Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
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ABSTRACT |
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Wang, Yong, Judith A. Strong, and Christie L. Sahley. Modulatory Effects of Myomodulin on the Excitability and Membrane Currents in Retzius Cells of the Leech. J. Neurophysiol. 82: 216-225, 1999. Ion channel modulation by the peptide myomodulin (MM) has been demonstrated in a wide variety of organisms including Aplysia, Lymnaea, and Pleurobranchaea. This neural and muscular modulation has been shown to be important for shaping and modifying behavior. In this paper, we report that MM modulates several distinct ionic channels in another species, the medicinal leech Hirudo medicinalis. Experiments have focused on the Retzius cell (R) because the R cell is a multifunction neuron that has been implicated in a number of behaviors including feeding, swimming, secretion, thermal sensing, and the touch elicited shortening reflex and its plasticity. Previous work had identified a MM-like peptide in the leech and demonstrated that this peptide modulated the excitability of the R cell. Using combined current- and voltage-clamp techniques to examine the effects of MM on the R cell, we found that in response to a step pulse, MM increased the excitability of the R cell such that the cell fires more action potentials with a shorter latency to the first action potential. We found that this effect was mediated by the activation of a Na+-mediated inward current near the cell resting membrane potential. Second, we found that MM differentially modulated the potassium currents IA and IK. No effect of MM was found on IA, whereas MM significantly reduced both the peak and steady-state amplitudes of IK by 49 ± 2.9% and 43 ± 7.2%, respectively (means ± SE). Finally we found that MM reduced the amplitude of the Ca2+ current by ~20%. The ionic currents modulated by MM are consistent with the overall effect of MM on the cellular activity of the R cell. An understanding of the cellular mechanisms by which MM modulates the activity of the R cell should help us to better understand the roles of both MM and the R cell in a variety of behaviors in the leech.
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INTRODUCTION |
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The myomodulin (MM) family of peptides has been
identified in a broad range of organisms including several molluscan
species (Fujiwara-Sakata and Kobayashi 1992;
Greenberg et al. 1997
; Santama et al.
1994a
,b
), arthropods (Christie et al. 1994
;
Evans 1994
; O'Brien and Taghert 1998
),
annelids (Keating and Sahley 1996
; Takahashi et
al. 1994
), and one mammal (Vilim and Ziff 1994
). Further, the MM peptides have been shown to play an important role in
the modulation of the properties of both muscles (Brezina et al.
1994a
,b
) and neurons (Critz et al. 1991
),
including those associated with specific behaviors such as feeding
(Cropper et al. 1987a
,b
), locomotion (Evans
1994
), reproduction (van Golen et al. 1996
), and
molting (O'Brien and Taghert 1998
). In
Aplysia, where MM first was isolated and purified
(Cropper et al. 1987b
), multiple forms of MM were found
to be localized in many neurons including feeding motoneurons
(Brezina et al. 1995
; Cropper et al. 1987b
,
1991
; Miller et al. 1993
). There they serve as
cotransmitters modulating neuromuscular transmission. The effects of MM
have been seen as a potentiation (Brezina et al. 1995
;
Cropper et al. 1987b
, 1991
) or depression of
neuromuscular transmission (Brezina et al. 1995
;
Cropper et al. 1988
, 1990
; Vilim et al.
1994
). The specific net modulatory effect of the neuropeptides
depends on the ratio of different MMs and other transmitters and
modulators involved (Brezina et al. 1995
, 1996
;
Cropper et al. 1987a
,b
). All nine MMs (A-I)
have a potentiation effect on the contraction of the accessory radular
closer muscle at low concentrations (10 nM), but seven of the nine MMs
(MMA and MMD-I) produce depression at
concentrations of 1-10 µM. Moreover,the potentiation and depression appear to have different time courses (Brezina et al.
1995
).
Voltage-clamp analysis has revealed that potentiation of buccal muscle
contraction in Aplysia is primarily due to the MM
enhancement of an L-type Ca2+ current
(Brezina et al. 1994a; Scott et al.
1997
). Depression of the contraction is due to the activation
of the modulator-induced K+ current
(Brezina et al. 1994a
; Scott et al.
1997
). Interestingly, MMs differ in their effectiveness in
activating K+ currents, whereas they are equally
effective in activating the Ca2+ current
(Brezina et al. 1994a
,b
, 1995
; Scott et al.
1997
). Different MMs activate the K+
current with variable efficacy at their corresponding maximal concentrations, and additive effects can be seen with selective combinations of different MMs (Brezina et al. 1995
).
Therefore the effect of MMs on the buccal muscle contraction is a
result of complex temporal interplay of the modulation of these various currents (Brezina et al. 1995
; Scott et al.
1997
).
In addition to its action on muscles, MM has been found to modulate
neurons. MM modulation of neural properties also has been analyzed. In
Aplysia, for example MM opens both the S-K current (IK,S) and the voltage-gated
K+ current
(IK,V) of the tail sensory neurons
(Critz et al. 1991), resulting in a decrease in the
excitability of these neurons. In the leech, MM transiently depolarizes
the resting membrane potential and increases the firing rate of the
Retzius cell (Wang et al. 1998
).
A set of putative MM-containing neurons in the CNS of the leech has
been identified (Keating and Sahley 1996). MM
immunoreactivity is distributed across cells within neural circuits
mediating several distinct behaviors including cardiovascular function
and the touch-elicited shortening reflex. Within the shortening reflex
circuit, the anterior Pagodas (AP), Leydig cells, longitudinal motor
neuons (L), S cells, and their coupling interneurons are all
immunoreactive for MM (Keating and Sahley 1996
). Thus MM
could potentially be important in the expression and modulation of the
touch-elicited shortening reflex. Moreover we recently observed a
putative peptidergic synapse between the S and the Retzius (R) cell
(Wang, unpublished observations). Given the MM immunoreactivity
observed in the S cell, R cell may be a physiologically relevant target
of MM modulation.
Several of the ionic currents in the R cell have been
characterized extensively in intact leech ganglia as well as in culture (Johansen et al. 1987; Schirrmacher and Deitmer
1991
; Stewart et al. 1989b
; for review, see
Kleinhaus and Angstadt 1995
). These include the
voltage-dependent Na+ current (Johansen
and Kleinhaus 1987
; Kleinhaus and Prichard 1976
;
Nicholls and Baylor 1968
), and two major components of
the K+ currents,
IA, a rapidly activating and
inactivating transient current, and
IK, a delayed rectifier current
(Johansen and Kleinhaus 1986
). In addition, a single
class of Ca2+ current has been revealed in R
cells by using both single-channel and voltage-clamp recordings
(Bookman and Liu 1987
; Johansen et al.
1987
; Schirrmacher and Deitmer 1991
). In this
report, we characterized the ionic basis of the MM-induced cellular
property changes in the R cell. We report that MM increases the
excitability of the R cell such that the cell fires more action
potentials or shortens the latency to the first action potential in
response to a step current pulse; MM induces a small
Na+-mediated inward current near the cell resting
membrane potential, which can account for the small depolarization
observed when cells are monitored in current-clamp mode; MM has little
or no effect on the rapidly activating and inactivating
IA current but significantly reduces
the delayed rectifier IK current; and
MM decreases the amplitude of the Ca current. Taken together, the
modulatory effects of MM on the ionic currents are consistent with the
excitatory effect of MM on R cells.
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METHODS |
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Adult leeches, Hirudo medicinalis (3-5 g) were obtained from Leeches USA (Westbury, NY) and maintained in plastic containers [28 (L) × 20 (W) × 8 (H) cm] filled to the depth of ~4 cm with artificial leech pond water [0.5 g of Hirudo salt (Leeches USA) dissolved in 1 L of ddH2O] at room temperature with 12/12 h light/dark cycle. Segmental ganglia, excluding the sex ganglia (ganglia 5 and 6), were dissected and pinned down in a custom-made Plexiglass (200 µl in volume) recording chamber containing silicone elastomer (Sylgard; Dow Corning, Midland, MI). The capsule covering each ganglion was removed carefully by fine scissors or forceps.
Cells were impaled with borosilicate microelectrodes (1 mm OD, 0.75 mm
ID) (Sutter Instrument, Novato, CA) filled with 3 M potassium acetate
with 100 mM KCl (10-20 M). An Axoclamp 2A (Axon Instruments, Foster
city, CA) was used for intracellular recording, current-clamp and
single- and two-electrode voltage clamp (SEVC and TEVC). Standard
intracellular recording in bridge mode was used to monitor the basic
effect of MM on the R cells. For the excitability test, conventional
discontinuous current clamp was used. The excitability was measured
~1.5-2 min after MM application, i.e., after the initial burst and
depolarization were over. The membrane potential of the R cell was
monitored and maintained between
55 and
65 mV by manually adjusting
the constant DC output of the current-passing electrode during the
excitability test. Bursts of action potentials were elicited by
injecting a series of 90-ms depolarizing current pulses. The amplitudes
of these depolarizing pulses range from 0.5 to 3 nA and were increased incrementally in steps of 0.5 nA. For all voltage-clamp experiments, the tips of the microelectrodes were coated with Sigmacote (Sigma, St
Louis, MO). SEVC (some with a ramp protocol) was used to characterize the MM-induced current. Sample rates were set between 3 and 7 kHz, and
the clamp gain was set at 25-50 nA/mV. For optimum capacitance neutralization, capacitance neutralization control was advanced until
the 10 mV/mV MONITOR waveform on a second oscilloscope decayed most
rapidly to a horizontal baseline without any overshoot or undershoot.
All K+ current studies were carried out using
standard TEVC. The gain for TEVC was set between 800 and 2,500 vol/vol.
The output bandwidth was set at 0.3 kHz. Both SEVC and TEVC were used
in Ca2+-current experiments. Data were digitized
by a Digidata 1200 converter (Axon Instrument). All voltage steps were
generated, stored, and analyzed by a Gateway P5-133 computer using
pClamp 6.0 software (Axon Instruments). Further filtering was applied
during data analysis and figure plotting. Data were accepted only if
the step changes in voltage-clamp potential were fast and no voltage
sag was detected (Critz et al. 1991
), and the electrode
drift at the end of the experiments was within ± 5 mV of the
starting value (Nadim and Calabrese 1997
). For
subtraction, leakage current was obtained by using hyperpolarizing
voltage steps. Leakage current from the hyperpolarizing pulse was
multiplied to the correct magnitude on the assumption of a linear
leakage and then used for subtraction.
All drugs and chemicals were purchased from Sigma unless noted
otherwise. Normal leech saline containing (in mM) 115 NaCl, 4 KCl, 1.8 CaCl2, and 10 HEPES with pH adjusted to 7.4 was
used for ganglia preparation, excitability, and MM-induced current experiments. Other solutions were made by equimolar substitution (unless otherwise noted) of the above formula with
N-methyl-D-glucamine replacing
Na+, and Co2+ (or
Mg2+) replacing Ca2+,
respectively. In some experiments, Ca2+ was
replaced by Ba2+ as charge carrier. Stock
solutions of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) were
made in ddH2O at 1 M each. TEA or/and 4-AP were
added to the working solutions so that their final concentrations were
25 and 8 mM, respectively. We were not able to block voltage-gated
Na+ current in the R cell with either
pharmacological methods (Kleinhaus and Prichard 1976) or
by somata ligation (Acosta-Urquidi et al. 1989
;
Johansen and Kleinhaus 1986
). Myomodulin A
(PMSMLRL-NH2) (Aplysia) peptide was
purchased from Peninsula Laboratories (Belmont, CA). Leech MM peptide
GMGALRL-NH2 (Wang et al. 1998
) was
synthesized by Research Genetics (Huntsville, AL). The peptides were
dissolved in ddH2O to make 1 M stock and kept at
80 or
20°C in small-volume aliquots. Because the effects of
Aplysia myomodulin A and leech MM were indistinguishable
(Wang et al. 1998
), the data for excitability and the
MM-induced current were pooled from experiments using either
Aplysia or leech MMs. Modulatory effects on
IA,
IK, and ICa were done using Aplysia
myomoduolin A. We found that the effect of MM was saturated between
25-50 µM, which was similar to the range of concentrations used to
study MM modulation of Aplysia neurons (Critz et al.
1991
). Before use, the stock solutions were thawed, diluted to
working concentration of 50 µM in appropriate recording solutions,
and applied to the cells by bath superfusion.
Statistical comparisons were of the within-preparation differences produced by the treatment, using a paired Student's t-test. The criterion for statistical significance was P < 0.05. All averaged data were expressed as means ± SE.
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RESULTS |
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MM effect on excitability
The response of the R cell to MM application is presented in Fig.
1Aa. As seen in the figure and
as reported previously (Wang et al. 1998) MM modulates
the activity of the R cell causing a small depolarization accompanied
by a train of action potentials. This response of the R cell to MM
application is subject to rapid desensitization. The responsiveness of
R cell to repetitive applications of MM decreased dramatically as shown
in Fig. 1Ab. In addition, as presented in Fig.
1B, MM appears to increase the excitability of the R cell.
That is, a given value of depolorizing current pulse elicited a
consistent increase in number of action potentials, a decreased latency
to fire, and a decreased interspike interval after MM application (Fig.
1B). For example, for a 2.5-nA current pulse, the average
number of action potentials before and after MM application were
1.8 ± 0.2 and 2.7 ± 0.4, respectively (t = 3.16, P < 0.05, n = 5). In addition,
there was a decrease of the latency time for the first spike from
25 ± 4.8 to 19 ± 4.8 ms, a reduction of 23 ± 5.5%.
There was also a consistent reduction in interspike interval time
between the first and the second spikes from 48 ± 6.0 to 28 ± 8.3 ms (n = 3). Excitability data from five cells
are plotted in Fig. 1C. No apparent changes in action
potential shape such as duration or afterhyperpolarization were
observed, although sometimes a small reduction of the action potential
amplitude was noticed.
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MM induced a small Na current
Using voltage-clamp procedures, we analyzed the specific ion
current modulation underlying the MM-induced changes in the R cell.
Desheathed leech ganglia were bathed in normal leech saline. R cells
were clamped at a holding potential of 60 mV, a potential slightly
hyperpolarized from rest. As seen in Fig.
2B, superfusion of 50 µM of
MM induced a small inward current (Fig. 2B). In the nine
cells studied with this protocol, the average MM-induced current was
0.70 ± 0.08 nA. The induced inward current was not completely
inactivated at 1 min after MM application. Because the currents near
the resting potential were so small, it was difficult to determine
whether the current was sustained or slowly decayed over longer time
periods.
|
As shown in Fig. 2C, in a Na-free saline, no MM-induced
inward current was observed (n = 7). An apparent, small
transient outward current was seen occasionally during the application
of MM. This small current was very similar to that sometimes observed during the application of control Ringer, thus it was considered to be
a superfusion artifact. The MM-induced current persisted in bath
solutions with lowered [Ca2+]
(Ca2+ = 0.9 mM) Ringer. The amplitude of the
MM-induced current in 0.9 mM Ca2+ Ringer solution
was relatively larger than the same current observed in normal Ringer,
1.11 ± 0.35 nA (VH = 60 mV,
n = 5), although that increase was not statistically
significant (t =
1.5, P > 0.05, df = 12). However, the MM-induced current was blocked by extracellular Co2+ ions. As shown in Fig.
2D, when the R cell was bathed in normal Ringer with 1.8 mM
Co2+ substituting for Ca2+,
the MM-induced current was reduced significantly to 0.23 ± 0.13 nA (n = 7; t = 3.2, P < 0.01, df = 14). MM-induced currents in four of seven cells
showed complete blockade by extracellular Co2+.
Results from the previous experiments revealed a lack of time
dependence in the MM-induced current. Thus we were able to study the
voltage dependence of the current using a ramp protocol. We applied a
voltage ramp from 85 to
55 mV in normal leech Ringer followed by an
identical ramp in MM containing Ringer solution. Figure
3A shows the total
current-voltage (I-V) curves before and after application of
MM. The MM-induced I-V relationship presented in Fig.
3B was acquired by subtracting the total current with MM
from the total current without MM.
|
MM effect on K currents
TOTAL K.
MM peptides have been demonstrated to differentially modulate several
distinct K+ currents in Aplysia
(Brezina et al. 1994a,b
, 1995
; Critz et al. 1991
; Scott et al. 1997
). Because modulation of
K+ currents has a dramatic effect on neuronal
excitability (Critz et al. 1991
), we also examined the
MM effect on the K+ currents in R cells. Using
two-electrode voltage clamp, we first examined the MM effect on the
total K+ current. Cells were bathed in both
Na+- and Ca2+-free saline.
The results from a series of step pulses from
70 to 40 mV in 10-mV
increments applied from the holding potential of
60 mV are presented
in Fig. 4. As seen in Fig. 4,
C and D, a general decrease of total
K+ current was observed. At 20 mV, there was a
25 ± 3.5% (t = 5.9, P < 0.005, n = 5) reduction in the peak current and a 27 ± 2.2% (t = 6.8, P < 0.005, n = 5) reduction in the steady-state current. The
difference current was obtained by subtracting the total current after
MM application from the total current before MM and presented in Fig.
4B. The difference current appears to resemble the
IK current (see following text) in
that it has a smaller peak current (compared with
IA) and a long-lasting steady-state
current.
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IA.
IA can be distinguished from
IK by its specific sensitivity to 4-AP
but not TEA. To study the effect of MM on
IA current, 25 mM of TEA was added to
the Na+-, Ca2+-free bath
solution to block IK. Results from a
set of incremental (10 mV) 300-ms depolarizing steps, preceded by a
500-ms conditioning prepulse to 90 mV from a holding potential of
60 mV are presented in Fig.
5B. Command voltage steps to
elicit IA were kept at or below 0 mV
to minimize the possible contribution of a small TEA-resistant fraction
of IK because there is potentially an
overlap of these two currents at VM
values more positive than 0 mV (Acosta-Urquidi et al.
1989
; Johansen and Kleinhaus 1986
). As seen in
Fig. 5B, MM had no effect on the peak amplitude of
IA (t = 0.92, P > 0.4, n = 5) within the voltage
range examined. For example at 0 mV, the peak
IA before and after MM application was
25.2 ± 1.4 and 25.9 ± 1.4 (t = 0.94, P > 0.3, n = 5). Moreover, no effect
on the inactivation time constant (
off) was
observed. Values of
off pre- and post-MM
application for currents elicited by voltage stepping to 0 mV from
VH of
60 mV were 42 ± 2.5 ms
and 37 ± 4.8 ms (t = 1.8, P > 0.1, n = 5). Representative unsubtracted sample records
before and after the application of 50 µM MM from a single cell were
shown in Fig. 5, C and D. An I-V plot
constructed from leak-subtracted currents in the presence (
) and
absence (
) of MM is plotted in Fig. 5B.
|
IK.
IK was separated from
IA by adding 8 mM of 4-AP to the
Na+-, Ca2+-free saline.
Current responses were evoked by command voltage steps in increments of
10 mV from a VH of 50 mV. In
contrast to the lack of effect on IA,
50 µM MM consistently suppressed both the peak and steady-state
amplitudes of IK in the R cells (Fig.
6). Sample records from a single cell
with and without MM are presented in Fig. 6, A and
B (leak currents not subtracted). The I-V plot
constructed from five cells for the peak current value for each voltage
step is presented in Fig. 6C. These data indicated that
there was a consistent decrease in peak current beginning at about
40
mV and reaching its maximum at 0 mV. The average reduction at 0 mV for
peak IK was 18.7 ± 4.3 nA
(control, 37.7 ± 8.3 nA; MM, 18.9 ± 4.2 nA). This
represents a mean decrease of 49 ± 2.9% (t = 4.3, P < 0.01, n = 5). Similarly,
there was a consistent decrease of steady state
IK for all the voltage steps examined.
Steady-state IK at 0 mV was reduced by
7.1 ± 2.0 nA (control, 15.7 ± 3.1 nA; MM, 8.6 ± 1.4 nA). This represents a mean decrease of 43 ± 7.2%
(t = 3.5, P < 0.05, n = 5; Fig. 6D).
|
MM effect on Ca current
Both patch-channel and voltage-clamp recordings have revealed only
a single type of voltage-gated Ca2+ current in R
cells of the adult leech (Bookman and Liu 1987; Johansen et al. 1987
). We examined the effect of MM on
this voltage-gated Ca2+ current. The effect of MM
on the Ca2+ current is presented in Fig.
7, C and D,
respectively. To isolate the Ca2+ current, we
used Na+-free Ringer with the addition of TEA (25 mM) and 4-AP (8 mM) to block the K+ currents with
Ca2+ or Ba2+ (at 2.8 mM) as
the charge carrier. Current traces were leak-subtracted using
conventional methods. Consistent with previous reports, we found that
from a holding potential of
60 mV, the current first appeared with
steps to about
20 mV, peaked between 5 and 10 mV, then decreased
until it reversed ~40 mV (Johansen et al. 1987
;
Schirrmacher and Deitmer 1991
). An I-V plot
from a sample recording is presented in Fig. 7B. As seen in
Fig. 7, C and D, MM induced a significant
decrease in peak current at 0 mV (VH =
60 mV). With calcium as the charge carrier, the current at 0 mV in
the presence of MM was reduced to 78 ± 4% (control, 23.5 ± 1.3 nA; MM, 18.0 ± 0.9 nA) of the pre-MM amplitude
(t = 4.6, P < 0.002, n = 9). With barium, the post-MM current at 0 mV was reduced to 83 ± 5% (control, 17.4 ± 1.6 nA; MM, 14.6 ± 1.7 nA) of the
pre-MM value (t = 3.5, P < 0.05, n = 5; Fig. 8). No change in the shape of ICa and
IBa before and after MM application
was apparent, indicating that the kinetics of the current was not altered.
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DISCUSSION |
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The results presented above extend the analysis of the mechanisms of the neuropeptide MM as a neuromodulator to an additional invertebrate species, the medicinal leech Hirudo medicinalis. Due to the peptide's localization to specific neurons within well-characterized and behaviorally relevant neural circuits in the leech, the role of MM in the expression of several specific behaviors can be analyzed. Understanding the actions of MM will facilitate our understanding as to how neuropeptides modulate behaviors.
Although we previously had purified a MM-like peptide from the leech
(Wang et al. 1998) and demonstrated that bath
application of MM produced an excitatory response in the R cell, the
ionic basis of this effect was not known. Here we present evidence that the MM effect on the R cell consists of the activation of a
Na+-mediated current near rest as well as the
modulation of IA and ICa2+.
The characteristics of the small, inward current induced by MM near rest are well suited to account for the initial, rapid depolarization seen when MM is applied in current-clamp conditions. Because the current disappeared in Na+-free Ringer and persisted in low Ca2+ Ringer, it appears that the current was carried predominantly by sodium, although we cannot rule out the possibility that calcium and/or potassium are also permeable.
Voltage steps around the resting potential did not reveal any new
time-dependent components induced by MM, hence we were able to use
voltage ramps study the current-voltage relation of this current.
Voltage ramps revealed an unusual current-voltage relationship for the
current. Over the narrow range of potentials examined, the current grew
larger at more positive potentials even though the driving force should
be smaller at these potentials. We were unable to examine this
phenomenon at potentials more positive than 50 mV because of our
inability to specifically block the voltage-gated, TTX-resistant sodium
current that underlies the action potential in the R cell.
A very similar current induced by the neuropeptide FMRFamide near the
cell resting potential was reported in the heart interneurons of the
leech (Schmidt et al. 1995). There are several striking similarities between the MM-induced current in the R cell and the
FMRFamide-induced current in the heart interneuron. Both currents have
similar I-V characteristics at the corresponding voltage range tested and both are blocked by extracellular
Co2+. Moreover effects of the peptides on the
corresponding cells desensitize rapidly. Finally, neuromodulator
currents with similar characteristics that are carried mainly by
Na+ have been reported in a variety of other
systems including proctolin in the lateral pyloric neurons of the crab
(Golowasch and Marder 1992
) and FMRFamide in the R14
neuron of Aplysia (Ichinose and McAdoo 1988
).
Interestingly, in these other systems, inward currents mainly mediated
by Na+ were blocked when extracellular
Ca2+ concentration was elevated (Gillette
and Green 1987
; Golowasch and Marder 1992
;
Ichinose and McAdoo 1988
). The effects of divalent cations on those currents suggests that there may be an instantaneous block of the currents by extracellular calcium. Although the MM-induced current in the R cell appears to be larger in low
Ca2+ saline, the difference is not significant.
However, the MM-induced current was blocked efficiently by
extracellular Co2+ ions. This effect is similar
to the FMRFamide-induced current in leech heart interneuron
(Schmidt et al. 1995
), the proctolin-induced current in
crab neurons (Golowasch and Marder 1992
), and the
serotonin-induced current in snail neurons (Gillette and Green
1987
; Sudlow and Gillette 1995
). The
I-V characteristics of the MM-induced current seem to
resemble the persistent Na+ current,
Ip, in the leech heart interneuron
(Opdyke and Calabrese 1994
). If the MM-induced current
and Ip were the same, then the effects
of Co2+ and MM on the current would be mutually exclusive.
The reduction of IK also could
contribute to the MM-induced increase in excitability. MM reduces
IK to about half its normal value. It
is likely that the modulation of IK
contributes to the increase in excitability because
IK begins to activate around 35 mV
(Figs. 4B and 6, C and D), a value
that is within the normal range of the R cell resting membrane
potential (
30 to
60 mV) (Hagiwara and Morita 1962
;
Leake 1986
). Although other investigators further divide
peak and steady-state IK into
IK1 and
IK2 (Hodgkin and Huxley
1952
; Simon et al. 1992
; Stewart et al.
1989b
), we did not attempt this distinction because we found
that MM reduced both peak and steady-state
IK equally effectively. No MM-induced kinetic alteration of IK was observed.
MM had no effect on IA both in terms of current amplitude and
inactivation time constant. Interestingly, the
IA off in the Hirudo R cells measured in these experiments is smaller than
what was reported in the R cell in Macrobdella
(Acosta-Urquidi et al. 1989
; Johansen and
Kleinhaus 1986
). This, perhaps, reflects the differences in the
voltage-gated IA between the two
species. In addition we found that the total K currents measured in
these experiments in Hirudo were larger than those in
Macrobdella (100 vs. 40 nA at 0 mV) (Johansen and
Kleinhaus 1986
).
As a multifunction neuron, activity in the R cell varies depending on
the particular behavior the animal is expressing. These behaviors range
from feeding (Lent and Dickinson 1984), to swimming (Kristan and Nusbaum 1983
; Willard 1981
),
to mucus secretion (Lent 1973
). It appears that changes
in the R cell may be the result of its modulation by a number of
neurotransmitters and neuropeptides including serotonin (5-HT)
(Acosta-Urquidi et al. 1989
), FMRFamide (Sahley
et al. 1993
; Strong et al. 1996
), and the small
cardiac peptide B (SCPB) (Kleinhaus and Sahley 1989
).
Analysis of the modulatory effects of these various neurotransmitters
indicates that several distinct and sometimes overlapping ion channels
are modified. One dramatic example of the relationship between R cell activity and behavior is the onset of patterned bursting of the R cell
that accompanies swimming (Friesen 1989
). This R cell
bursting can be mimicked by the application of FMRFamide (Sahley
et al. 1993
).
In contrast to MM and FMRFamide, serotonin has an immediate transient
inhibitory effect on Retzius cell because of the enhancement of a
chloride conductance (Walker and Smith 1973). In
addition, a more long-lasting effect of 5-HT has been characterized by
Acosta-Urquidi et al. (1989)
in which 5-HT enhances
IA while suppressing
IK. There is evidence that the effect
of 5-HT on leech neurons is mediated at least partially by elevated
cAMP level and accompanied by protein kinase A (PKA) activity
(Biondi et al. 1982
; Garcia-Gil et al.
1993
). Although a systematic characterization of the
second-messenger pathways underlying 5-HT and MM modulation is still
lacking, the fact that MM has different effect on the
IA and
IK compared with 5-HT indicates that a
different second-messenger mechanism may be involved.
Although cells express different types of voltage-gated
Ca2+ channels (Hille 1992),
single-channel and voltage-clamp recordings have revealed only one
class of Ca2+ channel in leech R cells
(Bookman and Liu 1987
; Johansen et al. 1987
; Schirrmacher and Deitmer 1991
). The R
cell Ca2+ channel permeability is
Sr2+>Ba2+>Ca2+,
and the Ca2+ current shows characteristic
Ca2+ dependent inactivation (Bookman and
Liu 1987
; Schirrmacher and Deitmer 1991
;
Stewart et al. 1989b
). Voltage-gated
Ca2+ (Ba2+) currents,
measured in Na+-free and
K+-channel-blocked solution, showed
characteristics of Ca2+ currents previously
measured in adult leech ganglion cells (Johansen et al.
1987
; Stewart et al. 1989b
). Activation occurs
at potentials more positive than
20 mV and peaks between 5 and 10 mV.
Our data showed a moderate but significant reduction of the
Ca2+ current after MM application. Peak
ICa reduction is ~10-20% of the
total ICa, with little alteration of
the activation and inactivation kinetics of the
Ca2+ current. The functional consequence of this
modulation on the excitability of the R cell is not known. The effect
of MM on ICa is expected to have
minimum effect on the cell excitability because R cell action potential
is exclusively Na+ dependent in the absence of
artificial perturbation of the endogenous Na+ and
K+ conductances (Kleinhaus and Prichard
1975
, 1976
). In addition, like others (Johansen and
Kleinhaus 1986
) we did not see evidence for a
Ca2+-activated
IK(Ca) component (data not shown) even
after prolonged step depolarization (>400 ms) although
IK(Ca) has been detected in cultured R
cells (Stewart et al. 1989b
) as well as in R cells from
specialized sex ganglia (Merz 1995
). However, it is
likely that the effect of MM on ICa
could have an impact on the Ca2+-mediated signal
transduction and transmitter releases (Stewart et al.
1989a
).
In the Aplysia buccal musculature, MM enhances the L-type
Ca2+ current by an average of 50-70% via a
cAMP-mediated second-messenger pathway (Brezina et al.
1994b; Hooper et al. 1994
; Scott et al. 1997
). FMRFamide, which does not activate cAMP in the buccal
muscle, causes a 10-20% reduction of the Ca current (Cropper
et al. 1994
; Scott et al. 1997
). We do not know
if the reduction of ICa in the R cell
by MM is mechanistically comparable with that in Aplysia by FMRFamide.
Taken together, our results on the MM modulation of the various ionic
currents are consistent with the overall excitable effect of MM on the
R cell. What could be the potential role of MM modulation on leech
behaviors? One possibility is that it may participate in modulating the
touch-elicited shortening reflex circuit in the leech. Recently we have
obtained evidence that indicates a peptidergic synapse exists between
the S and R cells (Wang, unpublished data). Previous work has indicated
that both the S cell (Modney et al. 1997; Sahley
et al. 1994
) and the R cell (Ehrlich et al. 1992
; Sahley 1994
) are important for the
expression of learning dependent changes in the behavior. Thus the S to
R synapse could be an important site of modulation. Studies are
underway to explore the role of modulation by MM in learning.
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ACKNOWLEDGMENTS |
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We thank Drs. B. Burrell, D. Ready, and K. Robinson for discussions of the data and for a critical reading of the manuscript.
This work was supported by a National Institute of Mental Health Grant RO1MH-44789 to C. L. Sahley.
Present address of J. A. Strong: Dept. of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati, College of Medicine, Cincinnati OH 45267-0521.
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FOOTNOTES |
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Address for reprint requests: C. L. Sahley, Dept. of Biological Sciences, Purdue University, West Lafayette, IN 47907.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 June 1998; accepted in final form 10 March 1999.
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REFERENCES |
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