Department of Physiology, Faculty of Medicine, Mie University, Mie 514-8507, Japan
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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
M) or filled with 2% Biocytin (Sigma) dissolved in 0.5 M K-acetate
(~150 M
). 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.
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RESULTS |
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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 M
, 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).
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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.
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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 M in Fig.
3A4, 66.1 M
in Fig. 3A5, 64.0 M
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 M
in Fig. 3B4, 73.9 M
in Fig. 3B5, 74.8 M
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.
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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).
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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).
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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 M; after application of apamin, 47.8 M
). 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 M; TTX, 96.0 M
) 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
(d
/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 M
and 64.0 M
).
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).
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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).
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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 M; Mn/Ca
substitution, 46.5 M
) 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 M
;
Fig. 7B5, 53.0 M
; Fig. 7B6, 60.0 M
) 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 M
;
after blocking Ca2+-influx and
IK(Ca), 59.0 ± 26.0 M
,
n = 10).
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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 M; blocker, 50.8 ± 20.5 M
; 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 M
; Fig. 8A5, 63.2 M
; Fig.
8A6, 63.2 M
). 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.
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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 M; Ca-0, 42.9 M
; washout, 38.1 M
).
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 M; Fig. 9A4, 96.0 M
). 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 M
;
Fig. 9B5, 64.0 M
; Fig. 9B6, 62.4 M
) 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 M
; and TTX, 57.2 ± 31.0 M
; n = 6).
|
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.
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DISCUSSION |
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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.
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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