Download High Safety Factor for Action Potential Conduction Along Axons But

High Safety Factor for Action Potential Conduction Along Axons But
Not Dendrites of Cultured Hippocampal and Cortical Neurons
PAUL J. MACKENZIE AND TIMOTHY H. MURPHY
Kinsmen Laboratory of Neurological Research, Departments of Psychiatry and Physiology, University of British
Columbia, Vancouver V6T 1Z3, Canada
INTRODUCTION
A number of recent studies contributed to the emerging
view that neurons are comprised of compartments of differing electrical excitability, both in the generation and propagation of active conductances. Dual-electrode and imaging
studies suggest that action potential (AP) generation does
not normally occur in dendrites (Häusser et al. 1995; Stuart
and Sakmann 1994); in contrast, the axon at or near the
hillock is the site of generation, suggesting greater excitability in the axon. APs that are initiated in the axon can invade
dendrites and back-propagate along them in a manner that
is sensitive to branching and decrements over distance (Stuart et al. 1997). In contrast, single APs conduct reliably
along axons to terminals, indicating that the distal axon is
also strongly excitable (Allen and Stevens 1994; Lüscher et
al. 1996; Mackenzie et al. 1996; Stevens and Wang 1995).
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Several lines of evidence suggest AP conduction may
fluctuate and be modifiable in an activity-dependent manner.
First, conduction block was observed in the spinal cord,
particularly after extended periods of high-frequency activity
(Lüscher et al. 1994; Wall 1995). Second, Na / channels,
which play a dominant role in AP generation and propagation, can be modulated by second messengers, including
protein kinases and G-proteins (Catterall 1993; Li et al.
1993; Ma et al. 1994). Third, several recent reports suggest
the possibility that blockade of AP conduction may contribute to fluctuations of synaptic responses. For example, intense activity in principal cells of the cerebellum and hippocampus results in decreased inhibition onto those cells from
presynaptic inhibitory cells (Llano et al. 1991; Pitler and
Alger 1994). In hippocampus, for example, intense postsynaptic activity can lead to failure of evoked but not spontaneous inhibitory postsynaptic currents through postsynaptic
Ca 2/ influx, a retrograde messenger, and possibly involving
presynaptic axonal conduction block (Alger et al. 1996). In
cerebellar Purkinje cells, fluctuations of inhibitory currents
can occur as a result of a presynaptic mechanism downstream of AP generation, possibly including fluctuations in
presynaptic depolarization (Vincent and Marty 1996).
Although membrane excitability can be modified, it is
unclear how such changes could affect neuronal function.
For example, Na / channel modification could influence AP
generation, the propagation of APs to terminals, or the local
depolarization of presynaptic terminals. Furthermore, it is
unclear whether different neuronal compartments are equally
sensitive to changes in Na / channel function. For example,
somatic and dendritic compartments are reported to have
similar Na 2/ channel densities and yet differ in excitability
(Hoffman et al. 1997; Magee and Johnston 1995; Schwindt
and Crill 1995; Stuart and Sakmann 1994). By using Ca 2/
imaging, we assessed the excitability of the CNS axon under
limiting conditions expected to reduce the safety factor (lowering of the external Na / concentration). These fine distal
axonal branches of mammalian CNS neurons are not directly
accessible to patch-clamp or intracellular electrodes. Although brain slice preparations would be preferable for this
study, Frenguelli and Malinow (1996) found that high concentrations of extracellular Ca 2/ and intracellular fura-2
were required to reliably view Ca 2/ dynamics in single boutons in response to a single AP in a slice preparation. Studies
performed in cerebellar slices (Callewaert et al. 1996) with
more physiological conditions were restricted to initial segments of axons and required averaging of trials and were
unable to resolve single boutons.
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Mackenzie, Paul J. and Timothy H. Murphy. High safety factor
for action potential conduction along axons but not dendrites of
cultured hippocampal and cortical neurons. J. Neurophysiol. 80:
2089–2101, 1998. By using a combination of Ca 2/ imaging and
current-clamp recording, we previously reported that action potential (AP) conduction is reliably observed from the soma to axonal
terminals in cultured cortical neurons. To extend these studies, we
evaluated Ca 2/ influx evoked by Na / APs as a marker of AP
conduction under conditions that are expected to lower the conduction safety factor to explore mechanisms of axonal and dendritic
excitability. As expected, reducing the extracellular Na / concentration from 150 to Ç60 mM decreased the amplitude of APs recorded
in the soma but surprisingly did not influence axonal conduction,
as monitored by measuring Ca 2/ transients. Furthermore, reliable
axonal conduction was observed in dilute (20 nM) tetrodotoxin
(TTX), despite a similar reduction in AP amplitude. In contrast,
the Ca 2/ transient measured along dendrites was markedly reduced
in low Na / , although still mediated by TTX-sensitive Na / channels. Dendritic action-potential evoked Ca 2/ transients were also
markedly reduced in 20 nM TTX. These data provide further evidence that strongly excitable axons are functionally compartmentalized from weakly excitable dendrites. We conclude that modulation of Na / currents or membrane potential by neurotransmitters
or repetitive firing is more likely to influence neuronal firing before
AP generation than the propagation of signals to axonal terminals.
In contrast, the relatively low safety factor for back-propagating
APs in dendrites would suggest a stronger effect of Na / current
modulation.
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By using cultures of cortical neurons in which Ca 2/ dynamics at single boutons can be reliably measured (Mackenzie et al. 1996), we observe that despite a 60% reduction
in somatic spike amplitude reliable conduction of single and
pairs of APs was maintained along main and collateral
branches of axons. In contrast, along dendrites, although
active propagation persisted, the Ca 2/ transient, an indicator
of the degree of depolarization, was greatly reduced. These
results suggest that along the axon there is a large degree
of conduction safety leading to reliable terminal depolarization and Ca 2/ influx. Consequently, agents that modify Na /
channel function, such as modulating neurotransmitters, are
unlikely to exert their effects by altering axonal conduction
reliability. In the dendrite, a graded relationship exists between the weakly propagating AP and Ca 2/ influx.
Hippocampal and cortical neurons and glia were dissociated
from 17- to 18-day gestation rat fetuses (separate cultures were
prepared from the 2 regions) placed in culture and allowed to
mature for 17–26 days in vitro as previously described (Murphy
and Baraban 1990), with the exception that the plating medium Lcystine concentration was supplemented to 300 mM. The whole
cell recording configuration in current-clamp mode (Hamill et al.
1981) was used to fill neurons with fluo-3/furaptra (Minta et al.
1989; Ragu et al. 1989) and to generate APs by current injection.
The patch pipette solution contained (in mM) 0.75 fluo-3 K / salt,
1 furaptra (used to view basal fluorescence), 122 K / MeSO4 , 20
NaCl, 5 Mg-ATP, 0.3 GTP, and 10 N-2-hydroxyethylpiperazineN *-2-ethanesulfinic acid (HEPES), pH 7.2. Imaging was performed with a 100 1 1.3 NA Zeiss objective on a Zeiss Axiovert
microscope with a custom-made stage that permits movement of
the preparation during patch-clamp recording. The following extracellular solution was used to isolate the effects of AP stimulation
from spontaneous synaptic activity (in mM): 137 NaCl, 5.0 KCl,
2.5 CaCl2 , 1 MgCl2 , 0.34 Na2HPO4 (7H2O), 10 NaHEPES, 22
glucose, 1 NaHCO3 , 20 mM picrotoxin, 100 mM D,L-2-amino-5phosphonovaleric acid, and 3 mM 6-cyano-7-nitroquinoxaline-2,3dione (CNQX), pH 7.4.
Experiments were performed in current-clamp mode with an
Axopatch 200A. To synchronize voltage records with video frames,
we used a sampling rate of 200 ms, which may have underestimated
the peak of the AP. Resting membrane potential ranged from 055
to 065 mV; a calculated liquid junction potential of 12 mV was
not corrected for (Neher 1995a). Membrane potential was adjusted
to 060 mV by injection of a small negative or positive current,
and overshooting APs were produced by injection of positive current (5- to 10-ms duration). In all trials, records of membrane
potential were inspected carefully to determine whether the AP
changed shape over time or whether stimulation failed to elicit
APs. Experiments were terminated if a progressive change in the
AP shape or the membrane potential occurred. After establishment
of whole cell configuration, data were not collected for 10–15
min to allow diffusion of the Ca 2/ indicators into fine processes.
Pyramidal-type neurons were used for imaging experiments and
were identified by their somatic size, shape, and prominent dendritic process. The axon of the neuron was identified according to
the criteria outlined by Mackenzie et al. (1996) and positioned
optimally for imaging.
Perfusion of low-sodium solution
Computer-triggered solenoid valves (Neptune Research, Northboro, MA) were used to control a gravity-fed double-barreled u
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Imaging and analysis
A fiberoptically coupled intensified charged-coupled device
camera with a Gen III intensifier tube was used for all experiments
(Stanford Photonics, Palo Alto, CA) in combination with a Epix
4 M12–64 MB frame grabber board (Northbrook, IL). Images
and electrophysiological data were analyzed together on a Pentium
computer with custom routines written in IDL (Research Systems,
Boulder, CO). Change in fluo-3 fluorescence (excitation 480/40
nm) was used to measure axonal and dendritic Ca 2/ dynamics in
response to Na / APs elicited at the soma (Jaffe et al. 1992; Markram et al. 1995). These intracellular Ca 2/ transients reflect AP
conduction through an imaged axonal segment because of the following observations: complete block of axonal Ca 2/ transients in
1 mM TTX (Fig. 8B), the Ca 2/ signal was proportional to the
number of APs delivered (Fig. 2C) (Mackenzie et al. 1996), the
Ca 2/ transients were blocked by removing extracellular Ca 2/
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METHODS
tube (900-mm diam; flow rate 0.75 ml/min) containing normal and
low Na / solutions. By using a 12.5 objective and a phenol red
containing test solution, the u tube was placed Ç4 mm from the
center of the chamber so that its contents rapidly exchanged the
bath solution of an area ( Ç4 mm2 ) surrounding the cell of interest.
As shown in Fig. 1A (not to scale), a compartment of the u tube
contained normal Na / (150 Na / ) Hanks balanced salt solution
(HBSS); the other side contained low Na / HBSS (40 mM; ionic
substitution with N-methyl-D-glucamine). Figure 1 illustrates that
the local perfusion system is able to alter the ionic composition of
the extracellular environment surrounding the neuron; this was
rapid and reversible. Examination of somatic records of membrane
potential revealed that the perfusion system established a low Na /
environment around the soma of the cell (Fig. 1B, note truncated
AP). However, it was also important that the solution was exchanged on distal axonal and dendritic processes. The following
points argue that distal axonal and dendritic processes experienced
rapid and reversible solution exchange. First, we used a largediameter u tube that perfused the solution and hence quickly exchanged it, over several millimeters, clearly encompassing the
areas where most distal measurements were made ( õ500 mm from
the soma). Second, after placement of the cell of interest into the
field of view but before establishing a whole cell recording, we
used a phenol red–containing HBSS in one barrel of the perfusion
system to confirm both rapid delivery from one barrel and efficient
clearance from the other. Third, the dendrite served as a positive
control because dendritic regions that were as distal as axonal
regions imaged were also affected by low Na / perfusion. Fourth,
a zero Ca 2/ solution used in the perfusion system blocked Ca 2/
transients evoked by APs in distal branches without altering AP
generation in six of six neurons (data not shown). Trials of 150
Na / and 60 Na / were alternated with a 15-s intertrial interval;
solution exchange for the following trial occurred at the beginning
of this 15-s period. (By using the phenol red–containing medium,
we calculated that the [Na / ]ext. after perfusion was 80 mM within
330 ms and reached a steady state of 60 mM within 2.5 s.) Between
trials of data acquisition, cells were bathed with buffer containing
150 Na / . By using the double-barreled perfusion system, we observed a small (2–5%) increase in basal fluo-3 fluorescence under
low Na / conditions (presumably caused by blockade of Na / /Ca 2/
exchanger). This increase in basal fluorescence was Ç10% of
the increase observed in response to 1 AP and therefore did not
appreciably saturate the Ca 2/ indicator. This is illustrated for one
axonal bouton in Fig. 1 (F), which shows changes in raw fluo-3
fluorescence in response to two somatic APs for an axonal site 174
mm from the soma. In contrast, in preliminary experiments in which
continuous (bath) application of low Na / medium was used, we
observed a robust rise in basal Ca 2/ concentration, thus requiring
the u tube application method.
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2091
(Mackenzie et al. 1996), and a lack of Ca 2/ transients after stimulation that was subthreshold for AP generation (Figs. 2C and 6A)
(Mackenzie et al. 1996). Furthermore, it is likely that, given the
high indicator concentrations used, the Ca 2/ signal is dominated by
Ca 2/ influx rather than handling (Neher 1995b). To view processes
under baseline conditions and to correct for process volume and
cell loading, the fluorescent Ca 2/ indicator furaptra (excitation 380
nm) was coinjected with fluo-3 into cells. Furaptra was chosen for
its high fluorescence signal at basal Ca 2/ , its low affinity for Ca 2/
that produces little buffering, and its well-distinguished spectra
that did not contaminate fluo-3 signals (Ragu et al. 1989). Plots
of fluo-3 fluorescence versus time were produced off-line from
single video frames corresponding to 33-ms intervals. After analysis, data were discarded if basal Ca 2/ levels were found to rise
significantly during the course of an experiment or if progressive
changes in the amplitude of the Ca 2/ transient were observed because of factors such as spike broadening. The integral of the
Ca 2/ transient was termed the Ca 2/ response. Ca 2/ responses were
calculated for individual trials for 1.4 1 1.4 mm2 regions along
dendrites and axons (including individual terminals). Percentage
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change in fluo-3 fluorescence ( DF/F) 1 100 was calculated by
dividing the change in fluo-3 fluorescence (mean F over 333 ms
after stimulation 0 baseline F) by the mean fluo-3 fluorescence
measured during baseline. This DF/F measurement was used for
analyses in which fluctuations were measured within individual
sites across trials. For analyses of the spatial profile of axonal and
dendritic Ca 2/ transients, responses were corrected for differences
in compartment volume and potential irregularities in illumination
or detection by dividing the change in fluo-3 fluorescence by background-subtracted basal furaptra fluorescence and expressed as a
ratio of change in fluo-3 divided by furaptra fluorescence ( DF480 /
F380 ). A single exponential fit was used to determine the decrement
of the Ca 2/ transient along a region of dendrite.
To detect possible conduction failure in 60 Na / , two analyses
were applied. First, for each site, we compared the average Ca 2/
response across trials in 150 Na / to average Ca 2/ response in 60
Na / (paired t-test). For a given axonal or dendritic site, this test
would be most sensitive to consistent decrements in the amplitude
of Ca 2/ transients across trials. At dendritic sites, a large (in some
cases complete) decrement of the Ca 2/ response in 60 Na / was
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FIG . 1. Local perfusion of 60 mM extracellular Na / reversibly reduces action
potential (AP) amplitude. A: cartoon of reversible perfusion system (not to scale). A
large-bore double-barreled u tube permitted
rapid exchange of the extracellular solution
surrounding the neuron of interest and its
processes. Calcium indicators were loaded
with intracellular dialysis, and records of
membrane potential were made in the current-clamp recording mode. B: low Na /
solution reversibly reduced the AP amplitude. Traces of membrane potential vs. time
overplotted for 38 alternating trials of 150
and 60 mM extracellular Na / . C: records
of membrane potential recorded at the soma
during a typical experimental sequence illustrating the reversible delivery of the low
Na / solution. Trials of 1 or 2 AP were
alternated between 150 and 60 mM external
Na / . D: input resistance was not significantly different between 60 and 150 mM
extracellular Na / . Records of somatic
membrane potential after delivery of 500ms current steps of 0400, 0200, 0, 200,
and 400 pA in 150 and 60 mM Na / and
wash (150 mM Na / ). E: voltage-current
relationship showing steady-state membrane potential as a function of current step
(incrementing by 100 pA). Input resistance
in 60 Na / was 98.8 { 2.7% of input resistance in 150 Na / (n Å 3 cells, range 144–
218 MV ). F: traces illustrating raw fluo-3
fluorescence vs. time in the axon 174 mm
from the soma after 2 somatic APs 50 ms
apart. The thick trace is the mean response,
and thin traces are 12 overplotted trials.
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observed. In contrast, at axonal sites, we observed a small albeit
significant decrease in Ca 2/ response in 60 Na / ( Ç90% of 150
Na / ). However, it is possible that tests evaluating differences in
the mean are not sensitive to a single or a few aberrant responses
and is therefore insensitive to possible intermittent failure in one
or a few trials. In contrast to the mean, the variance of the Ca 2/
response across trials is sensitive to single or a few outlying observations. We therefore applied an F-test (ratio of the variance across
trials in 150 Na / to the variance across trials in 60 Na / ) to detect
occasional conduction failures. To verify that this method was
sensitive to single failures, for each experiment, simulated data
sets were created for each site that were identical to Ca 2/ responses
in 150 Na / but with a known number of failures created in the
data set by subtracting the mean response from individual trials.
This ‘‘failure test’’ allowed us to confirm that, had conduction
failures (and consequent Ca 2/ transient failure) occurred, the analysis would have the sensitivity to detect them. This statistical ability to detect a single conduction failure was used as a criterion to
select axonal sites with adequate signal to noise properties for
analysis; sites that were excluded showed no sign of failure but
were simply too noisy to conclusively demonstrate a lack of failure
within the number of trials performed. Neighboring sites on small
contiguous segments of axon (3–20 mm in length) with low signal
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to noise were averaged in some experiments to improve the signal
to noise ratio.
RESULTS
Reliable axonal conduction in 150 and 60 mm Naext
Conduction reliability along axonal and dendritic processes was monitored by measuring Ca 2/ transients in these
compartments after AP generation at the soma (Fig. 1A).
Figure 1B illustrates 38 overplotted AP waveforms in 60
and 150 mM extracellular Na / (alternating trials). Delivery
of extracellular solution containing 60 Na / resulted in a
reversible 60% decrease in the amplitude of the AP. Figure
1C illustrates a typical experimental sequence, with alternating trials of one and two APs (1 and 2 AP) in 60 and 150
Na / . For two AP the interval between the steps was 50 ms.
In 60 Na / , input resistance to somatic current steps was not
significantly different from control solution (Fig. 1, D and
E), and basal fluo-3 fluorescence was not appreciably altered
in 60 mM Na / because of the rapid and reversible perfusion
(Fig. 1F, and see METHODS ).
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2/
FIG . 2. Reliable AP-evoked Ca
transients along the axon in low extracellular
Na / . A: basal fluorescence image of a region of axon 80–220 mm from the soma
of a cortical pyramidal neuron. B: records
of Ca 2/ response ( D fluo-3/basal furaptra;
DF480 /F380 ) vs. time for 3 axonal sites
(1.4 1 1.4 mm), overplotted for 7–9 trials
of 1 AP, 150 vs. 60 mM Na / (left panel);
2 AP, 150 vs. 60 mM Na / (right panel).
C: Ca 2/ response integral ( D fluo-3/basal
furaptra; DF480 /F380 ; y-axis) plotted over
repeated trials (x-axis) for axonal site 3.
Ca 2/ transients were evoked by 1 and 2
APs (top traces) in 150 mM Na / ( j ) and
60 mM Na / ( s ). Dashed line represents 2
SDs above baseline variation.
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FIG . 3. Reliable axonal conduction to distal release sites in 60 mM Na / . Left: basal fluorescence
(furaptra; F380 ) image illustrating varicose boutons
on a segment of tertiary axon ú250 mm from the
soma (cortical). Right: records of Ca 2/ response
( D fluo-3/basal furaptra; DF480 /F380 ) vs. time for
3 axonal sites, averaged over 8 trials of 1 AP, 150
vs. 60 mM Na / ; 2 AP, 150 vs.60 mM Na / .
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(P ú 0.05, unpaired t-test, distal 59 sites vs. 46 proximal
sites from 8 neurons).
Conduction reliability along axons was statistically tested
with two analyses (n Å 8 neurons). First, at each axonal
site, the difference in average Ca 2/ transient (60 vs. 150
Na / ) was evaluated with a t-test. Across 105 sites pooled
from 8 neurons, the mean Ca 2/ response in 60 Na / was
significantly lower than the response in 150 Na / (90 { 15%
of response in 150 Na / ; P õ 0.001, paired t-test). This is
illustrated in the plot of the mean Ca 2/ response in 60 versus
150 Na / shown in Fig. 4A for one AP, where a larger
number of observations is below the unity line (P õ 0.01,
x 2 ), indicating a general tendency toward smaller responses
in 60 Na / . The analysis by t-test thus indicated that the mean
AP-evoked Ca 2/ transients in 60 Na / were only minimally
affected. However, it was unclear whether this analysis
would detect a failure of conduction within a small number
of trials because the paired t-test is somewhat insensitive to
single outlying observations. In contrast, a comparison of
variance in 150 and 60 Na / would be sensitive to intermittent conduction failure (see METHODS ). Axonal sites with
high signal to noise ratio were identified, and an F-test was
applied to compare the variance in the amplitude of Ca 2/
transients across trials in 150 and 60 Na / at these high signal
to noise sites. This variance analysis revealed increased variance at only 3 of 105 axonal sites; hence intermittent axonal
conduction failure was not observed under the low Na /
condition expected to promote failure.
Axonal sites have sufficient signal to noise ratio to detect
intermittent failure
To determine whether the variance analysis would have
been sensitive to occasional conduction failure, we created
simulated failure data sets from 150 Na / data. For each
axonal site, the mean Ca 2/ response was subtracted from a
single trial to create data with one (artificial) failure (Fig.
4C). As predicted, adding one simulated failure to the con-
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To assess the reliability of AP propagation along axons
in 60 Na / , APs were generated and recorded at the soma,
and intracellular Ca 2/ transients were measured (monitoring
changes in fluo-3 fluorescence) at sites along the axon. Figure 2A shows a basal fluorescence image of a segment of
main and secondary axon. Figure 2B illustrates superimposed traces of fluo-3 fluorescence versus time at 3 axonal
sites in 150 versus 60 Na / . As we reported previously, in
150 mM Na / , single APs reliably evoked Ca 2/ transients
on every trial (Mackenzie et al. 1996). In 60 mM Na / ,
Ca 2/ transients persisted despite a truncation of the AP,
suggesting reliable axonal conduction in 60 Na / . Figure 2C
plots the integral of the Ca 2/ transient across trials for one
and two APs in 60 and 150 Na / for an axonal site. Ca 2/
responses in 150 and 60 Na / were ú2 SDs above baseline
variation (Fig. 2C). To investigate whether axonal Ca 2/
responses in 150 Na / varied with distance, we performed a
regression analysis (Ca 2/ response vs. distance) on 27 sites
from 4 cells that were within 90–175 mm of the soma. The
slope of the regression line was 0.08 { 0.1%/ mm (i.e., 8%
increase in DF480 /F380 over 100 mm), indicating a small but
nonsignificant tendency toward increased Ca 2/ responses at
more distal sites in 150 mM Na / (regression coefficient R Å
0.12, P Å 0.55).
Reliable AP conduction as assayed by Ca 2/ transients
was also observed at highly branched tertiary processes that
contained most of the synaptic boutons (Fig. 3). These varicose boutons, previously characterized as functional vesicular release sites, exhibited larger Ca 2/ transients than neighboring regions of axon (Mackenzie et al. 1996). Figure 3
plots the mean Ca 2/ transient at three presumed boutons
after one and two AP in both 150 and 60 mM extracellular
Na / . Consistent with the proximal axon, reliable conduction
was observed on synaptic bouton-rich tertiary axonal
branches, both in 150 and 60 Na / . A slightly reduced
( Ç10%) Ca 2/ transient in 60 Na / was observed at both
proximal and distal components ( ú160 mm) of the axon;
the degree of reduction did not differ between compartments
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P. J. MACKENZIE AND T. H. MURPHY
trol (150 Na / ) data set only minimally perturbed the mean
response (not reaching statistical significance by t-test). In
contrast to the analysis of the mean, creation of a single
failure resulted in a significant increase in variance at 38 of
105 axonal sites from 8 neurons (4 hippocampal and 4 cortical), indicating that it was possible to detect a single failure
at these sites had one occurred. This analysis indicated that
the 38 sites had signal to noise properties that were sufficient
to detect a single failure. When 60 Na / medium was compared with 150 Na / medium (simulated failure removed),
only 3 of the 38 axonal sites showed a significant increase
in variance (P õ 0.05). Thus, at most sites where we were
confident that we would have reliably measured increased
variance, no increase in variance was seen in low Na / ,
suggesting no increase in the number of conduction failures.
Analysis of conduction reliability of two APs was carried
out similarly, except that single failure simulated data sets
were created by subtracting one-half of the mean response
from one trial (equivalent to 1 AP). For 2 APs, 15 sites
from 5 neurons were found to have sufficient signal to noise
to permit detection of a failure of 1 AP. No significant difference in variance was observed when APs evoked in 60 and
150 Na / medium were compared at these sites. Therefore,
at most axonal sites that were determined to be capable of
detecting conduction failure, no indicative increase in variance was observed.
At many axonal sites, the signal to noise ratio was too
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poor to conclusively demonstrate no significant change in
variance with a single AP conduction failure (6–12 trials).
To compare variance in the two conditions, the coefficient
of variation (CV Å SD/mean) of Ca 2/ responses in 60 Na /
was plotted versus CV in 150 Na / , as shown in Fig. 4B.
We used the CV to control for the slight decrease in variance
expected in the 60 Na / group caused by the smaller average
Ca 2/ responses (Fig. 4A). A tendency toward increased
variance in 60 Na / would appear as a shift of the CV distribution above the unity line. CVs were distributed randomly
above and below the unity line (54 above, 51 below; P ú
0.05, x 2 ). Therefore, at all axonal sites, there existed no
general tendency toward a greater Ca 2/ response variance
in 60 Na / than in 150 Na / . We did not observe statistically
significant differences between cortical and hippocampal pyramidal neurons in the magnitude of the axonal Ca 2/ response in high or low Na / . Analysis of the variance across
trials thus suggested no increase in conduction failure in 60
or 150 mM Na / in both hippocampal and cortical axons
(data not shown). Therefore in both cell types we observed
Ca 2/ influx along the axon attributed to reliable and nonattenuating AP conduction in both 150 and 60 mM Na / .
Dendritic calcium transients evoked by APs are reduced
by lowering Naext
Because axonal conduction (as assayed by Ca 2/ imaging)
was reliable under conditions of lower external Na / , we
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FIG . 4. Group data showing reliable axonal
conduction in 60 mM Na / . A: mean Ca 2/ response ( DF480 /F480 1 100%) in 60 mM Na / (yaxis) vs. mean response in 150 mM Na / (xaxis). Pooled data from 105 axonal sites from 8
neurons for 1 AP. Diagonal line represents unity
(response in 60 mM Na / Å response in 150 mM
Na / ). B: coefficient of variation (CV Å SD/
mean) in 60 mM Na / vs. CV in 150 mM Na / .
Diagonal line represents unity (equal CVs in the
2 conditions). C: Ca 2/ response integrals evoked
by 2 APs at an axonal site plotted across 7 trials
in the following conditions: L, 150 Na / (real
data set); h, 60 Na / (real data set); m, simulated
failure data (constructed from 150 Na / data by
subtracting the mean Ca 2/ response, in 150 mM
Na / trials, from trial 6). A significant increase
in variance between 150 Na / (real) and the
‘‘failure’’ data set was observed (F-test, P õ
0.05) as indicated by the asterisk. Without the
simulated failure, no significant difference in
variance was observed between 150 and 60 Na /
at this axonal site, indicating reliable axonal conduction.
AXONAL AND DENDRITIC CONDUCTION
2095
next investigated whether propagation of single or pairs of
APs along dendrites was altered by similar manipulations.
Figure 5A, left panel, shows a basal fluorescence image
(furaptra excitation wavelength) of a representative pyramidal neuron indicating the main dendrite where Ca 2/ measurements were made. Ca 2/ transients evoked by single and
paired APs were measured along the length of the dendrite
(with the 480-nm excitation wavelength of fluo-3 for dynamic Ca 2/ measurements). Figure 5B illustrates traces of
average dendritic Ca 2/ transient (evoked by 2 AP) versus
time in 150 and 60 Na / . In 150 Na / , dendritic Ca 2/ transients decreased with distance from the soma with a length
constant of 150 mm (Fig. 5C). However, the decrement with
distance of the Ca 2/ response along the dendrite was greater
in 60 Na / than in 150 Na / (length constant 53 mm, Fig.
5D) after either one or two AP.
To investigate whether dendritic Ca 2/ responses for group
data in 150 Na / varied with distance, we performed a regression analysis (Ca 2/ response vs. distance) on 30 sites from
4 cells that were within 75–160 mm of the soma. The dendritic Ca 2/ response in 150 mM Na / was negatively correlated with distance (R Å 00.78, P õ 10 06 ; decrementing
/ 9k2d$$oc42 J152-8
43% over 100 mm). This differs from the spatial profile
of the group axonal Ca 2/ responses, which did not change
significantly with distance. Figure 6 presents group data
summarizing the Ca 2/ responses in 60 mM Na / (as a ratio
of response in 150 mM Na / ) in both the dendrite and axon.
Along the axon (Fig. 6A), Ca 2/ influx in response to both
one and two APs was unchanged in 60 mM Na / . In contrast,
along the dendrite (Fig. 6B), a reduction in the 60 Na / /
150 Na / response ratio was observed over a distance of 40–
160 mm for both one and two AP.
Ca 2/ transients in distal dendrites are mediated by TTXsensitive Na / channels
Because low Na / medium selectively decreased the Ca 2/
responses along the dendrite, we sought to further characterize the residual dendritic Ca 2/ response. To explore whether
the remaining Ca 2/ response in 60 Na / depended on regenerative potentials mediated by TTX-sensitive Na / channels,
dendritic Ca 2/ transients to subthreshold and suprathreshold
stimulation were measured in 1 mM TTX. Figure 7A illustrates the Ca 2/ response averaged over a region of dendrite
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FIG . 5. Spatial profile of action-potential
evoked Ca 2/ transients along dendrites in 150 and
60 mM extracellular Na / . A: basal fluorescence
image (F380 ) of a cortical spiny pyramidal neuron
illustrating the area where Ca 2/ transients were
imaged. B: Ca 2/ transients ( D fluo-3/basal furaptra DF480 /F380 ) evoked by 2 APs (50-ms interval)
for the indicated dendritic sites in 150 mM Na /
(thin traces) and 60 mM Na / (thick traces). Average of 9 trials in each condition. C: Ca 2/ response
( DF480 /F380 ) to 2 APs (y-axis) vs. distance from
soma along the dendrite (x-axis) in 150 mM Na /
( l ) and 60 mM Na / ( h ). D: Ca 2/ response along
the dendrite in 60 Na / expressed as a ratio of
response in 150 Na / .
2096
P. J. MACKENZIE AND T. H. MURPHY
that Ca 2/ responses to somatic current injection in TTX were
markedly reduced along the length of the dendrite (Fig.
7B, inset). Thus the spatially decreasing responses observed
under control and low Na / conditions were dependent on
TTX-sensitive Na / channels. Similar results were observed
in two other cells. A Ca 2/ spike did not contribute an appreciable component to the residual Ca 2/ response nor was
there evidence of a Ca 2/ spike in the somatic voltage records
(data not shown).
Conduction safety factor in axonal and dendritic
compartments in dilute TTX
FIG .
6. Group data illustrating the relative change in the action-potential
evoked Ca 2/ transient in 60 vs. 150 mM Na / along axons and dendrites.
A: axon. Average axonal Ca 2/ response in 60 mM Na / as a function of
distance from the soma, expressed as a ratio of response in 60 to response
in 150 mM Na / . h, 2 APs; l, 1 AP (8 cells). B: dendrite. Average
dendritic Ca 2/ response ratio (60 to 150 mM Na / ), as a function of distance
from the soma (6 cells). h, 2 APs; l, 1 AP.
30–80 mm from the soma (more distal regions would produce a weak signal). The threshold for AP generation in
this cell, determined before addition of TTX, and again after
washout, was Ç600 pA (5 ms of current injection). Figure
7A illustrates the average dendritic Ca 2/ response, in TTX,
of one or two 5-ms current steps of 300, 700, or 1,000
pA. Ca 2/ responses to 300-pA current injection were not
significantly different from baseline (P ú 0.30, t-test). After
700- and 1,000-pA stimulation, Ca 2/ responses were significantly above baseline variation (P õ 0.05, t-tests). After
washout of TTX, suprathreshold stimulation that now generated APs resulted in Ca 2/ responses that were 15- to 20fold greater than responses in TTX, as illustrated in Fig. 7A,
(note 6-fold change in scale on y-axis). Again, no measurable Ca 2/ responses were observed after one or two 300pA current steps (apparently below threshold). Figure 7B
illustrates the spatial profile of Ca 2/ responses along the
same region of dendrite after two current steps of 1,000 pA
in TTX, 700 pA after washout (suprathreshold: 2 AP), and
300 pA after washout (subthreshold). Figure 7B illustrates
/ 9k2d$$oc42 J152-8
Reliable axonal AP propagation in neurons from
hyperpolarized potentials
A-type K / channels have been shown to regulate excitability in dendrites of CA1 pyramidal neurons by limiting AP
generation and limiting AP back-propagation in dendrites
(Hoffman et al. 1997). A recent report also suggested that
A-type K / channel could regulate AP conduction in the
axon (Debanne et al. 1997). We therefore examined axonal
AP conduction from hyperpolarized potentials expected to
remove inactivation of the A-type K / channels and thereby
increase their effect (Segal and Barker 1984; Wu and Barish
1992), possibly leading to conduction failure. Single or pairs
of APs were elicited by somatic current steps from 060 or
0100 mV. Figure 9A illustrates a basal fluorescence image
of three representative secondary and tertiary axonal sites.
Figure 9B plots traces of fluo-3 fluorescence versus time in
response to pairs of APs from resting and hyperpolarized
potentials. No difference in the Ca 2/ transient was observed
at hyperpolarized potentials. Figure 9C illustrates traces of
membrane potential recorded at the soma after APs evoked
from 060 and 0100 mV. Figure 9D plots the average Ca 2/
transient elicited by 1 AP from 0100 mV versus average
Ca 2/ transient from 060 mV for 10 axonal sites from the
neuron illustrated in Fig. 9A. The average Ca 2/ response
from 0100 mV was not significantly different from the 060
mV response (P ú 0.05, t-test). Similar results were obtained in five of five neurons.
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To address potential concerns that the low Na / manipulation could also affect other Na / -dependent processes such
as pumps and exchangers (Bouron and Reuter 1996), we
used a low dose of TTX (20 nM) to attenuate the Na / spike
to a similar degree as 60 mM external Na / (range 45–57%
amplitude reduction in 20 nM TTX). Figure 8A illustrates
examples of Ca 2/ transients along the dendrite and axon in
response to one AP. In the dendrite, the Ca 2/ response at
distances ú100 mm from the soma was significantly reduced
in dilute TTX (compared with control) and decreased with
further distance. In contrast, the Ca 2/ response in the axon
at distances ú100 mm from the soma persisted in 20 nM
TTX. In the axon, the Ca 2/ response was reversibly blocked
by 1 mM TTX. After TTX washout, Ca 2/ transients were
fully restored in both the axon and dendritic compartments.
Figure 8B presents group data of the normalized Ca 2/ response from 23 sites from 5 cells (axon) and 39 sites from
5 cells (dendrite) that were ú100 mm from the soma.
AXONAL AND DENDRITIC CONDUCTION
2097
DISCUSSION
We explored the propagation of APs along axons and
dendrites of hippocampal and cortical neurons by measuring
Ca 2/ transients along distal regions of axon and dendrite
following somatic current steps. We previously reported that
single somatic APs reliably result in Ca 2/ influx along all
branches of axons. This Ca 2/ influx occurred predominantly
through high-voltage activated Ca 2/ channels and was enhanced at distal functional release sites along secondary and
tertiary axonal branches (Mackenzie et al. 1996). Here we
compared the reliability of axonal and dendritic propagation
under normal and limiting conditions expected to reduce
conduction safety factor. Axons and dendrites act as separate
compartments of differing excitability in normal external
Na / ; furthermore AP conduction is differentially sensitive
to a reduction in the flux of Na / ions along axonal and
dendritic compartments.
Reliable axonal conduction of truncated APs in low Na /
or dilute TTX
All axonal sites continued to show reliable Ca 2/ transients
after somatic APs in reduced extracellular Na / ; this was
observed on distal portions of axon as far as we could resolve
( ú400 mm from the soma) on both main and collateral
branches (Figs. 2 and 3). Despite a substantial decrease in
AP amplitude in 60 Na / , Ca 2/ transients were still reliably
observed at axonal terminals (Fig. 1, B and C). Two analyses
were used to test for the occasional failure of the axonal
/ 9k2d$$oc42 J152-8
Ca 2/ response to a single AP. First, we analyzed 38 sites
with sufficient signal to noise to detect intermittent failure
(Fig. 4C); at these sites variance analysis indicated that
intermittent AP failure does not occur, which is consistent
with Dityatev and Clamann (1996). Second, for all 105 sites
from 8 cells examined, we measured the CV in 150 Na /
versus the CV in 60 Na / and found no consistent tendency
/
(Fig. 4B), suggesting
toward increased variance in low Na ext
/
reliable AP conduction in 60 mM Na ext
.
At most axonal sites a small decrease in Ca 2/ transient
/
amplitude was observed in 60 mM Na ext
(Figs. 3 and 4).
However, this decreased Ca 2/ response was more likely due
to decreased Ca 2/ influx in the terminal than to conduction
failure for four reasons. First, Ca 2/ transients in 60 Na /
were only reduced by 10%, were significantly greater than
noise (no stimulation), and did not show failure (Figs. 2
and 4). Second, a response decrement with distance was not
observed (Fig. 2). Third, at all sites, Ca 2/ transients evoked
by two AP in 60 Na / were greater than those evoked by
one AP in 150 Na / (Figs. 2 and 3). Fourth, Ca 2/ responses
to one AP in 60 Na / were significantly above noise levels
(Figs. 2 and 3). Thus, in 60 mM Na / , APs successfully
conducted to axonal sites. Rather than reflecting conduction
failure, the decreased axonal Ca 2/ transient in 60 Na / may
have reflected a direct effect of the Na / substitute on the
Ca 2/ channels or could have been a result of a decreased
depolarization in 60 Na / . The reduction in AP amplitude
may have limited the activation of specific classes of Ca 2/
channels.
Axonal Ca 2/ responses to two APs were less than twofold
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2/
FIG . 7. Dendritic AP-evoked Ca
transients
depend on tetrodotoxin (TTX)-sensitive Na /
channels. A: change in dendritic Ca 2/ transients
( D fluo-3/furaptra; DF480 /F380 ) averaged along a
segment of dendrite 30–80 mm from the soma.
Left panel: Ca 2/ responses in the presence of 1
mM TTX to 5 ms somatic current steps of 300,
700, and 1,000 pA (1 current step: light bars; 2
current steps 50 ms apart: dark bars). Right panel:
Ca 2/ responses DF480 /F380 ) to current steps of
300 pA (subthreshold) and 700 pA (suprathreshold) after washout of TTX. Light bars are responses to 1 current step, and dark bars are responses to 2 current steps; note 6-fold change in
y-axis scale. B: spatial profile along the dendrite
of TTX-sensitive and -insensitive Ca 2/ transients
evoked by current steps of 1,000 pA in the presence of TTX ( m ), 300 pA after washout of TTX
( h, subthreshold), and 700-pA current steps after
washout ( l, 2 AP). Inset: expanded y-axis illustrating the spatial profile of the TTX-insensitive
component ( m, compared with subthreshold responses after washout ( h ).
2098
P. J. MACKENZIE AND T. H. MURPHY
larger than Ca 2/ responses to one AP ( Ç90% of an expected
doubling of the single AP response). However, it is unlikely
that this could be due to intermittent conduction failures
because the analysis of response variance (Fig. 4) showed
no increased variance in response to two APs versus one
AP, indicating reliable conduction. In addition analysis of
single sweeps (Figs. 2 and 4) indicates that with two APs
the Ca 2/ response does not drop to the level mediated by a
single AP. Therefore, although there is some attenuation of
the Ca 2/ signal mediated by a second AP, which could be
attributed to a variety of factors including dye saturation or
Ca 2/ channel inactivation (Forsythe et al. 1998), it cannot
be accounted for by intermittent failure.
To address potential concerns that the low Na / manipulation could also affect other Na / -dependent processes such
as pumps and exchangers, we also used dilute TTX (20 nM)
to attenuate the Na / spike to the same degree as 60 mM
external Na / . Ca 2/ responses after somatic APs also persisted under these conditions (Fig. 8), confirming reliable
axonal conduction.
Our results therefore suggest that modulation of Na /
channels may influence whether the neuron reaches threshold
as well as the degree of presynaptic terminal depolarization
/ 9k2d$$oc42 J152-8
rather than the conduction of APs along axons. However,
other ions such as Ca 2/ and K / may play a role in influencing axonal conduction safety, particularly during high-frequency trains, as demonstrated recently in dorsal root ganglion neurons (Lüscher et al. 1996). To investigate the role
of A-type K / channels, we compared AP-evoked Ca 2/ transients from resting and hyperpolarized potentials. Hyperpolarized potentials would be expected to remove steady-state
inactivation of A-type K / channels and increase their effect
(Segal and Barker 1984; Wu and Barish 1992). We observed
no change in the magnitude of Ca 2/ transients (Fig. 9),
suggesting that an (inactivated) A-type K / conductance
played little role in the modulation of axonal AP conduction.
This contrasts with a recent report that an A-type K / conductance regulates axonal AP conduction in hippocampal CA3–
CA1 cell pairs (Debanne et al. 1997). However, because
our method is limited to imaging only a portion of the axon
at a time, conduction failure could have been occurring on
other possibly more distal branches that we were not imaging
but that would have been detected in the study of Debanne
et al. Furthermore different patterns of axonal branching
could have contributed to the differences observed. As for
dendritic AP conduction, recent findings of Hoffman et al.
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FIG . 8. Selective impairment of dendritic but not
axonal AP evoked Ca 2/ response in 20 nM TTX.
A, top: records of average change in Ca 2/ response
D fluo-3/furaptra; DF480 /F380 ) vs. time in response
to 1 AP for 3 dendritic sites at varying lengths from
the soma. Baseline: dashed lines; TTX 20 nM: thick
solid lines; Washout: thin solid lines. Below: average change in fluo-3 fluorescence versus time in
response to 1 AP at an axonal segment 270 mm
from the soma. Line symbols as in dendritic traces.
Axonal response to current step in 1 mM TTX indicated by arrow. B: group data (mean { SE) for
axon (left, 5 cells) and dendrite ( right, 5 cells).
AXONAL AND DENDRITIC CONDUCTION
2099
(1997) demonstrated the regulation of dendritic excitability
by an A-type K / channel. We acknowledge that a potential
role for Ca 2/ ions could have been lessened by the high
concentration of fluo-3 that was required to a achieve a
sufficient signal to noise ratio in recordings. Therefore it is
possible that exogenous Ca 2/ buffering may have reduced
the activation of Ca 2/ -dependent K / channels, thereby increasing the reliability of AP conduction. To determine
whether conduction reliability was enhanced by Ca 2/ buffering, we increased the contribution of K / channels during
AP repolarization by using a low (1 mM) extracellular K /
medium (to increase the K / driving force). We did not
observe AP failure in the axon in three of three neurons
in preliminary experiments. Additional experiments in high
extracellular Ca 2/ medium (5 mM) were performed to directly increase Ca 2/ influx (and potential activation of K /
channels) with no apparent effect on conduction failure (n Å
4 neurons; data not shown).
Decreased dendritic AP propagation in low external
sodium
We measured Ca 2/ transients caused by back-propagating
APs along the main dendrite of cortical and hippocampal
/ 9k2d$$oc42 J152-8
neurons in 150 and 60 Na / . In proximal dendritic regions
( Ç30–50 mm from the soma), the Ca 2/ transients were
similar in magnitude to Ca 2/ responses in the axon (Figs.
2 and 5), consistent with reports by Calleweart et al. (1996)
and Llano et al. (1997). In the dendrite, the Ca 2/ response
decreased with distance from the soma with a length constant
of 150–220 mm (Figs. 5–7). The degree of attenuation of
the Ca 2/ transient evoked by a single AP along the apical
dendrite appears to vary according to the cell type, age, and
recording conditions (Schiller et al. 1995, 1997; Spruston et
al. 1995; Svoboda et al. 1997; Yuste et al. 1994), presumably
reflecting the differential distribution and activation of ion
channels and shunting conductances (Jaffe et al. 1992; Migliore 1996; Tsubokawa and Ross 1996; Westenbroek et al.
1992). Rather than focus on factors controlling dendritic
back-propagation, we chose to compare dendritic and axonal
sensitivity under conditions of low safety factor (low extracellular Na / ). In proximal dendritic regions ( Ç30 mm from
the soma), AP-evoked Ca 2/ responses in 60 Na / were reduced by Ç10% when compared with responses in 150 Na / .
This slight attenuation observed at the proximal dendrite is
likely due to the effects of Na / substitution and not conduction, as discussed previously. However, when more distal
regions were examined, the amplitude of the Ca 2/ response
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FIG . 9. Reliable axonal AP conduction from
hyperpolarized potentials. A: basal fluorescence
image illustrating secondary and tertiary axonal
branches and associated boutons ú250 mm from
the soma (hippocampal). B: traces of change in
D fluo-3/furaptra ( DF480 /F380 ) fluorescence vs.
time after 2 APs elicited from 060 mV (thin
trace) and 0100 mV (thick trace). Traces are
average of 8 ( 060 mV) and 11 trials ( 0100 mV)
for 3 axonal sites. C: traces of somatic membrane
potential illustrating 2 APs 50 ms apart elicited
from 060 mV (left) or from 0100 mV (right).
D: average Ca 2/ transient ( 0100 mV) vs. average Ca 2/ response ( 060 mV). Diagonal line represents unity where the 060 mV response equals
the 0100 mV response.
2100
P. J. MACKENZIE AND T. H. MURPHY
Electrical compartmentalization of the neuron
Previous studies contributed to the emerging view that the
neuron is composed of compartments of differing electrical
excitability (for review see Stuart et al. 1997). Cortical and
hippocampal dendrites are capable of local electrogenesis,
however this usually stops short of an all-or-none AP. In
contrast AP generation results in faithful propagation along
the axon (Allen and Stevens 1994; Lüscher et al. 1996;
Mackenzie et al. 1996; Stevens and Wang 1995) and backpropagation along dendrites where the amplitude decreases
with distance (Spruston et al. 1995; Stuart and Sakmann
1994; Stuart et al. 1997). We report here a marked difference
in conduction reliability between axons and dendrites of hippocampal and cortical neurons. The propagation of APs at
low frequency occurs in the axon even under conditions of
lower external Na / . This resistance to external 60 Na / was
unique to the axon and supports the suggestion that the axon
functions as a more excitable electrical compartment
(Mainen et al. 1995; Rapp et al. 1996). This considerable
degree of safety in the mechanism of AP propagation suggests that in the axon the AP, once generated, is indeed all
or none and that Na / channel modulation is not likely to
play a major role in AP conduction. However, as larger
current steps were required to generate an AP in 60 Na / ,
we acknowledge a possible role of Na / channel modulation
at the sites of AP initiation such as the axon hillock. Taken
together, this suggests that modulation of Na / channels or
leakage channels that affect membrane resistance may have
a greater impact on the AP generation or on the local depolarization of terminals than on conduction safety. In dendrites,
in low Na / , APs resulted in Ca 2/ transients over a more
limited distance along the dendrite. These results in the dendrite are consistent with previous results suggesting a graded,
modifiable dendritic AP (Jaffe et al. 1992; Spruston et al.
1995; Tsubokawa and Ross 1996). Our results support the
hypothesis that regulation of dendritic Na / channels can
control the spatial extent of dendritic electrogenesis, presumably for both forward synaptic integration and for retrograde
AP propagation (Colbert et al. 1997; Jung et al. 1997;
Mainen et al. 1995; Rapp et al. 1996).
We thank Drs. J. Seamans, S. Wang, and J. Winslow for helpful comments on the manuscript.
/ 9k2d$$oc42 J152-8
This work was supported by grants from the Medical Research Council
of Canada and the EJLB foundation to T. H. Murphy, an EJLB and Medical
Research Council scholar. P. J. Mackenzie is supported by a Medical Research Council studentship.
Address reprint requests to T. H. Murphy.
Received 3 March 1998; accepted in final form 9 July 1998.
REFERENCES
ALGER, B. E., PITLER, T. A., WAGNER, J. J., MARTIN, L. A., MORISHITA, W.,
KIROV, S. A., AND LENZ, R. A. Retrograde signaling in depolarizationinduced suppression of inhibition in rat hippocampal CA1 cells. J. Physiol. (Lond.) 496: 197–209, 1996.
ALLEN, C. AND STEVENS, C. F. An evaluation of causes for unreliability of
synaptic transmission. Proc. Natl. Acad. Sci. USA 91: 10380–10383,
1994.
AUGUSTINE, G. J. AND CHARLTON, M. P. Calcium dependence of presynaptic
calcium current and post-synaptic response at the squid giant synapse.
J. Physiol. (Lond.) 381: 619–640, 1986.
BARRON, D. H. AND MATHEWS, B.H.C. Intermittent conduction in the spinal
cord. J. Physiol. (Lond.) 85: 73–103, 1935.
BOURON, A. AND REUTER, H. A role of intracellular Na / in the regulation
of synaptic transmission and turnover of the vesicular pool in cultured
hippocampal cells. Neuron 17: 969–978, 1996.
CALLEWAERT, G., EILERS, J., AND KONNERTH, A. Axonal calcium entry
during fast sodium action potentials in rat cerebellar purkinje neurones.
J. Physiol. (Lond.) 495: 641–647, 1996.
CATTERALL, W. A. Structure and modulation of Na / and Ca 2/ channels.
Ann. NY Acad. Sci. 707: 1–19, 1993.
COLBERT, C. M. AND JOHNSTON, D. Axonal action-potential initiation and
Na / channel densities in the soma and axon initial segment of subicular
pyramidal neurons. J. Neurosci. 16: 6676–6686, 1996.
COLBERT, C. M., MAGEE, J. C., HOFFMAN, D. A., AND JOHNSTON, D. Slow
recovery from inactivation of Na / channels underlies the activity-dependent attenuation of dendritic action potentials in hippocampal CA1 pyramidal neurons. J. Neurosci. 17: 6512–6521, 1997.
DEBANNE, D., GUÉRINEAU, N. C., GÄHWILER, B. H., AND THOMPSON, S. M.
Action-potential propogation gated by an axonal IA-like K / conductance
in hippocampus. Nature 389: 286–290, 1997.
DITYATEV, A. E. AND CLAMANN, H. P. Reliability of spike propagation in
arborizations of dorsal root fibers studied by analysis of postsynaptic
potentials mediated by electrotonic coupling in the frog spinal cord. J.
Neurophysiol. 76: 3451–3459, 1996.
DODGE, F.A.J. AND RAHAMIMOFF, R. Co-operative action of calcium ions
in transmitter release at the neuromuscular junction. J. Physiol. (Lond.)
193: 419–432, 1967.
FORSYTHE, I. D., TSUJIMOTO, T., BARNESDAVIES, M., CUTTLE, M. F., AND
TAK AHASHI, T. Inactivation of presynaptic calcium current contributes
to synaptic depression at a fast central synapse. Neuron 20: 797–807,
1998.
FRENGUELLI, B. G. AND MALINOW, R. Fluctuations in intracellular calcium
responses to action potentials in single en passage presynaptic boutons
of layer V neurons in neocortical slices. Learn. Mem. 3: 150–159, 1996.
GROSSMAN, Y., SPIRA, M. E., AND PARNAS, I. Differential flow of information into branches of a single axon. Brain Res. 64: 379–386, 1973.
HAMILL, O. P., MARTY, A., NEHER, E., SAKMANN, B., AND SIGWORTH, F.
Improved patch-clamp techniques for high resolution current recordings
from cells and cell free membrane patches. Pflügers Arch. 391: 85–100,
1981.
HÄUSSER, M., STUART, G., RACCA, C., AND SAKMANN, B. Axonal initiation
and active dendritic propagation of action potentials in substantia nigra
neurons. Neuron 15: 637–647, 1995.
HEIDELBERGER, R., HEINEMANN, C., NEHER, E., AND MATTHEWS, G. Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature
371: 513–515, 1994.
HOFFMAN, D. A., MAGEE, J. C., COLBERT, C. M., AND JOHNSTON, D. K /
channel regulation of signal propagation in dendrites of hippocampal
pyramidal neurons. Nature 387: 869–875, 1997.
JAFFE, D. B., JOHNSTON, D., LASSER-ROSS, N., LISMAN, J. E., MIYAK AWA,
H., AND ROSS, W. N. The spread of Na / spikes determines the pattern
of dendritic Ca 2/ entry into hippocampal neurons. Nature 357: 244–246,
1992.
JUNG, H.-Y., MICKUS, T., AND SPRUSTON, N. Prolonged sodium channel
09-23-98 10:27:58
neupa
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 17, 2017
decreased with distance in 150 Na / and to a greater extent
in 60 mM Na / (Figs. 5 and 6).
To test whether the residual Ca 2/ transient in 60 Na /
depended on Na / channels, we measured Ca 2/ responses
along the dendrite to suprathreshold somatic current steps
in the presence of 1 mM TTX in 150 Na / . In TTX, Ca 2/
responses were considerably attenuated along the length of
the dendrite; responses were õ5% of the 150 Na / response
obtained after washout of TTX (Fig. 7). The spatial profile
in TTX contrasted with the profile in 60 Na / , where Ca 2/
transients at more proximal dendritic regions were considerable, up to 90% of their amplitude in 150 Na / (Fig. 5).
Therefore most of the Ca 2/ response that remained in 60
Na / depended on TTX-sensitive Na / channels. Dendritic
Ca 2/ responses to APs differed from axonal responses in
their spatial profile in normal external Na / and in their sensitivity to a reduction of Na / influx.
AXONAL AND DENDRITIC CONDUCTION
/ 9k2d$$oc42 J152-8
GABAergic inhibition in rat hippocampal pyramidal cells: G protein
involvement in a presynaptic mechanism. Neuron 13: 1447–1455, 1994.
RAGU, B., MURPHY, E., LEVY, L. A., HALL, R. D., AND LONDON, R. E. A
fluorescent indicator for measuring cytosolic free magnesium. Am. J.
Physiol. 256 (Cell Physiol. 25): C540–C548, 1989.
RAPP, M., YAROM, Y., AND SEGEV, I. Modeling back propagating action
potential in weakly excitable dendrites of neocortical pyramidal cells.
Proc. Natl. Acad. Sci. USA 93: 11985–11990, 1996.
SCHILLER, J., HELMCHEN, F., AND SAKMANN, B. Spatial profile of dendritic
calcium transients evoked by action potentials in rat neocortical pyramidal
neurones. J. Physiol. (Lond.) 487: 583–600, 1995.
SCHILLER, J., SCHILLER, Y., STUART, G., AND SAKMANN, B. Calcium action
potentials restricted to distal apical dendrites of rat neocortical pyramidal
neurons. J. Physiol. (Lond.) 505: 605–616, 1997.
SCHWINDT, P. C. AND CRILL, W. E. Amplification of synaptic current by
persistent sodium conductance in apical dendrite of neocortical neurons.
J. Neurophysiol. 74: 2220–2224, 1995.
SEGAL, M. AND BARKER, J. L. Rat hippocampal neurons in culture: potassium conductances. J. Neurophysiol. 51: 1409–1433, 1984.
SPRUSTON, N., SCHILLER, Y., STUART, G., AND SAKMANN, B. Activitydependent action potential invasion and calcium influx into hippocampal
CA1 dendrites. Science 268: 297–300, 1995.
STEVENS, C. F. AND WANG, Y. Facilitation and depression at single central
synapses. Neuron 14: 795–802, 1995.
STUART, G., SPRUSTON, N., SAKMANN, B., AND HÄUSSER, M. Action potential initiation and backpropagation in neurons of the mammalian CNS.
Trends Neurosci. 20: 125–131, 1997.
STUART, G. J. AND SAKMANN, B. Active propagation of somatic action
potentials into neocortical pyramidal cell dendrites. Nature 367: 69–72,
1994.
SVOBODA, K., DENK, W., KLEINFELD, D., AND TANK, D. W. In vivo dendritic
calcium dynamics in neocortical pyramidal neurons. Nature 385: 161–
165, 1997.
TSUBOK AWA, H. AND ROSS, W. N. IPSPs modulate spike backpropogation
and associated [Ca 2/ ]i changes in the dendrites of hippocampal CA1
pyramidal neurons. J. Neurophysiol. 76: 2896–2906, 1996.
VINCENT, P. AND MARTY, A. Fluctuations of inhibitory postsynaptic currents
in Purkinje cells from rat cerebellar slices. J. Physiol. (Lond.) 494: 183–
199, 1996.
WALL, P. D. Do nerve impulses penetrate terminal arborizations? A prepresynaptic control mechanism. Trends Neurosci. 18: 99–103, 1995.
WESTENBROEK, R. E., HELL, J. W., WARNER, C., DUBEL, S. J., SNUTCH,
T. P., AND CATTERALL, W. A. Biochemical properties and subcellular
distribution of an N-type calcium channel alpha 1 subunit. Neuron 9:
1099–1115, 1992.
WU, R.-L. AND BARISH, M. E. Two pharmacologically and kinetically distinct transient potassium currents in cultured embryonic mouse hippocampal neurons. J. Neurosci. 12: 2235–2246, 1992.
YUSTE, R., GUTNICK, M. J., SAAR, D., DELANEY, K. R., AND TANK, D. W.
Ca 2/ accumulations in dendrites of neocortical pyramidal neurons: an
apical band and evidence for two functional compartments. Neuron 13:
23–43, 1994.
09-23-98 10:27:58
neupa
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on June 17, 2017
inactivation contributes to dendritic action potential attenuation in hippocampal pyramidal neurons. J. Neurosci. 17: 6639–6646, 1997.
KRNJEVIC, K. AND MILEDI, R. Presynaptic failure of neuromuscular propagation in rats. J. Physiol. (Lond.) 149: 1–22, 1959.
LI, M., WEST, J. W., NUMANN, R., MURPHY, B. J., SCHEUER, T., AND CATTERALL, W. A. Convergent regulation of sodium channels by protein
kinase C and cAMP-dependent protein kinase. Science 261: 1439–1442,
1993.
LLANO, I., LERESCHE, N., AND MARTY, A. Calcium entry increases the
sensitivity of cerebellar Purkinje cells to applied GABA and decreases
inhibitory synaptic currents. Neuron 6: 565–574, 1991.
LLANO, I., TAN, Y. P., AND CAPUTO, C. Spatial heterogeneity of intracellular
Ca 2/ signals in axons of basket cells from rat cerebellar slices. J. Physiol.
(Lond.) 502: 509–519, 1997.
LÜSCHER, C., LIPP, P., LÜSCHER, H. R., AND NIGGLI, E. Control of action
potential propagation by intracellular Ca 2/ in cultured rat dorsal root
ganglion cells. J. Physiol. (Lond.) 490: 319–324, 1996.
LÜSCHER, C., STREIT, J., LIPP, P., AND LÜSCHER, H. R. Action potential
propagation through embryonic dorsal root ganglion cells in culture. II.
Decrease of conduction reliability during repetitive stimulation. J. Neurophysiol. 72: 634–643, 1994.
MA, J. Y., LI, M., CATTERALL, W. A., AND SCHEUER, T. Modulation of
brain Na / channels by a G-protein-coupled pathway. Proc. Natl. Acad.
Sci. USA 91: 12351–12355, 1994.
MACKENZIE, P. J., UMEMIYA, M., AND MURPHY, T. H. Ca 2/ imaging of
CNS axons in culture indicates reliable coupling between single action
potentials and distal functional release sites. Neuron 16: 783–795, 1996.
MAGEE, J. C. AND JOHNSTON, D. Characterization of single voltage-gated
Na / and Ca 2/ channels in apical dendrites of rat CA1 pyramidal neurons.
J. Physiol. (Lond.) 487: 67–90, 1995.
MAINEN, Z. F., JOERGES, J., HUGUENARD, J. R., AND SEJNOWSKI, T. J. A
model of spike initiation in neocortical pyramidal neurons. Neuron 15:
1427–1439, 1995.
MARKRAM, H., HELM, P. J., AND SAKMANN, B. Dendritic calcium transients
evoked by single back-propogating action potentials in rat neocortical
pyramidal neurons. J. Physiol. (Lond.) 485: 1–20, 1995.
MIGLIORE, M. Modeling the attenuation and failure of action potentials in
the dendrites of hippocampal neurons. Biophys. J. 71: 2394–2403, 1996.
MINTA, A., KAO, J., AND TSIEN, R. Y. Fluorescent indicators for cytosolic
calcium based on rhodamine and fluorescein chromophores. J. Biol.
Chem. 264: 8171–8178, 1989.
MINTZ, I. M., SABATINI, B. L., AND REGEHR, W. G. Calcium control of
transmitter release at a cerebellar synapse. Neuron 15: 675–688, 1995.
MURPHY, T. H. AND BARABAN, J. M. Glutamate toxicity in immature cortical neurons precedes development of glutamate receptor currents. Dev.
Brain Res. 57: 146–150, 1990.
NEHER, E. Voltage offsets in patch-clamp experiments. In: Single-Channel
Recording, edited by B. Sakmann and E. Neher. New York: Plenum,
1995a, p. 147–153.
NEHER, E. The use of fura-2 for estimating Ca buffers and Ca fluxes.
Neuropharmacology 34: 1423–1442, 1995b.
PITLER, T. A. AND ALGER, B. E. Depolarization-induced suppression of
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