnetic field were studied by manipulations that affect recurrent excitatory synaptic interactions among the pyramidal cells. High concentrations of Ca2+ in the bathing medium ([Ca2+]o) reduce recurrent synaptic interactions by increasing firing thresholds of the neurons via screening or binding of the membrane negative charge (Wong and Traub, 1983; Miles and Wong, 1987). As shown in Fig. 5,
increasing [Ca2+]o from 2.5 to 5.0 mM slightly reduced the initial spike and either reduced the amplitude or delayed the latencies of the subsequent spikes of the magnetic field and field potential (Fig. 5B, left). Also, the amplitude of the brief, early wave of the magnetic field was reduced. These results suggest that an elevated level of [Ca2+]o reduced the amplitude or probability of spikes generated in the pyramidal cells, leading to less depolarization in the cells. Elevated [Ca2+]o had the largest effect on the late slow wave. The reduced recurrent excitation, due to less firing in the pyramidal cell axons, probably resulted in reduced depolarization of the apical dendrites, leading to a diminution of the late slow wave. Bath application of CNQX (10 mM) abolished the synchronized spikes and the early wave as well as the late slow wave, revealing a response consisting of an initial spike followed by a slow wave resembling the depolarizing after-
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potential (DAP) commonly seen in intracellular recordings (see, for example, the intracellular record in Fig. 7 obtained with a 10 Hz stimulation rate). The right two traces in Fig. 5B compare the magnetic fields and field potentials obtained before and after adding CNQX to the bath. The spike in the magnetic signal was directed so that its underlying current was oriented from the soma toward the apical dendrites, just as for the large spike seen before adding CNQX. However, the peak latency of the spike (1 ms) was faster than the main spike seen in the elevated calcium medium. The spike amplitude was reliably larger in presence of CNQX, but its cause is unknown. The current for the subsequent slow wave was also directed from the soma toward the apical dendrites. Bath application of 0.5 mM TTX to the preceding solution abolished the responses seen in the presence of CNQX. The signals seen in the TTX medium reflect a discharge through the membrane capacitance at the end of stimulus application and the currents produced by activation of various voltagesensitive conductances other than gNa which was blocked
by TTX. These results indicate that the first large spike, as well as the superimposed brief wave and the late slow wave, seen with excitatory synaptic transmission intact were all synaptically mediated. The magnetic field seen in the presence of CNQX must be due to a direct, stimulus activation of gNa and fast potassium conductances. Voltage-sensitive sodium and potassium channels are numerous near the soma (Traub and Miles, 1991; Traub et al., 1991). Thus, opening of these channels can produce the intracellular currents directed from the soma toward the apical dendrites responsible for the spike and subsequent wave in the magnetic signal. The sensitivity of the late slow wave to an elevated level of [Ca2+]o and the apical-to-basal direction of the underlying currents indicate that this component was produced by depolarization in the apical dendrites produced by recurrent excitatory synaptic inputs. This notion is supported by the effect of an NMDA antagonist, d-APV (0.1 mM), on the slow wave shown in Fig. 6. As illustrated at the bottom of Fig. 6, APV did not strongly affect the spikes in the intracellular response
Fig. 6. Effects of NMDA receptor antagonist, APV. Simultaneously recorded magnetic fields and field potentials in normal Ringer with 0.1 mM PTX (control), after applying 0.1 mM APV and 20 min after washout of APV (30 epochs/ave). Intracellular potentials at the bottom were recorded from another slice under the same conditions.
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Fig. 7. Effects of stimulation frequency on three types of signal. Top five records: magnetic fields and field potentials were simultaneously recorded from a slice (30 epochs/ave). Bottom two records: simultaneously recorded field potential from the soma area and intracellular potential from another slice under the three conditions. Note: the time scale is the same for all records.
unlike CNQX, but it shortened the depolarization shift compared to the control (Ringer with 0.1 mM PTX). Consistent with this intracellular effect, the slow wave of the magnetic field and the corresponding wave of the field potential were both reduced in duration in the presence of APV. Evidently, blocking synaptic currents through NMDA channels, which appear to be located in the apical dendrites (Traub et al., 1993), shortened the duration of the apical depolarization, resulting in shortening of the duration and reduction of the amplitude of the late slow wave. As shown in Fig. 7, increasing stimulation frequency from 0.1 to 1 Hz reduced the amplitude of the magnetic field starting with the slower components. The slow wave of the magnetic field first showed reduction in its duration and gradually in its amplitude as well, this component disappearing at the 10 Hz stimulation rate. The spikes were also gradually reduced in amplitude and number. At 10 Hz, the early component partially remained. These changes are similar to the changes seen in the field potential and intracellular recordings. Just as for the magnetic field, the slow wave of the field potential was first reduced in duration, then
in amplitude with an increase in stimulation frequency. The spikes were reduced in number and amplitude by the increase in frequency. The depolarizing shift in the intracellular potential was also reduced in duration and amplitude with an increase in stimulation frequency. The spikes were reduced in number. A single spike followed by a DAP remained at 10 Hz. The reduction in response amplitude of these signals with stimulus frequency may be in part due to an increase in tonic inhibition mediated by GABAB (Traub et al., 1993), since the resting membrane became hyperpolarized with an increase in stimulus frequency even in presence of PTX which blocks GABAA inhibition (Fig. 7).
4. Discussion 4.1. Experimental analyses of the relationships among the magnetic field, field potential and intracellular potential Our aim was to elucidate the origins of spikes and waves
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in the evoked magnetic fields produced by a hippocampal slice preparation. An experimental analysis of this issue is feasible only if measured magnetic fields can be related to cellular events. Thus, we chose a preparation with a fairly simple cellular arrangement and contrived an experimental arrangement capable of generating highly synchronized activity. As in our previous study (Okada et al., 1997), the population activity was highly synchronized across the entire slice since the population spikes of the magnetic field were highly correlated with the field potentials and intracellular potentials measured in the pyramidal cells. This enabled us to interpret the physiological bases of the evoked magnetic fields at a single-cell level. Even in this type of simplified preparation, however, experimental analyses are not straightforward since the temporal waveform of the magnetic field is determined by a combination of intrinsic neuronal properties such as those of the active membrane conductances and network properties such as distribution and type of excitatory and inhibitory synaptic connections. Therefore, experimental analyses must be eventually combined with mathematical analyses for obtaining deeper and more definite insights into the physiological bases of MEG and EEG signals. We consider that an experimental analysis such as the one presented here is a necessary pre-requisite to such a more advanced analysis. It seems helpful to start with a brief theoretical account of the relationships between the magnetic field, field potential and intracellular potential before discussing the results presented above (cf. Swinney and Wikswo, 1980; Plonsey, 1981; Roth and Wikswo, 1985; Malmivuo and Plonsey, 1995). All three types of signals originate from transmembrane currents which flow in neurons as a result of changes in the conductance of either ligand- or voltage-gated channels. They in turn produce intra- and extracellular currents. Importantly, MEG and EEG signals measured far away from the active neurons are due to these intra- and extracellular currents along the inner and outer walls of the neuronal membrane. The transmembrane currents do not produce the signals because the neuronal membranes are thin and the currents are radial. Thus, the intra- and extracellular currents along the membrane surfaces are labeled as primary currents. The extracellular currents along any conductivity boundary surface other than the active neuronal membranes are labeled as secondary currents. The intracellular currents appear to account for about 75% of the magnetic signal in a CNS structure (turtle cerebellum) (Okada, 1989). Thus, we consider that both MEG and EEG signals are produced by intracellular currents (Okada et al., 1997). Both signals are linearly related to intracellular currents (Swinney and Wikswo, 1980; Roth and Wikswo, 1985; Barth and Sutherling, 1988; Barth and Di, 1991; Okada, 1989). The comparisons are, however, not necessarily straightforward for the data obtained from a slice. The magnetic field should be mostly due to the dipolar component of the
intracellular currents since it is measured far away from the cells (Swinney and Wikswo, 1980). The field potential measured in an extracellular space at some point along the longitudinal axis of the pyramidal cells within the slice is related to more local currents. Transmembrane currents as well as the intra- and extracellular currents should contribute to the field potential measured within an active tissue. The current distribution at any point in time along the axis may be complex, containing not only a dipolar, but also multipolar moments. This means that the magnetic field and field potential may not be always highly correlated if there are strong quadrupolar or higher moments in the distribution. A laminar field potential profile indicated that the magnetic field of a population spike may be quite weak even if there is a strong spike in the field potential because of a weak dipolar component (single current sink-source pair) and a strong quadrupolar component (two current sinksource pairs) along the longitudinal axis of the pyramidal cells (Okada et al., 1997). The latency of a component of a field potential may not always match with the corresponding component of the magnetic field (cf. Fig. 3A). This can occur, for example, when a spike is generated first in the axon hillock and then propagates to the soma as we suppose happened under the somatic stimulation (Fig. 3A). The currents associated with the spike in the axon hillock may have a weak dipolar component and a strong quadrupolar component, whereas the spike in the soma may have a stronger dipolar component. This can lead to a latency difference between the spike of the field potential measured in the basal area and the spike of the magnetic field. Similarly, the latency of a spike in the magnetic field may not match that of a spike of an intracellular potential because the potential is measured in one part of a cell, usually in the soma, and whereas the spike that gives rise to the magnetic field may be generated in another part of the cell. Nevertheless, our results here indicate that it seems possible to experimentally analyze the physiological bases of evoked magnetic fields from our slice preparation as long as one is cognizant of these possible complications. 4.2. Physiological bases of synchronized population spikes in the evoked magnetic field The initial spike in the intracelllular record from a pyramidal cell close to the stimulating electrodes was produced non-synaptically by a direct stimulus activation, since its peak latency was about 1 ms. The spike in the magnetic field had a peak latency of 1 ms, similar to that seen intracellularly. This spike was revealed in an experiment where the artifact was relatively small and the synaptic transmissions mediated by AMPA receptors were blocked by CNQX. The current giving rise to the spike in the magnetic field was oriented from the soma toward the apical dendrites and thus it was consistent with a notion that the spike was due to currents produced by opening of gNa in the soma. The spike was followed by a slow variation of the magnetic
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field which resembled the DAP of the intracellular potential. The mechanism for this DAP-like waveform must be elucidated in the future. The second spike in cells close to a pair of stimulating electrodes and the first spike in cells relatively far away from the stimulating electrodes were close together in their latencies. They were also highly time-locked to the first spike of the field potential and magnetic field clearly visible in a bath without synaptic transmission blockers. This component disappeared in a bath with CNQX (Fig. 5). Thus, it was produced synaptically. Judging from its latency, it was produced monosynaptically. The high degree of correlation with intracellular spikes suggests that it was produced by gNa and associated fast potassium conductances. The direction of the large spike in the magnetic field was opposite for the somatic and apical stimulations. In describing this result, we noted that the field potential was negative in the soma area for both types of stimulation. We concluded that the currents for spike ‘b’ was directed from the soma toward the apical dendrites in the case of the somatic stimulation and from the soma toward the basal dendrites in the case of the apical stimulation. How is it that the currents were directed toward the opposite directions even though the field potential was negative in the soma area? The field potentials along the longitudinal axis of the pyramidal cells (Fig. 3) had different profiles for these two types of stimulation. They were negative in both the basal and soma areas and positive in the apical area for the somatic stimulation, whereas they were positive on the basal and apical areas and negative in the soma area for the apical stimulation. Thus, the current sink was most likely closer to the apical side for the apical stimulation compared to the somatic stimulation. It is also possible that the currents may be directed oppositely even if the current sinks are the same. This can happen depending on the history of cellular activity just before a spike is initiated. For example, the basal dendrites may be more strongly depolarized than the apical dendrites when a spike is initiated in the axon hillock. Then, the current could flow toward the apical dendrites. Conversely, the currents could flow toward the basal dendrites if the apical dendrites were more strongly depolarized than the basal side, depending on the magnitude of the spatial gradient of the transmembrane potential along the longitudinal axis. Regardless of the exact locations of the current sink, the spikes of the magnetic field were opposite in polarity for the two types of stimulation. This was one of the most robust findings. Thus, it must be reconciled with earlier reports demonstrating the back-propagation of action spikes in pyramidal cells of the hippocampus (Traub et al., 1994) and the neocortex (Stuart and Sakmann, 1994). These reports have shown that an orthodromic excitation of the apical dendrites produces an action potential first in the soma followed by action potentials which propagate backward up the dendrites, even though the dendrites are depolarized initially. We hypothesize that the pyramidal cells can generate, depending on the exact stimulation parameters, either the
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back-propagation of this type or an action potential in the proximal apical dendritic trunk that is presumed to be the basis for the large spike in the magnetic field evoked by the apical stimulation. In our study, the depolarization in the apical dendritic trunk due to the electrical stimulation and the activation of gNa in this area must have been sufficiently high to maintain the trunk depolarized until the arrival of the monosynaptic recurrent synaptic inputs in the apical area. This can put the membrane potential in the trunk area near the threshold for firing, leading to an action potential in this area before an action potential in the axon hillock. 4.3. Physiological bases of the early wave and the slow wave of the magnetic field The early wave was eliminated by an application of CNQX. Thus, it was synaptically mediated. The currents underlying the brief early wave were directed from the basal dendrites and soma side toward the apical dendrites when the soma area was stimulated with a depolarizing negative potential extracellularly. It was directed toward the opposite direction when an apical area was depolarized with the extracellular electrodes. Furthermore, the direction of this current coincided with the direction of the spikes (Fig. 3). This suggests that the cellular events responsible for the early wave were related to the mechanism responsible for the spikes. They may reflect a depolarization in the soma in the case of the somatic stimulation and in the apical trunk in the case of the apical stimulation. The later slow wave was also synaptically mediated. It appears to be produced by currents originating in the apical dendrites since the magnetic field polarity indicated the underlying currents to have been directed from the apical to the basal side. The underlying currents were most probably produced by depolarization of the apical membrane due to both excitatory post-synaptic currents (EPSCs) at recurrent excitatory synapses and voltage-sensitive calcium conductances. This hypothesis was successfully used to account for the intracellular potentials observed in hippocampal slices bathed in PTX (Traub et al., 1993). Our study has obtained some independent evidence for this hypothesis. The elevated [Ca2+]o abolished the slow wave. This effect is probably due to reduced recurrent synaptic inputs, since a high level of [Ca2+]o raises firing threshold (Berry and Pentreath, 1976; Wong and Traub, 1983; Miles and Wong, 1987). APV reduced the duration of the late slow wave. This reduction correlated with the shortening of duration of the depolarizing shift in the intracellular record. This indicates that the slow wave was maintained in part by NMDA channels which remain open for 100–1000 ms (Traub et al., 1993). NMDA channels are localized in the recurrent excitatory synapses (Jefferys, 1989; Neumann et al., 1989). Thus, the slow wave appears to have been produced in part by the EPSCs carried by the NMDA channels. Swann et al. (1986) found a large extracellular current observed in both the apical and basal dendrite areas during
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a depolarizing shift in epileptiform bursts (Swann et al., 1986). Our results indicate that the current sink in the apical area was stronger than the sink in the basal area. The effects of stimulation frequency were also consistent with the above hypothesis. Increasing the stimulation frequency initially shortened the duration and finally suppressed the slow wave. This correlated with the shortening of the depolarizing shift in the intracellular potential seen with an increase in stimulation frequency. Increasing the stimulation rate can hyperpolarize the membrane by activating GABAB channels and thus shunt the membrane potential, reducing the depolarization caused by the recurrent synaptic inputs. In conclusion, the detection of the magnetic field associated with the non-synaptic, stimulus-activated spike in the intracellular potential indicates that it is feasible in future to study the way various intrinsic membrane conductances generate the magnetic field in a simplified situation without contributions from the network properties of the slice. The synchronized populations spikes seen clearly in the magnetic signals from slices with excitatory synaptic connections intact were synaptically mediated. The short duration of the spikes and a high degree of correlation with intracellular spikes indicate that they were produced by voltage-sensitive conductances with fast kinetics such as gNa and potassium conductances of the delayed rectifier type and A-type (Traub et al., 1991, 1994). It is necessary to verify whether the early wave reflects a relatively slow depolarization of the soma or apical trunk produced by activation of these fast conductances. The late slow wave of the magnetic field appears to be due to currents generated in the apical dendrites, probably by a combination of excitatory post-synaptic currents carried by NMDA and AMPA channels and calcium-mediated currents. It is necessary to verify this hypothesis and separate their relative contributions. These currents are evidently capable of producing magnetic fields with a slow onset and a long duration in the absence of GABAA inhibition, suggesting that the evoked magnetic fields produced by a single focal area of a CNS structure can be quite complex in temporal waveform. Acknowledgements This research was carried out at Veterans Affairs Medical Center, Albuquerque, NM 87108. The authors thank Professor Roger Traub for his encouragement and the VAMC for providing the laboratory space. We also thank Ms. AnaLorena Barrios for her assistance. This work was generously supported by US DHHS grant RO1-NS21149. References Barth, D.S. and Di, S. The electrophysiological basis of epileptiform magnetic fields in neocortex: spontaneous ictal phenomena. Brain Res., 1991, 557: 95–102.
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