Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5513
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ABSTRACT |
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Cameron, Jill S. and
Stuart E. Dryer.
BK-Type KCa Channels in Two Parasympathetic Cell
Types: Differences in Kinetic Properties and Developmental Expression.
J. Neurophysiol. 84: 2767-2776, 2000.
The intrinsic electrical properties of identified choroid and
ciliary neurons of the chick ciliary ganglion were examined by
patch-clamp recording methods. These neurons are derived from a common
pool of mesencephalic neural crest precursor cells but innervate
different target tissues and have markedly different action potential
waveforms and intrinsic patterns of repetitive spike discharge.
Therefore it is important to determine whether these cell types express
different types of plasma membrane ionic channels, and to ascertain the
developmental stages at which these cell types begin to diverge. This
study has focused on large-conductance Ca2+-activated K+ channels
(KCa), which are known to regulate spike waveform
and repetitive firing in many cell types. Both ciliary ganglion cell types, identified on the basis of size and somatostatin
immunoreactivity, express a robust macroscopic
KCa carried by a kinetically homogeneous population of large-conductance (BK-type) KCa
channels. However, the kinetic properties of these channels are
different in the two cell types. Steady-state fluctuation analyses of
macroscopic KCa produced power spectra that could
be fitted with a single Lorentzian curve in both cell types. However,
the resulting corner frequency was significantly lower in choroid
neurons than in ciliary neurons, suggesting that the underlying
KCa channels have a longer mean open-time in
choroid neurons. Consistent with fluctuation analyses, significantly
slower gating of KCa channels in choroid neurons
was also observed during macroscopic activation and deactivation at
membrane potentials positive to 30 mV. Differences in the kinetic
properties of KCa channels could also be observed
directly in single-channel recordings from identified embryonic
day 13 choroid and ciliary neurons. The mean open-time of
large-conductance KCa channels was significantly
greater in choroid neurons than in ciliary neurons in excised
inside-out patches. The developmental expression of functional
KCa channels appears to be regulated differently in the two cell types. Although both cell types
acquire functional KCa at the same developmental
stages (embryonic days 9-13), functional expression of
these channels in ciliary neurons requires target-derived trophic
factors. In contrast, expression of functional
KCa channels proceeds normally in choroid neurons developing in vitro in the absence of target-derived trophic factors. Consistent with this, extracts of ciliary neuron target tissues (striated muscle of the iris/ciliary body) contain
KCa stimulatory activity. However,
KCa stimulatory activity cannot be detected in
extracts of the smooth muscle targets of choroid neurons.
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INTRODUCTION |
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The electrophysiological
properties of neurons are typically tuned for a specific physiological
function. However, the factors controlling the developmental expression
of a specialized electrophysiological phenotype are not well
understood. The chick ciliary ganglion (CG) is a useful model system in
which to study this process, as it contains two populations of
mesencephalic neural crest-derived neurons that innervate different
target tissues in the eye (Marwitt et al. 1971).
Mature ciliary neurons innervate striated muscle cells in the iris and
ciliary body, whereas choroid neurons innervate smooth muscle cells in
the choroid layer. Both cell types are cholinergic, but choroid neurons
also express somatostatin whereas ciliary neurons do not, and choroid
neurons tend to be smaller than ciliary neurons (Darland and
Nishi 1998
; De Stefano et al. 1993
;
Epstein et al. 1988
). These differences provide
convenient criteria for identification of these neurons in dissociated preparations.
Choroid and ciliary neurons in the intact CG have markedly different
electrophysiological properties (Dryer 1994). Ciliary neurons fire spikes at a constant high frequency (>150 Hz) in response
to sustained injection of depolarizing current. The intrinsic firing
properties of ciliary neurons are well matched to those of their
striated muscle targets, which exhibit tetanic contraction only when
stimulated at 100-150 Hz (Pilar and Vaughan 1969
). In contrast, choroid neurons exhibit a maximum firing frequency of ~35
Hz and show substantial spike-frequency adaptation. Equally notably,
the spike duration at half-amplitude in choroid neurons is about twice
that of ciliary neurons (Dryer 1994
).
We have previously shown that the differentiation of excitability in CG
neurons is regulated by cell-cell interactions. Large CG neurons that
develop in vivo in the absence of normal target tissues fail to express
functional Ca2+-activated
K+ channels (KCa)
(Dourado et al. 1994). Moreover, large CG neurons developing in vitro fail to express KCa unless
iris extracts or iris-derived factors are included in the culture media
(Cameron et al. 1998
, 1999
;
Dourado and Dryer 1992
; Subramony et al.
1996
). The essential iris-derived factor appears to be an avian
ortholog of TGF
1. Thus application of TGF
1 can markedly
accelerate the functional expression of KCa in
vivo or in vitro, and a TGF
neutralizing antiserum blocks the normal
in vivo expression of KCa as well as the in vitro
actions of iris extracts (Cameron et al. 1998
). Macroscopic KCa in CG neurons is mediated by
large-conductance (BK-type) channels (Cameron et al.
1998
; Dryer et al. 1991
; Lhuillier and
Dryer 1999
) encoded by the slo gene (Dryer
1998
; Subramony et al. 1996
). Although other
types of KCa channels with lower unitary
conductance can be detected in inside-out patches excised from CG
neurons, these lower conductance KCa channels
cannot be detected on depolarization of intact cells and do not appear
to contribute to macroscopic currents (Lhuillier and Dryer
1999
). Their functional significance, if any, remains unknown.
However, macroscopic KCa contributes to spike
repolarization and afterhyperpolarization in CG neurons (Dryer
et al. 1991
), and therefore differences in the properties of
large-conductance KCa channels could contribute to the physiological differences between choroid and ciliary neurons.
Our previous studies on the developmental regulation of KCa channels and macroscopic KCa have focused on cells that, based on their size, are likely to consist primarily of ciliary neurons. Given the results in ciliary neurons, we hypothesized that KCa regulation in developing choroid neurons would follow a similar pattern, i.e., that expression of an identical population of KCa channels would require trophic factors secreted from target cells in the smooth muscle choroid layer. Here we show that both of these hypotheses are incorrect. Instead, developmental expression of macroscopic KCa in choroid neurons appears to be cell-autonomous, and certainly proceeds normally in the absence of target-derived factors. By contrast, functional expression of KCa in ciliary neurons is not cell-autonomous and requires exposure to trophic factors that are present in the iris, but that cannot be detected in the choroid smooth muscle layer. Moreover, the kinetic properties of large-conductance KCa channels, once they are present in the plasma membrane, are different in the two cell types. Specifically, choroid cells express large-conductance KCa channels with substantially slower kinetics than those of ciliary neurons. This conclusion is based on several independent kinetic methods, including analyses of macroscopic current fluctuation, activation, and deactivation, as well as more direct measurements in inside-out patches. These differences in macroscopic behavior can provide an explanation for at least some of the intrinsic differences in spike discharge between choroid and ciliary neurons. It is also possible that differences in gating properties and regulation of functional expression are consequences of a common molecular mechanism.
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METHODS |
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Cell isolation and culture
Ciliary ganglion neurons were dissociated at embryonic day
9 (E9) or E13 as described in detail
previously (Cameron et al. 1998, 1999
;
Dourado and Dryer 1992
; Subramony et al.
1996
). Cells isolated at E13 were used for
electrophysiology within 2 h of isolation. Cells isolated at
E9 were examined at various times up to 4 days after
isolation, as indicated. Dissociated cells were grown on
poly-D-lysine-coated glass coverslips in a culture medium
consisting of Eagle's minimal essential medium supplemented with 10%
heat-inactivated horse serum, 2 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 40 ng/ml recombinant rat ciliary neurotrophic
factor (R and D Systems, Minneapolis, MN). In some experiments,
E9 CG neurons were cultured for 12 h in media that
contained iris extracts, choroid extracts, or both, as indicated. To
prepare extracts, the iris and the choroid layer were dissected out of
chick embryos at E9, placed in ice-cold Earle's balanced salts (1 iris or choroid per ml), and homogenized in a glass
homogenizer. Extracts were centrifuged at 15,000 g for 1 h at 4°
to remove membranous fractions. Protein concentrations in all extracts
were adjusted to 230-260 µg/ml by addition of Earle's balanced
salts. Iris extracts were added to cell culture media at a
concentration of 3% (vol/vol). Choroid extracts were added to media at
various concentrations as indicated.
Immunocytochemistry
Somatostatin staining was performed essentially as described by
Darland and Nishi (1998). Briefly, CG neurons were
dissociated at E13, fixed for 30 min in Zamboni's solution
(4% paraformaldehyde, 15% picric acid, and 0.1 M sodium phosphate
buffer, pH 7.4), rinsed in PBS, and blocked overnight in 10% horse
serum, 0.5% Triton X-100, and 0.2% sodium azide in PBS. Cells were
then incubated overnight at room temperature (22°C) with monoclonal
rat anti-somatostatin (Accurate Chemical and Scientific, Westbury, NY,
cell line YC7; 1:100 dilution in blocking solution without detergent).
Cells were rinsed and incubated with PBS containing 10%
H2O2 and 30% ethanol to
inactivate endogenous peroxidase, and staining was revealed by the ABC
method (Vectastain kit, Vector Laboratories Burlingame, CA) using
diaminobenzidine as a chromogen according to the manufacturer's
directions. No signal was obtained from control CG cultures treated
with preabsorbed primary antibody or with no primary antibody.
Electrophysiology and data analysis
Whole cell recordings were made using standard methods as
described in detail previously (Cameron et al. 1998,
1999
; Dourado and Dryer 1992
;
Lhuillier and Dryer 2000
; Subramony and Dryer 1997
; Subramony et al. 1996
). In most
experiments, currents were evoked by depolarizing steps to 0 mV from a
holding potential of
40 mV in normal and
Ca2+-free external saline. Normal external saline
consisted of 145 mM NaCl, 5.4 mM KCl, 5.4 mM
CaCl2, 0.8 mM MgCl2, 5 mM
D-glucose, 13 mM HEPES-NaOH, and 250 nM tetrodotoxin, pH
7.4. Nominally Ca2+-free external saline was the
same except that it contained 5.8 mM MgCl2 and no
added CaCl2. Net
Ca2+-dependent outward currents
(KCa) were then obtained by digital subtraction
(control
Ca2+-free) and normalized for
cell size by measuring soma surface area for each cell as described
previously (Cameron et al. 1998
, 1999
;
Dourado and Dryer 1992
; Subramony et al.
1996
). For steady-state fluctuation analysis, currents in each
cell were evoked by a series of 10 depolarizing steps of 2.2 s
duration to various command potentials in normal saline, followed by 10 identical steps after application of Ca2+-free
saline. Data were digitized at 2 kHz, and the DC components of the
evoked currents were removed. The steady-state portions of the evoked
currents were cosine-tapered (w{n} = cos {
n/N}), and power spectra were
computed from 4,096 digital samples using routines implemented in
Pclamp software (Clampfit v. 8.0, Axon Instruments, Foster City, CA).
Spectra obtained for currents evoked by each of the 10 depolarizing
steps were averaged, and the resulting mean spectra were subtracted
(control spectrum
Ca2+-free spectrum).
This served to remove contributions from instrumentation noise and
other voltage-evoked ionic currents (except for
Ca2+ currents), and to thereby isolate
the mean power spectrum of macroscopic KCa. The
resulting KCa power spectra were smoothed by
adjacent point averaging and then fitted with Lorentzian curves of the
form S(f) = S(0)/[1 + (2
fc
)2]
as described in the text using a Levenburg-Marquardt least-squares algorithm (Microcal Origin, v 6.0, Northampton, MA). Single Lorentzian curves provided excellent fits to the KCa power
spectra at all command potentials, a finding consistent with our
previous studies with cell-attached patches in which we found that only
a single population of large-conductance KCa
channels became active on depolarization of intact cells, as well as
single-exponential macroscopic tail currents over a wide range of test
potentials (Lhuillier and Dryer 1999
). This spectral
subtraction procedure allows calculation of variance contributed by
those macroscopic currents that depend on external
Ca2+, which with these voltage-clamp protocols
consist of voltage-activated Ca2+ currents and
KCa. To determine whether
Ca2+ currents make a significant contribution to
subtracted power spectra, a series of similar protocols were carried
out in the presence of 10 mM tetraethylammonium, which causes a
complete blockade of KCa (Dryer et al.
1991
) and which therefore allows for isolation of spectral
components contributed by Ca2+ currents.
Increases in variance contributed by Ca2+
currents were undetectable in macroscopic measurements, a result consistent with the very low unitary conductance of these channels (Marrion and Tavalin 1998
). Consequently, the subtracted
power spectra reflect gating of BK type KCa
channels. Deactivation kinetics were analyzed from
Ca2+-dependent tail currents recorded at various
membrane potentials after a step pulse to 0 mV from a holding potential
of
40 mV (Lhuillier and Dryer 1999
). The decay phases
of tail currents were fitted with single-exponential curves using
Clampfit software.
Inside-out patch recordings were made by standard methods as described
in detail previously (Cameron et al. 1998;
Lhuillier and Dryer 1999
). Briefly, patches were excised
into Ca2+-free salines containing 10 mM EGTA, and
KCa channels were activated by bath application
of salines containing 5 µM free Ca2+. The
holding potential was +25 mV throughout. Single-channel data were
filtered at 2 kHz with a 4-pole Bessel filter and stored on magnetic
videotape in FM mode for off-line digitization (2 kHz) and analysis
using Pclamp software. Idealized traces were generated using a
half-threshold crossing procedure that ignored transitions of <0.5 ms
duration, and idealized data were used to construct open-time
histograms and to calculate Po. All
statistical analyses were carried out using Statistica software (v5.1,
Statsoft, Tulsa, OK). Throughout, error bars represent SE. Data were
analyzed by one-way ANOVA followed by Scheffé's multiple range
test (where comparisons were made between multiple groups), Student's
unpaired t-test (for comparisons between 2 groups), and
linear regression analysis. Cell size distribution data were analyzed
by the Kolmogirov-Smirnov one-sample test for deviations from a
unimodal Gaussian distribution. Throughout, P < 0.05 was regarded as significant.
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RESULTS |
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Identification of isolated choroid and ciliary neurons
In intact ganglia, the two populations of CG neurons can be
distinguished on the basis of size, ultrastructure, and somatostatin immunoreactivity. Choroid neurons express somatostatin, whereas ciliary
neurons do not, and the majority of choroid neurons are smaller than
ciliary neurons, although there is overlap in their size distributions
(Darland and Nishi 1998; De Stefano et al. 1993
; Epstein et al. 1988
). In particular,
De Stefano et al. (1993)
used ultrastructural criteria
to show that somatostatin is a highly specific marker for choroid
neurons. In our initial studies, we measured neuronal diameters in
acutely isolated preparations of embryonic day 13 (E13) CG neurons (Fig.
1A). The distribution of
neuronal diameters was then fitted with a Gaussian distribution or as
the sum of two Gaussians. Attempts to fit the observed distribution with a single Gaussian were unsatisfactory, and the raw data were significantly (P < 0.05) different from a unimodal
normal distribution (Kolmogorov-Smirnov 1-sample test). However, better
fits were obtained with the sum of two approximately equally weighted
Gaussians with means of 14.5 and 19.8 µm (Fig. 1A). To
determine whether these two groups correspond to choroid and ciliary
neurons, preparations of dissociated E13 CG neurons were
stained for somatostatin immunoreactivity (Fig. 1B), and the
size distributions of labeled and unlabeled neurons were determined.
The resulting distributions correspond closely to the two Gaussians
observed in unstained cells (Fig. 1C). In these
preparations, 114 of 116 neurons with diameters
14 µm expressed
somatostatin, a marker for choroid neurons. In contrast, none of the
108 cells with diameters
17 µm expressed somatostatin, indicating
that these represent an enriched population of ciliary neurons. To
minimize the possibility of sampling error in physiological
experiments, neurons with diameters
12 µm were considered to be
choroid neurons, neurons with diameters
20 µm were considered to be
ciliary neurons, and cells with intermediate diameters were not
studied.
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Differences in macroscopic KCa kinetics in choroid and ciliary neurons
We have previously shown that macroscopic
KCa in CG neurons reaches maximum current density
by E13 (Dourado and Dryer 1992). Here we have
observed that the current density of macroscopic KCa is not significantly different in
choroid and ciliary neurons isolated acutely at this developmental
stage (Fig. 2). However, the kinetic
properties of the underlying channels are different in the two cell
types. This was established in part by three different types of
macroscopic measurements. Whole cell recordings have the advantage of
sampling only those channels that contribute to macroscopic currents
[i.e., KCa channels that are properly co-localized with voltage-dependent Ca2+ channels
in the plasma membrane (see Gola and Crest 1993
;
Lhuillier and Dryer 1999
; Marrion and Tavalin
1998
)]. This recording configuration also avoids any changes
in KCa channel properties that could occur as a
result of patch excision.
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In the first approach, the kinetic properties of whole cell
KCa were determined by steady-state fluctuation
analysis. If one assumes that channels gate independently, then it is
possible to extract information about the underlying kinetics from
spectral density analysis of macroscopic current fluctuations
(Anderson and Stevens 1973). Although fine details of
the kinetics are masked by bandwidth limitations inherent in whole cell
recording, this type of analysis has been successfully used to resolve
kinetic differences associated with developmental changes in the
subunit stoichiometry of ligand-gated ionic channels (e.g.,
Fischbach and Schuetze 1980
; Vicini and Schuetze
1985
). In our experiments, currents in each CG cell were evoked
by a series of 10 depolarizing step pulses of 2.2-s duration to various
holding potentials from a holding potential of
40 mV in the presence
of Ca2+. These protocols were then repeated in
the absence of external Ca2+ (Fig.
3, A and B) as
described previously (Cameron et al. 1998
, 1999
; Dourado and Dryer 1992
;
Dourado et al. 1994
; Subramony et al.
1996
). The mean power spectra of macroscopic current
fluctuations were calculated for currents evoked in the presence and
absence of Ca2+, and the resulting spectra were
subtracted digitally to determine the
Ca2+-dependent component of the variance
associated with evoked steady-state outward currents (Fig. 3,
C and D). The subtracted power spectra were
fitted with Lorentzian curves of the form
S(f) = S(0)/[1 + (2
fc
)2],
where S(f) is power as a function of
frequency, S(0) is the extrapolated power at frequency = 0 Hz,
fc is the frequency at which power is
S(0)/2, and
is a time constant defined as 1/2
fc and related but not precisely equal
to the mean open-time. In both populations of neurons, single
Lorentzian curves provided excellent fits to the subtracted power
spectra, suggesting that a kinetically homogeneous population of
channels underlies macroscopic KCa (Fig. 3,
C and D). These panels show spectra calculated
for currents evoked by a depolarizing step to 0 mV, and they indicate that KCa channels have different kinetic
properties in the two populations of cells. The mean time constant of
fitted Lorentzian curves calculated from pulses to 0 mV in a large
number of choroid cells was more than twice that of ciliary cells (Fig.
3E). These differences are statistically significant and are
not a consequence of different probabilities of channel opening
(Po). It is possible to determine
Po during a depolarizing step by means
of binomial statistics (Anderson and Stevens 1973
). Thus
i = (1
Po)
I2/µI, where
i is the unitary current flowing through a single open KCa channel,
I2
is the variance of the macroscopic fluctuations associated with KCa gating, and µI is the
mean Ca2+-dependent outward current. Independent
measurements with cell-attached patches indicate that i = 6.5 pA at 0 mV with close to physiological ionic gradients
(Lhuillier and Dryer 1999
). Current variance
associated with KCa gating
(
I2) is simply the integral of the
fitted Lorentzian curve, which is given by S(0)
fc/2. The mean current
(µI) is the difference in net outward current
evoked in the presence and absence of Ca2+. From
these parameters, the mean Po during a
step pulse to 0 mV was calculated to be 0.92 ± 0.06 in ciliary
cells, and 0.95 ± 0.07 in choroid cells. These results are not
significantly different and indicate that step pulses to 0 mV evoke
comparable KCa activation in the two cell types.
In a separate set of experiments, similar analyses were carried out on
both cell types using a range of depolarizing steps (to
25 mV, 0 mV,
and +25 mV) from a holding potential of
40 mV. The resulting time
constants derived from power spectral analysis were significantly
(P < 0.05) longer in choroid cells than in ciliary
cells at all three membrane potentials (Fig. 3F). However,
these differences were more apparent at more depolarized potentials,
i.e., the voltage dependence of KCa gating appears greater in choroid neurons than in ciliary neurons.
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This pattern can also be seen in analyses of KCa
deactivation kinetics in the two cell types. For these experiments,
KCa was activated by a 25-ms step to 0 mV from a
holding potential of 40 mV, at which time the membrane was stepped
through a series of test potentials between
70 and
20 mV. This
protocol was repeated in normal and Ca2+-free
salines, and the resulting Ca2+-dependent
currents were obtained by digital subtraction (Fig. 4A). The decay phases of the
KCa tail currents were fitted with single-exponential curves, which provided good fits to the data in both
cell types as described previously for ciliary neurons (Dryer et
al. 1991
; Lhuillier and Dryer 1999
). As with
fluctuation analysis, the resulting deactivation time constants were
significantly longer in choroid neurons than in ciliary neurons,
but only at
30 and
20 mV. Tail-current decay time constants were
not significantly different in the two cell types at more negative
membrane potentials, an observation consistent with the trends noted
above in fluctuation analysis.
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Significant differences in macroscopic KCa
behavior in the two cell types also appeared in analyses of activation
kinetics derived from test pulses to 0 mV from a holding potential of
40 mV (Fig. 4B). The time course of activation was fitted
with a single-exponential. Activation of KCa was
several-fold slower in choroid neurons than in ciliary neurons, and the
differences in the resulting time constants are statistically
significant (P < 0.01). Macroscopic
KCa is the largest outward current that can be
evoked from normal resting potential in both cell types, and these data
therefore provide a mechanism for the different spike durations in the
two cell populations.
Differences in single-channel KCa kinetics in choroid and ciliary neurons
These analyses were carried out using two different single-channel
recording configurations. In one set of experiments, the kinetic
properties of KCa channels were determined in
inside-out patches excised from choroid and ciliary neurons. This
method has the advantage of examining KCa channel
gating more directly under controlled membrane potential and
[Ca2+]i, but has the
disadvantage of sampling all KCa channels in the patch membrane, whether or not they would normally contribute to
macroscopic currents. In these experiments, patches were excised into a
Ca2+-free bath saline (containing 10 mM EGTA) and
held at +25 mV. KCa channels were quiescent in
Ca2+-free bath saline but became active on bath
application of salines containing 5 µM free
Ca2+ (Fig. 5).
These conditions were chosen because they yielded a mean
Po > 0.90, comparable to the
gating conditions of the macroscopic measurements described above (Fig.
6, A and B).
Kinetic analyses were performed on patches that had one, or at most
two, large-conductance KCa channels, and that did
not contain other detectable channel types. In particular, patches that
contained intermediate-conductance KCa channels
were excluded, as these channels do not appear to contribute to
macroscopic currents and are not activated by depolarization of intact
CG neurons (Lhuillier and Dryer 1999). The unitary
conductance of single large-conductance KCa
channels, derived from all-points histograms, was indistinguishable in
ciliary and choroid neurons. In both cell types, this was 120 pS
with [K+]o = 37.5 mM and [K+]i = 150 mM
(data not shown). Open-time distributions were constructed from
idealized data ignoring transitions of <0.5 ms duration. Single
exponential curves provided excellent fits to the resulting distributions in both cell types. The open-time histograms shown in
Fig. 6, A and B, were constructed from patches
that contained only one KCa channel based on
maximum current amplitudes observed at high
Po. It should be noted that the time
constants of the fitted exponential curves were significantly longer in
choroid cells than in ciliary cells (Fig. 6, A and
B). Mean time constants pooled from several excised patches
were in good agreement with mean time constants obtained from
fluctuation analysis (Fig. 6C).
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Different modes of KCa regulation in developing choroid and ciliary neurons
We have previously shown that large CG neurons isolated at
E9 and allowed to develop in culture for 4 days do not
express macroscopic KCa at significant levels, in
distinct contrast to CG neurons isolated acutely at E13
(Cameron et al. 1998, 1999
; Dourado and Dryer 1992
; Subramony and Dryer
1997
; Subramony et al. 1996
). Application of
iris extracts (Subramony et al. 1996
) or
target-derived differentiation factors (Cameron et al.
1998
, 1999
) restores normal expression of
KCa in CG neurons developing in vitro. The
majority of those recordings were made from cells that, based on their
size, were likely to be ciliary neurons. To determine whether the two
cell types achieve their mature (E13) current density by
different developmental mechanisms, we initially determined whether the
two cell types acquire robust macroscopic KCa at
the same developmental stages. Approximately half of the CG neurons at
E9 are comprised of choroid cells (Landmesser and Pilar 1974
). Therefore robust expression of
KCa in E9 choroid neurons would
predict a distinctly bimodal distribution of current densities and an
inverse correlation between current density and cell size in the entire
population of CG neurons. This was not observed in a large
number of CG neurons isolated acutely at E9 (E9 + 0 in vitro cells), with care taken to sample neurons
spanning the full range of neuronal diameters apparent in these
preparations (Fig. 7A).
Instead, the distribution of current densities was unimodal, and there
was no correlation between current density and cell size either within
a group or when the entire population of CG neurons was considered.
These data indicate that very few cells of either type express
KCa at significant densities by E9, in
contrast to E13, when both cells express robust
KCa at a comparable current density (Fig. 2).
Does the developmental expression of KCa in
choroid neurons require inductive interactions, as observed previously
for ciliary neurons? To test this hypothesis, CG cells were isolated at
E9, a stage prior to significant synapse formation with
target tissues in either cell type. Neurons were then allowed to
develop in vitro for 4 days in standard culture medium (E9 + 4 in vitro cells), at which time KCa
expression was determined by means of whole cell recordings. As with
our previous studies, we found that KCa was
either undetectable or expressed at very low density in E9 + 4 in vitro ciliary neurons. In contrast, macroscopic KCa expression was robust in E9 + 4 in vitro choroid neurons and the current density was comparable
to that observed in CG neurons isolated acutely at E13 (Fig.
7B). These differences are statistically significant. As
expected from these data, the distribution of KCa
densities in the entire sample of E9 + 4 in vitro
CG neurons was clearly bimodal with a statistically significant inverse
correlation between cell size and KCa current
density when the entire population of CG cells was considered (Fig.
7C). There was no correlation between cell size and current
density within a group. In other words, qualitatively
different distributions of current density were observed in
E9 + 4 in vitro CG neurons compared with cells isolated
acutely at either E9 or E13. These results
support the hypothesis that KCa is regulated
cell-autonomously in small choroid neurons, and clearly indicate
different KCa regulatory mechanisms in developing
choroid and ciliary neurons.
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Differences in KCa stimulating activity of choroid coat and iris/ciliary body extracts
Given the differences in the functional regulation of
KCa expression in choroid and ciliary neurons, it
was of interest to determine whether choroid neuron target tissues in
the eye express KCa stimulatory factors. We have
previously shown that ciliary neurons that develop in vitro can be
induced to express macroscopic KCa by iris
extracts or by target-derived factors such as TGF1/4 (Cameron
et al. 1998
, 1999
; Subramony et al.
1996
). In the present experiments, we prepared extracts of the
choroid smooth muscle layer and the iris/ciliary body in parallel,
using procedures described previously (Cameron et al.
1998
, 1999
; Subramony et al.
1996
). CG neurons were isolated at E9 and cultured
for 12 h in the presence or absence of choroid extract and/or iris
extract. Recordings were then made from CG neurons, at least one
population of which express functional KCa in
response to iris extracts (Cameron et al. 1998
,
1999
; Subramony et al. 1996
). As
described previously, iris extracts evoked robust stimulation of
KCa expression in CG neurons, probably owing to
the ciliary neurons present in these cultures (Fig.
8). In contrast, application of choroid
extracts at several concentrations did not evoke
KCa expression in CG neurons.
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DISCUSSION |
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Choroid and ciliary neurons of the chick CG are both derived from
cranial neural crest and undergo key developmental events at the same
embryonic stages (reviewed in Dryer 1994). However, the
two cell populations of cells differ in several mature phenotypic traits (De Stefano et al. 1993
; Dryer
1994
; Epstein et al. 1988
; Marwitt et al.
1971
; Ullian et al. 1997
). Some of these, such as differences in neuropeptide expression, are regulated by cell-cell interactions during development (Coulombe and Nishi
1991
; Coulombe et al. 1993
; Darland and
Nishi 1998
; Darland et al. 1995
). In this study,
we have shown that the biophysical properties of large-conductance (BK-type) KCa channels, and the mechanisms
controlling their developmental expression, are different in choroid
and ciliary neurons of the chick CG. The unitary conductance of
KCa channels is the same in both cell types.
However, ciliary neurons express KCa channels with a relatively short open-time, which is associated with more rapid
macroscopic activation and deactivation kinetics in ciliary neurons.
Moreover, functional expression of KCa channels
in ciliary neurons is dependent on cell-cell interactions during
development, whereas choroid neurons regulate these channels by an
apparently cell-autonomous mechanism. Consistent with this,
KCa stimulatory factors are present in the iris,
one of the target tissues of ciliary neurons, but are not expressed in
the choroid smooth muscle layer, the target tissue of the choroid neurons.
This pattern may not be unique to the chick CG. Multiple cell
populations with distinct electrophysiological properties are present
in many autonomic ganglia and central nuclei. To cite just one example,
B-cells and C-cells within bullfrog paravertebral sympathetic ganglia
receive afferents from different spinal segments and innervate
different target tissues (Dodd and Horn 1983;
Horn et al. 1988
; Smith 1994
). These
cells also have different conduction velocities, action potential
waveforms, and intrinsic patterns of repetitive spike discharge
(Smith 1994
). Differences in the developmental
expression of ionic channels may often occur in neighboring neurons
that project to physiologically distinct target cells. In such cases,
differential regulation of ion channel expression by inductive
cell-cell interactions may provide a mechanism to ensure that the
intrinsic properties of pre- and postsynaptic elements are
appropriately matched. In this regard, we have observed that expression
of macroscopic KCa is regulated by soluble
trophic factors in essentially all chick lumbar paravertebral
sympathetic neurons, but that only a subpopulation of these respond to
nerve growth factor (Raucher and Dryer 1995
).
The intrinsic properties of ciliary and choroid neurons are well
matched to those of their respective target tissues. Specifically, ciliary neurons need to be able to discharge spikes at a sustained high
frequency (>100 Hz) because tetanic contractions of their striated
muscle targets in iris require stimulation at this range of frequencies
(Pilar and Vaughan 1969). The relationship between stimulus frequency and contraction has not been studied in choroid smooth muscle, but ocular smooth muscles in other species exhibit maximum contractions at stimulation frequencies as low as 10 Hz (Suzuki 1983
). It is not known whether
KCa kinetics can account for these differences in
CG neuron repetitive firing. However, the slower macroscopic kinetics
of KCa in choroid neurons would predict a longer
duration spike duration, as is observed (Dryer 1994
).
Other aspects of the behavior of choroid and ciliary neurons are quite
similar under voltage clamp, and this is the first consistent difference that we have observed over many years of investigation. In
this regard, it bears noting that neurons whose dominant outward currents exhibit more rapid activation and deactivation kinetics tend
to have shorter duration action potentials and are able to follow high
frequencies of synaptic stimulation (reviewed in Gan and
Kaczmarek 1998
; Rudy et al. 1999
).
The molecular basis for the different developmental and kinetic
properties of KCa channels in the two CG cell
types is not known, but it is very plausible that these observations
share a common mechanistic basis. One possibility is that the two cell types express different slo splice variants. Cell-specific
expression of different slo variants has been observed in
cochlear hair cells of the chick (Navaratnam et al.
1997; Rosenblatt et al. 1997
) and turtle
(Jones et al. 1999
), as well as in the mammalian
CNS (Tseng-Crank et al. 1994
) and in
Drosophila (Becker et al. 1995
). Differences
in KCa kinetics in hair cells are known to have
large effects on oscillatory membrane behavior (reviewed in
Fettiplace and Fuchs 1999
). We have previously detected
two different slo partial cDNAs in the chick CG. These cDNAs
correspond to transcripts that differ by the presence or absence of a
single 28 amino acid exon present in one isoform but not the other
(Subramony et al. 1996
). This alternative exon is
located close to the C-terminus, and variations in this portion of the
molecule do not produce particularly large differences in the kinetics
of SLO channel alpha subunits expressed in heterologous systems
(Ramanathan et al. 1999
, 2000
). However,
it is certainly possible that variability in this portion of the
channel molecule could result in differences in plasma membrane
targeting. Preliminary data from single-cell RT-PCR indicate that both
cell types express the smaller of these two slo splice
variants, but to date these experiments have been inconclusive as to
possible differential expression of the larger variant (data not shown).
It is also possible that the two populations of CG neurons
differentially express auxiliary (beta) subunits that result in different KCa kinetics (Ramanathan et al.
1999, 2000
) and/or developmental regulatory
mechanisms. This hypothesis is especially attractive because avian beta
subunits produce rather more substantial effects on
KCa kinetics (Ramanathan et al.
1999
, 2000
), and could also play a role in
membrane targeting. In this regard, we have recently obtained evidence
that a significant component of the trophic regulation of
KCa expression in ciliary neurons entails
trafficking of preexisting intracellular KCa
channels to the plasma membrane. Moreover, target-derived factors such
as TGF
1, which cause robust stimulation of functional
KCa expression in ciliary neurons, do not affect
expression of slo transcripts in those cells
(Lhuillier and Dryer 2000
). Therefore it is possible
that auxiliary subunits selectively expressed in one CG cell type
regulate plasma membrane insertion and the kinetics of
KCa channels. There is precedent in the
literature for these types of mechanisms. For example, two different
proteins isolated from Drosophila have been shown to
interact with SLO channels and inhibit their insertion into the plasma
membrane (Schopperle et al. 1998
; Xia et al.
1998
). One of these, known as dSLIP1, can also interact with
mammalian SLO channels (Xia et al. 1998
). Vertebrate
homologues of dSLIP1 have not been identified, but it is certainly
possible that multiple SLO interacting proteins exist, some of which
could act selectively on specific SLO isoforms. These types of
interactions are not unique to KCa channels. For
example, auxiliary subunits of various voltage-activated
K+ channels appear to regulate both the gating
kinetics and the efficiency of plasma membrane insertion in
heterologous systems (e.g., Fink et al. 1996
;
Salinas et al. 1997
; Shi et al. 1996
).
In summary, we have shown that two closely related populations of parasympathetic neurons that innervate different target tissues express large conductance KCa channels with different gating properties. These neurons regulate the functional expression of KCa channels by different mechanisms, which may allow for appropriate matching between the intrinsic firing properties of the neurons and the physiological properties of their target tissues.
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ACKNOWLEDGMENTS |
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We thank R. Nishi for assistance with somatostatin immunocytochemistry.
This work was supported by National Institutes of Health Grants NS-32748 to S. Dryer and MH-12758 to J. Cameron.
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FOOTNOTES |
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Address for reprint requests: S. E. Dryer (E-mail: SDryer{at}UH.EDU).
Received 13 April 2000; accepted in final form 10 August 2000.
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REFERENCES |
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