Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom
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ABSTRACT |
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Staras, Kevin, György Kemenes, and Paul R. Benjamin. Electrophysiological and behavioral analysis of lip touch as a component of the food stimulus in the snail Lymnaea. Electrophysiological and video recording methods were used to investigate the function of lip touch in feeding ingestion behavior of the pond snail Lymnaea stagnalis. Although this stimulus was used successfully as a conditioning stimulus (CS) in appetitive learning experiments, the detailed role of lip touch as a component of the sensory stimulus provided by food in unconditioned feeding behavior was never ascertained. Synaptic responses to lip touch in identified feeding motoneurons, central pattern generator interneurons, and modulatory interneurons were recorded by intracellular electrodes in a semi-intact preparation. We showed that touch evoked a complex but characteristic sequence of synaptic inputs on each neuron type. Touch never simply activated feeding cycles but provided different types of synaptic input, determined by the feeding phase in which the neuron was normally active in the rhythmic feeding cycle. The tactile stimulus evoked mainly inhibitory synaptic inputs in protraction-phase neurons and excitation in rasp-phase neurons. Swallow-phase neurons were also excited after some delay, suggesting that touch first reinforces the rasp then swallow phase. Video analysis of freely feeding animals demonstrated that during normal ingestion of a solid food flake the food is drawn across the lips throughout the rasp phase and swallow phase and therefore provides a tactile stimulus during both these retraction phases of the feeding cycle. The tactile component of the food stimulus is strongest during the rasp phase when the lips are actively pressed onto the substrate that is being moved across them by the radula. By using a semi-intact preparation we demonstrated that application of touch to the lips during the rasp phase of a sucrose-driven fictive feeding rhythm increases both the regularity and frequency of rasp-phase motoneuron firing compared with sucrose applied alone.
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INTRODUCTION |
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The nature and function of sensory pathways to
centrally located neuronal networks involved in feeding was examined in
a variety of molluscan systems, including Aplysia (e.g.,
Rosen et al. 1982a,b
), Limax (Delaney
and Gelperin 1990
), Lymnaea (Kemenes et al.
1986
), Pleurobranchaea (Bicker et al.
1982
), and Tritonia (Audesirk and Audesirk
1980
). A detailed understanding of these pathways is of
particular importance when they are activated by stimuli used in
conditioning experiments where changes in the strength of synaptic connections within the pathway may represent important mechanisms of
plasticity contributing to the learned response. The pond snail L. stagnalis is a significant model for studying cellular
mechanisms of learning because the neuronal network underlying feeding
is known in considerable detail (for review see Benjamin and
Elliott 1989
), and it was demonstrated that this animal can
undergo reliable appetitive conditioning. This was established at both
the behavioral level in intact animals (Audesirk et al.
1982
; Kemenes and Benjamin 1989a
,b
, 1994
;
Kojima et al. 1996
) and at the cellular level in semi-intact preparations (Kemenes et al. 1997
;
Staras et al. 1998b
, 1999
). In the most thoroughly
investigated of these paradigms the conditioning stimulus (CS), a
tactile input applied to the lips, is paired with the unconditioned
stimulus (US), sucrose (Kemenes and Benjamin
1989a
,b
). The effects of the unconditioned chemical stimulus on
the feeding network were examined in some detail at both the behavioral
and electrophysiological levels (Elphick et al. 1995
;
Kemenes et al. 1986
; Yeoman et al. 1995
), but the detailed function of the tactile input and the interactions between these two stimuli in normal unconditioned feeding behavior were
not investigated previously. Therefore in this study experiments were
first performed to characterize the synaptic inputs to identified motoneurons, central pattern generating (CPG) interneurons, and modulatory interneurons of the feeding system, resulting from tactile
stimulation of the lips in the absence of a chemosensory food stimulus.
Exactly the same types of tactile stimulus and semi-intact preparation
were used as in our recent conditioning experiments (Staras et
al. 1998b
, 1999
). A second type of experiment assessed the role
of tactile inputs after feeding was initiated by the chemosensory food stimulus.
Evidence for cross-modality integration of sensory information was
mainly obtained in Aplysia (Rosen et al.
1982b), where the touch component of food applied to the lips
reinforces the effects of the chemical stimulus to increase the biting
frequency and regularity. In this animal and other opisthobranch
mollusks, e.g., Tritonia (Audesirk and Audesirk
1980
) and Pleurobranchaea (Bicker et al.
1982
), mechanical stimuli alone appear insufficient to initiate
strong ingestion behavior. This was also thought to be the case in
Lymnaea, where it was demonstrated that touch to the lips
cannot elicit unconditioned feeding responses either in the intact
animal or in whole lip, semi-intact preparations where the
electrophysiological responses to lip touch were recorded on
motoneurons (Staras et al. 1998b
). Because sucrose can
successfully activate feeding in Lymnaea (Kemenes et
al. 1986
; Staras et al. 1998b
), it was assumed
that chemical cues were most important in initiating feeding, but a
subsidiary role for tactile input in the initiation of feeding could
not be definitely excluded. An alternative hypothesis is that the lip
touch has no role in the initiation of feeding but may interact with
feeding neurons to modulate a particular phase of the feeding cycle.
The results will show that touch to the lips in a semi-intact
preparation produces a complex sequence of inhibitory and excitatory
synaptic events in all the known neurons of the feeding circuit. The
main functional effect of these inputs is to reinforce rasp- and
swallow-phase activity once feeding was initiated by food. However, in
the absence of a chemical food stimulus, these inputs never led to the
initiation of feeding. This reinforcing rather than initiating function
for mechanosensory inputs from the lips was supported by analysis of
lip stimulation by the food substrate during feeding in freely moving
animals and by experiments testing the contribution of the tactile
stimulus to sucrose-driven fictive feeding activity in semi-intact
preparations. Taken together the data presented here suggest that the
tactile component of food provides a mechanical stimulus to the lip
structures during the rasp and swallow phases of feeding and through
specific connections with neurons of the feeding network mediates a
cycle-by-cycle reinforcement of fast, ongoing feeding.
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METHODS |
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General procedures
Wild-type specimens of adult L. stagnalis were obtained from suppliers (Blades Biological, Kent) and were maintained in large holding tanks containing Cu2+-free water at 18-20°C on a 12 h-12 h light:dark regime and fed lettuce three times a week. Before an experiment animals were moved into smaller tanks in the laboratory.
Whole lip CNS preparation
The preparation used in these experiments was developed to allow
intracellular recordings to be made from identified neurons of the
feeding network while simultaneously presenting a tactile stimulus or a
chemical stimulus, sucrose, to the lip structures (see Staras et
al. 1998b). Animals were dissected under a microscope in a
Sylgard-coated dish containing HEPES-buffered snail saline (Benjamin and Winlow 1981
). A dorsal body incision was
used to expose the CNS, and all peripheral nerves except for the median and superior lip nerves were cut. The buccal mass was excised, but a
short piece of esophagus was left attached to the brain to assist in
pinning out the preparation. The posterior end of the foot was removed,
and the preparation was arranged in a purpose-built Sylgard-coated
electrophysiology chamber containing saline. The lips were pinned down
so that they were accessible for sensory stimulation, and the buccal
ganglia were stretched clear of the head structures over a raised block
of Sylgard (to stabilize the preparation) and rotated 180° so that
the dorsal side was exposed for intracellular recording (Fig.
1A). On several occasions,
where it was necessary to record from ventrally located buccal neurons, one of the buccal ganglia was rotated around the buccal commissure to
expose the ventral surface (Fig. 1B). The whole lip CNS
preparation also made it possible to make recordings from feeding
neurons located in the cerebral ganglia. The outer ganglionic sheath of the cerebral and buccal ganglia was removed with a pair of fine forceps. The second, inner sheath was softened with a nonspecific solid
protease (Sigma, XIV, Sigma Chemical, Poole, UK). The enzyme treatment
was terminated by washing the preparation with fresh saline.
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Intracellular recording techniques
Glass microelectrodes (2 mm, Clark Electromedical, Redding, UK)
were pulled on a Narashigi vertical electrode puller and filled with 4 M potassium acetate to achieve a tip resistance of 10-50 M. The
electrode tips were marked with a black etching ink (Rotring, part no.
595617) to improve visualization. The chamber containing the
preparation was illuminated from a cold light source and viewed under a
zoom microscope. The chamber was connected to a peristaltic pump
permitting a rapid exchange of saline.
Micromanipulators with attached headstage preamplifiers (Neurolog, Digitimer, Welwyn Garden City, UK) arranged around the chamber allowed up to four simultaneous intracellular recordings. Signals were fed into amplifiers (NL102G, Digitimer) incorporating a bridge-balance circuit for current injection and then sent to a storage oscilloscope (GOULD 1604, Gould Instrument Systems, Hainault, UK), a chart recorder (GOULD TA240S), and a DAT recorder (BIOLOGIC DTR-1801, Biologic Science Instruments, Claix, France).
Identification and selection of cell types
The main objective of the electrophysiological experiments was
to monitor the synaptic inputs to identified neurons of the feeding
network evoked by tactile stimulation to the lips. The feeding network
is made up of CPG interneurons, motoneurons, and modulatory
interneurons (see Benjamin and Elliott 1989). The three behavioral phases of rhythmic feeding behavior, protraction, rasp, and
swallow, are generated by three main types of CPG interneurons known as
N1, N2, and N3 (Rose and Benjamin 1981b
) (Fig.
1B), each of which have two subtypes. These are the N1
medial (N1M) and N1 lateral (N1L) cells (Yeoman et al.
1995
), the N2 dorsal (N2d) and N2 ventral (N2v) cells
(Brierley et al. 1997
), and the N3 tonic (N3t) and N3
phasic (N3p) cells (Elliott and Benjamin 1985a
). All the
CPG interneurons occur as bilaterally symmetrical pairs of cells on the
dorsal surface of the buccal ganglia, except for the N2v cells, which
are on the ventral surface. The identity of the interneurons was
established by position, firing activity, and synaptic inputs they
receive (summarized by Brierley et al. 1997
;
Staras et al. 1998a
; Yeoman et al. 1995
).
The feeding motoneurons recorded in this paper (B1, B2, B3, B4, B4CL,
B5, B7a, B8, and B10; Fig. 1B) are located in bilaterally symmetrical pairs on the dorsal surface of the buccal ganglia (Benjamin and Rose 1979; Rose and
Benjamin 1979
). They receive synaptic inputs from the CPG
interneurons and are classified as protraction- (B1, B5, and B7a),
rasp- (B3, B4CL, and B10), or swallow- (B2, B4, and B8) phase neurons,
defined by the phase in which they become active (see Benjamin
and Elliott 1989
).
The three modulatory neurons examined were the slow oscillator (SO),
the cerebral ventral 1a (CV1a), and the cerebral giant cell (CGC)
interneurons (Fig. 1B). The SO is a single unpaired neuron
in either the left or right side of the dorsal surface of the buccal
ganglia (Rose and Benjamin 1981a). The CV1a cells, a
bilaterally symmetrical pair of neurons, are located on the ventral
surface of the cerebral ganglia (McCrohan 1984
). Both the SO and the CV1a cells can drive fictive feeding activity in the CPG
when they are depolarized, and they both fire in the protraction (N1)
phase. The CGCs, a bilaterally symmetrical pair of large neurons in the
anterior lobe of the cerebral ganglia, fires tonically showing moderate
entrainment to the feeding rhythm (McCrohan and Benjamin
1980
).
Salines
Two modified HEPES-buffered salines were used to investigate the
nature of the lip touch synaptic response. High Mg2+, low
Ca 2+ (HiLo) saline + EGTA (composition described by
Elliott and Benjamin 1989), which contains virtually no
Ca2+ and nine times the concentration of Mg2+
present in normal saline, blocks chemical synapses by replacing Ca2+ ions necessary for synaptic transmission with
Mg2+ and was used to test for the presence of electrotonic
synapses. The monosynaptic nature of chemical synapses was tested by
bathing the preparation with high Mg2+, high
Ca2+ (HiDi) saline. This saline is known to increase the
threshold of intermediate neurons (Elliott and Benjamin
1989
) and block polysynaptic pathways.
Tactile and chemical stimulation of the lips in semi-intact preparations
An electromagnetic coil-driven mechanical probe was used to
deliver the tactile stimulus to the lips of the animal. This was the
same stimulus used in previous experiments to test the survival of a
behaviorally conditioned response in semi-intact preparations derived
from trained whole animals (Staras et al. 1998b, 1999
) and was designed to closely mimic the CS used to train the whole animal
(see Kemenes and Benjamin 1989a
,b
; Staras et al.
1998b
, 1999
). The end of the probe consisted of a thin wedge of
soft flexible plastic. This stimulus, which was of standard duration and intensity, induced a small noise bar (at onset and offset) on the
recording equipment so that accurate information about touch response
latencies could be gathered.
A sucrose solution (0.01 M) was used to activate feeding in the
semi-intact preparations. This stimulus, which is the same as that used
in a previous study in Lymnaea to activate feeding behavior
(Staras et al. 1998b), was delivered from a thin plastic tube positioned at the front of the experimental chamber. Sucrose was
released from the end of the tube with a syringe and diffused passively
across the lip chemosensory structures. In this way the tactile
component of sucrose application could be minimized.
Behavioral function for lip tactile stimulus
The rasping movements of freely moving whole animals were recorded on videotape as they carried out consummatory feeding behavior in an inverted position at the water surface. This is the feeding position they normally adopt when they feed on floating pond weed in their normal environment or in aquarium tanks in the laboratory. Animals were presented with strips of fish food flake (AQUARIAN herbivore flakes, Pedigree Petfoods, Melton Mowbray, UK), which are presumed to have tactile properties similar to the vegetative diet of algae-covered pond weed or lettuce that they normally consume but had the advantage of being semi-transparent, allowing clear visualization of the lips, radula (rasping organ), and mouth during consummatory behavior. This behavior was recorded with a charge-coupled device camera (DXC-151P, Sony, Japan) mounted onto a dissecting microscope (Leica Wild M420, Heerbrugg, Switzerland), and still frames were captured from the video sequence with computer software.
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RESULTS |
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Touch-evoked synaptic responses on identified feeding neurons
The lip touch response in feeding-related neurons was investigated
in a semi-intact lip-CNS preparation that consisted of the head of the
animal attached to the CNS by two pairs of lip nerves (Fig.
1A). This allowed access to the buccal and cerebral ganglia
for intracellular recording and to the lips for tactile stimulation
(see METHODS). The tactile stimulus consisted of a brief
touch applied to the lip structures anterior to the mouth and most
closely resembled the area of the lips accessible for stimulation in
the freely behaving animal when conditioning experiments were performed
in vivo (Kemenes and Benjamin 1989a,b
, 1994
;
Staras et al. 1998b
, 1999
). The data will show that a
response to lip touch could be recorded on all the identified feeding
interneurons and key motoneurons. Each neuron type exhibited a complex
but characteristic lip touch synaptic response, the basic form of which
remained consistent between preparations. There were clear differences
in the overall effects of touch stimulation, depending on where the
neurons fire within the behaviorally defined feeding cycle. To
emphasize this finding, the lip touch responses on neurons of the
feeding system are categorized subsequently in terms of the phase of
the feeding cycle in which they fire, e.g., protraction, rasp, or swallow.
PROTRACTION-PHASE NEURONS. There are a variety of CPG interneurons and motoneurons (as well as modulatory interneurons) whose activity is largely confined to the N1 or protraction phase of the feeding rhythm. If tactile inputs from the lips were involved in initiating feeding protraction-phase neurons should be excited. One of the most important of these cell types is the CPG interneuron N1M. Its intrinsic excitability and endogenous bursting properties suggest that it is likely to be an important component in making the decision to feed and as such represents a pivotal element in the feeding circuit. For these reasons its synaptic response to tactile stimulation of the lip was used as a model for comparison with touch responses in other interneurons and motoneurons.
LIP TOUCH RESPONSE ON THE N1M PATTERN-GENERATING INTERNEURON. The lip touch response recorded in the N1M was complex. At resting membrane potential touch never evoked spike activity or drove fictive feeding rhythms in the N1M (n = 50 cells). Instead it evoked a complex sequence of synaptic events that were mainly inhibitory. A typical example of the N1M lip touch synaptic response is shown in Fig. 2Ai. The inputs are summarized in the schematic diagram in Fig. 2B. Initially a lip touch evoked a smooth hyperpolarization (input I) with a gradual recovery followed by a long-lasting slow depolarizing wave (input III, Fig. 2Ai). Superimposed on the recovery phase were large single inhibitory postsynaptic potentials (IPSPs, input II) and later a continuous series of small unitary IPSPs (input IV) that appear to continue throughout the period of sustained depolarization (input III).
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LIP TOUCH RESPONSE ON OTHER PROTRACTION-PHASE INTERNEURONS
(SO, N1L, AND CV1a).
The SO is a modulatory protraction-phase interneuron that like the N1M
is capable of driving the feeding CPG (Elliott and Benjamin
1985b). As was the case with the N1M, the SO received an
initial inhibitory touch-evoked input (n = 20 cells)
with a comparable latency but of a smaller amplitude (Fig.
3A). In the SO, there was also
evidence for the presence of the delayed subthreshold depolarizing
synaptic components seen on the N1M as well as small unitary IPSPs
(arrowed in Fig. 3A).
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LIP TOUCH RESPONSE ON PROTRACTION-PHASE MOTONEURONS (B5,
B7a, AND B1).
In addition to CPG and modulatory interneurons several identified
motoneurons are known to fire in the protraction phase of the fictive
feeding rhythm. Motoneurons B5 and B7a, as well as being involved in
activating buccal musculature, are also electrotonically coupled to the
N1M (Elliott and Kemenes 1992; Staras et al.
1998a
), and their activity closely follows that recorded in
this CPG interneuron. Theoretically, both cells could activate a
fictive feeding rhythm via the electrotonic connection with the N1M
cells. In fact, both show a lip touch response (B5, n = 3 cells; B7a, n = 4 cells) in which the initial
inhibition and subsequent mixed excitatory and inhibitory components
seen on the N1M appear to be present, although they can be variable in
amplitude (Fig. 3, D and E). It is unlikely that
the shared synaptic inputs could be due to electrotonic coupling alone
as this is insufficiently strong (unpublished observations;
k ~10-15%) to generate similar amplitude subthreshold synaptic events in all the coupled cells.
Rasp-phase neurons
The main effects of lip touch on the rasp-phase CPG interneurons were depolarizing, the opposite to the protraction-phase neurons. Although the responses were usually subthreshold for spike initiation, they could still be important in reinforcing the retraction phase of feeding.
LIP TOUCH RESPONSE ON RASP-PHASE CPG INTERNEURONS (N2v
AND N2d).
The N2v, a recently identified rasp-phase CPG interneuron, has
endogenous plateauing properties (Brierley et al. 1997).
The N2v lip touch response consisted of a single depolarizing input, the latency to onset of which was similar to the first phase of inhibition (input I) recorded in the N1-phase neurons (Fig.
4Ai). In most cells this
depolarizing wave on the N2 versus was subthreshold (n = 8 cells, Fig. 4Ai), but in one preparation it was
sufficient to consistently trigger a full-sized plateau with
superimposed truncated spike-like events characteristic of this cell
type (Brierley et al. 1997
) (Fig. 4Aii). The
N2d, a second type of rasp-phase CPG interneuron, also exhibited an
immediate depolarization in response to touch (Fig. 4B).
Usually this event had a slower rise time than that observed in the
N2v, which made the latency of its onset difficult to assess. The touch
input was never sufficient to trigger spikes in N2d (n = 9 cells). The N2d appeared to be hyperpolarized after the initial
depolarization, which often caused the membrane potential to fall below
the pretouch resting potential. This apparent inhibition had a very
slow waveform and had a similar duration to the input III depolarizing
wave on the N1M. Most of the depolarizing responses recorded on cells
were subthreshold for spike initiation so that they could not be
responsible for the synaptic inputs occurring on other cells such as
the N1Ms.
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LIP TOUCH RESPONSE ON RASP-PHASE MOTONEURONS (B3, B10, AND B4CL). The B3 motoneurons receive characteristic synaptic inputs after lip touch (n = 50 cells) that are identical in latency and waveform but are of opposite sign to those recorded in the N1M (Fig. 4C). The initial N1M inhibitory input I (Fig. 4C) appeared as an equal amplitude depolarizing component on B3, usually subthreshold for spike initiation. The unitary IPSPs recorded in the N1M were apparent as unitary EPSPs on the B3 (II, Fig. 4C) as were the smaller IPSPs (IV) occurring later during the long-duration hyperpolarization (Fig. 4D). The synaptic depolarizing input III in N1M (Fig. 4C) was seen as a mirror-image, long-duration IPSP in the B3, and the amplitude and time course were identical.
The B10 cell is a motoneuron of the radular tensor muscle (Staras et al. 1998aSwallow-phase neurons
Evidence from responses recorded in N1/protraction- and N2/rasp-phase neurons indicates that possible roles for the lip touch pathway are the reinforcement of the rasp phase and the inhibition of the protraction phase of the feeding cycle. In a feeding rhythm, the rasp phase is followed by activity in the swallow-phase neurons. Touch-evoked activity causes delayed excitation of swallow-phase neurons, which suggested that both retraction phases of the feeding cycle (rasp and swallow) were reinforced by lip touch.
LIP TOUCH RESPONSE ON SWALLOW-PHASE CPG INTERNEURONS
(N3p AND N3t).
The N3t fires a strong burst of spikes throughout the swallow phase of
the cycle (Elliott and Benjamin 1985a). Initially, the
lip touch response (n = 4 cells) causes a brief, weak
inhibition, after which the cell fires a series of spikes (Fig.
5Ai). No obvious depolarizing
input can account for this delayed spike activity, and it may be due to
postinhibitory rebound, a property that has previously been shown to be
present in the N3ts (Elliott and Benjamin 1985a
) with
hyperpolarizing stimuli that were similarly weak and brief as the
touch-evoked inhibitory input seen in this study.
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LIP TOUCH RESPONSE ON SWALLOW-PHASE MOTONEURONS (B2, B4, AND B8).
The B2 motoneuron, involved with gut contraction, exhibits only weak
entrainment to the feeding rhythm firing throughout the swallow phase.
In most recordings (n = 4 cells) the B2 bursts spontaneously, and in these instances a lip touch triggers an extra
burst of spikes (Fig. 5C). The B4 and B8 are the main
swallow-phase motoneurons. They receive a prolonged inhibition during
the N2 phase of feeding and so fire later than the B4CLs throughout the swallow phase of the feeding cycle. Their activity closely follows that
in the N3t interneurons to which they are electrotonically coupled
(Staras et al. 1998a). The lip touch responses on the B4
and B8 neurons compared with the B4CL neurons reflect these functional
differences (Fig. 5Di). The B4 and B8 neurons, like the
N3ts, initially show a brief inhibition, and this is followed by a
rebound excitation (n = 20 cells) often leading to full
spikes. This contrasts with the B4CL neurons discussed previously,
which are depolarized earlier and show no initial phase of inhibition. This is shown more clearly in the expanded time-base recording in Fig.
5Dii. One striking feature of the response to touch is that
the B8 cells always fire during the delayed depolarizing response to
touch. Electrotonic EPSPs occur on the B4 recorded at the same time,
but often they show no full spikes (Fig. 5Dii). These
results suggest that as well as depolarizing the N2 cells and promoting
rasp the touch CS also excites the swallow-phase motoneurons after a
delay. This suggests that touch during feeding would tend to strengthen
the rasp followed by swallow sequence of activity and would help to
coordinate the sequence.
Lip touch response on the modulatory CGCs
The CGCs are two large coupled serotonergic neurons, one
present in each anterior lobe of the cerebral ganglia (McCrohan
and Benjamin 1980). During fictive feeding they fire tonically
with a tendency to be excited during N1/protraction and inhibited
during N2/rasp. They have complex synaptic connections with most of the CPG interneurons and motoneurons and are known to be important in
modulating the feeding network (Tuersley and McCrohan
1989
; Yeoman et al. 1996
). In response to lip
touch (n = 10 cells) they show a burst of spikes of
~1 s in duration, which is followed by a recovery period during which
no action potentials occur (Fig. 6A). The onset of this
response is comparable with the latency recorded on most buccal
interneurons. Although it is clear from the relatively slow activation
that the CGCs are not responsible for the earliest response to touch
recorded on other feeding neurons, suppression experiments in which
both CGCs are hyperpolarized suggest that they may play some role in
the generation of secondary components of the touch-evoked activity in
buccal neurons. For example, Fig. 6B shows that the
suppression of CGC spike activity (in the same preparation as in Fig.
6A, n = 2 cells) does not abolish the
initial inhibitory component recorded on the N1M, but the subsequent
N3t inputs (input II) are markedly reduced in amplitude. CGC
suppression also apparently removes some of the depolarizing component
(input III) of the N1M touch-evoked response. This is reduced to the
point where the remaining input III inhibitory inputs hyperpolarize the
N1M cell below the potential seen before stimulation (Fig.
6B). Together these findings indicate that the normal burst
of touch-evoked activity in CGCs may assist the N3ts recovery from
inhibition to trigger spikes and also assist the recovery of the N1M
from its primary touch-evoked inhibition. These conclusions are
consistent with previous work demonstrating that CGCs have a
monosynaptic excitatory connection with the N3t interneurons and a slow
excitatory connection with the N1M (Yeoman et al. 1996
).
The ability of the CGCs to generate both unitary input II inhibition
via the CGC
N3t
N1M pathway and slow input III depolarization
via the CGC
N1M excitatory connection is confirmed in Fig.
6C, where instead of touch current induced bursts of CGC
spikes evoked these types of input (compare with Fig. 6A). CGCs also have synaptic connectivity to other CPG interneurons, SO, and
many motoneurons and so may also shape the general lip touch-evoked
response in all these cell types. This remains to be investigated.
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Behavioral function for the lip touch pathway
The electrophysiological data presented previously show that the
touch stimulus plays a role in reinforcing the rasp phase of the
feeding cycle, but whether touch to the lip as a component of a solid
food stimulus could provide a sensory input to the feeding system
during normal ingestion of food was unclear. This was tested by
videotaping four freely behaving animals that were feeding on solid
fish food flake in an inverted position at the water surface. An
example of one ingestion sequence is shown in Fig.
7. The sequence of seven frames
(approximately every 200 ms) shows one complete cycle of feeding with
protraction (P), rasp (R), and swallow (S) marked on each frame.
Several cycles of feeding already occurred before this sequence, so
part of the food piece was already inside the mouth. In frames 1-3,
the mouth opens during the first part of a new feeding cycle, and this
is known to be accompanied by protraction of the radula (see
Rose and Benjamin 1979). The food does not move during
this phase. However, during rasp (frames 4-5) the food is moved
further into the mouth. The first forward, then backward, and upward
rotating movement of the radula pulls food across the area of lip
anterior to the mouth (see Fig. 7, arrows), which provides a mechanical stimulus to the lip. This particular location on the lip is the same
one used for application of the mechanical probe in the semi-intact preparation. During swallow (Fig. 7, S, frames 6-7), the tactile stimulus is maintained as the food is being pulled even further into
the mouth. This sequence of feeding behavior was typical of all four
animals examined. Although food moving across the lip structures is
likely to be the primary source of lip tactile stimulation during
feeding, the maximal extrusion of the radula during late protraction
and rasp pushes the lips forward onto the food substrate (not shown),
and this would probably provide a second component of mechanical
stimulation. This observation is supported by early work of Hubendick
(1956)
, who examined the mechanics of feeding in Lymnaea. He
demonstrated clearly that the lip structures are pushed onto the
substrate during the rasp phase. This together with the movement of
food across the lips results in the tactile stimulation being strongest
for ~400 ms in the rasp phase of the feeding cycle.
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Interaction between tactile and chemical stimuli in fictive feeding rhythms
The food piece used in the behavioral experiments of Fig. 7
presumably also contained a chemical as well as a mechanical stimulus. This chemical component of the food stimulus must have been essential for initiating ingestion as simply stroking the lips with a mechanical probe never activates maintained sequences of feeding movements in the
whole animal. The relative roles of chemosensory and mechanical stimuli
in generating ingestion proved difficult to study quantitatively in the
intact snail by using food pieces, so electrophysiological experiments
were carried out instead with the semi-intact preparation (Fig.
1A). This type of preparation allowed both types of stimuli to be applied separately or together. By using a sucrose solution as a
chemostimulant (see Kemenes et al. 1986; Staras
et al. 1998b
) rather than solid food, it was possible to
minimize the normal tactile component. The lip tactile stimulus could
be made explicit by applying it with the same soft plastic mechanical
probe that was used to map the tactile inputs in quiescent
preparations. The duration of the stimulus was ~400 ms, the same as
the duration of the strongest tactile stimulation during feeding in
intact animals. In these experiments, a rasp-phase motoneuron (B3) was used to monitor fictive feeding activity in a sucrose-driven rhythm, and the effects of additional tactile stimulation were tested.
Purely sucrose-driven rhythms were often slow, and this is a common
observation in semi-intact preparations (Staras et al. 1998b). In the preparation shown in Fig.
8A, patterned CPG synaptic input was obvious with sucrose stimulation, but the B3 cell did not
fire consistently. However, with the addition of touch to the lip
during the rasp phase, the cell fired in a much more regular manner and
the frequency of the rhythm was higher (Fig. 8B). The brief
(~400 ms) tactile stimulus was provided at the onset of each rasp
phase during continuous sucrose stimulation to mimic the natural
stimulating effect of food revealed in the video sequence. An expanded
cycle from Fig. 8B (dashed box) is shown in Fig.
8D, illustrating that the touch stimulus is applied after
the onset of the rasp phase and so is contributing to the reinforcement of rasp, not its initiation. The ineffectiveness of lip touch stimulation alone in initiating and maintaining a fictive feeding rhythm was confirmed in the same preparation (Fig. 8C).
Periodic application of touch alone produced the expected weak
depolarizing response on B3, but this was never strong enough to evoke
bursts of spikes.
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We also tested for the effects of touch stimuli applied during interburst intervals rather than during bursts and for long-lasting effects of touch that might continue beyond the duration of a single feeding cycle. To achieve this we applied touch at various points during sucrose-activated fictive feeding. When touch stimuli were applied in the intervals between rasp-phase bursts, this had no effect on the duration of ongoing bursts or the frequency of the rhythm (Fig. 8Ei). In contrast, repeated application of touch during every other B3 burst increased the overall frequency of the rhythm. The first effect of the touch underlying this frequency increase was a reduction of the duration of the touch-stimulated bursts only (Fig. 8Eii). These were significantly shorter (3.7 ± 0.2 SE s, n = 3 bursts) compared with subsequent nontouch-stimulated bursts in the same rhythm (11.5 ± 0.3 s, n = 3 bursts) (paired t-test: df = 2, t = 17.8, P < 0.001). This first effect of the touch therefore did not last longer than a single cycle. The second effect of the touch was that the intervals that followed each burst (mean: 13.5 s, SE ±0.2, n = 6 bursts), including nonstimulated ones (13.0 ± 0.6 s, n = 3 bursts; Fig. 8Eii), were shorter when compared with the rhythm shown in Fig. 8Ei, in which touch was applied in the intervals between bursts or not applied at all (38.8 ± 2.6 s, n = 5 intervals; unpaired t-test: df = 6, t = 8.06, P < 0.001). The mechanism for the complex changes occurring during the touch stimulation of alternate B3 bursts and affecting interburst intervals is unclear but must be longer lasting than a single cycle.
The experiments comparing sucrose-driven fictive feeding rhythms with
those where touch stimuli were used to reinforce each rasp-phase burst
suggested that the addition of touch in a sucrose-driven rhythm
increased both the frequency and regularity of the fictive feeding
rhythm. The effect on frequency was analyzed quantitatively by
comparing the effects of sucrose alone and sucrose + touch in six
animals. The number of feeding cycles in the 2 min after the first
cycle after sucrose presentation was counted with and without tactile
stimulation. When analyzed statistically, the number of cycles with
sucrose + touch (median: 12.5, interquartile interval: 10.5-14.0) was
found to be significantly higher than with sucrose alone (median: 8.0, interquartile range: 5.5-10, Wilcoxon matched-pairs rank test:
Z = 0.02, P < 0.04), as illustrated in Fig. 9A.
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The effect of touch on the regularity of sucrose-driven fictive feeding cycles was examined in a frequency histogram (Fig. 9B). The frequency of occurrence of cycle periods as a percentage of the total observations for sucrose alone and sucrose + touch was compared in the same six preparations. In the sucrose + touch rhythms, 54.7% of cycle periods were within the 5- to 10-s bin, whereas with sucrose alone cycle periods were much more widely distributed with 90% spread over four bins between 5 and 25 s (Fig. 9B). Statistical analysis (F test) showed that in sucrose-alone rhythms the fictive feeding rate was significantly more variable than in sucrose + touch rhythms (F = 5.17, P < 0.001).
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DISCUSSION |
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Lip tactile responses in feeding neurons
One of the main objectives of this paper was to characterize
and functionalize the lip touch CS pathway in terms of the responses recorded on identified neurons of the feeding network. One possible role for the lip touch would be as part of the mechanism for initiation of the feeding response to potential food items. If this were the case,
touch to the lips would be expected to excite protraction-phase neurons, such as the SO or the N1M cells. These are capable of driving
a fictive feeding pattern of N1 N2
N3 activity in isolated
preparations (Elliott and Benjamin 1985b
; Rose
and Benjamin 1981a
). An alternative notion is that the lips may
be stimulated as the animal contacts the food substrate, and in this
case the tactile pathway may be more important in modulating ongoing
chemically driven feeding activity, particularly in the phase of
feeding where the lips receive most tactile stimulation. These
alternative hypotheses were examined by intracellularly recording
identified neurons in the feeding network and testing the touch
responses in each (summarized in Fig.
10).
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The data presented in this paper show that the protraction-phase CPG
interneurons (N1M and N1L) and motoneurons (B5 and B7a) received mainly
inhibitory inputs (input I, II, and IV, Fig. 10, P), and despite the
presence of a delayed depolarizing wave (input III) they were never
depolarized enough by the touch input to reach firing threshold. Thus
it seems unlikely that the type of lip touch used here is normally
involved in the sensory initiation of feeding. Previous experiments
with a different, split-lip preparation showed that over one-half of
them gave brief fictive feeding responses to touch, consisting of one
to three cycles of activity (Kemenes et al. 1997), but
these never occurred in the current whole lip preparation. The reason
for the difference in the responses in these two preparations is
unclear. It may be explained by a number of different factors such as
the absence of contralateral connections in the split-lip preparation,
which may be potentially important in the final shaping of sensory
signals arriving at the brain. However, in both whole and split-lip
preparations a maintained fictive feeding response to touch could only
be evoked after a lip touch CS was repeatedly paired with a US, which
was either the stimulation of an interneuron driving the feeding CPG
(Kemenes et al. 1997
) or a food stimulus (Staras
et al. 1998b
). In whole lip preparations, only the B1
protraction-phase motoneurons were excited by unconditioned touch, and
as these are probably salivary gland motoneurons and play no role in
pattern generation this is unlikely to be of major significance in the
sensorimotor organization of the buccal mass movements. Rasp-phase
interneurons (N2d and N2v) and motoneurons (B3, B10, and B4CL) all
showed depolarizing responses to touch (Fig. 10, R) although this
rarely evoked action potentials. However, during a CPG-driven rhythm,
touch arriving during the rasp phase of the feeding cycle would tend to
further depolarize the rasp-phase neurons, which were already
depolarized by the appropriate synaptic inputs and in this way
reinforce ongoing activity in both interneurons and motoneurons.
Swallow-phase interneurons (N3p and N3t) and motoneurons (B4, B8, and B2) all showed initial inhibition followed by depolarization that was often sufficient to drive them into spike activity (Fig. 10, S). Assuming that touch mainly occurs during rasp this would tend to prevent activity during the protraction phase of the feeding rhythm and then promote swallow. The overall effect of touch on the retraction phase of the feeding cycle in the intact snail would be to reinforce rasp-swallow neuronal activity in the correct sequence and inhibit protraction.
Touch-induced responses also occurred on modulatory neurons (Fig. 10,
M). These were complex and not always easy to interpret from the
functional point of view. The SO was inhibited by touch, and given that
it is a protraction-phase neuron that can contribute to the activation
of feeding (Yeoman et al. 1995) it was not surprising that like the N1M it showed this response. However, contrary to expectation was the touch-induced depolarization of the CV1a, another
protraction-phase modulatory neuron. This effect was always subthreshold but still interesting. It suggests that the CV1a may play
a different role in feeding to the SO, a neuron with which it is often
compared. The weak depolarizing tactile input to the CV1a may also be
potentially important because it may be strengthened during appetitive
training by using touch as the CS (Kemenes and Benjamin
1989
; Staras et al. 1998b
, 1999
). The CGCs,
another modulatory neuron type, which play an important role in the
feeding system as gating neurons as well as having effects on frequency
control of the feeding CPG (Yeoman et al. 1994a
,b
), were
also excited by touch. Activation of the CGCs would promote activation
of feeding in general, but in this context they were most interesting
because they were at least partly responsible for the long depolarizing
wave (input III) recorded on the N1M cells. This suggests that they
could be responsible for similar inputs on other cells recorded in this
paper, although this was not tested directly.
Functional role for lip tactile response in feeding neurons
The nature of the touch responses recorded electrophysiologically on feeding neurons supports the notion that this pathway may be important first in contributing to the rasp phase of feeding and then in activating neurons associated with the swallow phase. Although several neurons in the feeding network contribute to the touch response on other neurons through previously reported synaptic connections (e.g., N3ts and CGCs), none of the identified feeding neurons appears to be responsible for the primary synaptic response, the rasp-reinforcing component, which indicates that this probably arises from neurons in as-yet unidentified lip mechanosensory pathways.
The hypothesis that the touch CS pathway reinforced retraction-phase activity was supported by video analysis of feeding behavior showing that the lips received the strongest tactile stimulation during the rasp and swallow phases as solid food was taken into the mouth. The duration of the period while this strong tactile stimulation was maintained was ~400 ms, the same as the duration of the tactile stimulus used in the semi-intact preparations. By using a semi-intact whole lip-CNS preparation we were able to separate the tactile and chemical components of a food stimulus and test the effects of lip touch stimulation in sucrose-driven fictive feeding rhythms. We demonstrated that, when a lip touch equivalent to the proposed behaviorally relevant input was provided during each rasp phase, it led to more rhythmical rasp-phase bursts in retraction-phase motoneurons and a higher frequency of fictive feeding than sucrose alone, providing evidence that the lip stimulus is important both in reinforcing a particular phase of the rhythm and also as a general stimulus for maintaining a high-frequency rhythm. The mechanism by which touch increases the frequency of fictive feeding is unclear but seems to be based on a simultaneous shortening of both the duration of rasp-phase bursts and the intervals between them (see Fig. 8).
The current work relates to a previous study on the opisthobranch
mollusk A. californica, which provided evidence to show that
integration of tactile and chemical cues was most effective in
achieving high-frequency consummatory feeding behavior (Rosen et
al. 1982b). In the presence of a tactile and chemical stimulus, feeding was more regular and occurred at a higher frequency than when a
chemical stimulus was applied alone. In T. diomedea,
chemical stimulation alone is important in eliciting repeated biting
movements (Audesirk and Audesirk 1979
). However,
mechanical stimulation of the cavity of the buccal mass in the presence
of a chemostimlus was demonstrated to bring about inhibition of biting
responses and promotion of the swallow phase of feeding
(Audesirk and Audesirk 1979
). In this mollusk,
mechanosensory neurons with receptive fields in the buccal mass that
have the appropriate synaptic connectivity to bring about the
initiation of swallow phase were also identified (Audesirk
1979
). It is likely that mechanosensory neurons are present in
the lip structures of Lymnaea, which also have
phase-dependent effects. This results in the inhibition of protraction
and the promotion of the rasp and swallow phases of feeding. The
integration of chemical and tactile information in feeding at the
cellular level was not analyzed in detail in any system, although
higher-order neurons that may be sites of convergence of both chemical
and tactile cues were identified in several systems such as the
cerebral-to-buccal interneurons in Aplysia (Rosen et
al. 1991
), the cerebral to buccal interneurons in Limax
maximus (Delaney and Gelperin 1990
), and complex
mechanoreceptors in Tritonia (Audesirk and Audesirk
1980
). As we demonstrate in the present paper, in
Lymnaea the initial synaptic inputs arising from the lip
tactile stimulus can be recorded on the majority of feeding
motoneurons, CPG, and modulatory interneurons with approximately the
same latency, suggesting that convergence of tactile and chemical may
occur on all the feeding neuron types together rather than in a more
hierarchical arrangement where higher-order neurons act as sites of
sensory integration.
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ACKNOWLEDGMENTS |
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This work was supported by Biotechnology and Biological Sciences Research Council Grant GR/J33234.
Present address of K. Staras: Dept. of Physiology, Royal Free and University College Medical School, University College London, London NW3 2PF, United Kingdom.
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FOOTNOTES |
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Address for reprint requests: K. Staras c/o G. Kemenes, Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.
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 10 August 1998; accepted in final form 24 November 1998.
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REFERENCES |
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