1Center for Neurobiology and Behavior, New York State Psychiatric Institute and College of Physicians and Surgeons of Columbia University, New York, New York 10032; 2Institute of Neurobiology and Department of Anatomy, University of Puerto Rico Medical Science Campus, San Juan, Puerto Rico 00901; and 3Department of Physiology and Biophysics and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, New York 10029
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rosen, Steven C., Mark W. Miller, Elizabeth C. Cropper, and Irving Kupfermann. Outputs of Radula Mechanoafferent Neurons in Aplysia are Modulated by Motor Neurons, Interneurons, and Sensory Neurons. J. Neurophysiol. 83: 1621-1636, 2000. The gain of sensory inputs into the nervous system can be modulated so that the nature and intensity of afferent input is variable. Sometimes the variability is a function of other sensory inputs or of the state of motor systems that generate behavior. A form of sensory modulation was investigated in the Aplysia feeding system at the level of a radula mechanoafferent neuron (B21) that provides chemical synaptic input to a group of motor neurons (B8a/b, B15) that control closure and retraction movements of the radula, a food grasping structure. B21 has been shown to receive both excitatory and inhibitory synaptic inputs from a variety of neuron types. The current study investigated the morphological basis of these heterosynaptic inputs, whether the inputs could serve to modulate the chemical synaptic outputs of B21, and whether the neurons producing the heterosynaptic inputs were periodically active during feeding motor programs that might modulate B21 outputs in a phase-specific manner. Four cell types making monosynaptic connections to B21 were found capable of heterosynaptically modulating the chemical synaptic output of B21 to motor neurons B8a and B15. These included the following: 1) other sensory neurons, e.g., B22; 2) interneurons, e.g., B19; 3) motor neurons, e.g., B82; and 4) multifunction neurons that have sensory, motor, and interneuronal functions, e.g., B4/5. Each cell type was phasically active in one or more feeding motor programs driven by command-like interneurons, including an egestive motor program driven by CBI-1 and an ingestive motor program driven by CBI-2. Moreover, the phase of activity differed for each of the modulator cells. During the motor programs, shifts in B21 membrane potential were related to the activity patterns of some of the modulator cells. Inhibitory chemical synapses mediated the modulation produced by B4/5, whereas excitatory and/or electrical synapses were involved in the other instances. The data indicate that modulation is due to block of action potential invasion into synaptic release regions or to alterations of transmitter release as a function of the presynaptic membrane potential. The results indicate that just as the motor system of Aplysia can be modulated by intrinsic mechanisms that can enhance its efficiency, the properties of primary sensory cells can be modified by diverse inputs from mediating circuitry. Such modulation could serve to optimize sensory cells for the different roles they might play.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The transmission of sensory information from the periphery to the
nervous system is modulated both at the level of primary sensory
afferents (Brooke et al. 1997; Gu and MacDermott
1997
; Hill et al. 1997
; Passaglia et al.
1998
; Pasztor and Macmillan 1990
) and at various
stages of processing in the CNS (Blakemore et al. 1998
;
Filion et al. 1998
; Geyer and Braff 1997
;
Gottlieb et al. 1998
; Motter 1993
;
Steinman and Steinman 1998
). Modulation of sensory
information is involved both in basic sensory processing, such as
enhancement of contrast, and in complex cognitive processes, such as
attention. In both invertebrates and vertebrates there is evidence that
the gain of sensory signals can be modified either by direct or
indirect sensory-sensory interactions (Burrows and Matheson
1994
; El Manira et al. 1993
; Mar and
Drapeau 1996
; Wildman and Cannone 1996
), or by
inputs from pattern-generating circuitry (Dubuc et al.
1988
; Vinay et al. 1996
; Wolf and Burrows
1995
). As an approach toward understanding the basic processes
by which sensory signals are modulated, we have been investigating the properties of the radula mechanoafferent (RM) neurons of the mollusk Aplysia. The RM neurons are involved in the control of the
radula, the chitinous structure that grasps food during ingestive and egestive feeding responses. Some of the RM cells, such as B21, are
uniquely identifiable and contain small cardioactive peptide (SCP)
(Miller et al. 1994
; Rosen et al. 2000
).
Studies of the receptive fields, response properties, and synaptic
connections of B21 suggest that it functions to provide afferent
information during ongoing feeding behavior (Rosen et al.
2000
). B21 produces diverse synaptic outputs to other sensory
cells, interneurons, and motor neurons that control radula closure and
retraction. A striking feature of B21 and other RM cells is that they
receive substantial excitatory and inhibitory synaptic input from a
variety of sources (Miller et al. 1994
; Rosen et
al. 2000
). These multiple inputs, in principle, could function
to conjointly modify the gain of the sensory cell outputs and thus
improve the efficiency of sensory processing. In the present study we
explored 1) whether the synaptic inputs from the diverse
types of input cells to B21 can modulate its synaptic outputs,
2) the mechanisms and sites of action of possible synaptic
modulation of B21 outputs, and 3) the characteristics of the
motor programs during which B21 modulation might occur. The synaptic
outputs of B21 were found to be enhanced or depressed by the actions of
the various classes of cells (motor, sensory, or interneuronal) that
are active during different phases of ingestive and egestive motor
programs. The results suggest that just as the motor system of
Aplysia can be modulated so as to enhance its efficiency
(Kupfermann et al. 1997
), the properties of the primary
sensory cells can be modified so as to optimize their function for the
different behavioral roles they might play.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Subjects and preparations
The subjects were Aplysia californica weighing 300-450 g. Two types of preparations were used: a buccal mass and an odontophore preparation. The buccal mass preparation was used to identify motor neurons, to determine the synaptic connections of the RM neurons specifically to motor neurons, and to characterize the behavioral significance of buccal motor programs that were initiated by chemical and/or electrophysiological means. It consisted of a partially dissected buccal mass that was removed from the body wall by a series of anterior, posterior, and lateral cuts. The anterior end of the buccal mass was freed from the body cavity by a cut through the anterior part of the jaws. The posterior end was freed by a cut through the esophagus. A series of lateral cuts through the extrinsic muscles of the buccal mass, connective tissue, and the anterior aorta completed the dissection. The portion of the anterior aorta leading to the buccal artery was cannulated and tied off so that the buccal mass could be perfused with artificial seawater (ASW) to simulate normal blood pressure and blood flow. The buccal mass preparation included the buccal and cerebral ganglia and the radula with most of the attached muscles that form the odontophore. Particular care was taken to preserve the buccal mass innervation provided by the radula nerve and buccal nerve 3.
The odontophore preparation was used to determine the receptive fields,
response properties, and in some instances, the synaptic connections of
RM neurons (Rosen et al. 2000). The odontophore was cut
longitudinally to isolate it from the lower muscular wall of the buccal
mass. The excised odontophore included the chitinous radula, its
supporting membranes and muscles, and the innervation provided by the
buccal and cerebral ganglia via the radula nerve and cerebral-to-buccal
connective. The dissected odontophore rested on the cut surface,
leaving the entire radula facing upward. The attached ganglia were
pinned to a silicone elastomer (Sylgard) pedestal. A partial section of
the anterior muscles was sometimes made to visualize the I4, radula
closer muscles, and the I5, accessory radula closer (ARC) muscles.
Contraction of each of these muscles when its motor neurons were fired
was used to establish the identity of the motor neurons.
Electrophysiology
Neurons were impaled with double-barreled microelectrodes using
techniques previously described (Rosen et al. 2000). A
modified odontophore preparation was used (Fig.
1) in experiments aimed at examining the
mechanisms of synaptic modulation of the outputs of RM neurons and in
studies describing orthodromic and antidromic conduction of RM neuron
action potentials. In this preparation, the radula with its support
tissue was placed in a recording chamber along with the buccal and
cerebral ganglia. Only the radula nerve innervation was preserved. One
of the paired lateral branches of the radula nerve was cut, and the
proximal cut end was drawn into a suction electrode to obtain
extracellular recordings of action potentials in the nerve root
traveling either toward the periphery (centrifugally, away from the
center) or toward the ganglion (centripetally). Centripetal action
potentials could arise from the intact radula nerve branch when the
ipsilateral radula-half was mechanically stimulated. Centrifugal action
potentials could result from central activation of either the left or
right B21 RM neuron. Combinations of up to three neurons in the buccal ganglion were impaled with microelectrodes to electrically stimulate the cells or to make intracellular recordings of action potentials and
synaptic potentials. A fourth channel was used for extracellular nerve
recordings (A-M Systems, Differential Amplifier).
|
Mechanical and chemical stimulation
The recording chamber and the chemical and mechanical stimuli
used in this study were identical to those previously described (Rosen et al. 2000).
Morphology
Neurons were filled with one of four fluorescent dyes that were
delivered by iontophoretic ejection from microelectrodes. Aqueous
solutions of the following dyes were prepared: 1) a 3% solution of 5(6)-carboxyfluorescein dye (Kodak), 2) a 3%
solution of Lucifer yellow CH (Molecular Probes, Eugene, OR);
3) a 2% solution of Cascade Blue hydrazide; and
4) a 5% solution of Lissamine-Rhodamine (Molecular Probes,
Eugene, OR). To reduce the active transport of the dye out of the cells
(Steinberg et al. 1987), probenecid (10 mM final
concentration) was added to the ASW bathing medium, and the preparation
was kept for 24-48 h at 4°C (Rosen et al. 1991
). The
unfixed tissues were viewed with a fluorescence microscope (Nikon
Optiphot) using cubes (V-2A, B-2A, and G-1B) appropriate for
simultaneous visualization of the multiple dyes, which fluoresce at
different wavelengths. Confirmation of cell morphology was made with
Lucifer yellow fills, followed by fixation in paraformaldehyde, and
clearing in methyl salicylate.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Four types of input neurons evoke monosynaptic postsynaptic
potentials (PSPs) in RM neuron B21 (Rosen et al. 2000).
These include the following: 1) sensory neurons, e.g., B22;
2) interneurons, e.g., B19; 3) motor neurons,
e.g., B82; and 4) multifunction neurons, that have sensory,
motor, and interneuronal functions, e.g., B4/5 (Gardner
1971
; Jahan-Parwar et al. 1983
; Rosen et
al. 1982
). To determine whether each type of input neuron could
produce heterosynaptic modulation of the putative chemical synaptic
output of B21, we simultaneously monitored the synaptic outputs of B21
to motor neurons B8a (Church and Lloyd 1991
;
Gardner 1977
; Morton and Chiel 1993
) and
B15 (Cohen et al. 1978
). B8a innervates a part of the I4
muscle. B21 evokes a fast EPSP in B8a that shows marked synaptic facilitation, particularly if B21 is repetitively fired at relatively high frequency. B15 innervates the ARC (I5) muscle. Firing of B21
evokes fast electrotonic potentials and a slow, presumably chemical
excitatory postsynaptic potential (EPSP) in B15.
In the sections that follow, we first present data on dye fills of B21
and various neurons that provide synaptic input to it. These studies
were designed to help clarify the possible sites of action of the
modulatory inputs. We also show the effects of coactivation of B21 and
each of the various input neurons to B21 on the chemical EPSPs that B21
evokes in B8a and B15. Finally, we investigated the firing patterns of
selected input (modulator) neurons, during buccal motor programs,
particularly phase-specific activity produced during motor programs
driven by command-like interneurons CBI-1 and CBI-2 (Rosen et
al. 1991).
Morphology
A light microscopic examination of the morphological features of RM neuron B21 and of the identified buccal neurons that represent its inputs and outputs was undertaken to obtain basic anatomic information that might contribute to an understanding of the mechanisms of heterosynaptic modulation of B21. In a series of experiments (n = 18), pairs of cells located in the left buccal hemiganglion were identified by electrophysiological criteria and filled with different dyes. In each experiment, the B21 neuron was filled with 5-(6)-carboxyfluorescein (yellow-green) and the connected cell (i.e., B8a, B15, B4/5, B22, B19, or B82) was filled with Cascade Blue hydrazide. The appearance of the filled B21 cells was consistent with previously reported descriptions of B21 morphology (Fig. 2, A-F, topmost cell except in D). The cell is bipolar, having a lateral and medial process. The lateral process arises from the soma and extends to the lateral limit of the ipsilateral buccal hemiganglion. The medial process projects to the contralateral buccal hemiganglion via the buccal commissure. In the buccal commissure the medial process bifurcates and sends a major branch into the root of the radula nerve. This branch bifurcates at the point where the radula nerve root gives rise to the left and right radula nerves (the various processes and the somata are drawn and denoted by numbers in Fig. 1).
|
Within the limitations of light microscopy, visualization of the processes of B21 together with the motor neurons with which it makes chemical synaptic connections revealed that probable sites of contact were primarily located on the lateral process of B21 and the initial segment of the motor neuron. B8a is the lateral-most neuron of the ventral motor neuron cluster of the buccal ganglion. Dendritic processes of B8a arise from its initial axon segment as it courses medially through the hemiganglion toward the buccal commissure and radula nerve (Fig. 2A). The B8a dendrites are interspersed among the terminal branches of the lateral process of the B21 neuron, but are not seen in close proximity to the terminal branches of the medial process or soma of B21 (Fig. 2A). Similarly, dendrites arising from the initial segment of motor neuron B15 are interspersed with the terminal branches of the lateral process of B21, but not with the terminal branches of other portions of the cell (Fig. 2B). The soma of B15 is located in the medial portion of the buccal hemiganglion, in close proximity to the soma of B21. B15 projects an axon laterally toward the root of buccal nerve 3. The initial segment of B15 runs parallel to the B21 lateral process, and the fine processes of the two cells intermingle extensively.
In contrast to the relatively restricted spatial limitations on
possible contact sites between B21 and its follower motor neurons, with
which it makes chemical synaptic connections, the dye-fill experiments
indicated that the neurons that provide input to B21 could contact B21
at its lateral process, its medial process, its soma, or at some
combination of these regions. Neuron B4/5, which produces a chemically
mediated inhibitory postsynaptic potential (IPSP) in RM neuron B21
(Rosen et al. 2000), has numerous processes in close
proximity to the medial process and soma of B21 (Fig. 2C).
Moreover, as the axon of B4/5 traverses the buccal hemiganglion, from
the medially located B4/5 soma toward the root of buccal nerve 3, it
gives rise to additional branches in the vicinity of the lateral
process of B21. Most of these branches, however, pass above the lateral
process of B21 and do not appear to make contact with it. Neuron B22 is
a cell similar to B21, in that it is an SCP-containing radula
mechanoafferent neuron. It makes an electrical synaptic connection to
B21 (Rosen et al. 2000
). B22 has numerous filamentous
processes that appear to contact the medial and lateral processes of
B21 (Fig. 2D). Moreover, the soma of B22 often abuts the
soma of B21. Interneuron B19 (Fig. 2E) and motor neuron B82
(Fig. 2F) also make nonrectifying electrical synaptic
connections to B21 (Rosen et al. 2000
). The processes of
B19 and B82 appear to contact the lateral process and soma, but not the
medial process of B21.
Effect of firing of B4/5 on signal transmission of sensory cell B21
Previous work showed that B21 receives monosynaptic chemical inhibitory input from the multifunction neurons B4/5. Thus B4/5 activity might block signal transmission of B21 to one or more of its follower cells. The following experiments examined this hypothesis.
FIRING OF B4/5 CAN BLOCK PERIPHERAL SENSORY INPUT TO B21 AND ITS
FOLLOWER MOTOR NEURONS SUCH AS B82.
To examine the possible functional significance of the inhibitory
input that B4/5 evokes in B21, B21 was activated by means of
controlled, tactile stimuli applied to the radula surface. In the
modified odontophore preparation (see Fig. 1), a suction electrode
attached to a branch of the radula nerve was used to monitor the
afferent volley and ensure that the tactile stimulus evoked a
relatively consistent sensory response (Fig.
3). In the absence of the firing of B4/5,
the tactile stimulus evoked spikes in B21. B82, which receives
excitatory electrical input from B21 and perhaps other sensory neurons
(Rosen et al. 2000), depolarized and fired (Fig. 3,
left and right). When neuron B4/5 was fired in
the physiological range of 20 Hz or greater by injection of depolarizing intracellular current, the tactile stimulus either failed
to evoke spikes in B21 or evoked fewer spikes than in the control
condition (Fig. 3, middle). Under these conditions B82 showed a reduced excitatory input and often failed to spike. The results suggest that firing of B4/5 may block spikes originating in the
mechanosensitive peripheral processes of B21. The spikes presumably are
blocked at some point between their entrance into the ganglion and the
cell body. Based on the morphology of B21 (Fig. 2C), it is
possible that B4/5 could act to block B21 spikes via synaptic terminals
at the medial process and soma of B21 (Fig. 1, regions 2 and 3),
thereby preventing orthodromic spikes from reaching the soma and/or
lateral process of B21 (region 1) where the connections to B82 are
likely to be present (Fig. 2F).
|
FIRING OF B4/5 CAN BLOCK CENTRIFUGAL AS WELL AS CENTRIPETAL B21
SPIKES.
Consistent with the preceding interpretation are experiments in which
B21 was activated by central input and antidromic spikes were recorded
in the periphery. Central input to B21 was provided by depolarizing
B82, which is electrically coupled to B21. When B82 was stimulated so
that it fired at physiological rates (8-16 Hz), it evoked electrotonic
potentials in B21, which could trigger action potentials that
propagated to the periphery. Extracellular recording from the radula
nerve indicated a one-to-one correspondence between distal impulses and
intracellular spikes recorded in the soma of B21 (Fig.
4, left and right).
When neuron B4/5 was fired at 20 Hz, at the same time that B82 was
stimulated, the B82 spikes no longer evoked action potentials that
could be recorded either in the soma of B21 or at its peripheral
processes in the radula nerve (Fig. 4, middle). Thus B4/5
activity appears to block spikes in B21 that are propagated either
toward or away from the cell body.
|
|
Chemical synaptic output of B21 is dependent on the membrane potential of the cell
The inhibitory synaptic input of B4/5 to B21 not only could
function to block spikes in B21, but could, in principle, modulate the
synaptic output of B21 even in the absence of spike blocking. This
might be a consequence of the fact that synaptic outputs typically are
dependent on the tonic membrane potential of the presynaptic cell. In
Aplysia, tonic depolarization of the presynaptic terminal
results in a graded increase of the transmitter released by a
presynaptic spike (Shapiro et al. 1980). To further
explore the effects of presynaptic membrane potential on the synaptic output of B21, we altered the steady-state membrane potential of B21
over a range of 15 mV and examined the consequences on the chemical
synaptic output of B21 to B8a. Spikes in B21 were evoked by means of
brief depolarizing pulses. Over the range of steady-state holding
potentials examined, progressive depolarizations resulted in a graded
increase in the EPSP B21 evoked in B8a (Fig. 6). The size of the B21 spike appeared to
be largely unchanged over the range of membrane potentials studied,
suggesting that the effect was not due to blockage of the B21 spike.
|
Effect of the firing of B4/5 on the chemical PSPs that B21 evokes in B8a and B15
When a train of action potentials was produced in B4/5 at the same time that a train of high-frequency spikes was elicited in B21, the excitation that B21 normally evoked in its follower motor neurons B8a and B15 was reduced or completely blocked (Fig. 7), even though the soma spikes in B21 were not blocked. Spikes were not blocked because B21 was directly fired by strong, repetitive depolarizing pulses. In the absence of the firing of B4/5, the firing of B21 at 12 Hz evoked facilitating EPSPs in B8a that produced action potentials (Fig. 7, left). Firing of B21 also produced small electrotonic potentials and a slow EPSP in neuron B15. Simultaneous firing of both B21 and B4/5 resulted in abolition of the facilitating EPSPs and spikes evoked in B8a/b and a substantial reduction of the slow EPSP evoked in B15 (Fig. 7, middle). The responses evoked by B21 activity were restored on termination of B4/5 stimulation (Fig. 7, right). Note that B4/5 abolished the slow EPSP, but not the electrotonic potentials recorded in B15. Given that the chemical synaptic output of B21 has been shown to be dependent on its maintained membrane potential, a reasonable explanation of the present observations is that the IPSPs evoked in B21 by B4/5 act presynaptically and reduce transmitter release, but do not substantially alter the electrical coupling between the cells. We cannot completely rule out an alternative hypothesis, which is that the IPSPs evoked in B21 by B4/5 may have relatively little presynaptic action, and the reduction of synaptic transmission between B21 and motor neurons B8a and B15 is due to a postsynaptic effect. The possibility that the effects of B4/5 are mediated polysynaptically cannot be excluded in these experiments.
|
Incorporation of the firing of B4 into buccal motor programs driven by CBI-1 and CBI-2
To begin to understand the functions of the inhibitory synaptic
input produced in B21 by neurons B4/5, we examined the timing of the
phasic firing of B4/5 during two different feeding motor programs:
namely an ingestive program driven by command-like interneuron CBI-2
and an egestive motor program driven by mechanosensory interneuron CBI-1 (Rosen et al. 1991). In previous studies that
utilized a buccal mass preparation (Rosen et al. 1988
),
it was shown that depolarizing, constant current, intracellular
stimulation of CBI-2 produced a robust buccal motor program that
usually generated rhythmic biting-like movements of the buccal mass.
Simultaneous video recording of the buccal mass movements and CBI-2
activity, indicated that during the initial phase of each biting cycle, accelerated firing of CBI-2 was correlated with the opening of the jaws
and the protraction of the odontophore with the radula open
(Rosen et al. 1988
). During the inhibitory phase of
CBI-2 activity, the odontophore was retracted and the radula and jaws closed. It has not proven practical to record from B4/5 during buccal
mass movements, because the buccal ganglion that contains these cells,
unlike the cerebral ganglion that contains CBI-2, is securely attached
to the posterior surface of the buccal mass by nerves and connective
tissue and is therefore subject to vigorous displacements during
feeding behaviors. As a means of obtaining information about the timing
of B4/5 firing during an ingestive motor program, we monitored the
activity of B4/5 at the same time that CBI-2 was fired to drive a motor
program in the odontophore preparation. Moreover, we used the
termination of the CBI-2 burst as a marker of the phase transition
between protraction and retraction. As suggested by previous reports
(Church and Lloyd 1994
; Rosen et al.
1991
), we found that B4/5 exhibited vigorous rhythmic bursting when CBI-2 received inhibition. A detailed analysis, however, indicated
that in five of six preparations examined, B4/5 began to fire 1 s
or more before the spiking in CBI-2 was terminated (Fig.
8A). Although B4/5 fired in
phase with interneuron B19, which fires during the retraction phase of
the CBI-2 driven program, B4/5 typically began to fire before the onset
of firing in B19. Early firing of B4/5 relative to B19 was similar to
the relatively early firing of B4/5 when compared with the firing
patterns of numerous retraction phase motor neurons (Church and
Lloyd 1994
). As indicated in Fig. 8B and elsewhere
(Rosen et al. 2000
), B21 receives phasic inhibitory and
excitatory input when a motor program driven by moderate levels of
CBI-2 stimulation is initiated and maintained. To help determine
whether the inhibitory input correlated with the onset of the B4/5
burst, a slower motor program was driven by relatively weak firing of
CBI-2 (Fig. 9). Under these conditions the interval of time between the onset of intense B4/5 bursting and the
onset of B19 bursting increased, as did the duration of the bursting of
each of these cells. The resulting motor program showed that the onset
of the inhibitory input correlated with the onset of the B4/5 burst and
is presumably due at least in part to the IPSPs that B21 receives from
B4 and B5. Despite the fact that B4/5 produced inhibition in B21, late
during a burst of firing of B4/5, B21 exhibited an abrupt return to
resting potential, or a depolarization beyond resting potential. The
depolarizing potentials corresponded to the time that B19 exhibited its
peak of bursting activity and are consistent with the fact that B19 activity excites B21 (Rosen et al. 2000
). The data
suggest that during the feeding cycle, the activity of B4/5 occurs at
or about the phase transition from protraction to retraction, and this should not be equated with the transition from radula opening to radula
closing, which is not necessarily in exact synchrony with retraction
(Rosen et al. 1998
).
|
|
Unlike the relatively late burst of B4/5 activity evoked by CBI-2,
CBI-1 evoked a nonrhythmic rejection program characterized by a
short-latency burst of B4/5, often followed by a second burst (Church and Lloyd 1994; Rosen et al.
1991
). In recordings of the synaptic potentials evoked in B21
during the motor program driven by CBI-1, an inhibitory burst of input
was observed (Fig. 10, left arrow). The latency of this burst is similar to that previously shown for the burst of B4/5 (Rosen et al. 1991
). The
burst occurred in the absence of firing of radula closure motor neuron
B8a, a pattern expected for a rejection response. There were also
indications of a second inhibitory burst (Fig. 10, right
arrow), which might be related to the second B4/5 burst that is
often evoked by CBI-1.
|
Excitatory and inhibitory effects of interneuron B19 on chemical EPSPs that B21 evokes in B8a and B15
In addition to intraganglionic interneurons such as B4/5, the
buccal ganglion contains interganglionic interneurons that project their axons to the cerebral ganglion via the cerebral-buccal
connective. To determine whether this type of neuron might modulate the
synaptic output of B21 we examined buccal-to-cerebral interneuron B19
(Fig. 2E). Firing of B19 evoked electrotonic EPSPs in B21
(Rosen et al. 2000). B19 receives a weak IPSP from B4/5
(Fig. 11). Based on the sensitivity of
B21 synaptic output to its membrane potential, it was predicted that
depolarization and firing of B19 would enhance the synaptic outputs of
B21. Indeed, when B19 was fired by means of a depolarizing current
pulse, the chemical EPSPs that B21 normally evoked in its follower
motor neurons B8a and B15 (Fig.
12A) were greatly increased
in amplitude (Fig. 12B). Conversely, when B19 was
hyperpolarized by a constant current pulse, the EPSPs that B21 evoked
in its follower motor neurons were decreased in amplitude (Fig.
12C, compare with A and D). Under
conditions in which B21 was fired at high frequency, the EPSPs it
produced in B8a exhibited temporal summation, and it was not clear to
what extent changes in the magnitude of the summed potential were due
to changes in the size of the individual PSPs rather than to changes in
the rate of decay of the PSPs. By firing B21 at a relatively low rate (10 Hz), it was possible to see discrete PSPs it evoked in B8a (Fig.
13A), and these were
revealed more clearly at a fast sweep speed (Fig. 13B).
Under this condition, when B19 was depolarized 20 mV above its resting
potential, the amplitude of individual EPSPs could increase by
150-200% of controls (Fig. 13A, left; B, top). The
increase in EPSP amplitude persisted as long as B19 was depolarized and
almost immediately returned to control levels on termination of the B19
current injection. On the other hand, when B19 was hyperpolarized, the
individual EPSPs that B21 evoked in B8a decreased to 40-60% of
control (Fig. 13A, right; B, bottom). Again the effect was
short-lived.
|
|
|
Incorporation of the firing of B19 into the motor program driven by CBI-2
B19 was consistently found to exhibit rhythmic bursting activity during the ingestion-like motor program driven by CBI-2 (Figs. 8A and 9). As previously noted, its activity was confined to the odontophore retraction phase (n = 20 preparations). Unlike B4/5, its bursting did not bridge the transition from protraction to retraction.
Excitatory and inhibitory effects of the firing of motor neuron B82 on PSPs that B21 evokes in B8a/b and B15
It was previously shown (Rosen et al. 2000) that
B21 has a particularly strong electrical synaptic input from motor
neuron B82. As was the case for B19, depolarization and firing of B82 increased the summated EPSPs that B21 evoked in follower motor neurons
B8a and B15 (Fig. 14B,
compared with controls, Fig. 14, A and D).
Conversely, hyperpolarization of B82 decreased the summated EPSPs that
B21 evoked in follower motor neurons B8a and B15 (Fig. 14C,
compared with controls, Fig. 14, A and D). The
effects of hyperpolarization or depolarization of B82 could be seen in
individual EPSPs when B21 was repetitively fired at 8 Hz and B82 was
either depolarized or hyperpolarized 20 mV from its resting membrane
potential (Fig. 15). In the control
condition when B82 was at rest (Fig. 15, middle pair of
traces), the amplitude of the EPSPs that B21 evoked in B8a went
from <1.0 mV for the first few EPSPs to an average of 8.2 mV. When B82
was depolarized by 20 mV (Fig. 15, top pair of traces), the
amplitude of the EPSPs that B21 evoked in B8a went from <1.0 mV for
the first few EPSPs to an average of 10.4 mV. When B82 was
hyperpolarized by 20 mV (Fig. 15, bottom pair of traces), the amplitude of the EPSPs that B21 evoked in B8a went from <1.0 mV
for the first few EPSPs to an average of 6.2 mV. The final amplitude
for the summated potentials was 30 mV for the control, 45 mV for the
depolarized, and 15 mV for the hyperpolarized conditions.
|
|
Incorporation of B82 into the motor programs driven by CBI-1 and CBI-2
In an attempt to understand the possible functional significance of the modulatory actions of motor neuron B82 on the outputs of sensory neuron B21, variations of the membrane potential of B82 were recorded during bite-like motor programs driven by firing CBI-2. During such motor programs, B82 received phasic depolarizing synaptic inputs that caused it to fire periodic bursts of spikes (Fig. 16). In the example shown, CBI-2 exhibited a typical pattern of bursting interrupted by strong inhibitions. The onset of the inhibition is coincident with the onset of odontophore retraction, which occurs when the radula is fully closed. Each B82 burst occurred during the same interval that B21 received its rhythmic excitatory synaptic input. B82 was also found to fire during motor programs driven by CBI-1 and during similar spontaneous programs associated with egestive movements of the buccal mass (Fig. 17). B82 received excitatory synaptic inputs during a cycle of the egestive program sufficient to cause it to fire late in the program, in conjunction with radula closer motor neurons (see spikes in the radula nerve recording). B82 spiking also occurred when B21 received excitatory input during the motor program.
|
|
Other radula mechanoafferent neurons can produce phase-dependent modulation of the synaptic output of B21
To determine whether other sensory cells could modify the output
of B21, we examined the effects of other immunoreactive RM neurons,
which are known to be electrically coupled to B21 (Rosen et al.
2000). We found that the effects of membrane potential changes
of RM neuron B22 on the outputs of B21 were similar to that of other
cells that are electrically coupled to B21 (e.g., B19 or B82).
Specifically, when a RM neuron was depolarized and fired, the chemical
EPSPs that B21 evoked in the follower motor neuron B8a and B15 were
increased in amplitude (Fig.
18B) compared with control
conditions (Fig. 18, A and D). When B22 was
hyperpolarized, the EPSPs that B21 evoked in its follower motor neurons
were decreased in amplitude (Fig. 18C) compared with the
other conditions.
|
Incorporation of RM activity into the motor program driven by CBI-2
As noted in a previous report (Miller et al. 1994)
SCP-containing RM neurons receive inputs from central and peripheral
sources. The central sources can cause them to depolarize and fire
rhythmically during various buccal motor programs. During the motor
program driven by command-like interneuron CBI-2, RM neurons, including B22, receive phasic depolarizing synaptic inputs, which sometimes evoke
spikes (Fig. 19). The inputs
predominantly occur during the retraction phase of the buccal motor
program.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In a previous study we found that the synaptic output of B21
exhibits a type of plasticity that is the outcome of previous activity
of the synapse. This type of plasticity has been termed "homosynaptic
plasticity" to distinguish it from heterosynaptic plasticity, which
results from the action of one synapse on another synapse
(Kandel 1976). The data in the present paper indicate that the various synaptic outputs of RM neuron B21 can be enhanced or
depressed by means of extensive heterosynaptic inputs provided by
remarkably diverse categories of other neurons. The heterosynaptic modulation differs in a number of respects from the well-known examples
of presynaptic depression and facilitation, extensively documented for
sensorin-containing mechanosensory neurons involved in defensive
reflexes in Aplysia (Byrne and Kandel 1996
).
First, unlike cells mediating defensive responses, the modulation of B21 is very short lasting and appears to be related to the alterations of membrane potential that the heterosynaptic activity produces in it.
Second, the modulation of B21 can occur rhythmically, and in phase with
motor output to the structure (radula) that contains the B21 sensory
endings. The gain of the B21 transmission can be either increased or
decreased at different times, because the cell receives both excitatory
as well as inhibitory synaptic inputs.
Inputs and outputs of B21
B21 is in intercommunication with at least three major classes of
neurons involved in generating buccal mass movements: 1) other radula sensory neurons, 2) buccal motor neurons, and
3) pattern-generating interneurons and/or premotor cells
(Fig. 20, left). In
addition, B21 receives a depolarizing input from at least one
modulatory neuron, the metacerebral cell (MCC) (Alexeeva et al. 1998). The extensive synaptic inputs from the various
classes of neurons provide an opportunity for different types of
neuronal processing to adjust the gain of the sensory signals coming
from the radula (see also Fischer and Carew 1993
). The
inputs from motor neurons and interneurons provide a simple means for a
corollary discharge (Grusser 1995
) to functionally
adjust sensory input. Inputs from other sensory cells can serve to
ensure that sensory flow into the nervous system only occurs in
response to appropriate stimuli, e.g., those contacting at least a
minimal amount of the receptive field. In addition, as has been
suggested for electrically coupled mechanoafferents in the crayfish
(El Manira et al. 1993
), the coupling could serve to
facilitate sensory transmission conveyed by the conjoint activation of
functionally related afferents.
|
Interconnections with motor neurons provides a means of enhancing the output of B21
B21 makes diverse types of synaptic connections with motor neurons
such as B8a/b, B15, and B82 (Fig. 20, right). The electrical connections to motor neurons such as B15 and B82 provide a source of
depolarizing input to B21 directly related to the excitation and firing
of the motor neurons. It should be noted that not all motor neurons
provide excitatory input to B21, and that input from motor neurons that
fire during the phase of radula closing is particularly prominent.
Because B21 has effects on pattern-generating neurons (see
Reciprocal interactions with higher order control elements), modulation of its outputs by motor neurons
provides one means by which the activity of motor neurons can influence pattern generation in mollusks (Hurwitz et al. 1994;
Staras et al. 1998
).
Multifunction neurons B4/5 mediate phasic inhibition of B21
A major source of inhibition that serves to decrease the synaptic
output of B21 is provided by the firing of B4/5 (Fig. 20, right), which is a paired multifunctional interneuron that
produces synaptic outputs to numerous buccal motor neurons
(Gardner 1971; Jahan-Parwar et al. 1983
;
Ono 1989
; Rosen et al. 1982
). The IPSPs that B4/5 and homologous neurons produce on motor neurons appear to
alter the timing of the firing of the motor neurons (Nagahama and Takata 1990
), but an additional role for the inhibitory
outputs of B4/5 may be to suppress or gate sensory information to the feeding network by actions on RM cells. Our evidence suggests that this
gating might be due to a direct effect of membrane potential of the RM
cells on transmitter release. Gating may also involve spike failure,
which is consistent with morphological data indicating that the
terminals of B4/5 contact B21 at its soma and along its thick medial
axon (Fig. 2C), which is a likely site of spike failure. An integrative role for spike failure of the axons of afferent processes has been suggested for a variety of systems (Chiel et al. 1990
; Mar and Drapeau 1996
; Van Essen
1973
).
B4/5 activity appears to block spikes in B21 that are propagated either
toward or away from the cell body. The functional significance of
centrifugally directed spikes is unknown. The findings that B21
contains neuroactive peptides and that the fine peripheral processes of
the cell in the subradula tissue contain varicosities (Miller et
al. 1994; Rosen et al. 2000
) suggest the possibility that centrifugal spikes might evoke the release of bioactive peptides in the subradula tissue.
B4/5 appears to fire intensely just before the transition from
odontophore protraction to retraction. The inhibition of B21 produced
by B4/5 may help ensure that sensory stimulation of the radula during
ingestive behavior (e.g., biting) does not result in RM outputs that
can contribute to premature radula closure during the transition from
protraction to retraction. Later, in the retraction phase of a biting
cycle, heterosynaptic inhibition of the RM cells is terminated or
replaced by heterosynaptic facilitation, and this may enable radula
stimuli to enhance radula closing on food objects, or perhaps
adaptively regulate the force of closure, according to the mechanical
properties of the food. Thus the alterations of the gain of the sensory
signal may function as a form of attentional device that permits the
transfer of information only at moments of time that the information is
functional. Movement-related inhibitory input to primary sensory
mechanoafferents is a prominent feature in arthropods, in which diverse
roles for this type of gating has been suggested (Burrows
1996; Krasne and Byran 1973
). Similar functions
are also served by gating of higher order sensory information, as in
saccadic suppression in vertebrates (Lee and Malpeli
1998
).
It should be noted that B4/5 may also be active during egestive
(rejection) movements as well as ingestive movements, and that its
firing phase relative to motor neurons is different for the different
behaviors (Church and Lloyd 1994). It may be that during
the interval in which the open radula moves backward toward the
esophagus to grasp inedible objects, it is crucial that RM sensory
signals produced by objects contacting the radula be suppressed so that
the excitatory input to radula closer neurons decreases. When the
radula then closes on the object and moves forward, excitation and
termination of suppression of B21 could facilitate the regulation of
radula closure. When the radula opens and releases the object, it would
be useful to again suppress B21 function with a second burst of B4/5
activity, so that the object is not grasped and drawn back into the
esophagus. Just as motor neurons and muscles can be engaged in multiple
behaviors, sensory neurons also typically function in different
behaviors. Thus it makes functional sense for their response properties
and outputs to be optimized according to the specific needs of the
behavior they are engaged in. The heterosynaptic inputs from motor
neurons, interneurons, and other sensory cells, may function to provide
this optimization.
Reciprocal interactions with higher order control elements
B21 has electrical synaptic connections to pattern-generating
interneurons such as B19, and it has been shown that B21 is also
coupled to pattern-generating neurons such as B64 and B51 (Evans
and Cropper 1998; Rosen et al. 2000
; C. G. Evans and E. C. Cropper, personal communication). These neurons
fire during the radula closure/retraction phase of the ingestive motor
program, suggesting that a sensory neuron such as B21 by virtue of its central connectivity might also contribute to, and therefore be a part
of, the underlying mechanisms of the feeding pattern generation (Evans and Cropper 1998
; Pearson 1993
).
Pattern generator interneuron B19 can provide input to, as well as
receive input from B21 by means of electrical connections. B19 also
projects an axon to the cerebral ganglion and provides phase-specific
inputs to cerebral-to-buccal interneurons, which are involved in the
initiation and patterning of buccal ganglion programs (Rosen et
al. 1991
). It is interesting that for mammalian rhythmic
feeding motor programs, there is also evidence that primary sensory
neurons generate both antidromic and orthodromic spikes and may be
involved in the dual roles of sensory processing as well as motor
pattern generation (Lund et al. 1998
).
Output states of B21
Figure 21 is a cartoon
illustration of our thinking of how the outputs of B21 are regulated
(darkened portions of cells in Fig. 21 indicate regions of active
spikes; synaptic output is represented by dots). B21 can be thought of
as operating in three overlapping states in which its synaptic output
is either low, completely blocked, or enhanced. In the resting state,
during which the radula is partially open (Fig. 21, rest/open), and
neuronal activity is minimal, peripheral afferent spikes, at least at
low frequency, invade only part way to the main branches and terminals
of the cell (see Fig. 7 in Rosen et al. 2000). Thus in
this condition little or no synaptic output from the cell is present.
During the initial part of the radula closing phase (Fig. 21, early
close), activity of cells such as B4/5 hyperpolarizes B21, resulting in a direct reduction of its synaptic outputs, as well as an indirect reduction that results from blockade of afferent spikes (see Fig. 3).
Finally, during the late phase of radula closing (Fig. 21, late close),
excitatory inputs from cells such as B19, result in increased invasion
of the peripheral spikes as well as enhanced transmitter release (see
Figs. 6 and 13).
|
Optimization of sensory processing
The synaptic inputs that impinge on B21 provide the cell with a
means of altering its gain as a function of context that is defined by
other sensory inputs as well as by signals provided by circuitry
associated with motor pattern generation. In addition, the transmission
of information by the RM neurons is conditioned by local contraction of
the subradula tissue (Cropper et al. 1996). The
modulatory affects at the RM cells are provided, in part, directly by a
corollary discharge of the motor neurons and various classes of
interneurons that constitute the mediating circuitry that controls the
generation of movements. In the crayfish, sensory gain is decreased by
central signals to protect sensory cells from reafferent activity that
can produce depression of the synaptic output of the sensory cells
(Grusser 1995
; Krasne and Byran 1973
). This is unlikely to be a function of the depression of RM gain in
Aplysia because the outputs of the RM cells do not exhibit homosynaptic depression. Furthermore, because the modulation at the RM
cells is rhythmic, it is unlikely to serve a function such as has been
suggested to occur in the crayfish in which an extrinsic modulatory
system associated with a particular behavioral state serves to ensure
that incompatible classes of responses (e.g., feeding and escape) do
not occur simultaneously (Krasne and Lee 1988
;
Krasne and Wine 1975
). It has been suggested that in
mammals sensory inputs are adaptively modified by the highly complex
circuitry of the cerebellum, which acts as an extrinsic input to the
mediating circuitry that generates the behavior (Bower
1997
; Courchesne 1997
; Miall et al.
1996
). The current results indicate that in Aplysia,
considerable modulation of sensory signals can be accomplished by the
sensory, motor, and interneuronal circuitry that actually mediates
behavior. Considerable data already indicate that the efficiency of
motor systems in Aplysia is improved not only by the action
of specialized extrinsic modulatory systems that are not involved in
generating behavior, but also by peptidergic co-transmission within the
intrinsic circuitry that generates responses. The outputs of the radula
sensory neurons are also affected by extrinsic modulatory systems, in
particular, the serotonergic metacerebral cells (Alexeeva et al.
1998
). The array of inputs to the RM sensory neurons of Aplysia is an example of a parallel, distributed
organization that provides an opportunity for different classes of
mediating neurons to directly influence, and in turn, be influenced by
sensory processes.
![]() |
ACKNOWLEDGMENTS |
---|
This study was supported by National Institutes of Health Grants MH-51393, GM-32099, MH-50235, MH-35564, and K05 MH-01591 and National Science Foundation Award IBN 9-722349.
![]() |
FOOTNOTES |
---|
Address for reprint requests: S. C. Rosen, Center for Neurobiology and Behavior, 1051 Riverside Dr., Box 87, New York, NY 10032.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 June 1999; accepted in final form 15 November 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|