Marine Biomedical Institute, University of Texas Medical Branch, Galveston, Texas 77555-1069
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ABSTRACT |
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Laurienti, P. J. and
J. E. Blankenship.
Properties of Cholinergic Responses in Isolated Parapodial Muscle
Fibers of Aplysia.
J. Neurophysiol. 82: 778-786, 1999.
The parapodial neuromuscular
junction in the marine snail Aplysia brasiliana is a
model synapse for the investigation of neural modulation. The
parapodial muscle fibers are innervated by cholinergic motoneurons and
by serotonergic modulatory cells. The physiological properties of
voltage-gated currents of the muscle membranes and the effects of
serotonin on these currents have been published previously. However,
the pharmacological properties of the cholinergic receptors have not
been investigated. Acetylcholine (ACh) applied exogenously to
dissociated muscle fibers produces a response with a reversal potential
of about 52 mV; the resting membrane potential of the average muscle
fiber is approximately
56 mV. ACh induces variable responses
(depolarizations or hyperpolarizations) in individual cells, but the
transmitter never causes a depolarization adequate to produce muscle
contraction. We demonstrate that the ACh response is the result of the
activation of two distinct receptors. One receptor is linked to a
chloride channel and induces a hyperpolarization with a reversal
potential near
70 mV. This receptor is activated selectively by
suberyldicholine and by nicotine and is antagonized by curare but not
by hexamethonium. The second response, presumably caused by increased
conductance to mixed cations, results in muscle fiber depolarization
with a reversal potential near
35 mV and does induce muscle
contraction. This receptor is activated by methylcarbamylcholine and
selectively blocked by hexamethonium; atypically, this receptor is not
activated by nicotine nor by carbachol. The depolarizing,
cation-selective receptors likely are associated with identified
excitatory cholinergic motoneurons the activity of which typically
results in muscle contractions because the reversal potential for this
ACh response is more depolarized than the activation threshold for
voltage-gated calcium channels in these fibers. The hyperpolarizing,
chloride-selective receptors may be associated with inhibitory
motoneurons; such motoneurons have yet to be identified, but their
presence is inferred because of the occurrence of spontaneous
inhibitory junctional potentials recording from muscle fibers in situ.
Muscle fiber responses to exogenously applied ACh reflect the relative
contribution of each receptor type in each muscle fiber.
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INTRODUCTION |
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The parapodia of opisthobranch molluscs comprise a
bilateral pair of flaps, or wing-like extensions, of the dorsolateral
body wall that fold over the back of the animal to cover the mantle cavity. In a small number of aplysiids, the parapodia have become modified to serve also as well-muscularized swimming appendages that
are flapped up and down rhythmically to propel the animal through the
water. We have been studying the motor control of the parapodia in the
swimming species Aplysia brasiliana (McPherson and
Blankenship 1991a,b
). Identified excitatory motoneurons in the
pedal ganglia produce 1:1 excitatory junctional potentials (EJPs) in
parapodial muscle fibers, and the muscle contracts in a graded fashion
in proportion to the amount of summation and facilitation of EJPs;
parapodial muscle fibers do not express action potentials. The EJPs are
blocked by hexamethonium, a well-known specific blocker of
cation-depended cholinergic responses in Aplysia (e.g.,
Kehoe 1979
), and acetylcholine-esterase has been
identified histochemically in the muscle beds of the parapodia,
suggesting that these motoneurons use acetylcholine as their
transmitter (McPherson and Blankenship 1991a
).
Inhibitory junctional potentials (IJPs) occasionally are observed
during in situ recordings from parapodial muscle fibers, but only
excitatory motoneurons have been identified (McPherson and
Blankenship 1991a
,b
).
Parapodial muscle fibers also are innervated by serotonergic modulatory
neurons known as parapodial opener-phase (POP) cells (McPherson
and Blankenship 1991c; Parson and Pinsker 1988
).
The amplitude of the cholinergic EJPs and the magnitude of the
subsequent muscle contraction are both increased in the presence of
exogenously applied serotonin or with concurrent activity in the
modulatory POP cells (McPherson and Blankenship 1991c
).
Neither POP cell activity nor serotonin has any direct effect on muscle
fiber resting potential or conductance; the amine works instead to
modulate cholinergic transmission and muscle fiber calcium current
(Laurienti and Blankenship 1996a
,b
, 1997
).
We have studied the physiology of this neuromuscular synaptic complex as a model for aminergic modulation of synaptic transmission. However, the pharmacological properties of the cholinergic response have not been reported in detail. This paper considers this issue and develops evidence for a dual role of acetylcholine (ACh) at the neuromuscular junction (NMJ) in the parapodia of intact, swimming animals. Muscle fiber ACh receptors associated with excitatory motoneurons open cation-selective channels that lead to muscle depolarization and contraction. Other ACh receptors that are chloride selective and associated with putative inhibitory motoneurons could lead to fiber hyperpolarization and relaxation. Exogenous application of ACh to isolated muscle fibers activates both receptor types simultaneously and produces a voltage response that is relative small and variable and never produces muscle contractions.
Physiological, pharmacological, biochemical, and histochemical evidence
clearly indicates that ACh is a common neurotransmitter in aplysiid
molluscs, acting at a variety of neural-neural (Blankenship et
al. 1971; Giller and Schwartz 1971
; Kehoe
1972a
,b
; McCaman et al. 1973
), neuroglandular
(Rayport et al. 1983
; Tritt and Byrne 1982
), and NMJs. The latter include NMJs in the gill
(Carew et al. 1974
), the buccal mass (Cohen et
al. 1978
; Ram et al. 1994
), the vasculature
(Liebeswar et al. 1975
; Sawada et al.
1982
), and the body wall (McPherson and Blankenship
1991a
; Sugi and Susuki 1978
). Comparable
findings have been made in a large variety of other molluscan families
(see reviews by Ascher and Kehoe 1975
; S.-Rozsa
1984
; Walker 1986
). Kehoe
(1972a
-c
) performed one of the first detailed, systematic
analyses of the cholinergic responses in Aplysia neurons.
She described three pharmacologically distinct responses (Kehoe
1972b
) with differing physiological profiles and ionic
dependencies (Kehoe 1972a
). Two of the cholinergic
responses were inhibitory, with one response classified as rapid and
the other as slow. Each of the inhibitory responses could be isolated pharmacologically: the rapid response was mimicked by nicotine and
suberyldicholine (Ascher and Erulkar 1983
; Kehoe
1979
) and carried by a chloride current, and the slow response
was mimicked by arecoline and carried by potassium. Each of the
inhibitory responses was selectively antagonized, with respect to the
other, by certain compounds. The rapid response was blocked by curare, strychnine, and dihydro-
-erythroidine and also is blocked by
-bungarotoxin (Kehoe et al. 1976
; Kozak et al.
1996
; but also see Shain et al. 1974
). The slow
response was blocked by tetraethylammonium (TEA),
phenyltrimethylammonium (PTMA), and methylxylocholine. The third
cholinergic response recorded in Aplysia neurons was excitatory and activated by carbamylcholine (carbachol), which also
activated both inhibitory responses, and by nicotine, which also
activates the fast inhibitory response. However, the excitatory response was selectively blocked by hexamethonium (and, although less
effectively, by atropine). Muscarine and its related agonists and
antagonists have not been shown to be very effective at molluscan cholinergic synapses, and it is not clear that the equivalent of a
G-protein-coupled ACh receptor with "muscarinic" properties exists in molluscan neurons or muscle (see e.g., Kehoe
1972b
).
Most studies of cholinergic molluscan neuromuscular junctions have
indicated that the physiology and pharmacology of these synapses is
quite similar to that at neural-neural synapses. Most muscle
preparations appear to have both a chloride-dependent "fast" inhibitory response and a cation-dependent excitatory cholinergic response. [A slow, K+-dependent
hyperpolarization has been seen in molluscan cardiovascular muscle
(Liebeswar et al. 1975; Sawada et al.
1982
) but has not been reported in other types of molluscan
muscle fibers.] One extensively studied muscle preparation,
Aplysia buccal accessory radula closer (ARC) muscle, has
been shown to have such a two-component ACh response but both receptor
types are activated simultaneously by any single motoneuron
(Kozak et al. 1996
). The inhibitory component is carried
by chloride, mimicked by suberyldicholine, and blocked by curare and
-bungarotoxin. The excitatory response is carried by mixed cations
(mainly Na+) and is blocked selectively by
hexamethonium. Kozak et al. (1996)
demonstrated that in
this preparation, ACh has a net depolarizing effect and lowers the
muscle membrane potential below the threshold for contraction.
Therefore even though it activates both receptor types, ACh, whether
released by motoneurons or added exogenously, produces a net
depolarization of muscle fibers and induces muscle contraction. A
chloride-dependent IJP-like response is never seen in buccal fibers,
and apparently these fibers receive no input from "inhibitory
motoneurons." Similar reports of cholinergic activity in other
Aplysia buccal muscle preparations have been published by
Ram et al. (1994)
. In their work, they demonstrated that
hexamethonium, atropine, and mecamylamine all antagonized ACh-induced muscle contractions. However, they did not record the
associated changes in membrane potential. Results from other molluscan
buccal muscle preparations indicate that ACh is an excitatory transmitter and its contractile actions can be selectively blocked by
hexamethonium (Yoshida and Kobayashi 1991
; Zoran
et al. 1989
).
By using pharmacological compounds with a demonstrated high probability
of selective and specific activity in molluscan preparations, we
developed a pharmacological profile of two separate cholinergic responses in Aplysia parapodial muscle, an excitatory,
cationic response and a rapid, hyperpolarizing, chloride-dependent
response. In general, the properties of these two responses resemble
those reported by others in Aplysia neurons and muscle.
However, some significant differences occur that continue to obscure a
clear pattern of ACh responses among molluscan excitable cells and
their relationships to nicotinic ACh responses in vertebrate neurons and muscle. We further discuss the implication of the physiological properties of each response in the context of a behaving animal. An
abstract containing preliminary data from some of these experiments has
been reported previously (Laurienti and Blankenship
1994).
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METHODS |
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Specimens of A. brasiliana were collected from Laguna Madre near Port Isabel, Texas. They were housed in our Institute's aquarium facility in large aquaria with recirculating artificial seawater (ASW) at room temperature and fed dried seaweed daily.
Dissociated parapodial muscle fibers
Animals used for these studies ranged in size from 30 to
300 g. Animals were anesthetized by injecting isotonic
MgCl2 into the foot sinus (dose, 33 ml/100 g body
wt). The muscle tissue was dissociated using a modified version
(Laurienti and Blankenship 1996a) of previously proposed
methods (Brezina et al. 1994
; Ishii et al.
1986
). Briefly, the skin covering a parapodium was removed, and
small pieces of muscle tissue were dissected out and placed into a vial
containing 0.2-0.4% Type I collagenase. The vials were incubated at
30-33°C in a shaking water bath for 4-7 h. The enzyme solution then
was removed with pipettes and replaced with ASW containing penicillin
and streptomycin. Dissociated muscle fibers were stored
4 days in a
chilled water bath at 13 °C.
Isolated muscle fibers were embedded in agarose gel in a recording
chamber before electrophysiological recordings according to the methods
reported by Brezina et al. (1994). The chamber was
placed on an inverted, phase-contrast microscope and attached to a
perfusion system. The perfusion system allowed flow rates
10 ml/min;
this provided rapid exchange of the chamber solution, which was ~200
µl. Previously published data have demonstrated rapid response times
and adequate washout using this perfusion system (Brezina et al.
1994
; Laurienti and Blankenship 1996a
).
Electrophysiological methods
Conventional electronics were employed for intracellular
recordings using an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). Signals were recorded on a Gould (Valley View, OH) chart
recorder and converted to digital signals on a Digidata 1200 (Axon
Instruments) and stored on a PC computer using pClamp software (Axon
Instruments). Dual-beam oscilloscopes (Tektronix, Beaverton, OR) were
used for continuous monitoring of neural activity and to monitor the
quality of the voltage clamp. Intracellular electrodes were pulled from
capillary glass on a horizontal puller. Electrodes were filled with KAc
(3 M) and had resistances that ranged from 20 to 50 M. The bath was
grounded through an agar bridge.
Experimental data were collected under current- and voltage-clamp
conditions using single electrodes. Current-clamp experiments were used
to monitor membrane voltage responses to various pharmacological compounds. During all current-clamp experiments, 10-ms negative constant-current pulses were injected through the recording electrode to monitor changes in cell resistance. Muscle fiber current-voltage (I-V) relationships were obtained under voltage clamp using
1-s voltage ramps. Cells were clamped at a holding potential of 60 mV, and a voltage ramp was induced from
120 to 0 mV in the presence or absence of cholinergic compounds. These experiments allowed the
determination of reversal potentials for specific cholinergic agonists.
The physiological properties of the muscle membranes, including
characterization of voltage-dependent and serotonin-induced currents,
and the quality of the voltage clamp have been discussed previously in
detail; these dissociated fibers are adequately spaced-clamped under
our recording conditions (Laurienti and Blankenship 1996a
,b
,
1997
). Because of the limited gain attainable with
intracellular voltage-clamp recordings, the command voltage and the
actual membrane voltage may slightly differ. However, the actual
membrane voltage was recorded and was used in all data interpretation
and figure preparation.
Solutions and drugs
Normal ASW was used to store isolated muscle fibers and to
perform all experiments unless otherwise specified. ASW contained (in
mM) 427 Na+, 499 Cl, 10 K+, 10 Ca2+, 48 Mg2+, 3 HCO3
, and 26 SO42
. In some experiments Na propionate was used as a
substitute for NaCl to produced 15% Cl
(propionate substituted)-SW. ACh, nicotine, hexamethonium, and Type I
collagenase were obtained from Sigma; tubocurare, methylcarbamylcholine (MCC), and suberyldicholine were obtained from Research Biochemicals International (Natick, MS). All other chemicals used were obtained from
either Sigma or Fisher. All drugs were applied by bath perfusion. Drugs
typically were made in a stock solution in either ASW or distilled
water and added to the bath chamber to yield the desired concentration.
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RESULTS |
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Application of ACh (5-10 µM) to isolated muscle fibers results
in an increase in membrane conductance that produces a voltage response
that is quite variable in amplitude. ACh-induced responses, unlike
motoneuron-induced EJPs, vary from hyperpolarizations in a few fibers
to depolarizations in most fibers; an occasional fiber shows no voltage
response to applied ACh but demonstrates a decrease in membrane
resistance. This variability results from the simple fact that the
resting potential and ACh reversal potential in these fibers are close
to one another and vary independently such that in different fibers the
ACh reversal level may be more negative or more positive than the
resting level. On average, ACh produces a response with a reversal
potential of 52 ± 2.1 (SE) mV (see following text and Fig. 8).
The average resting membrane potential of isolated parapodial muscle
fibers was
56 ± 1 mV (n = 40) (see
Laurienti and Blankenship 1996a
). Although the
cholinergic response varies among muscle fibers, the response never
depolarizes cells to contraction threshold. In fact, most responses
result in membrane voltages between
50 and
60 mV. Such potentials
are suggestive of a chloride component to the ACh-induced response.
To determine if chloride contributes to the hyperpolarizing ACh
response, we compared the reversal potential for large ACh-induced hyperpolarizations in normal ASW and in low-chloride ASW. Figure 1A demonstrates a typical
current-clamp experiment where a muscle fiber is held at varying
membrane potentials and ACh is applied. Under such conditions, an
extrapolated reversal potential can be determined. In the example
shown, the ACh reversal potential is 68 mV. In the presence of 15%
Cl
(propionate substituted)-SW, the response is
shifted such that the new reversal potential is extrapolated to
40
mV. Figure 1B shows data from three cells in which similar
cholinergic responses were obtained in ASW and in low-chloride sea
water. These data demonstrate that, on average, the reversal potential
for such ACh responses in ASW is
69 mV, and in 15%
Cl
-SW, the cholinergic reversal potential
shifts to
41 mV, a displacement of 28 mV. The predicted Nernstian
shift for a purely chloride-dependent response with a change in
extracellular [Cl
] of this magnitude is 48 mV. The chloride reversal potential we have estimated is somewhat more
negative than that measured in most other molluscan neurons
(Gardner and Moreton 1985
; Kehoe 1972a
)
and muscle fibers (Blankenship et al. 1977
; Kozak
et al. 1996
and references therein). This could indicate that
parapodial fibers have a lower internal resting chloride concentration
than that of most other neurons and muscle or that potassium ions as well as chloride are contributing to membrane current during the cholinergic response. The fact that the cholinergic response does not
shift to the extent predicted by the Nernst equation also might suggest
that chloride is not the sole ion contributing to the cholinergic
response, but it also may reflect a passive reduction in
[Cl
]i when
[Cl
]o is reduced
greatly or that propionate has some permeability through the chloride
channel (see Blankenship et al. 1977
; Kehoe 1972a
; Kozak et al. 1996
). Most importantly, we
believe that those muscle fibers that produce a relatively large
hyperpolarizing response to ACh represent fibers that are expressing a
preponderance of chloride-sensitive receptors. ACh responses in most
fibers were, however, small- to moderately sized depolarizing responses or small hyperpolarizations and could represent activation of additional receptors.
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Several pharmacological compounds were identified that allowed us to distinguish two components to the cholinergic response. The pharmacological properties of the first component are illustrated in Fig. 2. This response, like that in Fig. 1, is another example of a relatively pure, uncontaminated version of a chloride-sensitive hyperpolarization. This component is activated by ACh and selectively mimicked by suberyldicholine (n = 42, Fig. 2A), nicotine (n = 36; Fig. 2B), and carbachol (n = 12; data not shown here; see following text). This hyperpolarizing response is blocked by curare (n = 30; Fig. 2C) but not by hexamethonium (n = 13; Fig. 2D). Nicotine consistently produces, like suberyldicholine, only hyperpolarizing responses in these muscle fibers and these responses are blocked by curare (data not shown, but see following text).
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Many muscle fibers respond to ACh with a net depolarization, but the use of suberyldicholine reveals that this response is composed of both a depolarizing component and an occult chloride-dependent hyperpolarization that apparently is masked by the simultaneously occurring large depolarizing response (Fig. 3A).
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The second component, represented by the net depolarizing response to
ACh, is blocked by hexamethonium (n = 13; Fig.
3B). Hexamethonium also blocks motoneuron-induced EJPs in
reduced muscle preparations (McPherson and Blankenship
1991a). The depolarizing response also is mimicked selectively
by MCC (n = 23; Fig. 4). As seen in Fig. 4A, increasing the dose of MCC produces a
physiological dose response. All subsequent experiments were conducted
using the 500 µM concentration of MCC as this is the lowest dose to induce a maximal depolarization. Neither nicotine nor carbachol was
ever seen to activate this second, depolarizing component of the ACh
response (see following text). The MCC-induced response is blocked
partially by hexamethonium (Fig. 4B) and by curare (Fig.
4C).
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The muscle-fiber responses to ACh were only occasionally purely of
either the depolarizing or hyperpolarizing type. Most responses to ACh
were found by use of selective agonists (Fig. 4D) or
antagonists (Fig. 4E) to comprise both a hyperpolarizing
(chloride-dependent) and a depolarizing (cationic-dependent) component,
these occurring in different proportions from cell to cell. The
depolarizing component is activated selectively by MCC and blocked
selectively by hexamethonium. The hyperpolarizing, chloride-dependent
response is activated selectively by suberyldicholine (and,
unexpectedly, by nicotine, which in other molluscan preparations also
activates the cationic, depolarizing response). Curare blocks both
responses. Others have reported that -bungarotoxin selectively
blocks the chloride-dependent fast hyperpolarizing response
(Kehoe at al. 1976
; Kozak et al. 1996
).
We have not yet tested whether
-bungarotoxin blocks the hyperpolarizing response.
Using voltage clamp it is possible to determine the reversal potential
of the pharmacological agonists by inducing voltage ramps in the
presence and absence of drug. Such data are plotted on an
I-V curve with the crossing points of the two curves being the reversal potential for the agonist. Figure
5 demonstrates I-V curves for
suberyldicholine, MCC, carbachol, and nicotine, with the lower portion
of each panel representing difference currents. The difference currents
were obtained mathematically by subtracting the current recorded in
control ASW from that recorded in the presence of the cholinergic
agonist. These difference currents represent the current that is
activated by the specific agonists and are free of any leak currents or
voltage-gated currents. As demonstrated in this figure,
suberyldicholine induces a current that reverses near 70 mV (near the
range of the chloride reversal potential). Carbachol and nicotine
induce currents that reverse near
60 mV, again indicating predominant
activation of a chloride current. MCC, however, as an agonist more
selective for the depolarizing component of ACh responses, induces a
current that reverses near
30 mV, which is below (more positive than)
the contraction threshold for these cells. This current presumably is
carried by a mixed cationic current, probably including sodium,
potassium, and calcium.
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Figure 6 shows an experiment in which the
effects of ACh, suberyldicholine, and MCC were all recorded in the same
cell. The experiment then was repeated in the presence of hexamethonium which selectively antagonizes the depolarizing current. This figure shows that in the presence of hexamethonium all three compounds activate a current with a reversal potential near 65 mV; this is
consistent with the chloride reversal potential. As previously demonstrated, suberyldicholine is selective for the hyperpolarizing response, ACh is nonselective and activates both the hyperpolarizing and depolarizing response with differing selectivities across different
cells, and, finally, MCC predominantly activates the depolarizing
response but does activate the chloride response to some degree. It can
be seen from the Fig. 6, right, that in the presence of
hexamethonium, the amplitude of the MCC current is smaller than that
activated by ACh or by suberyldicholine.
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To more clearly demonstrate the individual components of the ACh-
induced current, we used computer-aided subtraction protocols. In Fig.
7 the chloride and cationic components of
the ACh current have been isolated. The chloride component
(ACh(Cl)) was derived by subtracting the control
current in Fig. 6B from the current recorded in response to
suberyldicholine in the presence of hexamethonium (Fig. 6B).
This trace represents the hexamethonium-insensitive current free of
leak and background currents, or the pure inhibitory response. The
cationic current [ACh(cat)] was isolated by
subtracting the MCC-induced current in the presence of hexamethonium
(Fig. 6B) from the MCC current obtained in normal ASW (Fig.
6A). Therefore this trace represents only the
hexamethonium-sensitive current and any chloride component has been
subtracted out. As is evident from Fig. 7, the chloride current
reverses at 75 mV, which is near the reversal potential for the
chloride ion. The presumptive cationic current reverses near
20 mV,
which is well below (more positive than) the contraction threshold for
these cells.
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We consistently observed muscle contractions when MCC was applied to
the chamber bath (data not shown). However, no contractions were
observed in response to either ACh or suberyldicholine. Figure 8 is a graph of the reversal potential of
these three cholinergic agonists. The reversal potentials were
determined using voltage-clamp experiments; each compound was tested in
at least nine different cells. The graph clearly demonstrates that MCC
has an average reversal potential below (more positive than) the
contraction threshold (40 mV) (Laurienti and Blankenship
1996a
) for the muscle fibers, whereas ACh and suberyldicholine
have reversal potentials above (more negative than) the contraction
threshold.
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DISCUSSION |
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We have presented the pharmacological properties of the
cholinergic response of isolated parapodial muscle fibers. In the whole
muscle, motoneurons induce 1:1 EJPs that are antagonized by
hexamethonium, which also blocks motoneuron-induced muscle contractions. Of interest, however, is the fact that application of ACh
to the isolated muscle cells resulted in mixed responses, relatively
small hyperpolarizations or depolarizations but never contractions. The
average reversal potential of ACh as determined in voltage-clamp
experiments was 52 ± 2.1 mV, which is above (more negative
than) the contraction threshold for these cells (see Fig. 8) and near
the normal resting potential for these fibers (
56 mV)
(Laurienti and Blankenship 1996a
).
The ACh-induced response has two pharmacologically distinct components;
one component is carried predominantly by chloride and the other
component is likely to be carried by mixed cations. The
chloride-dependent response is activated by suberyldicholine and
closely mimicked by carbachol and nicotine, and has a reversal potential near 70 mV (
68.3 mV, ±1.5 mV, SE). This response can be
antagonized by curare but is insensitive to hexamethonium. The second
component observed is an MCC-activated depolarization that is
insensitive to suberyldicholine, nicotine, and carbachol. The
depolarizing response is antagonized by hexamethonium but also is
blocked partially by curare. This response has a reversal potential of
34.3 ± 1.3 mV), which is below (more positive than) the
contraction threshold for the muscle fibers. We consistently observed
muscle contractions in response to MCC and these contractions were
blocked in the presence of hexamethonium (data not shown).
In keeping with the findings of other investigators (Ascher and
Kehoe 1975; Kehoe 1972b
, 1979
; Kozak et
al. 1996
), the properties of the chloride-dependent receptor in
parapodial muscle fibers closely resemble those of vertebrate skeletal
nAChRs. However, the lack of sensitivity of the receptors controlling
the cationic response to nicotine and carbachol in these fibers appears
to set them apart from other molluscan neurons and muscle fibers and
makes their relationship to vertebrate neuronal nAChRs unclear. Although there are some small differences in the pharmacological profiles of buccal and parapodial cation-related receptors, it is clear
that each muscle type expresses both a cation- and chloride-dependent receptor in dissociated fibers. Despite this, the fibers respond differently to exogenously applied ACh that simultaneously activates both receptors, and the fibers appear to be innervated in different ways in the intact animal.
In buccal muscle of A. californica, application of ACh
always results in a depolarization and one sufficient to induce a
muscle contraction (Kozak et al. 1996). These findings
are in keeping with observations that ACh applied to whole buccal
muscle causes contractions (Cohen at al. 1978
). In
parapodial muscle fibers, this is not the case. Furthermore we have
observed in several experiments using intact parapodial muscle in
reduced preparations of both A. californica and A. brasiliana that direct application of ACh, either by superfusion
onto skinned muscle preparations or by perfusion through parapodial
blood vessels, even in the presence of cholinesterase inhibitors, never
produces contractions of parapodial muscle. Furthermore in buccal
muscle, both intact and dissociated from both species, we have always
found the muscle to respond to exogenous ACh with brisk contractions
(unpublished observations). On the other hand, the application of MCC,
which is more selective for the cationic response, does consistently induce contractions in dissociated parapodial muscle fibers.
Furthermore the MCC response is antagonized by hexamethonium, the same
antagonist that blocks motoneuron-induced EJPs and contractions in the
whole muscle. These data pose an interesting contrast concerning the physiological roles of the two ACh receptors in the intact animal. To
account for the discrepancy, we propose two models for the arrangement
of the cholinergic receptors in the behaving animal (Fig.
9).
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The first model, the one we believe most likely applies to intact
parapodial muscle, involves receptor segregation where the excitatory
receptors are clustered in the synaptic regions of excitatory
motoneurons and the inhibitory receptors are clustered separately at
the synaptic regions of inhibitory motoneurons. Although no inhibitory
motoneurons have been identified, IJPs have been recorded in muscle
fibers in intact parapodial preparations (McPherson and
Blankenship 1991a). This model also requires that the
excitatory and inhibitory motoneurons be activated selectively and that
contractions would occur in response to selective activation of
excitatory motoneurons, which is in fact the case (McPherson and
Blankenship 1991a
,b
).
The second model, one believed to obtain in buccal muscle (Kozak
et al. 1996), does not require selective receptor clustering but instead suggests that the two receptor types are mingled close together and are activated by a single class of excitatory motoneuron. This model does require that the number or efficacy of functional excitatory receptors be greater than the number or efficacy of inhibitory receptors so that the net effect of ACh and of all motoneuron activity is to depolarize the muscle cell. The role of the
chloride-dependent response in buccal muscle is to limit the amount of
depolarization ACh can produce in a single fiber, and to do so by being
colocalized and co-activated at a common synaptic site with the
cationic receptor not separated from the excitatory site as a unique
and independent locus for IJPs (Kozak et al. 1996
).
This model is less likely to obtain in parapodial muscle because the
ACh reversal potential is near the resting potential, and exogenous ACh
application never induces a contraction and often produces a net
hyperpolarization of fibers. In other words, application of ACh to
isolated parapodial fibers does not mimic the response of fibers to
excitatory motoneuron input as is the case in buccal fiber responses.
Furthermore unlike the situation in buccal muscle (Cohen et al.
1978), IJPs are observed in parapodial muscle and serve as
candidates for the physiological response of a unique
chloride-dependent input from cholinergic inhibitory motoneurons.
However, it is possible that enzymatic dissociation procedures may
alter either the excitatory cholinergic receptors or the inhibitory
ones so as to confer different response properties on isolated fibers
compared with those in situ. It is also possible that either of two
types of clustered receptor could respond differently with repetitive
activation due to desensitization or sensitization or either type, as
has been observed in certain cholinergic neural synapses in
Aplysia (Wachtel and Kandel 1971
). In
preliminary experiments where we have made repetitive applications of
ACh to isolated fibers, we have seen no evidence of significant
alterations in response amplitude with multiple stimulations. Thus it
is not possible at this time to be certain whether two separate
receptor populations are activated by specific motoneurons, but this
seems the most likely case. The presence of separate excitatory and inhibitory inputs to parapodial muscle, using the cationic and chloride
channels, respectively, could serve at least two roles: inhibitory
inputs to one set of muscles could complement excitation of antagonists
during the alternating up-and-down flaps of the parapodia during
swimming, and direct inhibitory input may play a special role in muscle
control when the parapodia are not utilized for swimming and instead
are folded quietly over the back of the animal to enclose the mantle cavity.
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ACKNOWLEDGMENTS |
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This work was supported in part by National Institute of Neurological Disorders and Stroke Grant T32 NS-07185.
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FOOTNOTES |
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Address for reprint requests: J. E. Blankenship, Marine Biomedical Institute, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069.
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 27 October 1998; accepted in final form 30 March 1999.
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REFERENCES |
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