Download Presynaptic Inhibition of Exteroceptive Afferents by Proprioceptive

JOURNAL
OF NEUROPHYSIOLOGY
Vol. 76. No. 2, August
1996. Printed
in U.S.A.
Presynaptic Inhibition of Exteroceptive Afferents by Proprioceptive
Afferents in the Terminal Abdominal Ganglion of the Crayfish
PHILIP L. NEWLAND,
HITOSHI
Animal Behaviour and Intelligence,
060 Sapporo, Japan
SUMMARY
AND
AONUMA,
MOTOAKI
SATO, AND TOSHIKI
NAGAYAMA
Division of Biological Sciences, Graduate School of Science, Hokkaido
CONCLUSIONS
1. Exteroceptive hairs that are sensitive to water displacement
and touch are distributed over the surface of the tailfan of crayfish.
We show that the sensory neurons innervating these hairs receive
a primary afferent depolarization
(PAD) from sensory neurons
innervating a proprioceptor that monitors movements of the endopodite and protopodite of the tailfan. This PAD occurs only during
high-velocity
movements of the exopodite, which are similar to
those that occur during swimming. The effects that the proprioceptor mediate are widespread, so that afferents in four sensory nerve
roots of the terminal abdominal ganglion, innervating hairs on the
protopodite, exopodite, endopodite, and telson, receive a PAD. The
PAD is unlikely to be mediated through monosynaptic pathways
because there is no anatomic overlap between the central projections of chordotonal afferents and many of the exteroceptive afferents. The depolarization
is associated with a conductance increase
and can be increased by the injection of hyperpolarizing
current
or reversed ( - 10 mV above resting potential) by injection of depolarizing current. The properties of the presynaptic input are, therefore, consistent with being mediated through chemical synapses.
This is supported by the observation in the electron microscope
that the exteroceptive afferents receive chemical input synapses.
The depolarization
is mimicked by y-aminobutyric
acid and reduced by bath application of picrotoxin or bicuculline, suggesting
that it is a depolarizing inhibitory postsynaptic potential. The PAD
reduces the amplitude of exteroceptive afferent spikes, an action
that is thus likely to reduce transmitter release and the efficacy of
synaptic transmission.
INTRODUCTION
Sensory information is modified as it passesthrough the
many layers of neurons in local circuits and can even be
modified within the terminals of the sensory neurons themselves,by meansof presynaptic inhibition. Presynaptic inhibition alters the ability of an action potential to causetransmitter releaseand, in both vertebrates and invertebrates, may
result from the activation of other sensory afferents in the
periphery (Blagburn and Sattelle 1987; Burrows and Laurent
1993; Schmidt 1971) or from a centrally generated rhythmic
drive (Cattaert et al. 1992; Gossard et al. 1990; Sillar and
Skorupski 1986). The effect of this inhibition is to reduce
the excitability of the sensory neurons so that sensory inflow
can be matched to the ongoing activity of an animal.
Few studies, however, have analyzed in detail the inhibition of one sensory modality by another at a local level.
Those that have done so have failed to identify the source
of that inhibition. For example, wind-sensitive hairs are distributed over the terminal appendagesof insects, the cerci.
0022-3077/96
$5.00 Copyright
University,
These hairs can detect wind or sound over a range of frequencies (Bentley 1975; Nicklaus 1965; Orida and Josephson
1978; Plummer and Camhi 1981; Shimozawa and Kanou
1984). Large depolarizations occur in the central terminals
of these wind-sensitive hairs in the locust when a sensory
neuron, thought to monitor cereal movements, is stimulated
(Boyan 1988). The identity of this presumedproprioceptive
sensory neuron is unknown. The depolarization that it evokes
in other sensory afferents does, however, reduce the amplitude of their spikes and hence their ability to release transmitter. Thus the outputs of the sensory neurons will depend
on the voluntary movements made by the locust.
In this study, we examine in detail the inhibition of exteroceptive signalsby proprioceptive signals. Water-motion-sensitive exteroceptive hairs are distributed over the surfaces of
the appendages of the tailfan of the crayfish. These hairs
have a wide range of responseproperties, varying from those
that have low thresholds and respond to the velocity of an
imposed movement to those that have high thresholds and
respond to acceleration (Ebina and Wiese 1984). The sensory neurons that innervate thesehairs project to the terminal
abdominal ganglion where they make synaptic connections
with local interneurons (Nagayama et al. 1993) and intersegmental inter-neurons(Nagayama and Sato 1993; Sigvardt et
al. 1982) and are involved in the production of local avoidance reflexes of the uropods in responseto tactile stimulation
or to water movements (Nagayama et al. 1986). Some of
the sensory neurons excite indirectly, via a population of
intersegmental interneurons, the lateral giant fibers (LGs)
that are involved in the production of an escaperesponses,
called a “tail-flip”
(Zucker 1972). The LGs activate fast
flexor motor neurons that innervate fast muscles in each
abdominal segment; the activity of these muscles leads to a
rapid flexion of the abdomen that pitches the animal upward
and forward away from the source of stimulation (Wine
1984; Wine and Krasne 1972). The synapsesbetween the
exteroceptive hair afferents and intersegmental inter-neurons
are prone to depression when they are used repeatedly
(Bryan and Krasne 1977a,b; Krasne 1969; Krasne and Bryan
1973). This depression contributes to a behavioral habituation to nonthreatening stimuli, but this would also reduce
sensitivity to subsequentthreatening stimuli. To counter this
depression,the afferent to interneuron synapsesare protected
by neuronal pathways that presynaptically inhibit the sensory
afferents during giant fiber-mediated tail flips. This presynaptic inhibition in the central terminals of the afferents takes
the form of a primary afferent depolarization (PAD), which
0 1996 The American
Physiological
Society
1047
1048
P. L. NEWLAND,
H. AONUMA,
M.
SATO,
AND
T. NAGAYAMAA
is accompanied by a conductance increase (Kennedy et al.
1974,198O; Kirk and Wine 1984). Ionophoresis
of y-aminobutyric acid (GABA) into the terminal ganglion produces a
depolarization that mimics the effects of PAD and has a
stained by injecting Lucifer yellow dye using 500 ms duration
hyperpolarizing
current pulses of 5 - 10 nA at 1 Hz for 1O- 15 min
The Lucifer yellow was allowed to diffuse through the sensory
afferents for a further 15 min in situ before the terminal ganglion
was removed and fixed for 15 min in 10% formalin, dehydrated
similar reversal potential
to PAD. Moreover,
it has been
in an alcohol series, and cleared in methyl salicylate. Afferents
suggestedthat a GABA-mediated chloride conductance un- then were photographed in whole mount under a fluorescence miderlies the giant-fiber-mediated PAD in the water-motion- croscope for subsequent reconstruction. Current-voltage curves of
sensitive afferents (Kennedy
et al. 1980).
hair afferents were measured using single-electrode current clamp
Other, so-called ‘ ‘nongiant’ ’ interneurons, can also cause (Nihon Kohden CEZ-3 100). GABA and its antagonists, picrotoxin
escape movements with similar viscous drag and thrust and bicuculline, were obtained from Sigma.
All physiological
recordings were stored on a PCM data reforces acting on the hairs (crayfish, Wine and Krasne 1972;
lobsters, Newland and Neil 1990). Thus the depression cording system for later analysis and display on an IBM-compatible
prone synapsesmust be protected during all forms of swim- computer using a GPIB interface. Results described here are based
on the successful recording and filling of 57 afferents from 49
ming. The aim of this study was, therefore, to identify and
analyze pathways that contribute to the presynaptic inhibi- crayfish.
tion of water-motion- and touch-sensitive afferents innervating hairs on the telson and uropods.
We show that the stimu-
lation of a chordotonal organ that monitors movements of
two segmentsof the tailfan, the endopodite and exopodite
(Field
et al. 1990),
produces
depolarizing
inhibitory
post-
synaptic potentials in the terminals of sensory neurons that
innervate exteroceptive hairs on the tailfan.
METHODS
Adult male and female crayfish, Procambnrus
clarkii (Girard),
of 7 to 9 cm body length (from rostrum to telson) were obtained
from a commercial supplier, maintained in running freshwater
aquaria, and fed weekly on a diet of chopped potato and liver.
The abdomen was isolated and pinned ventral side up in cooled
physiological saline (van Harreveld 1936) at 18°C. The swimmerets were removed and the terminal (6th) abdominal ganglion exposed by removing the sixth sternite, the surrounding soft cuticle
and the ventral aorta. The ganglion was stabilized on a silver platform and treated with protease (Sigma type XIV) for 20 s to
facilitate penetration with glass microelectrodes.
The exopodite was fixed at a 60” angle relative to the midline
of the abdomen and the protopodite overlying the third ganglionic
root, the hypodermis, connective tissue, and muscle were all removed to expose the chordotonal organ at the proximal ventral
edge of the endopodite (Fig. 1 A). Sensory afferents from the
chordotonal organ enter nerve root 3 (Field et al. 1990), and their
spikes were monitored by an oil-hook electrode placed on the nerve
near the proximal edge of the protopodite.
Physiologye
The chordotonal organ was stimulated by displacing it using a
fine pin attached to a vibrator (Ling Dynamic Systems), through
distances equivalent to 10” of exopodite movement (see Nagayama
and Newland 1993 ) . The vibrator was driven by a Shoshin (OI8 > computer-controlled
stimulator, which produced variable duration and amplitude ramp waveforms. GABA was pressure injected
into the neuropil, close to the afferent terminals, using a WPI
picopump (PV830) with glass microelectrodes of external tip diameter of 3-5 pm. Intracellular recordings were made from watermotion-sensory afferents and chordotonal afferents either in nerves
l-4 immediately as they enter the terminal ganglion, or in the
neuropil of the ganglion (Fig. 1) . Recordings were made with
glass microelectrodes
filled with either 3% Lucifer yellow CH
dissolved in 0.1 M lithium chloride (Stewart 1978) or with a 4%
solution of horseradish peroxidase (HRP). The hair afferents had
a mean resting potential of -74.8 I~I 1.9 mV (mean t SE, n =
20), and the spikes had an amplitude of -70 mV in their axons
in the ganglion. After physiological characterization, afferents were
Electron microscopy
Intracellular recordings for electron microscopy were carried out
with glass microelectrodes filled with a 4% solution of HRP (Sigma
Type VI) in a 0.1 M Tris buffer (pH 7.4) containing 0.25 M
potassium chloride (DC resistances of 60-80 Mf2). After HRP
injection ganglia were incubated for 1 h in cold saline to allow the
dye to diffuse throughout the neuron, fixed in situ using glutaraldehyde (2.5%) in 0.05 M phosphate buffer containing 0.15 M sucrose
at pH 7.4, dissected out and placed in fresh fixative at 4°C for >4
h. HRP development was carried out using the cobalt chloridediaminobenzidine-glucose
oxidase method (Itoh et al. 1978; Watson and Burrows 1981). After fixation, ganglia were rinsed with
a phosphate buffer and a 0.2 M Tris buffer, bathed in 0.5% CoCl,
in Tris buffer for 10 min, returned to the Tris buffer, and washed
with phosphate buffer. Ganglia then were incubated for 1.5 h at
38°C in a medium containing 50 ml of 0.05 M phosphate buffer,
25 mg 3.3-diaminobenzidine,
20 mg ammonium chloride, 100 mg
P-D glucose, and 25 units glucose oxidase (Sigma type V). After
incubation, ganglia were rinsed with phosphate buffer, and the
HRP-filled neurons examined under the light microscope. This was
then followed by osmication ( 1% in phosphate buffer) for 1 h,
dehydration in acetone, and embedding in epoxy resin (Quetol
8 12, Nissin EM).
Ganglia were sectioned horizontally (50 ,um thickness), and the
neurons drawn with the aid of a camera lucida attached to a light
microscope. Sections containing HRP-labeled processes were then
glued to an epoxy resin block to section serially (70 to 90 nm
thickness). The series of ultrathin sections was collected on Formvar-coated single slot grids. These were viewed and photographed
using a Hitachi H-800 transmission electron microscope.
RESULTS
Synuptic potentials in water motion sensitive afferents
The sensory neurons innervating the water-motion-sensitive hairs on the uropods project to the terminal abdominal
ganglion through the first five of the six pairs of nerve roots
(Calabrese 1976; Kondoh and Hisada 1987). Intracellular
recordings from these axons, close to the ganglion, show
that they supported spikes when the receptors on the tailfan
were
stimulated.
For example,
gentle jets of saline directed
toward the telson evoked bursts of spikes in an afferent
recorded in its axon in root 4 just as it entered the terminal
abdominal
ganglion
(Fig. 1 B). Other afferents could only
be excited by stronger jets of saline or by gently moving
groups of hairs directly with a fine paintbrush,
presumably
due to their higher stimulus thresholds. Not only did the jets
PRESYNAPTIC
INHIBITION
OF HAIR AFFERENTS
1049
microelectrode
pin
1 OmV
500ms
2oow
I,,
I’
1 oow
.._.
-T
1
0.5mV
250ms
1
ll
CO moy+ment
FIG. 1. A: diagram of the experimental arrangement for stimulating and recording hair sensory neurons. Ventral view of
the left half of the tailfan showing the proto-, endo-, and exopodites of the tailfan. Hair afferents were recorded in their
axons in roots l-4 (Rl -R4) of the terminal (6th) abdominal ganglion (G6). The exopodite-endopodite chordotonal organ
(CO) was stimulated by moving the strand with a small pin fixed to a Ling vibrator. B: puffing water onto the hairs on the
uropods (arrows) evoked depolarizations (*) and spikes in an afferent recorded intracellularly in root 4. C: stimulation of
a chordotonal organ that monitors the angle between the exopodite and the endopodite, evoked spikes in its afferent recorded
in root 3 and primary afferent depolarizations (PADS) in the same water-motion-sensitive afferent as in B. An upward
deflection of the movement monitor trace is equivalent to an opening of the exopodite. Presynaptic inputs were only present
at stimulus velocities >200” SK’. The amplitude of PADS depended on the stimulus velocity, becoming larger as the stimulus
velocity was increased, and decrement rapidly on repetitive stimulation. Stimulus amplitude was 10”.
of saline evoke bursts of spikes in the hair afferents, but
they also gave rise to small depolarizing potentials in the
same afferents (Fig. 1B, asterisks; see later).
Exteroceptive afferents received a depolarizing synaptic
input (PAD) when the chordotonal organ between the endopodite and the exopodite was stimulated. This chordotonal
organ has 12 sensory neurons whose axons travel in root 3
to the terminal abdominal ganglion. Ramp displacements of
the chordotonal organ, equivalent to opening and closing
movements of the exopodite relative to the endopodite,
evoked bursts of spikes in its afferents and depolarizing
potentials in a water-motion-sensitive hair afferent recorded
in its axon in root 4 (Fig. 1 C) . These depolarizations were
seen typically only at stimulus velocities >200” s -’ . Above
this velocity the amplitude of the depolarization increased
with increasing stimulus velocity up to the maximum that
could be generated of 800” s-’ (Fig. 1 C) . The potentials
did not give rise to spikes in the water-motion-sensitive afferents. Upon repetitive stimulation,
the depolarization
evoked in an afferent adapted rapidly between the first and
second cycles of stimulation but still persisted at a smaller
amplitude for >25 cycles (Fig. 1C) .
Axons of hair afferents in the first four of the five nerve
roots of the terminal abdominal ganglion all received depolarizing inputs during stimulation of the chordotonal organ
(Fig. 2). Many attempts to penetrate these afferents in the
smaller diameter fifth root failed, however, so we do not
know whether they too receive depolarizing inputs from the
chordotonal organ.
Properties
of the depolarizing
synaptic input
The depolarizations could be evoked in water-motion-sensitive afferents during both stretch and release movements
of the chordotonal organ at 800” s-* (Fig. 3A). The direction
of movement that evoked the largest PAD varied in different
afferents but did not appear to be dependent upon the sensitivity of the receptor itself. Superimposed sweeps triggered
from the onset of ramp displacements of the chordotonal
organ show that the depolarizations occur with short but
variable latency of 5-7 ms from the onset of the stimulus
to the chordotonal organ. Because the stimulation of the
chordotonal organ evoked high-frequency bursts of spikes
from its afferents, it was not possible to use signal averaging
1050
P. L. NEWLAND,
H. AONUMA,
foot 1 afferent
[--+JJ
A
root 2 afferent
M.
SATO,
AND
T. NAGAYAMAA
were observed in these areas and synaptic vesicles were
densely concentrated in many of the profiles. Synapses were
identified by one or more of the following characteristics: a
presynaptic density, a clustering of vesicles, a thickening of
the postsynaptic membrane, and a widening of the gap between the pre- and postsynaptic membranes. Input synapses
(Fig. 4, C, Cl, and D) were usually associated with two
postsynaptic profiles, with the HRP-labeled process of a water-motion-sensitive
hair being one of those profiles. Similar
dyadic arrangements were also found for output synapses
(Fig. 4, B and E). The input and output synapses were
intermingled on these small branches of the afferent and on
occasion within 0.5 pm of each other (Fig. 4E).
Aflerent depolarization
by GABA antagonists
FIG.
2. Exopodite-endopodite
chordotonal
organ has widespread
effects
on hair afferents. Hair afferents from receptors on the tailfan have axons
that travel to the terminal
ganglion through 5 sensory nerve roots. Hair
afferents in 4 of the 5 sensory roots receive PAD during chordotonal
organ
stimulation.
A trigger pulse used to trigger ramp stimuli was used as a
trigger for signal averages of the depolarization
in the hair afferents. Each
trace is an average of 16 sweeps. The chordotonal
organ was moved with
a ramp displacement
at 800” s --’ with an amplitude
of 10”.
to determine the mean latency of the response from the
afferent spikes and hence whether the connection between
the chordotonal afferents and the hair afferents was monoor polysynaptic.
The potentials in the hair afferents were accompanied by
conductance increases as revealed by injecting 0. I-nA current pulses at 100 Hz through the recording electrode (Fig.
3B). Hyperpolarizing
a hair afferent while stimulating the
chordotonal organ increased the amplitude of the PADS (Fig.
3C). Conversely, depolarizing the hair afferent reduced the
amplitude of the PAD and at currents of >5 nA, reversed
it. The input resistance of the hair afferents, measured using
single-electrode current clamp, at +5 nA was 2.0 t 0.2 MS2
(mean t SE, y2 = 13; range 0.9-3.3 MO; Fig. 3D), and it
was therefore estimated that the potentials evoked by chordotonal organ stimulation reversed at a potential just above
rest, at -66 2 1.3 mV. This is a potential far lower than
that of spike threshold in the axons of these hair afferents.
The results are consistent with these potentials being produced by the release of a chemical transmitter.
Hair afferent ultrastructure
A hair afferent with an axon in root 3 enters the terminal
ganglion and sends branches anteriorly into both lateral and
medial areas of neuropil (Fig. 4A). Input and output synapses were found on the small diameter branches that were
devoid of glial cells (Fig. 4, B-E). Many synaptic structures
is mimicked by GABA and blocked
Bath application of 200 PM picrotoxin, a GABA antagonist, reduced the amplitude of the PAD in a hair afferent
evoked by chordotonal organ stimulation (Fig. 5, A and
C). In all tests, picrotoxin reduced the amplitude of the
depolarization to approximately ~20% within 15 min (n =
6). This slow reduction presumably reflects the time taken
for the exchange of bathing solutions and the time taken for
the picrotoxin to cross the ganglionic sheath and enter the
neuropil. The effect of picrotoxin was completely reversible,
with the PAD recovering to its control value within 40 min
of washing in normal saline (Fig. 5, B and C). Bath application of 200 pm bicuculline, a GABA, receptor blocker, also
reduced the amplitude of the depolarization
(data not
shown). This effect was also reversed on wash with normal
saline.
Pressure injection of 1 mM GABA into the neuropil, close
to the hair afferent terminals, evoked depolarizations in hair
afferents (n = 2) (Fig. 50). These depolarizations were
accompanied by conductance increases.
Anatomic
organization
of hair and chordotonal
afferents
.
There is little or no anatomic overlap between high-velocity threshold chordotonal afferents and many of the hair
afferents. Chordotonal afferents that respond to displacement
of the chordotonal organ with low-velocity
thresholds project anteriorly in the ganglion, whereas afferents with highvelocity thresholds project more posteriorly in the ganglion
(Nagayama and Newland 1993). PAD was observed only
in hair afferents at stimulus velocities >200” s -I and chordotonal afferents with similar, or higher, thresholds do not
overlap with all hair afferents receiving PAD. For example,
a chordotonal afferent with a velocity threshold of 400” s-’
(Fig. 6A) projects posteriorly in the ganglion to an area of
neuropil at a level where root 1 emerges from the ganglion
(Fig. 6B).
Individual hair afferents project to different areas in the
ganglion based on the location of their receptors on the
tailfan and the nerve root containing their axons. For example, an afferent in nerve root 1, which contains afferents that
innervate hairs on the postero-lateral edge of the protopodite
(Calabrese 1976; Kondoh and Hisada 1987)) has an axon
that enters the ganglion in a postero-lateral
position and
projects anterio-medially and branches midway between the
PRESYNAPTIC
INHIBITION
OF
HAIR
AFFERENTS
1051
25mV
C
-6nA
D
-54-
I
-8
I
-6
1
-4
0
l
l
1
2
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-84 -
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4
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6
I
8
Current (nA)
0
l
z
-94 -
5
a
IOms
0>
iL*4-
-114-
FIG. 3.
Properties of PAD. A : ramp displacements
of the chordotonal
organ at 800” s-’ could evoke depolarizations
in
water-motion-sensitive
afferents during opening and closing movements
of the ramp. Superimposed
sweeps show variability
in the latency but this may come from a number of sources, including
variability
in spike threshold
of the chordotonal
afferents, as well as being indicative
of a polysynaptic
connection.
B: potentials were accompanied
by a large conductance
increase. One example is shown (top) of PAD during current injection
of -0.1 nA at 100 Hz while another is shown
(bottom)
of a PAD for comparison
to illustrate the time course. C: constant current hyperpolarization
increased the amplitude
of PAD and depolarization
decreased and eventually
reversed it. D: plot of membrane
potential of a root 3 afferent against
applied current, measured using a single-electrode
voltage clamp. Outward
rectification
is present at membrane
potentials
above -85 mV. Resting potential of this afferent was -74 mV. Input resistance of this afferent was therefore
-2 MQ.
anterior and posterior boundaries of neuropil (Fig. 6C). An
afferent with an axon in root 2, which contains afferents that
innervate hairs on the exopodite, branches in a similar
antero-posterior position, overlapping with some of the
branchesof root 1 afferents but having a number of branches
that project more medially (Fig. 6C). Root 3 afferents,
which innervate the endopodite, project to an area slightly
posterior to that of root 2 afferents and again closer to the
midline (Fig. 6C). Finally an afferent with its axon in root
4, which contains afferents that innervate hairs on the telson,
has the most medial branches in the terminal ganglion (Fig.
6C). Thus the projections of many of these hairs are anterior
and lateral to the branches of the chordotonal afferents. The
absenceof an overlap between the chordotonal afferents and
hair afferents suggeststhat the PAD in the hair afferents is
mediated through another layer, or layers, of interneurons.
Spike amplitude reduction
To determine the effects of presynaptic inhibition on spike
height, electrical stimulation of root 4 afferents was carried
out at different times relative to ramp movements. Spikes
were reduced in amplitude by 3-6% (n = 3 crayfish) if
they occurred at the same time as the chordotonal organ
evoked depolarization of their terminals. For example, a
spike evoked in a root 4 afferent by electrical stimulation
had an amplitude of 67 mV when recorded in the ganglion
(Fig. 7A). Stimulation of the chordotonal organ at 800” s -’
to evoke PADS, followed by stimulation of root 4 to evoke
a spike in a hair afferent that was coincident with the peak
of the PAD, produced a PAD of 4 mV and a 3-mV reduction
in the peak voltage of the spike when recorded close to its
terminal in the ganglion. This shows an overall reduction in
spike amplitude of 7 mV. No reduction in spike height occurred in hair afferents recorded in their axons in the nerve.
Instead a spike simply summed with the PAD evoked by
nerve 4 stimulation. Thus the change in spike height close
to the afferent terminals is not due simply to a local change
in membrane potential at the electrode because it is greater
than could be accounted for by the PAD alone. Thus it must
1052
P. L.
NEWLAND,
H.
AONUMA,
A
’
D
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c
B
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\
C,Ci
/
1
M.
SATO,
AND
T. NAGAYAMAA
PRESYNAPTIC
200pM
INHIBITION
OF HAIR AFFERENTS
1053
picrotoxin
wash
40ms
C
FIG. 5. PAD is reversibly blocked by picrotoxin. A : bath
application of 200 PM picrotoxin, a y-aminobutyric acid
(GABA) antagonist, reduces the amplitude of the chordotonal organ evoked PAD in a hair afferent. Picrotoxin reduced
the amplitude of the PAD within 15 min (n = 6). B : effect
of picrotoxin was completely reversible with the chordotonal
organ-evoked PAD recovering to its control value within 40
min of washing in normal saline. C: time course of picrotoxin
effect on synaptic potentials of another root 3 afferent. D:
pressure injection of 1 mM GABA into the neuropil depolarizes a root 2 afferent.
7
6
0
0
!!* 4 1
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nl
2
2
n 1
0
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0
0
0
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I
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10
20
Time (min)
30
40
50
D
J
5mV
2s
GABA
FIG. 4. Electron micrographs of a root 3 hair afferent filled with horseradish peroxidase (asterisks). A: reconstruction of
projections from serial sections of the afferent shown in B-E. After entering the ganglion, the afferent branches at a level
anterior to the emergence of nerve root 1 where it sends projections medially and laterally. B: an output synapse (double
arrowhead) from a thin neurite in an area indicated in A, showing dyadic arrangement of profiles (scale 1 pm). Labeled
afferent is indicated (asterisk). C: a dyadic input synapse (single arrowhead) near the output synapse shown in B (scale 1
pm) and shown at higher magnification in Ci (scale 0.1 pm). Note the prominent synaptic density in the smaller presynaptic
process. D : an input synapse from a larger diameter neurite (scale 0.5 pm). E: input and output synapses located within 1
,wm of each other on another small diameter branch (scale 0.2 pm).
P. L. NEWLAND,
H. AONUMA,
M.
SATO,
AND
T. NAGAYAMAA
800°s-’
root 3 intracellular
FIG. 6.
Responses
and central projections of a root 3 chordotonal
afferent. A :
responses of a chordotonal
afferent to ramp
stimuli of different
velocities
and peak displacement
of 5”. The afferent had a spike
threshold at a stimulus velocity of 400” s -’ .
B: drawing
of the chordotonal
afferent in
the terminal
ganglion
in whole mount. C:
drawings
of 4 hair afferents with axons in
different
nerve roots superimposed
on the
same outline of the ganglion showing their
medio-lateral
mapping. Note that there is little overlap of the chordotonal
and hair afferents.
C
200pm
result from a shunting conductance change elsewhere in the
neuropil.
Presynaptic inputs from other hair afferents
Saline directed toward the tailfan evokes depolarizations
in the terminals of the samehair afferents that receive input
from chordotonal afferents (Fig. 1B). As all appendages
were firmly fixed to prevent movement, thesedepolarizations
must be the result of activity in the sameor other hair afferents and not from chordotonal afferents.
When a root 4 afferent that received chordoton al input
(Fig. 7B) was stimulated extracellularly at levels subthreshold for spike production in the lateral giant fibers, the sti.mulus evoked depolarizing potentials in the afferent that occurred with a latency of 2-3 ms (Fig. 7C). These depolarizing potentials increasedin amplitude asthe stimulus intensity
was increased so that when the stimulus was suprathreshold
for spike initiation, the spike occurred simultaneously with
the depolarizing potential.
These results clearly demonstrate that hair afferents are
not only inhibited by proprioceptive inputs, but that they
also receive inputs from other exteroceptive afferents. The
nature of these inputs have not been analyzed in detail.
DISCUSSION
Synaptic potentials in hair afferent terminals
The terminals of water-motion-sensitive hair afferents
from receptors on the tailfan receive synaptic inputs during
stimulation of a small chordotonal organ that monitors the
movements of th.e exopodite relative to the endopodite. The
effects mediated by the chordotonal organ are widespread
so that hair afferents in four of the five sensory nerve roots
of the terminal ganglion receive synaptic inputs. These in-
PRESYNAPTIC
INHIBITION
OF
HAIR
AFFERENTS
1055
1 OmV
25ms
B
I
ImV
IOms
foot 4 afferent
CO movement
0.5mV
20ms
PAD leads to a reduction
in spike amplitude.
A : root 4 afferents could be electrically
stimulated
( l ) to evoke
spikes. Spike amplitude was reduced if it occurred at the same time as the chordotonal
organ evoked PAD. Two superimposed
sweeps are shown. One sweep shows the afferent spike occurring
without PAD (stimulus marked with black dot), the other
in spike height was 3%. C: water-motion-sensitive
afferents
with PAD caused by moving the CO at 800” s-l. Reduction
also evoke PAD in other water-motion-sensitive
afferents. When a root 4 afferent that receives chordotonal
organ input (B)
is stimulated extracellularly
at levels subthreshold
for spike production,
the stimulus evokes PAD in the afferent. This PAD
increases in amplitude
as the stimulus intensity is increased so that when the stimulus is suprathreshold
for spike initiation,
the spike occurs simultaneously
with PAD from other root 4 afferents.
FIG. 7.
puts are associatedwith a conductance change in the membrane and reverse at potentials - 10 mV above resting potential and are thus far lower than the potential for spike threshold. They are blocked by the application of picrotoxin and
can be mimicked by pressure injection of GABA into the
neuropil. The synaptic potentials never give rise to spikes.
These results suggestthat the potentials are depolarizing and
inhibitory and are mediated by the release of the transmitter
GABA onto the afferent terminals. Our results are similar
to those of other studies on primary afferents such as locust
FCO afferents (Burrows and Laurent 1993; Burrows and
Matheson 1994)) crayfish coxo-basipodite CO afferents
(CBCO) (Cattaert et al. 1992), and identified crayfish tailfan
afferents (Fricke and Kennedy 1983). Our measuresof reversal potential, while similar to those described by Burrows
and Laurent ( 1993) in locust FCO afferents, by Kennedy et
al. ( 1974, 1980) for crayfish tailfan afferents and by Glantz
et al. ( 1985) for ascendinginterneurons in the crayfish brain,
but contrast with those of crayfish CBCO afferents examined
by Cattaert et al. ( 1992) that reverse up to 40 mV more
positive than rest.
Synaptic inputs from afferents of a different modality
We already know that some afferents in the terminal ganglion receive descending presynaptic inhibition. Fricke and
Kennedy ( 1983) showed using the sucrose block technique
that mechanosensory afferents with axons in the fourth root
of the terminal abdominal ganglion of crayfish receive a
PAD when a ventro-medial region of the abdominal connective is stimulated. This region of the nerve cord is thought
to contain interneurons carrying proprioceptive information
from the walking legs (Fricke et al. 1982). Thus when an
animal walks or a leg is passively moved, proprioceptive
input from unidentified proprioceptors in the legs is thought
to reduce the afferent input from the hairs on the uropods
1056
P. L. NEWLAND,
H. AONUMA,
and telson. Our results show that inhibition of hair afferents
on the tailfan occurs locally within the terminal abdominal
ganglion. Moreover, we have identified the source of this
presynaptic input as a small proprioceptor that monitors the
angle of the exopodite relative to the endopodite (Field et
al. 1990) that contains - 12 afferents with different velocity
thresholds (Nagayama and Newland 1993). The descending
presynaptic inhibition described by Fricke and Kennedy
( 1983 ) is likely to occur during different behaviors from the
presynaptic inhibition described in this study. This is because
the input to the afferents that we have analyzed only occurs
when the exopodite moves faster than 200” s-’ . Such velocities are achieved during the power and return strokes of the
tail flip escape response (Cooke and MacMillan 1985) when
they reach velocities in excess of 1,000” s -I. During swimming, however, the limbs are held in a streamlined position
(Cooke 1985 ) so that they are unlikely to provide a rhythmic
drive to the water-motion-sensitive
afferents that could reduce unwanted input as the exopodites and endopodites
move.
Are the connections
pathways?
mediated through monosynaptic
Afferent-to-afferent
interactions are responsible for presynaptic inhibition between leech mechanosensory
neurons
(Baylor and Nicholls 1969)) where they sharpen the boundaries in receptive fields, and between muscle receptor organ
afferents in the crab (Wildman and Cannone 199 1 ), where
they increase the dynamic ranges of the afferents. For a
number of reasons, direct afferent-to-afferent
interactions are
not thought to be responsible for the inhibition of the watermotion-sensitive
afferents on the tailfan by the chordotonal
organ afferents. Chordotonal organ afferents make monosynaptic excitatory chemical connections with many spiking and
nonspiking local interneurons and with ascending intersegmental interneurons ( Newland and Nagayama 1993 ) . Moreover, no proprioceptive afferents inhibit, monosynaptically,
their postsynaptic
targets. Instead presynaptic
inhibition
must result from a layer, or layers, of interposed interneuTons. Only in a few studies have interneurons responsible for
mediating the PAD been described. The giant fiber-mediated
PAD in root 4 afferents is mediated by at least three different
classes of interneuron: ascending and descending intersegmental interneurons and local interneurons (Kirk 1985). Giant interneurons, however, cannot be responsible for the effects in the water-motion-sensitive
afferents we describe
here because electrical stimulation of the nerve roots of the
terminal abdominal ganglion was subthreshold to evoke
spikes in the giant interneurons, even though PAD was present in the afferent terminals. This suggests the pathway mediating PAD during chordotonal organ stimulation can be quite
separate from that produced during giant fiber-mediated tail
flips.
The PADS occur in the crayfish water-motion-sensitive
afferents even at high frequencies of chordotonal organ stimulation, suggesting that they must be mediated through very
reliable pathways with high gain connections, similar to presynaptic inhibition of chordotonal afferents of locusts (Burrows and Laurent 1993; Burrows and Matheson 1994).
M.
SATO,
AND
T. NAGAYAMAA
Synaptic inputs from other hair afferents
In addition to the PAD received from proprioceptive afferents, the same water-motion-sensitive
afferents also receive
inputs from other hair afferents, although the function of
these inputs has not been examined in this study. In vertebrates, cutaneous afferents receive PAD from neighboring
cutaneous afferents (Janig et al. 1968; Schmidt 197 1) where
it is thought to increase spatial discrimination and to limit
excitation. In crayfish this may also be true, but one other
possibility may be likely. Water-motion
hairs are dually innervated so that one of the afferents responds only to one
direction of movement of the hair shaft while the other responds to movements of the hair in the opposite direction
(Wiese 1976). The presynaptic inhibition could act to enhance directional coding of water movement through mutual
inhibition. Moreover, water-motion-sensitive
hairs have different physiological properties (Plummer et al. 1983 ) ; some
respond phasically whereas others tonically to imposed
movements of the hair shaft. Presynaptic inhibition could act
to enhance discrimination of different movement parameters,
such as velocity or acceleration, of the stimulus. It is possible
that the hair afferent-mediated
depolarizing potentials observed in this study may be the result of electrical coupling
between the central terminals of these afferents, as has been
demonstrated between afferents of a chordotonal organ at
the coxo-basipodite joint of a crayfish leg (El Manira et al.
1993). This, however, remains to be examined.
Functional
implications
for PAD
Presynaptic inhibition is thought to have many functions.
For example, during rhythmic movements, presynaptic inhibition can act to reduce inappropriate sensory feedback
caused by limb movements that would be disruptive to a
particular movement produced by central pattern generators
(El Manira et al. 199 1; Gossard et al 1991; Sillar and Skorupski 1986). Lateral inhibition between cutaneous afferents
in vertebrates is thought to enhance spatial discrimination
(Janig et al. 1968). More recently it has been suggested that
presynaptic inhibition results in a form of automatic gain
control (Burrows
and Matheson 1994) such that the effectiveness of the output of a synapse of one afferent depends
on the collective activity of the remaining afferents from
the same receptor. Moreover sensory neurons coding one
modality of a stimulus may presynaptically inhibit afferents
coding a completely different modality. Such presynaptic
inhibition has been shown to occur in locust cereal windsensitive hair afferents, which are inhibited by proprioceptive afferents that monitor cereal movement. The result of
this is that the pattern of inputs from the wind-sensitive
hairs
will vary depending upon the voluntary movements of the
locust (Bernard 1987; Boyan 1988).
In adult crayfish, hair afferents excite the lateral giant
neurons via monosynaptic electrical synapses and via disynaptic pathways involving interneurons. The afferent synapses onto the interneurons depress very rapidly, leading to
a behavioral habituation of tail flipping. This habituation has
been the subject of a number of studies, most recently leading to the finding that it is not present in juvenile lobsters
(Edwards et al. 1994) and that a third pathway, a monosyn-
PRESYNAPTIC
INHIBITION
aptic chemical connection between the afferents and LG
fibers exists in juveniles. Moreover, Krasne and Teshiba
( 1995 ) have suggested that descending tonic inhibition from
higher centers also plays a role in preventing depression.
Nevertheless, as crayfish mature the monosynaptic chemical
connections are gradually masked by the disynaptic pathway.
Thus in adults, to prevent depression or habituation, the
afferent-to-interneuron
synapse is protected presynaptically
during giant fiber activation (Krasne and Bryan 1973). No
mechanisms have been put forward, however, to explain
how these depression prone synapses might be protected
during non-giant swimming, when it is clear that the velocity
of movement is similar to that during giant tail flips (2.5
ms -I ) (Webb 1978) and, as such, will generate similar drag
forces on the sensory hairs. This will produce a similar volley
of afferent spikes as during a giant fiber tail flip and this in
turn will excite the same interneurons that have depression
prone synapses with the afferents. It is clear from our results
that the inhibition of the hair afferents during movements
of the uropods may modify afferent input during rapid movements and also protect the afferent-to-interneuron
synapses
from depression.
We are grateful to Prof. Masakazu
Takahata for his hospitality
and the
use of laboratory
facilities. We thank M. Burrows,
T. Matheson,
T. Friedel,
H. Schuppe, M. Takahata, and M. Wildman
for comments on earlier drafts
of this manuscript.
P. L. Newland was supported by a fellowship
from the Human Frontiers
Science Program
and by a grant from the Wellcome
Trust to Malcolm
Burrows.
T. Nagayama
was supported by a grant-in-aid
from the Japanese
Ministry
of Education,
Science, and Culture.
Present address of M. Sato: Laboratory
of Biology,
Rakunou-Gakuen
University,
069 Ebetsu, Japan.
Present address and address for reprint requests: P. L. Newland,
Dept.
of Zoology,
University
of Cambridge,
Downing
St., Cambridge
CB2 3EJ,
United Kingdom.
Received
7 December
1995; accepted
in final form
21 February
1996.
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