Download Axons, but not cell bodies, are activated by electrical stimulation in

 Springer-Verlag 1998
Exp Brain Res (1998) 118:489±500
RESEARCH ARTICLE
Lionel G. Nowak ´ Jean Bullier
Axons, but not cell bodies,
are activated by electrical stimulation in cortical gray matter
II. Evidence from selective inactivation of cell bodies and axon initial segments
Abstract The results presented in the companion paper
showed that extracellular electrical stimulation of the gray
matter directly activates axons, but not cell bodies. The
second set of experiments presented here was designed
to separate the contribution of the axon initial segments
and cell bodies from that of the axonal branches to the
pool of presynaptic neuronal elements activated by electrical stimulation. For that purpose, N-methyl-d-aspartate
(NMDA) iontophoresis was used to induce a selective inactivation of the cell body and of the adjoining portion of
the axon by depolarization block, without affecting axonal branches that lack NMDA receptors. After NMDA
iontophoresis, the neurons located near the iontophoresis
electrode became unable to generate action potentials in
an irreversible manner. When the NMDA-induced depolarization block was performed at the site of electrical
stimulation, an unexpected increase in the amplitude of
the orthodromic responses was observed. Several control
experiments suggested that the field potential increase
was due to changes of the local environment in the vicinity of the iontophoresis pipette, which led to an increased
excitability of the axons. After the period of superexcitability, the orthodromic responses displayed an amplitude
that was 15Ð20% lower than that observed before the
NMDA-induced depolarization block, even though cell
bodies and axon initial segment at the site of stimulation
could not be activated by electrical stimulation. This result shows a low contribution for axon initial segments
to the pool of neuronal elements activated by the electrical stimulation. Altogether, these experiments demonstrate that the postsynaptic responses obtained after electrical stimulation of the cortical gray matter result almost
exclusively from the activation of axonal branches. Since
the neocortex is organised as a network of local and long-
)
L.G. Nowak1 ´ J. Bullier ( )
INSERM UnitØ 371, ªCerveau et Vision,º
18 Avenue du Doyen LØpine, F-69500 Bron, France
Present address:
Section of Neurobiology, Yale University School of Medicine,
C303 Sterling Hall of Medicine, 333 Cedar Street, New Haven,
CT 06510, USA
1
range reciprocal connections, great attention must be paid
to the interpretation of data obtained with electrical stimualtion.
Key words Visual cortex ´ Brain slice ´ Intracortical
microstimulation ´ NMDA ´ Rat
Introduction
The results reported in the companion paper (Nowak and
Bullier 1997) suggest that, when a post synaptic response
is observed after an extracellular electrical stimulation,
axons, rather than cell bodies, are the presynaptic elements activated by extracellular electrical stimulation.
This, we argued, results from the different biophysical
properties of axons and cell bodies, endowing axons with
a lower threshold for activation. However, the relative
contribution of axon initial segments and that of axonal
branches could not be determined from the chronaxie
measurements used in that study. The aim of the experiment reported in the present paper was to determine in
what proportion axon initial segments and axonal branches are activated by extracellular electrical stimulation.
For that purpose, we chose to compare the effects of
electrical stimulation before and after the inactivation of
one of these two groups. To achieve this comparison,
we used a method that prevents spike initiation at the level of the cell body and initial segment, while leaving undisturbed the generation and propagation of action potentials in axonal branches. This method consisted in applying NMDA by iontophoresis at the site of stimulation. It
may look somewhat paradoxical to use NMDA to inactivate neurons, but the following explanation gives the rationale for such a choice. Dendrites and possibly cell bodies possess glutamate receptors that are activated by
NMDA (reviewed in Huntley et al. 1994b). The reversal
potential for NMDA-gated channels is about 0 mV (Crunelli et al. 1984; Nowak et al. 1984). On the other hand,
sodium channels of neocortical neurons are inactivated
at a potential of Ÿ40 mV (Huguenard et al. 1988). There-
490
fore, NMDA can depolarize neurons by such an amount
that sodium channels are inactivated. In that situation,
dendrites and cell bodies are in a state of ªdepolarisation
block.º It follows that extracellularly applied electrical
stimulation is unable to elicit any action potential. The
initial segment should also be blocked, and presumably
also the first node of Ranvier, because they are electrotonically close to the cell body (Gogan et al. 1983). On the
contrary, axonal branches, which do not possess glutamate receptors, should not be affected. If electrical stimulation is applied during NMDA-induced depolarization
block, only axonal branches should be activated. Then,
by comparing results obtained with electrical stimulation
applied before, during, and after the NMDA-induced depolarization block, it was possible to determine whether
initial segments on the one hand, or axonal branches on
the other hand, are the elements activated by electrical
stimulation. The conclusion we finally reached is that
the postsynaptic effects obtained after electrical stimulation in the cortical gray matter result almost exclusively
from the activation of axonal branches.
Materials and methods
Brain slice preparation
The procedure for the preparation of cortical brain slices is the same
as the one described in the preceding paper.
Recording and stimulation
Recording and stimulation were performed in areas 17 and 18a of rat
visual cortex. Some of the results presented in this paper were obtained from neurons involved in corticocortical connections between
these two areas, identified as described in Nowak et al. (1997). Extracellular recording of field potentials (Figs. 3Ð5) was made with
glass micropipettes filled with 1 M NaCl (d.c. resistance 1Ð5 MW).
Field potentials were amplified on an amplifier (Biologic VF 180)
followed by a Neurolog device containing amplifiers and filters.
The field potentials were low- and high-pass filtered between 0.1
or 1 Hz and 1 khz.
The extracellular recordings of the action potentials and field potentials presented in Figs. 1 and 2 were obtained with tungsten-in-glass
microelectrodes (Merrill and Ainsworth 1972) with 15 to 25-m exposed tips and plated with platinum black (impedance less than 0.5
MW at 1000 Hz).Amplification and filtering were achieved through
a Neurolog recording system. Filtering was adjusted for individual recordings to give a proper signal-to-noise ratio. Tungsten-in-glass microelectrodes plated with platinum black were also used for electrical
stimulation. The glass was removed over a length of 15Ð25 m. Electrical stimulation consisted of monopolar, cathodal pulses of 0.2 ms
duration. Single pulses were delivered at a frequency of 0.5 Hz or
0.3 Hz through a stimulation isolation unit (Neurolog).
Iontophoresis
NMDA (Sigma) was dissolved in distilled water to a final concentration of 200 mM (pH adjusted to 8.0 with NaOH). Iontophoresis
was made through double-barreled glass micropipettes. One barrel
was filled with NMDA and the other, used for return current, with
1 M NaCl.
Tungsten-in-glass microelectrodes, used for stimulation and/or
recording, were glued to iontophoresis micropipettes with dental ce-
ment. The distance between the tips varied between 20 and 150 m.
For small tip separations, we noticed that the leakage of NMDA
could induce action potentials and eventually a depolarization block.
These effects were avoided by using a retaining current of 20 nA.
Results
Distance at which NMDA induces a depolarisation block
Before starting the experiments themselves, two sets of
control experiments had to be done. Since NMDA was
iontophoretically applied, the first series of control experiments was designed to determine the radius of the sphere
within which neurons are in depolarization block for a
given current of iontophoresis.
The set-up for such experiments is shown in Fig. 1A.
Action potentials were recorded through tungsten-in-glass
microelectrodes that were glued to the iontophoresis micropipettes. The lateral separation between the microelectrode and the pipette tips was 60, 120, and 150 m for
three electrode assemblies from which the results shown
in Fig. 1 were obtained. For the recordings obtained with
the electrode assembly of 150 m tip separation, electrical
stimulation was applied in the white matter or in the lower
cortical layers to induce orthodromic or antidromic action
potentials. For the other two electrode assemblies, action
potentials were produced by the leakage of NMDA or by
applying small amounts of ejection current. Once action
potentials were elicited by one of these two modes, the
iontophoresis current was raised in steps of 5 nA or 10
nA until action potentials were suppressed.
One example is shown in Fig. 1B. Each trace corresponds to the superposition of 20 sweeps. Electrical stimulation was applied in the white matter and elicited orthodromic action potentials. The large action potentials disappeared with a iontophoresis current of 30 nA. They recovered after the iontophoresis apparatus was switched
back to retaining mode.
Another example is illustrated in Fig. 1C. An antidromic action potential was produced by white matter
stimulation. The antidromic action potential was suppressed by a iontophoresis current of 40 nA. The recovery
trace shows that the action potentials have a smaller amplitude and a slightly longer latency than in the control
situation. This is not unexpected, since both latency and
amplitude of antidromic action potentials can be modified
by changes of the neuron input resistance and resting potential (see Lipski 1981), both effects that NMDA is likely to produce before its complete washout. Figure 1B and
C shows in addition that, during the NMDA-induced depolarization block, the action potential is entirely suppressed, not simply reduced in amplitude. This indicates
that the axon initial segment also lost its ability to generate action potentials.
Figure 1D presents the distribution histogram of the
iontophoresis current leading to a depolarization block,
measured with the electrode assemblies having separations of 120 m and 150 m. The pale gray bars corre-
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Fig. 1A±D Distance at which NMDA produces a depolarization
block. A Set-up. A tungsten-in-glass microelectrode for extracellular
recording and a micropipette for NMDA iontophoresis were glued
together, such that the separation between their tips was perfectly
controlled. Recording and iontophoresis were limited to the supragranular layers. The axis defined by the line passing at the tip of
both electrodes was parallel to the pial surface, so that recording
and iontophoresis were performed in the same cortical layer. B
One example of NMDA-induced depolarization block. The separation between the tips of the recording microelectrode and the iontophoresis pipette was 150 m. Orthodromic action potentials were
elicited by white matter stimulation (upper trace). NMDA applied
with an iontophoresis current of 30 nA led to their suppression (middle trace). They recovered (lower trace); however, they are less
grouped than during the control period. Although they were not tak-
spond to the electrode assembly for which the tip separation was 120 m. In that case, the neurons were activated
by the NMDA itself. The dark gray and the hatched bars
correspond to the electrode assembly for which the tip
separation was 150 m. The action potentials in that case
were induced by electrical stimulation. These were either
en into account owing to uncertainty relative to their separation from
both iontophoresis and recording electrodes, one can observe that
the small long-latency action potentials visible in the control period
did not recover. Another example of NMDA-induced depolarization
block appears in C. In that case the white matter stimulation produced an antidromic action potential. About ten individuals sweeps
were averaged for the different periods and are shown superimposed. The antidromic action potential was suppressed with a iontophoresis current of 40 nA. D Histogram illustrating the number of
neurons in which NMDA induced a depolarization block as a function of the iontophoresis current required to produce this effect. Data
presented are from two electrode assemblies having 150 m or 120
m separation between the recording and the iontophoresis tips. In
this and the following figures, positive potential is upward
antidromic action potentials (hatched bars) or orthodromic action potentials (dark gray bars).
The histogram shows that one-half of the cells were in
a state of depolarization block with iontophoresis current
equal to 30 nA when the separation was 120 m, and 40
nA when the separation was 150 m. It further shows that
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Fig. 2A, B Time course of the
NMDA-induced depolarization
block. A Example of an antidromic population spike and its
suppression by NMDA. Each
trace corresponds to the mean of
ten sweeps. One trace was obtained every 5 min. Time is indicated on the right of the series
of field potentials every two
traces. The control period was
established between 0 and 30
min. NMDA was iontophorized
between 30 and 40 min with an
ejection current of 80 nA. The
NMDA led to an almost complete suppression of the antidromic population spike. The
recovery period corresponds to
time 40 to 110 min. The antidromic population spike did not
recover. B Line chart summarizing the effect of NMDA on
antidromic population spikes in
seven experiments. The control
period was always 30 min. The
period of NMDA application
(30±40 min) is indicated by the
two vertical dashed lines. The
recovery period was at least 30
min (40Ð70 min) but was longer for two experiments. The
bars show 1 SEM. The horizontal dashed line corresponds
to the mean control value
(100%). NMDA led in all cases
to a strong reduction of the
population spike amplitude,
without recovery
all neurons located at a distance of 150 m from the iontophoresis electrode were inactivated with a current of 80
nA. With the electrode assembly having a tip separation
of 150 m, the action potentials that were antidromically
elicited were suppressed with an iontophoresis current
similar to that required for the suppression of orthodromic
action potentials (Mann-Whitney U-test, P=0.9). With the
electrode assembly having a tip separation of 60 m (not
shown in the histogram), all the neurons became unable to
generate action potentials with a iontophoresis current of
20 nA only.
Time course of the NMDA-induced depolarization block
The second control experiment consisted in determining
the duration of the NMDA-induced depolarization block.
The set-up used for these experiments was the same as the
one presented in Fig. 1. The only difference was that the
filtering of the recording obtained through the tungstenin-glass microelectrode was modified to allow the measurement of antidromic population spikes (the simulta-
neous antidromic activation of a large number of cells)
produced by electrical stimulation of the white matter of
the lower cortical layers.
One example appears in Fig. 2A. A control baseline
was first established, corresponding to the antidromic
population spikes obtained from time 0 to 30 min. After
the last recording of the control period (30 min), the iontophoresis was switched from its retaining mode to the
ejection mode. The ejection current was 80 nA and was
maintained for 10 min. During the iontophoresis of
NMDA, the antidromic population spikes were almost
completely suppressed (traces at 35 min and 40 min). After the 10 min of NMDA application, the ionotophoresis
was switched back to its remaining mode. Recordings obtained at time 45±110 min in Fig. 2A show that there was
no recovery of the initial population spikes.
We determined the time course of the NMDA-induced
depolarization block in seven cases. The distance between
the tip of the iontophoresis micropipette and that of the
recording microelectrode was 24 m in one case, 35 m
in three cases, 60 m in two cases, and 120 m in one
case. Figure 2B illustrates the pooling of these seven ex-
493
periments. The maximum amplitude of the antidromic
population spikes was first measured for each individual
case. The mean of the control values (obtained between
0 and 30 min) was thereafter calculated and used to normalize the amplitudes. Once expressed as percentages, the
mean of the seven amplitudes obtained at a given time
was calculated.
Figure 2B shows that the iontophoresis of NMDA led
to a very strong decrease in the amplitude of antidromically elicited population spikes in all the cases. This decrease persisted during the whole period following iontophoresis. For five of the seven cases, this period lasted 30
min (40Ð70 min in Fig. 2B). For another case, it lasted
60 min, and for the seventh, which is illustrated in Fig.
2A, it lasted 70 min.
Field potentials were not completely suppressed, however. There remained about less than 10% of the control
amplitude. This remaining activity could represent a contribution of action potentials recorded from axons. Alternatively, it could correspond to the contribution of action
potentials from neurons that are far enough to be unaffected by NMDA. If this was the case, the neurons generating
these action potentials must have been located at a distance larger than 150 m from the iontophoresis micropipette, since, as shown in Fig. 1, an iontophoresis current
of 80 nA suppressed the single-unit activities up to that
distance at least.
The lack or recovery is at variance from the examples
shown in Fig. 1B and C. However, the recovery of singleunit action potentials was observed only if the iontophoresis current was applied for a short period corresponding
to the time necessary to check that the action potentials
were suppressed. If the iontophoresis was extended to a
few minutes, there was no recovery of single-unit activities. Therefore, when iontophorized for 10 min with 80
nA, the action of NMDA was not simply a transient inactivation, since neurons definitively lost their ability to
generate action potentials. Rather, NMDA induced a microlesion of the neurons, which proved to be irreversible.
Stimulation during NMDA-induced depolarization block
The set-up of these experiments is shown in Fig. 3A. The
electrical stimulation was applied at the site of NMDA
iontophoresis (labeled ªstim.º in Fig. 3A). In all the cases
stimulation and iontophoresis were made in the supragranular layers. The separation between the tips of the
stimulating electrode and iontophoresis pipette was between 20 and 35 m. For this series of experiments
(n=12), electrical stimulation intensity was 15 A in nine
cases and 12 A in the three other cases.
The electrical stimulation must activate neuronal elements contained in an area smaller than the area in which
NMDA induces a depolarization block. According to experiments reported in another paper (Nowak and Bullier
1996), a stimulating intensity of 15 A should activate
neuronal elements contained within a sphere having a
maximum radius of 82 m. The dark gray area surround-
ing the stimulating and iontophoresis electrodes in Fig. 3A
schematizes this area. The light gray area in Fig. 3A schematizes the area in which neurons are in a state of depolarization block with 80 nA of iontophoresis current. According to the experiments illustrated in Fig. 1, this area has a
minimum radius of 150 m.
Electrical stimulation was used to elicit orthodromic
field potentials. All recordings were made in the supragranular layers. The sets of connections that were activated by
electrical stimulation in these experiments were either the
intrinsic horizontal connections (n=6, separation between
recording and stimulating sites ranging from 0.5 to 0.9
mm) or the corticocortical connections (n=6, separation between recording and stimulating sites ranging from 1.1 to
1.5 mm).
If electrical stimulation effectively activates axon initial
segments, cell bodies, and dendrites, then the amplitude of
the orthodromic field potential should be reduced when the
stimulation is applied during the NMDA-induced depolarization block. Unexpectedly, the opposite was observed.
One series of orthodromically elicited field potentials is presented in Fig. 3B. After a control period of 30 min, NMDA
(80 nA) was applied for 10 min. There was not a decrease,
but an increase in the field potential amplitude during the
NMDA induced depolarization block (time 35 min and 40
min). In other words, when neurons (dendrites, cell bodies,
and initial segments) are in such a state that they cannot be
activated by the electrical stimulation, the amplitude of the
field potential is larger than when they could contribute to
the pool of neuronal elements activated. During the following 30 min of recovery, the amplitude of the field potential
declined and reached its initial control value at time 60 min.
The data pooled from the 12 experiments are shown in
Fig. 3C. The maximum amplitude of the field potential
was measured and normalized as already described. Due
to the small stimulation intensity and the large separation
between stimulating electrode and recording micropipette,
the field potential amplitude was often small and averaged
Ÿ160109 (SD) V during the control period. In all the
cases, the amplitude increased during the NMDA iontophoresis, by a factor larger than 1.5 on average. The increase in
amplitude was highly significant.
The amplitude of the field potentials returned to its control value 20 min after the end of the iontophoresis. The amplitudes measured at 60, 65 and 70 min are not significantly
different from those of the control period (unpaired t-test,
P>0.05). For 5 of the 12 cases, however, the recovery period was prolonged from 30 to 50 min. This extended recovery period is illustrated by the gray dots in Fig. 3C. It can be
seen that, when the amplitude reaches a stable value, it is
significantly smaller than the control value (unpaired t-test,
P<0.05 for time 75Ð90 min).
These experiments show that, when there was hardly any
cell body or initial segment that could be activated at the site
of stimulation (Fig. 2), the amplitude of the orthodromic field
potential was decreased by 15Ð20% only compared with
what it was before the NMDA induced microlesion. This
suggests that the contribution of dendrites, cell bodies, and
axon initial segments to the pool of presynaptic neuronal el-
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Fig. 3A±D Stimulation during NMDA-induced depolarization
block. The set-up is illustrated in A. The stimulation (Stim) was applied at the site of NMDA iontophoresis. The dark gray circle near
the tip of the stimulating-iontophoresis electrode assembly schematizes the sphere in which neuronal elements are activated with a current of 15 A or less, which is smaller than the sphere in which neurons are led to a depolarization block with a iontophoresis current of
80 nA (schematized by the light gray area). An example of the
changes of an orthodromic field potential when stimulation is applied at the site of NMDA ejection before, during and after the iontophoresis appears in B. The recordings obtained between 0 and 30
min correspond to the control period and were used to establish the
baseline value. Iontophoresis of NMDA (80 nA) was performed between 30 and 40 min. Time 40±70 min corresponds to the recovery
period. C Line chart summarizing the evolution of the orthodromic
field potential amplitude measured every 5 min in 12 experiments.
The dots represent the mean amplitude expressed as percentage of
the baseline value. The bars indicate SEM. The gray dots correspond to five experiments for which the recovery period was prolonged from 30 to 50 min. D Evolution of the peak latency of the
orthodromic field potentials. It appears unaffected by the NMDA
iontophoresis
ements activated by electrical stimulation was much lower
than the contribution of axonal branches, which accounts
for 80Ð85% of the postsynaptic response observed.
Origin of the transient increase in amplitude
Because the increase in amplitude of field potentials observed during the iontophoresis of NMDA was unexpected, we designed additional experiments to determine its
origin. We first reasoned that the NMDA, spreading from
the iontophoresis pipette, could reach neurons located
outside the region of depolarization block and depolarize
them in a tonic fashion. This depolarization could facilitate their orthodromic activation by the axons stimulated
near the stimulating electrode that project onto them. If,
in addition, these neurons were sending projections to
the neurons we were recording, their activation would
lead to the activation of additional synapses, thereby to
an increase in field potential amplitude. Nevertheless, this
495
Fig. 4A±C Evolution of orthodromic field potentials when
electrical stimulation is applied
at a site remote from the site of
NMDA-induced depolarization
block. Set-up illustrated in A.
The stimulation (Stim.) was applied at the site remote from the
site of NMDA iontophoresis. B
Time series of a field potential
obtained when the electrical
stimulation was applied in the
infragranular layers. The pooled
data from five experiments are
presented in C. In all five cases
the stimulation was applied in
the infragranular layer in an interleaved fashion with those applied at the site of NMDA iontophoresis (5 of the 12 cases
reported in Fig. 3C). The dots in
C represent the mean of the
amplitude after normalization
with respect to the baseline value. The bars indicate 1 SEM.
The increase in field potential
amplitude was not observed
when stimulation was applied at
a site remote from the site of
NMDA iontophoresis
increased amplitude would result from the recruitment of
a disynaptic pathway, implying that the latency of the
maximum amplitude of the field potential should increase. Therefore, to determine whether NMDA led to
the recruitment of a disynaptic pathway, we measured
the latency of the maximal amplitude of the field potentials (peak latency). The case illustrated in Fig. 3B does
not show the expected change in latency. The pooled data
for the 12 experiments is illustrated in Fig. 3D. The mean
latency remains very close to 100%, during both the
NMDA iontophoresis and the recovery period. The peak
latency during and after the NMDA iontophoresis was, indeed, not significantly different from its control value
(unpaired t-test, P>0.05). This suggests that the transient
increase in field potential amplitude was not the consequence of the recruitment of a polysynaptic pathway.
Two other possibilities were tested in the same experiments. These two possibilities have in common some
properties of cortical neurons, which are their inward retification properties. These properties are such that, when a
neuron is depolarized from rest, the amplitude of the
EPSP can increase, contrary to what would be predicted
if the amplitude of the EPSPs was solely determined by
the driving force. These rectifying properties are related
to the presence of some voltage-dependent conductances
(Stafstrom et al. 1985; Deisz et al. 1991) and to the behavior of glutamate receptors of the NMDA subtype
(MacDonald et al. 1982; Flatman et al. 1983; Nowak et al.
1984; Thomson 1986a, b).
For these rectifying properties to be turned on, it is required that the neurons we were recording from underwent a tonic subthreshold depolarization during the ionto-
496
Fig. 5A±C Evolution of the
amplitude of antidromic population spikes evoked by electrical stimulation applied before,
during, and after NMDA-induced depolarization block.
A Set-up. The stimulating-iontophoresis electrode assembly
was placed in the infragranular
layers. The stimulation was used
to elicit antidromic population
spikes that were recorded in the
supragranular layers. An example appears in B. Time 0±30 min
corresponds to the control period. NMDA was iontophorized
(80 nA) between 30 and 40 min.
C Line chart summarizing the
time course of the antidromic
population spike amplitude before, during, and after the iontophoresis of NMDA. Six experiments are pooled. The dots
correspond to the mean amplitude expressed as percentage of
baseline value and the bars to
1 SEM. This shows that antidromic population spikes resulting from axonal activation
were increased in a way similar
to orthodromic field potentials
by the iontophoresis of NMDA
at the stimulating site
phoresis of NMDA. Such tonic subthreshold depolarization could have occurred if the NMDA was spreading
from the site of iontophoresis to the recording site, although such a spread may be considered unlikely given
the large separation between recording and iontophoresis
sites. Alternatively, NMDA could have activated neurons
that were in the periphery of the area of depolarization
block, which would induce a tonic firing at their level.
If these neurons were projecting to the neurons we were
recording from, their tonic firing could have induced a
subthreshold depolarization at the recording site. In both
cases, the depolarization could have triggered the mechanisms responsible for the anomalous rectification. This
hypothesis implies that the changes were occurring at
the level of the neurons we were recording from Therefore, the amplitude of field potentials should have increased also if the electrical stimulation was applied at
a position remote from the iontophoresis micropipette.
This hypothesis was tested and the results are presented in Fig. 4. The stimulating electrode was in a site distant
from that of iontophoresis, in the lower cortical layers below the recording site (Fig. 4A). A series of field poten-
tials obtained before, during, and after the NMDA iontophoresis is shown in Fig. 4B. These field potentials were
obtained in an interleaved fashion with those shown in
Fig. 3B (This explains why the timing is deplayed by
2.5 min with respect to the timing of the measurements
of Fig. 3B). Contrary to those of Fig. 3B, however, the
amplitude of the field potentials of Fig. 4B did not increase during the iontophoresis of NMDA, even though
the same neurons were recorded. Figure 4C summarizes
the results obtained in five similar experiments. In no case
did the field potentials amplitude increase during the
NMDA iontophoresis. This excludes the subthreshold depolarization and the associated activation of inward rectification in the neurons located at the site of recording as
the mechanism responsible for the increase in field potential amplitude that was observed when stimulation and
iontophoresis were given at the same site. Neither the
spread of NMDA to the site of recording nor the depolarization of neurons at the recording site by a tonic drive
from the neurons located at some distance from the iontophoresis electrode are responsible for the transient increase.
497
Therefore, the origin of the transient depolarization
must be located at the site of the iontophoresis itself.
The massive depolarization caused by NMDA must have
led to a modification of the local environment. In particular, there should have been an increase in the extracellular concentration of potassium. It is known that increasing
the extracellular concentration of potassium increases the
excitability of neurons (Hablitz and Lundervold 1981;
Balestrino et al. 1986). It is also known that excitatoxic
lesions are accompanied by a swelling of the neuronal
processes (Garthwaite et al. 1986; Choi 1987). This swelling might have increased the extracellular impedance,
leading to an increased extracellular potential for a current of the same intensity. In both cases, applying NMDA
would, indirectly, have acted as if the threshold for activation of neuronal elements was decreased.
Figure 5 shows that, in fact, the excitability of axons
was increased by the application of NMDA. In these experiments, the set-up of which appears in Fig. 5A, the
electrical stimulation and the NMDA were applied in
the lower cortical layers while the recordings were obtained in the supragranular layers. Unlike in the preceding
cases, we recorded antidromically elicited population
spikes that resulted from the activation of axons passing
near the stimulating electrode (note the different shape
and time course of the responses illustrated in Fig. 5B
compared with Figs. 3B, 4B). An example of a time series
of population spikes is presented in Fig. 5B. After the
control period of 30 min, NMDA was iontophorized (80
nA) for 10 min. Similarly to what was observed with
the orthodromic field potentials of Fig. 3B, the application of NMDA also led to an increase in amplitude for
the antidromic population spikes.
Summary data after pooling of six experiments is given in Fig. 5C. In all the cases, the amplitude of the antidromic population spikes increased when electrical stimulation was applied during the iontophoresis of NMDA.
After the end of the iontophoresis, the amplitude decreased toward the control value. However, the control
value was not reached after 70 min. This is due to two
cases, which retained an amplitude higher at 70 min than
during the control period. In one of these two cases, the
recovery was prolonged and it was found that the amplitude remained stable at this higher value.
In summary, these different experiments suggest that
the local environment surrounding the iontophoresis micropipette was modified by the NMDA application. The
most likely explanations for the transient increase in both
orthodromic and antidromic field potentials are the following: NMDA application might have induced a massive
depolarization of the neurons. This would have led to an
increase in the extracellular concentration of potassium.
This extracellular potassium would have increased the excitability of neuronal elements present in the neighborhood. Alternatively, the application of NMDA might have
led to a swelling of the neuronal processes that would
have resulted in an increase in the extracellular impedance, hence to a decreased threshold for axonal activation.
The consequence of that increased excitability was an in-
crease in the amplitude of the responses. After the iontophoresis, as the NMDA was washed out, the amplitude of
the responses returned to its baseline value as the concentration of potassium was equilibrated with that of the
bathing medium or as the neuronal swelling was resumed.
Discussion
The experiments based on chronaxie measurements suggested that, when electrical stimulation is applied close
to a cell body, its axon initial segment is preferentially activated and problably also a number of axonal branches
located in the vicinity of the electrode (Nowak and Bullier
1997). However, the chronaxie measurement did not allow the differentiation between the activation of axonal
branches and that of axon initial segments. It could be argued that initial segments are more excitable than axonal
processes and may contribute for a large part to the postsynaptic responses elicited by extracellular electrical
stimulation. The experiments based on stimulation during
NMDA-induced depolarization block, designed to determine this contribution, show that the postsynaptic responses obtained after stimulation of the cortical gray
matter results not from the activation of cell bodies or initial segments but almost exclusively from the activation
of axonal branches.
NMDA suppresses action potential electrogenesis
The first requirement for these experiments was that axonal branches are not affected by NMDA. Several immunohistochemical studies have shown that axonal trunks
lack NMDA receptors (Aoki et al. 1994; Huntley et al.
1994a; Petralia et al. 1994). That axons are not sensitive
to glutamate has also been shown with electrophysiological methods (Christian and Dubek 1988). This is substantiated by the results of Fig. 5, showing no attenuation of
antidromic population spike after their transient increase,
which also indicate that the current used for iontophoresis
did not lead to any axonal damage.
The second requirement for these experiments was that
NMDA induced a depolarization block in all neurons.
Electrophysiological studies have shown that all neocortical pyramidal cells bear NMDA receptors: all respond to
NMDA applied iontophoretically and all display components in their EPSPs that are mediated by NMDA receptors (Thomson 1986a, b; Sutor and Hablitz 1989a, b;
Thomson et al. 1989; Artola and Singer 1990; Armstrong-James et al. 1993). These electrophysiological evidences are paralleled by several recent labeling and immunohistochemical studies (Benke et al. 1993; Aoki et al.
1994; Huntley et al. 1994a; Petralia et al. 1994) that indicate that the very large majority of cortical neurons possess NMDA receptors. In fact, the only type of neuron
that may lack NMDA receptors corresponds to a subpopulation of inhibitory neurons (Huntley et al. 1994a; see
also Thomson et al. 1993; Thomson and Deuchars
498
1994). That some inhibitory neurons lack NMDA receptors was not a problem in our case, since we were interested in inactivating excitatory neurons having long-range
projections. All the neurons became unable to elicit action
potentials after the iontophoresis of a sufficient amount of
NMDA (Fig. 1). Our conclusion that NMDA leads to a
depolarization block was based on extracellular recordings. A more precise measure of the sodium channel inactivation induced by NMDA would have been obtained using intracellular recording. However, several studies
based on intracellular recording have already shown that
small amounts of glutamate or NMDA effectively depolarize cells to well above the threshold for action potential
initation (Schwarztkroin 1975; Hablitz 1982; Sutor and
Hablitz 1989b).
The third requirement was that the initial segment was
blocked. We also showed that action potentials were not
only partially, but completely, suppressed by NMDA.
This indicates that the depolarization block induced in
the cell body spread to the initial segment that become unable to elicit action potentials. It has been suggested that
the action potential initiation site is not necessarily the
initial segment, but the first node of Ranvier on the axon
(Gogan et al. 1983; Golbert and Johnston 1996). In our
experimental situation, it has not been possible to test
whether the NMDA-induced depolarization block extended to the first node of Ranvier, although this could have
occurred given the long space constant of the axon (Gogan et al. 1983). If this occurred, however, it would have
led to an underestimate of the proportion of axons activated by electrical stimulation, which is already of 80Ð85%.
One of the reasons why we chose NMDA is that, being
an artificial agonist, its spread is not limited by uptake
systems. We indeed observed that small amounts of
NMDA were able to suppress action potential generation
at a relatively large distance from the iontophoresis micropipette (Fig. 1). There was no recovery of action potential electrogenesis after 10 min of NMDA iontophoresis (Fig. 2) even after 1 h of recovery. That excessive glutamate or NMDA concentration is neurotoxic has been
demonstrated in a number of studies (Rothman 1985;
Garthwaite et al. 1986; Choi 1987; Lehmann and Jacobson 1990; McNamara and Dingledine 1990). The effects
produced by NMDA corresponded to a chemical microlesion in a restricted part of the cortex.
Stimulation during and after NMDA-induced
cortical microlesion
When NMDA was used to suppress action potential generation in cell bodies and axon initial segments at the site
of stimulation, there appeared to be an unexpected increase in the orthodromic field potential amplitude. The
control experiments (Figs. 4, 5) showed that the increase
was due to changes at the level of the iontophoresis electrode. The most probable change that occurred was a
modification of the local concentration of ions or a
change in the local impedance of the extracellular medi-
um, which led to an increased excitability for the neuronal
elements present in the vicinity.
The transient increase in the field potentials observed
during the NMDA iontophoresis obscured the effect of
the inactivation. Another reason why we chose NMDA
is the irreversibility of its effects. After the period of superexcitability, the stimulation yielded field potentials
that were lower by 15Ð20% only than those obtained
during the control period (Fig. 3). This strongly suggests
that the activation of axonal branches is responsible for
the bulk (around 80Ð85%) of the orthodromic responses
observed after electrical stimulation in the cortical gray
matter.
Our conclusions are at variance from those obtained by
Hirsch (1995). To determine the contribution of axonal
branches to the pool of neuronal elements activated by
electrical stimulation, she used local application of
GABA near the stimulating electrode to raise the activation threshold for cell bodies. In her Fig. 1 (upper part),
the GABA application led to the disappearance of evoked
synaptic responses. Nevertheless, these synaptic responses clearly appear as polysynaptic. That polysynaptic responses are suppressed by GABA is not so surprising
and does not exclude that axonal branches were first activated to trigger orthodromic responses in the inserted neurons. The lower part of Hirschs Fig. 1 shows suppression
of an excivatory postsynaptic current (EPSC) that presents features more typical of a monosynaptic response,
which was also suppressed by GABA application. It could
be that this EPSC resulted from the direct activation of an
initial segment.
The small contribution of axon initial segments to the
pool of neuronal elements activated to produce orthodromic responses can be understood given the difference in
density for axonal branches and initial segments (or cell
bodies). The density in cell body of rodent visual cortex
is around 100000/mm2 of cortical surface (Peters 1987;
Schüz and Palm 1989; Beaulieu 1993). Data for axonal
density are not available (to our knowledge) for rat visual
cortex, but those obtained for mouse area 17 should give a
correct approximation: according to data presented by
Braitenberg and Schüz (1991), the density of axonal
branches should be 4100000/mm2. In other words, there
are 41 times more axonal branches than initial segments
in a given volume of cortical gray matter. If initial segments and axonal branches display the same threshold
for activation, then the initial segments activation would
account for only 2.4% of the postsynaptic response obtained. This is even lower than the 15Ð20% determined
from our experiments. This discrepancy can be related
to different parameters: first, it could reflect a higher excitability for the initial segments compared to axonal
branches. This could be related to the larger diameter of
the axon initial segment (Farinas and DeFilipe 1991)
and of the main axonal trunk compared with the diameter
of axon collaterals, especially the unmyelinated axon collaterals (Peters and Sethares 1996). Second, a more precise evaluation of the contribution of axonal branches
compared with that of initial segment might be obtained
499
if the density of the nodes of Ranvier was known, although not all axons are myelinated in the cortical gray
matter. Finally, the axon of cortical neurons branches extensively in the vicinity of the cell body as well as in distant patches, such that the axon from a single cell might
contribute several times to the number of axons encountered in one sections: this might lead to an overestimation
of the true proportion of axons compared with that of cell
bodies.
Conclusion
Whenever a potsynaptic effect is elicited after an electrical stimulation in the cortical gray matter, this effect results mainly from the activation of axonal branches. The
biophysical properties of the axon favor their activation
by extracellular electrical stimulation (Nowak and Bullier
1997), while the number of axon initial segments is too
small with respect to the number of axonal branches to represent a significant component in the responses elicited
by electrical stimulation.
That electrical stimulation mainly activates axonal
branches is not so much a problem if the aim is just to
elicit a postsynaptic potential. However, when electrical
stimulation is used to activate a given set of connections
in order to elicit a response in a particular target, there is a
problem that should not be neglected. Examination of the
literature shows that, in general, this has not been the
case. Electrical stimulation of the cortical gray matter
can lead to erroneous interpretation, because, first, the
connections linking two points in the cortex are always
reciprocal, and second, because all pyramidal cells of
the neocortex give rise, in addition to their projection axon, to a number of local axon collaterals arborizing in the
vicinity of the cell body. When a stimulation is applied,
for example in area 17, it will activate orthodromically efferent axons from this area that project onto area 18. But
the stimulation will also induce action potentials in the
axons of area 18 neurons that project to or travel near
the stimulation site in area 17. The action potentials will
propagate antodromically to area 18, where they will invade the intrinsic collaterals. At the end of the collaterals,
they will elicit a postsynaptic potential. What happens in
that case may sound paradoxical: the resulting postsynaptic response produced in a neuron of area 18 comes, at
least partially, from other neurons of area 18, although
the stimulation was applied in area 17.
Another problem arises when electrical stimulation is
used to study the vertical, intracolumnar connections. Responses obtained, for example in layer 3 after electrical
stimulation applied in layer 4, result from the activation
of axons efferent from layer 4 neurons, but also from
the activation of axons that travel through layer 4. These
axons have a number of different origins: from other cortical areas, from lower cortical layers, from subcortical
structures, and from layer 3 neurons as well. To avoid
such problems, the only alternative is to use stimulation
methods that activate only the cell body. One is the acti-
vation of a single presynaptic neuron with intracellular
stimulation. The drawbacks of that technique are that only
one neuron is activated and that it is difficult to use in vivo due to its requirement for a good stability.
Chemical activation, with glutamate or other excitatory neurotransmitters, appears to be a mode of stimulation
that specifically activates cell bodies in large numbers;
but the very useful timing of electrical stimulation is lost.
In addition, results obtained with chemical stimualation
may suffer interpretation problems linked to the phenomenon of depolarization block.
Acknowledgements We thank Pascale Giroud and Naura Choulamountri for help during the experiments, also Pierre-Marie Chorrier for help with the mechanics, Christian Urquizar for help with the
electronics. Thanks to Guy Chouvet for advice and help with iontophoresis. Thanks to G.E. Loeb for bibliographical references.
Thanks to visitors to the Neuroscience meeting (Washinton 1993),
to M. Abeles, H.A. Swadlow, W.T. Newsome, and E.J. Tehovnik
for their comments and criticisms on this work while it was on progress. This work was supported by H.F.S.P. RG 55/94. L.G.N. was
supported by a fellowship from the Minist›re de la Recherche et de
la Technologie and by a fellowship from the Fyssen foundation.
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