Committee on Neurobiology and Department of Neurobiology, Pharmacology, and Physiology, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Fox, Lyle E. and Philip E. Lloyd. Glutamate is a Fast Excitatory Transmitter at Some Buccal Neuromuscular Synapses in Aplysia. J. Neurophysiol. 82: 1477-1488, 1999. Studies of the modulation of synaptic transmission in buccal muscle of Aplysia were limited because the conventional fast transmitter used by a number of large buccal motor neurons was unknown. Most of the identified buccal motor neurons are cholinergic because they synthesize acetylcholine (ACh) and their excitatory junction potentials (EJPs) are blocked by the cholinergic antagonist hexamethonium. However, three large identified motor neurons (B3, B6, and B38) do not synthesize ACh and their EJPs are not inhibited by hexamethonium. To identify the fast excitatory transmitter used by these noncholinergic motor neurons, we surveyed putative transmitters for their ability to evoke contractions. Of the noncholinergic transmitters tested, glutamate was the most effective at evoking contractions. The pharmacology of the putative glutamate receptor is different from previously characterized glutamate receptors in that glutamate agonists and antagonists previously used to classify glutamate receptors had little effect in this system. In addition, glutamate itself was the most effective agent tested at reducing EJPs evoked by the noncholinergic motor neurons presumably by desensitizing glutamate receptors. Finally, immunocytology using an antiserum raised to conjugated glutamate in parallel with intracellular fills indicated that the varicose axons of these motor neurons were glutamate-immunoreactive. Taken together, these results indicate that the fast transmitter used by the noncholinergic neurons is almost certainly glutamate itself. This information should help us understand the role of transmitters and cotransmitters in the generation of feeding behaviors in Aplysia.
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INTRODUCTION |
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Neuronal mechanisms that generate feeding
behaviors have been heavily studied in Aplysia. In
particular, peripheral modulation at neuromuscular synapses in buccal
muscles has been examined extensively (Fox and Lloyd
1998; Weiss et al. 1992
, 1993
; Whim et
al. 1993
). These studies have been limited because the identity of the conventional transmitter used by a group of buccal motor neurons
was unknown. Here we attempt to identify the conventional fast
excitatory transmitter used by these motor neurons.
Many buccal motor neurons have been identified based on a combination
of criteria including their size, location, the muscles they innervate,
and expression of modulatory peptide cotransmitters (Church and
Lloyd 1991, 1994
; Cohen et al. 1978
). The fast
excitatory transmitters used by many of these neurons is acetylcholine
(ACh). These neurons contain choline acetyltransferase activity, as
determined by the synthesis of ACh from intracellularly injected
labeled choline, and their excitatory junction potentials (EJPs) are
blocked by the cholinergic antagonist hexamethonium (Church and
Lloyd 1994
; Cohen et al. 1978
). However, three
large identified motor neurons (termed B3, B6, and B38) are not
cholinergic by these criteria. They do not synthesize ACh from injected
choline, and their EJPs are not blocked by hexamethonium (Church
et al. 1993
; Lloyd and Church 1994
;
Lotshaw and Lloyd 1990
). All three of these neurons
express modulatory peptide cotransmitters and innervate different but
overlapping regions of intrinsic buccal muscle 3 (I3). B38 expresses
the small cardioactive peptides (SCPs) and innervates the
anterior region of the I3 muscle (I3a), B3 expresses FMRFamide and
innervates the anterior and medial regions of the muscle, and B6
expresses the SCPs and innervates the medial and posterior regions
(Church and Lloyd 1991
). This muscle also is innervated
by two identified excitatory cholinergic motor neurons (termed B9 and
B10) that innervate medial and posterior regions. I3 is a nonspiking
muscle, and both the cholinergic and noncholinergic neurons evoke
graded contractions that result from the summation of EJPs.
In the present paper, we present evidence implicating glutamate as the fast excitatory transmitter used by the identified noncholinergic motor neurons. Three lines of evidence support this conclusion. A survey of transmitters, transmitter agonists, and antagonists indicated that glutamate was one of only a few substances that evoked contractions of isolated I3a muscles. In addition, of the many substances tested, glutamate itself was most effective at reducing the EJPs evoked by the noncholinergic motor neurons presumably by desensitizing glutamate receptors. Finally, immunocytology using an antiserum raised to conjugated glutamate in parallel with intracellular fills indicated that the varicose axons of these motor neurons were glutamate-immunoreactive.
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METHODS |
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Animals
Aplysia californica (50-150 g) were obtained from Marinus (Long Beach, CA), maintained in circulating artificial sea water (ASW) at 16°C, and fed dried seaweed every 3 days.
Measurement of I3a muscle contractions
AGONIST TEST PROTOCOL. A few bundles of the I3a muscle were isolated in these experiments (to enhance penetration of tested substances), mounted in a small chamber (total volume 300 µl), and superfused with ASW at a flow rate of 1.5 ml/min. Test substances in ASW (5-10 µl) were injected into the superfusion, just before it entered the chamber, to determine if they evoked contractions. Because the superfusion was aimed at the muscle fibers, the initial concentration of the test substance applied to the muscle was similar to the concentration in the bolus; however, it was immediately diluted. Contractions were measured with an isotonic transducer.
ANTAGONIST TEST PROTOCOL. Reproducible submaximal contractions were evoked by periodic bolus injections of ACh or glutamate. Test substances were applied via the superfusion to determine their effects on these contractions. Because the amplitude of the evoked contractions sometimes changed during the experiments, results were quantified by comparing the amplitude of the contractions evoked during treatment with the test substance to the average of the contractions evoked before treatment and after washout. Boluses for both experiments were applied at 5- to 7-min intervals. Most substances tested (Table 1) were obtained from Sigma or RBI.
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Motor neuron stimulation experiments
Animals were immobilized with an injection of isotonic
MgCl2 and the dissection carried out in either
low Ca2+ (0.5 mM; 0.05× normal), high
Mg2+ (110 mM; 2× normal) ASW (termed low Ca ASW)
or high Ca2+ (33 mM; 3× normal), high
Mg2+ (165 mM; 3× normal) ASW (termed high Ca, Mg
ASW). The buccal mass/buccal ganglia complex was removed, bisected
along the midline, all nerves severed except ipsilateral buccal nerve 2 (nerve designations from Gardner 1971; muscle
nomenclature from Howells 1942
; also see Lloyd
1988
), and pinned in a dish with a silicone elastomer (Sylgard,
Dow Corning) base. The hemiganglion was desheathed and selectively
superfused with either low Ca ASW to suppress synaptic transmission in
the ganglion or high Ca, Mg ASW to raise the firing thresholds of
neurons in the ganglion. The remainder of the bath containing the I3
muscle was separated by a barrier (except when a perfusion electrode
was used, see following text) through which the intact nerve 2 ran and
was superfused with ASW. The test substances were applied in ASW
selectively to the bath containing the muscle so the ganglion was not
exposed to them. Neurons were usually impaled with two microelectrodes
(2-4 M
; filled with 3 M K acetate, 0.1% Fast Green), one to inject
current and one to monitor membrane potential. Motor neurons were
identified by their position, size, nature of synaptic input, and
muscle innervation patterns (see Church et al. 1993
).
Measurement of I3 EJPs
Individual spikes in motor neurons were driven by brief (10-20
ms) depolarizing current pulses. EJPs were recorded with an intracellular electrode (~20 M; filled as described in the
preceding text) or via a perfusion electrode (see following text). For
intracellular muscle fiber recordings, short bursts of two to eight
action potentials at 10-12.5 Hz were used to ensure that a burst did
not evoke contractions. Intracellular EJPs were quantified by comparing
the EJP amplitude during application of the test substance to the EJP
amplitude before its application because it was difficult to maintain
intracellular recordings until the effects of the test substances
completely reversed. The perfusion electrode consisted of a small
chamber (100 µl) and aperture (~1.5 mm), which was positioned to
press firmly down on a portion of the muscle (see Fig.
1 in Church et al. 1993
). The
inside of the chamber was superfused rapidly with ASW (1.5 ml/min). The
remainder of the muscle outside of the recording chamber was superfused
with low Ca ASW to suppress synaptic transmission and muscle
contractions. This procedure confined the contractions to the small
area of the muscle covered by the recording chamber and thus markedly
reduced movement artifacts in the recordings. The earliest evoked
muscle contractions occur after the sixth EJP so the early EJPs in a
burst are recorded in the absence of any movement. Stimulation at 16 Hz
was used routinely in these experiments. EJPs were recorded by
extracellular electrodes placed inside and just outside the wall of the
perfusion apparatus. Signals were amplified using a Grass P15D AC
amplifier. The test substances were applied in ASW to the inner chamber
of the perfusion electrode so the ganglia were not exposed to these
substances. Typical application periods were 20 min to ensure adequate
penetration into the muscle. Experiments were performed at room
temperature (~22°C). In most experiments, long interburst intervals
(100 s) were used to minimize release of endogenous peptide
cotransmitters from the motor neurons and to minimize posttetanic
potentiation (Church et al. 1993
; Lotshaw and
Lloyd 1990
; Whim and Lloyd 1990
). Recordings
obtained with the perfusion electrode were quantified by comparing the EJP amplitude during treatment with the test substance to the average
amplitude of EJPs evoked before treatment and after washout.
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Intracellular labeling
The buccal mass/buccal ganglia complex was dissected and pinned
in a dish as described in the preceding text except that the ganglia
were not desheathed. Neurons were impaled by tapping a single
microelectrode [15-30 M; filled with 5% biocytin (Sigma) in 0.5 M
K chloride, 0.1% Fast Green] through the sheath. Motor neurons were
identified, and biocytin was ionophoresed into a single identified
neuron by hyperpolarizing the neuron 10 mV from rest and injecting
20-mV depolarizing steps (0.5 s, 1 Hz) for 2-8 h. The preparation then
was incubated at 4°C with a continuous superfusion of ASW containing
0.1 mg/ml glucose and 1% (vol/vol) penicillin-streptomycin-fungizone
(GIBCO). After waiting 48-96 h for the biocytin to diffuse to
terminals in the muscle, the tissue was fixed and processed for
histology (see following text).
Tissue preparation and staining
Immunocytology was carried out using the methods of
Longley and Longley (1986) as modified by Pearson
and Lloyd (1989)
. Freshly isolated tissue or tissue in which
neurons had been labeled intracellularly were washed with low
Ca2+ high Mg2+ ASW and
fixed in 4% paraformaldehyde, 30% sucrose in 0.1 M phosphate buffer
(PB, pH 7.4) for 1 h at room temperature and then overnight at
4°C. After fixation, the tissue was washed with 30% sucrose in PB
for 24 h at 4°C. Buccal nerve 2 was cut so the muscle and ganglion could be processed separately. The biocytin was visualized in
the ganglion (as described in the following text) without sectioning to
verify that only one neuron was labeled. Frozen sections (7-10 µm)
were cut from the muscle in a cryostat and transferred to nylon
mesh-bottomed wells containing PB saline (PBS; 0.14 M NaCl, 0.01 M
phosphate, pH 7.4) for staining. The sections were washed with PBS
(4 × 10 min each) and then cleared overnight with PB containing
0.2% Triton X-100, 0.1% sodium azide (PB-Triton-X) at 4°C. The
biocytin was visualized with a 1:250 dilution of either strepavidin-Texas Red (BRL) or avidin-Texas Red (Vector) in
PB-Triton-X. After 4 h, the sections were washed with PB-Triton-X
(4 × 10 min each), and then nonspecific binding was blocked by
incubating the tissue in a blocking solution of PB with 0.5% Triton
X-100, 0.1% sodium azide, and 5% normal goat serum (NGS; GIBCO)
overnight at 4°C. Primary and secondary antiserum were diluted in
this blocking solution. Antiglutamate antiserum (Sigma) was used at a
dilution of 1:1000. Sections were incubated with the antiserum
overnight at 4°C, washed with the blocking solution, and the staining
visualized with a fluorescent secondary antiserum, BODIPY FL goat
anti-rabbit (Molecular Probes) used at a dilution of 1:250. This tissue
was mounted on gelatin-coated slides, dried, and coverslipped in 2% n-propyl gallate in a 1:6 PBS:glycerol solution (pH 8.5).
The fill was not visible with the fluorescein filter set and
the immunofluorescence was not visible with the Texas Red/rhodamine
filter set.
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RESULTS |
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Pharmacological studies
ISOLATED MUSCLE.
Conventional transmitters were screened to test if they caused
contractions of isolated segments of the I3a muscle. Of the transmitters tested (Table 1), only bolus application of glutamate, ACh, dopamine, and histamine evoked contractions at concentrations of
10 mM. We concentrated our studies on glutamate for the following reasons. 1) ACh is clearly not the conventional transmitter
used by B3, B6, and B38. These neurons do not contain choline
acetyltransferase activity and their EJPs are not inhibited by
hexamethonium. The contractions evoked by ACh could be due to the
presence of excitatory cholinergic receptors that normally respond to
ACh released by the cholinergic motor neurons that innervate I3.
2) It is unlikely that B3, B6, and B38 are dopaminergic.
Dopaminergic neurons in the CNS of Aplysia have been
identified by glyoxylic acid treatment and the
formaldehyde-glutaraldehyde-induced histofluorescence technique
(Goldstein and Schwartz 1989
; Hawkins
1989
; Rathouz and Kirk 1988
). Although there
appears to be two or three midsized dopaminergic neurons in each buccal
ganglion, none of these were large enough or positioned correctly to be
the noncholinergic motor neurons. 3) It is unlikely that B3,
B6, and B38 are histaminergic. The Aplysia CNS and
peripheral tissue were stained with an antiserum directed against
histamine (Elste et al. 1990
; Soinila et al. 1990
). No histamine-like immunoreactivity was observed in any large buccal neurons or in buccal muscle.
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EXTRACELLULAR EJPS.
We next studied the effects of the pharmacological agents on EJPs
evoked by stimulation of motor neurons. A perfusion electrode was used
to record extracellular EJPs in a small portion of the I3 muscle that
was perfused with ASW while the remainder of the muscle was superfused
with low Ca ASW to suppress synaptic transmission and muscle
contractions (Church et al. 1993). This procedure
permitted stable long-term recordings, rapid solution turnover, and
simultaneous recordings from a population of fibers thereby reducing
sampling bias. Results from this technique are qualitatively similar to those obtained with intracellular electrodes (Fox and Lloyd
1997
). First we verified that EJPs evoked by B3, B6, or B38
were not inhibited by application of hexamethonium to the muscle. By
contrast, evoked EJPs were inhibited when glutamate was applied to the
muscle. Superfusion with 1 mM glutamate reduced EJPs to 44 ± 3%
for B3 (n = 19), to 7 ± 4% for B6
(n = 3), and to 33 ± 12% of control for B38
(n = 3) (Fig. 3) These
effects presumably reflected desensitization of the glutamate receptor
as there was little effect on cholinergic EJPs evoked by B9 (reduced to
86 ± 6% of control, n = 4). Even at higher
concentrations (10 mM), glutamate had little effect on cholinergic
neuromuscular synapses in I5, indicating again that glutamate's action
was selective. Most of the glutamate agonists that evoked contractions
(D- and L-aspartate, cysteate, CSA, and HCA;
Fig. 4) and substances that specifically
reduced glutamate-evoked contractions (ASBA and HCA; Fig.
5) were tested for their ability to
inhibit B3-evoked EJPs. All of these substances had little effect on
B3-evoked EJPs. The effects of ASBA and HCA on EJPs were much smaller
than their effects on contractions evoked by glutamate boluses. These
differences might be a consequence of the procedures used to test the
substances. Bolus application of glutamate might activate extrasynaptic
receptors that are not normally activated by glutamate released from
the motor neurons. These substances may act on other components of the
excitation-contraction coupling mechanism and not directly effect
synaptic transmission. Although there were some differences in the
effects of the test substances on neuronally evoked EJPs and glutamate
evoked contractions, glutamate was the most effective substance tested
at reducing both of these responses. In addition, glutamate was
selective in that it only inhibited EJPs evoked by noncholinergic motor neurons.
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INTRACELLULAR EJPS.
Although it was technically difficult to do, it was necessary to
examine the effects of glutamate on intracellularly recorded EJPs. The
reduction in the amplitude of the extracellularly recorded EJPs could
have been due to a tonic depolarization of the muscle fibers that would
bring the resting potential of the muscle fibers closer to the synaptic
reversal potential and thus reduce the amplitude of the EJP. The
perfusion electrode would not have detected a slow tonic depolarization
as it is an extracellular recording technique. I3 muscle fibers have a
resting potential of 75 ± 1 mV (n = 14). Bath
application of glutamate transiently depolarized the muscle fibers
18 ± 2 mV, but the fibers potential nearly returned to rest
(
70 ± 2 mV) within 20 min. Thus the depolarizing response to
glutamate appears to desensitize. Indeed, application of 1 mM glutamate
to I3 reversibly inhibited EJPs evoked by B3, B6, or B38 (reduced to
64 ± 3% for B3, n = 5; to 27 ± 6% for B6,
n = 3; to 16 ± 9% of control for B38,
n = 3; Fig. 7). The
effects of glutamate were measured after the reversal of the transient depolarization. If glutamate functions by desensitization only and if
glutamate is also the fast transmitter of the noncholinergic motor
neurons, it should reduce the size of EJPs evoked by these neurons
without decreasing muscle fiber input resistance. We used the time
constant for the decay of EJPs that could be fitted with a single
exponential as an indirect measure of changes in input resistance. In
glutamate, the time constant of the muscle fibers, measured after the
transient depolarization, increased (to 120 ± 10% of control,
n = 11), indicating that, if anything, muscle fiber
input resistance increased slightly. These results indicate that
glutamate desensitizes the receptors for the noncholinergic fast
transmitter. Finally, the effects of glutamate were selective because
B9-evoked cholinergic EJPs recorded in the same fibers were essentially
unchanged (reduced to 95 ± 4%, n = 3; Fig. 7; see following text).
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Immunocytological studies
Glutamate-like immunoreactivity was visualized using a primary
antiserum directed toward glutamate and a fluorescent (BODIPY) secondary antibody. No immunoreactive neuronal somata were observed in
the ganglia, and no immunoreactive large caliber axons were observed in
the muscle, nerve, or ganglia. Indeed, preliminary analyses of primary
amines present in individual identified neuronal somata by
high-performance liquid chromatography indicate that glutamate
levels in cholinergic and noncholinergic neurons were similar
(unpublished observation). These findings are similar to those observed
in lobster where glutamate levels in excitatory motor axons and somata
that use glutamate as a fast transmitter were similar to those in
inhibitory motor axons and somata that use GABA as a fast transmitter
(Kravitz and Potter 1965; Otsuka et al.
1967
). In our study, immunoreactivity was associated with fine-diameter varicose axonal fibers in the neuropil of ganglia or in
muscle sections. Numerous immunoreactive fibers and varicosities were
present in the I3 muscle (Fig. 8). The
immunoreactivity appears to be quite specific to glutamate. In blinded
observations, staining with the antiglutamate antiserum was eliminated
by incubation with 1 mM L-glutamate but was unaffected by
incubation with 10 mM aspartate, glutamine, glycine, glucose, cysteate,
or homocysteate.
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Immunoreactive fibers also were observed in the I5 muscle, which
receives only cholinergic excitatory motor input (Cohen et al.
1978). However, the immunoreactive fibers were much sparser than those observed in I3. The immunoreactivity in I5 may be due to the
presence of sensory neuron fibers, which are thought to use glutamate
as their transmitter. Consistent with this interpretation, glutamate-like immunoreactive fibers in I5 were concentrated in the
region where the muscle attaches to the cartilage, an area that is
likely to have heavy sensory innervation. Staining in I3 was more
uniform and was similar to the pattern observed when immunocytology was
carried out using antiserum directed toward peptide cotransmitters
expressed in motor neurons (Lloyd et al. 1985
;
Miller et al. 1992
).
To determine if the varicose axons of B3, B6, or B38 contained glutamate-like immunoreactivity, double label experiments were carried out. Neurons were identified physiologically, and their cell bodies were injected intracellularly with biocytin and incubated for several days to allow for diffusion of biocytin to terminals in the I3 muscle (~30 mm distance). Only a single motor neuron was injected in each preparation. Muscles then were fixed and sectioned, and biocytin was visualized with either avidin- or strepavidin-Texas Red. These experiments had inherent limitations. Large-caliber axons in the muscle filled well but, as expected from the results described in the preceding text, were not immunoreactive, whereas small-caliber varicose axons were immunoreactive but filled less efficiently presumably due to their diffusional distance from the filled cell bodies. Nevertheless, double staining in axonal fibers and varicosities in I3 muscle was detected for B3, B6, and B38, indicating that the terminals of each motor neuron were indeed immunoreactive (Fig. 9). Observation of the ganglia at the end of the incubation period indicated that biocytin was confined to the injected neuron, indicating that there was no significant dye coupling to other neurons.
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Individual muscle fibers receive fast excitatory EJPs from both cholinergic and noncholinergic motor neurons
We previously had determined that individual muscle fibers
in the anterior region I3 were innervated by excitatory noncholinergic motor neurons (B3 and B38) and an inhibitory cholinergic motor neuron
(B47) (Church et al. 1993). Similar results also have
been observed in A. kurodai where posterior and medial
buccal muscle fibers were innervated by excitatory noncholinergic motor
neurons and either an inhibitory or dual component
(inhibitory/excitatory) cholinergic motor neuron (Nagahama and
Takata 1990
). We now report that individual muscle fibers in
the medial portion of I3 are innervated by excitatory noncholinergic
motor neurons (B3 or B6) and excitatory cholinergic motor neuron (B9)
(Lloyd and Church 1994
). As expected from previous
results, B9-evoked EJPs are inhibited by hexamethonium, whereas those
of B3 and B6 are not (Fig. 10). These
results are similar to observations made in the Aplysia gill
where individual muscle fibers also receive both cholinergic and
noncholinergic EJPs (Carew et al. 1974
). However, in
both systems it is important to emphasize that we are describing
functional innervation; because of electrical coupling between muscle
fibers (Lotshaw and Lloyd 1990
), the anatomic
innervation need not be on the same fiber.
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DISCUSSION |
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Evidence implicating glutamate as the fast transmitter of the noncholinergic motor neurons
Overall, the pharmacological evidence is consistent with glutamate being the fast excitatory transmitter used by the noncholinergic motor neurons. Except for ACh, glutamate itself was the most potent substance tested at producing contractions. The actions of glutamate were selective for I3. Buccal muscle I5, which has a purely cholinergic motor innervation, did not contract in response to the application of glutamate at any concentration. Glutamate was also the most effective of all substances tested at reducing the amplitude of EJPs evoked by noncholinergic motor neurons apparently through desensitization of receptors. These results were obtained even though many other substances were tested, some with structures very similar to glutamate. Glutamate had no effect on EJPs evoked by cholinergic neurons recorded in the same fibers nor did it reduce the EJP relaxation time constant. These results indicate that glutamate did not decrease the input resistance of the muscle fibers.
Receptors activated by AMPA and kainate in vertebrate CNS and at
arthropod neuromuscular junctions desensitize rapidly with a time
constant of <50 ms (Dudel et al. 1988, 1990
;
Trussell and Fischbach 1989
), whereas NMDA-activated
receptors in the vertebrate CNS desensitize with a slower time constant
of 1-10 s (Zorumski et al. 1989
). Desensitization of
glutamate receptors in I3 was very slow compared with that observed for
glutamate receptors in the vertebrate CNS and in arthropods. However,
much of the delay is due to the time required for solution exchange and
diffusion of glutamate to receptors deep in the fiber bundles. The
magnitude of the desensitization appeared to differ between the three
noncholinergic neurons. B3-evoked EJPs, recorded with either
intracellular or extracellular electrodes, were reduced less than EJPs
evoked by stimulating the other neurons. Although this difference could be due to B3's terminals being deeper in the bundle and less
accessible to bath applied glutamate, it is also possible that it
represents a real physiological difference between receptors at these
neuromuscular synapses. Different subtypes of the glutamate receptor
may be expressed at B3's synapses. The family of ionotropic glutamate receptors is large and diverse, currently including at least 22 cloned
subunits (Hollmann and Heinemann 1994
). We cannot
distinguish between these possibilities based on our results.
Perhaps the most convincing evidence implicating glutamate as the fast
excitatory transmitter was the immunocytology using an antiserum raised
to conjugated glutamate. First, we were able to identify the
immunoreactive varicose axons as being those of B3, B6, or B38 by
filling their cell bodies with biocytin. Second, the immunoreactivity
was reduced substantially by 1 mM glutamate while even 10-fold higher
concentrations of several closely related compounds did not detectably
affect the immunoreactivity, suggesting that it was highly selective
for glutamate. We were not able to find neuronal cell bodies that
contained glutamate-like immunoreactivity in the Aplysia
CNS. Recently this has been accomplished in the pedal-pleural ganglia
using semithin sections to improve the penetration of the antiserum and
reduce the background staining (Levenson et al. 1997).
Even then only weak to moderate staining was observed in the cell
bodies compared with the intense staining of axonal processes in the
neuropil. The observation that glutamate-like immunoreactivity was weak
in cell bodies and large-caliber axons is consistent with biochemical
measurements made in lobster that indicate that glutamate levels are
similar in neuronal cell bodies and large-caliber axons of motor
neurons that use glutamate and motor neurons that use GABA as their
fast transmitters (Kravitz et al. 1963
; Otsuka et
al. 1967
). This may reflect the selective expression of
high-affinity uptake sites and the localization of vesicles containing
glutamate to synaptic regions (Nicholls 1993
).
Taken together, the evidence that glutamate is the fast excitatory
transmitter at these neuromuscular synapses is quite convincing. However, other conventional transmitters such as ACh, dopamine, and
histamine evoked contractions of the isolated I3 muscle. We do not
believe that any of these are the conventional transmitter used by the
noncholinergic motor neurons. ACh caused contractions in the isolated
muscle, but B3, B6, or B38 do not contain choline acetyltransferase
activity and their EJPs are not blocked by hexamethonium, a cholinergic
antagonist in Aplysia (Church et al. 1993;
Lloyd and Church 1994
; Lotshaw and Lloyd
1990
). We assume that the contractions evoked by ACh were due
to the presence of excitatory cholinergic receptors for cholinergic
motor neurons (e.g., B9) that functionally innervate the same I3 muscle
fibers as the noncholinergic motor neurons. Dopaminergic neurons in the
CNS of Aplysia have been identified by glyoxylic acid and
formaldehyde-glutaraldehyde induced histofluorescence techniques
(Goldstein and Schwartz 1989
; Hawkins 1989
; Rathouz and Kirk 1988
). The dopamine
positive neurons are all much smaller than any of the identified
noncholinergic motor neurons. Also, histamine-like immunoreactivity
also has been localized to the CNS and peripheral tissue in
Aplysia (Elste et al. 1990
; Soinila et
al. 1990
). Four histaminergic neurons were detected in the
buccal ganglia and were identified as premotor sensory neurons
(Evans et al. 1999
). Because none of the identified
motor neurons that innervate I3 appear to use dopamine or histamine as
transmitters, we do not know why these substances evoke contractions. There are several possible explanations. 1) There could be
unidentified dopaminergic or histaminergic neurons that innervate
buccal muscle from other ganglia. For example, some motor neurons for
buccal muscles are located in the cerebral ganglia (Chiel et al.
1986
; Jahan-Parwar and Fredman 1983
).
2) Dopamine or histamine might have indirectly evoked
contractions by stimulating the release of the conventional
transmitters from the terminals of identified motor neurons to I3.
3) Dopamine or histamine could be released from modulatory
neurons that innervate the muscle or from neuroendocrine organs and act
as hormones. Bath application of SCP or serotonin, which are purely
modulatory transmitters, can evoke contractions of isolated I3 muscle
(Fox and Lloyd 1997
). Although dopamine and histamine do
not appear to be the fast transmitters used by the noncholinergic motor
neurons, our results suggest that they may be transmitters in this
system and may warrant further investigation.
Why are two fast excitatory transmitters used at buccal neuromuscular synapses?
Although the EJPs evoked by the cholinergic and the noncholinergic
motor neurons were quite variable among preparations, we observed no
consistent differences in the amplitude and kinetics of EJPs evoked
from either of the two neuronal groups. Of course it is possible that
the fast transmitter used by a motor neuron is a consequence of
developmental or evolutionary factors and not its function.
Alternatively, two fast excitatory transmitters might be important for
the optimization of behaviors or the transition between different
behaviors. Feeding is highly modulated. The sources of the modulation
include: peptides in the motor neurons, modulatory neurons that
innervate the muscle, and sensory feedback by proprioceptive neurons.
If either the cholinergic or noncholinergic synaptic transmission is
targeted selectively by a modulator, it could be important
behaviorally. For example, noncholinergic motor neuron B3 and
cholinergic motor neuron B9 functionally innervate the same fibers, but
FMRFamide potentiates B3-evoked EJPs and contractions while it inhibits
B9-evoked EJPs and contractions (Keating and Lloyd
1997). Selective effects of modulators could allow the animal
to optimize the feeding movements to the physical characteristics of
the food being ingested. It is important to emphasize that this
innervation may not represent anatomic synapses on the same muscle
fibers because the muscle fibers are coupled electrically.
Comparisons with other systems
Across phyla, ACh and glutamate appear to be by far the most
common, and perhaps the only, transmitters used at fast excitatory neuromuscular synapses (Gerschenfeld 1973;
Johnson and Stretton 1985
; Kehoe and Marder
1976
; Segerberg and Stretton 1993
). Of course,
the fast excitatory transmitter at vertebrate neuromuscular junctions
is ACh (Dale et al. 1936
). In crustaceans and insects, the predominant fast excitatory transmitter at neuromuscular synapses is very likely to be glutamate, although ACh appears to be used in a
limited number of cases (Johansen et al. 1989
;
Kawagoe et al. 1982
; Kravitz et al.
1970
; Marder 1976
; Shupliakov et
al. 1995
). Finally, at least in buccal motor neurons in
Aplysia, ACh and glutamate are the fast transmitters.
The evidence implicating glutamate as a transmitter in mollusks is
constantly growing. In the central ganglia, an excitatory amino acid
recently has been implicated as the fast excitatory transmitter used by
sensory neurons in Aplysia. One group concluded that
homocysteic acid was the likely transmitter (Trudeau and Castellucci 1993), whereas another group suggested it was
likely glutamate itself (Dale and Kandel 1993
). If our
conclusion is correct and the fibers containing glutamate-like
immunoreactivity in the cholinergic I5 muscle are indeed those of
sensory neurons, then our finding that glutamate but not homocysteate
blocked staining suggests that glutamate is the more likely transmitter
at least for buccal sensory neurons. The staining of the pedal-pleural ganglia sensory neurons with an antiserum directed against glutamate also supports this conclusion (Levenson et al. 1997
).
Glutamate also appears to be an important transmitter in molluscan
feeding systems. It has been identified as the fast transmitter for
retraction phase interneurons in Lymnaea
(Brierley et al. 1997
) and it is the fast transmitter
for the radula mechanosensory neuron B21 in Aplysia
(Klein et al. 1998
). However, the pharmacology of the synapses formed by these neurons is different from what we observed in
that the postsynaptic actions of glutamate are blocked by kainate/AMPA antagonists (i.e., CNQX or DNQX).
Buccal muscle in A. kurodai also is innervated by
excitatory noncholinergic motor neurons that have an unusual
pharmacology (Nagahama and Takata 1990). The
pharmacology of these neurons cannot be directly compared with the
glutamate receptors in the CNS of A. californica because
different antagonists were used in these studies. It is also difficult
to compare them to the buccal motor neurons in A.
californica. Although GDEE inhibited EJPs and contractions
evoked by stimulating A. kurodai noncholinergic neurons,
in A. californica it was not specific in that it
inhibited ACh-evoked contractions more effectively than
glutamate-evoked contractions.
A peripheral synapse between neuron B4 and salivary cells of
Helisoma has a similar pharmacological profile to what
we observed (Bahls et al. 1995). None of the classic
subtype-specific glutamate agonists or antagonists were effective in
either system. Indeed, the pharmacology of the two systems differ in
only two respects: D-glutamate is an antagonist in
Helisoma, whereas it was essentially without effect on
the Aplysia buccal muscle, and L-aspartate
was not an agonist in Helisoma, whereas it was the
second most effective glutamate-like agonist in Aplysia.
However, it is not clear if these differences in pharmacology indicate
that they use different glutamate receptors. It is possible that the
differences in the pharmacology are due to the fact that the salt
concentration in Aplysia blood and physiological
solutions is ~10-fold higher than in Helisoma blood.
In conclusion, our studies of peripheral modulation have been limited because the identity of the conventional transmitter used by some buccal motor neurons has been unknown. Here we have shown that the fast transmitter used by identified noncholinergic motor neurons (B3, B6, and B38) is an excitatory amino acid and very likely glutamate itself. This information should help us understand the mechanisms that underlie feeding in Aplysia by allowing us to continue to dissect physiologically the role of transmitters and cotransmitters at the synapses of identified motor neurons.
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ACKNOWLEDGMENTS |
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This work was supported by National Research Service Awards 1-F31-MH10656 to L. E. Fox and IBN-9728453 to P. E. Lloyd.
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FOOTNOTES |
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Address for reprint requests: P. E. Lloyd, Committee on Neurobiology, University of Chicago, 947 E. 58th St., Chicago, IL 60637.
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 6 April 1999; accepted in final form 24 May 1999.
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REFERENCES |
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