Feeding in Helisoma trivolvis: The Morphological and
Physiological Bases of a Fixed Action Pattern
STANLEY B. KATER
Department of Zoology, The University of Iowa, Iowa City, Iowa 52242
SYNOPSIS. This report is a description of feeding in the pulmonate snail, Helisoma
trivolvis and provides a detailed account of: (i) the behavior, (ii) the muscular system,
(ill) the muscle activity patterns, (i\) the neurouiusculai iclationships, (v) the niuioneurons to specific muscles, and finally, (vi) an introduction to the premotor system.
Feeding is the result of the integration of sensory information onto components of a
central program which is deiived from connections within the buccal ganglia. This
report emphasizes the analysis of the centrally programmed components of the feeding
output by characterizing interactions among three classes of neurons (approximately
50 cells) in the buccal ganglia: protractor motoneurons, retractor motoneurons, and
the neurons of an electrically coupled neural network which produces both the timing
and driving of motoneuron activity.
INTRODUCTION
Because of the magnitude of the problem
of describing the neuronal events underlying any behavioral act, many investigators
have selected experimental subjects whose
nervous systems are composed of relatively
small numbers of nerve cells. Many of these
neurons can be identified routinely by visual inspection of the living nervous system
(see review by Kater et al., 1974). Since we
are attempting to relate the activity of such
identified neurons to behavioral events, it is
useful to have the opportunity to record
neural activity from relatively intact animals. By employing animals meeting these
criteria we increase our potential for investigating both the biophysical and morphological relationships of neurons mediating
specific subsets of behavioral output.
About five years ago, I selected a particular experimental animal, Helisoma
trivolvis, as the basis for my studies on
neurobehavioral organization. This choice
was based upon the promise that this animal held for allowing implementation of a
broad spectrum of experimental approaches. To date my colleagues and I have
emphasized some of these approaches (e.g.,
physiological, morphological, behavioral)
in our work and reconfirmed the feasibility
of employing others (e.g., developmental
and genetic manipulations). This paper introduces general features of Helisoma trivolvis and presents specific findings on the
neuronal mechanisms underlying a discrete
behavioral act—feeding.
THE EXPERIMENTAL ANIMAL,
Helisoma trivolvis
I am particularly grateful to Mr. Jack Rued
whose technical expertise and dedication greatly
facilitated this work. I thank Drs. Fountain, Kaneko,
Kollros, Llinas, and Nicholson for helpful comments
throughout the course of these investigations and
the preparation of this manuscript. Special thanks
are due Ms. Carolyn Heyer for her expert assistance
with the morphology of the musculature of the
buccal mass as well as her skillful comments on this
manuscript. Thanks also to Freddy Uahls for corroboration of the muscle activity patterns. Professor
Emeritus Gordon Marsh is most gratefully acknowledged for the derivation of the term "cyberchron." This research was supported in part by
PHS grant NS 09696 and NSF University Development Crant GU 2591.
Helisoma trivolvis is a pulmonate mollusc of the order Basommatophora, which,
along with closely related species, is found
in freshwater lakes and ponds throughout
the world. Helisoma is readily raised in the
laboratory and has a generation interval of
approximately 5 weeks. For our routine
studies we employed either animals collected from specific sites and maintained in
the laboratory, or their offspring which
have been raised in the laboratory. Specific
investigations have made use of one of the
1017
1018
STANLEY B. KATER
100 pm
FIG. 1. A photograph of the living buccal ganglia
of Helisoma trivolvis. The rather asymmetric appearance of neuronal somata in the two ganglia is
an artifact caused by the pressure of a cover slip.
several inbred (approaching isogenic) lines
which we have produced by full sib inbreeding procedures over the past 3 years.
The central nervous system of Helisoma
is composed of six closely approximating,
but not fused, major ganglia (or pairs of
ganglia). The majority of the neuronal
somata composing these ganglia are arfanged cortically as a rind on the outer
surface of each ganglion and are readily
observed in living animals with the aid of a
low-power dissecting microscope (Fig. 1).
The yellow, or in some cases blue-white,
color of the somata is sharply offset by the
red color of the extraneuronal components
of the ganglia. Consistent differences in
cellular pigmentation, coupled with size,
shape, and location have proven excellent
criteria for routine identification of many
neurons (Kater and Kaneko, 1972; Kater
and Rowell, 1973). We have concentrated
the majority of our attention on the paired,
buccal ganglia which are located on the
posterior aspect of the ventral surface of the
buccal mass and are connected with the
remainder of the CNS by the cerebrobuccal
connectives. Our observations have led to
BRN
FIG. 2. A map of the characteristic location of
identified neuronal somata in the buccal ganglia of
Helisoma. BRN, buccal retractor nerve; CBC, cerebrobuccal connectives; ET, esophageal trunk; HBN,
heterobuccal nerve; PBN, posteriobuccal nerve;
PJN, posteriojugalis nerve; VBN, ventrobuccal
nerve. Inset: A map depicting an aberration from
the more usual case. Even when neurons appear
to be displaced on the surface of these ganglia,
functional clusters of neuronal somata retain their
normal associations.
FEEDING IN
Helisoma trivolvis: A
the conclusion that the neuronal somata
are arranged in functional groups on the
surface of each of the bilaterally symmetrical ganglia (Fig. 2). Note the grouping of
protractor motoneurons (cells 17-21), retractor motoneurons (cells 28, 33, and 9-14),
as well as interneuron groups "forty" and
"fifty." While absolute position of a given
neuron is variable with respect to its location on the surface of the ganglion, the relative position of the neurons, within a group,
remains quite constant. An extreme deviation from the standard neuronal map is
shown as an inset (Fig. 2). In this case there
has been a nearly complete inversion of
group positions, but the location of neurons
within each group remained consistent with'
the more usual observations. Thus, while
there is some variability in the juxtaposition of neuron clusters, as a rule, neurons
maintain fixed orientations within their
functional groups. We, therefore, use cell
location within a group as major criterion
of the identification of individual nerve cell
bodies. It should be noted that on the basis
of light microscope observations, more than
75% of the neurons in the buccal ganglia
are located on the dorsal surface. Thus,
with the simple procedure described previously (Kater and Kaneko, 1972), we are
able to gain access to and penetrate a large
proportion of buccal ganglion neurons in
situ.
THE FEEDING BEHAVIOR
Helisoma possesses a number of discrete
acts in its behavior repertoire (Kater and
Kaneko, 1972). I have been most attracted
to those behaviors which are composed of
cyclically recurring events. Such outputs, by
virtue of their continued repetition, facilitate our ability to examine in detail all
components involved in each phase of the
motor output.
Our ethological studies on Helisoma
closely parallel those made by others on the
related basommatophoran snail, Lymncea
stagnalis. Snails may display "spontaneous"
feeding behavior; when not feeding, they
can be induced to feed by the presence of
food or specific chemostimulants to either
FIXED ACTION PATTERN
1019
the lips or the cephalic tentacles (Bovbjerg,
1968; Weis, 1972). Conversely, feeding can
be abruptly inhibited by the presentation
of any of a number of aversive stimuli.
Control and modulation of the feeding output may reside in several of the ganglia
comprising the CNS. On the other hand,
the actual neuronal machinery responsible
for the feeding movements is confined, in
large part, to the buccal ganglia. This has
been demonstrated by observing the feeding
movements generated by isolated buccal
mass/ buccal ganglion preparations. Under
these conditions, radular movements are
identical to those seen in intact animals
(within, of course, the limits placed on
these movements by mechanical deformation due to the excision of the structures).
Furthermore, one can demonstrate that the
buccal ganglia provide the necessary information for feeding in intact animals by
severing all other connections (i.e., cerebrobuccal connectives) to the CNS and observing the postoperative snails in normal feeding behavior (Goldschmeding and Jager,
1973).
There is a clear succession of steps required for understanding the feeding behavior of Helisoma. We shall first define
the behavior in terms of its component
movements and then describe the musculature moving the buccal mass. We shall
relate the temporal sequence of the activity
in individual muscles to the feeding cycle,
and then identify the motoneurons producing these activity patterns. With this
level of information we can examine the
neuronal machinery underlying the generation of the cyclical motor output driving
the feeding movements. Finally, we can begin to explore the sensory feedback and
higher-order systems which modulate and
perhaps determine whether or not feeding
will occur.
Feeding movements
The feeding activity of Helisoma may
be regarded as a fixed action pattern (FAP)
composed of at least five events. The muscular activity required for feeding can be
divided into the four stages defined by
1020
STANLEY B. KATER
FIG. 3. A diagrammatic representation of the feeding movement of Helisoma trivolvis. Positions 1
through 5 are stages in the confluent movements
which compose a single feeding cycle. Both intact
animals and dissected preparations can display continuous recurrence of this cycle for several hours at
a time. 1, Rest position of the buccal mass, radula
(RAD) and odontophore cartilage (CART). 2,
Buccal mass in initial protraction. } , Buccal mass
fully protracted as well as independent protraction
of odontophore and radula. 4, Slight retraction of
odontophore (as well as independent radular movement, see text). 5, Completion of retraction of the
buccal mass, radula, and odontophore.
Carriker (1946) for Lymnaea stagnalis:
(i) movements of the lips and mandibles
resulting in the opening and closing of the
mouth, (ii) back and forth movements of
the odontophore, (iii) reciprocating movements of the radula over the odontophore,
and (iv) concomitant movements of the entire buccal mass. To these we can add, from
our studies on Helisoma, (v) cyclical activity
of the salivary glands (see section on motoneurons).
Ingestion of food in Helisoma is accomplished primarily through cyclical
scraping action of the rasp-like radula (Fig.
3). A cycle begins as the odontophore and
radula are moved anteriorly and ventrally
(from position 1 to position 3) so as to
appose the radula against the substratum.
During this movement, in which there is
a forward tilting of the buccal mass (protraction), there may be some scraping of
FIG. 4. A view of the intact buccal mass of Helisoma
trivolvis shows eight major muscles, (i) The anterior jugalis (aj) is a large sheet-like external
muscle spanning the anterior two-thirds of the
dorsal and lateral aspects of the buccal mass. Contraction of this muscie facilitates the return of the
odontophore cartilage to its rest position within
buccal mass, (ii) The buccal retractor muscles (br)
are two, stout, bilaterally symmetrical strands of
muscle having their origin at the columellar muscle
and insert on the lateral surface of the buccal mass.
These major extrinsic muscles are primarily responsible for returning the buccal mass to its rest
position with a retraction stroke, (iii) The dorsolateral protractors (dlP) originate on the floor of
the body wall at either side of the mouth and insert
on the dorsal medial external surfaces of the buccal
mass. These muscles tilt the buccal mass approximately 60° in the anterior ventral plane during the
protraction phase of the cycle, (iv) The posterior
jugalis muscle (pj) is a sheet of fibers enveloping
the posterior third of the buccal mass. It has its
origin along the dorsal midline, along the radular
sack, and margins of the odontophore cartilage.
These muscles act to tilt the odontophore forward
(i.e., stage 2-3 of Fig. 3), by raising the posterior
edge of this structure, (v) The postventral levator
(poL) inserts on the posterior ventral surface of
the mass and has its origin on the ventral lateral
body surface. This muscle acts to provide a whiplike thrust to the return stroke of the buccal mass
and odontophore during the retraction phase of
the feeding cycle, (vi) The postventral protractor
(poP) (actually a "retractor"; see text) inserts just
posterior to the preventral levator and just external
to the supralateral radular tensor and has its origin
on the ventral body wall. This muscle provides a
major thrust for retraction of the buccal mass due,
at least in part, to its point of insertion, which
provides a great mechanical advantage with respect
to the distant fulcrum at the mouth, (vii) The
preventral levator (prL) inserts on the ventral floor
of the buccal mass near the midline. Its origin is
as in (v) above. This muscle acts to provide a final
FEEDING IN
Helisoma trivolvis: A
the substratum by the lateral margins of the
radula.
In the next portion of the cycle, movement of the radula relative to the odontophore is the subject of some controversy in
Lymnaea. In Helisoma, we have examined
feeding movements in albino strains, dissected specimens, and embryos about to
hatch. Under all of these conditions we
found that the radula can: (i) maintain
constant position with respect to the odontophore for several cycles of feeding (cf.
Hubendick, 1956), (ii) move independently
of the odontophore during each cycle of
feeding (cf. Carriker, 1946), or (iii) move
back and forth for several diminutive rasps
during a long period of odontophore protraction (position 3). As yet we have not
defined the situations which underlie these
variations in the feeding cycles; therefore,
for the purposes of this paper, we shall deal
with the simplest and most frequently observed case, that of maintained radula/
odontophore apposition.
During the retraction stroke the major
rasping of the substratum occurs and food
is Drought into the buccal cavity. Initially
the odontophore and radula begin a posterior-ventral rotation toward rest position.
Almost simultaneously the buccal mass
itself begins a vigorous thrust in the same
direction. The conclusion of these movements results in the buccal mass, odontophore, and radula being restored to their
rest position. From this point food passes
to the stomach as a result of the beating
activity of cilia on the dorsal roof of the
protractor force to the odontophore. (viii) The
supralateral radular tensors (slT) are the most
massive muscles of the buccal apparatus. They have
their origins on the margins of the odontophore
cartilage and insert near the origin of the radular
membrane. Contraction of these muscles causes a
tight apposition of the radula to the odontophore
cartilage, provides anteriorly directed force to the
odontophore, and also produces a relatively solid
structure against which other muscles can exert
effective tension.
Other neural, glandular and muscular elements
of the buccal mass include: dD, dorsomandibular
dilator; E, esophagus; GB, buccal ganglion; M,
mouth; mA, mandibular approximator; prP, preventral protractor; RS, radular sac; sD, suboral
dilator; SG, salivary gland.
1021
FIXED ACTION PATTERN
«j
voP
FIG. 5. A view of the buccal mass in which the
anterior jugalis and all muscles external to this
sheet have been removed reveals two further muscles of importance. The first of these is the dorsal
odontophore flexor (doF) which has its origin
near the anterior dorsal midline of the buccal mass
and its insertion along the lateral margins of the
cartilage. The second muscle is the ventral odontophore protractor (voP) which has its origin on the
lateral sides of the mouth and inserts near the
ventral midline of the cartilage. It participates in
the final protraction stroke of the odontophore (between stages 2 and 3 of Fig. 3). The infraventral
odontophore protractor (ioP) is also shown. (See
also Fig. 4.)
buccal cavity and peristaltic contractions of
the esophagus.
Structure of the buccal mass
The radula (Fig. 3) is a rather thin, semirigid chitinous ribbon approximately 4 to
7 mm long and 2 mm wide. Its surface is
covered by many rows of sharp denticles
which are capable of scoring a variety of
organic surfaces (including the shells of
other snails). One of the forces against
which the muscles may develop tension is
the turgidity of the buccal mass (produced
as a result of direct arterial blood pressure).
The radula is held firmly against the substratum by virtue of the closely apposed,
so-called, odontophore "cartilage." (The
term "cartilage" is a malacological "carryover" since, in pulmonates, this structure is
composed of muscle and connective tissue
[see Hubendick, 1956].)
We recognize 19 bilaterally symmetrical
pairs of muscles associated with the buccal
mass (Figs. 4, 5, 6). Carriker's classical de-
STANLEY B. KATER
RAD
COL
doF
CART
icT
FIG. 6. A view of the left side of the buccal mass
as seen after a longitudinal section just to the right
of the midline reveals the supramedial radular
tensor (smT). This muscle has its origin on the
ventrolateral margins of the odontophore "cartilage" (CART) and inserts near the anterior surface
of the radular sac. Contraction of this muscle results
in the final protraction thrust of the odontophore
against the substratum. Also shown: BC, buccal
cavity; COL, collostyle; icT, infralateral cartilage
tensor; irT, inframedial radular tensor; RAD,
radula; Th, tensor of hood. (See also Figs. 4, 5.)
scription of the pulmonate feeding apparatus of Lymnaea (1946, 1947) has
facilitated enormously our own observations in Helisoma. There is a high degree
of similarity between the musculature of
the feeding apparatus of Lymnaea and that
of Helisoma, and we have thus adopted
much of Carriker's nomenclature. One difference that should be noted is that the
muscle termed the "postventral protractor"
by Carriker is, in fact, active during the
retraction phase in Helisoma and thus
classified as a retractor muscle in our present scheme.
Sequence of muscle activity
We employed standard electromyographic procedures (Kater et al., 1971) during feeding to determine the activity phase
relationships of each of the major muscles
comprising the buccal mass (Fig. 7). This
is possible because the muscles composing
the buccal mass are among the most discrete
in the phylum Mollusca and the neuromuscular junctions are rather simple, nonintegrating synapses (Kater et al., 1971;
Heyer et al., 1973). By recording simultane-
ously from two, three or four different
muscles of the buccal mass, we have developed a comprehensive picture of the
activity phase relationships of the major
muscles of the feeding apparatus. For convenience, the feeding cycle can be divided
into retraction and protraction phases, with
the rasp stroke beginning at the interface
of the protraction and retraction phases
(Fig. 8). It should be noted that these data
(i.e., Fig. 8) and all subsequent data have
been concerned primarily with feeding
cycles which vary between 2 and 5 sec in
duration. Longer cycles or cycles in which
independent odontophore/radular movements occur are more difficult to interpret
and will not be the main subject of this
paper.
Neural control of buccal mass musculature
—motoneurons
The neuromuscular relationships of the
muscles forming the buccal mass of
Helisoma are among the simplest in the
phylum Mollusca (Heyer et al., 1973). For
the muscles we will deal with in this paper
we observe a single basic pattern of innervation comparable to the vertebrate motor
unit (Kater et al., 1971). During the feeding
behavior, all of the muscle activity we have
observed can be accounted for on the basis
of the firing of the specific motoneuron
identified for a given muscle (Table 1).
A variety of techniques have been applied to the identification of motoneurons
(Kater et al., 1971). Among our most valuable criteria is that obtained by simultaneous electrical recordings from individual
nerve cell bodies and muscles. Under favorable recording conditions (Fig. 9), an extracellular muscle electrode also records the
activity of the axon innervating the muscle,
and we find one-to-one correspondences
among intracellularly recorded neural action potentials, extracelluarly recorded action potentials from the axon of that
neuron, and the extracellularly recorded
muscle activity. At firing frequencies below
10/sec this association is essentially invariant (Fig 9B), but at high repetition rates
there is a failure of the muscle potentials
FEEDING IN
Helisoma trivolvis: A
FIXED ACTION PATTERN
1023
aj-L
dt****"'^'-'1"'""*^^
FIG. 7. Simultaneous myogram recordings from
selected pairs of muscles of the buccal mass. Records
were obtained from several animals at a variety of
cycle periods and show the wide latitude of intercycle interval observed in the feeding behavior of
Helisoma. A, The activity of a protiactor muscle,
the posterior jugalis (pj) compared with that of a
retractor muscle, the anterior jugalis (aj). At cycle
durations as long as these, there is often an extended protraction stroke accompanied by independent radular movements. B, Two protractor
muscles, the ventral odontophore protactor (voP)
and the supralateral radular tensor (slT) which
characteristically fire slightly out of phase with one
another. The activity of adjacent muscles is often
observed in such recordings. Between the second
and third bursts of junction potentials of voP one
can see the activity of the overlying retractor muscle, aj. The source of additional potentials such as
these can be checked precisely by relocating the
recording electrode. In this manner one can obtain
up to six muscle recordings from two recording
electrodes. C, Retractor muscle poL (the postventral levator) and retractor muscle aj (the anterior jugalis) firing in phase with one another.
D, A demonstration of the synchronous firing of
junction potentials in bilaterally homologous muscles; right and left anterior jugalis muscles. The
variability of junction potential amplitude seen
throughout these records is primarily a reflection
of displacements of the recording electrodes during
the vigorous feeding movements. Time calibration
at the bottom of each pair of records equals 1 sec.
(Fig. 9C). Such failures are consistent with
the view that neuromuscular transmission
in these muscles is the result of chemical
synaptic transmission as found at the vertebrate neuromuscular junction (Kater et
al., 1971).
Another class of effector neuron is represented by buccal ganglion neuron 4 (both
right and left) which directly innervates
the large secretory cells of the salivary
gland. An action potential in neuron 4
ipsilateral to the site of recording gives rise
to a chemically mediated EPSP which can
result in an all-or-none, over-shooting action potential in salivary gland cells (Fig.
10).
1024
STANLEY B. KATER
Rasp
sIT
Pi
doF
dIP
smT
voP
prL
POP
br
pot
O|
FIG. 8. A summary of the activity patterns of individual buccal mass muscles. Mo\emails are schematically represented as protraction (P) and
retraction (R) strokes (see Fig. 3). The interval
between protraction and retraction (Rasp) can be
highly variable as compared with the interval between retraction and protraction. The bars opposite the name of each muscle indicate duration
of myogram activity, with the shaded portions
giving an indication of the degiec of variability
characteristic in these recordings. The period of the
cycle depicted is about 3.5 sec. aj, atiteiinr jugalis;
br, buccal retractor, dIP, dorsolaleral piotiactor;
doF, dorsal odontophore fle\oi; pj, posterioi jugalis;
poL, poswentral levato); poP, poslventral "piutioctor" (see text); prL, jnevenlral levator; sIT, supialaleral radular tenso>: smT, supramedial rarlulm
tensoi; voP, vential udontophore protractor.
Classification of neurons of the buccal
ganglia
We recognize three major classes of neurons within the buccal ganglia on the basis
of electrical activity recorded intracellularly
during the generation of feeding output.
While there are undoubtedly other neurons
(and perhaps even other classes) in these
ganglia, the three classes described below
are responsible for the generation of the
basic feeding movements.
The diagnostic feature of the protractor
motoneuron group is the marked hyperpolarization which precedes each primary
burst of action potentials (Figs. 9, 11, 12).
To date, we have identified five protractor
motoneurons in each of the paired buccal
ganglia. On the basis of one-to-one motoneuron-muscle relationships and muscle
activity patterns, this number represents
only about two-thirds of the protractor
motoneurons present in the buccal ganglia.
In fact, intracellular recordings from some
neurons whose functions are as yet undefined (e.g., the small cell beneath 19 in
Fig. 2) can reveal activity characteristic of
this class and electrical coupling to known
protractor motoneurons (see below). Accordingly, such cells are provisionally regarded as additional members of the protractor motoneuron class.
The second class of neurons in the buccal ganglia is characterized by bursts of
action potentials occurring in antiphase to
bursts in protractor motoneurons. Such activity is observed in retractor motoneurons
and the salivary effector neurons. We have
thus far identified eight retractor motoneurons in each buccal ganglion, but again this
must represent only about two-thirds of
those present.
The final class of neurons which we
recognize forms the core of the central program underlying feeding. By virtue of the
connectivity of its members, this group produces the intrinsic timing of the feeding
cycle and also drives the motoneurons.
Rather than refer to this kind of a network
of neurons in terms which may connote
mechanism (e.g., "oscillator"), we have
adopted the term "cyberchron" (Gk, kybern—to steer, direct, and chronos—time)
TABLE 1. Motoneurons to major muscles of the
buccal mass.
Neuron
Muscle or Effector
OR, !)L
10-R, 10-L
Right and left postvential levatoi
Right and left anterior jugalis (ventral)
Right and left anterior jugalis (lateral)
Right and left anterior jugalis
(dorsal)
Right and left dorsolateral protractor
Right and left ventral odontophorc
protractor
Right and left supralateral radular
tensor
Right and left supramedial radular
tensor
Right and left posterior jugalis
Right and left postventral protractor
Right and left salivary gland cells)
Il-R, 11-L
12-R, 12-L
17-R, 17-L
18-R, 18-1.
19-R, 10-1.
20-R. 20-1.
21-R, 21 -L
28-R. 28-L
(4-R. 4-L
FEEDING IN
Helisoma trivolvis: A
FIXED ACTION PATTERN
1025
B
FIG. 9. Some of the criteria used in identification
of a motoneuron. A, Simultaneous intracellular
record from the soma of neuron 21-R (upper trace)
and extracellular myogram from the right posterior
jugalis muscle (lower trace) during the generation
of feeding output. B, Multiple superimposed sweeps
showing action potentials in neuron 19-L evoked by
intracellular depolarizing current injections (upper
trace) and simultaneous extracellular recordings
(lower trace) from the peripheral axon of neuron
19-L (first aiiow) and the supralatcral radulai
tensor muscle (second arrow). Repetition rate was
appioximatcl) 8/sec. C, The same as shown in 13,
but repetition rate was increased to about 20/sec.
Under these conditions there is a failure of the
muscle potential (second arrow), which probably
indicates fatigue at the ncuromuscular junction.
Calibiation: A, 4"> mv and 4."i sec; B and C, 150
inv and 25 msec.
as a more general term. Such a network is
defined operationally in that it not only
drives motoneurons, but also provides
timing for the motor output as a direct
consequence of the connectivity inherent in
the network (see "Output of the Cyberchron
Network").
motor output, while the sensory feedback
acts to regulate the firing frequency and
duration of motoneuron bursts (Kater and
Rowell, 1973). The remainder of this report
will deal with the aspects of central connectivity which drive and time motoneuron bursting, while subsequent papers will
consider other inputs to the central program (Fountain, Kaneko, and Kater, unpublished; Kaneko, Kater, and Fountain,
unpublished).
The essential features responsible for
precisely timed burst generation in motoneurons can be recognized by simultaneous
intracellular recordings from pairs of neurons. The first point to note is that all
neurons within a particular group fire action potentials in close correspondence
with one another (Fig. 11). The second
essential feature of the timing of motor
THE GENERATION OF PATTERNED ACTIVITY
IN MOTONEURONS
We have previously demonstrated that
the motor output for feeding is the result
of integration of specific sensory input onto
retractor and protractor motoneurons,
which are themselves part of a centrally
programmed network. The centrally programmed component of the motor output
(entirely derived from connections within
the buccal ganglia) sets the timing of the
1026
FIG 10. Simultaneous intracellular recordings from
buccal ganglion neuron 4-R and one of the lobular
cells of the salivary gland (SG). An action potential
in neuron 4 (evoked by intracellular depolarizing
current injection) gives rise to a chemically mediated EPSP in salivary gland cells. In some cases the
EPSP is supratheshold and results in an all-or-none
action potential in the salivary gland cell. Calibration: 50 mv and 1 sec.
FIG. 12. Simultaneous intracellular recordings from
protractor and retractor motoneurons. A, Both retractor motoneuron 28 (upper trace) and protractor
motoneuron 19 (lower trace) are held hyperpolarized (by microelectrode-injected biasing current) to
retard the generation of action potentials and reveal
underlying synaptic potentials. B, Normal activity
of protractor motoneuron 17 (lower trace) and retractor motoneuron 11 (upper trace). Calibration:
A, 25 mv and 1 sec; B, 25 mv and 5 sec.
output is the reciprocal firing of retractor
and protractor motoneurons (Fig. 12). Thus,
in order to dissect the mechanisms underlying this patterned motor output for feeding we must understand: (i) why motoneurons within a group fire in phase with one
another, and (ii) why retractor and protractor motoneurons fire in antiphase to
one another. We can approach these problems by analyzing the connectivity patterns
and biophysical properties of individual,
identified neurons of the buccal ganglion.
Inputs to retractor motoneurons
FIG. 11. Nearly simultaneous activity in protractor
motoneurons (A) and retractor motoneurons (B).
A, Simultaneous intracellular recordings from protractor motoneuron 20-R (upper trace) and 21-L
(lower trace). The dorsobuccal nerve on the right
side has been cut and the bursts in protractor
motoneurons on that side are accordingly lengthened (see Kater and Rowell, 1973). B, Simultaneous
intracellular recordings from retractor motoneurons
(action potentials clipped) 10-R (upper trace) and
28-L (lower trace). Calibration: A, 45 mv and 3.5
sec; B, 30 mv and 3.5 sec.
The phasic generation of action potentials in retractor motoneurons is the result
of three classes of summating, subthreshold,
depolarizing, postsynaptic potentials (Fig.
12/4). The initial slow depolarization underlying each burst is the result of summating chemical EPSP's driven by a
member of the cyberchron network, neurons 41-R and 41-L (see "Output of the
Cyberchron Network"). The synchronous
arrival of this input to all members of the
FEEDING IN
Helisoma trivolvis: A
B_
FIG. 13. A demonstration of electrical coupling
between two retractor motoneurons. A, An action
potential in neuron 28 (lower trace) gives rise, at
very short latency, to an EPSP in neuron 11 (upper
trace). Such potentials, as well as DC injected currents, show no directional rectification (DC coupling
coefficients are inexact due to slight bridge imbalance). B and C, Passage of DC current from neuron
28 to neuron 10 (B) and from neuron 10 to neuron
28 (C). Calibration bar at end of each record: 10
mv and 50 msec; B and C, upper trace, 10 nA.
retractor class (e.g., Fig. 1123) figures prominently in the tight phase locking of the
neurons.
Two additional excitatory inputs, of
lesser magnitude, are involved in burst
generation by retractor motoneurons (neither of which has, as yet, been demonstrated for neuron 4). One of these is the
excitatory sensory feedback previously described by Kater and Rowell (1973), and
the other input is the result of mutual
excitatory electrical synapses (Fig. 13).
FIXED ACTION PATTERN
1027
hyperpolarizations and smaller EPSP's following the hyperpolarizations. A third,
somewhat less obvious class of PSP's is seen
as small IPSP's immediately preceding and
during the large hyperpolarization.
As is the case for retractor motoneurons,
all protractor motoneurons are electrically
coupled to one another (Fig. 14) in a nonrectifying fashion. Although the individual
EPSP's are small, the time constants of these
neurons are long enough that, at high frequencies of firing, one protractor motoneuron can evoke action potentials in other
coupled members of this class. All EPSP's
known to occur in protractor motoneurons
can be traced to the mutual electrical
coupling among these neurons (Fig. 15).
The smaller class of IPSP's observed in
protractor motoneurons is of short duration and has two origins: the mechanoreceptors previously described by Kater and
Rowell (1973) and a member of the cyberchron network (either 50 or 51). The
mechanoreceptor-evoked IPSP regulates
burst duration. The role of the smaller
IPSP's generated by the cyberchron neuron
is as yet undetermined. IPSP's from these
two sources have not been distinguishable
on the basis of the wave forms recorded
postsynaptically. They have a reversal po-
Inputs to protractor motoneurons
The synaptic inputs responsible for the
patterned generation of action potentials
in protractor motoneurons are somewhat
more complex than those found for retractor motoneurons. The following section
will suggest that bursts of action potentials
result from post-inhibitory rebound from
the characteristic large hyperpolarizations
seen in these neurons during feeding activity.
The synaptic potentials underlying burst
generation on protractor motoneurons can
be examined in neurons in which action
potentials are blocked by hyperpolarizing
DC current injection (Fig. 12). Two obviously different PSP's are present: Large
FIG. 14. A demonstration of electrical coupling
between protractor motoneuron 18-R (second trace)
and protractor motoneuron 19-R (lower trace). The
top trace indicates the timing of each current injection into neuron 19-R. Within the limits of precision of the bridge apparatus used for these
experiments, there is no rectification between any
of the protractor neurons tested. Calibration: middle trace, 10 mv and 1 sec (peak of action potential
is clipped); lower trace, 20 mv and 1 sec.
1028
STANLEY B. KATER
18R
19R
TH*V^vv*vVt(»>>«
FIG. 15. Simultaneous intraccllular recordings from
protractor motoneurons 19-R and 18-R during the
generation of aberrant feeding activity resulting
from damage to a cyberchron neuron. There are
two classes of obviously different IPSP's evident in
thcse records. The only EPSP's observed are corlelatcd with action potentials in the second neuron.
Calibration: bars at end of record, 10 mv; bottom
trace, 1 sec.
tential (as measured in the soma) which is
significantly closer to resting potential than
that of the large IPSP's (compare Figs. 16
and 17), and are reversed by intracellular
injections of chloride ions suggesting that
the ion providing the driving force is
chloride.
Another cyberchron neuron (52) synapses
on each of the protractor motoneurons and
produces large IPSP's, summating to produce the hyperpolarizing potential characteristic of protractor motoneuron activity
during feeding. Conductance changes of
large magnitude are observed in all protractor motoneurons during these hyperpolarizing potentials (Fig. \7A,B). The
cyberchron neuron apparently synapses on
each protractor motoneuron, since the hyperpolarizing potential can be indepen-
dently reversed in any of the protractor
motoneurons without reversing it in others
(Fig. 17C). All factors producing the rather
large value for the reversal potential of
these IPSP's have not been clarified. Nonetheless, it is clear from the magnitude of
this IPSP that the ions supplying the
driving force for this IPSP have an equilibrium potential significantly greater than
the maximum reversal potential of the
small, chloride-mediated IPSP's. These
large IPSP's are significantly diminished by
increasing extracellular potassium and are
unaffected by intracellular chloride injections and may therefore be similar to the
potassium-component IPSP's described by
Kehoe (1972) in abdominal ganglia of
Aplysia.
The lack of excitatory synaptic input
FEEDING IN
Helisoma trivolvis: A
mV
? 57
**J
67
77
87
97
lOnistc
FIG. 16. Determination of ihc reveisal potential of
the small, spontaneously occurring IPSP's observed
in protractor inoioiicuruu 17-L during ongoing
feeding activity. Pulse at the onset of each trace,
10 mv and 10 msec.
exogenous to this group suggests that these
neurons generate action potentials out of
post-inhibitory rebound. This can be tested
by releasing neurons from a biasing, hyperpolarizing current injection (anode break).
To demonstrate that this capability was
inherent to the neuron tested, these tests
were performed in high magnesium, calcium free Ringer's solution, a treatment
known to block chemical synaptic transmission in this animal (Kater and Kaneko,
1972). These neurons do indeed generate
action potentials from anode break and the
number of action potentials produced is a
direct function of the magnitude ot the hyperpolarization from which the neuron is
released (Fig. 18).
We offer the following provisional mechanism for the generation of bursts of action
potentials in this class of neurons. The
precise timing is derived from the cy-
FIXED ACTION PATTERN
1029
berchron network, which generates action
potentials giving rise to the large hyperpolarizing potential characteristic of protractor motoneurons. As the burst of action
potentials in the cyberchron network subsides, protractor motoneurons are released
from hyperpolarization and each generates
a number of action potentials in proportion
to the magnitude of the hyperpolarization
from which it has been released. These
action currents are transmitted electrotonically throughout the network of five
to ten motoneurons in each of the paired
buccal ganglia (as well as between ganglia);
they act as a positive feedback system, resulting in the explosive burst of action
potentials characteristically observed immediately after each hyperpolarization during normal feeding output. Bursts are
normally terminated by inhibitory synaptic
input from mechanoreceptors (Kater and
Rowell, 1973). In deafferented preparations
we can see an indication of some degree of
continued positive feedback among protractor motoneuions in the prolonged nature
of the periods of spike generation (compare
activity of neuron 20-R with 21-L in Fig.
]\A; for details, see Kater and Rowell,
1973).
A further point of interest with respect
to protractor motoneuron phase relationships is that not all protractor motoneurons
fire precisely in phase with one another (see
Fig. 8). The maximum difference in the
onset of firing between particular motoneurons ranges from 75 to 100 msec. While the
onset of the large IPSP hyperpolarization
occurs nearly synchronously in all protractor motoneurons (Fig. 19), there are significant differences in the rate of rise out of
hyperpolarization. Such differences between
various protractor motoneurons can be accounted for, in large part, by their different
time constants. For instance, protractor
motoneuron 19 begins generation of action
potentials significantly before 18. Likewise,
19 returns to resting potential before 18.
The characteristic time constant for 19 (obtained during periods of low-level synaptic
activity) is about 20 msec whereas that for
18 is 15 msec. Such differences in time
constant may be a major factor in account-
1030
60
8O
E12O
140
160
>
FIG. 17. Observations on the large IPSP seen in
protractor motoneurons during ongoing cyclical activity. A and B, Conductance changes (note open
arrows) measured independently in neuron 18-R
(lower trace) and 20-R (upper trace) during the
large hyperpolarization. An interesting feature of
such records is the marked reduction in electrical
coupling (black arrow marks one of the few instances of measurable coupling) observed during
the ongoing feeding output (compare with Fig. 14).
This is apparently due to the shunting effects of
ongoing synaptic activity (see Spira and Bennett,
1972). C, A simultaneous pair of recordings from
19-R (upper trace) and 19-L (lower trace) from
another preparation. Black dots mark the occurrence of the large IPSP. By injecting hyperpolarizing
current (using the technique of Brennecke and
I.indemann, 1971) in increasing magnitudes (black
arrows) a value of —120 mv was obtained for the
reversal potential of the PSP. Calibration: bars at
end of each trace, 10 mv; interval between pulses
on the bottom traces, 1 sec.
ing for the variability of onset of spike
output in different protractor motoneurons.
other. It is therefore impossible, under
normal conditions, to determine which particular neuron of the network drives synaptic potentials in motoneurons. However, by
reducing coupling between the cyberchron
neurons with EDTA (Asada and Bennett,
1971; Bennett, 1973), we ascertained that
the large IPSP's in protractor motoneurons
are driven by neurons 52-R and 52-L and
that the slow EPSP's in retractor motoneurons are driven by neurons 41-R and 41-L
(Fig. 20).
It would be of interest to understand the
mechanisms by which the cyberchron network generates cyclically patterned output.
Cyclical motor output, of variable duration,
can be evoked by a short pulse of depolarizing current injected into a single
THE OUTPUT OF THE CYBERCHRON NETWORK
Alternation between retractor and protractor motoneuron firing is dependent
upon the phase relationships of cyberchrondriven slow EPSP's producing retractor
bursts and large hyperpolarizations causing
protractor bursts (Fig. 20). We will conclude this report with a brief discussion
of the cyberchron network (details will be
presented later by Kater and Kaneko, unpublished).
All members of the cyberchron network
(i.e., neurons 40 through 43, 50 through 52
and 60) are electrically coupled to one an-
FEEDING IN
Helisoma trivolvis: A
FIG. 18. A demonstration of the ability of protractor motoneurons to generate action potentials out
of anode break. A through D show the effects of
increasing magnitudes of current injection (upper
trace) on the number of action potentials generated
in protractor motoneuron 17-R. Recordings were
obtained in high magnesium, calcium free Ringer's
solution. Calibration: 1 sec, 10 mv (voltage traces),
and 5 nA (current traces).
19L
FIG. 19. Simultaneous intracellular recordings from
two protractor motoneurons displayed at high sweep
speed to show differences in their onset of firing.
Calibration: vertical bars, 60 mv; interval between
pulses on the bottom trace, 1 sec.
member of the cyberchron network (Figs.
21, 22). Once initiated by the generation of
action potentials in one of its members,
positive feedback among the neurons of the
network can continue to produce cyclically
FIXED ACTION PATTERN
1031
FIG. 20. A source of inputs to retractor (A) and
protractor (/{) motoneurons. A, Action potentials
generated in a member of the cyberchron network,
neuron 41, by intracellular injection of depolarizing
current result in EPSP's, at short latency (< 10
msec), in a retractor motoneuron (held hyperpolarized to block action potentials). B, Another
cyberchron neuron, number 52, drives IPSP's at
short latency which can summate to produce marked
hyperpolarizations (i.e., greater in magnitude than
the undershoots of action potentials in protractor
motoneurons). Calibration: lower traces of A and
B, 40 mv; upper trace A, 10 mv; upper trace B,
60 mv; time, 1 sec.
FIG. 21. Activation of the cyberchron network by
the generation of action potentials in one of its
members. A short depolarizing current (upper
trace) injected into neuron 51 results in the generation of a series of rhythmic action potentials which
are correlated with hyperpolarization in protractor
motoneuron 19 (which has been held hyperpolarized
by current injection). Calibration: 10 mv and 1 sec.
recurring bursts of action potentials in the
majority of members of this network. The
1032
STANLEY B. KATER
I L ^ ^ - s . ^
-MA
MA___1
B
43
FIG. 22. Characteristic activity of members of the
cyberchron network. A, A short depolarizing current
injection (signaled in the upper trace) into a
cyberchron neuron can result in a scries of cyclical
spike discharges which drive cyclical motor output.
II, In other cases cyclical activity is represented in
the form of baseline oscillations as opposed to
bursts of action potentials. This sort of activity is
not characteristic of a particular cyberchron neuron,
but rather can be observed in any of these neurons
at various times. C, The bursts observed in cyber-
chron neurons (whether as a result of a short
depolari/ing current [upper trace] or during spontaneous activit)) are composed of extremely complex wave forms, in large part due to decrementing
electronic conduction through the electrical synapses
of this system. In this case a short depolarization of
neuron 51-L evoked a barrage of activity in this
neuron, neuron 51-R in the opposite ganglion, and,
based on our cumulative experience, in the majority of neurons forming the cyberchron. Calibration: 10 mv and 1 sec.
complexity of this interaction can be seen
by the intricacy of the wave forms which
compose each burst (Fig. 22C).
We can modify motor output in a predictable fashion by specific manipulations
of cyberchron neurons. Injection of hyperpolarizing currents into individual neurons
of the cyberchron network in normal preparations produces an increase in the cycle
period of both the cyberchron network and
the following motor output. This effect is
directly related to the magnitude of current
injected. In addition, when the electrical
coupling between members of the cyberchron network is significantly reduced
by (i) EDTA treatment, (ii) replacement
of external chloride by propionate, or (iii)
mechanical shock, there is no cyclical output produced by this system.
All cyclical subthreshold activity observed
in members of the cyberchron network (in
isolated buccal ganglion preparations) can
be traced to other members of the network.
Not all neurons of the cyberchron network
need generate action potentials during each
burst of the network (Fig. 22B); rather,
nearly any member of this class may show
baseline oscillations in phase with retractor
motoneuron bursts. Under such conditions,
these neurons are, in fact, incapable of
generating action potentials at normal
levels of depolarization due to some as yet
FEEDING IN
Helisoma trivolvis: A
undefined form of decreased excitability.
These oscillations appear to be the result
of the activity in the other electrically
coupled neurons of this network. This explanation is plausible since the electrical
junctions of this network act as low-pass
filters. The amplitude of the oscillations
observed in one cyberchron neuron can be
diminished by hyperpolarizing a second,
spiking cyberchron neuron, to reduce the
latter's electrotonic contribution.
Taken together, these features have led
us to the conclusion that the cyberchron
network is responsible for the production
of the cyclical timing of the motor output,
and that the ability to produce rhythmic
output is not likely to be derived from
the property of a single neuron in this
group, but rather the result of the connectivity of the cyberchron network as a whole
(Fig. 23).
DISCUSSION
This paper describes the individual components of the feeding behavior of
Helisoma. Our aim has been to provide a
foundation for continued investigation of
the neuronal basis of the central program
underlying the consumatory behavior of
this animal. Accordingly, we have directed
a series of probes at successively more central aspects of the control system of this
behavior. Our goal at this stage of our investigations is to have sufficient information
on the neuronal basis of the motor output
system to allow us to inquire into the mechanisms by which higher-order control is
exerted on this system.
We had previously demonstrated that the
motor output for feeding results from the
integration of mechanoreceptor information onto the cyclical timing produced by a
central program (Kater and Rowell, 1973).
These mechanoreceptors are, however, not
the sole source of sensory input to the central program. Other inputs, some of which
can act at the level of the cyberchron, will
be described in future reports (Fountain,
Kaneko, and Kater, unpublished; Kaneko,
Kater, and Fountain, unpublished). More
important, however, is the fact that we now
FIXED ACTION PATTERN
1033
recognize that the sensory feedback described by Kater and Rowell (1973) probably exerts its major role at cycle periods
in excess of about 5 sec. During shorter
cycle periods, the phase correspondence between the termination of protractor activity
and the onset of retractor activity is such
that the mechanoreceptor input can produce little modification of the basic centrally programmed output. This situation
is quite similar to that described for cockroach walking, where the centrally programmed motor output predominates at
high frequencies and the role of sensory
feedback is increased at lower frequencies
of motor output (Pearson and lies, 1973).
In the present report we have attempted
to elucidate central control systems by examining the factors which mediate motoneuron firing patterns. We can account for
retractor motoneuron activity in a rather
straightforward manner on the basis of excitation produced by the summating EPSP's
underlying each burst of action potentials.
Protractor motoneuron activity presents a
somewhat more complex situation. It appears that bursts of action potentials in
these neurons are the result of post-inhibitory rebound with subsequent reinforcement by positive feedback among the
members of this group.
We have introduced the elements of the
premotor system which form the cyberchron
network in order to provide a more complete picture of the neural substrates of the
feeding behavior. The characterization and
mechanism of function of the cyberchron
network requires more rigorous treatment,
but even on the basis of the information
presented in this report we can appreciate
its central role in the generation of feeding
behavior. From our own comparative studies we now recognize that the cyberchron
network of Helisoma is identical with the
system of small, electrically coupled neurons in the buccal ganglia of the closely
related snail, Planorbis (Berry, 1972).
The interpretation of neuronal events
underlying cyclical motor outputs requires
great care. A limited view of the system as
a whole could produce bias in the interpretation of the underlying mechanisms. For
1034
STANLEY B. KATER
R
MR
ILJL
FIG. 23. A summary of our present view of the
neural organization untlcrlying the feeding behavior
of Helisoma trivolvis. Individual neurons are represented as circles and the buccal mass mechanoreceptors (Kater and Rowell, 1973) as a box.
Schematized recouls opposite each ncuronal element depict characteristic activity observed in these
elements during the generation of feeding activity
with a cycle period of about 3 sec. Protractor
motoneurons (P) fire in antiphase to retractor
motoneurons (R) as a result of synaptic input from
neurons of the cybeichron network (C). Resistor
symbols denote electrical coupling between neurons
with the presence of additional elements implied
by resistors leaving each group (the protractor
group actually contains at least 7 neurons per
ganglion; the retractor gioup, at least 11 neurons
per ganglion; and the cybcrchron, at least 8 neurons
per ganglion). Open circles are excitatory chemical
synapses, closed circles arc inhibitory chemical
synapses, and the variable resistor symbol between
cybcrchron neurons refers to the probable ability
of higher-order inputs to decrease the coupling between individual neurons of the network and thus
inhibit generation of feeding output.
FEEDING IN
Helisoma trivolvis: A
instance, on several occasions we have presented individual cyberchron neurons showing only baseline oscillations correlated
with motor output. Hyperpolarization (by
means of intracellular current injection)
can result in a decrease in the frequency of
occurrence of both the oscillations and the
motor bursts. Greater magnitudes of hyperpolarization can completely inhibit both
the oscillations and motor output. Are the
neuronal events underlying the production
of cyclical motor output the result of specific properties of an individual neuron, or
are they the result of specific connectivity
of a network of neurons? For the cyberchron network underlying the feeding behavior of Helisoma, we believe the latter to
be the case; timing is the result of specific
neuronal interactions. At first sight, however, the situation described above is
strikingly similar to the single neuron "oscillator" described by Mendelson (1971).
Despite the fact that highly suggestive recordings can be obtained from single neurons, final judgment on underlying mechanisms must be deferred until these neurons
can be examined under situations which
effectively block network interactions. Under such conditions, continued cyclical
output would lead us to considerations of
single neuron membrane properties.
The series of steps we have taken from
the muscles effecting feeding towards the
control center for this, behavior is schematically summarized in Figure 23. We
still must look at inputs to the central program. Specific sensory inputs which can
effect the output of the central program
(e.g., Kater and Rowell, 1973; Weis, 1972;
Fountain, Kaneko, and Kater, unpublished)
are known and must be related to our present understanding of the central program.
We must also examine a class of input
which is best categorized as higher-order
control (i.e., making the decision whether
or not feeding will occur). We know of at
least one major input in the form of synapses on cyberchron neurons which reduce the
electrical coupling among these neurons
(Kaneko, 1973; Kaneko, Kater, and Fountain, unpublished). Accordingly, we have
schematically represented the coupling be-
FIXED ACTION PATTERN
1035
tween the cyberchron neurons by the symbol for a variable resistor (Fig. 23).
In closing, I would like to consider what
future developments we might expect from
this area of inquiry. If present trends continue, we will not only have information
on the neuronal basis of feeding in Helisoma, but also on the physiological and
morphological substrates of this behavior
in a number of additional members of the
class Gastropoda (to name a few: Aplysia,
Gardner, 1971; Anisodoris, Gorman and
Mirolli, 1969; Archidoris, Rose, 1971;
Navanax, Levitan et al., 1970, and Woollacott and Moore, 1972; Planorbis, Berry,
1972; Pleurobranchia, Davis et al., 1973).
With this compendium of data we will have
the unique opportunity to examine the interspecific diversity of the neuronal mechanisms underlying a specific behavior. This
approach will be the reductionistic counterpart to the ethological analysis of phylogenetic ritualization (Lorenz, 1966) and
should provide significant insight into the
evolution of neuronal circuitry.
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