Department of Neuroscience and Center for the Neural Basis of Cognition, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
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ABSTRACT |
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González-Burgos, Guillermo and German Barrionuevo. Voltage-Gated Sodium Channels Shape Subthreshold EPSPs in Layer 5 Pyramidal Neurons From Rat Prefrontal Cortex. J. Neurophysiol. 86: 1671-1684, 2001. The role of voltage-dependent channels in shaping subthreshold excitatory postsynaptic potentials (EPSPs) in neocortical layer 5 pyramidal neurons from rat medial prefrontal cortex (PFC) was investigated using patch-clamp recordings from visually identified neurons in brain slices. Small-amplitude EPSPs evoked by stimulation of superficial layers were not affected by the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoic acid but were abolished by the AMPA receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione, suggesting that they were primarily mediated by AMPA receptors. AMPA receptor-mediated EPSPs (AMPA-EPSPs) evoked in the apical dendrites were markedly enhanced, or increased in peak and duration, at depolarized holding potentials. Enhancement of AMPA-EPSPs was reduced by loading the cells with lidocaine N-ethylbromide (QX-314) and by local application of the Na+ channel blocker tetrodotoxin (TTX) to the soma but not to the middle/proximal apical dendrite. In contrast, blockade of Ca2+ channels by co-application of Cd2+ and Ni2+ to the soma or apical dendrite did not affect the AMPA-EPSPs. Like single EPSPs, EPSP trains were shaped by Na+ but not Ca2+ channels. EPSPs simulated by injecting synaptic-like current into proximal/middle apical dendrite (simEPSPs) were enhanced at depolarized holding potentials similarly to AMPA-EPSPs. Extensive blockade of Ca2+ channels by bath application of the Cd2+ and Ni2+ mixture had no effects on simEPSPs, whereas bath-applied TTX removed the depolarization-dependent EPSP amplification. Inhibition of K+ currents by 4-aminopyridine (4-AP) and TEA increased the TTX-sensitive EPSP amplification. Moreover, strong inhibition of K+ currents by high concentrations of 4-AP and TEA revealed a contribution of Ca2+ channels to EPSPs that, however, seemed to be dependent on Na+ channel activation. Our results indicate that in layer 5 pyramidal neurons from PFC, Na+, and K+ voltage-gated channels shape EPSPs within the voltage range that is subthreshold for somatic action potentials.
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INTRODUCTION |
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In cortical pyramidal
neurons, excitatory postsynaptic potentials (EPSPs) are generated in
the dendrites, at places distant from the action potential initiation
site, located in proximal segments of the axon (Colbert and
Johnston 1996; Stuart et al. 1997
, 1999
). In a
purely passive neuron, dendritic cable filtering attenuates strongly
the excitatory postsynaptic potentials (EPSPs), reducing significantly
the efficacy of most synapses to depolarize the axon (Henze et
al. 1996
; Spruston et al. 1994
; Stuart
and Spruston 1998
). However, voltage-dependent ion channels are
present not only in the axon but throughout the pyramidal cell membrane (Magee 1999
). Therefore during propagation the EPSPs
could generate subthreshold Na+ and
Ca2+ currents to amplify the EPSPs, counteracting
cable filtering (Crill 1999
). Potassium- and
hyperpolarization-activated conductances also act to shape EPSPs in
ways different from the amplification by Na+ and
Ca2+ inward currents. Interestingly, recent
studies have shown that blockade of Na+ and
Ca2+ currents affects significantly small
subthreshold EPSPs only if the EPSPs are evoked at membrane potentials
depolarized from rest, near firing threshold (Andreasen and
Lambert 1999
; Deisz et al. 1991
; Fricker
and Miles 2000
; Gillessen and Alzheimer 1997
; Hoffman et al. 1997
; Lipowsky et al.
1996
; Markram and Sakmann 1994
; Schwindt
and Crill 1995
; Stuart and Sakmann 1995
;
Urban et al. 1998
).
EPSP amplification remains largely unexplored in pyramidal cells from
cortical regions other than CA1 hippocampus and sensorimotor cortex.
Across cortical regions, expression of ion channel genes and sorting of
channel proteins to specific membrane regions could be different in
modes that may affect EPSP propagation. For example, the
somatodendritic membrane distribution of K+
channels differs significantly in CA1 hippocampal (Hoffman et al. 1997) compared with layer 5 somatosensory cortex pyramidal neurons (Bekkers 2000a
; Korngreen and Sakmann
2000
).
Here, we examined the role of voltage-dependent channels in shaping
subthreshold EPSPs in layer 5 pyramidal neurons from rat medial
prefrontal cortex (PFC). In vivo, PFC neurons are able to sustain
firing at 20-50 Hz for seconds after a brief trigger stimulus ceases.
This persistent, stimulus-outlasting firing is also found in other
regions of cortex, but it is more robust and more frequently observed
in PFC. Thus it is thought to be the cellular substrate of working
memory (Fuster 1997; Goldman-Rakic 1995
).
Until now, it is not entirely clear whether sustained firing relates to
unique electrophysiological properties of individual PFC neurons or to
network interactions.
The intrinsic firing properties of PFC pyramidal neurons in vitro are
consistently similar to those of neurons from other cortical regions
(Ceci et al. 1999; Geijo-Barrientos 2000
;
Geijo-Barrientos and Pastore 1995
; Gulledge and
Jaffe 1998
; Henze et al. 2000
; Yang and
Seamans 1996
; Yang et al. 1996
; Zhou and
Hablitz 1999
). In contrast to firing properties, only a few
studies investigated EPSP propagation in PFC neurons. Simulations in a
model of layer 5 PFC cells have shown that the geometry of their
dendritic tree indeed determines a substantial EPSP attenuation during
passive propagation (Jaffe and Carnevale 1999
). Seamans
and colleagues focused on the role of Ca2+
channels by simultaneously blocking Na+ and
K+ conductances (Seamans et al.
1997
). Their study showed that the proximal apical dendrite of
layer 5 PFC neurons in vitro generates subthreshold
Ca2+-dependent potentials that can amplify distal
EPSPs or initiate Ca2+ spikes prior to the soma.
Interestingly, in layer 5 neurons from somatosensory cortex,
Ca2+-dependent potentials are absent in proximal
segments and restricted to the most distal portions of the apical
dendrite (Larkum et al. 1999a
,b
; Schiller et al.
1997
; Stuart et al. 1997
; Zhu
2000
).
We investigated whether Na+ or
Ca2+ currents play a predominant role in shaping
subthreshold EPSPs in neocortical layer 5 pyramidal neurons from rat
PFC. We also tested if activation of K+ channels
participate in shaping EPSPs in these cells. PFC neurons were visually
identified in brain slices using infrared differential interference
contrast video microscopy (Stuart et al. 1993), which allowed localized application of channel blockers and also to perform
dendritic injections of synaptic-like current (Stuart and
Sakmann 1995
) to test the effect of channel blockade on EPSP propagation independently of effects on transmitter release.
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METHODS |
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Slice preparation
Male Sprague-Dawley rats (22-33 days old; mean: 24.7) were deeply anesthetized and perfused intracardiacally with a solution of the following composition (in mM): 230.0 sucrose, 1.9 KCl, 1.2 NaH2PO4, 25.0 NaHCO3, 20.0 glucose, 0.5 CaCl2, and 6.0 MgCl2, at 4°C. Coronal slices (300- to 400-µm thick) were cut from the frontal cortex using cold sucrose-based solution.
Electrophysiological recordings
For recording, slices were transferred to a submersion
recording chamber and superfused with the following solution (in mM): 125 NaCl, 2.5 KCl, 1.25 Na2HPO4, 25 NaHCO3, 2.0 CaCl2, 1.0 MgCl2, and 20 glucose [with
D-2-amino-5-phosphonopentanoic acid (D,L-AP5) 50 µM added, when specified]. Bath solution temperature was set at
30-32°C when field-potential or whole cell voltage recordings were
performed only from the soma. Experiments with simultaneous somatic and dendritic electrodes were made either at room temperature (20-24°C) or at 30-32°C. The effects of tetrodotoxin,
Cd2+, and Ni2+ on simulated
EPSPs (simEPSPs) were identical when tested at any of these
temperatures. TEA and 4-aminopyridine (4-AP) were tested only at
30-32°C. Field potentials were recorded with electrodes filled with
0.5 M NaCl (3-5 M), using an Axoclamp-2A amplifier (Axon
Instruments, Foster City, CA). A field potential recording electrode
was first placed on the surface of layer 1 and lowered into the slice
at a depth at which maximal field potential amplitude was observed.
Then, using low magnification and a calibrated video monitor, the
electrode was placed at similar depth into the slice, to record the
field potential every 50 µm from the initial position away from the pia.
For whole cell recordings, pyramidal neurons were identified visually
with infrared illumination and differential interference contrast
optics (Stuart et al. 1993). Somata of recorded neurons were located in the prelimbic or infralimbic regions of the rat medial
PFC (Fig. 1A), at ~500
to ~900 µm from the pial surface, corresponding to layer 5 at the
postnatal age range (van Eden and Uylings 1985
). Patch
pipettes were filled with (in mM): 120 K-methylsulphate, 10 KCl, 10 HEPES, 0.5 EGTA, 4.5 ATP 0.3 GTP, and 14 phosphocreatine. Simultaneous
somatic and dendritic recordings were obtained with Axoclamp-2A
amplifiers (Axon Instruments) operating in bridge mode. Somatic (3-6
M
) and dendritic (10-18 M
) patch electrodes were pulled from
borosilicate glass. Membrane potential was not corrected for liquid
junction potential (experimentally measured to be 6-7 mV). Dendritic
voltage recordings were rejected for analysis if the series resistance
exceeded 80 M
or when it changed rapidly, precluding adequate bridge
balance control. Signals were low-pass filtered at 3 kHz, digitized at
10 or 20 kHz and stored on disk for off-line analysis. Data acquisition
and analysis were performed using LabView (National Instruments).
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Synaptic stimulation and generation of simulated EPSPs
For stimulation of distal inputs to layer 5 PFC neurons, a
vertical cut was made in the slice from the border between layers 1 and
2 through the white matter (Fig. 1B), as described by
Cauller and Connors (1994). Synaptic potentials were
elicited applying extracellular current pulses (20-300 µs; 40-300
µA, 0.1 Hz) with bipolar stimulation electrodes (50-µm-diam Formvar
wire) placed in layer 1, 250-400 µm away from the vertical cut (Fig.
1B). The recording electrodes were placed 200-300 µm
lateral to the opposite side of the cut. In some experiments with
simEPSPs, to verify the action of Na+ and
Ca2+ channels blockers, synaptic responses were
evoked by electrical stimulation applied to deep layer 3 or layer 5.
To elicit simulated EPSPs, dendritic current injections were generated
using customized programs written in LabView as described previously
(Urban and Barrionuevo 1998). In most experiments, EPSPs were simulated by injecting current with the shape of an alpha function: I(t) = Io(t/
)e(
t).
In other experiments, EPSPs were generated by injecting current with a
double-exponential time course of the form:
I(t) = Io[1
e(
t/
on)] * [e(
t/
off)], with
on = 1-5 ms and
off = 5-20 ms.
Io represents the maximal current.
Both types of current waveforms reproduced accurately the EPSPs evoked
by synaptic stimulation. Because drug effects using both current
waveforms were identical, results were pooled.
In many cases, access resistance was high and unstable at the site of dendritic current injection. Therefore adequate dendritic voltage recording was not possible because series resistance and capacitance could not be adequately compensated by the bridge and capacitance compensation controls of the Axoclamp 2A amplifier. We report dendritic voltage recorded only for cases in which compensation was adequate. Fortunately, the access resistance at the dendritic site (even if large, not stable, and not well compensated) does not affect the voltage change recorded by the somatic electrode, unless it increases up to the point of reducing significantly the current-passing ability of the dendritic electrode. This was controlled by periodically monitoring the actual injected current or the somatic voltage change for a given set of parameters. Typically, data were excluded of analysis if a consistent decrease of 10% in the peak of the somatic simEPSP was observed for constant injection parameters that initially elicited a simEPSP with 2-3 mV of somatic peak amplitude.
Statistical analysis
Statistical significance of differences between group means were assessed by paired t-test or repeated-measures ANOVA followed by Dunnett's t-test contrasts. In all cases differences were considered significant if P < 0.05.
Drugs
D,L-AP5 and 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX) were purchased from RBI (Natick, MA). All other drugs were from Sigma Chemical (St. Louis, MO). Drugs were daily prepared as concentrated stock solutions in water and diluted in external recording solution shortly before application. When localized application was employed, pipettes with tip diameters of ~1-2 µm were filled with freshly oxygenated external solution containing the drugs as indicated. Flux of the external solution in the chamber was oriented so that the puffs did spread in a perpendicular direction away from the primary apical dendrite. When millimolar concentrations of 4-AP and TEA were employed, NaCl concentration was reduced to preserve osmolality constant.
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RESULTS |
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Isolation of synaptic responses evoked by layer 1 stimulation
In layer 5 pyramidal neurons, the most distal EPSPs are predicted
to be the most strongly attenuated by dendritic cable filtering (Jaffe and Carnevale 1999; Stuart and Spruston
1998
). Therefore if ion channels provide currents that amplify
the EPSPs, removal of these currents should have its strongest effect
on distal EPSPs. To determine whether voltage-dependent ion channels
amplify EPSPs in layer 5 PFC neurons, we employed a slice preparation
(Fig. 1A) in which a vertical cut sectioned all horizontal
connections except those in layer 1, to restrict the synaptic responses
to the distal apical dendrites (Cauller and Connors
1994
; Zhu 2000
). This preparation also
facilitates to apply channel blockers locally without altering
transmitter release (see following text).
We examined the spatial pattern of activation in slices prepared in this way by recording extracellular field potentials across layers 1-5 and evoked by stimulation of layer 1. In layers 1 and 2, field potentials were composed by a monophasic extracellular negativity, consistent with the presence of synaptic current sinks (Fig. 1B). Field potentials recorded in deeper layers (layer 3 and superficial layer 5) usually reversed in sign, consistent with the current sinks being present predominantly in superficial layers (Fig. 1B). However, in layers 3 and 5, the field potentials often had complex waveforms, consistent with the presence of a mixture of current sinks and sources in these laminae (Fig. 1B). Therefore the spatial pattern of activation confirms that in this preparation the activated synapses are mainly restricted to layers 1 and 2 and thus to distal portions of the apical dendrite of layer 5 pyramidal neurons, although activation of some proximal synapses could not be completely ruled out.
Patch-clamp electrode voltage recordings were obtained from the soma of
visually identified layer 5 PFC pyramidal neurons (n = 89). Resting membrane potential (RMP) and input resistance (at RMP)
measured at the soma were 67.8 ± 3.5 mV and 58 ± 7 M
(means ± SD). In response to somatic injection of depolarizing current, the majority of pyramidal cells (81/89) exhibited regular spiking firing mode; cells with weak bursting (5/89) or strong bursting
firing modes (3/89) were found but were less common. No significant
differences were observed in subthreshold EPSP amplification in cells
with different firing modes, therefore results were pooled. In slices
prepared as shown in Fig. 2A,
only 57% of the layer 5 neurons yielded satisfactory responses to
layer 1 stimulation (Fig. 2B). The remaining cells showed no
responses or responded only to high stimulation intensities with
potentials that were usually composed by multiple excitatory and
inhibitory events, some of which had prolonged and highly variable
latency and thus were likely polysynaptic. Synaptic inhibition was
revealed as a fast hyperpolarization that truncated the EPSPs when
recorded at depolarized potentials below spike threshold (
50 to
45
mV). The GABAA receptor antagonist bicuculline
could not be employed to isolate EPSPs because, in its presence (5-10
µM), layer 1 stimulation caused epileptiform activity
(n = 6 slices tested, data not shown). Responses with
polysynaptic and inhibitory components were excluded from data
analysis. We focused on monophasic responses with a latency that was
short and exhibited very small trial-to-trial variability (Fig.
2B). Responses with these characteristics had small peak
amplitudes (mean peak amplitude, <4 mV at RMP). In the absence of
bicuculline, no inhibition could be detected in the majority of these
responses, which therefore were considered as EPSPs. If an inhibitory
component overlapping with the EPSP was detected, the cell was excluded
from analysis.
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The peak of EPSPs evoked by layer 1 stimulation and recorded at RMP was
reduced to 11.7 ± 4% of control (Fig. 2C) 15 min
after bath application of 10 µM of the AMPA/kainate glutamate
receptor antagonist CNQX (n = 4 cells). In contrast, in
five of five neurons recorded at RMP in our standard bath solution
(external Mg2+ concentration: 1 mM), the NMDA
receptor antagonist AP5 (50 µM) did not affect the peak and area of
EPSPs recorded at the cells' RMP (Fig. 2D). When the EPSPs
were evoked at subthreshold depolarized potentials (60 to
50 mV),
AP5 had nonsignificant effects on EPSPs evoked by layer 1 stimulation
(peak, 110.6% and area, 98.45% of control, n = 5).
These results show an absence of significant voltage contribution of
NMDA receptor activation to EPSPs evoked by layer 1 stimulation and
recorded at subthreshold membrane potentials in vitro, as also shown
previously for some neocortical and hippocampal synapses
(Emptage et al. 1999
; Koester and Sakmann
1998
). These results are also in agreement with recent findings
suggesting that, within individual layer 5 neocortical pyramidal cells,
NMDA and non-NMDA receptors are differentially distributed across the somatodendritic axis, with the NMDA/AMPA response ratio dropping markedly with increasing distance from the soma (Dodt et al.
1998
). Although distal EPSPs in layer 5 PFC neurons seem to
lack a significant NMDA component, the remaining experiments (unless
otherwise stated), were performed in the presence of AP5 to study the
role of voltage-dependent ion channels in EPSP amplification
independently of the voltage-dependent relief from
Mg2+ block of synaptic NMDA channels.
Effects of inhibition of Na+ and Ca2+channels on the synaptic responses evoked by layer 1 stimulation
In previous studies, it was shown that inhibition of voltage-dependent channels affects EPSPs more significantly, if not exclusively, when evoked at potentials depolarized from rest and close to action potential threshold. At these potentials, the peak and duration of the EPSPs is enhanced, or amplified, in control conditions and the increase is reduced by blockade of voltage-dependent channels. Therefore we first examined whether AMPA-EPSPs evoked by layer 1 stimulation in layer 5 PFC pyramidal neurons exhibited a similar voltage-dependent enhancement. Indeed, in contrast to what was expected if the AMPA-EPSPs propagated passively (EPSP reduction due to decrease in driving force or no change in the EPSP if the driving force at the distal synapse is not altered), the peak and area of the AMPA-EPSPs were markedly increased at depolarized potentials (Fig. 3A). EPSPs were enhanced maximally when evoked near spike threshold and, although the magnitude of maximal increase varied significantly across cells, it was consistently observed in all layer 5 PFC neurons (Fig. 3, A and B). Hyperpolarization below RMP reduced slightly, but not significantly, the EPSP peak and area (Fig. 3, A and B).
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To test if, as in pyramidal neurons from other cortical areas, the
voltage-dependent enhancement of EPSPs in PFC neurons is mediated by
voltage-dependent channels, we first loaded the PFC cells with low
concentrations of the Na+ channel blocker
lidocaine N-ethylbromide (QX-314) (2-5 mM). In this condition, neither
EPSP peak nor area were enhanced when evoked at depolarized potentials
(at 75 mV, peak: 6.3 ± 0.3, area: 607 ± 62 mV*ms; at
45
mV, peak: 1.7 ± 0.2 mV; area: 50 ± 43 mV*ms). The marked
effects of QX-314 suggest that the voltage-dependent amplification
requires activation of voltage-gated Na+
channels. However, QX-314 also blocks low- and high-voltage-activated Ca2+ currents (Talbot and Sayer
1996
), K+ currents (Pare and Lang
1998
; Svoboda et al. 1997
) and
hyperpolarization-activated currents as well (Perkins and Wong
1995
). Therefore the relative contribution of
Na+ channels versus other QX-314-sensitive
channels in shaping the EPSPs is not clear.
To examine the effects of selective inhibition of
Na+ or Ca2+ channels, we
employed tetrodotoxin (TTX) or a mixture of Ni2+
and Cd2+, respectively. In response to layer 1 stimulation in our slice preparation, transmitter release presumably
occurred at distal synapses (Fig. 1), therefore channel blockers could
be applied locally to the apical dendrite and soma without altering
glutamate release. Drugs were delivered by applying positive pressure
to pipettes filled with oxygenated bath solution containing either 1 µM TTX or 2 mM Ni2+ plus 200 µM
Cd2+ and placed near to the soma or the apical
dendrite (Fig. 4A). In every
tested neuron, application of TTX to the somatic region, abolished the
initiation or strongly reduced the amplitude of action potentials
elicited by somatic current steps. At the same time, the increase in
EPSP peak and area at depolarized potentials was strongly and
reversibly reduced by TTX applied to the soma (Fig. 4, B and
C). In contrast, the layer 1-evoked small AMPA-EPSPs recorded at RMP or more hyperpolarized potentials (80 to
75 mV)
were not affected by TTX (Fig. 4, B and C). In
marked contrast to TTX, somatic application of
Cd2+ and Ni2+ had no
detectable effects on the AMPA-EPSPs at any membrane
potential tested (Fig. 4, B and C).
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Since application of TTX to the somatic region strongly reduced the
area of EPSPs recorded at depolarized potentials, activation of
Na+ channels located at or near the soma seems to
account for a significant fraction of the voltage-dependent EPSP
amplification, as reported previously. It has also been reported,
however, that dendritic Na+ and
Ca2+ channels contribute to EPSP amplification in
addition to somatic Na+ channels
(Gillessen and Alzheimer 1997; Lipowsky et al.
1996
). To examine the contribution of dendritic
Na+ and Ca2+ channels to
EPSP propagation in layer 5 PFC neurons, channel blockers were locally
applied to the apical dendrite, 200-300 µm from the soma. In most
cells (5 of 7 tested), application of TTX to the apical dendrite had no
significant effects on the AMPA-EPSP at any membrane potential tested
(Fig. 4C), although dendritic TTX attenuated strongly the
AMPA-EPSPs regardless of membrane potential in two neurons, probably by
acting on presynaptic Na+ channels. As shown in
Fig. 4C, application of Cd2+ and
Ni2+ to the apical dendrite generally had no
effects on the EPSPs in six of seven tested neurons
(Cd2+ and Ni2+ attenuated
the EPSP peak recorded at
75 mV to 25.3% of control in the remaining cell).
The kinetics of activation of some voltage-gated
Ca2+ channels is slow, relative to that of
Na+ channels (McCobb and Beam
1991; Mermelstein et al. 2000
). Thus it
is possible that single AMPA-EPSPs are too fast to gate
Ca2+ channels. If so, then a more prolonged
EPSP-induced depolarization could reveal a Ca2+
channel contribution. Indeed, it was previously shown that very prolonged depolarizations (0.5-1 s) in some cases activate
Ca2+ in addition to Na+
conductances (Schwindt and Crill 1997
). Synaptic input
more prolonged than single EPSPs can inactivate
K+ channels that could be acting to dampen the
EPSPs, thus preventing the activation of some
Ca2+ channels by the EPSP. The presence of an
NMDA EPSP component prolongs the EPSP decay time and thus could also
reveal a contribution of Ca2+ channels not
observed with AMPA-EPSPs (Calton et al. 2000
;
Mermelstein et al. 2000
; but see de la Peña
and Geijo-Barrientos 2000
). Our results, however, suggest a
weak or absent contribution of NMDA channels to EPSPs evoked by layer 1 stimulation in layer 5 PFC neurons (see Fig. 2).
To test if more prolonged EPSP-induced depolarizations, could reveal a
contribution of Ca2+ channels, we applied layer 1 stimulation (Fig. 1) by delivering trains of five stimuli at
inter-stimulus intervals of 25 or 50 ms. Also, to favor any possible
contribution of NMDA receptor activation, the NMDA antagonist AP5 was
omitted from the external solution. Figure
5A shows that the amplitude of
subsequent EPSPs recorded at a somatic membrane potential of 75 mV
displayed marked depression despite some degree of temporal summation.
When evoked at depolarized potentials, the amplification of individual
EPSPs lead to a pronounced summation and enhancement of the
depolarization induced by the EPSP train (Fig. 5A).
Simultaneous application of Cd2+ and
Ni2+ (200 µM and 2 mM, respectively) to either
the apical dendrite (140-230 µm from the soma) or the soma did not
significantly alter the EPSP trains recorded at
75 mV nor at
depolarized potentials (Fig. 5B). In contrast, localized
application of TTX (1 µM) to the somatic region, removed almost
completely the voltage-dependent amplification of the EPSP trains by
membrane depolarization. During application of TTX to the soma, the
overall depolarization induced by the EPSP trains recorded at
55 to
50 mV was not significantly different from that recorded at
75 mV
(Fig. 5C). A similar voltage-dependent enhancement of EPSP
trains by voltage-dependent Na+ channels was
reported for layer 5 pyramidal cells in somatosensory cortex
(Williams and Stuart 1999
, 2000
).
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Overall, these results indicate a predominant contribution of somatic
or proximal Na+ channels, but not of
Ca2+ channels, in shaping the EPSPs. However, the
role of Na+ or Ca2+
channels that may be located in the apical dendrite is not entirely clear. First, because responses were evoked without blockade of inhibitory transmission, we could not completely rule out that either
evoked or spontaneous inhibitory postsynaptic potentials (IPSPs)
prevented some form of Na+- or
Ca2+-dependent dendritic electrogenesis
(Kim et al. 1995; Larkum et al. 1999a
,b
;
Miles et al. 1996
; Tsubokawa and Ross
1996
). Second, localized application of drugs to the dendrites
may have been ineffective at producing the necessary level of blockade
of the relevant dendritic channel types. To address these issues, we performed the experiments described in the next sections, using dual
patch pipette recordings.
Effects of Na+ and Ca2+channel blockers on the EPSPs simulated by dendritic current injection
To gain further insight into the mechanisms and types of
voltage-dependent conductances involved in shaping EPSP waveforms in
layer 5 PFC neurons, we obtained simultaneous current-clamp recordings
from the soma and primary apical dendrite of these neurons (Fig.
6A). As described originally
by Stuart and Sakmann (1995), one electrode was used to
inject, into the apical dendrite, current waveforms similar to
excitatory postsynaptic currents (see METHODS) while
simultaneously recording the somatic depolarization, referred to as
simEPSP. As shown in Fig. 6B, dendritic current injection
could generate simEPSPs with time courses and amplitudes within the
range observed for EPSPs evoked by extracellular stimulation of layer
1. This approach allowed us to test the effects of blockade of
Na+ and Ca2+ channels
throughout the membrane by bath applied drugs, independently of their
effects on synaptic transmission. In addition, the simEPSPs were
recorded during blockade of GABAA IPSPs by 10 µM bicuculline and thus propagated without interference of evoked or
spontaneous IPSPs. In 56% of the experiments, AMPA and NMDA receptors
were blocked by CNQX (10 µM) and AP5 (50 µM), also eliminating the potential effects of spontaneous excitatory synaptic transmission. Interestingly, no differences were observed in the effects of bath-applied Na+ and Ca2+
channel antagonists on the simEPSPs with or without CNQX and AP5.
Dendritic current injections were performed into the primary apical
dendrite of layer 5 PFC neurons at 30 to ~280 µm from the soma,
which corresponds to the proximal to mid segments of the primary apical
dendrite (Fig. 6A). Within these compartments, the effects
of Na+ and Ca2+ channel
blockers were independent of the distance between the dendritic
injection site and the soma.
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As shown for AMPA receptor-mediated EPSPs, simEPSPs were markedly
enhanced when evoked at depolarized membrane potentials (Fig. 6,
C and D). The increase in simEPSP area was also
larger than the increase in peak, and amplification also was maximal when the simEPSPs were near action potential threshold (Fig. 6, C and D). In some experiments (n = 5), the simEPSPs were successfully recorded at the site of dendritic
current injection (>100 µm from the soma) and at the soma. The
dendritic resting potential determined shortly after rupture of the
patch was 60.6 ± 3.0 mV (mean ± SE, n = 5), a value not significantly different from that measured at the soma
in the same neurons (
66.7 ± 2.5 mV). These dual voltage recordings showed that for current injections at distances >100 µm
from the soma, the simEPSPs attenuated significantly as they propagated
between the site of current injection and the soma with the cells at
rest (Fig. 6E). For injections at very proximal dendritic
sites (<40 um from the soma) little attenuation was observed (not
shown). Indeed, these proximal dendritic regions appear to be
essentially isopotential with the soma. When the cells were depolarized
by somatic current injection, the area of both dendritic and somatic
simEPSPs increased significantly (Fig. 6E). In contrast, the
peak of the dendritic simEPSP did not change with depolarization,
although the somatic simEPSP peak increased (Fig. 6E). In
addition, the increase in area with depolarization was in general
slightly larger for the somatic than the dendritic simEPSP (Fig.
6E). These results are consistent with the idea that the
voltage-dependent EPSP amplification takes place by activation of
TTX-sensitive Na+ channels located at or near the
soma. Activation of these Na+ channels, appears
to generate a subthreshold potential that prolongs the decay of the
somatic EPSP and propagates, with some attenuation, back into the
apical dendrite to prolong the decay of the dendritic EPSP.
Similarly to the AMPA-EPSPs evoked by stimulation of layer 1, the
increase in simEPSP peak and area by depolarization was strongly
reduced when the layer 5 PFC neurons were loaded with QX-314 (2-5 mM).
Once fast Na+ spikes were abolished by QX-314,
the amplitude and area of the simEPSPs did not change significantly
with depolarization of the cell elicited by simultaneous injection of
current into the soma (simEPSP peak at 75 mV: 2.77 ± 0.09 mV;
simEPSP peak at
50 mV: 2.70 ± 0.03 mV; simEPSP area at
75 mV:
106.81 ± 9.31 mV*ms; simEPSP area at
50 mV: 110.66 ± 17.08 mV*ms; n = 3 cells; differences not significant
by paired t-test, P > 0.05).
We next bath-applied TTX to examine the effect of selectively blocking voltage-activated Na+ channels on the shaping of simEPSP waveforms by depolarization. For each neuron, simEPSPs with small amplitudes at the soma when recorded at RMP were evoked at a range of hyperpolarized and depolarized membrane potentials, first in control bath solution and then with TTX (1 µM) in the bath. The action of TTX was verified by the blockade of evoked excitatory synaptic transmission or action potentials (APs) elicited by somatic current steps. Figure 7A shows that the peak and area of small simEPSPs recorded at hyperpolarized membrane potentials, were unaffected by TTX. In contrast, TTX strongly attenuated or removed completely the enhancement of the peak and area of small simEPSPs evoked at depolarized membrane potentials in the same neurons (Fig. 7A).
|
The removal of voltage-dependent EPSP amplification by bath-applied TTX suggests that activation of Na+ channels accounts completely for the voltage-dependent enhancement of EPSPs in PFC neurons. However, it was still possible that activation of Ca2+ channels, secondary to Na+ channel activation, also was required. To test this possibility, we bath-applied a mixture of the Ca2+ channel blockers Cd2+ and Ni2+, at concentrations sufficient to block most of the available voltage-dependent Ca2+ channels, regardless of subtype (200 µM each or 200 µM Cd2+ plus 2 mM Ni2+). As in the case of TTX, small simEPSPs recorded at RMP or more hyperpolarized potentials were not affected by application of Cd2+ and Ni2+ (Fig. 7B). In contrast to TTX, however, enhancement of simEPSPs by depolarization was not affected during extensive block of Ca2+ channels by bath application of Cd2+and Ni2+ (Fig. 7B). The action of Ca2+ channel blockers was verified by its block of excitatory synaptic responses or of the afterhyperpolarizations (see following text). In three of these neurons, 1 µM TTX was applied after washout of Cd2+ and Ni2+ and strongly reduced the voltage-dependent amplification of simEPSPs at depolarized potentials (data not shown).
Previous studies have shown that T-type nickel-sensitive
Ca2+ channels contribute to EPSP waveforms in
both hippocampal and neocortical pyramidal neurons (de la
Peña and Geijo-Barrientos 2000; Deisz
1991
; Urban et al. 1998
). Because T-like
currents inactivate rapidly with depolarization, it is possible that a contribution of these currents to EPSPs was underestimated when EPSPs
were evoked at depolarized holding potentials. Small EPSPs (<5 mV
peak) evoked at hyperpolarized potentials that would remove T-channel
inactivation were unaffected by Ni2+, but their
small amplitudes may have not been sufficient to gate the
Ca2+ channels. To test if
Ca2+ channels could contribute to EPSPs with
large amplitudes, we compared the effects of Cd2+
and Ni2+ on small and large EPSPs. In our
experimental conditions, large amplitude EPSPs (peak >4 mV) evoked by
superficial layer stimulation were in most cases associated with
polysynaptic EPSPs and IPSPs. Therefore the effect of channel block on
EPSPs with large amplitude was examined in small and large simEPSPs,
generated in the same neuron by adjusting the size of the synaptic-like
current injected into the apical dendrite. The kinetics of large and
small simEPSPs waveforms was generally similar, except for a tendency
of the decay phase of the large EPSPs to hyperpolarize a few millivolts below the cells' resting potential (Fig.
8A). By increasing the size of
the current injected by the dendritic electrode, the amplitude of the
somatic simEPSPs could be progressively incremented up to the point of
reaching firing threshold from a hyperpolarized membrane potential of
70 mV (Fig. 8B). When the same current injection protocol
was repeated after applying TTX (1 µM) to the bath solution, the
amplitude of small EPSPs was unaffected, whereas that of large simEPSPs
was reduced significantly (Fig. 8B). Interestingly, in three
of three cases in which more than three magnitudes of dendritic current
injection could be employed, simEPSP peak was observed to increase
linearly with injected current in control bath solution and sublinearly
in the presence of TTX (Fig. 8C). As shown in Fig. 8,
C and D, neither small nor large simEPSPs were
significantly affected by adding Ca2+ channel
blockers (2 mM Ni2+ plus 200 µM
Cd2+) to the external solution. Similar results
were found when the cells were held at a potential of
80 mV, thus
further removing channel inactivation (data not shown). These results
suggest that, within the middle-proximal compartment of the apical
dendrite of layer 5 PFC neurons, Ca2+ channels do
not contribute to propagation of EPSPs that had amplitudes in the whole
subthreshold range of membrane potentials. In contrast, TTX- sensitive
Na+ channels do contribute to the depolarization
elicited by large simEPSPs evoked at
70 mV. This contribution,
however, was less prominent than that found for small EPSPs evoked at
depolarized potentials, suggesting that different mechanism may be
involved in shaping large and small EPSPs, at least regarding the
effect of Na+ channels.
|
Overall, our results seem different from those reported in a previous
study of layer 5 PFC pyramidal neurons, in which it was suggested that
a Ca2+-dependent potential generated in the
proximal apical dendrite or soma amplifies single distal EPSPs
(Seamans et al. 1997). However, in the previous study
the recordings were performed after Na+ and
K+ channels were inhibited by application of
QX-314 and Cs+ or TEA. Therefore altogether the
results open the interesting question of whether
Ca2+ channel contribution to EPSPs is secondary
to a reduction of K+ currents. In pyramidal and
nonpyramidal neurons in the CA1 region of hippocampus, inhibition of
K+ currents uncovers or enhances EPSP
amplification (Andreasen and Lambert 1999
;
Fricker and Miles 2000
; Hoffman et al.
1997
). In neocortical layer 5 pyramidal neurons, it has not
been examined whether a similar balance between inward and outward
currents takes place.
To examine this possibility, we tested the effects of inhibiting
K+ currents on the voltage-dependent
amplification of EPSPs. When applied in the presence of
K+ channel antagonists, layer 1 stimulation
elicited epileptiform activity, therefore the effects of
K+ channel inhibition were tested only on
simEPSPs that were elicited by injection of current into the proximal
apical dendrite (up to 100 µm from the soma), with excitatory and
inhibitory synaptic transmission blocked by CNQX (10 µM), AP5 (50 µM), and bicuculline (10 µM). In layer 5 neurons from somatosensory
cortex, transient and sustained K+ conductances
found in dendrites and soma are blocked by millimolar concentrations of
4-AP and TEA, respectively (Bekkers 2000a,b
; Korngreen and Sakmann 2000
). In the presence of 4-AP and
TEA (2 mM each), the peak and amplitude of simEPSPs elicited at resting or more hyperpolarized potentials were not significantly different from
those recorded in control bath solution (Fig.
9A). However, in the presence
of the K+ channel blockers, the enhancement of
simEPSPs by depolarization was increased, relative to control
conditions, at potentials equal to or more depolarized than
65 mV
(Fig. 9A). As a consequence of this enhanced amplification
by K+ channel blockers, EPSPs reached firing
threshold at potentials more hyperpolarized than in control conditions
(Fig. 9A).
|
If the EPSP enhancing effect of K+ current
inhibition results from a contribution of voltage-dependent
Ca2+ channels, then the presence of
K+ channel blockers should reveal an effect of
the Ca2+ channel blockers
Cd2+ and Ni2+. To test this
possibility, we examined the effects of Cd2+ and
Ni2+ (200 µM Cd2+ plus 2 mM Ni2+) when applied in the presence of 4-AP and
TEA (2 mM each). As shown in Fig. 9B, the enhanced
amplification of simEPSPs by 4-AP and TEA was not significantly
affected by the presence of Cd2+ and
Ni2+. In contrast, the effect of the
K+ channel antagonists was strongly inhibited by
bath application of 1 µM TTX, which actually removed completely the
voltage-dependent amplification (Fig. 9B). These results
suggest that K+ currents counteract the enhancing
effect of Na+ channels but do not enable a
contribution of Ca2+ channels to the EPSPs.
However, at 2 mM, 4-AP and TEA do not fully inhibit
K+ currents, therefore it is possible that the
residual K+ current was sufficient to shunt a
depolarization otherwise generated by Ca2+
currents. To test this possibility, in a separate series of
experiments, we employed 4-AP and TEA at concentrations (4-AP: 5 mM;
TEA: 20 mM) above those that inhibit 50% of the
K+ currents mediated by a variety of
K+ channels (Coetzee et al. 1999),
including transient and sustained K+ currents
found in the soma and dendrites of hippocampal and neocortical pyramidal neurons (Bekkers 2000a
; Hoffman et al.
1997
; Korngreen and Sakmann 2000
). SimEPSPs were
elicited by dendritic current injection at distances from the soma of
120 µm, first in control conditions and then in the presence of 5 mM 4-AP and 20 mM TEA. Small simEPSPs were not significantly affected
by the K+ channel blockers when recorded at
hyperpolarized potentials. In contrast, at potentials equal to or more
depolarized than
60 mV, the simEPSPs were enhanced relative to
control conditions (Fig. 9C), and the enhancement was larger
than that observed with the lower concentrations of 4-AP and TEA (Fig.
9B). With 5 mM 4-AP and 20 mM TEA, most cells fired bursts
of action potentials triggered by the simEPSPs at holding potentials of
55 mV or more positive. When a mixture of Cd2+
and Ni2+ (200 µM Cd2+
plus 2 mM Ni2+) was applied in the presence of 5 mM 4-AP and 20 mM TEA, the Ca2+ channel blockers
reduced the increase in simEPSP area at depolarized potentials (Fig.
9C). At
55 mV, simEPSPs were still enhanced more than in
control conditions, but burst firing was absent at this holding
potential. This suggests that the enhanced amplification observed under
extensive K+ channel block results in part from
contribution of Ca2+ currents. Interestingly,
bath application of TTX (1 µM) together with 4-AP and TEA (5 and 20 mM) removed almost completely the EPSP amplification (Fig.
9C), suggesting that the contribution of
Ca2+ channels to EPSPs is secondary to
Na+ channel activation.
The secondary role of Ca2+ channels in shaping
the EPSPs seemed not to be due to significant run-down of postsynaptic
Ca2+ currents. As illustrated in Fig.
10A, APs were followed by a
fast afterhyperpolarization (fAHP) and a medium-duration AHP (mAHP) that were observed when recording at prolonged times after break-in (30 min and up to ~90 min in some cases). In neocortical pyramidal cells (Pineda et al. 1998
; Schwindt et al.
1988
), the mAHP results from activation of
Ca2+-sensitive K+ channels
coupled to Ca2+ influx through voltage-gated
Ca2+ channels (Marrion and Tavalin
1998
). Therefore the presence of the AHPs suggests that
Ca2+ currents involved in its generation,
probably N, P, and Q (Pineda et al. 1998
), were well
preserved in our recording conditions. Consistent with this, bath
application of Cd2+ and
Ni2+ reduced strongly the mAHPs in every PFC
neuron tested (Fig. 10A). Also, during application of
Ca2+ channel inhibitors, the excitability of PFC
neurons was enhanced (data not shown), an effect previously attributed
to blockade of L-type channels (Pineda et al. 1998
). The
presence of Ca2+ currents was also confirmed in
experiments in which an external solution containing 4-AP (5 mM), TEA
(20 mM) and TTX (1 µM) was applied to the slices 20-30 min before
obtaining the recordings. In these neurons, depolarizing somatic
current steps delivered shortly after break-in (8-25 s), elicited
high-threshold regenerative events that resembled
Ca2+ spikes. Amplitude of these spikes showed
little change (98.3 ± 4.5%) after the first 25-30 min, the time
window in which EPSPs were usually studied, but was strongly reduced
soon after bath application of Cd2+ and
Ni2+ (Fig. 10B). Calcium spikes were
not observed if TTX was applied in the absence of
K+ channel blockers (see also Svoboda et
al. 1997
), suggesting that K+ currents
normally restrict the initiation of Ca2+ spikes
in the soma and proximal dendrites. In contrast to TTX applied alone,
when Na+ channels were inhibited by loading the
cells with 5-10 mM QX-314, depolarizing steps evoked small-amplitude
Ca2+ spikes (Fig. 10C), indicating
that QX-314 inhibits the K+ currents that
restrict Ca2+ spike initiation in these neurons,
as also shown previously (Svoboda et al. 1997
).
|
Finally, developmental changes in the expression and subcellular
distribution of voltage-gated channels seem not to be related to the
weak effects of Ca2+ channel block in PFC
neurons. We compared the effects of Na+ and
Ca2+ channel blockers on simEPSPs evoked in
neurons obtained from rats in the two extremes of the range of
postnatal days employed in our study. The results showed no significant
differences between the age groups: the area of simEPSPs recorded at
depolarized potentials (55 to
50 mV) was, at 22-23 days postnatal:
29.8 ± 12.2% of control (n = 5) in the presence
of 1 µM TTX and 121.5 ± 19.0% of control (n = 5) in the presence of Cd2+ and
Ni2+ (200 µM and 5 mM, respectively); at 31-33
days postnatal, 25.4 ± 8.2% of control (n = 4)
in the presence of TTX and 97.5 ± 4.1% of control
(n = 3) in the presence of Cd2+
and Ni2+. These results suggest that between
postnatal days 22 and 33, developmental changes in
Na+ and Ca2+ channel
distribution within the proximal and middle compartments of the apical
dendrite examined here are not significant regarding propagation of
EPSPs. Indeed, the recent study of Zhu (2000)
showed that the main changes in morphology and electrophysiology of layer 5 neocortical pyramidal neurons happen between postnatal days 0 and 14 and are near or at plateau around 30 days. In addition, the
developmental changes described by Zhu (2000)
take place
in the most distal portions of the apical dendrite.
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DISCUSSION |
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By selectively inhibiting currents mediated by Na+, Ca2+, and K+ channels, independently of their effects on glutamate release, we have investigated the role of these channels in shaping subthreshold EPSPs in neocortical layer 5 neurons from rat medial PFC. We found that in cells resting at membrane potentials typical for pyramidal neurons in vitro, EPSPs with small amplitude at the soma and evoked in the apical dendrite were unaffected by inhibition of Na+, Ca2+, or K+ channels. At depolarized subthreshold potentials, both peak and area of EPSPs were enhanced by Na+ currents. Potassium currents also participated in shaping the EPSPs during depolarization. Calcium current contribution, in contrast, occurred only when K+ currents were strongly inhibited by high concentrations of 4-AP and TEA and seemed to be secondary to Na+ channel activation.
Ionic mechanisms of EPSP amplification
Our results suggest a predominant role of
Na+ channels in shaping subthreshold EPSPs in
layer 5 pyramidal neurons from rat PFC. The contribution of
Na+ currents was revealed under conditions
(depolarized holding potentials) similar to those required for
pyramidal cells in other brain regions, namely CA1 (Andreasen
and Lambert 1999; Hoffman et al. 1997
;
Lipowsky et al. 1996
) and layer 5 somatosensory neurons
(Stuart and Sakmann 1995
). Moreover, in layer 5 PFC
neurons, Na+ channels shaping the EPSPs appear to
be located near the soma, as in other cell types (Andreasen and
Lambert 1999
; Stuart and Sakmann 1995
;
Urban et al. 1998
). Pyramidal neurons in PFC exhibit Na+-dependent plateau potentials and persistent
Na+ currents (Geijo-Barrientos and Pastore
1995
; Gorelova and Yang 2000
; Maurice et
al. 2001
; Yang et al. 1996
). Thus it is likely that voltage-dependent enhancement of EPSPs in PFC neurons, similarly to other neurons, is mediated by Na+ channels in
a persistent or slowly inactivating gating mode (Alzheimer et
al. 1993
; Andreasen and Lambert 1999
;
Lipowsky et al. 1996
; Schwindt and Crill
1995
; Stuart and Sakmann 1995
). However,
subthreshold EPSPs can also gate fast Na+
channels (Magee and Johnston 1995
), which are activated
during depolarization-dependent amplification of EPSPs, since
full-amplitude action potentials are triggered frequently during the
decay of the amplified EPSPs. Because both fast and persistent
Na+ channels are TTX-sensitive, at present it is
difficult to discern the relative contribution of these two channel
gating modes.
Compared with the ubiquitous role of Na+ currents
in shaping subthreshold EPSPs, the role of Ca2+
currents seems controversial. Calcium channels were reported to amplify
EPSPs in CA1 and CA3 hippocampal (Gillessen and Alzheimer 1997; Urban et al. 1998
) and neocortical
(de la Peña and Geijo-Barrientos 2000
;
Deisz et al. 1991
; Markram and Sakmann
1994
) pyramidal neurons. In contrast, no role of
Ca2+ channels in shaping small subthreshold EPSPs
was suggested for neocortical layer 5 (Stuart and Sakmann
1995
) or CA1 neurons (Andreasen and Lambert
1999
; Magee and Johnston 1995
; Magee et
al. 1995
) in other studies. Blockade of
Ca2+ channels attenuates EPSPs in layer 5 somatosensory neurons from 14- to 18-day-old (Markram and
Sakmann 1994
; Stuart and Sakmann 1995
) but not
from 28 day-old rats (Schiller et al. 1997
;
Stuart and Sakmann 1995
), suggesting that maturation of
cell properties can explain the different results in some cases.
Inhibition of inward currents affected the small EPSPs mainly when they
were evoked at subthreshold depolarized holding potentials. If the
voltage dependence and kinetics of inactivation of
K+ channels in layer 5 PFC neurons is similar to
that in layer 5 cells from somatosensory cortex (Bekkers
2000a; Korngreen and Sakmann 2000
), then part of
the effect of depolarized holding potentials might be due to
inactivation of K+ channels. Consistently with
this idea, single EPSPs with large peak amplitudes (20-25 mV at the
soma) evoked at hyperpolarized holding potentials were affected much
less by blockade of Na+ channels than small EPSPs
evoked at depolarized potentials. Pharmacological blockade of
K+ currents enhanced the amplification of EPSPs
by increasing the contribution of Na+ channels.
In our experiments, Ca2+ currents did not
contribute to either single or trains of EPSPs unless
K+ currents were extensively reduced by
pharmacological inhibition, and this contribution seemed secondary to
the activation of Na+ channels. In the previous
study of Seamans and colleagues (1997)
, nickel-insensitive Ca2+ channels were found to
amplify EPSPs in these same neurons after Na+
channels were blocked. The differences between the results could be
explained if the experimental conditions of Seamans and colleagues (1 M
Cs+, and 80-100 mM QX-314 in sharp-electrode
internal solution, plus 20 mM TEA in the bath solution) produced a
reduction of K+ currents greater than that
attained by 5 mM 4-AP and 20 mM TEA in our experiments. Perhaps an even
larger inhibition of K+ currents enables a
contribution of Ca2+channels independent of
Na+ channels. Another possibility is that the
contribution of Ca2+ channels found in the
previous study (Seamans et al. 1997
) is somehow tightly
coupled to the presence of a significant NMDA component in the EPSPs.
Indeed, whereas in the previous study pure NMDA EPSPs were recorded,
for EPSPs evoked in our experimental conditions, the NMDA component was
either absent or extremely weak. This suggests that the synaptic
population studied by Seamans and colleagues differed from the synaptic
population studied here with respect to the NMDA/AMPA conductance
ratio. A small NMDA/AMPA ratio in distal synapses, as found in our
experiments, is consistent with recent findings on the distal to
proximal distribution of NMDA and AMPA receptors in neocortical layer 5 pyramidal neurons (Dodt et al. 1998
). Therefore a
plausible explanation for the differences in NMDA component is that the
synapses stimulated in the previous study were located in dendritic
compartments significantly more proximal than those studied here.
Surprisingly, in both studies electrical stimulation was applied to
superficial layers 1 and 2 to activate synapses distally. The
mechanisms by which Ca2+ channel contribution to
EPSPs would be related to activation of NMDA receptors are at present
unclear. Duration of the depolarizing synaptic input seems not to be
the cause, since Ca2+ channels still did not
contribute to amplify trains of EPSPs lasting ~500 ms. Another
difference with the recordings from the previous study, is the presence
of Cs+, which is a blocker of the
hyperpolarization-activated H conductance. Until now, the role of H
channels in shaping EPSPs has not been examined in PFC neurons. In
other neurons, H channels shorten the EPSP duration, thus deactivation
of this conductance at depolarized potentials may explain in part the
effect of depolarization on EPSPs. It is possible that a contribution
of Ca2+ channels is favored by the combined
inhibition by Cs+ and deactivation by
depolarization of H channels, but this remains to be addressed.
Functional implications
Our present results confirm previous findings in other neuronal
types, showing that, at rest, neither Na+ or
Ca2+ channels amplify small EPSPs in cortical
pyramidal neurons. The depolarization-dependent enhancement of EPSPs by
Na+ channels found here and in previous studies
is dependent on somatic or axonal Na+ channels.
Thus it can boost EPSPs only after they invade the soma/axon region,
therefore not counteracting dendritic filtering. Because our drug
applications and dendritic current injections were done at
proximal/middle portions of the primary apical dendrite (280 µm
from the soma), we cannot rule out that in more distal dendritic
compartments Na+ or Ca2+
channels act to counter dendritic filtering of EPSPs. In other cell
types, activation of Na+ and
Ca2+ channels in the distal dendrites provides an
all-or-none electrogenesis mechanism that seems to detect coincident
activation of multiple synaptic connections, rather than amplifying
subthreshold EPSPs (Larkum et al. 1999a
,b
;
Zhu 2000
). Interestingly, recent experiments have shown
that, in CA1 pyramidal neurons, distal EPSPs are not usually amplified
by voltage-gated channels but that the AMPA synaptic conductance
actually increases with distance from the soma, compensating for the
dendritic attenuation of the EPSPs (Magee and Cook
2000
). Whether a similar scaling of distal synaptic conductance
occurs in pyramidal neurons from neocortex remains to be demonstrated.
The ionic mechanisms found here to shape subthreshold EPSPs may be
important to PFC-specific patterns of activity in a number of ways. The
EPSP decay time determines the degree of temporal summation
(Koch et al. 1996), which must be important to synaptic excitation and sustained firing in the PFC during working memory. Computer simulations in biophysically realistic network models (for a
review, see Durstewitz et al. 2000
) suggest that this
may be indeed the case. Specifically, Wang and colleagues have shown that EPSPs with kinetics, such as those determined by just an AMPA
synaptic conductance, decay too fast for the network to generate stable
sustained firing at rates consistent with delay period activity
(Compte et al. 2000
; Wang 1999
).
Increasing the EPSP decay time constant, by adding NMDA synaptic
conductance, made sustained firing more stable (Compte et al.
2000
; Wang 1999
). Here we demonstrate that, in
addition to the synaptic conductance, in PFC neurons the EPSP decay is
shaped by voltage-gated channels at subthreshold potentials whereby a
large fraction of synaptic NMDA channels would be blocked by
Mg2+ (Hestrin et al. 1990
). Thus,
we predict that incorporation into network models of the
voltage-dependent slowing of EPSP decay by active conductances would
make less critical the contribution of NMDA channels, perhaps
eliminating the requirement (for the models to reproduce delay-related
firing) of synapses with very high NMDA/AMPA receptor ratio
(Durstewitz et al. 2000
; Lisman et al.
1998
), which are uncommon in the adult neocortex.
Shaping of EPSPs by voltage-gated channels also suggests an important
mechanism by which neuromodulatory systems can regulate activity in
PFC. Input from midbrain dopamine neurons is particularly strong in the
PFC and other regions of frontal cortex but is very weak or absent in
posterior cortical regions, like somatosensory and visual areas
(Fuster 1997). Dopamine signaling in PFC is necessary for correct performance in working memory tasks (Brozoski et al. 1979
; Sawaguchi and Goldman-Rakic 1991
).
Dopamine, via G-protein-linked receptors, may modulate the
voltage-dependent channels that shape subthreshold EPSPs, having a
critical effect on temporal summation, and thus on the stability of
sustained firing in PFC. If neuromodulators were to affect EPSP decay
and temporal summation, then our results clearly point to
Na+ channels as a crucial target. In most
neurons, dopamine downregulates Na+ currents;
however, results obtained up to date with PFC cells are contradictory
since different groups report up- or downregulation (Geijo-Barrientos and Pastore 1995
; Gorelova and
Yang 2000
; Maurice et al. 2001
; Yang and
Seamans 1996
). Modulation of some K+
currents also would affect EPSP kinetics and summation and could potentially enable contribution of Ca2+ channels.
It remains to be demonstrated if neuromodulation can reduce
K+ currents to the levels found to be necessary
to reveal an effect of Ca2+ channels in shaping
EPSPs in vitro.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Nathaniel N. Urban for helpful comments and discussions during the course of the experiments and Drs. James G. Dilmore and Jeremy K. Seamans for comments on an earlier version of the manuscript.
This work was supported by National Institute of Mental Health Grants MH-45156 and MH-51234.
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FOOTNOTES |
---|
Address for reprint requests: G. González-Burgos, Dept. of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260 (E-mail: burgos{at}bns.pitt.edu).
Received 5 February 2001; accepted in final form 5 June 2001.
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REFERENCES |
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