Department of Anatomy and Neurobiology, University of Tennessee, Memphis, Tennessee 38163
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ABSTRACT |
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Stewart, Ansalan E. and Robert C. Foehring. Effects of Spike Parameters and Neuromodulators on Action Potential Waveform-Induced Calcium Entry Into Pyramidal Neurons. J. Neurophysiol. 85: 1412-1423, 2001. Neocortical pyramidal neurons express several different calcium channel types. Previous studies with square voltage steps have found modest biophysical differences between these calcium channel types as well as differences in their modulation by transmitters. We used acutely dissociated neocortical pyramidal neurons to test whether this diversity extends to different activation by physiological stimuli. We conclude that 1) peak amplitude, latency to peak, and the total charge entry for the Ca2+ channel current is dependent on the shape of the mock action potential waveforms (APWs). 2) The percent contribution of the five high-voltage-activated currents to the whole cell current was not altered by using an APW as opposed to a voltage step to elicit the current. 3) The identity of the charge carrier affects the amplitude and decay of the whole cell current. With Ca2+, there was a greater contribution of T-type current to the whole cell current. 4) Total Ba2+ charge entry is linearly dependent on the number of spikes in the stimulating waveform and relatively insensitive to spike frequency. 5) Current decay was greatest with Ca2+ as the charge carrier and with minimal internal chelation. 6) Voltage-dependent neurotransmitter-mediated modulations can be reversed by multiple spikes. The extent of the reversal is dependent on the number of spikes in the stimulating waveform. Thus the neuronal activity pattern can determine the effectiveness of voltage-dependent and -independent modulatory pathways in neocortical pyramidal neurons.
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INTRODUCTION |
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Electrophysiological studies of
calcium channel currents using prolonged square voltage steps to evoke
the current have revealed that the soma/proximal dendrites of
neocortical pyramidal neurons express robust currents of at least six
types. These include T-type (Sayer et al. 1990;
Tarasenko et al. 1998
), and five high-voltage-activated (HVA) currents: L, N, P, Q, and R type (Brown et al.
1994
; Foehring et al. 2000
; Lorenzon and
Foehring 1995a
; Mermelstein et al. 1999
; Sayer et al. 1990
; Ye and Akaike 1993
).
Ca2+ influx through voltage-gated
Ca2+ channels serves an important role in
neuronal integration by regulating Ca2+-dependent
second messengers, gene expression, neurotransmitter release, and
repetitive firing behavior (Bertolino and Llinas 1992;
Bito et al. 1997
; Kasai and Peterson
1994
; Mermelstein et al. 2000
; Pineda et
al. 1998
). For example, Ca2+-dependent
transcription factors are preferentially supported by L-type currents
in hippocampal pyramidal neurons (Bito et al. 1997
;
Mermelstein et al. 2000
). Neocortical pyramidal cell
Ca2+ currents are differentially involved in
repetitive firing, activation of Ca2+-dependent
K+ currents underlying afterhyperpolarizations
(AHPs), and spike frequency adaptation (Pineda et al.
1998
). N-, P-, and Q-type channels activate AHPs as well as
provide inward current, but L-type currents act only as an inward
charge carrier (Pineda et al. 1998
). HVA currents in
neocortical pyramidal neurons are also differentially modulated by
transmitters (Choi and Lovinger 1996
; Foehring
1996
; Sayer 1998
; Sayer et al.
1992
; Stewart et al. 1999
). It is unknown,
however, whether Ca2+ channel types are activated
differently in response to physiological stimuli.
Under physiological conditions, voltage-gated
Ca2+ channels are activated by excitatory
postsynaptic potentials and action potentials (APs). Since AP
parameters show variation under physiological conditions in pyramidal
neurons (Connors et al. 1982; Lorenzon and
Foehring 1993
; McCormick and Prince 1987
;
Stafstrom et al. 1984
; Wheeler et al.
1996
), we used AP waveforms (APWs) as the voltage stimuli to
elicit Ca2+ channel currents in acutely
dissociated neocortical pyramidal neurons (cf. Brody et al.
1997
; Jackson et al. 1991
; Llinas et al.
1982
; McCobb and Beam 1991
; Park and
Dunlap 1998
; Patil et al. 1998
;
Pennington et al. 1992
; Schiller et al.
1995
; Scroggs and Fox 1992
; Toth and
Miller 1995
; Williams et al. 1997
). We tested
hypotheses concerning the effects of pyramidal cell AP parameters on
Ca2+ entry, the role of
Ca2+ current inactivation and facilitation in
regulating Ca2+ entry during trains of spikes,
and the influence of APs on modulation by transmitters.
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METHODS |
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Acute isolation of pyramidal cells
Two- to 6-week-old Sprague-Dawley rats were anesthetized with
methoxyfluorane. Under anesthesia, the rats were decapitated and the
brains extracted. The brains were then sectioned into 400-µM slices
using a vibrating tissue slicer (Cambden Instruments) in an oxygenated
high-sucrose solution that contained (in mM) 250 sucrose, 2.5 KCl, 1 NaH2PO4, 11 glucose, 4 MgSO4, 0.1 CaCl2, and 15 HEPES (pH = 7.3 adjusted with 1 N NaOH; 300 mOsm/l). The slices
were held for a minimum of 1 h in a carboxygen (95%
O2-5% CO2) bubbled
artificial spinal fluid (ACSF). The sensorimotor cortex (combined
primary motor and primary somatosensory cortices) was placed in
ice-cold ACSF and dissected from the slices with the aid of a
stereomicroscope. The ACSF contained (in mM) 125 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 20 glucose, 1 kynurenic acid, 1 pyruvic
acid, 0.1 nitro-arginine, and 0.05 glutathione (pH = 7.4 adjusted
with 1 N NaOH; 310 mOsm/l). The dissected cortex was then incubated at
32°C for 20-30 min in an oxygenated ACSF containing Pronase E (Sigma
protease type XIV, 1.0 mg/ml) (modified from Lorenzon and
Foehring 1995a). Following the incubation period, the tissue
was first rinsed in a sodium isethionate solution that contained (in
mM) 140 Na isethionate, 2 KCl, 1 MgCl2, 23 glucose, 15 HEPES, 1 kynurenic acid, 1 pyruvic acid, 0.1 nitro-arginine, and 0.05 glutathione (pH = 7.3, adjusted with 1 N
NaOH; 310 mOsm/l), then triturated in the same solution using
fire-polished Pasteur pipettes. The supernatant was collected and
poured into a plastic petri dish (Lux) positioned on the stage of an
inverted microscope (Nikon Diaphot 300). The cells were allowed several
minutes to adhere to the petri dish, and then the background flow of
HEPES-buffered saline solution (HBSS) was initiated (~1 ml/min). HBSS
contained (in mM) 10 HEPES, 138 NaCl, 3 KCl, 1 MgCl2, and 2 CaCl2 (pH = 7.3, adjusted with 1 N NaOH, 300 mOsm/l).
Recording solutions and pharmacological agents
The external recording solution used to isolate the
Ca2+ channel currents (TEA-free solution)
consisted of (in mM) 125 NaCl, 20 CsCl, 1 MgCl2,
10 HEPES, 5 BaCl2, 0.001 TTX, and 10 glucose (pH = 7.3 adjusted with TEA-OH; 300-305 mOsm/l). The standard internal recording solution included the following (in mM): 180 N-methyl-D-glucamine (NMG), 4 MgCl2, 40 HEPES, 10 ethylene glycol-bis (-aminoethyl ester)-N,N,N',N'-tetraacetic acid (EGTA),
0.1 leupeptin, 0.4 GTP (GTP), 2 ATP, and 0.007-0.015 phosphocreatine
(pH = 7.2, adjusted with 0.1 N
H2SO4; 265-275 mOsm/l).
Ten to 20 mM
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA) replaced EGTA in several experiments. For experiments where
minimal chelation was desired, EGTA was replaced with 0.1 mM BAPTA or
no chelator was used.
The stock solutions of muscarine chloride,
±8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide
(8-OH-DPAT), and the calcium channel antagonists, with the exception of
nifedipine, were dissolved in water. Stock solutions of -conotoxin
GVIA (CgTx GVIA; 500 µM),
-conotoxin MVIIC (CgTx MVIIC; 500 µM)
and
-agatoxin-IVA (AgTX; 100 µM) were aliquoted and frozen. Each
of the stocks were diluted to the appropriate concentration in the
external recording solution immediately prior to the experiment.
Nifedipine was dissolved in 95% ethanol before being added to the
external solution. The final ethanol concentration was <0.05%. This
concentration has previously been shown to have no effect on
Ca2+ currents in these cells (Lorenzon and
Foehring 1995a
). Nifedipine was protected from ambient light.
Cytochrome C, at a final dilution of 0.01%, was combined with
solutions containing AgTX to prevent nonspecific binding of AgTX to
glass and plastic (Bargas et al. 1994
; Lorenzon
and Foehring 1995a
).
Whole cell recording
Whole cell recordings were acquired at room temperature using a
DAGAN 8900 or an Axopatch 200A electrometer. The recordings were
monitored and controlled by pCLAMP6 (Axon Instruments) installed on a
486 computer. The electrodes were pulled from 7052 glass (Garner) and
fire polished. Leak currents and capacitative artifacts were subtracted
on-line with a p/4 procedure. In addition, currents obtained in the
presence of 400 µM Cd2+ were subtracted from
the data. Series resistance compensations of 70-80% were employed.
The average whole cell capacitance for recorded cells was 14.6 ± 0.7 (SE) pF (median = 13.6 pF, n = 64). Uncompensated series resistance averaged 1.7 ± 0.1 M. For a
typical peak current of 2-6 pA, this would lead to an estimated series resistance error of 3-10 mV (calculated using Ohms Law,
V = I*R: uncompensated series
resistance multiplied by peak current). Uncompensated series resistance
was estimated from dial readings for series resistance and percent
compensation on the Axopatch 200A. Voltage control was also assessed by
observing tail currents after brief voltage steps (Lorenzon and
Foehring 1995a
). Cells with broad or variable tail current
decay were discarded. A gravity-fed parallel array of glass tubes was
used to apply the drugs. Voltage data were uncorrected for liquid
junctional potential (8 mV).
SYSTAT (SYSTAT, Evanston, IL) software was used to carry out all
statistical calculations. Unless otherwise stated, the sample data are
represented as median and mean ± SE. Data are also presented graphically as scatter plots or box plots. In the box plots, the internal line represents the median while the outer edges of the box
represent the inner quartiles of the data set. The bars extending from
the box depict the two outer quartiles. Data points more than two times
the difference between the box edges were considered outliers and are
indicated by a small asterisk in the plots (Tukey 1977).
Statistical differences were determined with the Mann-Whitney U test (
= 0.05) unless otherwise stated.
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RESULTS |
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Voltage steps (30 ms) elicit robust Ba2+
currents through Ca2+ channels in neocortical
pyramidal cells (Lorenzon and Foehring 1995a
,b
;
Sayer et al. 1990
), and the current evoked with 5 mM Ba2+ has similar I-V relationships to
currents using the more physiological 2 mM Ca2+
(Lorenzon and Foehring 1995a
). In this study, we
examined Ca2+ and Ba2+
currents in response to action potential waveforms.
We first elicited Ba2+ currents in voltage clamp,
using as the voltage command an AP recorded and digitized from a
pyramidal cell at room temperature in the slice preparation
(intracellular current clamp) (for methods see Lorenzon and
Foehring 1993) (Fig. 1A; n = 5). We
also evoked currents with mock APs (APWs) consisting of a series of two
or three voltage ramps, which had amplitude, base width, and
polarization rates similar to the digitized AP (Fig. 1,
B-D). In some cases, the APW included an AHP phase (Fig. 1,
B and D) (cf. Wheeler et al. 1996
)
that increased current amplitude in a given cell (n = 6) but did not qualitatively alter the form of currents compared with
those elicited by APWs that returned to
70 mV (Fig. 1C).
The mock APs had the advantage of allowing precise control of stimulus
parameters.
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In response to either digitized or mock APs, the
Ba2+ current was first evident during the rising
phase of the AP or APW, peaked during the repolarization phase, and
decayed rapidly during the AHP phase (Fig. 1, n = 91).
These results are in agreement with similar studies in various other
neuron types (e.g., Llinas et al. 1982; McCobb
and Beam 1991
; Park and Dunlap 1998
;
Scroggs and Fox 1992
; Spencer et al.
1989
; Wheeler et al. 1996
).
Effect of APW parameters on Ba2+ entry
The shape of the AP is altered under various physiological conditions (i.e., ontogeny, repetitive spiking, modulation, cell injury). Therefore, to test how changing the shape of the AP affects the amplitude and timing of Ba2+ entry, we systematically varied the parameters (e.g., holding potentials, widths, and depolarization/repolarization rates) of the mock AP. Experimental estimates of Ca2+ channel availability under a range of physiological conditions are necessary for modeling postsynaptic integration by pyramidal cells.
HOLDING POTENTIAL.
We first compared the current evoked from a holding potential (HP) of
70 mV to that from an HP of
50 mV. These potentials were chosen to
reflect the "up" and "down" states observed in in vivo cortical
recordings from anesthetized (Cowan and Wilson 1994
;
Stern et al. 1997
) or sleeping (Steriade et al.
1993
) rats. The cells were held for >1 min at a given holding
potential before testing for currents. Voltage protocols are shown in
Fig. 2. Peak currents were smaller
(significant: P < 0.04) in all cells tested when
elicited from a HP of
50 versus
70 mV (Fig. 2, A and
B, n = 5; median peak at
70 mV: 1.6 nA;
median peak at
50 mV: 1.5 nA). Total charge entry (measured as the
time integral of the current) was also reduced at a HP of
50 versus
70 mV (median area at
70 mV: 2.5 pC; median area at
50 mV: 2.3 pC).
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AP SHAPE. We next examined the effects of spike width and rates of change in voltage (depolarization and repolarization). The rates were chosen to reflect an approximate physiological range of values (adjusted for temperature). Over the range examined, as the rate-of-rise was increased (repolarization rate kept the same: Fig. 3A1), there was a linear decrease in the peak current (Fig. 3B1) and total charge entry decreased exponentially (Fig. 3B2; n = 16). (In Fig. 3, "normalized" = all data points divided by the largest value.) Accelerating the repolarization rate (rise rate kept the same) led to a linear increase in the peak amplitude (Fig. 3B1) and an exponential decrease in the total charge entry for the range of values examined (Fig. 3B2; n = 19). The faster the repolarization rate, the more rapid the change in driving force, thus the greater the peak amplitude of the current. The largest currents were obtained with an instantaneous jump from peak depolarization to the final voltage (cf., tail currents after voltage steps). The peak also came earlier in time with increasing repolarization rate (Fig. 3A2). Total charge entry decreased as the repolarization rate was increased because the spikes became narrower.
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Ca2+ current types
We have previously used voltage steps to study the kinetics and
voltage dependence of activation and inactivation for specific Ca2+ current types (Lorenzon and Foehring
1995a,b
; Mermelstein et al. 1999
). In
neocortical pyramidal neurons, we found only modest differences among
N-, P-, and L-type Ba2+ currents in terms of
current time-to-peak, voltage dependence of activation, inactivation
kinetics, percent inactivation, and deactivation kinetics
(Foehring et al. 2000
; Lorenzon and
Foehring 1995a
; Mermelstein et al. 1999
).
The largest differences in neocortical pyramidal cells were that Q-type
current inactivated faster than N-, P-, and L-type (Mermelstein
et al. 1999
) and R-type current activated at more negative
potentials and inactivated faster and more completely than L-, P-, N-,
and Q-type Ca2+ currents (Foehring et al.
2000
).
Since the biophysical differences are small and primarily related to
inactivation kinetics (which are slow compared with an APW), we
hypothesized that the percentage that each current type contributes to
the peak whole cell current would not differ between currents elicited
by APWs or 30-ms steps. We used selective pharmacological antagonists
to determine the percent that each current type contributed to the
whole cell current evoked by either an APW or a voltage step (Fig.
4). The Ca2+
channel blockers were added sequentially in the order: 5 µM
nifedipine, 25 nM AgTX, 1 µM CgTx GVIA, 1 µM CgTx MVIIC, and
Cd2+ to block L-type (nifedipine sensitive),
P-type (AgTx sensitive), N-type (CgTx GVIA sensitive), Q-type (CgTx
MVIIC sensitive, after prior block of N and P type), and R-type
(resistant to organic blockers, Cd2+ sensitive)
HVA currents, respectively. A 30-ms voltage step from 90 to
10 mV
was used to evoke the current during the application of each toxin.
Peak currents were measured and plotted versus time. Once the block by
the antagonist reached steady state (- - - in Fig. 4B),
the stimulating waveform was switched to an APW (from
70 to +30 mV,
3.6-ms base width). This procedure allowed for the comparison of
current types in the same cell evoked by both steps and APWs. We found
that although individual cells showed considerable variability in the
percentage of each current subtype, on average a similar percentage of
each Ba2+ current type constituted the whole cell
current when either stimuli was used (Fig. 4; n = 7).
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The currents carried by each of the specific channel types were obtained by subtracting the antagonist-sensitive current from the whole cell current. For the APW, the five HVA currents were then compared with respect to their base width, half-width, latency-to-peak, half decay time, and amplitude at 10 and 90% of rise. For the step-induced current, we measured the activation time constant and the decay time constant. On average, we found no significant differences between calcium channel types for these measures (Kruskal-Wallis test; data not shown). The biophysical differences between the current types are apparently too small to alter the percent contribution to the peak current or affect the contribution to different temporal portions of a brief stimulus such as an AP.
Ba2+ versus Ca2+
Ba2+ (5 mM) was used as the charge carrier
in the previous experiments to avoid complications of
Ca2+-dependent inactivation or activation of
Ca2+-dependent currents. After substituting 2 mM
Ca2+ for 5 mM Ba2+, the
peak current evoked by an APW was reduced, and a second slowly decaying
component was evident in the current decay (21% of total decay; Fig.
5A; n = 7). We
compared 2 mM Ca2+ to 5 mM
Ba2+ because these concentrations display a
similar voltage dependence (Lorenzon and Foehring 1995a)
and because physiological
[Ca2+]o is thought to be
1-2 mM. The peak amplitude of the current was larger when
Ba2+ was used as the charge carrier because
Ba2+ is more permeant than
Ca2+ through HVA Ca2+
channels (Tsien et al. 1988
) and a smaller concentration
of Ca2+ than Ba2+ was used.
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Since T-type Ca2+ currents have slower
deactivation kinetics than HVA currents (Matteson and Armstrong
1986) and T-type currents are more prominent in
Ca2+ than Ba2+ in pyramidal
cells (Lorenzon and Foehring 1995a
,b
; Sayer et
al. 1990
), we hypothesized that the slow component to the
Ca2+ current decay represented T-type current. If
this hypothesis is correct, holding the cell at
50 mV should
eliminate this component since T-type channels in pyramidal cells are
inactivated at this voltage (Huguenard 1996
;
Sayer et al. 1990
; Tarasenko et al. 1998
; Ye and Akaike 1993
). We tested five cells that displayed
both fast and slow components of the Ca2+ current
decay following an APW from a holding potential of
70 mV. When the
holding potential was maintained at
50 mV for >1 min, the slow
component was eliminated in three of five cells and reduced (53%,
86%) in the remaining cells (Fig. 5B). Nickel ions
(Ni2+: 50 µM), a relatively specific antagonist
for T-type currents of the
1H type (Lee et al. 1999
;
Sayer et al. 1990
; Tsien et al. 1988
),
blocked the slow component in four of five cells tested, yet only
caused a minor change in the peak current (Fig. 5C). These
results are consistent with the slow tail current being due to T-type channels.
Facilitation and inactivation
The preceding results suggest that single APWs are too brief for differences in biophysical properties of different types of HVA currents to be evident in our cells. We next examined whether multiple spikes lead to either facilitation or inactivation of Ca2+ currents. These processes could potentially alter the additive effects of multiple spikes on Ca2+ entry during prolonged firing at physiological rates.
FACILITATION.
Facilitation refers to an increase in the current recorded during a
test pulse that was preceded by a depolarizing prepulse (Dolphin
1996; Elmslie and Jones 1994
; Ikeda
1991
; Kasai 1992
). We tested whether multiple
APWs in sequence would lead to facilitation, defined here as the
percent increase in the peak amplitude of the current evoked by the
final spike over that evoked by the first spike in the train. We
observed no increase in peak Ba2+ or
Ca2+ current (no facilitation) during trains of
2, 4, 8, or 16 spikes at 50, 100, or 200 Hz (n = 12, Fig. 6). This result is consistent with
data obtained with voltage steps where in the absence of a
G-protein-mediated modulation, Ba2+ current can
be facilitated by only ~12% in pyramidal cells of the sensorimotor
cortex (Foehring 1996
; Stewart et al.
1999
).
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INACTIVATION.
Percent current decay was determined by comparing the peak amplitude of
the current entering in response to the first spike to that entering
with the final spike in trains of APWs at various frequencies. Both
voltage-dependent and Ca2+-dependent inactivation
of calcium channel currents occur in various types of neurons (e.g.,
Forsythe et al. 1998; Gutnick et al.
1989
; Jones and Marks 1989
; Nagerl and
Mody 1998
). We first used Ba2+ as the
charge carrier and chelated Ca2+ with internal
EGTA (10 mM) to emphasize voltage-dependent inactivation. We compared
these data to that from cells where Ca2+ was the
charge carrier and minimal chelation (0.1 mM BAPTA) was employed to
allow both Ca2+- and voltage-dependent processes.
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Modulation
N- and P-type Ca2+ currents in various cell
types are modulated via voltage-dependent, membrane-delimited pathways
(Hille 1992; Jones and Elmslie 1997
). In
neocortical pyramidal cells, N- and P-type Ca2+
channels are modulated by both serotonin (5HT1A)
and muscarinic (M2) receptor activation via
membrane-delimited pathways (Foehring 1996
;
Stewart et al. 1999
). Such modulations have been
proposed to play important roles in regulating neurotransmitter release at synaptic terminals (Wu and Saggau 1997
).
In many voltage-dependent, membrane-delimited modulations, activation
kinetics are slowed. When this slowing is present and a voltage step is
used to evoke the current, the modulation is greatest early in the
trace and reduced later in the step (Bean 1989). Thus
when a brief stimulus like an APW is used, the modulation should be
larger compared with that determined at the end of a long voltage step.
We previously found that activation kinetics were not slowed in the
presence of muscarine (100 µM) (Stewart et al.
1999
), and slowing was only noted at high concentrations of the
5HT1A agonist 8-OH-DPAT (e.g., 100 µM)
(Foehring 1996
). Percent modulation was determined by
calculating the absolute amplitude of current blocked by agonists, then
dividing by the amplitude of the control current. Our criteria for
kinetic slowing were the presence of two
s to current activation or
a slower single
(Foehring 1996
; Stewart et
al. 1999
).
We first compared the magnitude of the modulation by 2 µM muscarine (n = 6) or 2 µM 8-OH-DPAT (n = 8) when current was evoked by an APW or by a voltage step (measured at peak current). At this dose, slowing of activation kinetics was not evident (Fig. 9, A and C), and the Ca2+ channel current elicited by an APW was modulated to a similar extent as the current evoked by a voltage step (Fig. 9, B and D: no significant difference). Interestingly, one cell exhibited slowed activation kinetics in 2 µM 8-OH-DPAT, and this cell was modulated more in the APW than at peak current in response to the step (Fig. 9B). Modulation of APW Ba2+ currents by 100 µM 8-OH-DPAT caused slowing of activation kinetics, and APW-induced currents were modulated to a greater extent than peak currents elicited by a 30-ms square wave (n = 6; Fig. 9, E and F; P < 0.05). The differences in percent modulation were lost if step-induced currents were compared at 1.8 ms into the step (corresponding to peak current in response to an APW; data not shown).
|
Another characteristic of voltage-dependent, membrane-delimited
modulations is that they can be reversed by long (e.g., 30 ms)
depolarizing prepulses (e.g., to +100 mV) (Bean 1989;
Elrlich and Elmslie 1995
; Kasai 1992
). In
a previous study in cortex, the 5HT1A-mediated
modulation was reversed by such prepulses by ~50% (Foehring
1996
) and the M2-like modulation was
100% voltage dependent (Stewart et al. 1999
).
AP-like steps were reported to reverse G-protein-mediated modulations
in basal forebrain neurons (Williams et al. 1997) and chick DRG cells (Park and Dunlap 1998
) but not dorsal
raphe neurons (Pennington et al. 1991
). We asked whether
a train of APs at physiological firing rates can effectively reverse
membrane-delimited, voltage-dependent modulations in neocortical
pyramidal neurons (Figs. 10 and
11).
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|
We tested the voltage-dependent 5HT1A and
M2-receptor-mediated modulations (target N- and
P-type currents) and the voltage-independent M1
pathway (targets L-type current) (Stewart et al. 1999).
The 5HT1A effect was elicited with the specific
agonist 8-OH-DPAT. The rapid M2-mediated
modulation could be isolated using 10 mM EGTA in the internal recording
solution to block the M1 pathway (the modulation
is Ca2+ dependent) (Stewart et al.
1999
). The M1 modulation was examined by
using minimal chelation (0.1 BAPTA) and 50 µM N-ethyl
maleimide (blocks the M2 pathway) (Stewart et al.
1999
).
We used two different protocols. In the first protocol, we used a 15 ms
test step to 30 mV (HP =
90 mV). Multiple brief voltage steps
with parameters similar to an AP (holding potential of
70 mV, peak of
+30 mV, 2-ms duration) were employed as the prepulse. We measured the
peak current response to the test pulse following 0, 10, 20, or 50 AP-like steps (at 100 Hz) and found that the
5HT1A and M2 modulations
were reduced by the AP-like steps (Fig. 10B) and that the
amplitude of the modulation depended on the number of steps used in the
prepulse. Following a 50-step prepulse, the
5HT1A- (Fig. 10B; n = 5) and M2-mediated modulations (n = 4; data not shown) were almost completely reversed [for both transmitters, median reversal = 100% (e.g., median modulation after steps = 0%)] (see also Williams et al.
1997
). In contrast, the isolated M1
pathway was not affected by a prepulse of multiple AP-like steps
(n = 4, data not shown).
Our second protocol elicited current with 16 APWs at 200 Hz. The amplitude of current entering in response to the first spike in control solution was compared with the amplitude of the current entering with the first spike and the 16th spike in the presence of the modulator. The modulation was considered completely reversed if the amplitude of the current entering in response to spike 16 in the presence of transmitter was equal in amplitude to the current entering with spike 1 under control conditions. We found the 5HT1A-mediated modulation to be completely reversed by this protocol in five of six cells tested and partially reversed in the sixth cell (by 44%; Fig. 10A). The fast (M2) muscarinic modulation was likewise completely reversed in four of five cells and partially reversed in the fifth (by 30%; Fig. 11, A and B). The slow muscarinic modulation (M1) was not reversed in any of the four cells tested (Fig. 11, C and D).
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DISCUSSION |
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Calcium-dependent mechanisms influence the integrative behavior
(e.g., repetitive firing, gene regulation) of neocortical pyramidal
neurons (Bito et al. 1997; Connors et al.
1982
; Lorenzon and Foehring 1993
; Pineda
et al. 1998
; Schwindt et al. 1988
). Understanding these mechanisms requires knowledge of
Ca2+ entry in response to physiological stimuli.
Accordingly, we varied the parameters of mock APWs in the physiological
range to examine how variations in holding potential, and action
potential shape, numbers, and frequency influence
Ca2+ entry into acutely dissociated neocortical
pyramidal neurons. In addition, we examined currents during spike
trains and the influence of spike number and frequency on the
modulation of Ca2+ currents by transmitters.
HOLDING POTENTIAL.
In vivo, neocortical pyramidal cells shift between two preferred
membrane potential ranges, 45 to
55 mV ("up state") and
60 to
90 mV ("down state") (Cowan and Wilson 1994
;
Steriade et al. 1993
; Stern et al. 1997
).
In slices, resting potentials are typically about
65 to
80 mV
(Lorenzon and Foehring 1993
; McCormick et al.
1985
). We found that AP-induced Ca2+
channel currents were somewhat sensitive to holding potential in these
potential ranges: current amplitude and charge entry were greater
from negative potentials, indicating that more
Ca2+ channels are available for activation when
the neuron resides in the down state versus the up state.
SPIKE PARAMETERS.
During prolonged repetitive firing in neocortical pyramidal cells,
action potentials become smaller and broader (rise rate decreases)
(Connors et al. 1982; Stafstrom et al.
1984
). During postnatal development, pyramidal cell action
potentials become narrower and increase in amplitude and in rates of
rise and repolarization with age (Lorenzon and Foehring
1993
; McCormick and Prince 1987
).
PHARMACOLOGY.
We found that HVA Ca2+ current types contribute a
similar percentage to the whole cell current when evoked by either a
voltage step or an APW. Furthermore we found no significant differences on average between the properties of APW-evoked currents for the five
HVA currents, suggesting that the biophysical differences found with
step protocols are too slight to be detected with short stimuli. In
contrast, in cultured hippocampal pyramidal cells, L-type current
comprises a much higher proportion of currents in response to voltage
steps or EPSP-like waveforms versus APWs (underlying greater activation
of CREB phosphorylation by EPSPs vs. APs) (Mermelstein et al.
2000). In those cells, (unlike neocortical pyramidal neurons)
(Lorenzon and Foehring 1995a
), L-type currents activate more slowly and at more negative potentials than other HVA
current types (Mermelstein et al. 2000
).
CHARGE CARRIER.
Previous studies using voltage steps and Ba2+
found little T-type current in acutely dissociated neocortical
pyramidal cells (Lorenzon and Foehring 1995a,b
;
Ye and Akaike 1993
; but see Sayer et al.
1990
). With Ca2+ as the charge carrier,
we found that T-type current contributed to a prolonged tail current
after APWs. This finding is consistent with data from dorsal root
ganglion (DRG) neurons (McCobb and Beam 1991
;
Scroggs and Fox 1992
), where T-type current contributed more than expected to the whole cell current elicited by an APW and
caused prolonged decay of the current. In neocortical pyramidal cells,
T-type current activation is first detected at about
65 mV (in the
subthreshold range for Na+ spikes). T-type
currents can be completely inactivated by holding the cell at
potentials depolarized to about
70 mV (Sayer et al. 1990
). Therefore T-type current would contribute to
subthreshold inward current when action potentials depolarize the
membrane from the down state, but not from the up state.
SPIKE TRAINS.
Pyramidal cells in awake animals in vivo exhibit highly irregular low
firing rates (<10 Hz) which are increased to 30-50 Hz with sensory
stimulation (Mountcastle et al. 1969; Simons et
al. 1992
). In response to sensory input, they often fire at
rates of >100 Hz for prolonged periods (>1 s) in awake behaving
rats (Burne et al. 1984
) and can sustain firing rates of
300 Hz in monkeys (Cheney and Fetz 1980
; Knierim
and Van Essen 1992; Newsome et al. 1989
). We
examined the effects of spike trains at 50-200 Hz.
MODULATION.
Voltage-dependent and -independent modulation of
Ca2+ currents has been observed in many cell
types (Jones and Elmslie 1997). Activation of
5HT1A and M2 receptors
leads to reduction in N- and P-type currents in neocortical pyramidal
cells (Foehring 1996
; Stewart et al.
1999
). We found that peak modulation was greater for APW-
versus step-evoked currents when kinetic slowing was evident (high
doses of 8-OH-DPAT). If slowing did not occur, peak modulation was
similar for each protocol. These data are similar to findings in dorsal
raphe and sympathetic neurons (Pennington et al. 1992
;
Toth and Miller 1995
). A recent study on chick ciliary ganglion (Artim and Meriney 2000
) reported no change in
the kinetics of APW-induced Ca2+ currents loaded
with GTP
s, suggesting that modulated channels are not activated
during single APWs.
Conclusion
We found that several parameters of APWs influence
Ca2+ entry in neocortical pyramidal cells. With
Ba2+ as the charge carrier, there was no evidence
for differential involvement of Ca2+channel
subtypes in the AP or for current facilitation or inactivation. With
Ca2+ as charge carrier, T-type current was
unmasked and current decay was increased, suggesting a possible role
for Ca2+-dependent inactivation. The magnitude of
membrane-delimited modulations mediated by 5HT1A
or M2 receptors were greater for AP-induced currents (vs. peak currents in steps) if activation slowing was evident. APWs were efficient in reversing these voltage-dependent pathways (see also Brody et al. 1997; Park and
Dunlap 1998
; Williams et al. 1997
) but not the
voltage-independent M1 pathway, suggesting that
neuronal activity can selectively alter the effectiveness of different
modulatory pathways.
In synaptic terminals, G-protein modulation of
Ca2+ channels is thought to be a major mechanism
underlying presynaptic inhibition (Wu and Saggau 1997).
Even small changes in spike parameters (e.g., width, amplitude) would
cause large changes in neurotransmitter release (Sabatini and
Regehr 1997
) because of the power relationship between
Ca2+ entry and release (Dodge and
Rahamimoff 1967
). Variable relief of this modulation by spike
trains may underlie some forms of synaptic facilitation (Brody
and Yue 2000
). If similar Ca2+ channels
are expressed in synaptic terminals of pyramidal neurons (cf.
Choi and Lovinger 1996
; Wheeler et al.
1996
) as we have observed in soma/proximal dendrites, our
findings would also have implications for transmitter release. Calcium
channel inactivation would seem to play little role for single APs and
a limited role during repetitive activity. Likewise, facilitation
appears unimportant unless transmitters activate G-protein modulation
of Ca2+ channels. Finally, reduction of
transmitter release by voltage-dependent modulatory pathways would only
be effective at low rates of activity.
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ACKNOWLEDGMENTS |
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The authors thank C. Wyndham and Dr. M. Fowler for technical assistance and Drs. Reese Scroggs and Shelly Timmons for a critical reading of an earlier version of the manuscript.
This work was supported by National Institutes of Health Grant NS-33579 (to R. C. Foehring) and NIH Predoctoral Fellowship 5T32MH-19547 (to A. E. Stewart).
Present address of A. E. Stewart: Dept. of Pharmacology, The George Washington University Medical Center, 634 Ross Hall, 2300 Eye St., N.W., Washington, DC 20037.
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FOOTNOTES |
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Address for reprint requests: R. C. Foehring, Dept. of Anatomy and Neurobiology, University of Tennessee, 855 Monroe Ave., Memphis, TN 38163 (E-mail: foehring{at}nb.utmem.edu).
Received May 24, 2000; accepted in final form December 22, 2000.
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REFERENCES |
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