Ionic Mechanisms Underlying Burst Firing of Layer III Sensorimotor Cortical Neurons of the Cat: An In Vitro Slice Study

Yoshihiro Nishimura, Masaru Asahi, Koichi Saitoh, Hirofumi Kitagawa, Yuichi Kumazawa, Kunio Itoh, Min Lin, Takanobu Akamine, Hiroshi Shibuya, Toshihiro Asahara, and Tetsuro Yamamoto

Department of Physiology, Faculty of Medicine, Mie University, Mie 514-8507, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nishimura, Yoshihiro, Masaru Asahi, Koichi Saitoh, Hirofumi Kitagawa, Yuichi Kumazawa, Kunio Itoh, Min Lin, Takanobu Akamine, Hiroshi Shibuya, Toshihiro Asahara, and Tetsuro Yamamoto. Ionic Mechanisms Underlying Burst Firing of Layer III Sensorimotor Cortical Neurons of the Cat: An In Vitro Slice Study. J. Neurophysiol. 86: 771-781, 2001. We examined the ionic mechanisms underlying burst firing in layer III neurons from cat sensorimotor cortex by intracellular recording in a brain slice. Regular spiking was observed in 77.4% of 137 neurons in response to constant intracellular current pulses of 0.5- to 1-s duration. The rest of the neurons showed burst firing. An initial burst followed by regular-spike firing was seen in 71.0% of 31 bursting neurons. The rest of the bursting neurons (n = 9) exhibited repetitive bursting. In the bursting neurons, spikes comprising the burst were triggered from the afterdepolarization (ADP) of the first spike of the burst. We examined the ionic mechanisms underlying the ADP by applying channel-blocking agents. The ADP was enhanced (rather than blocked) by Ca2+ channel blockade. This enhancement of the ADP by Ca2+ channel blockade was apparent even after blockade of the afterhyperpolarization by apamin or intracellular Ca2+ chelation by EGTA. The firing rate of the regular-spiking cells was increased by apamin, intracellular EGTA or Ca2+ channel blockers. In 17.9% of the neurons examined (n = 56), these agents switched the regular-spiking pattern into a bursting one. Burst firing could not be changed to regular spiking by these agents. Four neurons that responded with a single initial burst in control solution responded with repetitive bursting after application of these agents. We conclude that the main function of Ca2+ influx in layer III neurons is to activate Ca2+-dependent K+ conductance, which prevents or limits burst firing. At a time when spike amplitude was unchanged, the ADP was blocked and the burst firing changed to regular spiking by extracellularly applied tetrodotoxin (TTX) or intracellularly applied N-(2,6-dimethylphenylcarbamoylmethyl) triethyl ammonium bromide (QX314). We concluded that a TTX- and QX314-sensitive Na+ current underlies the ADP and therefore contributes to the burst firing of layer III neurons from the cat cortex.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major function of the motor cortex is an execution of skilled movements that is achieved through information processing in the columnar organization of the cortical neurons (Porter 1981). To understand this information processing, the integrative function of the major projecting pyramidal neurons should be considered. The integration of information in the pyramidal neurons is performed through the intrinsic mechanisms underlying the transformation of the graded synaptic inputs into the encoded action potentials, that is, the mechanisms controlling the input-output relation. Injected current pulses mimic the summed synaptic currents arriving at the soma. Therefore the input-output relation in the neuron may be understood by examining the firing properties induced by current injections.

The firing patterns of cortical pyramidal neurons in rodents were classified into regular spiking and intrinsic bursting (Connors et al. 1982). It was also reported that the bursting neurons were found in layers IV and V (Connors et al. 1982), and they play a specific role in information processing (Chagnac-Amitai and Connors 1989; Mason and Larkman 1990). Our previous study revealed the presence of bursting neurons in layer III pyramidal neurons of the cat sensorimotor cortex (Nishimura et al. 1996).

Kandel and Spencer (1961) hypothesized that burst firing results from afterdepolarization (ADP) "summation," which maintains spike recruitment until the accumulated spike inactivation terminates the discharge. Furthermore, a number of other investigators have suggested that the voltage-gated inward currents, which are larger than the outward currents shortly after a spike, generating the ADP will produce burst firing (Azouz et al. 1996; Friedman and Gutnick 1987, 1989; Jensen et al. 1996; McCormick et al. 1985; Silva-Barrat et al. 1992; Wong and Prince 1978, 1981). We also found that the ADP following a spike plays an important role in generation of burst firing in cat layer III sensorimotor cortical neurons (Nishimura et al. 1996). Therefore it is important to study the mechanisms underlying the ADP to understand the mechanisms generating the burst-firing pattern of these neurons.

A body of evidence has implicated Ca2+ currents as playing a pivotal role in the generation of the ADP and/or burst firing (Friedman and Gutnick 1989; Jahnsen and Llinas 1984; Kobayashi et al. 1997; McCormick and Pape 1990; Silva-Barrat et al. 1992; Wong and Prince 1978). In contrast, recent studies showed that the ADP and/or the burst firing were controlled mainly by activation of Na+ currents (Azouz et al. 1996; Deisz and Prince 1987; De Waele et al. 1993; Franceschetti et al. 1995; Guatteo et al. 1996; Hoehn et al. 1993; Jensen et al. 1996; Montoro et al. 1988). Moreover, recently Brumberg et al. revealed the contribution of persistent Na+ currents (INaP) to the generation of ADP and burst firing in the ferret visual cortical neurons (Brumberg et al. 2000). To investigate the ionic basis of the ADP and the burst firing in layer III pyramidal neurons in this study, we examined the role of both Ca2+ and Na+ currents by observing the effect of specific channel-blocking agents on the ADP amplitude and on the evoked firing pattern.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The procedures for making and maintaining slices and identifying the layer III pyramidal neurons were described in detail previously (Nishimura et al. 1996) and mentioned here briefly. A block of the brain including the precruciate cortex was removed after dissecting the skull in the cat, which was previously anesthetized with ketamine hydrochloride (20 mg/kg im). Parasaggital sections (400-500 µm) were made using a vibratome (DTK 1000, DOSAKA EM, Kyoto, Japan) and then stored in a chamber filled with artificial cerebrospinal fluid (ACSF, see following text) bubbled with 95% O2-5% CO2 continuously and maintained at 35°C. The slices were transported to the recording chamber in which the slice was maintained at the interface between ACSF and 95% O2-5% CO2 saturated with water vapor. The composition of the ACSF was (in mM) 130 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 dextrose. The ACSF was bubbled with 95% O2-5% CO2 to maintain pH at 7.4.

Intracellular recording and staining were performed using microelectrodes (1.5 mm OD) filled with 3 M-KCl (DC resistance of ~40 MOmega ) or filled with 2% Biocytin (Sigma) dissolved in 0.5 M K-acetate (~150 MOmega ). In some experiments, microelectrodes containing 2.7 M KCl plus 0.1 M ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA, Fluka Chemie AG, Switzerland) or containing 25 mM N-(2,6-dimethylphenylcarbamoylmethyl) triethyl ammonium bromide (QX314, Alamone Labs, Jerusalem, Israel) in 3 M KCl were used. Electrodes were inserted in the gray matter at ~700 µm below the pial surface, which is estimated to be layer III (Hassler and Muhs-Clement 1964). Just after the cell impalement, steady hyperpolarizing currents were injected to stabilize the cell. The recording of electrical activity, however, was done without steady hyperpolarizing currents. In 60 cases, the recorded neurons were verified to be layer III pyramidal neurons with intracellular staining using methods described previously (Nishimura et al. 1996).

Voltage responses were evoked by current injections (5-ms or 0.5- ~ 1-s pulses) through the microelectrode using an active bridge (MEZ 8201, Nihonkohden, Tokyo or Axoclamp 2B, Axon Instrument, Foster City, CA) or a discontinuous current clamp (Axoclamp 2B, Axon Instrument). Data were recorded on a multichannel videocassette recorder with pulse code modulation (sampling rate; DC ~ 44 kHz in 2 channels, Neurocorder, Neuro Data Instrument, New York, NY). The recorded data were analyzed by a personal computer after off-line digitization (McLab 4, Analog Digital Instrument, Castle Hill, Australia). Spike height was measured from the baseline (the resting membrane potential). Input resistance (Rn) was calculated from a linear portion of current-voltage relations obtained by plotting steady-state subthreshold voltage responses against injected currents.

To block voltage-dependent Ca2+ channels, we employed the following procedures: application of CdCl2 (400 µM) in HEPES-buffered solution or NiCl2 (100 µM); Ca2+-free solutions in which equimolar (2 mM) Co2+ (Co/Ca substitution) or Mn2+ (Mn/Ca substitution) replaced Ca2+ in NaH2PO4-free ASCF; Ca2+-free solution containing EGTA (0.5 mM) and raised Mg2+ (5 mM) (Ca-0). When employing the Ca2+-channels blockade, electrical properties were examined >= 20 min, a time judged sufficient to substantially block voltage-gated Ca2+-channels (see Fig. 2). Intracellular application of EGTA or application of apamin in the ACSF was used to block Ca2+-mediated K+ currents. In initial experiments, EGTA was injected iontophoretically, but in most experiments, EGTA entered the neuron by diffusion from the intracellular recording electrodes. Apamin (SIGMA, 300 nM) and tetrodotoxin (TTX, SIGMA, 1 µM) were applied in the perfusate. In some experiments, TTX (170 µM) and apamin (10 µM) were applied in a droplet in the recording chamber away from the slice in the case of TTX or on the slice surface near the recording electrode in the case of apamin. Tetraethylammonium chloride (TEA, 20 mM) was applied by substitution for equimolar NaCl. When the voltage responses were compared before and after application of the chemicals, the membrane potential was maintained at the control value by DC-current injections (-0.3 to +0.2 nA). Data were analyzed statistically by Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General properties

Intracellular recordings were obtained from 137 layer III pyramidal neurons from the sensorimotor cortex of 53 cats. Layer III was estimated to lie ~700 µm below the pial surface. Sixty of these neurons were verified to be layer III pyramidal neurons by intracellular staining. The mean values of resting membrane potential (RP), spike height, and input resistance (Rn) of the sampled 137 neurons were -73.1 ± 10.6 mV, 87.3 ± 14.9 mV, and 55.0 ± 34.1 MOmega , respectively (means ± SD). Because the mean values of these parameters were not different between stained and unstained neurons, we assume that all sampled neurons were layer III pyramidal neurons.

Firing patterns

We examined the firing patterns of the layer III neurons evoked by 0.5- ~1-s injected current pulses. We classified current-evoked firing pattern into two types: regular spiking (77.4%, n = 106) and bursting (22.6%, n = 31). Regular spiking indicates continuous repetitive firing, and bursting represents the generation of an endogenous burst of three to five action potentials. Moreover, the bursting pattern was grouped into two subtypes, burst-and-regular-spike firing (71.0% in 31 layer III bursting neurons) and repetitive bursting. In the former group, an initial burst at the onset of the current pulse was followed by regular spiking. As reported previously (Nishimura et al. 1996), the mean values of RP, spike height, and Rn were not significantly different between regular-spiking and bursting neurons.

The firing patterns evoked by constant current injection in layer III pyramidal neurons are shown in Fig. 1. The regular-spiking neurons exhibited spike frequency adaptation (Fig. 1A). The firing reached its steady-state firing-adapted rate in 63.6 ± 51.8 ms (n = 106) after the pulse onset. The mean value of the steady-state firing rate evoked by a current pulse of 0.7 nA was 75.0 ± 50.0 Hz (n = 106). Action potentials evoked by 5-ms current pulses in regular-spiking neurons were followed by a fast afterhyperpolarization (fAHP, Fig. 4, A and C), an ADP (Fig. 4C), and a medium AHP (mAHP, Fig. 4, A1 and B1). The regular-spiking pattern did not change to a bursting pattern if the injected current intensity was increased or when RP was changed by DC-current injections (data not shown).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1. Firing patterns of layer III cortical neurons. A: regular spiking induced by 0.5-s depolarizing current of 0.4 nA. B: subthreshold responses induced by 0.28-nA current pulse of 0.5 s (B1). Burst-and-regular-spike firing was induced in an all-or-none manner by 0.5-s depolarizing current of 0.3 nA (B2) and of 0.35 nA (B3). In this neuron, 5-ms current pulse evoked the afterdepolarization (ADP) following an action potential and an extraspike on the ADP in an all-or-none-manner (B4, 0.35 nA; B5, 0.39 nA; B6, 0.41 nA). C: repetitive burst due to 0.5-s current pulse injection (C1, subthreshold response by 0.23 nA; C2, repetitive burst by 0.25 nA). The initial burst in C2 is shown in C3. *, the afterhyperpolarization following the burst. The resting membrane potentials were -84 mV for A, -60 mV for B, and -90 mV for C. Voltage calibration is identical in B, 1-3, and C, 1-3, and voltage scale in B6 applies to B, 4-6. Time scale in B2 applies to A, B, 1-3, and C, 1 and 2. Time scale is identical in B, 4-6. Injected currents are not shown.

The burst-and-regular-spike firing pattern of layer III neurons is represented in Fig. 1B. In these neurons, a burst was followed by a train of regular spikes (Fig. 1B3). In 18 of 22 burst-and-regular-spike firing layer III neurons the burst was evoked in an all-or-none manner (Fig. 1B, 1 and 2), but 4 neurons produced regular spikes to just-suprathreshold current pulses. The average intraburst firing rate in burst-and-regular-spike firing neurons was 157.6 ± 52.7 Hz (n = 22), which was much higher than the firing rate during regular spiking that followed the initial burst.

The other type of bursting, repetitive bursting, is shown in Fig. 1C. Some of the sampled repetitive-bursting neurons were similar to "chattering cells" reported in cat visual cortex (Gray and McCormick 1996). The burst was generated in all-or-none manner in all the repetitive bursting neurons (n = 9, see Fig. 1C, 1 and 2). The frequency of the repetitive bursts evoked by the current at the threshold was 13.9 ± 9.3 Hz (n = 9) and became faster as the current was increased. The intraburst firing rate was similar to that in the burst-and-regular-spike firing neurons.

In both subtypes of bursting neurons, the burst consisted of spikes that incompletely repolarized (Fig. 1C3), and the burst was followed by a prominent hyperpolarization as indicated by an asterisk in Fig. 1, B3 and C3. The average value for the membrane potential of the ADP in the individual spike during the firing (see "ADP amplitude" in Fig. 1C3) was -34.1 ± 11.4 mV (n = 31) in burst neurons. In burst neurons, current pulse (5 ms) evoked a prominent ADP that sometimes reached threshold and evoked an extra spike in an all-or none manner (Fig. 1B, 4-6). Such an ADP was seen in 22 of 31 bursting neurons and in 52 of 106 regular-spike firing neurons. The amplitude of ADP was significantly (P < 0.05) larger in burst neurons (21.9 ± 9.3 mV, n = 22) than in regular-spike-firing neurons (13.8 ± 7.7 mV, n = 52) although RPs were not different in these groups. The activation of an extra spike on the ADP was seen in 31.8% of the burst neurons examined (n = 31), but no spikes were seen on the ADP in regular-spiking neurons.

We hypothesized that the ADP plays an important role in generation of the burst firing. Thus we studied ionic mechanisms underlying the ADP. Because a contribution of voltage-dependent Ca2+ currents to the ADP was implied in past studies (Friedman and Gutnick 1987; Kobayashi et al. 1997; Wong and Prince 1981), we first examined the effects of Ca2+ channel blockade on the ADP.

Effects of Ca2+ channel blockade on ADP

We used a variety of divalent cations to block voltage-gated Ca2+ channels, as well as Ca2+-free solution to reduce Ca2+ influx through these channels, to ensure that the primary effects observed on the ADP were due to reduction of Ca2+ influx rather than a nonspecific effects of one of the divalent cations. To test the effectiveness of Ca2+ blocking solutions (see METHODS), we first verified that the Ca2+-channel -blocking agents used in this study readily block Ca2+ spikes at the concentrations employed and over the recording times employed. Ca2+ spikes were evoked by depolarizing current pulses in TTX (1 µM)- and TEA (20 mM)-containing solution. All the agents used to block Ca2+ channels abolished the Ca2+ spikes reversibly without any significant (P > 0.05) changes of Rns in ~20 min: Co-2 mM/Ca-2 mM substitution, 18.0 ± 6.6 min, n = 3; Mn-2 mM/Ca-2 mM substitution, 18.0 ± 1.7 min, n = 3 (Fig. 2A); Ca-0, 26.3 ± 14.9 min, n = 6 (Fig. 2B); Ni2+ (100 µM), 22.0 ± 3.5 min, n = 3; Cd2+ (400 µM), 20.4 ± 2.2 min, n = 5. Next, we examined the effect of these Ca2+-channel-blocking agents on the ADP that followed Na+ spikes evoked by brief current pulses.



View larger version (8K):
[in this window]
[in a new window]
 
Fig. 2. Ca2+ spikes induced by depolarizing current pulses (A, 10 ms; B, 30 ms) in TTX (1 µM)- and TEA (20 mM)-containing solution (trace 1, control; trace 2, after application of Ca2+ channel blockers; trace 3, after washout of blockers). Blockade of Ca2+ spikes by Mn (2 mM)/Ca (2 mM) substitution (A) and Ca-0 solution (B). The resting membrane potentials were -78 mV for A and -70 mV for B. Scales are identical in A and B.

Figure 3A shows an enhancement of the ADP and activation of an action potential on it (Fig. 3A2) in Ca2+-free solution containing EGTA and raised Mg2+ solution (Ca-0). These changes were reversible (Fig. 3A3) and without a significant change in Rn (64.5 MOmega in Fig. 3A4, 66.1 MOmega in Fig. 3A5, 64.0 MOmega in Fig. 3A6). Enhancement of the ADP was seen in eight of nine neurons, and activation of an extra spike on the ADP was seen in one of nine neurons examined in the Ca-0 solution. The ADP was increased in six of seven neurons examined after Cd2+ was added in the solution. In one of these seven neurons, an action potential was activated on the enhanced ADP (Fig. 3B2). Twenty-five minutes after the washout of Cd2+, the ADP remained still large but the extra spike on the ADP disappeared (Fig. 3B3). These changes were also observed without a significant alteration in Rn (74.6 MOmega in Fig. 3B4, 73.9 MOmega in Fig. 3B5, 74.8 MOmega in Fig. 3B6). Co-2 mM/Ca-2 mM substitution blocked the mAHP in three neurons having a control mAHP and enhanced the ADP or evoked an extra spike on the enhanced ADP in all the neurons having a control ADP (n = 7). Mn-2 mM/Ca-2 mM substitution also blocked mAHP in one neuron. It enhanced the ADP (including extra spike activation in 2 neurons) in all the neurons having control ADP (n = 6). Application of Ni2+ (100 µM) to the perfusate increased the ADP or generated an extra spike on the enhanced ADP in five of eight neurons having a control ADP.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. Effects of blockade of Ca2+ influx on the ADP. A: reversible enhancement of ADP and activation of a spike on it in Ca2+-free solution containing EGTA and raised Mg2+ (Ca-0). Bottom traces in A4 and traces in A, 5 and 6, indicate voltage responses against current pulses shown in top traces in A4. B: Cd2+ (400 µM) increased the ADP and evoked an extra spike on it (B2). An extra spike disappeared by washout of Cd2+ (B3). Hyperpolarizing responses on the current pulses (top traces in B4) are shown in B, 4-6. Calibrations in A, 3 and 6, and B, 3 and 6, apply to A, 1-3 and 4-6, and B, 1-3 and 4-6, respectively. Current calibration in B4 applies to A4 and B4. The resting membrane potentials are -83 mV for A and -64 mV for B.

The agents used in this study to block Ca2+ influx increased the ADP in amplitude significantly (P < 0.05) as shown in Table 1. The Rns did not change significantly (P > 0.05) by using these pharmacological agents (see Table 1).


                              
View this table:
[in this window]
[in a new window]
 
Table 1. Effects of blocking Ca2+ influx on ADP and Rn

It is reported that the mAHP in layer V pyramidal neurons in sensorimotor cortex is generated by activation of Ca2+-mediated K+ current, IK(Ca) (Lorenzon and Foehring 1992; Schwindt et al. 1988, 1992). We hypothesized that the enhanced ADP amplitude observed after Ca2+ channel blockade was secondary to the blockade of IK(Ca). We tested this possibility by examining the ADP after blockade of IK(Ca) by intracellular Ca2+ chelation or by extracellular apamin.

Effects of intracellular EGTA and extracellular apamin on the ADP

Figure 4 shows the afterpotentials recorded by an electrode filled with EGTA-containing solution. After diffusion of EGTA into the neuron (40 min after impalement) the mAHP (Fig. 4A1, arrow) was blocked and the ADP increased (Fig. 4A2). This enhancement of the ADP, including the activation of an extra spike or the alteration of an mAHP to an ADP, was seen in all the neurons examined (n = 17), but the fAHP (Fig. 4A2, arrows) remained present. In 15 of the neurons having a control ADP, the ADP increased significantly (P < 0.05) without any significant (P > 0.05) changes in Rn (see Table 2). We presume that the mAHP was eliminated by preventing the rise of intracellular Ca2+ that normally activates IK(Ca).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4. Effects of blockade of Ca2+-mediated K+ current (IK(Ca)) on spike afterpotentials. A: fast afterhyperpolarization (fAHP), ADP, and medium AHP (mAHP) recorded just after penetration with EGTA-filled electrode (A1). After diffusion of EGTA into the neuron the ADP increased in amplitude (A2). Ni2+ (100 µM) evoked an extra spike on the enhanced ADP. B: spike afterpotentials before (B1) and after application of apamin (B2) in a droplet. C: effects of apamin (300 nM) applied in the perfusate on the ADP. Apamin enhanced the ADP (C2) without a change in input resistance (Rn, C4). Bottom traces in C, 3 and 4, are induced by current pulses shown in top traces in C3. D: spike afterpotentials recorded after apamin-application (300 nM, D2) and Ni2+(100 µM) application in apamin-contaning solution (D3). Resting membrane potentials were -70 mV (A), -88 mV (B), -74 mV (C), and -86 mV (D). Scales are identical for A, 1-3, for B, 1 and 2, for C, 1 and 2, for C, 3 and 4, and for D, 1-3.


                              
View this table:
[in this window]
[in a new window]
 
Table 2. Effects of blocking IK(Ca) and Na+ currents on ADP

Application of apamin in the recording chamber in a droplet (10 µM) also blocked the mAHP (Fig. 4B). In Fig. 4C, apamin (300 nM) enhanced the ADP (Fig. 4C, downward arrow) without a significant change in Rn (control, 51.0 MOmega ; after application of apamin, 47.8 MOmega ). The blockade of mAHP by apamin was seen in all of four neurons, which showed a control mAHP. By apamin (300 nM), enhancement of the ADP was seen in 9 neurons without a significant (P > 0.05) change in Rn (see Table 2) and evoked an extra spike on the enhanced ADP in 2 neurons of 11 neurons having a control ADP. As in the case using EGTA-filled electrodes, the fAHP was not blocked by apamin (Fig. 4C, upward arrow). Thus IK(Ca) activation normally limits ADP amplitude.

The pharmacological experiments described in the preceding text indicate that blockade of IK(Ca) enhances the ADP. We then examined whether the enhanced ADP could be blocked by Ca2+ channel blockade. It is possible that a reduction of the ADP by Ca2+ channel blockade in our first set of experiments was masked by the ADP-enhancing effect of blocking IK(Ca). In three neurons, we applied Ni2+ or Cd2+ in the perfusate after first eliminating the mAHP by intracellular chelation of Ca2+. In all three neurons examined, the amplitude of the enhanced ADP was not reduced. The addition of 100 µM Ni2+ after intracellular EGTA became effective in activating an extra spike on the ADP (Fig. 4A3). Effects of Ni2+ or Cd2+ on the ADP enhanced by apamin (300 nM) were also examined in five neurons. In each of these neurons, application of Ni2+ or Cd2+ could not reduce the enhanced ADP (see Fig. 4D in the case of application of Ni2+).

In these experiments, all the agents used to block the influx of Ca2+ enhanced the ADP rather than blocking it. It is apparent that the inward currents other than Ca2+ currents generate the ADP. We hypothesized therefore that Na+ currents contribute to the ADP.

Effects of TTX on the ADP

We examined the effects of Na+ channel blockade on the ADP by applying TTX in a droplet near the slice (4 neurons) and by using 1 µM TTX-containing solution (other 3 neurons). In the former case, we put 3 µl of 170 µM TTX in the recording chamber at a distance from the slice to see the time course of the changes in the ADP as TTX gradually diffused into the slice and progressively blocked Na+ currents.

After TTX application, the amplitude of the ADP decreased, and the action potential evoked on the ADP disappeared (Fig. 5A2) at a time when the spike height and Rn (see Fig. 5A, 5 and 6; control, 104.0 MOmega ; TTX, 96.0 MOmega ) did not change significantly. Corresponding records of the time derivatives (Fig. 5A, 3 and 4) of the spikes shown in Fig. 5A, 1 and 2, also did not differ greatly (dupsilon /dt for initial spike: 390.0 mV/ms in Fig. 5A1; 364.3 mV/ms in Fig. 5A2). Such a reduction of the ADP was seen in all five neurons showing a control ADP without a significant change in Rns (see Table 2). An extra spike on the ADP was seen in two neurons, and it also disappeared after TTX application. During these changes, the rate of rise of the action potentials did not change significantly (control: 365.1 ± 223.2 mV/ms, TTX: 355.5 ± 221.6 mV/ms, n = 7, P > 0.05). To observe the effects of TTX on the ADP clearly, we examined the effects of TTX on the enhanced ADP due to blockade of Ca2+ influx in five neurons. The ADP enhanced by application of Cd2+ (400 µM, Fig. 5B1) was also reduced by TTX reversibly (see Fig. 5B, 2 and 3) without a significant change in Rn (see Fig. 5B, 4 and 5, 60.0 MOmega and 64.0 MOmega ). In all five neurons showing enhancement of the ADP by the blockade of IK(Ca), TTX decreased the ADP significantly (P < 0.05), but Rns (Table 2) and derivatives of action potentials were not changed significantly (control: 442.4 ± 144.6 mV/ms, TTX: 440.4 ± 140.9 mV/ms, P > 0.05).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. Effects of TTX and N-(2,6-dimethylphenylcarbamoylmethyl) triethyl ammonium bromide (QX314) on the ADP. A: a drop application of TTX (170 µM) decreased the ADP and eliminated an activation of an extraspike on the ADP (A2). Time derivatives of the spikes in A, 1 and 2, are shown in A, 3 and 4, respectively. Time derivatives of artifacts due to the onset and the finish of current pulses were eliminated. Rn was not changed before and after application of TTX (A, 5 and 6). B: enhanced ADP induced by Cd2+ (400 µM, B1) was decreased by application of TTX (B2) reversibly (B3). Hyperpolarizing responses against current pulses indicated in top traces in A5 are shown in B, 4-6. C: intracellular application of QX314 (25 mM) blocked the ADP (C2). The resting potentials were -73 mV (A), -76 mV (B), and -70 mV (C). Calibrations are identical in A, 1 and 2, in A, 3 and 4, in A, 5 and 6, in B, 1-3, in B, 4-6, and in C, 1 and 2. Time scale in A2 applies to A, 1-4.

It is reported that intracellular application of QX314 could abolish the persistent Na+ current (INaP) while the rate of rise of spikes reflecting spike Na+ conductance was not completely reduced (Stafstrom et al. 1985). We then examined the effects of intracellular application of QX314 using electrodes filled with 3 M KCl plus 25 mM QX314 on the ADP. Figure 5C1 shows an action potential followed by the ADP recorded just after penetrating a cortical neuron with an electrode containing QX314. In ~2 min after a penetration, the ADP was blocked (Fig. 5C2). Such a complete blockade of ADP due to diffusion of QX314 could be observed in all of four neurons examined.

We next examined the effects of blocking Ca2+-influx, IK(Ca) and Na+ channels on the firing patterns evoked by long-lasting current pulses. If the ADP was responsible for a bursting behavior, we would not expect bursting to be abolished by Ca2+ channel blockade. Rather we would expect bursting to be enhanced because the Ca2+ channel blockade enhanced the ADP. Furthermore as the ADP was blocked by application of TTX, it is expected that TTX would also eliminate bursting.

Effects of blockade of Ca2+ currents and IK(Ca) on regular-spiking neurons

The rate of regular spiking evoked by a depolarizing current pulse was increased by blockade of Ca2+ influx or IK(Ca) (Co-2 mM/Ca-2 mM substitution, 7 neurons; Mn-2 mM/Ca-2 mM substitution, 5 neurons; Ca-0, 4 neurons; 100 µM Ni2+, 4 neurons; 400 µM Cd2+, 5 neurons; intracellular diffusion of EGTA, 12 neurons; apamin, 9 neurons; see Table 3). Figure 6A shows an example of the reversible effects of Ni2+ on regular spiking and B indicates the increment of firing rate by apamin. Concomitantly, the AHP following the repetitive firing was decreased by these pharmacological agents (Table 3). These changes in the repetitive firing were observed without a significant (P > 0.05) change in Rn (see Table 3). These results indicate that the firing rate in a regular-spiking neuron is restricted by activation of IK(Ca).


                              
View this table:
[in this window]
[in a new window]
 
Table 3. Effects of Ca2+ influx and IK(Ca) on firing rate, AHP following firing and Rn



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6. Effects of blockade of Ca2+-influx and IK(Ca) on the regular-spiking patterns. A: Ni2+ (100 µM) increased the firing rate induced by 0.33-nA current pulse (A2) reversibly (A3). B: apamin (300 nM) increased the firing rate evoked by a current pulse of 0.4 nA. The resting membrane potentials were -65 mV in both neurons. Calibrations in B2 apply to A and B.

The blockade of Ca2+-influx or IK(Ca) switched the regular-spiking pattern into a bursting pattern in ~17.9% (n = 10) of neurons examined. Figure 7A shows the alteration of the regular-spiking pattern into the repetitive bursting without a significant change in Rn (control, 52.6 MOmega ; Mn/Ca substitution, 46.5 MOmega ) after replacement of Ca2+ (2 mM) with Mn2+ (2 mM). In Fig. 7B, a regular-spiking pattern reversibly changed to a repetitive bursting without a significant alteration of Rn (Fig. 7B4, 60.0 MOmega ; Fig. 7B5, 53.0 MOmega ; Fig. 7B6, 60.0 MOmega ) by recording in Ca-0 solution. Cd2+ (400 µM) and Ni2+ (100 µM) also could alter regular-spiking pattern to repetitive bursting. The blockade of IK(Ca) due to intracellular Ca2+ chelation by EGTA-filled electrode or application of apamin induced a change of a regular-spiking pattern to a bursting pattern (data not shown). In these experiments using blocking agents for IK(Ca) and Ca2+-influx, alteration of the regular-spiking patterns to the bursting patterns was observed without any significant changes (P > 0.05) in Rns (control, 56.5 ± 38.9 MOmega ; after blocking Ca2+-influx and IK(Ca), 59.0 ± 26.0 MOmega , n = 10).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 7. Effects of blockade of Ca2+ influx on the regular-spiking patterns. A: firing by 0.5-s current pulse of 0.4 nA before (A1) and after (A2) Mn (2 mM)/Ca (2 mM) substitution and after recording in Ca2+-containing solution (A3). Hyperpolarizing responses on current pulses shown in the top trace in A4 were not changed before (A4) and after (A5) Co/Ca substitution and after recording in Ca2+-containing solution (A6). B: firing evoked by 0.5-s current pulse of 0.5 nA in control (B1), in Ca2+-free solution containing EGTA and raised Mg2+ (Ca-0, B2), and after returning to Ca2+-containing solution (B3). Hyperpolarization due to current injections (B4, top) are shown in B, 4-6. The resting potentials were -62 (A) and -75 mV (B). Scales are identical in A, 1-3, in A, 4-6, in B, 1-3, and in B, 4-6. Time scale in B1 applies to A, 4-6, and B, 1-6.

We attribute these effects of Ca2+ channel blockade to blockade of IK(Ca). Because the blockade of IK(Ca) resulted in burst firing in some regular-spiking neurons and increased excitability in the rest, we suppose IK(Ca) normally prevents bursting and reduces excitability in regular-spiking neurons. These results are consistent with our finding that Ca2+ blockade increases ADP, which we hypothesize to underlie bursting. We next examined the effects of blocking Ca2+-influx and/or IK(Ca) on bursting neurons.

Effects of the blockade of Ca2+ currents and IK(Ca) on the burst

If Ca2+ influx was responsible for bursting, we could expect the bursting pattern to change into a regular-spiking pattern after blockade of Ca2+ influx or IK(Ca), but this was not observed in any of 17 bursting neurons tested. Instead the results were consistent with our finding of an enhanced ADP after blockade of Ca2+ influx or IK(Ca). Mn-2 mM/Ca-2 mM substitution, Ca-0, or blockade of Ca2+ channels by Cd2+ induced repetitive bursting in four burst-and-regular-spike firing neurons without any significant changes in Rns (control, 49.5 ± 20.5 MOmega ; blocker, 50.8 ± 20.5 MOmega ; n = 4, P > 0.05). In these cases, after returning to the control solution, the burst-and-regular-spike firing pattern recovered. In Fig. 8A, the burst-and-regular-spike firing pattern (Fig. 8A1) changed to the repetitive burst (Fig. 8A2) reversibly by Mn/Ca without a significant change in Rn (Fig. 8A4, 70.2 MOmega ; Fig. 8A5, 63.2 MOmega ; Fig. 8A6, 63.2 MOmega ). In the rest of 13 burst-and-regular-spike firing neurons tested, the bursting pattern was not changed by blocking agents of Ca2+ influx or IK(Ca) even though a stable recording was maintained over ~1-2 h after using these agents.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 8. Effects of blockade of Ca2+ influx and IK(Ca) on the bursting patterns. A: effects of Mn (2 mM)/Ca (2 mM) substitution on the burst-and-regular-spike firing. The burst-and-regular-spike firing recorded in control solution (A1). The repetitive burst induced by Mn (2 mM)/Ca (2 mM) substitution (A2). The burst-and-regular-spike firing recorded after returning to the Ca2+-containing solution (A3). Hyperpolarizing responses induced by current pulses shown in top traces in A4 (A, 4-6). B: effects of Ca2+-free solution containing EGTA and raised Mg2+ (Ca-0). The bursting patterns recorded in control solution (B1), in Ca-0 solution (B2), and after returning to Ca2+-containing solution (B3). Hyperpolarizing responses against the current pulses shown in top traces in B4 (B, 4-6). The resting potentials were -71 (A) and -80 mV (B). Voltage calibrations are identical in A, 1-3, and B, 1-3, and in A, 4-6, and B, 4-6. Current calibration in A4 applies to B4. Time scales are identical in A, 1-3, and in A, 4-6, and B, 1-6.

In all of four repetitive bursting neurons examined, blockade of Ca2+ influx or IK(Ca) did not change the bursting pattern even though the firing pattern was examined over 1-2 h after an application of the blockers, a time far in excess that needed to block Ca2+ spikes by these agents. Figure 8B shows increased intraburst firing and bursting rates after recording in Ca-0 solution (Fig. 8B2), but the bursting pattern did not convert into a regular-spike-firing pattern even in a long-period recording in this solution (~90 min). Both of these rates decreased after returning to Ca2+-containing solution (Fig. 8B3). These changes occurred without a significant change in Rn (see Fig. 8B, 4-6; control, 44.0 MOmega ; Ca-0, 42.9 MOmega ; washout, 38.1 MOmega ).

The effects of Ca2+ influx or IK(Ca) blockade on bursting were consistent with the effects on the ADP. In contrast to Ca2++-influx blockade, we found that TTX or QX314 reduced the ADP, and we next examined whether TTX or QX314 blocked the bursting pattern.

Effects of TTX on the burst

In two of four bursting neurons tested, TTX application eliminated the burst. Figure 9A shows the change of the repetitive burst to the regular-spike-firing pattern without a significant change in Rn (Fig. 9A3, 104.0 MOmega ; Fig. 9A4, 96.0 MOmega ). In all of four bursting neurons tested after Cd2+ application (2 burst-and-single-spike firing neurons and 2 repetitive bursting neurons) TTX application induced regular-spiking pattern reversibly without a significant change in Rn (Fig. 9B4, 60.0 MOmega ; Fig. 9B5, 64.0 MOmega ; Fig. 9B6, 62.4 MOmega ) as shown in Fig. 9B. The elimination of burst firing by TTX could be observed without significant (P > 0.05) changes in Rns (control, 56.5 ± 30.5 MOmega ; and TTX, 57.2 ± 31.0 MOmega ; n = 6).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 9. Effects of TTX and QX314 on the bursting firing. A1: the repetitive bursting firing in control. A2: the regular-spike firing after TTX application. A, 3 and 4: hyperpolarizing responses on the currents in A3. B1: the repetitive burst induced in Cd2+-containing solution. B2: a regular-spike firing induced by application of TTX in the Cd2+-containing solution. B3: the repetitive burst induced after washout of TTX. B, 4-6: voltage responses on currents in A3. C1: repetitive burst recorded in control solution. C2: regular-spike firing recorded after intracellular application of QX314 (25 mM). C, 3 and 4: action potentials underlined by dotted lines in C1 and C2 are shown in a faster sweep. The resting potentials are -73 mV (A), -76 mV (B) and -70 mV (C). Voltage calibrations are identical in A, 1 and 2 and B, 1-3, in A, 3 and 4, in B, 4-6 and in C, 1-4. Time scales are identical in A, 1 and 2, in A, 3 and 4, in B, 1-3, in B, 4-6, in C, 1 and 2 and in C, 3 and 4.

Intracellular application of QX314 (25 mM) also changed the repetitive burst into a regular-spike firing as shown in Fig. 9C. Figure 9C, 3 and 4, shows records, in a faster sweep, of action potentials indicated in Fig. 9C, 1 and 2 (- - -), respectively. Coincident with a blockade of ADP by TTX or QX314, Na+-channel blockade eliminated the burst firing.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we obtained the following results: the ADP was enhanced rather than blocked by Ca2+ channel blockade in layer III cortical neurons; Ca2+ channel blockade or IK(Ca) blockade induced burst firing in regular-spiking layer III neurons; burst firing in layer III neurons was enhanced rather than blocked by Ca2+ channel blockade; TTX or QX314, which minimally altered the spike, blocked the ADP and burst firing in layer III neurons. Each of the pharmacological result described in the preceding text was obtained in at least one neuron verified to be a layer III pyramidal neuron by intracellular staining. Thus we suppose our results are representative of cat layer III pyramidal neurons. These results are mainly based on pharmacological experiments employing agents to block Ca2+ influx, which change composition and concentrations of extracellular divalent cations. Therefore such procedures induce alterations of resting membrane properties including input resistances probably due to surface potential changes (Hille 1991). We certified that the changes in ADP or firing pattern are not merely due to a change in input resistance by assuring the consistency in input resistances before and after application of chemical agents. We discuss here the ionic mechanisms underlying the ADP and burst firing in cat sensorimotor cortical neurons.

Many authors have pointed out the importance of the ADP following a spike in the generation of the burst (Azouz et al. 1996; Friedman and Gutnick 1987, 1989; Jensen et al. 1996; Kandel and Spencer 1961; McCormick et al. 1985; Silva-Barrat et al. 1992; Wong and Prince 1978, 1981). In our experiments, the bursting neurons are characterized by activation of an extra spike on the ADP. Neurons with an ADP sufficiently large to reach threshold generate extra spikes on the ADP following a preceding spike, and this causes the burst. AHPs also followed the spike, and the outward current generating the AHP opposes the inward current generating the ADP. We proposed that these neurons having an inward ADP current large enough to overcome the outward AHP current generate a large enough ADP to evoke extra spikes and the burst firing.

In many central neurons, including sensorimotor cortical neurons, the mAHP following a spike is mainly generated by the activation of IK(Ca) (Lorenzon and Foehring 1992; Schwindt et al. 1988, 1992). The mechanism underlying the ADP is controversial: 1) Ca2+-dependent mechanisms including the activation of the voltage-dependent Ca2+ channels (Friedman and Gutnick 1987; Kobayashi et al. 1997; Wong and Prince 1978, 1981), Ca2+-activated cation currents (Haj-Dahmane and Andrade 1997), Ca2+-dependent Cl- currents (Higashi et al. 1993), and electrogenic Ca2+ extrusion (Friedman et al. 1992); 2) Na+-dependent mechanisms (Azouz et al. 1996; Brumberg et al. 2000; Guatteo et al. 1996; Hoehn et al. 1993); and 3) the somatic spread of dendritic excitation (Mainen and Sejnowski 1996).

In this study we examined whether the ADP in layer III cortical neurons is related to Ca2+ currents by using external Ca2+-free solutions, Cd2+ and Ni2+. In all cases, however, these agents did not block the ADP but rather enhanced ADP amplitude. These results are not likely due to insufficiency of blocking Ca2+ influx by pharmacological agents used in this study because of the following reasons. These agents sufficiently blocked Ca2+ spikes (Fig. 2) and AHP following a spike. Their effects on the ADP and the bursting pattern were examined after a sufficient time for Ca2+ spikes to be blocked had passed (sometimes 2 h). We found that the enhancement of the ADP in the Ca2+-free solutions was due to the blockade of IK(Ca) because an application of apamin or intracellular EGTA diffusion, agents that eliminate IK(Ca) (Hugues et al. 1982; Krnjevic et al. 1978), also enhanced the ADP (Fig. 4, A2 and C2). The enhancement of the ADP after intracellular Ca2+ chelation by EGTA might be due to the removal of Ca2+-dependent inactivation of Ca2+ conductance (Eckert and Tillotson 1981). This is, however, unlikely the case for our results because enhanced ADP by EGTA diffusion could be neither reduced nor blocked by blocking Ca2+ influx (Fig. 4A3).

Thus it is likely that primary effects of the blockade of Ca2+ influx in our preparation was the blocking IK(Ca), which secondarily enhanced the ADP. That the ADP was not reduced by the Ca2+-channel blockers might be simply because the AHP (i.e., IK(Ca)) was more sensitive to reduction of Ca2+ influx, but our results reject this idea. Even after IK(Ca) was blocked by apamin or by intracellular Ca2+ chelation, Cd2+ or Ni2+ could neither block nor reduce the ADP but rather further increased the amplitude of ADP enough to evoke extra spikes (Fig. 4, A3 and D3).

Neither Ca2+-activated cation currents nor Ca2+-dependent Cl- currents seem to play an important role in generation of the ADP in cat layer III cortical neurons because these Ca2+-dependent currents would have been reduced or blocked by Ca2+ channel blockade or by EGTA injection. Neither do our results with EGTA support the possibility that electrogenic Ca2+ extrusion generates the ADP because it is reported that the ADP due to electrogenic Ca2+ extrusion was sensitive to intracellular EGTA (Friedman et al. 1992).

In contrast to the ineffectiveness of Ca2-channel blockers at layer III cortical pyramidal neurons, application of TTX or injection of QX314 decreased or blocked the ADP (see Fig. 5) at a time when the amplitude of the action potential was not decreased. The fact that the ADP was blocked without a significant change in spike height is particularly important. This means that voltage-gated Ca2+ channels (or any other voltage-gated channel activities during the spike) experienced the same voltage range as in the control solution and the same influx of Ca2+ occurred. The slower rise time of the spike in TTX may have allowed slightly more Ca2+ channels to have been activated than in control.

The blockade of the ADP by TTX was also reported in the rat cortical pyramidal neurons (Guatteo et al. 1996), the hippocampal CA1 pyramidal neurons (Azouz et al. 1996), and the ferret visual cortical neurons (Brumberg et al. 2000). Layer V cortical neurons have two functionally different Na+ currents, the transient Na+ current (INaT) underlying the spike upstroke and a persistent Na+ current (INaP) that is activated below spike threshold (Brown et al. 1994). Either or both currents could generate the ADP: INaP because it is activated at the membrane potential of the ADP and does not inactivate or INaT due to rapid recovery from partial inactivation at the membrane potential of ADP. Because the membrane potential of the ADP during the firing (-34.1 ± 11.4 mV) in burst neurons was more depolarized than needed for complete inactivation of INaT in rat neocortical neurons (Huguenard al. 1988), it is more likely that INaP generates the ADP, and although the membrane potential of the ADP following the last spike in each burst (-34 mV) was more depolarized than the threshold of a spike evoked from resting potential (-55 mV). The ability of the ADP to reach this membrane potential without triggering another spike is likely due to inactivation of INaT. INaT inactivation would be favored by the initiation of the high-rate preceding spikes with their progressively shallower AHPs and the slow rise of the membrane potential envelope during the burst.

A computer simulation study has indicated that dendritic fast Na+ channels can cause the generation of an ADP by the mechanism of somatic spread of dendritic excitation (Mainen and Sejnowski 1996). However, in our study TTX abolished the ADP without a significant change in fast Na+-spikes amplitude. Thus the ADP is unlikely to be caused solely by the somatic spread of dendritic excitation although we cannot exclude these mechanisms completely.

Considering the pharmacological experiments done in this study, the generation of the burst is unlikely due to influx of Ca2+. The alteration of the burst-and-regular-spike firing pattern into repetitive bursting by Ca2+-channel blockade (Fig. 8) is likely due to the blockade of IK(Ca) and the enhancement of the ADP. This idea is supported by the results, which showed a similar alteration of the firing patterns by the blockade of IK(Ca) with intracellular diffusion of EGTA or apamin application. The switch of the regular-spiking pattern into the bursting pattern by Ca2+-channel blockade and IK(Ca) blockade shown in Fig. 7 also supports this idea. Such a change of regular spiking into the bursting by blockade of IK(Ca) or mAHP was reported in several neurons (Azouz et al. 1996; Deisz 1996; Deisz and Prince 1987; De Waele al. 1993; Schwindt et al. 1988). Thus the main role of Ca2+ influx in neocortical neurons seems to be the suppression or limitation of burst firing by activation of IK(Ca).

Coincident with the reduction of the ADP, the bursting pattern induced in normal solution or Cd2+-containing solutions could be blocked by low concentration of TTX or intracellular injection of QX314 (Fig. 9). Based on these results, we propose that INaP plays a major role in generation of the bursting pattern normally. The contribution of INaP to the generation of burst firing was reported in the rat neocortical neurons (Franceschetti et al. 1995; Guatteo et al. 1996), the hippocampal pyramidal neurons (Azouz et al. 1996), the medial vestibular neurons (De Weale et al. 1993), and the visual cortical neurons (Montoro et al. 1988). Recent studies by Brumberg et al. revealed the dependence of ADP and bursts on Na+ currents but not Ca2+ currents (Brumberg et al. 2000). And they also indicated that neither blocking of Ca2+ influx nor the intracellular chelation of free Ca2+ inhibited the generation of bursts, but that pharmacologically blocking Na+ currents with TTX or QX314 inhibited the burst (Brumberg et al. 2000). The present study also supports these results in cat layer III cortical neurons and especially demonstrates that the mechanisms postulated by Brumberg et al. (2000) are also true in the cat sensorimotor layer III neurons. They also reported that incidence of bursts is higher in in vitro preparation bathed in extracellular solution with a physiological concentration of Ca2+ (1 mM) to compare with that in the conventionally used extracellular solution ([Ca]o = 2 mM) (Brumberg et al. 2000). The present study is also consistent with it as the incidence of bursts was increased in Ca-0 solution in our experiments.

As the bursting was blocked by TTX in this study, the TTX-insensitive Na+ current that was reported to generate burst firing in guinea pig neocortical neurons (Deisz 1996) is unlikely to be related to burst generation in cat layer III cortical neurons. Although the present results do not allow us to completely exclude the contribution of the other mechanisms except an activation of INaP, we conclude that the activation of INaP may be one of the major contributors to the generation of the ADP and the burst.

In cat layer III pyramidal neurons, depolarizing inputs activate both INaT and INaP. The depolarization induced by these currents induce the Ca2+ influx through voltage-dependent Ca2+ channels, which in turn activate IK(Ca). In the neurons in which INaP surpasses IK(Ca), the large ADP develops and the burst is induced. During intraburst firing the further influx of Ca2+ induces an enhancement of AHP leading to produce the larger AHP and terminate the burst. On the other hand, mechanisms to cease the burst are complicated in the burst firing induced by IK(Ca) blockade. Several mechanisms are available to contribute to repolarize the burst after blockade of IK(Ca): voltage-gated IK (e.g., M current), apamin insensitive IK(Ca), Ih (McCormick and Pape 1990), electrogenic Na/K pump (Angstadt and Friesen 1991), Na+-dependent K+ current (Schwindt et al. 1989). Further investigation is needed to reveal the mechanisms of burst termination after IK(Ca) blockade.


    ACKNOWLEDGMENTS

We express our gratitude to Profs. P. C. Schwindt (University of Washington) and R. C. Foehring (University of Tennessee) for reading the manuscript and giving useful comments. We thank J. Kobayashi and K. Kawamura for excellent assistance.

Part of this work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (0668080, 11680806) and by a grant from Epilepsy Research Foundation.

Present addresses: M. Asahi, Dept. of Neurology, Faculty of Medicine, Mie University, Tsu, Mie 514-8507, Japan; K. Saitoh, Dept. of Neurosurgery, Faculty of Medicine, Mie University, Tsu, Mie 514-8507, Japan; K. Itoh and T. Akamine, Dept. of Ophthalmology, Faculty of Medicine, Mie University, Tsu, Mie 514-8507, Japan.


    FOOTNOTES

Address for reprint requests: Y. Nishimura, Dept. of Physiology, Faculty of Medicine, Mie University, Tsu, Mie 514-8507, Japan (E-mail: b612west{at}doc.medic.mie-u.ac.jp).

Received 25 September 2000; accepted in final form 4 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society