1Center for Neurobiology and Behavior, New York State Psychiatric Institute and College of Physicians and Surgeons of Columbia University, New York, New York 10032; 2Institute of Neurobiology and Department of Anatomy, University of Puerto Rico Medical Science Campus, San Juan, Puerto Rico 00901; and 3Department of Physiology and Biophysics and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, New York 10029
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ABSTRACT |
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Rosen, Steven C., Mark W. Miller, Colin G. Evans, Elizabeth C. Cropper, and Irving Kupfermann. Diverse Synaptic Connections Between Peptidergic Radula Mechanoafferent Neurons and Neurons in the Feeding System of Aplysia. J. Neurophysiol. 83: 1605-1620, 2000. The buccal ganglion of Aplysia contains a heterogeneous population of peptidergic, radula mechanoafferent (RM) neurons. To investigate their function, two of the larger RM cells (B21, B22) were identified by morphological and electrophysiological criteria. Both are low-threshold, rapidly adapting, mechanoafferent neurons that responded to touch of the radula, the structure that grasps food during ingestive and egestive feeding movements. Sensory responses of the cells consisted of spike bursts at frequencies of 8-35 Hz. Each cell was found to make chemical, electrical, or combined synapses with other sensory neurons, motor neurons and interneurons involved in radula closure and/or protraction-retraction movements of the odontophore. Motor neurons receiving input included the following: B8a/b, B15, and B16, which innervate muscles contributing to radula closing; and B82, a newly identified neuron that innervates the anterodorsal region of the I1/I3 muscles of the buccal mass. B21 and B22 can affect buccal motor programs by way of their connections to interneurons such as B19 and B64. Fast, chemical, excitatory postsynaptic potentials (EPSPs) produced by RM neurons, such as B21, exhibited strong, frequency-dependent facilitation, a form of homosynaptic plasticity. Firing B21 also produced a slow EPSP in B15 that increased the excitability of the cell. Thus a sensory neuron mediating a behavioral response may have modulatory effects. The data suggest multiple functions for RM neurons including 1) triggering of phase transitions in rhythmic motor programs, 2) adjusting the force of radula closure, 3) switching from biting to swallowing or swallowing to rejection, and 4) enhancing food-induced arousal.
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INTRODUCTION |
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Sensory neurons are not mere passive conduits of
afferent information. They also perform important computational
operations on that information. The role of the integrative functions
of sensory neurons can be advantageously studied in certain
invertebrates (e.g., Baccus 1998; Burrows and
Matheson 1994
; Chiel et al. 1990
; Mar and
Drapeau 1996
; Van Essen 1973
). In some cases
both the sensory cells, and the circuitry to which they connect, are
readily accessible for study. For example, in the feeding system of the marine mollusk Aplysia, some of the relevant sensory neurons
have large cell bodies that are located in the CNS (Chiel et al.
1986
; Miller et al. 1994
; Rosen et al.
1979
, 1982
; Weiss et al. 1986
), and there is considerable information about the identified neurons that
comprise the central circuitry that controls feeding responses (Hurwitz et al. 1997
; Hurwitz and Susswein
1996
; Plummer and Kirk 1990
; Rosen et al.
1991
; Susswein and Byrne 1988
; Teyke et
al. 1993
). One set of sensory neurons in the feeding system of
Aplysia, the radula mechanoafferent (RM) neurons, are
particularly interesting because they have been found to contain
neuroactive peptides. Furthermore, the activity and excitability of the
RM cells can be affected by excitatory and inhibitory synaptic inputs
(Miller et al. 1994
), and, in addition to responding to
external mechanical stimulation of the radula, the cells respond to
stretch and contraction of a subradula tissue (Cropper et al.
1996
). A broad aim of this, and a companion study (Rosen
et al. 2000
), was to investigate the integrative actions of
sensory neurons and the mechanisms by which sensory neurons interact
with pattern-generating elements and higher order neurons that generate behavior.
Initial attempts to analyze the function of RM sensory cells were
complicated by indications that the RM neuron population was
heterogeneous. The neurons differed greatly in size and in morphological characteristics. Some had a monopolar shape, whereas others had a bipolar appearance. Furthermore, many, but not all of the
cells, were found to be immunopositive for small cardioactive peptide
(SCP). In the present study, the two largest RM neurons were
investigated and shown to be distinctly identifiable based on
morphological and electrophysiological criteria. The cells, B21 and
B22, were both found to be immunopositive for SCP, but had different
types of synaptic interconnections to specific motor neurons,
interneurons, and other sensory cells. This paper focuses on the nature
of the diverse synaptic outputs and inputs of B21 and B22. We also
describe different forms of homosynaptic plasticity that occur in the
connections between these cells and certain identified follower
neurons. Elsewhere (Rosen et al. 2000), we report the
results of the study of a variety of sources of inputs that can produce
phase-specific heterosynaptic modulation of the outputs of a RM neuron
during ongoing feeding motor programs.
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METHODS |
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Subjects
The subjects were Aplysia californica weighing
300-450 g. They were maintained in 14°C, aerated, artificial
seawater (ASW). Two types of preparations were used: an odontophore
preparation and a dissected odontophore preparation. The odontophore is
the tongue-like structure arising from the floor of the cavity of the
buccal mass (feeding apparatus). It is capped by the radula, the
chitinous, rasp-like, grasping surface that is divided into two halves
by a medial longitudinal groove about which the halves fold and unfold
during closing and opening movements (Howells 1942).
Internally, the odontophore consists of a semirigid core made up of
continuations of the radula, the radula stalk, and the radula sac
(including the collistylar cap) to which attach the: I4 muscles; I5
accessory radula closer (ARC) muscles; and the I7-I10 complex of radula
opener muscles (Evans et al. 1996
). The side walls of
the odontophore consist of a spongy support tissue (the rotella) that
contains bolsters (fluid-filled channels) that contribute to the
strength, shape, and flexibility of the structure (Drushel et
al. 1997
; Eales 1921
).
The odontophore preparation, with innervation provided by the cerebral
and buccal ganglia, was used chiefly to determine the identity of the
RM neurons (Fig. 1A). The
general properties of RM neurons, including their receptive fields,
response characteristics, and synaptic connections to identified higher
order neurons were determined with this preparation. The preparation
included the structures comprising the core of the odontophore (see
above), the rotella, the muscles known to be involved in radula opening and closing, and some of the muscles involved in forward and backward rotations of the radula. A number of the larger muscles that lead into
the odontophore were cut to free it from the floor of the buccal
cavity. Particular care was taken to preserve innervation provided by
the buccal ganglion via the radula nerve. In selected experiments,
innervation provided by buccal nerves 2 and 3 was also preserved. The
nomenclature of the nerves follows previous convention (Gardner
1971). The cerebral-buccal connectives and cerebral ganglion
were retained in this preparation, while the remainder of the nervous
system was discarded.
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The dissected odontophore preparation was used in experiments in which
radula motor neurons needed to be identified, and for the anatomic and
physiological experiments on the subradula tissue (SRT), a thin, flat
muscle sheet (Cropper et al. 1996) that lies immediately
below the full extent of the chitinous radula. The SRT contains the
terminal fields and terminal specializations of the RM neurons
(Miller et al. 1994
). For the dissected odontophore preparation a partial cut was first made through the rotella muscles forming the anterior wall of the odontophore. Additional small cuts
through the dorsal part of the radula permitted the rotella halves to
be splayed open to visualize the I4 and I5 muscles (ARC) muscles (see
Fig. 1B and legend). In some experiments additional dissection isolated the SRT. The bottom of the radula sac (collistylar cap) was sectioned and removed. The subradula tissue covering the
interior surface of the radula stalk-radula sac complex was then
peeled off, starting from the anterior ventral region and moving into
the posterior dorsal region. Continued peeling of the tissue separated
it from the internal face of the radula proper. The insertions
(attachment sites) of the I4 and I5 muscles to the subradula tissue
were preserved, as were the muscles and their nerves. The subradula
tissue was separated from the edges of the rotella by cutting across
the borders of these tissues. The dissected tissues of the odontophore
and attached ganglia were pinned to the silicone elastomer (Sylgard)
floor of a recording chamber.
Electrophysiology
Neurons were impaled with double-barreled microelectrodes for
intracellular recording and stimulation. The electrodes were made of
thin-walled glass that contained 2 M potassium acetate. They were
beveled so that their impedances ranged from 10 to 15 M. To identify
cells and examine their morphologies, the potassium acetate in the
stimulating electrode was replaced by a 3% solution of
5(6)-carboxyfluorescein dye in 0.1 M potassium citrate, titrated to pH
8.0 with KOH (Rao et al. 1986
). The impedance of the
dye-containing electrode was 30-40 M
. In other experiments, aimed
at obtaining accurate measurements of the resting potentials of
identified cells, the electrodes were filled with 2 M potassium
chloride and beveled so that their impedances were again between 10 and 15 M
. Up to four simultaneous intracellular recordings were obtained using conventional electrometers. The recording chamber (Lucite) was
divided into two compartments that were interconnected by several
petroleum jelly (Vaseline)-filled notches, allowing for the passage of
nerves or connectives and selective infusions of ASW solutions
containing controlled concentrations of relevant ions (e.g.,
K+, Mg2+, and
Ca2+) and chemostimulants to the central ganglia
or the peripheral tissues. Unless otherwise indicated, all multiple
cell recordings were from cells located in the same buccal hemiganglion.
Mechanical and chemical stimulation
Tactile stimuli were provided manually by a fire-polished
Pasteur pipette or a set of flexible von Frey hairs consisting of plastic fibers that were heated and pulled to various diameters. The
"hairs" were calibrated for delivery of punctate stimuli with forces ranging between 0.1 and 10 g. Automatically controlled stimuli were provided by a custom made tapper consisting of a wooden
rod, 1 mm diam, attached to the membrane of a minispeaker that was
driven by a Grass S4 stimulator (Cropper et al. 1996). Chemical stimuli consisted of a piece of moistened, commercial dried
seaweed (Laver, Roland Foods), presented by a hand-held forceps, or
graded concentrations of seaweed extract gently perfused over the
radula (Susswein et al. 1976
). Other chemical stimuli consisted of 4 M NaCl and solutions of amino acids found in seaweed, e.g., glutamic acid (Jahan-Parwar 1972
).
Morphology
Neurons were filled with 5(6)-carboxyfluorescein dye (Kodak) by
iontophoretic ejection from microelectrodes. To reduce the active
transport of the dye out of the cells (Steinberg et al. 1987), probenecid (10 mM final concentration) was added to the ASW bathing medium, and the preparation was kept for 24-48 h at 4°C
(Rosen et al. 1991
). The unfixed tissues were viewed
with a Nikon fluorescence microscope. Confirmation of cell morphology was made with Lucifer yellow ejection, followed by fixation in paraformaldehyde, and clearing in methyl salicylate.
Immunocytochemistry
Previously described methods for indirect immunofluorescence,
whole-mount mapping of Aplysia ganglia were followed
(Longley and Longley 1986; Miller et al.
1991
). In brief, individual neurons were identified by
electrophysiological criteria and/or their responses to mechanosensory
stimuli. Cells were filled with Lucifer yellow (4%) by iontophoretic
ejection from microelectrodes (
5 to
10 nA; pulses 0.5 s on,
0.5 s off; 30 min), and ganglia were fixed for 2-4 h (room
temperature) in 4% paraformaldehyde. Nonspecific antibody binding was
blocked by preincubating tissues in a phosphate buffer solution
containing 0.8% normal goat serum (NGS; Miles Sci., Naperville, IL)
and 2% Triton X-100 for 2 h at room temperature. Primary rabbit
antiserum against small cardioactive peptide B (SCPB) (provided by A. Mahon) (see Lloyd et
al. 1985
) was then applied (1:50 to 1:200). After incubation
and rinsing (1-2 days each), secondary antibody (goat anti-rabbit
immunoglobulin G rhodamine conjugated Fab fragment; Cappel, Malvern,
PA) was applied at a 1:50 or 1:100 dilution. After incubation and
washing, tissues were mounted on depression slides in phosphate buffer:
glycerol (1:6), viewed under a Leitz microscope equipped with
epifluorescence (filterpack N-2 for rhodamine, D for Lucifer yellow),
and photographed with Tri-X film.
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RESULTS |
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SCP-containing radula mechanoafferent neurons can be identified as individuals
The cluster of SCP-containing RM neurons, located on the rostral
surface of each buccal hemiganglion, was found to consist of a
heterogeneous population of ~40 cells of varying position, size,
shape, and axon distribution pattern (Miller et al.
1994). Typically the SCP-positive cluster of cells contained
three or four neurons that had a distinctive bipolar morphology,
whereas the remainder were monopolar or multipolar cells. Other nearby sensory neurons were found to have similar response properties, but
were not SCP-immunoreactive. To expedite analysis, unique individuals
were sought that consistently showed SCP immunoreactivity and that
could also be unambiguously identified by morphological and
electrophysiological criteria (Fig. 2).
We found that it was possible to identify the two largest, bipolar,
SCP-containing RM neurons, that are found at the medial aspect of the
rostral RM cluster. In each of four preparations, in which the cells
were filled with Lucifer yellow dye, the identified cells, B21 and B22,
tested positive for SCP immunoreactivity (Fig.
3). In 48 of 50 preparations only a
single neuron with the distinctive properties of B21 was found. B22 on
the other hand shared a number of properties with two to three other
bipolar, although smaller, SCP-immunoreactive cells.
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Although B21 and B22 share a number of common electrophysiological
properties (e.g., resting potential, spike amplitude, spike duration,
sensory threshold, and receptive fields), the cells differed in
morphological appearance, axon distribution, and pattern of synaptic
connectivity. A summary of some of their similarities and differences
is shown in Table l. As detailed below,
key differences used to routinely distinguish the cells include the
findings that B21 receives a conventional, monosynaptic inhibitory
postsynaptic potential (IPSP) from identified neurons B4 and B5. B4 and
B5 are virtually identical cells that are located next to one another, and have combined sensory (Jahan-Parwar et al. 1983),
motor (Evans et al. 1996
; Rosen et al.
1982
), and interneuronal functions. By convention we refer to
the more medially located cell as B4, but either cell individually can
be termed B4/5. B22 does not receive an input from B4/5. Furthermore,
B21 produces a slow excitatory postsynaptic potential (EPSP) in neuron
B15, but B22 does not. B22 is invariably electrically coupled to B4/5
and B16, but B21 is not. Routine dye-fills showed consistent
morphological differences correlated with the physiological findings.
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Morphological properties of B21 and B22
B21 is the largest cell (long dimension, 80-120 µm diam in 400-g animals, n = 16) within the rostral cluster of SCP-containing RM neurons (Figs. 2 and 3). It is usually found at the ventromedial edge of the cluster. B21 is a bipolar cell with a rounded soma that projects a lateral process into the ipsilateral buccal hemiganglion and a thick (10-20 µm diam) medial process into the contralateral buccal hemiganglion (Fig. 4, top). In the buccal commissure the medial axon bifurcates and sends another main branch into the radula nerve. The radula axonal branch itself divides at the first major bifurcation of the radula nerve, thereby innervating both the left and right sides of the radula. Both the lateral and medial B21 axons periodically give rise to tufts of short processes that terminate in the neuropil of the buccal ganglion.
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B22 is an oblong shaped, bipolar cell, that is considerably smaller than B21 (Fig. 3B). Its soma size measured along its long dimension is 60-90 µm. It is found adjacent to B21 in a more dorsal position. Like B21, B22 has a lateral and a medial main axon. The medial axon sends a branch into the radula nerve that innervates both the left and right sides of the radula. The main axons give rise to long filamentous processes that extensively ramify in the neuropil of the ipsilateral buccal hemiganglion (Fig. 4, bottom).
Fields of terminal specializations
When either a B21 or a B22 neuron was filled with
5(6)-carboxyflourescein and incubated with probenicid for 72 h
(n = 6), it was possible to follow the peripheral axons
of the cells in the main branches of the radula nerve (Fig.
5A) to the radula surface.
Each axon entered a membranous sheath (the SRT) that lies just below
the chitinous radula. The subradula tissue adheres to the matrix of
support material that holds the teeth of the radula. Along the surface
of the SRT, which is apposed to the support material (external
surface), the finest axonal branches of the RM neurons terminated in
varicose specializations (Fig. 5, B and C). The
opposite (internal) surface of the SRT is the site of attachment of the
buccal muscles of the odontophore implicated in control of radula
closure, in particular the I4 muscle leaflets (Eales
1921; Howells 1942
; Scott et al.
1991
), and I5 (accessory radula closer, ARC) muscle
(Cohen et al. 1978
). No varicosities of B21 or B22
processes were observed along the internal surface of the SRT.
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Receptive fields
B21 and B22 were found to have bilateral mechanosensory receptive fields that generally coincided with their fields of terminal specializations that lie just below the radula surface. The receptive fields were confined chiefly to the chitinous radula surface and to the posterior surface of the odontophore immediately adjacent to the radula (Fig. 6, left). The fields generally included invaginations of the radula that form the median groove and the posterior (trailing) edges of the radula halves that contact food that is grasped by the radula. The sensitivity to tactile stimuli within the receptive field was variable. Loci of maximal sensitivity were found along, and within, the median groove and also along the trailing edge of the radula. The receptive fields of B21 and B22 were mapped in 14 preparations in which both cells were found. The overall shape and size of the fields of individual B21 and B22 neurons were similar, although B21 neurons typically exhibited larger areas of maximal sensitivity.
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Response properties
B21 and B22 are low-threshold, rapidly adapting,
mechanoafferent neurons that respond vigorously to punctate pressure
stimuli applied to the surface of the radula (Fig. 6, right
arrows). The responses recorded from each cell's soma consisted
of bursts of A-spikes (axon spikes, which reflect action potentials
that fail to invade the soma) having instantaneous spike rates ranging
between 8 and 35 Hz. The number of evoked spikes and the spike
frequency of the response bursts generally increased with the force of
the stimulus, but repeated applications of the same stimulus often evoked variable responses. The cells exhibited sensory adaptation when
the stimulus was maintained for 1 s (Fig. 6, right horizontal lines). There was never an "off-response" when maintained
stimuli of 1 s were terminated (n = 100; 45 preparations). Moving stimuli, pulling stimuli, or mechanical stimuli
that exerted shearing forces on the radula were ineffective in
eliciting sensory responses, as were a variety of chemical stimuli
including seaweed extracts and 4 M NaCl (Miller et al.
1994
). Sensory responses of B21 could also be observed in the
dissected odontophore preparation when mechanical stimuli were applied
directly to the SRT that was stripped from the radula support matrix.
Under these conditions, recordings from the somata of the cells
revealed full spikes, as well as the smaller potentials that appeared
to be blocked spikes (A-spikes) (Weiss et al. 1986
; see
Van Essen 1973
). To study this in greater detail we used
an electromechanical device to present brief, reproducible, suprathreshold mechanical stimuli. The stimuli were presented at 6 Hz,
which is below the rate at which the cells typically discharge in
response to a maintained stimulus. At this low rate, the stimuli evoked
two types of A-spikes: small A-spikes with an amplitude of <10 mV or
large A-spikes with an amplitude in excess of 20 mV (Fig.
7A). Moreover, when a
succession of large A-spikes was evoked in B21, they invariably showed
a further gradual increase in amplitude. In contrast to what is seen
when full spikes are evoked by a maintained stimulus, the A-spikes
failed to evoke discernible EPSPs in postsynaptic (follower) cells such
as motor neuron B8a. When, however, RM neuron B21 was depolarized 20 mV from its resting membrane potential by injection of intracellular current, all the threshold mechanical stimuli applied at the periphery evoked full soma spikes (evident by the high spike amplitudes, 8-10 mV
larger than large A-spikes, and the presence of hyperpolarizing afterpotentials), and EPSPs were recorded from follower neuron B8a.
Axon spikes rapidly returned when the current injection was terminated.
When the strength of the brief mechanical stimulus was increased well
above threshold (Fig. 7B), the repeated stimuli at 6 Hz had
an increased likelihood of evoking the large-type A-spike in B21, but
the A-spikes still failed to evoke postsynaptic responses in B8a. These
results suggest that action potentials generated in the periphery of
the RM neurons may be vulnerable to branch block at peripheral, as well
as at central branch points of the processes of the RM neurons.
Moreover, the membrane potential at, or near, the soma may be important
in regulating the blockade. The mechanism by which strong or maintained
stimuli promote the generation of large-amplitude spikes is not known
but may involve enhanced depolarizations at branch-block points, due to
summation of depolarizing afterpotentials or electrical interactions
(see SENSORY NEURONS) between mechanosensory
neurons with overlapping receptive fields. In addition to responses
evoked by peripheral mechanical stimuli contacting the radula surface,
the cells are known to fire in response to contractions of the
subradula tissue (Cropper et al. 1996
).
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Synaptic connectivity
In addition to their activation by mechanostimulation of the radula, B21 and B22 also receive synaptic input from at least four types of neuronal sources (Fig. 20 is a summary diagram of the neurons found interconnected with B21 and B22). Many of the connections of B21 and B22 consist of nonrectifying electrical synapses. Depending on the context, these connections can be considered as either input or output pathways.
SENSORY NEURONS.
One type of synaptic interconnection to B21 and B22 includes electrical
connections with other SCP-immunoreactive RM neurons. As described in a
previous report (Miller et al. 1994), SCP-immunoreactive neurons are part of an electrically coupled network of sensory cells.
Neurons B21 and B22 were found to make strong electrical synaptic
connections to each other (Fig. 8,
A and B), and to their homologues in the
contralateral buccal hemiganglion (Fig. 8, A-C). Moreover,
many other monopolar and bipolar neurons comprising the
SCP-immunoreactive RM cell clusters make weak electrical synaptic connections to B21 and B22. For the majority of cases of pairs of cells
tested (14 of 25 or 56%), the electrical coupling ratio was similar in
both directions of current flow.
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INTERNEURONS.
A second source of synaptic interconnection to identified neurons B21
and B22 is the bilateral pair of interganglionic interneurons, B19s.
Each B19 sends its main axon from one buccal hemiganglion to the
cerebral ganglion via the ipsilateral cerebral-buccal connective (Fig.
2A). B19 provides bilateral chemical synaptic input to
cerebral-to-buccal interneurons (e.g., CBI-2), and to bilateral groups
of cerebral motor neurons that control the lips and the extrinsic
muscles of the buccal mass (Chiel et al. 1986;
Rosen et al. 1991
). Each B19 makes a strong electrical
synaptic connection to RM neuron B21 in the ipsilateral buccal
hemiganglion (Fig. 8, C and D). Injection of
hyperpolarizing or depolarizing current pulses into the soma of a B19
neuron produced hyperpolarizing or depolarizing electrotonic potentials
in the ipsilateral and contralateral B21 and B22 neurons, respectively
(Fig. 8, A-D). The responses in the ipsilateral B21 and B22
cells were always greater than those in the contralateral cells
(n = 20 pairs).
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MULTIFUNCTION NEURONS. Another source of synaptic input to B21 and B22, and the only known source of inhibitory chemical synaptic input to one of the RM neurons, is provided by multifunction neurons B4 and B5. B4 and B5 make inhibitory, chemical, synaptic connections to B21, but not B22 (Fig. 10). Repetitive spikes elicited in these cells produce nondecrementing, fast IPSPs in RM neuron B21 (Fig. 10, A1, B1, and B4) and small, depolarizing electrotonic potentials in neuron B22 (Fig. 10A). Indicative of monosynaptic connections, the synaptic potentials follow the presynaptic spikes, one for one, with constant delay, even at high frequencies of firing of B4 or B5. In addition, the IPSPs in B21 were eliminated in ASW solutions containing a high concentration of Mg2+ and a low concentration of Ca2+ (Fig. 10B2) but were not blocked by raising the concentration of Mg2+ and Ca2+, which would increase the threshold of interposed interneurons (Fig. 10B3).
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MOTOR NEURONS.
B21 and B22 were found to have synaptic interconnections with many
motor neurons found in the ventral motor neuron cluster of each buccal
hemiganglion. The investigation focused on those identified motor
neurons that innervated muscles of the odontophore, with the exception
of a cell that was an example of a motor neuron with a strong
electrical connection to the RM sensory neurons. The motor neurons on
the ventral surface that receive RM connections appear to be involved
in one or another aspect of the radula closure phase of buccal motor
programs. One pair of motor neurons consists of previously identified
neurons B8a and B8b (Church and Lloyd 1991;
Gardner 1977
; Morton and Chiel 1993
) that
innervate portions of the I4 muscle. A second pair consists of motor
neurons B15 and B16 that innervate the I5 or ARC muscle (Cohen
et al. 1978
). We have identified a third type of cell that
appears to be involved in radula closure. This cell we provisionally
identify as B82. B82 is a motor neuron that innervates the anterodorsal
I1/I3 buccal muscles that are located over the jaws. Elsewhere
(Rosen et al. 2000
) we show that during a buccal motor
program driven by the firing of CBI-2, B82 fires in a phase similar to
that of radula closer motor neurons such as B16 and B8a/b. The type of
synaptic potential that each B21 and B22 evoked in each of their
follower motor cells was different (see summary in Fig. 20). Some of
the synapses are electrical, others are chemical, and some are mixed electrical-chemical. As described in detail in the following sections, some of the sensory cell connections exhibit very marked homosynaptic plasticity at particular identified motor neurons.
RADULA CLOSURE MOTOR NEURONS B8A AND B8B.
Two motor neurons with axons in the radula nerve have been implicated
in the control of radula closure (Church and Lloyd 1994; Morton and Chiel 1993
). Consistent with earlier
descriptions, we refer to the cells as B8 neurons, namely B8a and B8b.
B8a is the most lateral, and B8b the next most lateral, ventral cluster motor neuron that receives an IPSP from multifunction neuron B4/5 (Fig.
10B). In our experiments, the sister cell B8b was found in the vicinity of B8a in 30% of 12 preparations examined. We have found
that B8a innervates portions of the leaflets of the I4 muscle, that
insert into the ventral regions of the odontophore and attach to the
SRT in the vicinity of the grasping surface of the radula. B8a/b was
identified by observing that it produced closure of the radula in the
odontophore preparation. B8a/b was also identified by the
characteristic EPSP it receives from identified RM neuron B21 (Figs.
11
13). Both B8a and B8b receive a
nondecrementing EPSP from neuron B21, which exhibits marked synaptic
facilitation and summation (Figs. 11-13), such that brief bursts of
B21 spikes, within normal physiological rates, are sufficient to
trigger action potentials in the B8a and B8b motor neurons. The
synaptic facilitation is most clearly evident when B21 is fired between
8 and 12 Hz (Fig. 12). An individual
B21 neuron is capable of producing facilitating EPSPs in both the
ipsilateral and contralateral B8a motor neurons, although the response
of the ipsilateral follower cell is consistently larger than that of
the contralateral follower. The latter finding was evident when
simultaneous recordings were made from the left and right B21 neurons,
as well as from the left and right B8a motor neurons (Fig.
13, A and B). The
result is consistent with the morphological findings indicating that
the medial axon of each B21 neuron projects to the contralateral buccal
hemiganglion and that the number and density of putative terminal
branches of the B21 cells are greater in the region closest to the soma of B21 (Fig. 4).
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ARC MOTOR NEURONS B15 AND B16.
B15 and B16 are the two motor neurons that innervate the ARC (or I5)
muscle (Brezina et al. 1994; Cohen et al.
1978
; Probst et al. 1994
; Vilim et al.
1996
, Weiss et al. 1992
; Whim and Lloyd 1990
). When a single B21 RM neuron was intracellularly
stimulated so that it fired a burst of action potentials, one for one
EPSPs were evoked in neuron B16 (Fig.
14, A and C). B15
exhibited a multiphasic EPSP consisting of rapid potentials that arose
on a slowly rising tonic depolarization that slowly decayed when the
firing of B21 was terminated (Fig. 14A). Responses in B15
and B16 were observed in the absence of any responses in B4 (Fig.
14A), suggesting that B21 was probably not evoking a buccal
motor program that, in turn, evoked synaptic inputs to B15 and B16. To
determine which, if any, of the evoked potentials was due to chemical
synaptic transmission, the preparations were bathed in a high
Mg2+, low Ca2+ ASW
solution. When B21 was fired under these conditions, the synaptic
potentials in B16 were completely eliminated (Fig. 14B). The
gradually increasing slow synaptic potential in B15 was eliminated, but
the cell still exhibited fast potentials and a tonic depolarization, reflecting electrical coupling. The existence of electrical coupling was supported by injecting hyperpolarizing current pulses into B21 and
B22, which resulted in hyperpolarizing electrotonic potentials in
neuron B15 (see Fig. 15, C
and D). Using the above protocol, B22 was found to produce a
weak electrical EPSP in neuron B16. B22 was also found to be
electrically coupled to neurons B4 (Fig. 10A), B15 (Fig. 15,
B and D) and B19 (Fig. 8, B and
D).
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B82 NEURONS.
Motor neuron B82 is one of a pair of bilateral, medium-large neurons
found among cells comprising the ventral cluster of motor neurons in
the buccal ganglion (see Fig. 2F of Rosen et al.
2000). It is accessible from the rostral surface and is found
near the lateral edge of the cluster in the vicinity of B8a and B8b.
B82 sends an axon out ipsilateral buccal nerve 2, and its firing
produces contraction of middorsal I1 muscles of the buccal mass. The
contraction results in a shortening of the muscles above the jaws and
in a forward movement of the pharyngeal tissue comprising the dorsal wall of the buccal cavity. Buccal nerve 2 trifurcates as it enters the
buccal mass. The B82 axon passes in the dorsal branch of the trifurcation (termed BNc) (Warman and Chiel 1995
). B82
does not receive any input from B4, but it does receive substantial
input from unidentified interneurons, which cause it to fire
rhythmically during buccal motor programs. B21 makes a nonrectifying
electrical synapse with B82 (Fig. 16)
as does B22. A neuron, previously identified as B45 produces jaw
shortening (Church and Lloyd 1991
, 1994
)
and is in a similar position to B82. B45, however, has a relatively small cell body (P. J. Church, personal communication) and sends its axon into buccal nerve 3 rather than 2.
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Homosynaptic neuromodulation of the synaptic connections of B21
Two of the chemical synaptic connections of B21 show intrinsic
(homosynaptic) modulation, that results in larger EPSPs
(facilitation) when B21 is fired at increasing rates of firing. As
shown in Fig. 12, when B21 was stimulated with repetitive intracellular
current pulses so that it fired at 4 Hz (Fig. 12A), fast
electrotonic potentials were evoked simultaneously in neurons B15 and
B82 (2 cells that make electrical synapses with B21), but not in B8a (a
cell that receives chemical synaptic input from B21). Firing B21 at 6 Hz (Fig. 12B) produced small, chemical EPSPs in B8a and
electrotonic potentials again in B15 and B82. However, when B21 was
fired at 8 Hz (Fig. 12C), it produced strongly facilitating
EPSPs in B8a and a slowly rising EPSP that accompanied the one for one
electrotonic potentials evoked in B15. Only electrotonic potentials
were seen in B82. Firing B21 at 10 Hz (within its normal frequency
range) produced facilitating EPSPs in B8a that exceeded B8a's spike
threshold and also produced a marked slow EPSP in neuron B15 that was
particularly evident during the latter part of the EPSP and during the
decay phase (Fig. 12D). Elsewhere we present evidence that
the transmitter release from B21 is dependent on its baseline membrane
potential (Rosen et al. 2000).
Although the chemical synaptic connection of B21 to B8a can exhibit very marked facilitation, the duration of the facilitation appeared to be remarkably brief, persisting for <1 s (Fig. 17) as studied in three preparations.
|
B21 activity affects the excitability of ARC motor neuron B15
Previous work has shown that the excitability of B15 is increased
following exogenous applications of the peptide SCP, which modulates
several ionic currents and elevates levels of second messengers in the
cell, including cAMP (Sossin et al. 1987; Taussig et al. 1989
). Because B21 contains SCP, we tested whether the slow EPSP produced by B21 in neuron B15 might be associated with an
alteration of the excitability of B15. B15 was repeatedly depolarized by intracellular current pulses of fixed duration and intensity so that
a constant number of action potentials were elicited. Firing of B21
resulted in an increase in the number of B15 spikes that were elicited
by the current pulses (Fig. 18,
A and C). The effect was strongest when current
pulses in B15 were presented during the tail of the evoked slow EPSP in
B15 (Fig. 18A). At least part of the effect may be due to
membrane depolarization of B15 because depolarization of B15 alone can
increase its excitability (Fig. 18B). Nevertheless, even
when stimulation of B21 did not produce any obvious long-lasting
membrane depolarization in the B15, a small excitability increase in
the cell could still be observed (Fig. 18C).
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Outputs to pattern-generating neurons
In a previous section, B21 was shown to make a nonrectifying
electrical synapse with interganglionic interneuron B19 (Fig. 8). The
synapse allows for the possibility of bidirectional communication between the cells. In a companion paper we show that activity in B19
can affect the chemical synaptic output of B21 to motor neurons B8a and
B15 (Rosen et al. 2000). B19 is a powerful regulator of
the biting command-like interneuron CBI-2 (Rosen et al.
1991
) and produces a complex inhibitory-excitatory monosynaptic
EPSP that contributes to the timing of the firing of CBI-2, and thereby can contribute to pattern generation. Because B21 affects B19, we
predicted that firing of B21 might have an indirect effect on CBI-2 and
consequently might have an effect on pattern generation. When CBI-2 was
intracellularly stimulated with a maintained, constant current pulse,
such that the rate of the spiking that was elicited was insufficient to
drive a motor program, the additional firing of a burst of B21 spikes
evoked several cycles of a motor program (Fig.
19). The effect was seen in four of
four preparations that were tested and suggests the B21 might provide
important input to the feeding pattern generating circuitry.
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DISCUSSION |
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Identification of unique SCP-containing RM neurons in the buccal ganglion
This study establishes the identity of two neurons (B21 and B22)
that are constituents of the cluster of SCP-containing RM neurons found
on the rostral surface of each buccal hemiganglion (Lloyd et al.
1985; Miller et al. 1994
). It appears as if
there is only one, or at most two, B21 neurons in each hemiganglion. However, B22 shares some properties with other smaller, bipolar, SCP-immunoreactive cells.
Comparisons with other mechanoafferents in Aplysia
B21 and B22 exhibit rapidly adapting responses to mechanical
stimuli that are similar to other types of mechanoreceptor neurons that
have been studied in the cerebral (Chiel et al. 1990;
Rosen et al. 1979
, 1982
; Weiss et
al. 1986
), abdominal (Byrne et al. 1974
;
Dubuc and Castellucci 1991
), and pleural ganglia
(Walters et al. 1983
) of Aplysia. Two types
contain a peptide that may function as a cotransmitter. One contains
the neuropeptide sensorin A (Brunet et al. 1991
;
Gapon and Kupfermann 1996
), whereas RM neurons contain
the neuropeptide SCP and do not appear to contain sensorin A
(Miller et al. 1994
).
The two types of peptidergic mechanoafferent cells differ in several
respects. Elsewhere we provide evidence that diverse synaptic inputs
received by some RM cells (Rosen et al. 2000) provide
rapid modulation of the magnitude of the synaptic outputs of the cells.
The output of the sensorin mechanoafferents also can be modulated by
heterosynaptic input, but this modulation occurs over a relatively long
time course and does not appear to involve fast, conventional synaptic
inputs (Byrne and Kandel 1996
; Rosen et al.
1989
). Furthermore, all the known synaptic outputs of the RM
cells are nondecrementing or facilitating, whereas the sensorin sensory
cells all produce decrementing synaptic potentials, although
high-frequency firing of these cells can result in posttetanic potentiation (Walters and Byrne 1984
). The excitatory
chemical synapses between B21 and motor neurons B8a and B8b exhibit a
remarkably prominent synaptic facilitation (homosynaptic modulation)
that strongly depends on the frequency of firing of B21, but exhibits little posttetanic potentiation. The facilitation might provide a
mechanism by which very weak, or short-duration, sensory information may be prevented from having an effect on the CNS. The short duration of posttetanic potentiation may function to diminish cycle to cycle
variation of the synaptic potential.
Possible functions of B21 and other RM neurons
The RM peripheral endings terminate in the subradula tissue
(Miller et al. 1994) that underlies the tooth-lined
chitinous radula, the structure that grasps and releases food during
feeding movements. The RM receptive fields are located chiefly on the grasping surfaces of the radula. The relatively low firing thresholds for tactile stimuli contacting the radula make RM neurons suitable for
detecting the position or movement of the radula halves and/or detecting the presence of food or other objects contacting the radula.
During feeding, the grasping surfaces of the radula open and close, and
in addition, they protract and retract between the jaws and the
esophagus. Mechanosensory information arising from the strategically
located receptive fields of B21 and B22 can be conveyed
monosynaptically to various identified motor neurons and interneurons
that control the muscles producing radula closure (Fig.
20). The motor neurons include B8a and
B8b, which control the leaflets of the I4 muscle; B15 and B16, which
control the I5 (ARC) muscle; and B82, which controls dorsal muscles of
the buccal mass that act conjointly with radula movements to move food
between the jaws and the esophagus. These muscles are involved in both
ingestive behaviors (e.g., biting, swallowing), and egestive behaviors
(rejection), which can be elicited by nonfood stimuli contacting the
radula.
|
The synaptic connections and sensory responsiveness of B21 and B22
suggest involvement in one or more sensory functions that occur during
either ingestion or egestion. These possible functions include
1) initiation of phase transitions during rhythmic
behaviors, such as between radula opening and radula closing;
2) adjustment of the force or timing of muscle contractions
so movements of structures, such as the radula, are appropriate for the
particular food that is being grasped; 3) behavioral
switching, e.g., from biting to swallowing, or swallowing to rejection;
and 4) regulation of behavioral warm-up and other
manifestations of food-induced arousal. The cells could perform these
functions by means of the various types of synaptic connections they
make to their follower neurons. The cells may perform different roles
as a function of the state of the CPG or of the pattern or rate of the
firing of the sensory neurons, as has been shown for the AGR
mechanosensory neuron in the lobster stomatogastric system
(Combes et al. 1999).
The suggestion that B21 might be involved in the phase transition
between protraction and retraction is supported by the finding that B21
excites interneuron B64. B64 appears to be active in ingestive programs
during the phase of radula closure, and previous evidence suggests that
it contributes importantly to the phase transition between radula
opening/protraction and radula closing/retraction by, among other
actions, inhibiting radula opener motor neurons (Evans et al.
1996) as well as protractor motor neurons (Hurwitz and
Susswein 1996
). The idea that B21 is involved in adjustments of
the force of muscle contractions is supported by the observation that
B21 directly excites motor neurons. A role in behavioral switching from
biting to swallowing is supported by the observation that B21 excites
radula closer motor neurons, in particular B15, which is highly active
during swallowing while the radula retracts. ARC muscle activity and
the firing rate of B15 strongly increase when food is grasped during a
bite, and the motor program switches from bite to swallow
(Cropper et al. 1990
). The capacity of B21 to enhance
the excitability of B15 might contribute to behavioral arousal and
increased firing of B15 during swallowing when food stimuli contact the
radula surface. Indeed the slow excitatory potential that B21 evokes in
B15 might be mediated by the release of the peptide SCP, that is
present in the mechanoafferent neurons. Previous data indicate that SCP
can evoke modulatory actions in B15, mediated by the activation of
second messengers (Sossin et al. 1987
; Taussig et
al. 1989
). Thus a neuropeptide such as SCP, which has been
shown to modulate the properties of muscles (Weiss et al.
1993
), may also modulate the motor neurons controlling the
muscles. The RM sensory neurons may be an example of the growing number
of neural elements that are intrinsic to the sensorimotor pattern-generating circuitry, but that can exert modulatory effects (Cropper et al. 1987
; Katz and Frost
1996
; Katz and Harris-Warrick 1991
).
In addition to connections to motor neurons, radula mechanoafferents excite interneurons, including buccal-to-cerebral-interneuron B19 and buccal interneuron B64. These interneurons might contribute to behavioral switching and phase transitions within a program. The activity of these interneurons could also promote behavioral warm-up and other features of behavioral arousal. B21 activity could potentiate feeding motor programs and muscle contractions via its connections to B19, which provides complex synaptic input to cerebral-to-buccal interneurons such as CBI-2, that are capable of driving ingestive motor programs.
A surprising finding of this study is that RM neurons receive synaptic
input from one or more representatives of every major class of
intrinsic neuron that is involved in the generation of feeding motor
patterns. These classes include motor neurons, pattern-generating interneurons, and other sensory cells. Although the connections are
extensive, they are not indiscriminate. Some connections are inhibitory, but many are excitatory and due to electrical coupling. As
shown elsewhere (Rosen et al. 2000), the various
synaptic inputs of the intrinsic feeding circuitry can modulate the
synaptic outputs of the RM cells and are thus likely to have functional
significance. Thus the RM neurons exhibit short-duration forms of both
homosynaptic plasticity (e.g., facilitation) and heterosynaptic
plasticity (Kandel 1976
), and these cells provide an
example of an intrinsic neuromodulatory element that is itself subject
to intrinsic sources of neuroplasticity.
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ACKNOWLEDGMENTS |
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This study was supported by National Institutes of Health Grants MH-51393, GM-32099, MH-50235, MH-35564, and K05 MH-01591 and National Science Foundation Award IBN-9722349.
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FOOTNOTES |
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Address for reprint requests: S. C. Rosen, Center for Neurobiology and Behavior, 1051 Riverside Dr., Box 87, New York, NY 10032.
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 14 June 1999; accepted in final form 15 November 1999.
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REFERENCES |
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