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The Journal of Neuroscience, February 1, 1998, 18(3):1047-1055
A Role for L-Type Calcium Channels in Developmental Regulation of
Transmitter Phenotype in Primary Sensory Neurons
Teresa A.
Brosenitsch1,
Delanthi
Salgado-Commissariat2,
Diana L.
Kunze1, 2, and
David M.
Katz1
1 Department of Neurosciences, Case Western Reserve
University, School of Medicine, Cleveland, Ohio 44106-4975, and
2 Rammelkamp Center for Research, Cleveland, Ohio
44109-1998
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ABSTRACT |
To examine the influence of activity-dependent cues on
differentiation of primary afferent neurons, we investigated the short- and long-term effects of depolarization and calcium influx on expression of transmitter traits in sensory ganglion cell cultures. We
focused on expression of tyrosine hydroxylase (TH), a marker for
dopaminergic neurons, in developing petrosal ganglion (PG), nodose
ganglion, and dorsal root ganglion neurons grown in the presence or
absence of depolarizing concentrations of KCl. Exposure to 40 mM KCl increased the proportion of TH-immunoreactive
neurons in all three ganglia in a developmentally regulated manner that corresponded to the temporal pattern of dopaminergic expression in vivo. PG neurons, for example, were most responsive
to elevated KCl on embryonic day 16.5 (E16.5), the age at which the
dopaminergic phenotype is first detectable in vivo.
However, KCl was relatively ineffective at increasing TH expression in
neonatal PG, indicating a critical period for induction of this
phenotype by depolarization. Detailed analysis of TH induction in PG
neurons demonstrated that, although N-type calcium channels carried the
majority of the high voltage-activated calcium current, only L-type
calcium channel blockade inhibited the effect of elevated KCl. Further
studies revealed that after removal of high KCl, neurons remained
sensitized to subsequent stimulation for >1 week. Specifically,
cultures exposed to KCl beginning on E16.5 (the conditioning stimulus), then returned to control medium, and subsequently re-exposed to elevated KCl after 9 d (the test stimulus) contained fourfold more
TH-positive neurons than did cultures exposed to the test stimulus
alone. Moreover, blockade of L-type calcium channels during the
conditioning stimulus completely abolished long-term potentiation of
the TH response to elevated KCl. These findings demonstrate a novel
role for L-type calcium channels in activity-dependent plasticity of
transmitter expression in sensory neurons and indicate that exposure to
depolarizing stimuli during early development may alter neuronal
response properties at later ages.
Key words:
Bay K-8644;  -conotoxin GVIA; depolarization; dopaminergic; long-term potentiation; nimodipine; petrosal ganglion; primary sensory neurons; transmitter plasticity; tyrosine hydroxylase; voltage-dependent calcium channels
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INTRODUCTION |
Activity of developing primary
sensory neurons plays a critical role in regulating maturation of
postsynaptic target cells in the CNS. For example, blocking the
activity of vestibulocochlear afferents alters gene transcription,
protein synthesis, and the size of neurons in the brainstem cochlear
nucleus (Sie and Rubel, 1992 ; Garden et al., 1995), and the activity of
primary olfactory afferents is thought to regulate developmental
expression of dopaminergic transmitter properties in the olfactory bulb
(Baker and Farbman, 1993). Although the importance of afferent activity
in the development of second-order sensory neurons seems clear, it is
unknown whether maturation of primary afferents themselves is also
regulated by activity-dependent cues.
In mature primary sensory neurons, activity regulates quantitative
expression of transmitter properties, such as preprotachykinin mRNA
(Noguchi et al., 1988). Therefore, to explore the possibility that
activity may also play a role in sensory neuron differentiation, we
have been examining the influence of depolarizing stimuli on development of sensory transmitter phenotypes (Fan, 1995; Hertzberg et
al., 1995). We have focused in particular on dopaminergic traits, which
are expressed by subpopulations of sensory neurons in cranial and
spinal ganglia (Katz et al., 1983; Price and Mudge, 1983). In
vivo, the magnitude and time course of dopaminergic phenotypic expression vary widely among different sensory ganglia. In adult dorsal
root ganglia (DRG), for example, only 1-4% of the total neuronal
population is dopaminergic, depending on axial level (Price and Mudge,
1983; Vega et al., 1991). Tyrosine hydroxylase (TH) immunoreactivity
and dopamine histofluorescence are first detectable in lumbar DRG
neurons 1-2 weeks after birth (Price and Mudge, 1983). In contrast,
dopaminergic afferents in cranial sensory ganglia are more abundant and
differentiate earlier. In the adult glossopharyngeal petrosal ganglion
(PG), for example, at least 10-20% of the neuron population is
dopaminergic (Katz and Black, 1986), and TH immunoreactivity is first
detectable in these cells on embryonic day 16.5 (E16.5) (Katz and Erb,
1990).
Recently, we found that expression of dopaminergic properties by
developing cranial and spinal sensory neurons is highly regulated by
depolarizing stimuli in vitro, indicating that
activity-dependent mechanisms may influence the number of sensory
neurons that express a dopaminergic phenotype (Fan, 1995; Hertzberg et
al., 1995). Specifically, treatment of fetal cranial sensory neurons
with depolarizing concentrations of veratridine (10 µM)
or KCl (40 mM) increased the proportion of ganglion cells
that expressed TH and elevated dopamine synthesis by up to 10-fold
(Hertzberg et al., 1995). Thus, although dopaminergic properties are
normally expressed by a relatively small subset of primary sensory
neurons in vivo (Katz et al., 1983), exposure to
depolarizing stimuli can unmask a widespread potential for dopaminergic
phenotypic expression in these cells, raising the possibility that
activity plays an important role in sensory neuron differentiation.
The present study was designed to examine the relationship between
sensory neuron responsiveness to depolarizing stimuli in vitro and dopaminergic development in vivo and to begin
defining mechanisms that underlie depolarization-mediated TH
expression. We found that KCl-induced depolarization increases the
proportion of dopaminergic neurons in sensory ganglia in a
developmentally regulated manner that corresponds to the temporal
pattern of dopaminergic expression in vivo. In addition, we
made the unexpected discovery that transient exposure of sensory
neurons to depolarizing stimuli during fetal development leads to
long-term changes in the regulation of TH expression. Moreover, calcium
influx through L-type channels is required for both short-term
regulation and long-term potentiation to occur.
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MATERIALS AND METHODS |
Cell culture
Pregnant dams (Sprague Dawley rats; Zivic-Miller, Zelienople,
PA) were rapidly killed by exposure to carbon dioxide. The uterine horns were removed and placed in PBS containing 10% glucose, and the
embryos were excised. To assign gestational ages, we designated the day
after mating E0.5. Newborn (P0) and 1-week-old (P7) pups were killed
with an overdose of sodium pentobarbital (6.0 gm/kg, i.p.). Dissociate
cultures of E13.5, E14.5, E16.5, E19.5, P0, and P7 PG, nodose ganglia
(NG), and cervical DRG were grown in Leibovitz's L-15/CO2
medium containing 10% NuSerum (Collaborative Biomedical Products,
Bedford, MA), 5% heat-inactivated rat serum, fresh vitamin mixture
(Mains and Patterson, 1973), penicillin (50 IU/ml; GIBCO BRL,
Gaithersburg, MD), and streptomycin (50 µg/ml; GIBCO BRL). Embryonic
ganglia were digested in Dispase (Collaborative Biomedical Products;
diluted 1:1 in PBS) for 1 hr at 37°C followed by trituration through
fire-polished Pasteur pipettes; P0 and P7 ganglia were digested in
0.5% trypsin (Worthington, Freehold, NJ) for 20 min at 37°C followed
by trituration. Embryonic and newborn cells were plated onto glass
coverslips coated with poly-D-lysine (0.1 mg/ml) and
laminin (0.3 mg/ml), whereas P7 cells were grown on growth
factor-reduced Matrigel matrix (Collaborative Biomedical Products;
diluted 1:5). All cultures were supplemented with recombinant human
brain-derived neurotrophic factor (BDNF; Regeneron Pharmaceuticals,
Inc., Tarrytown, NY) at a concentration of 10 ng/ml. DRG cultures were
supplemented with, in addition to BDNF, 10 ng/ml nerve growth factor
(NGF; Dr. Kenneth Neet, Chicago Medical College) and 10 ng/ml
neurotrophin-3 (NT-3; Regeneron Pharmaceuticals, Inc.). Depolarizing
conditions were produced by supplementing cultures with 34 mM KCl to achieve a final concentration of 40 mM. Control cultures were not adjusted to isosmolarity
because we found previously that TH expression in these cells was
unaffected when the concentration of NaCl, rather than KCl, was raised
to 40 mM (Hertzberg et al., 1995). In most experiments,
neurons were cultured for a total of 3 d in the presence or
absence of depolarizing concentrations of KCl. In some experiments,
neurons were cultured for 2 d in control medium containing 6 mM KCl and then transferred for 24 hr to medium containing
40 mM KCl either with or without a selective calcium
channel antagonist. Specifically, the N-type calcium channel antagonist
-conotoxin GVIA (1 µM; Sigma, St. Louis, MO) and the
L-type calcium channel antagonists nifedipine (1 µM;
Calbiochem, San Diego, CA), nimodipine (2 µM; a gift from Miles Pharmaceuticals, West Haven, CT), and verapamil (10 µM; Sigma) were used. In other experiments, an L-type
calcium channel agonist, (±)-Bay K-8644 (1 µM;
Calbiochem), was added at the beginning of the 3 day culture period. In
long-term experiments (15 d), neurons were cultured on growth
factor-reduced Matrigel. During the initial 3 d of culture,
cytosine  -D-arabinofuranoside (10 µM;
Sigma) was added to the culture medium to eliminate non-neuronal cells.
Immunocytochemistry
All cultures were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, overnight at 4°C.
Double immunostaining was performed using polyclonal anti-TH
(Pel-Freeze Biologicals, Rogers, AR), polyclonal anti-substance P (SP)
(Incstar Corporation, Stillwater, MN), monoclonal anti-neurofilament
protein (NF160,68; Sigma), goat anti-rabbit IgG-FITC
(for TH staining, Boehringer Mannheim, Indianapolis, IN; for SP
staining, Cappel, West Chester, PA), and goat anti-mouse IgG-rhodamine
(Cappel) antibodies.
TH immunostaining. Cells were (1) incubated overnight at
room temperature in anti-TH (1:200) and anti-NF (1:100) diluted in PBS
containing 0.5% Triton X-100, (2) washed three times in PBS, (3)
incubated for 1 hr at room temperature in goat anti-rabbit IgG-FITC
(1:200) plus goat anti-mouse IgG-rhodamine (1:200) diluted in
PBS-Triton containing 10% goat serum and 10% rat serum, (4) washed
in PBS, (5) incubated in  -phenylenediamine (1 mg/ml) for 1 min, (6)
washed in PBS, and (7) coverslipped with glycerol gel.
Substance P (SP) immunostaining. The same protocol described
for TH immunostaining was used except that the primary incubation was
performed in anti-SP (1:2000) and anti-NF (1:50) diluted in PBS
containing 0.5% Triton X-100, 2% rat serum, and 2% goat serum.
Cell counts and statistical analysis. The two fluorophores
were distinguished using an Olympus fluorescence microscope (model BH-2) with rhodamine and FITC filter cubes. The number of TH- and
NF-immunoreactive neurons in each culture was estimated by counting all
cells in a measured area of each coverslip. All experiments were
performed at least three times with three cultures per experimental group. Percentages were normalized (arcsine transformation), and values
were compared using ANOVA followed by Scheffé's multiple comparison procedure (Kleinbaum and Kupper, 1978). p < 0.05 was considered significant.
Electrophysiology
Calcium currents in dissociated E16.5 PG neurons were examined
using conventional whole-cell recording techniques (Hamill et al.,
1981). Experiments were performed at room temperature (25°C), ~20
hr after dissociation. Currents were amplified (80 dB/decade low-pass
Bessel filter set at a  3 dB frequency of 10 kHz; Axopatch-1C; Axon
Instruments, Inc., Foster City, CA), digitized (100 µsec/point), and
analyzed using Clampfit (pClamp v. 6.0; Axon Instruments, Inc.),
whereas whole-cell current measurements were made in response to
voltage protocols controlled by pClamp v. 5.6 (Axon Instruments,
Inc.).
Solutions. The compositions of solutions used to isolate
calcium currents were as follows (in mM): for the pipette
solution, 124.0 CsCl, 11.0 EGTA, 1.0 CaCl2, 2.0 MgCl2, and 10.0 HEPES, pH 7.4, and for the bath
solution, 140.0 TEA (tetraethylammonium chloride), 5.0 4-AP
(4-aminopyridine), 15.0 HEPES, 2.0 CaCl2, and 20.0 glucose, pH 7.4. In one series of experiments, membrane potentials were
examined in a bath solution containing L-15 serum-free media and
varying concentrations of KCl (6, 15, and 40 mM). In these
experiments, the pipette solution contained (in mM): 145 K-Asp, 5.0 HEPES, 1.3 CaCl2, 2.2 EGTA, 2.0 MgCl2, 5.0 NaCl, and 10.0 glucose, pH 7.2. The
membrane potential was defined as the voltage (in millivolts) inside,
relative to outside, the cell and was measured at zero current in
current-clamp mode.
Calcium channel modulators. For electrophysiological
measurements, S()-Bay K-8644 (Research Biochemicals,
Natick, MA) was dissolved in ethanol and stored in a 1 mM
stock solution, nimodipine was dissolved in methanol and stored at a
concentration of 0.5 mM, and -conotoxin GVIA was
dissolved in distilled water and stored at a concentration of 100 µM.
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RESULTS |
Sensory neuron responsiveness to depolarizing stimuli is
developmentally regulated
We found previously that chronic exposure of E16.5 PG, NG, or DRG
neurons to depolarizing stimuli in vitro can markedly
increase both the percentage of ganglion cells that express TH and
dopamine synthesis (Hertzberg et al., 1995). However, the proportion of neurons in which TH expression could be evoked varied from ~8% in
the DRG to almost 100% in the PG (Hertzberg et al., 1995). These
differences could reflect an underlying heterogeneity in phenotypic
potential among different sensory neuron populations. Alternatively,
responsiveness to depolarizing stimuli may be temporally regulated and
exhibit a different time course in each ganglion. To explore this issue
further, we compared the effect of KCl-mediated depolarization on TH
expression in PG, NG, and DRG neurons cultured at various ages between
E13.5 and P7. Neurons were grown for 3 d in the presence of
control medium (6 mM KCl) or medium containing a
depolarizing concentration of KCl (40 mM) and subsequently
were processed for TH and NF immunostaining. An exposure time of 3 d was chosen because preliminary experiments demonstrated that, although 6 hr of KCl treatment was sufficient to induce TH expression, a 3 day exposure produced the greatest induction. The membrane potential of neurons grown for 3 d in control medium was between 62 and 66 mV (n = 6), compared with 18 to 20 mV
(n = 6) in neurons grown for the same period of time in
40 mM KCl.
The percentage of TH-positive (TH+) neurons in all
three ganglia was significantly increased by KCl treatment; however,
the magnitude and time course of the response were different in each population (Fig. 1). PG neurons exhibited
the largest and earliest peak response (92% TH+
neurons on E16.5), followed by the NG (55% TH+
neurons on E19.5) and the DRG (22% TH+ neurons on
P0). In addition, KCl treatment seemed to define a window of
responsiveness in TH regulation; in the PG, for example, KCl treatment
was 2.5- and 5-fold less effective at inducing TH expression in E13.5
and P7 cultures, respectively, than in E16.5 cultures.
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Figure 1.
The developmental time course of
depolarization-induced TH expression in PG, NG, and DRG neurons.
Ganglia were removed at the ages indicated and grown in dissociate cell
culture for 3 d in the absence (Control) or
presence (KCl) of 40 mM KCl. Each bar indicates the percentage of neurons exhibiting TH
immunoreactivity. Data are presented as the mean ± SEM. P7
ganglia were grown on Matrigel, whereas all others were grown on
laminin (see Materials and Methods). Each KCl-treated group is
significantly different from its corresponding control.
Comparisons between ages were made using ANOVA followed by
Scheffé's test; *p < 0.05;
**p < 0.01; ***p < 0.001.
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To determine whether depolarization increased the percentage of
TH+ cells by raising TH expression per cell or by
selectively increasing either proliferation or survival of
TH-immunoreactive neurons, we compared total neuron numbers in the
absence or presence of depolarizing concentrations of KCl. In NG and
DRG cultures, neuronal survival was unaffected by KCl treatment at all
ages examined (Fig. 2). In the PG, KCl
treatment had no effect on neuronal survival at E13.5, E14.5, E16.5,
and P7 but significantly increased survival in E19.5 and P0 cultures
(Fig. 2). However, when P0 cultures were grown on Matrigel, a basement
membrane-rich substrate that augments neuronal attachment and survival,
KCl evoked a comparable increase in the percentage of
TH+ neurons without altering neuronal survival
[%TH+ neurons, 16.1 ± 1.7 (control) vs
51.2 ± 4.1 (KCl-treated); number of NF-positive
(NF+) neurons, 235 ± 36 (control) vs 247 ± 50 (KCl-treated)]. Thus, altered survival or proliferation of
TH-immunoreactive neurons cannot explain the increase in the percentage
of TH+ cells in KCl-treated cultures compared with
age-matched controls. Moreover, PG neurons are postmitotic by E13-E14
(Altman and Bayer, 1982), i.e., before the age at which we observed the
greatest percentage of TH+ neurons in the presence
of high KCl.
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Figure 2.
Neuron survival in PG, NG, and DRG. Cultures were
grown for 3 d in the absence (Control) or
presence (KCl) of 40 mM KCl.
Bars represent the mean number (± SEM) of
neurofilament-positive cells per ganglion. Significant differences
between KCl and corresponding control values were detected using ANOVA
followed by Scheffé's multiple comparison;
*p < 0.05; **p < 0.01.
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Substance P expression in NG and PG neurons is unaffected
by depolarization
To determine whether depolarizing stimuli act selectively to
upregulate dopaminergic traits, we examined expression of another neurotransmitter, the neuropeptide SP in PG, NG, and DRG cultures. SP
is normally expressed by a subset of neurons in all three ganglia; however, TH and SP are not localized in the same cells (Price, 1985;
Kummer et al., 1990; Finley et al., 1992). In marked contrast to TH,
the percentage of PG and NG neurons exhibiting SP immunoreactivity (SP+) was unchanged by KCl treatment at all ages
examined (Fig. 3), indicating that
depolarization does not increase transmitter traits in these ganglia in
a nonselective manner. KCl treatment did, however, increase the
percentage of SP neurons in E16.5 DRG cultures.
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Figure 3.
The developmental time course of
depolarization-induced SP expression in PG, NG, and DRG neurons.
Ganglia were removed at the ages indicated and grown in dissociate cell
culture for 3 d in the absence (Control) or
presence (KCl) of 40 mM KCl. Each bar indicates the mean percentage (± SEM) of neurons
exhibiting SP immunoreactivity; n.d., not detectable.
Comparisons with corresponding controls were made using Student's
t test; **p < 0.01.
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Transient exposure to high KCl leads to long-term potentiation of
TH expression
The KCl-mediated increase in TH expression in E16.5 PG cultures is
completely reversed after a return to control medium for 12 d
[compare Figs. 1 and 4, 91.6 ± 0.9% (3d KCl) vs 2.1 ± 0.3% (3d KCl + 12d Con) TH+ neurons]. To determine whether
transient depolarization altered neuronal responsiveness to subsequent
stimulation, E16.5 PG cultures were initially depolarized for 3 d
(conditioning stimulus), returned to control medium for 9 d, and
then depolarized a second time for 3 d (test stimulus). TH
expression in these cultures was compared with that in cultures that
received only the test stimulus during the last 3 d of the 15 d culture period. Controls included neurons cultured for the entire
15 d in medium without additional KCl, as well as neurons that
received only the initial conditioning stimulus. Cultures grown in
either of these control conditions contained few TH-immunoreactive
neurons at the end of the culture period (<2.5% of the total
population; Fig. 4, 15d Con and 3d KCl + 12d
Con). Conversely, cultures that received the conditioning stimulus
contained 29.7 ± 2.1% TH-positive neurons, a fourfold increase
compared with cultures exposed to the test stimulus alone (Fig. 4,
7.8 ± 1.3%, 12d Con + 3d KCl). Total
neuronal survival was equivalent in all experimental groups
[NF+ cells/ganglion, 114 ± 6.5 (15d
Con), 132 ± 10.3 (3d KCl + 12d Con), 124 ± 17.2 (12d Con + 3d KCl), and 130 ± 16.1 (3d KCl + 9d Con + 3d KCl); p > 0.05]. Therefore, although the increase in TH expression induced by
the conditioning stimulus alone was reversible, this initial period of
depolarization led to a long-term potentiation of the TH response to a
subsequent depolarizing stimulus. However, conditioned cultures, after
re-exposure to elevated KCl, did not contain as high a percentage of
TH+ neurons as did E16.5 cultures examined
immediately after 3 d of KCl treatment (Fig. 1). These data
indicate that at least some neurons became refractory to the effect of
KCl depolarization with time.
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Figure 4.
Long-term potentiation of TH expression by KCl
depolarization. Dissociated E16.5 PG neurons were cultured for a total
of 15 d as indicated. Each bar shows the mean
percentage (± SEM) of neurons exhibiting TH immunoreactivity at the
end of the culture period. Con, Control;
n.d., not detectable. Comparisons were made using ANOVA
followed by Scheffé's test; ***p < 0.001.
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N-type channels carry a majority of the high voltage-activated
calcium current in embryonic PG neurons
Previous studies using the pheochromocytoma cell line PC12
demonstrated that calcium entry into the cell is required for
depolarization-mediated gene expression, including regulation of TH
(Kilbourne and Sabban, 1990; Menezes et al., 1996; Bito et al., 1997).
To determine whether calcium entry is also important for short-term
regulation and long-term potentiation of TH expression in developing
sensory neurons, we first characterized the calcium currents present in E16.5-dissociated PG neurons using whole-cell recording techniques. We
focused on PG neurons because these cells exhibited the greatest potential to express dopaminergic traits in response to depolarization (Fig. 1). Depolarizing voltage steps (400 msec in duration) were applied from a holding potential of 100 mV to potentials ranging from
80 to +40 mV. The current-voltage relationship consisted of two
major components: a small, low threshold (50 mV) rapidly activating
and inactivating T-type current and a larger, high threshold (30 mV)
partially inactivating current. The average peak calcium current evoked
at 0 mV from the holding potential of 100 mV was 648 ± 169 pA
(Fig. 5A). To further
characterize the calcium currents, we repeated these experiments in the
presence of selective calcium channel antagonists. -Conotoxin (1 µM), an N-type calcium channel antagonist, reduced the
average peak current by 68% to 196 ± 36 pA (Fig. 5C).
In contrast, nimodipine (2 µM), which blocks L-type
calcium currents, reduced the average peak current by only 16% to
539 ± 140 pA (Fig. 5B).
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Figure 5.
Representative recordings of inward calcium
currents from an E16.5 PG neuron. Depolarizing voltage steps from 80
to +40 mV were applied in 10 mV increments from a holding potential of
100 mV. A, A recording from a neuron in the control
bath solution. B, A recording from the same neuron in
the presence of nimodipine (2 µM). C, A
recording from the same neuron after washing with control bath solution
to remove nimodipine and superfusing with -conotoxin (1 µM).
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To examine the voltage-dependent inactivation of calcium currents in PG
neurons, we applied conditioning voltage steps ranging from 120 to 0 mV for 2 sec, before pulsing the neurons with a test voltage of 0 mV.
The holding potential in these experiments was 100 mV. Inactivation
of the calcium current began gradually but became steeply voltage
dependent at conditioning voltages between 70 and 10 mV (Fig.
6). At the 0 mV step, most of the current
was inactivated with only 6.7% of the peak current remaining. In the
presence of -conotoxin (1 µM), a profound inhibition
was observed at all conditioning voltages, whereas nimodipine (2 µM) had only a small inhibitory effect on total current
at each step (Fig. 6).
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Figure 6.
Voltage-dependent inactivation of high threshold
calcium current in E16.5 PG neurons. Conditioning pulses ranging from
120 to 0 mV were applied for 2 sec before applying a test voltage of
0 mV. Points represent normalized data of percentages of
maximum control current (± SEM; n = 5).
Closed circles, Control inactivation curve;
closed triangles, inactivation in the presence of
-conotoxin (1 µM); open circles,
inactivation in the presence of nimodipine (2 µM).
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Calcium entry via L-type channels mimics depolarization-dependent
induction of TH
To determine whether calcium entry through a specific channel type
is important for short-term regulation of TH expression, we exposed
E16.5 PG neurons to KCl for 24 hr in the presence or absence of
specific calcium channel antagonists. Application of -conotoxin (1 µM) had no effect on TH induction by KCl (Fig. 7). However, application of nimodipine (2 µM), nifedipine (1 µM), or verapamil (10 µM), L-type calcium channel antagonists, significantly inhibited depolarization-dependent induction of TH. Nimodipine completely abolished the TH response to elevated KCl (Fig. 7), whereas
nifedipine and verapamil resulted in 37 and 51% inhibition, respectively (data not shown).
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Figure 7.
The effect of selective calcium channel
antagonists on depolarization-induced TH expression. Dissociated E16.5
PG neurons were cultured for 2 d and subsequently exposed
(KCl) or not exposed (Control) to 40 mM KCl in the
presence or absence of either -conotoxin (1 µM) or
nimodipine (2 µM) for 24 hr. Each bar
indicates the percentage (± SEM) of neurons exhibiting TH
immunoreactivity; n.s., not significantly different;
ANOVA followed by Scheffé's test.
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To examine whether activation of L-type channels was
sufficient to increase TH expression, we exposed E16.5 PG
neurons to Bay K-8644, a selective L-type channel agonist (Nilius et
al., 1985; Nowycky et al., 1985). Because Bay K-8644 is most effective when neurons are slightly depolarized, we first determined a KCl concentration that depolarized the neurons without altering TH expression. In the presence of 15 mM KCl, neurons were
depolarized to approximately 42 mV (range between 41 and 43 mV;
n = 4), but TH expression remained unchanged (Fig.
8). However, Bay K-8644, in the presence
of 15 mM KCl, induced an increase in the percentage of
TH+ neurons that was similar in magnitude to that
produced by 40 mM KCl alone (Fig. 8). Moreover, at 40 mV,
Bay K-8644 produced a substantial increase in calcium current (Fig.
9), indicating that the increase in TH
expression observed in the presence of Bay K-8644 was indeed associated
with an increase in calcium entry.
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Figure 8.
Selective activation of L-type calcium
channels mimics the effect of 40 mM KCl. E16.5-dissociated
PG cultures were grown for 3 d in medium containing 6 mM KCl (control), 15 mM KCl, 40 mM KCl, or 15 mM KCl plus 1 µM Bay K-8644
(black bar). Each bar indicates the mean
percentage (± SEM) of neurons exhibiting TH immunoreactivity. Comparisons were made using ANOVA followed by Scheffé's test; ***p < 0.001
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Figure 9.
Current-voltage relationship derived from
recordings from an E16.5 PG neuron in the presence of Bay K-8644.
Depolarizing voltage steps from 80 to +40 mV were applied in 10 mV
increments from a holding potential of 100 mV in the absence
(diamonds) or presence (squares) of 1 µM Bay K-8644. The effect of Bay K-8644 is greatest between 40 and 20 mV.
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Long-term potentiation of TH regulation requires calcium entry
through L-type channels
Because activation of L-type channels can mimic
depolarization-dependent induction of TH expression in PG neurons, we
wondered whether calcium entry was required for KCl-induced long-term
potentiation of TH expression. Therefore, we examined whether blockade
of L-type channels by nifedipine during the conditioning stimulus would alter the TH response to a subsequent KCl test stimulus. These experiments followed the same protocol as the conditioning experiments described above. At the end of the 15-day culture period, the percentage of TH neurons in cultures exposed to nifedipine (1 µM) during the conditioning stimulus alone was not
significantly different from that in cultures exposed to only the test
stimulus [Fig. 10, 12.4 ± 1.5%
(nifedipine plus 3d KCl + 9d Con + 3d KCl) vs
10.6 ± 2.1% (12d Con + 3d KCl)
TH+ neurons; p = 0.96], indicating
that L-channel blockade completely abolished the effect of the
conditioning stimulus. Moreover, exposure to nifedipine alone during
the initial 3 d in vitro did not significantly change
the percentage of TH+ neurons in cultures that
received only the test stimulus (Fig. 10, compare 12d Con + 3d
KCl with nifedipine plus 12d Con + 3d KCl),
indicating that the nifedipine treatment did not alter the ability of
PG neurons to respond to high KCl at the end of the culture period.
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Figure 10.
Inhibition of long-term potentiation of TH
expression by L-type calcium channel blockade. Dissociated E16.5 PG
neurons were grown for a total of 15 d as indicated. Each
bar shows the mean percentage (± SEM) of neurons
exhibiting TH immunoreactivity at the end of the culture period.
Black bars represent the percentage of TH-positive
neurons in cultures exposed to 1 µM nifedipine during the initial 3 d of culture only. Comparisons were made using ANOVA followed by Scheffé's test; *p < 0.05; ***p < 0.001.
|
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| |
DISCUSSION |
Our data demonstrate that depolarizing stimuli and subsequent
activation of L-type calcium channels can produce both short- and
long-term changes in dopaminergic phenotypic expression in developing
primary sensory neurons in vitro. These observations raise
the possibility that activity-dependent mechanisms and transmitter differentiation may be linked. In particular, our findings indicate a
critical period during which exposure to depolarizing stimuli can
unmask a relatively widespread potential for dopaminergic expression,
most notably in developing nodose and petrosal cranial sensory neurons.
Moreover, the time course of neuronal responsiveness to depolarizing
stimuli in vitro correlates well with developmental expression of dopaminergic traits in vivo. For example, TH
expression in PG neurons was most sensitive to the effect of elevated
KCl when neurons were cultured on E16.5, the same age at which a stable dopaminergic phenotype is first detectable in vivo (Katz and
Erb, 1990; dopaminergic expression in NG neurons also begins
prenatally; however, the time course of this expression has not been
studied in detail). Similarly, DRG neurons, in which dopaminergic
traits do not appear in vivo until after birth, were most
sensitive to elevated KCl in culture at P0 (Fig. 1). These
correlations, therefore, are consistent with a role for neuronal
activity in potentiating dopaminergic expression in vivo.
This possibility is further supported by our finding that, in the
absence of depolarizing stimuli in vitro, PG neurons did not
express TH (Figs. 4, 10, 15d Con). In addition, these cells
lost the ability to express TH in response to depolarization after 2 weeks in culture (Figs. 4, 10, 12d Con + 3d KCl). In
contrast, cultures conditioned by an initial exposure to elevated KCl
(Figs. 4, 10, 3d KCl + 9d Con + 3d KCl) exhibited fourfold
more TH+ neurons after a second depolarizing
stimulus compared with unconditioned cultures. This result demonstrates
that depolarization is required for fetal PG neurons to maintain their
ability to express TH, at least in vitro. Moreover, we found
previously that target contact alone, unlike depolarization, does not
induce expression of dopaminergic traits (Hertzberg et al., 1994). On
the basis of these findings, we hypothesize that acquisition of the
dopaminergic phenotype in vivo requires that neurons become
electrically active during the critical period of responsiveness to
depolarizing influences defined by our in vitro studies.
In fact, several lines of evidence indicate that in vivo the
dopaminergic subset of PG neurons undergoes transmitter differentiation coincident with the onset of tonic electrical activity. The timing of
these events is related to the selective projection of dopaminergic PG
neurons to the carotid body (Katz and Black, 1986), a chemoreceptive organ that responds to changes in arterial pO2,
pCO2, and pH; tonic activation of chemoafferent PG
neurons is mediated by excitatory transmitter release from presynaptic
glomus cell receptors in the carotid body (Gonzalez et al., 1992).
First, synapse formation between PG neurons and their glomus cell
targets, which is required for trans-synaptic activation of afferent
terminals, increases rapidly beginning on E16.5 (Kondo, 1975), the same
time at which a stable dopaminergic phenotype is first detectable in PG
neurons (Katz and Erb, 1990). Carotid body receptors exhibit
differentiated properties at this age (Kondo, 1975) and are capable of
responding to changes in arterial pO2 in utero
(Blanco et al., 1984; Dawes, 1984). The number of dopaminergic neurons
in the PG then gradually increases (Katz and Erb, 1990), coincident
with proliferation of synaptic contacts in the carotid body (Kondo,
1975). Indeed, recordings of action potentials from carotid body
afferents in fetal sheep demonstrated that these neurons are active by
the third trimester of gestation (Biscoe et al., 1969). Together, these
data indicate that the carotid body afferent pathway is functional in
the fetus at the time when the dopaminergic phenotype first begins to
appear. In contrast, most other PG neurons innervate taste buds in the
tongue, which do not undergo final differentiation until the second
week after birth (Torrey, 1940; Hosley and Oakley, 1987). TH expression
in PG neurons is relatively insensitive to depolarizing influences by
this time (Fig. 1), and gustatory afferents fail to develop a
dopaminergic phenotype in vivo [a subset of carotid body
afferents is also nondopaminergic (Finley et al., 1992); however, at
least some, such as those that express SP, do not form synapses with
chemoreceptor cells (Chen et al., 1986; Kummer et al., 1989)]. We
hypothesize, therefore, that selective expression of dopaminergic
traits by carotid body afferents, and not gustatory afferents, is
related to the asynchronous functional maturation of these two
populations of cells (Fig. 11). In
particular, we propose that the early, tonic activity of carotid body
afferents in vivo potentiates TH expression in a manner
analogous to the effect of chronic depolarization in vitro.
However, depolarization alone is unlikely to account completely for
neuronal commitment to the dopaminergic phenotype, because the increase
in TH expression induced by high KCl in vitro is reversible
after return to control medium. Recent experiments in our laboratory
support a role for activity-dependent mechanisms in regulating
development of the sensory dopaminergic phenotype in vivo.
Specifically, exposure of newborn rats to mild hypoxia, which increases
carotid body chemoafferent impulse activity (Bisgard and Neubauer,
1995), significantly increases the number of PG neurons that express TH
(J. T. Erickson, T. Hertzberg, and D. M. Katz, unpublished
observations).
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|
Figure 11.
Proposed model for activity-dependent development
of the dopaminergic (DA) phenotype in carotid body
afferent neurons. On E16.5, all PG neurons have the potential to
express the DA phenotype (shading). However, during late
fetal development (middle panel), this potential is
gradually lost (light shading), except in those neurons
that become sufficiently depolarized (dark shading). We hypothesize that a subpopulation of PG neurons that innervate the
carotid body become active during this window of transmitter plasticity
and, as a consequence, develop a stable DA phenotype. In contrast,
gustatory afferents, whose receptors are not terminally differentiated
until after birth, do not become sufficiently depolarized during this
critical period to induce DA phenotypic traits.
|
|
Our data indicate that activation of L-type calcium channels is
sufficient to reproduce the effects of KCl depolarization on TH
expression in developing sensory neurons. However, we cannot exclude a
potential role for other channel subtypes as well. For example, the
inability of -conotoxin to block depolarization-dependent induction
of TH in PG neurons may reflect an inactivation of N-type channels
during long-term exposure to high KCl. Specifically, neurons treated
with 40 mM KCl exhibited membrane potentials of approximately 20 mV, and after a 2 sec conditioning pulse of 20 mV,
only 28% of the total calcium current remained (Fig. 6). Alternatively, the induction of TH expression by depolarization might
require calcium entry through specific channel subtypes, such as
the L-type, and not through others, because of activation of
channel-specific intracellular signaling cascades. Greenberg and
colleagues have proposed such a mechanism based on studies of
hippocampal neurons (Gallin and Greenberg, 1995; Ghosh and Greenberg,
1995). In these cells, calcium entry through either NMDA or L-type
channels increases expression of the immediate-early response gene
c-fos (Bading et al., 1993). However, in each case distinct
signaling cascades are recruited to induce this expression; calcium/calmodulin-dependent (CaM) kinase is required for
c-fos induction after activation of L-type channels but not
after stimulation of NMDA receptors (Bading et al., 1993). In addition,
unique promoter elements in the c-fos gene are required for
calcium-induced c-fos expression, depending on whether
calcium enters through L-type channels or NMDA receptors (Bading et
al., 1993).
Our finding that calcium entry through L-type channels is
required for the long-term effect of transient depolarization on TH
induction suggests possible mechanisms by which this potentiation may
occur. Early depolarization could change the sensitivity of second
messenger pathways to subsequent calcium influx, a mechanism proposed
by Schwartz and Greenberg (1987) as a molecular substrate for memory.
CaM kinase II, which is required for some forms of long-term
potentiation in the hippocampus (Malinow et al., 1989; Silva et al.,
1992), has been proposed as a memory store because it can remain active
in the absence of external stimuli (Lisman, 1994). Alternatively,
depolarization of PG neurons may produce a long-lasting change in the
transcription rate of the TH gene and thereby potentiate TH induction
by subsequent stimulation. Experiments in progress are designed to
differentiate among these potential mechanisms.
Our finding that exposure to depolarizing stimuli can produce long-term
phenotypic changes in developing sensory neurons is reminiscent of the
sensitization observed in mature nociceptive afferents. During
nociceptor sensitization, neuronal excitability is enhanced for many
days after an intense stimulus (Levine et al., 1993). Coincident with
this change in excitability, transmitter expression and release are
increased (Levine et al., 1993). The mechanisms underlying these
long-term changes are not well understood, although there is evidence
that alterations in ion channels may be involved. For example, agents
that sensitize sensory neurons increase Na+ current
in DRG cells (Gold et al., 1996) and inhibit calcium-dependent K+ current in nodose neurons (Weinreich and
Wonderlin, 1987). Our finding that L-type calcium channels are
important for long-term potentiation of TH expression in developing
sensory neurons raises the possibility that these channels may
contribute to sensitization of transmitter expression in adult sensory
neurons as well.
| |
FOOTNOTES |
Received Sept. 15, 1997; revised Nov. 3, 1997; accepted Nov. 6, 1997.
This work was supported by National Institutes of Health Grants
HL-25830 (Project 4, D.M.K.; Project 5, D.L.K.), T32NS07118 (T.A.B.),
and American Heart Association of Northeast Ohio 233F (D.S.C.).
Correspondence should be addressed to Dr. David M. Katz, Department of
Neurosciences, Case Western Reserve University, School of Medicine,
10900 Euclid Avenue, Cleveland, OH 44106.
| |
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Abstract
Introduction
