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INTRODUCTION |
A striking feature of behavior is its plasticity. Even relatively simple motor acts are often continuously adjusted to accommodate changes in the external environment. To characterize cellular mechanisms that are likely to mediate this type of plasticity, interactions between sensory neurons and central pattern generators (CPGs) have been studied both in vertebrate and invertebrate preparations (e.g., Burrows 1997
; Buschges 1995
; Clarac and Cattaert 1996
; Laurent 1991
; Pearson and Ramirez 1997
; Sillar 1991
; Wallen 1997
). From this work, it has become apparent that sensory input can play an important role in determining the characteristics of physiologically relevant motor programs. An important aspect of sensory control of rhythmic movement, however, is that in many systems sensory-induced changes in rhythmic activity must occur in a manner that is appropriate for the ongoing behavior. In these cases therefore afferent-CPG interactions are often not just unidirectional transfers of information. Instead, as afferent activity can influence CPG activity, CPG activity can often in turn regulate afferent activity (e.g., El Manira et al. 1991a
; Sillar and Skorupski 1986
; Skorupski and Sillar 1986
; Vinay et al. 1996
; Wolf and Burrows 1995
). Although progress has been made in characterizing mechanisms by which CPG activity can modify afferent transmission, the range of these mechanisms appears to be extensive (e.g., Sillar 1991
) and is just beginning to be appreciated (Pearson and Ramirez 1997
).
In recent work we have begun to address this issue within the context of feeding in the marine mollusk Aplysia. One potential source of modulation of sensory neurons are the serotonergic metacerebral cells (MCCs). The MCCs (or homologous neurons) have been well characterized in mollusks (e.g., Berry and Pentreath 1976
; Cottrell and Macon 1974
; Gerschenfeld and Paupardin-Tritsch 1974
; Gerschenfeld et al. 1978
; Weinreich et al. 1973
) and have been extensively studied within the context of feeding (e.g., Gillette and Davis 1977
; Granzow and Kater 1977
; McCrohan and Audesirk 1987
; Weiss and Kupfermann 1976
; Weiss et al. 1982
; Yeoman et al. 1994a
,b
, 1996
). These cells exert widespread actions that are manifested both centrally and peripherally, i.e., on the feeding circuitry and on feeding musculature. Peripheral effects of serotonin (5-HT) or MCC activity have been extensively investigated in Aplysia in a few model neuromuscular systems (e.g., Fox and Lloyd 1997
; Lotshaw and Lloyd 1990
; Scott et al. 1997
; Weiss et al. 1978
). Although central effects of the homologous CGCs have recently begun to be investigated in a related mollusk, Lymnaea (Yeoman et al. 1996
), central effects of MCC activity in Aplysia have not been well characterized (although see Weiss et al. 1978
). Sensory neurons with central or peripheral somata that are modulated by MCC activity have not been identified.
The sensory neuron in this study is the SCP-containing radula mechanoafferent B21 (Miller et al. 1994
; Rosen et al. 1992
). This neuron is of interest because B21 and other radula mechanoafferents appear to play an important role in ingestive feeding behavior in Aplysia (Cropper et al. 1996b
; Miller et al. 1994
; Rosen et al. 1992
-1994
). For example, it was suggested that these neurons are partially responsible for producing the adjustments in feeding motor programs that are necessary to convert bites (where food is not ingested) to bite-swallows (where food is deposited in the esophagus) (Cropper et al. 1996b
; Rosen et al. 1992
). This conversion is accomplished by changes in phase and amplitude relationships between radula closing/retraction and radula opening/protraction. For example, radula closing/retraction is prolonged and enhanced, and radula opening/protraction is delayed to accommodate the enhanced closing/retraction (Cropper et al. 1990
). That B21 could play a role in inducing these types of changes in ingestive responses is suggested by the fact that it is activated when the biting surface of the radula is touched, e.g., by food (Miller et al. 1994
; Rosen et al. 1992
). Additionally, it is either directly or indirectly connected to many of the identified motor neurons and interneurons active during ingestive motor programs (Rosen et al. 1992
-1994
). Many neurons active during closing/retraction are directly excited by radula mechanoafferent activity, and many neurons active during opening/protraction are indirectly inhibited by radula mechanoafferent activity.
Although B21 clearly functions as a sensory neuron it is additionally rhythmically depolarized by the feeding CPG (Miller et al. 1994
; Rosen et al. 1993
, 1994
). It is likely that these CPG-induced depolarizations in B21 play an important role in gating the effectiveness of centripetal activity in this cell (Rosen et al. 1993
, 1994
). The mechanism utilized in this context is, however, not the mechanism commonly observed in situations where CPGs induce primary afferent depolarizations (PADs). Thus PADs often exert a local inhibitory effect because they are often mediated by a presynaptic conductance increase that decreases the effectiveness of afferent transmission to postsynaptic neurons (e.g., Clarac and Cattaert 1996
; Nusbaum et al. 1997
). In contrast, CPG-induced depolarizations in B21 are not strictly presynaptic, i.e., they can be recorded in the soma of B21, and they exert a potentiating effect on afferent transmission (Rosen et al. 1993
, 1994
). Thus when B21 is depolarized centripetal spikes are more, rather than less, effective at eliciting PSPs in follower cells. During ingestive motor programs B21 is rhythmically depolarized during radula closing/retraction (Miller et al. 1994
; Rosen et al. 1993
, 1994
). This depolarization will therefore potentiate afferent transmission during this phase of behavior.
In investigating the effects of MCC activity on B21 we therefore sought to determine whether MCC activity was likely to affect the central mechanism that gates radula mechanoafferent activity. Thus we sought to determine whether the MCCs would directly depolarize B21 and cause it to spike and/or whether the MCCs would simply increase the excitability of B21. Additionally, radula mechanoafferent activity is influenced by contractions of the subradula tissue (SRT) (Cropper et al. 1996b
), which contains the processes of B21 (Miller et al. 1994
). The SRT is innervated by at least one motor neuron that fires rhythmically during ingestive feeding motor programs (Borovikov et al. 1997
). When the SRT contracts, neuron B21 is activated, and its responsiveness to touch is increased (Cropper et al. 1996b
). Thus in this study we additionally sought to determine whether contractile properties of the SRT were altered by MCC activity and whether changes in the contractile properties of the SRT were likely to affect radula mechanoafferent activity. Some of these data were published as an abstract (Cropper et al. 1996a
).
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METHODS |
Animals
Experiments were conducted with 250-350 g A. californica that were maintained in holding tanks containing 14-16°C artificial seawater (ASW). Animals were anesthetized by injection of isotonic MgCl2 and then dissected to create reduced preparations.
Identification of neurons
The nomenclature used in this study follows that of Gardner (1971)
. The radula mechanoafferent cluster of neurons was identified by criteria described by Miller et al. 1994
. It consists of a centrally located group of cells on the rostral surface of the buccal ganglion. Radula mechanoafferent neurons are electrically coupled to each other, and peripherally generated spikes are elicited when the radula is touched. Neurons B21 and B22 are the largest cells of the cluster (Rosen et al. 1992
). Neuron B21 differs from neuron B22 in that 1) it is generally larger, 2) it is typically more spherical, neuron B22 is typically more elongate, 3) it is generally more ventrally located, and 4) IPSPs are recorded from neuron B21 when the B4/B5 neurons are stimulated. In contrast, neuron B22 receives excitatory synaptic input from neurons B4/B5 (Rosen et al. 1992
).
Serotonergic input to the SRT and the buccal ganglion
Localization of 5-HT-immunoreactive (5-HT-IR) fibers was determined by standard whole-mount methods (Longley and Longley 1986
; Miller et al. 1994
). The primary antiserum (rabbit host, Incstar, Stillwater MN) was applied for 48 h (1:200 dilution, room temperature) and the second F(ab')2 tetramethyl rhodamine isothiocyanate-conjugated antibody (anti-rabbit IgG heavy and light chain, donkey host; Jackson Immunoresearch, West Grove, PA) was applied for 24 h (1:200 dilution, room temperature). Tissues were viewed with a Nikon microscope equipped with epifluorescence and photographed with Tri-X (ASA 400) film. Within the buccal ganglion, B21 and B22 were identified by using the criteria listed above. They were injected with 3% Lucifer yellow (LY) dye (20-30 min, 1 s on, 1 s off pulses) before fixation (4% paraformaldehyde, 4 h, room temperature) and processed for 5-HT immunocytochemistry. Ganglia were examined on a scanning confocal microscope (NORAN Odyssey) equipped with an argon laser and barrier filters suitable for viewing the LY (band pass 520-560 nm) and rhodamine (long pass 590 nm). Images were generated with the Metamorph Imaging System (West Chester, PA). They were transferred as TIFF bitmap files for processing with Adobe Photoshop and labeled with CorelDraw.

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| FIG. 1.
The metacerebral cell (MCC) is a source of serotonergic input to the SRT. A: intracellular stimulation of the MCC (bottom traces) elicits extracellular responses (top traces) in distal sections of the radular nerve. B: serotonin (5-HT) immunoreactivity in the radular nerve secondary branch that innervates the subradula tissue (SRT). A large smooth immunoreactive fiber (arrow) is present in the nerve. Small varicose fibers appear to be associated with the nerve sheath. Calibration bar 100 µm. C: 6-8 fine immunoreactive fibers pass in parallel (arrow) in the distal radular nerve branch within the SRT. Near the terminus of this branch, the serotonergic fibers bear off in various directions and subdivide, reaching a substantial portion of the tissue. Calibration bar 100 µm. D: lower magnification shows the high-density of serotonergic innervation in the region of the SRT adjacent to the insertion of the accessory radula closer (ARC) muscle. The density of immunoreactive fibers tapers off within the ARC muscle itself. Calibration bar 250 µm.
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| FIG. 2.
5-HT-IR fibers in the buccal ganglion. A: low-power confocal image of the rostral surface of the buccal ganglion. Ganglion is oriented with the buccal commissure directed toward the top right corner of the image. Neurons B21 and B22 in the radula mechanoafferent cluster were identified by using morphological and electrophysiological criteria and injected with Lucifer yellow (LY, arrows) before processing the ganglion for 5-HT immunoreactivity (TRITC-conjugated second antibody). In this image, the barrier filter used to view LY allowed "bleedthrough" of the rhodamine-labeled 5-HT-IR fiber system. B: same field as A using a filter that permits viewing the rhodamine-labeled 5-HT fibers exclusively. Positions of the cell bodies of B21 and B22 are indicated as in A. Neurons with asterisks are likely to be B4/B5. Calibration bar of 100 µm applies to A and B. C: higher magnification of the midline region of ganglion using the same filter as A. B21 and B22 are spindle-shaped cells with prominent processes directed medially and laterally. Note that after the immunocytochemistry process LY retention within the nuclei (n) of injected cells exceeds that of the cytoplasmic regions, which exhibit a "halo" appearance. Arrows indicate the large initial segments of B21 and B22 that are directed toward the midline. Calibration bar 50 µm. D: higher-power image of the region shown as a dotted box in C. Varicose 5-HT-IR fibers (arrows) are associated with the initial segment and cell body of B21. Calibration bar 20 µm.
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| FIG. 3.
Central effects of MCC stimulation. A: depolarizations of a few millivolts were observed in neuron B21 when the MCC was stimulated at relatively high firing frequencies. Because neuron B21 is generally 10 mV from threshold, MCC stimulation did not directly elicit action potentials in B21. These depolarizations could be observed when experiments were conducted in a 3× Mg2+; 3× Ca2+ artificial seawater (ASW). B: when B21 was repeatedly depolarized with subthreshold fixed current pulses, the pulses evoked spikes when the MCC was stimulated. These effects on excitability could be observed when the MCCs were fired at frequencies that did not produce central depolarizations.
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To determine whether serotonergic input to the SRT did indeed arise from the MCCs, electrophysiological techniques were used to determine whether intracellular MCC stimulation elicited extracellular spikes in peripheral sections of the radular nerve. Intracellular stimulation techniques used were standard. Electrodes were beveled to relatively low impedances (i.e., <10 M
) and were filled with 2 M potassium acetate. Extracellular recordings were made with suction electrodes.
Effects of MCC stimulation and 5-HT on B21 afferent activity and contractile properties of the SRT
These experiments were conducted in reduced preparations that were described in detail elsewhere (e.g., Cropper et al. 1996b
) (see also Fig. 4A). Briefly, the SRT was removed from the chitinous radula, and other muscles, nerves, and ganglia were dissected away. Particular dissections depended on the purpose of the experiment. For example, the cerebral ganglion was only present in experiments where effects of the MCCs were studied. In experiments where SRT contractions were elicited by motor neuron stimulation, both the radular nerve and buccal nerve 3 were intact. In experiments where SRT contractions were elicited by stretch, only the radular nerve was intact. In all cases, preparations were transferred to Sylgard-lined dishes, and the SRT was attached to a semiisotonic force transducer (Harvard Apparatus). A Lucite subchamber was then placed around the SRT to pharmacologically isolate it from the nervous system (Fig. 4A). Contractions of the SRT were elicited either by stretch or with intracellular stimulation of B66 (the SRT motor neuron). Stretch was induced by counterweighting muscles. Thus, the SRT was not directly attached to the transducer (Fig. 4A). It was attached to one end of a lever. The middle of this lever made contact with the transducer and was therefore immobilized and served as a pivot point. Counterweights were applied by placing metal washers on the end of the lever that was not attached to the SRT. In experiments where afferent activity of B21 was studied, the SRT was not attached to the force transducer. It was pinned to a Sylgard "stage" so that it was approximately perpendicular to the device used to apply mechanical stimuli. Mechanical stimuli were delivered by means of a minispeaker (Quam) that had a wooden probe (tip diameter 1 mm) that was perpendicularly attached to the speaker membrane. Reproducible movements of the membrane were regularly elicited by driving the speaker with a Grass stimulator.

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| FIG. 4.
5-HT affects contractions of the SRT elicited by stretch. A: reduced preparation used to study effects of stretch on the SRT. In these preparations the SRT was separated from the chitinous radula, placed in a subchamber pharmacologically isolated from the nervous system, and attached to a semiisotonic force transducer. The tissue was stretched by adjusting the position of a counterweight. B: stretch-induced contractions of the SRT produce centripetal spikes in the left and right B21 neurons (LB21 and RB21). C: 5-HT increases the frequency and amplitude of stretch-elicited contractions. In C1 the ganglion was bathed in normal ASW. The counterweight was adjusted to produce small rhythmic contractions, and 10 7 M 5-HT was applied to the SRT subchamber. Stretch-induced contractions increased in amplitude and frequency. C2: to determine whether the effect shown in C1 was mediated centrally or peripherally, the experiment shown in C1 was repeated with the ganglion bathed in 0.5× Ca2+; 4× Mg2+.
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A second type of preparation was used in experiments where contractions of the SRT were elicited with extracellular nerve stimulation. The SRT was left attached to the chitinous radula, except that, to gain access to the SRT, the I2 muscle and the radula sac were removed. Additionally, connections between the SRT and muscles I4, I5, and I7 were severed to reduce the likelihood that movements not specifically related to the contraction of the SRT would be transduced. The radular nerve was generally left intact; all other buccal nerves were cut close to the buccal ganglion so that stimulating electrodes could easily be applied. Transducers were attached to the SRT and chitinous radula with a piece of thread (as described previously). Buccal nerves were stimulated with suction electrodes and a Grass stimulator.
cAmp measurements in the SRT
Animals were anesthetized and dissected, and the SRT and attached musculature were removed from the chitinous radula. To specifically isolate the part of the SRT that contains the peripheral processes of B21, we removed as much of the I4 and I5 muscles as possible. The SRT was trimmed and left for 2 h in ASW to allow for recovery from adenosine 3',5'-cyclic monophosphate (cAMP) increases induced by the dissection. Exogenous 5-HT was applied for 10 min. The SRT was then homogenized in 65% ethanol, boiled for 15 min, and spun at 10,000 g for 15 min. cAMP levels were measured with a commercially available radioimmunoassay (RIA; Amersham). cAMP levels are expressed as picomoles per milligram of tissue (wet weight).
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RESULTS |
Source of serotonergic input to the SRT
Previous studies have suggested that the serotonergic MCCs innervate the SRT. At least in proximal sections of the radular nerve, extracellular spikes can be recorded when the MCCs are stimulated intracellularly (Weiss and Kupfermann 1976
). The radular nerve does, however, branch several times before it reaches the SRT. We therefore placed extracellular electrodes as close to the SRT as possible and stimulated the MCCs. Extracellular responses were recorded in peripheral branches of the radular nerve (Fig. 1A).
Additionally, we used immunocytochemical techniques to determine whether there are 5-HT-IR fibers in regions of the SRT that contain the receptive fields of radula mechanoafferent neurons. 5-HT-IR fibers were most obvious in the region of the SRT where the accessory radula closer muscle inserts (Fig. 1D). This is also a region that is innervated by B21 (Rosen et al. 1992
). Thus the current physiological and anatomic observations suggest that the MCCs are likely to be the source of serotonergic input to the SRT.
The MCCs branch extensively within the buccal ganglion, so it was of interest to determine whether they have processes that terminate on B21 centrally, i.e., within the buccal ganglion. To determine the relation between 5-HT-IR fibers and radula mechanoafferent neurons, B21 and B22 neurons were injected with LY dye, and buccal ganglia were processed for 5-HT immunocytochemistry (Fig. 2). 5-HT-IR fibers were observed in the vicinity of B21/B22, particularly in the region of the medially directed initial segment of B21 (Fig. 2D). In the following physiological experiments we therefore sought to determine whether the MCCs act centrally and/or peripherally to modulate radula mechanoafferent activity.
Central effects of MCC stimulation
To determine whether MCC input is likely to directly cause B21 to spike we stimulated MCCs and recorded the resulting change in membrane potential in B21 (Fig. 3). Firing the MCC at relatively high frequencies resulted in a slow depolarizing response of a few millivolts in B21 in five of six preparations (e.g., Figs. 3A and Fig. 6). B21 is generally
10 mV from threshold in quiescent preparations (e.g., Fig. 3B), and the depolarization evoked by the MCC failed to evoke spikes. It is difficult to unequivocally determine whether the effect of the MCCs on B21 is direct because neurons that are electrically coupled to B21 such as B15 and B16 are also depolarized by MCC stimulation (Weiss et al. 1978
). Effects of MCC stimulation on B21 could, however, still be observed when experiments were conducted in a 3× Mg2+; 3× Ca2+ ASW (Fig. 3A).
We next sought to determine whether MCC stimulation could produce an increase in the excitability in neuron B21, such that it exhibited enhanced firing during an input that occurs during feeding motor programs. Depolarizing current pulses were repeatedly injected into B21 before, during, and after MCC stimulation (Fig. 3B). Current pulses that were subthreshold before MCC stimulation elicited action potentials in neuron B21 during MCC stimulation and just after stimulation in three of four preparations. These data suggest therefore that, although the MCCs are not likely to directly cause radula mechanoafferent neurons to fire, MCC activity may alter B21 excitability and thereby alter its response during feeding motor programs.
Effects of 5-HT on contractile properties of the SRT
One method of eliciting contractions of the SRT is by stretch (Cropper et al. 1996b
). To determine whether 5-HT affects stretch-induced contractions of the SRT, we used preparations in which the SRT was separated from the chitinous radula and the rest of the buccal mass. The SRT was placed in a subchamber and attached to a semiisotonic force transducer (Fig. 4). The transducer arm was adjusted so that the SRT was counterweighted with loads that elicited small infrequent contractions. 5-HT was then applied directly to the SRT subchamber. Stretch-induced contractions increased in size and frequency at low, presumably physiological, concentrations (i.e., 10
8-10
6, n = 3; Figs. 4 and 5). These effects were seen when activity in the central nervous system was blocked by incubating the nervous system in 0.5× Ca2+; 4× Mg2+ solutions (e.g., Fig. 4C2). In general the effects of 5-HT were long lasting, and it generally took an hour for preparations to recover from higher concentrations of 5-HT.

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| FIG. 5.
Effects of 5-HT on stretch-induced contractions of the SRT are concentration dependent. The preparation used for this experiment was similar to the one shown in Fig. 4A except that the nervous system was not present. All 4 records are from the same preparation. The preparation was washed for 30 min between 5-HT applications (not shown). 5-HT was applied directly to the SRT subchamber. At low concentrations 5-HT increased the amplitude and frequency of stretch-induced contractions of the SRT. At 10 5 M, however, contractions were inhibited.
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Although it is not known if the counterweight used in these experiments simulated a physiological load, the radula undergoes vigorous movements and shape changes during feeding in intact animals (Drushel et al. 1997
), and the SRT is likely to experience considerable loads. We found that the effects of 5-HT could be seen even with very light counterweights (e.g., 45 mg), suggesting that 5-HT could induce contractions of the SRT in the absence of activity of the SRT motor neuron. To determine whether 5-HT is released from the MCCs in concentrations that are sufficient to exert this type of effect on the SRT, the SRT was attached to the force transducer with a counterweight that was just barely subthreshold for eliciting contractions, and the nervous system was placed in a 4× Mg2+; 0.5× Ca2+ ASW. Under these conditions MCC stimulation caused the SRT to contract, but the contractions were much smaller than those elicited by direct application of 5-HT to the SRT (n = 5; Fig. 6). These data suggest that MCC-induced contractions are not therefore likely to produce vigorous radula movements.

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| FIG. 6.
Effects of MCC stimulation on a SRT subjected to stretch. The preparation used for these experiments is shown in Fig. 4A. The SRT was counterweighted in a manner that was just subthreshold for eliciting contractions, and the MCC was stimulated. Contractions that were much smaller than those elicited by exogenous 5-HT were elicited (arrows). This was the case even when the MCCs were fired in different patterns, e.g., in repeated bursts. Because MCC stimulation produces excitability increases centrally (e.g., note the depolarization in B21), it was possible that elicited contractions resulted from the activation of a SRT motor neuron. The experiment was therefore repeated with the ganglion, but not the SRT, bathed in 4× Mg2+; 0.5× Ca2+. Small contractions were still elicited with MCC stimulation.
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In addition to stretch, SRT contractions can be elicited by firing of a SRT motor neuron, B66 (Borovikov et al. 1997
). We therefore next determined whether 5-HT could affect SRT contractions evoked by stimulation of buccal nerve 3, which contains the axon of the SRT motor neuron. Experiments were conducted in relatively intact buccal mass preparations. 5-HT applied directly to the SRT increased nerve 3-evoked contraction size in a concentration-dependent manner (n = 3; Fig. 7). We next determined whether MCC activity could modulate parameters of muscle contractions evoked by direct firing of B66. Contractions of the SRT were therefore elicited with a fixed number of spikes before, during, and after MCC stimulation (n = 4; Fig. 8A). Contraction size, rate of rise, and relaxation rate all increased. The MCCs do therefore modulate parameters of motor neuron-elicited contractions of the SRT.

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| FIG. 7.
5-HT modulates contractions of the SRT elicited by nerve shock. Contractions of the SRT were repeatedly elicited by shocking buccal nerve 3 (bars under recordings). 5-HT increased contraction amplitude at 10 7 and 10 6 M.
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| FIG. 8.
A: MCC stimulation potentiates contractions of the SRT elicited by motor neuron stimulation. Contractions of the SRT (middle trace) were elicited by stimulating the SRT motor neuron B66 in bursts (top trace). MCC stimulation altered parameters of B66-elicited muscle contractions. B: changes in the parameters of B66-elicited SRT contractions affect activity in B21. When B66 is stimulated at higher frequencies SRT contractions increase in size and contract at a faster rate. Both effects are seen when SRT contractions are modulated by MCC activity (only the effect on size is apparent at the slow chart speed shown in A). When parameters of SRT contractions are altered in this manner, afferent activity in neuron B21 is increased.
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To determine whether MCC-induced changes in the contractile properties of the SRT could alter radula mechanoafferent activity, contractions of the SRT were elicited with different parameters of motor neuron stimulation, and changes in B21 responsiveness were determined (Fig. 8B). When the intraburst stimulation frequency was increased contractions were altered in the most MCC-like manner, i.e., contractions produced with higher intraburst stimulation frequencies were larger in amplitude and had a faster rate of contraction. Under these conditions the number of centripetal spikes in neuron B21 did in fact increase. Taken together therefore the data shown in Fig. 8, A and B, suggest that effects of the MCCs on contractile properties of the SRT are in fact likely to alter radula mechanoafferent activity.
Mechanism of action of 5-HT on the SRT
The relatively slow onset and long-lasting nature of the effects of 5-HT on contractile properties of the SRT suggested that 5-HT might be acting via a second messenger. Specifically, we hypothesized that effects might be cAMP mediated because it has been commonly demonstrated that 5-HT elevates cAMP in other muscles of the buccal mass of Aplysia (e.g., Lloyd et al. 1984
; Lotshaw and Lloyd 1990
; Weiss et al. 1979
). To test this hypothesis exogenous 5-HT was applied to the SRT for 10 min, and resulting cAMP levels were measured. 5-HT did indeed increase SRT cAMP levels in a concentration-dependent manner (Fig. 9A). Additionally we tested a membrane permeable cAMP analogue (8 CPT-cAMP) on physiological preparations. 8 CPT-cAMP exerted 5-HT-like effects on parameters of contractions of the SRT (Fig. 9C). Exogenous 5-HT was still effective at elevating cAMP levels in a Ca-free ASW (Fig. 9B), i.e., under conditions where transmitter release presumably cannot occur, suggesting that the 5-HT effect was direct.

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| FIG. 9.
Effects of 5-HT on the SRT are at least partially mediated by cAMP. A: in this experiment the SRT was isolated and incubated in 5-HT solutions for 10 min. 3',5'-Cyclic monophosphate (cAMP) levels are expressed in picomoles of cAMP per milligram of tissue (wet weight; for each point n = 4; SEs are plotted). B: to determine whether 5-HT increases cAMP levels in the SRT under conditions where synaptic transmission cannot occur, the SRT was incubated for 10 min in 10 6 M 5-HT in Ca2+-free ASW. Significant increases in cAMP levels were still observed (n = 3; SEs plotted). C: membrane-soluble cAMP analogue 8-CPT-cAMP produces 5-HT-like effects on the SRT. Left: stretch-induced contraction of the SRT before application of the cAMP analogue. Middle and right: in the presence of the cAMP analogue stretch-induced contractions of the SRT increase in amplitude.
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Effects of 5-HT and the MCCs on the peripheral excitability of B21
One type of experiment that suggests that 5-HT can act outside the buccal ganglion and affect the peripheral excitability of B21 is shown in Fig. 10. Here 5-HT was sequentially applied to the SRT subchamber at different concentrations while B21 responses were recorded. At 10
8 M SRT contractions were elicited, and centripetal spikes were recorded in neuron B21. At 10
7 M, contractions were similar in size, but fewer centripetal spikes were recorded in neuron B21. Finally at 10
6 M large SRT contractions still occurred, but centripetal spikes were no longer recorded from neuron B21. As the 5-HT is washed out, however, B21 responses returned (Fig. 10B). Although in this experiment it cannot be determined whether 5-HT exerted an excitatory action on the responsiveness of B21 at lower concentrations, an inhibitory effect was observed at 10
6 M.

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| FIG. 10.
5-HT exerts inhibitory effects on sensory responses. A and B are records from the same preparation. The preparation was washed for 30 min between applications of 5-HT (not shown). A1: 10 8 M 5-HT induces contractions of the SRT (bottom trace) and centripetal spikes in neuron B21 (top trace). A2: in 10 7 M 5-HT contractions are slightly larger, yet fewer centripetal spikes are elicited in B21. A3: in 10 6 M 5-HT contractions are still larger, yet centripetal spikes are no longer observed in neuron B21. B shows the initial part of the washout of 10 6 M 5-HT. As the 5-HT was washed out centripetal spikes became apparent in neuron B21 despite the fact that contractions did not initially change in size. This experiment suggests that 5-HT exerts inhibitory effects on B21 peripheral processes. These inhibitory effects appear to be apparent at concentrations that are not yet high enough to produce inhibitory effects on SRT contractile properties.
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To determine whether 5-HT can affect responsiveness of B21 to peripherally applied mechanical stimuli, a stimulus that was barely suprathreshold was repeatedly applied at fixed intervals (n = 3). One centripetal spike occurred for every three or four stimuli applied (Fig. 11); 10
9 M 5-HT was then directly applied to the SRT subchamber, and one centripetal spike was now recorded for every one or two stimuli applied. The 5-HT concentration was then increased to 10
8 M, and two centripetal spikes occurred every time a stimulus was given. Finally, the 5-HT concentration was increased to 10
7 M, and centripetal spikes were no longer recorded. When synaptic transmission was blocked by bathing the buccal ganglion in 0.5× Ca2+; 4× Mg2+ seawater (Fig. 11B), 5-HT still increased responsiveness to peripherally applied stimuli. Experiments in Figs. 10 and 11 indicate therefore that 5-HT can affect the peripheral excitability of B21.

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| FIG. 11.
5-HT increases the sensitivity of B21 to peripherally applied mechanical stimuli. A and B are from the same preparation. Mechanical stimuli were delivered by means of a minispeaker that had a wooden stick perpendicularly attached to the speaker membrane. Reproducible movements of the membrane were regularly elicited by driving the speaker with a Grass stimulator. Arrows indicate when mechanical stimuli were applied. The preparation was washed for 30 min between 5-HT applications (not shown). 5-HT was directly applied to the SRT subchamber. A1: under control conditions a weak stimulus elicited centripetal spikes in B21 every 4th or 5th time the stimulus was applied. In 10 9 and 10 8 M 5-HT response frequency increased. In 10 7 M 5-HT, inhibitory effects were observed. In B the ASW bathing the ganglion was replaced with 0.5× Ca2+; 4× Mg2+ ASW.
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To determine whether MCC activity can affect B21 responsiveness a subthreshold mechanical stimulus was repeatedly applied before, during, and after MCC stimulation. Mechanical stimuli that were subthreshold before the MCC was stimulated became suprathreshold during MCC stimulation (n = 4). These experiments were then repeated when the normal ASW bathing both the buccal and cerebral ganglia was exchanged for 0.5× Ca2+; 4× Mg2+ (Fig. 12B). Again MCC stimulation caused subthreshold stimuli to act as suprathreshold stimuli. These data show therefore that MCC activity can alter the sensitivity of B21 to peripherally applied mechanical stimuli. Note, however, that effects of MCC stimulation are not very dramatic in the sense that they do not elicit afterdischarges in B21. Thus, although the MCCs may produce increases in B21's sensitivity to a peripheral stimulus while it is actually present, they are not likely to cause B21's activity to outlast stimulus presentation.

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| FIG. 12.
Stimulation of the MCC increases responsiveness of B21 to peripherally applied mechanical stimuli. Arrows indicate when mechanical stimuli were applied. A and B are from the same preparation. A: a subthreshold mechanical stimulus did not elicit centripetal spikes in neuron B21 until the MCC was stimulated. B: effects seen in A persisted when the normal ASW bathing the buccal and cerebral ganglia was replaced with 0.5× Ca2+; 4× Mg2+ ASW.
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DISCUSSION |
The MCCs have been well characterized in Aplysia and other mollusks and have been extensively investigated within the context of feeding behavior (Berry and Pentreath 1976
; Cottrell and Macon 1974
; Gerschenfeld and Paupardin-Tritsch 1974
; Gerschenfeld et al. 1978
; Gillette and Davis 1977
; Granzow and Kater 1977
; McCrohan and Audesirk 1987
; Weinreich et al. 1973
; Weiss and Kupfermann 1976
; Weiss et al. 1982
). Although previous work has indicated that the MCCs exert effects that are widespread, there has been no indication that the MCCs have effects on sensory neurons. Thus C2, a mechanoafferent that provides excitatory drive to the MCCs (Weiss et al. 1986b
), is not reciprocally excited by MCC activity (K. Weiss, personal communication). Although cerebral mechanoafferent neurons respond to 5-HT, the physiological source of the serotonergic input to these cells has not, however, been identified (Rosen et al. 1989a
). This study therefore adds to current conceptualizations of the role of the MCCs in that it demonstrates that effects of the MCCs are not confined to neuromuscular systems utilized during feeding behavior. Activity of at least one group of sensory neurons is modulated by the MCCs.
Previous work in Aplysia has indicated that the MCCs are silent in quiescent animals but begin to fire when animals are exposed to food (Kupfermann and Weiss 1982
; Weiss et al. 1978
). As animals ingest food the level of MCC activity remains correlated with the general state of arousal. For example, initially bite speed is high (indicating that animals are aroused) (Susswein et al. 1978
; Weiss et al. 1982
), and the MCCs fire at relatively high frequencies (Kupfermann and Weiss 1982
; Weiss et al. 1978
). As a meal progresses, bite speed and MCC activity decrease (Kupfermann and Weiss 1982
; Susswein et al. 1978
; Weiss et al. 1982
). Therefore these data and data from other studies, e.g., lesion experiments (Rosen et al. 1989b
) and experiments where the MCCs were stimulated in semiintact feeding preparations (Weiss et al. 1986a
), suggested that the MCCs play a role in inducing the motivational state of food-induced arousal in Aplysia.
In Aplysia an essential feature of MCC actions at both the behavioral and synaptic level is that they are modulatory, i.e., they are relatively prolonged and their effects are contingent on the occurrence of some other event (e.g., Weiss et al. 1982
). Thus at the behavioral level MCC activity does not initiate feeding. It does, however, alter responsiveness to an appropriate stimulus, i.e., food (Weiss et al. 1986a
). Similarly, at the synaptic level, MCC stimulation does not directly activate neurons. Instead excitatory effects of other inputs can be enhanced (Weiss et al. 1978
). Data obtained in these experiments are in keeping with this general idea. Centrally, we observed that, although MCC activity can elicit a slow depolarization in neuron B21 (i.e., act at site 1 in Fig. 13), it does not cause B21 to spike. MCC activity can, however, enhance radula mechanoafferent responsiveness to depolarization from some other source, e.g., from phasic inputs associated with elements of the feeding CPG.

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| FIG. 13.
Data obtained in this study indicate that the MCCs exert at least 3 types of effects that modify radula mechanoafferent activity. 1) They act centrally, i.e., in the buccal ganglion, and increase radula mechanoafferent excitability. 2) They act peripherally and increase SRT contractility. Increases in the size of B66-induced SRT contractions are likely to indirectly alter radula mechanoafferent activity. When SRT contractions are increased in amplitude and rise time more centripetal spikes are evoked in B21. 3) MCCs also act outside the buccal ganglion, presumably in the periphery, and increase radula mechanoafferent responsiveness to peripherally applied mechanical stimuli.
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Peripherally the MCCs enhance the contractility of the SRT (i.e., act at site 2 in Fig. 13). This effect will also be manifested in a phase-dependent manner because it is dependent on activity of the SRT motor neurons, which fire rhythmically during ingestive motor programs (Borovikov et al. 1997
). The enhanced contractility of the SRT will affect radula mechanoafferent activity in two ways. If contractions are large enough, radula mechanoafferent activity will be elicited in the absence of other mechanical stimulation (Cropper et al. 1996b
). If contractions are smaller, centripetal spikes may not be directly elicited, but radula mechanoafferent neurons will be more sensitive to externally applied mechanical stimuli (Cropper et al. 1996b
). This mechanism may therefore serve two functions. It may generate radula mechanoafferent activity or act as a peripheral way of gating or adjusting radula mechanoafferent gain. In either case it will be phase dependent.
Other experiments show that the MCCs can act outside the buccal ganglion, presumably in the periphery, and alter radula mechanoafferent responsiveness to mechanical stimuli (i.e., act at site 3 in Fig. 13). These types of effects are, however, not very dramatic in the sense that the MCCs do not induce afterdischarges in B21. Afterdischarges were observed as a result of noxious stimulation in other mechanosensory neurons in Aplysia (Clatworthy and Walters 1993
). Because neuronal activation outlasts stimulus presentation when afterdischarges are observed, this phenomenon would presumably disrupt the phasic nature of radula mechanoafferent activity. Thus at a cellular level 5-HT may exert effects on peripheral processes of B21 that will produce subtle increases in B21's sensitivity to a stimulus without causing B21 to fire in a manner that is temporally unrelated to the presence of a stimulus.
In future experiments it will be interesting to characterize these cellular mechanisms of action in more detail. In particular, it is interesting to note that experiments with exogenous 5-HT indicate that inhibitory effects on the peripheral excitability of B21 are apparent at lower concentrations of 5-HT than inhibitory effects of 5-HT on contractile properties of the SRT (cf. Figs. 5 and 11). Although physiologically released 5-HT is not likely to reach concentrations that will be purely inhibitory, physiological concentrations of 5-HT may produce inhibitory effects that are masked by excitatory effects. This type of observation was made in the well-studied accessory radula closer neuromuscular system (e.g., Brezina et al. 1994
). If this is the case it is interesting that inhibitory effects of 5-HT appear to be the most pronounced on the tissue where the most "control" may be needed, i.e., on sensory terminals.
The central and peripheral effects of the MCCs on radula mechanoafferent activity appear therefore to be similar in that they are both likely to be phase specific. Interestingly, however, they may be manifested during two antagonistic phases of behavior. For example, central effects of the MCCs will be effective when radula mechanoafferents receive depolarizing input from the CPG, i.e., during the radula closing/retraction phase of ingestive motor programs (Rosen et al. 1993
, 1994
). In contrast, the effects of the MCCs on contractile properties of the SRT are likely to be most effective when SRT motor neurons are active, i.e., during the opening/protraction phase of ingestive motor programs (Borovikov et al. 1997
).
In this respect radula mechanoafferents appear therefore to be similar to stretch receptor neurons in lamprey (Grillner et al. 1984
). Lamprey stretch receptors are peripherally excited when contralateral motor neurons are active (Viana Di Prisco et al. 1990
). Additionally, stretch receptors receive depolarizing input from the CPG during the phase of behavior in which the peripheral drive to stretch receptors is decreased, i.e., during ipsilateral motor neuron activity (Vinay et al. 1996
). It was hypothesized that this arrangement may be analogous to the efferent control of muscle spindles by
-motor neurons in higher vertebrates (Sjostrom and Zangger 1976
). Thus CPG-induced depolarizations may serve to prolong stretch receptor sensitivity because they may at least partially compensate for the decreases in peripheral drive that will occur when the ipsilateral musculature contracts (Vinay et al. 1996
).
In lamprey, lateral movements that mimic swimming movements will efficiently entrain centrally generated motor programs (e.g., Grillner et al. 1981
). It appears therefore that sensory activity can control phase transitions during locomotory activity. Presumably this is due to the fact that there are actually two types of receptor neurons, those that project ipsilaterally and excite network neurons and those that project contralaterally and inhibit network neurons (Viana Di Prisco et al. 1990
). Possibly, radula mechanoafferents can act in a similar manner. Only one type of radula mechanoafferent has been described, but, as discussed above, these neurons 1) make excitatory connections with motor neurons active during radula closing/retraction and 2) make excitatory connections with an interneuron that inhibits motor neurons active during radula opening/protraction. Thus radula mechanoafferents may do more than simply prolong radula closing/retraction and delay radula opening/protraction when bites are converted to bite-swallows. Additionally they may play a role in causing opening/protraction to end and closing/retraction to begin.
If this is the case, MCC activity in turn may have dual effects. Because the MCCs increase SRT contractility they may increase the number of centripetal spikes that are generated in radula mechanoafferents during opening/protraction as a result of SRT contractions. In this manner MCC activity may influence transitions between radula opening/protraction and radula closing/retraction. Consistent with this idea, MCC activity does increase the rate at which ingestive feeding responses are made (e.g., Weiss et al. 1986a
). Second, during the closing/retraction phase of behavior, MCC activity may increase radula mechanoafferent excitability and may increase radula mechanoafferent responsiveness to food. These latter effects may partially underlie increases in biting strength that occur when animals are food aroused (Weiss et al. 1978
).
In summary, afferent activity can be influenced by modulatory neurotransmitters in many systems (e.g., Billy and Walters 1989
; El Manira et al. 1991b
; Mar and Drapeau 1996
; Pasztor and Bush 1989
; Rossi-Durand 1993
; Wenning and Calabrese 1995
). To address the physiological significance of this type of regulation, we studied interactions between the MCCs and B21. We show that 5-HT can modulate B21 activity in several ways; its central excitability can be increased, its peripheral excitability can be increased, and contractile properties of the SRT can be enhanced (which will indirectly enhance B21 activity). Experiments with the MCCs suggest that this modulation will occur in a phase-dependent manner. Previous studies have shown that MCC activity is likely to contribute importantly to food-induced arousal. Considerable evidence indicates that this effect is mediated by central and peripheral actions on motor neurons and feeding musculature. We now show that activity of a class of sensory neurons is additionally affected.