Biology Department, University of Victoria, Victoria, British Columbia V8W 3N5, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Paul, Dorothy H. and
Jan Bruner.
Receptor potentials and electrical properties of
nonspiking stretch-receptive neurons in the sand crab Emerita
analoga (Anomura, Hippidae). Four nonspiking, monopolar
neurons with central somata and large peripheral dendrites constitute
the sole innervation of the telson-uropod elastic strand stretch
receptor in Emerita analoga. We characterized their
responses to stretch and current injection, using two-electrode current
clamp, in intact cells and in two types of isolated peripheral
dendritic segments, one that included and one that excluded the
dendritic termini (mechanosensory membrane). The membrane potentials of
intact cells at rest (mean ± SD: 57 ± 4.4 mV,
n = 30), recorded in peripheral or neuropil processes,
are similar to the membrane potentials of isolated dendritic segments
and always less negative than membrane potentials of motoneurons and
interneurons recorded in the same preparations. Ion substitution
experiments indicate that the membrane potential is influenced strongly
by Na+ conductance, probably localized in the
mechanotransducing terminals within the elastic strand. The form of the
receptor potential in response to ramp-hold-release stretch remains the
same as stretch amplitude is varied and is not dependent on initial
membrane potential (
70 to
30 mV) or recording site: initial
depolarization (slope follows ramp of applied stretch), terminated by
rapid, partial repolarization to a plateau (delayed depolarization)
that is intermediate between the peak depolarization and the initial
potential and sustained for the duration of the stretch. Responses to
depolarizing current pulses are similar to stretch-evoked receptor
potentials, except for small amplitude stimuli: an initial peak occurs
only in response to stretch and probably reflects elastic recoil of the
extracellular matrix surrounding the dendritic terminals. The rapid,
partial repolarization depends on holding potential and is abolished by
4-aminopyridine (4-AP; 10 mM), implicating a fast-activating,
fast-inactivating K+ conductance; TEA (60 mM) abolishes the
remaining slow repolarization to the plateau. In intact cells, but not
dendritic segments, regenerative depolarizations can arise in response
to stretch or depolarizing current pulses; they are reduced by
CdCl2 (10 µM) in the saline containing TEA and 4-AP and
probably reflect current spread from Ca2+ influx at
presynaptic terminals in the ganglion. We found no evidence for other
voltage-activated conductances. Unlike morphologically similar
"nonspiking" thoracic receptors of other species, E. analoga's nonspiking neurons are electrically compact and do not
boost the analogue afferent signal by voltage-activated inward
currents. The most prominent (only?) voltage-activated extra-ganglionic conductances are for potassium; by reducing the slope of the
stretch-plateau depolarization curve, they extend each neuron's
functional range to the full range of sensitivity of the receptor.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The capacity of a neuron to convey information generally is
assumed to be related to the nature of the information. The ubiquity of
coding by frequency and temporal patterning of action potentials (Perkel 1970) made the discovery of nonimpulsive, graded
signals in one class of crustacean mechanosensory neurons
(Ripley et al. 1968
) unexpected. Analogue signals in
nonspiking local interneurons, as well as graded transmission between
neurons capable of generating action potentials, are now well
documented in regionally localized networks where distances are short
(Burrows and Laurent 1989
; Burrows and Siegler
1976
; Graubard 1978
; Graubard et al.
1980
; Laurent 1993
; Mendelson
1971
; Paul and Mulloney 1985a
,b
; Wolf and
Büschges 1995
). The "nonspiking" stretch-receptive
neurons (NSRs) in decapod crustaceans, on the other hand, must transmit signals from the periphery, where their mechanotransducing channels are
located, to their output synapses in the CNS. All are unusual for
arthropod sensory neurons in having monopolar, centrally located somata. They all monitor position and movement of the basal joint of
the limb (or the equivalent in the case of the oval organ) (Pasztor and Bush 1982
), and although some are truly
nonspiking, incapable of generating action potentials (Cannone
1987
; Paul 1972
; this study), others employ
graded or constant amplitude action potentials in addition to
electrotonic spread of the receptor potential depolarizations
(Bush and Pasztor 1983
; Cannone and Nijland
1989
; Lin and Llinas 1993
; Lowe et al.
1978
; Mirolli 1981
; Pasztor and Bush
1982
; Roberts and Bush 1971
). The analogue
signaling by these proprioceptors is made more enigmatic by the
occurrence of morphologically similar (except for the smaller diameter
peripheral process) spiking stretch receptors in comparable positions,
mediating apparently similar reflexes, at the homologous joints in
other species (Miyan and Neil 1986
; Paul and
Wilson 1994
). Nevertheless, NSRs have evolved repeatedly
because they occur variably in different body segments in at least half
a dozen families, which suggests recurrent selection for analogue
signaling in different species.
The receptor potentials and physiological properties of the
thoracic members of this small group of mechanosensory neurons have
been investigated in some detail (Blight and Llinas
1980; Bush 1976
; Bush and Pasztor
1983
; Cannone 1987
; Cannone and Nijland 1989
; Lin and Llinas 1993
; Lowe et al.
1978
; Mirolli 1979
, 1981
, 1983
; Pasztor
and Bush 1982
; Ripley et al. 1968
;
Wildman and Cannone 1996
), whereas the more caudal NSRs,
associated with abdominal appendages (Heitler 1982
;
Paul 1972
), are less well characterized. The absence of
a receptor muscle and the more stereotyped movements of the associated
appendages (swimmerets in crayfish, uropods in hippid sand crabs)
should make the abdominal NSRs more accessible to functional analysis
of analogue signaling in this morphologically distinctive type of
sensory neuron.
We used standard intracellular microelectrode techniques and
pharmacological methods to characterize the responses to stretch and to
current injection of the four NSRs innervating the telson-uropod stretch receptor in the sand crab Emerita analoga. The
somata of these monopolar neurons are in the terminal ganglion of the CNS (Paul 1972) (Fig. 1)
and their large (20-40 µm, depending on size of animal) peripheral
dendrites extend
2 mm to an elastic strand in which their
mechanosensitive termini are embedded (Wilson and Paul
1990
). E. analoga's peculiar mode of swimming (with
the uropods) and their nonspiking telson-uropod stretch receptor
evolved together (Paul and Wilson 1994
), suggesting a
direct functional correlation that, if understood, might provide
insight into the significance of analogue signaling in NSRs in general.
Preliminary reports of some of this work have been published
(Paul and Bruner 1991
, 1993
).
|
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
E. analoga were collected from beaches of
Monterey Bay, CA, and held in a recycling sea water facility at the
University of Victoria. Large specimens (carapace length 2.7-3 cm)
provided the receptors used in two-electrode current-clamp experiments; receptors for the other experiments came from smaller animals (carapace
length 1.6 cm).
The crabs were anesthetized by chilling and perfused with cold saline, to minimize clotting of blood cells around the receptors, before removing the caudal abdominal nerve cord with, usually, both telson-uropod stretch receptors attached to the terminal ganglion (G6) of the ventral nerve cord. By keeping sufficient tissue on either side of the region of the elastic strand occupied by the NSRs' dendritic termini (Figs. 1, C-E, and 2A), the latter were not damaged or compressed when pinning the tissue on silicone elastomer (Sylgard) resin (Dow Corning) in the experimental chamber.
|
The strand's attachment to the telson is between muscles and had to be cut, but a cotton thread ligature placed around this end of the elastic strand (lig. in Fig. 1E) facilitated manipulation of this delicate structure and provided a sturdy site for attaching forceps to apply stretch stimuli. Minimum and maximum lengths of the portion of the elastic strand containing the NSRs' terminals measured in situ, with the uropods in rest and fully extended positions, were used to gauge the appropriate range of stretch amplitudes to use after the dissection.
For intracellular recording experiments on portions of the NSRs' peripheral dendrites, we placed additional ligatures. One ligature around the proximal end of the receptor nerve isolated the distal segments (DS) of all four peripheral dendrites from the central portions of the cells (l1 in Fig. 2A). This nerve branches from the large nerve leaving G6 about half-way between G6 and the elastic receptor strand. Note that DS include the mechanotransducing membrane of the dendritic termini, which are embedded in the elastic strand. Another ligature around an individual dendrite just outside the elastic strand (l2 in Fig. 2A) excluded the stretch-activated channels, generating a distal segment without strand (DSWS) 425-500 µm in length.
Controlled stretches of the receptor were made via fine forceps that
gripped the elastic strand close to or on the ligature posterior to the
NSR terminations (see preceding text; Fig. 1E). The forceps
were glued to one end of the core of a DC-operated linear variable
differential transformer (LVDT; Schaevitz, Pennsauken, NJ), which gave
a record of the stretch stimuli delivered; the other end of the LVDT
core was fixed to the center of an 8- speaker cone. Ramp
stretch-hold-release commands to drive the actuator for the speaker
were shaped either by a Phillips PM5153 function generator or by one of
the analog out-channels in the Clampex program (PClamp software, Axon
Instruments, Foster City, CA) programmed to generate ramp
stretch-hold-release sequences.
Microelectrodes were pulled from 1.2 mm, standard-wall, glass capillary
(Clarke Electromedical Instruments) and filled with 2.7 M KCl.
Resistances of electrodes for two-electrode current clamp were between
6 and 10 M. Electrodes for recording in bridge mode from sites close
to or inside the ganglion had higher resistance (15-35 M). In the
latter experiments, dye-filled electrodes sometimes were used to reveal
the identity (by serial order of termination along the elastic strand)
and central morphology of the NSR. Injected dyes move preferentially
toward the periphery in these neurons, so very small amounts suffice
for identification, but larger amounts are needed to make visible their
central structure (Fig. 1). Either Lucifer yellow (3-5% in 0.1 M
LiCl) or Texas Red (sulforhodamine 101 acid chloride; 3% in 0.1 M KCl)
was injected by 1-Hz, 4- to 10-nA, 500-ms, hyperpolarizing current pulses.
Ganglia were fixed in 10% formaldehyde in saline for 1-2 h or for 20 min followed by 1 hovernight in 10% methyl formcel in methanol,
dehydrated (or directly into 100% ETOH from methyl formcel), and
cleared and mounted in methylsalicylate in a depression slide for
viewing and photographing through a Leitz Aristoplan epifluorescence microscope.
Signals were recorded via an Axoclamp 2A amplifier (Axon Instruments) and displayed on an oscilloscope. Initially, we recorded data photographically on film or paper, using a Nihon-Kohden camera mounted on a Textronix 565 oscilloscope; these records were later digitized or scanned for plotting and preparation of figures. In later experiments, we used PClamp software (Clampex, Clampfit programs; Axon Instruments), stored data on computer, and used origin technical graphics and analysis software (MicroCal Software, Northampton, MA) for preparing graphs.
Aerated solutions were superfused continuously through the experimental
chamber (full volume 2 ml) at a rate of 2.8-3.0 ml/min. The
temperature at the inflow was kept at 14 ± 0.5°C by a Peltier cooling device. During pharmacological experiments, the volume was kept
minimum (~0.8 ml) for rapid exchange of solution. A minimum of 20 min
of superfusion with normal saline separated the delivery of each test
solution, which was superfused through the chamber for 6-10 min while
the test stimulations were made. Emerita saline (ES)
contains (in mM) 460 NaCl, 12.7 KCl, 13.7 CaCl2, 10 MgCl2, and 14 Na2SO4, buffered with
N-[2-hydroxyethyl]
piperazine-N'-[2-ethylsulfonic acid] (HEPES) at pH 7.5. Glucose (0.9 g/l) sometimes was added to the saline, although
preparations in saline without glucose showed no signs of deterioration
for many hours. Salines of different ionic compositions were prepared
from 1 M stock solutions according to the protocol of Gola and
Selverston (1981). Solutions for pharmacological experiments
were made up fresh the day of the experiment; concentrations to produce
maximum, reversible effects were used in the experiments reported here.
NaCl was reduced in saline containing tetraethylammonium chloride
(TEA+, final concentration 60 mM) by equivalent millimoles;
4-aminopyridine (4-AP; final concentration 10 mM) was dissolved in ES
shortly before use. Veratridine (100 µg) was dissolved in 10 µl
DMSO and 10 µl 100% ETOH, then diluted to 10
5 M in ES.
A 3 × 10
3 M stock solution of tetrodotoxin (TTX)
was diluted in ES before use. The efficacy of the veratridine and TTX
solutions in blocking action potentials was verified by recording
extracellularly from a motor nerve or connective and observing
reversible blocking of tonic activity. All chemicals were from Sigma.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The four nonspiking stretch receptors (NSRs I-IV) of E. analoga's telson-uropod stretch receptor have monopolar somata
located medial to the principle neurites, two in a relatively posterior position and two more central, within the last abdominal ganglion (Fig.
1, A and B) (Paul and Wilson
1994). The principal neurite of each neuron gives off short
branches into medial and lateral neuropil before expanding to become
the large dendrite that exits the ganglion. In the periphery, the
dendrites terminate sequentially (I-IV, anterior to posterior) in an
elastic strand strung between the inner dorsal telson and the
medial-ventral rim of the uropod propodite (Figs. 1,
C-E, and 2A) (Paul 1972
).
We studied the membrane responses of the four NSRs to stretch and to
intracellular current injection in three types of preparation: intact
neurons; the distal one-third to one-half of the peripheral dendrite
(DS), isolated from the proximal portion of the dendrite and soma by a
tight ligature around the receptor nerve (l1 in Fig.
2A); and similar distal segments that were isolated from
their stretch-sensitive terminals in the elastic strand (DSWS) by a
second ligature (l2 in Fig. 2A).
NSRs at rest
When the elastic strand is relaxed, the membrane potentials of the
NSRs recorded in or near the ganglion are typically 55 to
64 mV:
[
57.8 ± 4.4 (SD) mV; n = 30] and usually 3 to
~8 mV lower (depolarized) than those of motoneurons and most
interneurons (neuropil recordings) in the same preparations. The
membrane potentials recorded in the distal segments were similar: NSR
I,
63.4 ± 3.4 mV (n = 5); NSR II,
59.9 ± 4 mV (n = 8); NSR III,
58.6 ± 4.8 mV
(n = 14); NSR IV,
62 ± 3.3 mV
(n = 4); and no different when the distal segments were
separated by ligature from the neurons' proximal portions (
58.9 ± 4.9; n = 9).
We did not thoroughly investigate the ionic dependence of the NSRs'
membrane potential, but three ion-substitution experiments produced
similar results and revealed that this potential is influenced strongly
by Na+ conductance, probably because of their
mechanotransducing terminals. In one of these experiments, for example,
substituting sucrose for Na+ increased the membrane
potential from 58 to
76 mV within minutes, whereas Li+
in place of Na+ decreased it somewhat (to
53 mV).
Increasing Ca2+ fourfold had little effect, but the
membrane potential in Ca2+-free saline was lowered to
54
mV when Na+ replaced Ca2+ and to
49 mV when
Ba2+ replaced Ca2+. In every case except after
Ba2+ in place of Ca2+ in the saline, the
resting potential quickly returned to the control value of
58 mV;
recovery after exposure to Ba2+ was slower and incomplete.
Receptor potential
By impaling an NSR's dendrite close to or inside the ganglion, the elastic strand can be stretched without dislodging the microelectrode and the resultant receptor potential recorded intracellularly (Fig. 2). When the tissue had been pinned to allow stable recordings from more distal sites, close to the branch point of the receptor nerve, the receptor potentials recorded in response to small to moderate stretch were similar in form to those recorded routinely from penetrations close to or inside the ganglion. Figure 2B, inset, shows the receptor potentials recorded simultaneously, with dye-filled microelectrodes, from NSR II, penetrated just inside the ganglion, and NSR III, penetrated well outside of the ganglion, close to where the receptor nerve branches from the main nerve. The rapid, synchronous oscillations of their membrane potentials during the plateau phase were the result of lateral vibrations of the forceps gripping the elastic strand which, at certain positions of the LVDT, are unavoidable with rapid stretch. (These lateral movements are visually apparent at ×20 magnification, but not detected by the LVDT because they are perpendicular to its core.) This dual recording is interesting for both the similarity in form and amplitude of the receptor potentials recorded in the ganglion (NSR II) and >500 µm outside the ganglion (NSR III) and for the illustration of how precisely small, rapid signals are transmitted by these neurons.
The general form of the receptor potential remains the same as stretch
amplitude is varied: rapid depolarization (of a few millivolts from
initial potential, for the smallest stretches, to depolarizations to
about 10 mV for the largest stretches), which is terminated abruptly
by a rapid, partial repolarizing swing of the membrane potential that
merges with a stable plateau depolarization (called delayed
depolarization) intermediate between the peak depolarization and the
initial potential (Fig. 2B). The peak and subsequent delayed
depolarizations increase with increasing stretch amplitude, but neither
ever reaches 0 mV (Fig. 3). For moderate
to large stretches, the rapid repolarization phase (which ends the
initial "peak") begins before the end of the ramp (Figs. 2B and 3C, inset), but note that even the
smallest stretches produce a small peak (Figs. 2B and
3A). No peak occurs with low-velocity stretches of any
amplitude, rather the receptor potential follows the stimulus wave
form.
|
Neither the form nor amplitude of the receptor potential is influenced
strongly by resting potential. Low membrane potentials (less than 50
mV and as low as
30 mV), whether due to poor penetration, tonic
stretch of the elastic strand, or pharmacological treatment, do not
significantly modify the form of the receptor potentials generated in
response to stretch. The "negative dynamic" mimics the ramp
relaxation phase of the stretch stimuli, being an approximate mirror
image of the depolarizing phase, except that a substantial "negative
peak" (afterhyperpolarization) occurs only when the elastic strand
is initially under slight tension; this implicates elastic properties
of the strand rather than voltage-sensitive conductances as its cause.
Regardless of the membrane potential at rest in ion-substituted saline (see preceding section), the amplitudes of both peak and delayed depolarization of receptor potentials were unaffected when Li+ replaced Na+ in the saline but were reduced to one-third of control amplitudes when Na+ was replaced by sucrose. Na+ in place of Ca2+ increased both peak and delayed phase ~150%. The peak was unchanged but the delayed depolarization dramatically increased (>300%) when Ba2+ replaced Ca2+ in the saline.
Electrical properties of NSRs
In both whole cell and isolated distal segments of NSRs,
depolarization by current injection through the second intracellular electrode produces an initial, fast depolarization, which, when sufficiently large, is terminated by a fast, partial repolarization that, like the partial repolarization after stretch-induced initial peaks, merges with a second, delayed phase of depolarization to achieve
a steady plateau (Fig. 4). Steady-state
current-voltage relations (measured at 40 ms, by which time maximum
responses had been reached) are linear for hyperpolarizing current
pulses but show rectification for depolarizatations, starting ~15 mV depolarized (at about 40 to
45 mV). Input resistances measured in a
distal segment were initially between 0.6 and 1.5 M
in intact cells
and 1-2 M
in ligated distal segments and usually increased over
time to 2-3 M
.
|
The fast repolarization depends on the holding potential. Figure
5A shows responses of an
isolated distal segment of NSR III to two series of depolarizing pulses
applied from different holding potentials, 60 mV (the resting
potential in this cell) and
75 mV. The peak depolarizations reached
the same absolute level (
10 mV) for the largest current pulse in both
series (Fig. 5A). For all NSRs studied, shifting the holding
potential toward progressively less negative values reduced, and then
eliminated, the repolarizing phase (Fig. 5B). A
hyperpolarization immediately preceding a depolarizing current pulse
from a holding potential of
60 mV increased the repolarization after
the peak (Fig. 5C).
|
Ionic dependence of the NSRs' responses
Potassium-channel blockers have pronounced effects on whole cell responses. The partial repolarizing swing of receptor potentials in response to stretch of the elastic strand, recorded proximally at the edge of the ganglion, is abolished (Fig. 6, A, C, and D1). The effect is similar on responses to injected depolarizing current, recorded in the distal dendrite (Fig. 6B2). [The small dip in the rising phase of the receptor potential (Fig. 6, A and C) reflects elastic adjustment in the receptor strand after the end of the ramp stretch.] The resultant increase in both delayed depolarizations (produced by stretch or current injection), can become regenerative and outlast the stimulus (Fig. 6B). These delayed depolarizations are reduced by CdCl2 (10 µM) in the saline containing 60 mM TEA+ (Fig. 6, A and C). This suggests that this depolarization, which can overshoot 0 mV by a few millivolts and become regenerative (Fig. 6B), results from a voltage-activated Ca2+ conductance added to the stimulus (stretch or injected current)-induced depolarization. In normal conditions, it is at least partially antagonized, probably by an outward current during the delayed phase of the response. CdCl2 (10 µM) in normal saline produces a small, reversible increase in plateau amplitude (not shown), implicating a small contribution to the normal delayed response from a Ca2+-activated K+ conductance.
|
Spontaneous fluctuations and oscillations of membrane potentials are common in relaxed, unstimulated NSRs in TEA+ saline and are particularly prevalent immediately after the receptor has been stretched repeatedly; at times they resemble synaptic potentials recurring singly or in short bursts (Fig. 6D2), implicating current spread from synaptic terminals as their source.
The effects of K+-channel blockers on dendritic segments and on whole cells differ. The fast repolarization phase in both whole cells and dendritic segments is eliminated by 4-AP (10 mM), the initial depolarization being instead reversed by a slower, partial repolarization to the plateau level (Figs. 6A, trace 2, and 7A). In dendritic segments, TEA+ (in addition to 4-AP) abolishes the repolarization phase and thereby eliminates the broad transient, so the response mimics the square waveform of the current pulse (Fig. 7B1). However, dendritic segments (DS and DSWS) do not develop the regenerative depolarizations observed in whole cells when K+-channel blockers are present. This is evidence that these depolarizations arise in proximal, presumably presynaptic terminal, portions of these neurons, which the morphology (Fig. 1, A and B) suggests are electrically close to the large, peripheral dendrite.
|
We found no evidence for voltage-activated sodium inward current in
E. analoga's NSRs. The amplitude of the depolarization reached by the peak increases progressively with increasing amplitudes of stretch or injected depolarizing current. There is no evidence of an
inflection or step increase in the rising phase of the response, such
as would be expected were there a voltage-activated sodium inward
current. The absence of voltage-activated Na+ channels also
is indicated by the facts that neither the slope nor amplitude of the
peak depolarization in response to stretch is dependent on initial
membrane potential (Fig. 6A, traces 1 and 4) and
the slope of the peak potential in response to depolarizing current
pulses is not modified by shifting the membrane potential to different
values (Fig. 5). Finally, bathing the preparation in TTX
(106 M in saline containing 10 mM 4-AP) or in veratridine
(10
5 M) had no obvious effect on the responses to current
pulses that depolarized the NSR up to 0 mV from a holding potential of
65 mV (data not shown).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of this study reinforce the impression from functional
and ultrastructural data (Paul 1972; Wilson and
Paul 1990
; Paul, unpublished observations) that the four NSRs,
which constitute the entire innervation of the elastic strand of
E. analoga's telson-uropod stretch receptor, are not
differentiated from each other and that each NSR responds over the full
range of the receptor. The responses to stretch and depolarizing
current pulses are very similar to each other, except for small
amplitude stimuli: the initial peak depolarization preceding the
sustained plateau occurs only in response to stretch and not to current
injection (Figs. 2B and 4A). The partial
repolarization during stretches of such small amplitudes could be due
to one or more of the following: activation of voltage-dependent
outward conductance, adaptation of the mechanosensory channels, and
relaxation of the extracellular matrix in the elastic strand.
Our current-clamp data provide no evidence that the first mechanism could be involved. In fact, the depolarization of the membrane at the recording site by only a few millivolts from rest potential apparently did not activate the outward conductances. The ability of the NSRs to sustain plateau depolarizations for minutes militates against adaptation of mechanoelectric transducing channels, which leaves decline in the mechanical stimulus received by the dendritic termini, due to compliance of the elastic strand, as probable cause of these partial repolarizations. The elastic properties of the receptor strand most likely also underlie the small peak that persists in receptor potentials elicited in the presence of potassium-channel blockers (Fig. 6, A and C), as well as the independence from resting potential of the peak-plateau form of receptor potentials.
The dye-fills confirmed that the terminations of the four NSRs occupy
approximately similar-sized, nonoverlapping regions in the elastic
strand. Wilson and Paul (1990) detailed how, inside the
elastic strand, the large branches of each NSR's dendrite give rise to
a very large number of short, uniform (1 × 10 µm) dendritic
termini, all parallel to the long axis of the elastic strand and
surrounded by a distinctive extracellular matrix. These termini undergo
differential compression of their distal portion and expansion of their
proximal portion when the strand is stretch, and Wilson and Paul
(1990)
proposed that it is the expansion of the proximal
portions that opens stretch-activated channels. The ultrastructural
appearance of the dendritic terminations and surrounding extracellular
matrix is similar for other NSRs (reviewed in Wilson and Paul
1990
); presumably similar events in the peripheral terminals of
all members of this morphological type of neuron link the mechanical stimulus to the mechanoelectric transducers, and, as modeled by Berger and Bush (1979)
, account for the gross similarity
in form of their receptor potentials.
Our data indicate that, in addition to their mechanotransducing
channels, E. analoga's NSRs have a remarkably simple
complement of voltage-activated conductances. A fast-activating,
fast-inactivating outward conductance, which is blocked by 4-AP and
resembles KA-conductances in a wide spectrum of other cells
(Rogawski 1985) including nonspiking interneurons
(Laurent 1993
), mediates the fast, partial
repolarization that, except for small amplitude stimuli, cuts short the
depolarizing swing of the membrane potential in response to stretch or
depolarizing current. Its effect is to narrow the peak, i.e.,
accelerate the slower repolarization due to elastic recoil of the
extracellular matrix in the receptor strand. This restrains the
depolarization until a slower, TEA+-sensitive conductance
develops that maintains the stable, delayed depolarization phase in the
face of the continuing depolarizing drive due to the stretch or current
injection. We could find no indication of any other voltage-activated
conductance in the extraganglionic portion of E. analoga's NSRs.
The sometimes overshooting, regenerative responses that develop in the
presence of TEA+ and are blocked by cadmium occur only in
intact cells, not in dendritic segments that exclude synapses-bearing
neuropil processes. This suggests that they result from the spread of
current generated by Ca2+ influx through voltage-sensitive
Ca2+ channels at the presynaptic terminals (Blight
and Llinas 1980). CdCl2 (10 µM) in saline
produced a modest reduction in amplitude of large stretch-induced
plateau depolarizations (not shown). A calcium-activated, outward
(K+) conductance also may be present in the neuropil segments.
Unlike all of the thoracic NSRs that boost their initial transients by
a voltage-activated Na+ conductance (Bush and
Pasztor 1983; Cannone and Nijland 1989
; Lowe et al. 1978
; Mirolli 1979
, 1981
,
1983
; Pasztor and Bush 1982
), with the possible
exception of the D neuron (Cannone 1987
), E. analoga's NSRs appear to have no voltage-activated
inward-conducting channels in their peripheral dendrite. This
correlates with the relatively short distance (2 mm maximum in large
individuals of 3 cm carapace length) over which the receptor potentials
must spread to reach the output synapses in the ganglion compared with several centimeters for some of the thoracic NSRs. For the latter, it
has been suggested that the active component of their responses is
needed as much for compensating capacitative slowing as for boosting
amplitude of decrementing, analog signals that must spread over long
distances (Mirolli 1983
).
Although we have been unable to maintain stable microelectrode
penetrations close to the periphery while stretching the elastic strand
to record the receptor potential close to its source, it is clear that
neither attenuation nor distortion is substantial over the full
distance that the receptor potential of E. analoga's NSRs
must spread. Small stretches applied to a taught (unstretched) elastic
strand produce ~1 mV receptor potentials recorded in the ganglion,
which effect transmitter release (Paul 1989a); also, at
these central recording sites, rapidly varying mechanical stimuli are
followed with little distortion by fluctuations in membrane potential.
Without outward conductances to restrain the amplitude of
depolarizations reaching the ganglion in NSRs as short as E. analoga's, saturation at the output synapses could occur with even small stretch stimuli. By lowering the slope of the
stretch-depolarization curve, the voltage-sensitive K+
conductances extend these neurons' functional range to include the
full range of sensitivity of their peripheral mechanotransducing termini, which is equal to that of the receptor as a whole
(Wilson and Paul 1990
); the latter role of
K+ conductance has been identified in other sensory cells
(Pepose and Lisman 1978
). The superior ability of graded
potentials to transmit information (de Ruyter and Laughlin
1996
) may explain their common occurrence in sensory systems
where the amount/rate of information transfer needs to be high. This
exigency seems unlikely for proprioceptors at basal limb joints (not
all of which are nonspiking) and suggests that other explanations for
analog signals in some members of the small group of crustacean stretch receptors with central somata should be considered (Paul
1989b
).
These comparative data suggest that the nonspiking members of this
morphological class of sensory neuron are primarily nonspiking (rather
than having lost voltage-activated depolarizing conductances) (Mirolli
1981) and secondarily have added some inward conductance when needed to
transmit signals over longer distances as in the thorax. The
combination of analog and spiking transmission was exploited for
further enrichment of signaling in ways appropriate for each situation
(Cannone and Nijland 1989
; Pasztor and Bush 1982
; Wildman and Cannone 1996
).
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by Natural Science and Engineering Research Council, Canada Research Grant OGP08183 to D. H. Paul. J. Bruner's travel between Paris and Victoria was paid by the Center National de la Recherche Scientifique, France.
Permanent address of J. Bruner: Laboratoire de Neurobiologie Cellulaire et Moleculaire, CNRS, Gif-sur-Yvette, France.
![]() |
FOOTNOTES |
---|
Address for reprint requests: D. H. Paul, Biology Department, University of Victoria, P.O. Box 3020 STNCSC, Victoria, B.C. V8W 3N5, Canada.
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 13 October 1998; accepted in final form 11 January 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |