Test of the mechanotactile hypothesis: neuromast morphology and response dynamics of mechanosensory lateral line primary afferents in the stingray
Department of Zoology and Hawai'i Institute of Marine Biology, University of Hawai'i at Manoa, 2538 The Mall, Honolulu, HI 96822, USA
* Author for correspondence (e-mail: maruska{at}hawaii.edu)
Accepted 14 June 2004
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Summary |
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Key words: canal, elasmobranch, frequency response, hair cell, neuromast, lateral line, stingray, Dasyatis sabina
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Introduction |
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The extensive non-pored canals of sharks and batoids are located primarily
on the ventral body surface, the rostrum and around the mouth
(Chu and Wen, 1979;
Maruska, 2001
). The absence of
skin pores indicates that pressure differences caused by localized weak
hydrodynamic flow will not directly produce canal fluid motion, as occurs in
pored canal systems. Although the non-pored system of batoids was used to
model the sensitivity of lateral line neuromasts to fluid velocity
(Sand, 1937
), the response
properties of the non-pored canal system in relation to natural behaviors such
as prey localization are unknown.
The mechanotactile hypothesis was proposed to explain one function for
non-pored canals in elasmobranch fishes
(Maruska and Tricas, 1998).
This hypothesis states that the non-pored canals on the ventral surface of the
stingray function as tactile receptors that facilitate localization and
capture of small benthic invertebrate prey. The mechanotactile hypothesis
generates several testable, though not mutually exclusive, predictions about
the stimulus encoding properties of this system. First, direct coupling of the
skin and canal fluid should result in sensitivity to the velocity of skin
movement. Thus, primary afferents that innervate neuromasts in non-pored
canals should show characteristics more consistent with detectors of the
velocity rather than the acceleration of skin depression. Second, without
direct connection to the environment, non-pored canals should have a lower
sensitivity to dipole water motion compared to direct tactile stimulation.
Third, if non-pored canals represent a specialized tactile system, they may
show neurophysiological adaptations that enhance the discrimination of prey
such as silent units to facilitate detection of phasic stimuli. In addition,
they should show greater tactile sensitivity than the general cutaneous
somatosensory system that has a displacement threshold of about 20 µm
(Murray, 1961
). Fourth, if
non-pored canals function as touch receptors to detect transient skin
movements adjacent to the canal, then non-pored canals may have a greater
proportion of hair cells oriented orthogonal to the canal axis compared to
pored canals. These non-axial hair cell orientations shown in some
chondrichthyan fishes (Roberts,
1969
; Roberts and Ryan,
1971
; Ekstrom von Lubitz,
1981
; Maruska,
2001
) could encode lateral cupular deflections to expand the
tactile receptive field to include areas adjacent to the canal.
This study tests these predictions of the mechanotactile hypothesis by determination of the frequencyresponse properties of primary afferent neurons that innervate neuromasts in pored and non-pored canals in the stingray. In addition, we assess hair cell orientations among canal groups to test the prediction that non-pored canals have a greater proportion of hair cells oriented orthogonal to the canal axis compared to pored canals. Our results provide neurophysiological support for the mechanotactile hypothesis and are interpreted in relation to the natural behavior and ecology of this elasmobranch fish.
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Materials and methods |
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Extracellular single unit recording experiments used glass microelectrodes
(1550 M, 4 mol l1 NaCl) visually guided under
a microscope to the nerve surface. Lateral line primary afferents were
distinguished from electrosensory afferents by their phasic response to a
water movement stimulus delivered to the tank with a pipette. Neuromast
locations were identified by probing the skin with a small water jet or probe.
Dipole hydrodynamic stimuli were produced by a plastic sphere attached to an
18-guage stainless steel shaft and sinusoidally driven by a function generator
and minishaker. Sphere diameter was either 6 mm (ventral recordings) or 9 mm
(dorsal recordings), both of which are within the size range of natural
invertebrate prey (Cook,
1994
). The sphere was positioned with an XYZ slider
system and the dipole axis fixed at a 45° angle to the skin surface
23 mm above the skin for pored canals, or in direct contact with the
skin above the canal for non-pored canals. Receptive fields for Dp and Vp
canals were located over pores on the pectoral fins, while the receptive field
for Vnp canals was centered over the sub-epidermal canal located along the
ventral midline. To test the prediction that non-pored canals are most
sensitive to touch, the response of primary afferents from non-pored canals to
direct tactile stimulation was compared to that of a hydrodynamic flow source
23 mm above the canal.
Displacement amplitude of the sphere was controlled by a function generator
and a servocontrol feedback system in order to maintain constant source
peak-to-peak (PTP) stimulus amplitudes. Peak sphere displacement at each
stimulus amplitude and frequency was calibrated under a microscope and showed
a linear relationship over this stimulus range. The amplitude of the water
displacement at the skin surface (d) was estimated by
d=U(R/D)3, where U is the
amplitude of sphere displacement, R is the radius of the sphere, and
D is the distance between the center of the sphere and the skin
(Kroese and Schellart, 1992).
Estimated water displacement amplitudes at the skin surface (d)
ranged from approximately 0.1270 µm (for 6 and 9 mm spheres). This
estimation method is limited because it does not take into account the
influence of the nearby skin and effect of the boundary layer. However,
boundary layer effects were probably not significant in this study because all
neuromasts were located inside canals and not on the skin surface (see
Kroese and Schellart, 1992
).
Stimulus frequencies ranged from 1 to 220 Hz with a minimum of 3 s of rest
activity between each stimulation trial. For each individual afferent, the
amplitude of sphere displacement remained constant as frequency was changed.
Frequency sweeps began at 30 Hz, followed by testing of higher frequencies up
to 220 Hz and then a return to test frequencies <30 Hz. For each stimulus
frequency a minimum of 500 spikes were collected for peristimulus periods.
Neural activity was monitored visually on an oscilloscope and acoustically on
a loud speaker. Analog neural discharge signals were amplified, filtered at
3003000 Hz, and stored on tape.
The tactile receptive field for non-pored canal primary afferents was
estimated by lightly probing the skin with an 800 µm diameter sphere
attached to a thin insect pin shaft at a frequency of 1 Hz above and
adjacent to the canal. The distance between the points of maximum neural
excitation (directly over the canal) and no response (neuron returns to
spontaneous rate or silent), S, was measured to the nearest mm
orthogonal to the canal axis. Receptive field area was then calculated as
(2S)2. This is a conservative estimate of receptive field
area because the response distance directly along the canal axis was on
average at least twice as great as the response distance S that was
orthogonal to the canal axis.
Analyses of single unit responses were conducted off-line. Analog spikes were discriminated and converted to digital event files via a Cambridge Electronic Design 1401 and Spike 2 software (Cambridge, UK). Interspike interval (ISI) histograms of resting discharges were generated from 500 consecutive spikes compiled in 2 ms bins. Resting discharge ISI histograms were used to classify units as regular (unimodal with near identical median and mode) or irregular (Poisson-like distribution). Silent afferents showed no spontaneous activity and discharged only when stimulated. Resting discharge variability was expressed as the coefficient of variation (CV), which is the dimensionless ratio of standard deviation (S.D.) to mean ISI. A regular unit was defined by a unimodal distribution and CV<0.40, and an irregular unit by a distribution skewed to the right and CV>0.40. Resting discharge characteristics that were not normally distributed were compared among canal subsystems by non-parametric KruskalWallis one-way analysis of variance (ANOVA) on ranks and differences determined by Dunn's multiple comparisons test. Data that passed normality and equal variance tests were compared with a one-way ANOVA and subsequent Tukey's test.
To determine whether the neural responses were linear, several afferents
were tested at multiple stimulus amplitudes for each frequency. Linearity was
examined by plotting response amplitude (peak discharge average
resting rate) as a function of stimulus amplitude. These preliminary
experiments confirmed that the neural responses are linear relative to
stimulus amplitude, as found in other lateral line systems (e.g.
Kroese et al., 1978;
Münz, 1985
; see Results
below).
Neural sensitivity, frequency and phase responses of units were determined by construction of period histograms (128 bins) and period analyses. A Fourier transformation of the period histogram was used to generate coefficients for mean resting rate (DC), peak discharge rate, and the phase relationship between the peak unit response and the stimulus peak. Neural sensitivity for individual units was calculated as [(peak discharge rate DC)/stimulus amplitude]. In order to compare neural responses among units, sensitivity was converted to relative neural gain (dB) calculated as 20xlog(neural sensitivity).
Best frequency (BF) of each neuron was defined as the frequency that evoked
the greatest increase in the number of spikes above mean resting rate (peak
discharge rate DC). Data used to generate frequencyresponse
curves were normalized by assigning a value of 0 dB to BF in order to control
for absolute differences in sensitivities among afferents. d at the
skin surface was estimated as described above and converted to velocity
(u) and acceleration (a) values with the relationships
u=2fd and
a=4
2f2d, where f
is the sphere vibration frequency and d is estimated peak-to-peak or
peak displacement at the skin surface
(Coombs and Janssen, 1990a
).
Neural sensitivity for units from pored canals was estimated from PTP stimulus
and response, whereas peak stimulus and response values were used for units
from non-pored canals because the sphere was in contact with the skin for only
half of the sinusoidal stimulus cycle. To illustrate the difference in
relative sensitivity between tactile and hydrodynamic stimuli, afferent
responses to tactile stimuli were normalized relative to responses to
hydrodynamic flow. Neural sensitivity to hydrodynamic flow was assigned a gain
value of 0 dB, and relative neural gain (dB) to tactile stimuli calculated as
20xlog(tactile neural sensitivity/hydrodynamic neural sensitivity). The
phase relation between the stimulus and neural discharge response was
expressed as the difference in arc degrees between the peak discharge rate and
peak stimulus amplitude.
Hair cell orientations
Hair cell sensitivity to fluid flow in lateral line canals is dependent
upon the orientation of kinocilia and stereocilia relative to the longitudinal
axis of the canal. Hair cell polarities were determined to test the prediction
that a greater number of hair cells are oriented orthogonal to the
longitudinal canal axis in non-pored canals compared to pored canals. Adult
stingrays were euthanized with an overdose of MS222, the epidermis removed,
canals opened to expose the neuromasts, and cupulae mechanically dislodged
from neuromasts by a gentle water jet. Approximately 25 neuromasts were
removed from Dp, Vp and Vnp hyomandibular canals in each animal, fixed for
12 h in 2% glutaraldehyde in Millonig's buffer, and soaked in
Millonig's buffer overnight. Tissue was then rinsed in 0.05 mol
l1 phosphate buffer (PB), postfixed in 1% osmium tetroxide,
rinsed again in PB and dehydrated in an ethanol series (50100%).
Neuromasts were dried in an LADD (Burlington, VT, USA) critical point dryer
with carbon dioxide as a transitional fluid and sputter-coated with
goldpalladium alloy. Samples were viewed on a Hitachi S-2700 scanning
electron microscope (SEM) at an accelerating voltage of 810 kV and
images recorded on VHS tape for analysis.
Individual hair cell orientation was determined by the semicircular angular deviation (from 0180°) from the axis of maximum excitation of the hair cell (towards the kinocilium) to the longitudinal axis of the neuromast (canal axis). Orientations were measured for 10 randomly selected hair cells from several neuromasts in each of the three canals (Dp, Vp and Vnp) in each animal. Hair cell orientation data failed tests of normality and could not be normalized by transformation. Thus, the non-parametric KruskalWallis one-way ANOVA on ranks was used to test whether hair cell orientations differed among neuromasts located in different canal subsystems.
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Results |
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There were no differences in resting discharge activity, ISI or CV among the total population of afferents that innervate Dp and Vp canals (KruskalWallis one-way ANOVA on ranks, P>0.05). However, resting discharge characteristics of lateral line primary afferents are further categorized into regular, irregular and silent units within each canal type and are summarized in Table 1. The plots in Fig. 4 show a clear separation between regular and irregular afferents among canal types with respect to CV, but some overlap and greater variability with respect to ISI. Primary afferents classified as regular differed only between Dp and Vnp canals in CV (one-way ANOVA, Tukey's test, P<0.05), resting discharge activity (one-way ANOVA, Tukey's test, P<0.05), and interspike intervals (KruskalWallis one-way ANOVA on ranks, Dunn's test, P<0.05). Thus, regular primary afferents in Dp canals had a greater CV and resting discharge activity, but lower ISI compared to Vnp regular afferents (Fig. 4). In contrast, primary afferents classified as irregular did not differ with respect to these characteristics (KruskalWallis one-way ANOVA on ranks, P>0.05).
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Frequencyresponse
Frequencyresponse characteristics were determined for a total of 77
lateral line primary afferent neurons (Dp=40, Vnp=28 and Vp=9) in 14
stingrays. Sinusoidal stimulation of the lateral line system produced
modulation of primary afferent spontaneous activity, and evoked discharges
from silent units. Recordings were made at stimulus amplitudes where there was
a linear relationship between peak neural response and stimulus intensity. The
amplitude of the response was proportional to the amplitude of the stimulus
across the range of frequencies used (Fig.
5). The gain and phase of the response of individual afferents was
independent of the stimulus amplitude, and confirms linearity for this
system.
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When neural responses of units from Dp canals were plotted as a function of
water velocity, they showed band-pass characteristics with a 6dB
bandwidth of 2090 Hz (Fig.
6Ai). Neural responses of afferents from Vp canals showed low-pass
characteristics to hydrodynamic stimuli re: velocity with a response above
6dB maintained <70 Hz (Fig.
6Bi). When the skin was stimulated above Vnp canals, primary
afferents from Vnp canals had a flat low-pass characteristic <30 Hz
re:velocity with a measured best frequency of 8.6±1.3 Hz (mean ±
S.E.M.;
Fig. 6Ci). Relative to
acceleration, primary afferents from Dp and Vp canals showed a flat frequency
response to hydrodynamic stimuli re: acceleration below 30 Hz
(Fig. 6Aii,Bii). Thus, primary
afferents from Dp canals show frequency response characteristics consistent
with acceleration detectors as per Coombs and Janssen
(1989). The response of
primary afferents from Vp canals showed properties of both an acceleration and
a velocity detector, which may be partially due to the large variation and
small sample size of this group of afferents. In contrast, primary afferents
from Vnp canals showed low-pass characteristics to tactile stimuli re:
acceleration with a 6 dB drop in neural gain achieved by 5 Hz
(Fig. 6Cii). Further, primary
afferents from Vnp canals show frequency-independent response characteristics
to tactile stimuli re: velocity below 30 Hz, which is consistent with velocity
detectors (Fig. 6Ci). Although
frequencyresponse differences were identified among canal subsystems,
there was no obvious relationship between neuromast location on the body and
best frequency of primary afferents.
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The low frequency slope of the frequencyresponse curve relative to
displacement for afferents that innervate Dp canal neuromasts
(x=37.3±7.8 dB decade1, mean ±
S.D., N=19 units) was higher than that of
afferents that innervate Vnp canal neuromasts (x=19.4±8.6 dB
decade1, N=19 units) (one-way ANOVA,
P<0.001). These values agree with the expected amplitude slopes of
40 dB decade1 for an acceleration detector and
20
dB decade1 for a velocity detector
(Kroese et al., 1978
),
respectively. The low frequency slopes of afferents from Vp canal neuromasts
(x=19.8±4.8 dB decade1, mean ±
S.D., N=3 units) were similar to those from Vnp
units, but the low sample size precludes conclusions from these data.
Phase relationships of the peak neural response relative to peak sphere displacement confirm these different sensitivities to velocity and acceleration (Fig. 7). Many afferents from Dp canal neuromasts showed a phase lead of approximately 180° at low frequencies (<20 Hz), while afferents from Vnp canals had a phase lead near 90° at low frequencies (<20 Hz) (Fig. 7). Phase increases at higher frequencies are at least partially due to differential primary afferent conduction times between the various stimulus and recording sites. Phase relationships of afferents from Vp canals were not analyzed, due to small sample size and high variation among units.
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Primary afferent BF also differed among canal subsystems (Table 2; Fig. 8). Best frequency of acceleration-sensitive Dp primary afferents ranged from 140 Hz (x=21.7±1.7 Hz, mean ± S.E.M.), but the mode was at 30 Hz (Fig. 8). In contrast, while the range of BF for Vnp velocity sensitive afferents was also broad (130 Hz, x=8.6±1.3 Hz, mean ± S.E.M.), maximum BF mode was lower at 10 Hz (Fig. 8). Best frequency of the total population of afferents from Dp canals was higher than that of Vnp canals when all fiber responses were tested within a single category of displacement, velocity or acceleration (KruskalWallis one-way ANOVA on ranks, Dunn's test, P<0.05). However, BF of the total population of afferents from Vp canals was higher than that of Vnp canals when tested within a single category of velocity or acceleration (KruskalWallis one-way ANOVA on ranks, Dunn's test, P<0.05), but not displacement (KruskalWallis one-way ANOVA on ranks, Dunn's test, P>0.05). Thus, in the context of biologically significant stimuli for these canal subsystems (velocity and acceleration), the population of afferents from pored canals were more sensitive to higher stimulus frequencies than those in non-pored canals. There was no difference in neural sensitivity (gain) at BF among canal subsystems in terms of velocity or acceleration (KruskalWallis one-way ANOVA on ranks, Dunn's test, P>0.05; Table 3). The relationship between neural discharge and hydrodynamic acceleration for three individual Dp primary afferents shows that neural discharge increases as a function of stimulus intensity (Fig. 5). The most sensitive primary afferent in this example (0.31 spikes s1/mm s2) had a more than fourfold greater average peak discharge slope than the least sensitive afferent (0.07 spikes s1/mm s2). Mean neural sensitivity at best frequency ranged from 0.01 to 1.2 spikes s1/mm s2 for Dp, 0.04 to 1.4 spikes s1 per mm s2 for Vp, and 5 to 86 spikes s1/mm s2 for Vnp canals (Table 3).
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Mechanotactile sensitivity
The prediction that non-pored canals should have a greater sensitivity to
tactile stimuli than to hydrodynamic stimuli was supported. Afferents from Vnp
canals showed phasic responses to skin depression velocities of approximately
30630 µm s1 from 120 Hz, and velocities as
low as 63 µm s1 to >5 mm s1 at a
frequency of 10 Hz. Primary afferents that innervate neuromasts in the
non-pored ventral hyomandibular canal respond to direct tactile stimulation as
well as to hydrodynamic flow several mm above the canal. However, afferents
were an average of 210 times more sensitive to tactile stimulation than
to water movements directly above the tactile stimulus location
(Fig. 9). This difference is
most prominent at lower frequencies (1020 Hz) where the mean change in
neural sensitivity between tactile and hydrodynamic stimuli was
6.3±0.92 spikes s1/mm s2 at 20 Hz
(mean ± S.E.M.).
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The tactile receptive field was also determined for 26 primary afferents
that innervate neuromasts in the non-pored ventral hyomandibular canal.
Receptive fields on the skin above the non-pored canals ranged from
0.254.0 cm2 with a mean of 1.4±0.3 cm2
(mean ± S.E.M.). Thus, rays are
sensitive to tactile stimulation at least 2.5 mm lateral to the main canal on
either side, which encompasses the size of benthic invertebrate prey
(210 mm) found in their diet.
Hair cell orientations
The prediction that non-pored canal neuromasts have a higher proportion of
hair cells oriented off the longitudinal canal axis compared to hair cells on
neuromasts within pored canals was not supported by SEM analyses. The majority
of hair cells were oriented within 45° of the longitudinal canal axis in
Dp (94%), Vp (93%) and Vnp (86%) canals
(Fig. 10). These orientations
indicate that about 90% of hair cells in all canals will respond with at least
70% (cos45°) of the maximum response to fluid flow along the longitudinal
canal axis. Although there was no difference in hair cell orientations among
Dp, Vp and Vnp canal neuromasts (KruskalWallis one-way ANOVA on ranks,
P=0.285), about 614% of all hair cells were oriented within
45° of the orthogonal canal axis. Thus only a small percentage of hair
cells would be most sensitive to localized lateral depressions of the canal
wall.
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Discussion |
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Resting discharge activity
Lateral line primary afferents that lack any resting discharge (silent
units) were encountered 310 times more frequently in Vnp canals
compared to Vp or Dp canals. Silent units in the lateral line of the cichlid
fish were relatively insensitive to sinusoidal stimuli and were sometimes
localized to injured neuromasts
(Münz, 1985
). This is not
the case in the stingray because all silent units had sensitivities within the
range of spontaneously active neurons. Silent afferents that discharge only in
response to stimulation may be advantageous in the non-pored ventral canals of
the stingray that encode transient movements of underlying skin caused by
stimuli such as small excavated prey. Thus, this physiological subpopulation
may serve as a discriminator of localized skin movements.
Spontaneous discharge activities of primary afferent neurons in the
stingray are similar to those reported for lateral line systems in other
fishes (Roberts, 1972;
Münz, 1985
;
Tricas and Highstein, 1991
;
Kroese and Schellart, 1992
).
Interspike interval distributions within each class (regular and irregular)
were variable, similar to those seen in lateral line afferents of the cichlid
and other teleosts, and may be a function of fiber diameter and different
conduction velocities (Münz,
1985
). Primary afferents that innervate Dp canal neuromasts had
higher resting discharge activity and shorter interspike intervals than
afferents from Vnp canal neuromasts. Primary afferents that innervate canal
neuromasts in most teleosts have higher discharge rates than those from
superficial neuromasts, which is suggested to be a function of the increased
size and greater hair cell to afferent convergence ratio of canal neuromasts
(Münz, 1985
,
1989
). This contradicts
results from the present study because primary afferents from Vnp canals of
the stingray have a slower discharge rate, but innervate larger neuromasts and
have a greater hair cell to afferent ratio than those in the Dp canal, which
has afferents with a faster discharge rate
(Maruska and Tricas, 1998
;
Lowrance, 2000
). Thus, the
difference in spontaneous discharge activity between Dp and Vnp canals cannot
be explained by variations in convergence ratios, but rather, discharge
regularity may be due to postsynaptic mechanisms, as suggested for the
vestibular system (Goldberg et al.,
1984
; Boyle and Highstein,
1990
).
Detection of lateral line stimuli requires central nervous system
recognition of a change in primary afferent resting discharge rate or pattern.
Afferents with fast regular discharge rates would show enhanced temporal
resolution for encoding external stimuli, especially at low frequencies, due
to the low endogenous variance in resting interspike intervals. In contrast,
irregular discharging units would better encode higher frequency information.
Moving objects generally produce complex water motions, which may contain both
high and low frequency components
(Bleckmann et al., 1991). Thus,
the mix of fiber types in all canal locations of the stingray can allow
detection of a range of different frequencies potentially available to the
lateral line system. Regular and irregular primary afferents within each canal
subsystem differed in discharge variability (CV), but had similar best
frequencies. Thus the functionality of regular versus irregular
discharge patterns at the primary afferent level is unclear, but may be more
important in central processing.
Frequencyresponse
Frequencyresponse characteristics of primary afferents indicate that
pored canal systems function as acceleration detectors. Neural responses to
sinusoidal stimulation at frequencies below best frequency increase in
relative neural gain at a rate of 37 dB decade1 and have a
phase lead of about 180°, a typical response feature for an acceleration
detector (Kroese and Schellart,
1992). Historically, frequencyresponse properties of
lateral line primary afferent neurons are interpreted in terms of displacement
of the stimulus source (see Kalmijn,
1989
). However, canal neuromasts are known to be sensitive to
water acceleration such that the flow velocity inside the canal is
proportional to the net acceleration between the fish and surrounding water
(for reviews, see Kalmijn,
1989
; Coombs and Janssen,
1990b
; Coombs and Montgomery,
1999
) and there must be a pressure gradient across the canal pores
to generate fluid flow inside the canal. Therefore, when
frequencyresponse data are plotted in terms of acceleration, they
exhibit broader tuning curves with relatively constant gain up to
4050 Hz and low-pass characteristics (BF<30
Hz)(Coombs and Janssen, 1989
;
Kalmijn, 1989
). This is also
true for the frequencyresponses recorded from the stingray, which is
consistent with those observed in teleosts and further illustrates the
behavioral importance of low frequency velocity and acceleration information
to the lateral line system.
The neural response of primary afferents to water motion is influenced by
the morphologies of the canal, neuromast and cupula (Denton and Gray,
1988,
1989
; van Netten and Kroese,
1989a
,b
).
The lateral line canal system of elasmobranchs contains a main canal with a
nearly continuous sensory epithelium and lateral neuromast-free tubules that
terminate in pores on the skin (see Fig.
1). The frequencyresponse properties of lateral line
primary afferents in the pored canals of the stingray are similar to those
reported for teleosts in which only a single neuromast is found between two
adjacent pores (Münz,
1985
; Coombs and Janssen,
1990a
; Kroese and Schellart,
1992
). Thus, differences in neuromast, canal and pore
organizations between bony and cartilaginous fishes are not reflected in
overall response properties of primary afferent neurons. A comparable example
in a single species exists in the Antarctic fish, Trematomus
bernacchii, which has large variations in peripheral canal morphology but
relatively homogeneous frequency response properties
(Coombs and Montgomery, 1992
).
In the stingray dorsal hyomandibular canal system, the increase in the number
of pores associated with a single lateral tubule increases the receptive field
area on the distal pectoral fins, similar to that observed for the
prickleback, Xiphister atropurpureus
(Bleckmann and Münz,
1990
). These branched tubules on the dorsal surface indicate an
increased sensitivity to water motions, possibly at a loss of spatial
resolution, but the physiology data from the present study shows no difference
in neural sensitivity among canal types. Thus, any functional significance of
multiple neuromasts between adjacent pores or branched tubule patterns in
elasmobranchs requires further investigation of parameters such as hair cell
afferent innervation; canal, neuromast and cupulae organization and
mechanics; and projection patterns for central processing.
The dorsal hyomandibular canal in the Atlantic stingray is best positioned
to detect water movements near the disk margin that may be generated by
predators, conspecifics during social interactions, epifaunal or swimming prey
items, and distortions in the animal's own flow field for object localization
(Maruska, 2001). Such water
movements are often transient and complex. Detection of the acceleration
component of water motion by Dp canals is advantageous in that acceleration
precedes the actual displacement of the object, thus resulting in an earlier
response (Wubbels, 1992
). The
amplitude of acceleration is also relatively large at onset and offset of a
movement, which would cause a strong response at the peripheral lateral line
provided the stimulus is within the receptor bandwidth. Strong and quick
responses at the periphery are essential for lateral line-mediated behaviors
such as prey capture and predator detection in all aquatic species. Recordings
from the brain of the batoid Platyrhinoidis triseriata show that fast
transient events (high acceleration) best stimulate midbrain and forebrain
lateral line regions (Bleckmann et al.,
1989
). Thus, the ability of the peripheral and central lateral
line system to detect and encode transient acceleration stimuli supports its
hypothesized biological functions in the stingray.
In contrast to pored canal systems, the response properties of primary
afferents from non-pored canals of the stingray are not interpreted in terms
of hydrodynamic stimuli. Neuromasts are enclosed within the canal and internal
fluid motion is created by movement of the skin rather than by pressure
gradients across skin pores. Frequencyresponse properties of afferents
in non-pored canals show a low-frequency roll-off of 19 dB
decade1 and phase lead of about 90° to tactile
stimulation, which is within the expected range for a velocity detector
(Kroese and Schellart, 1992).
However, because the hydrodynamic force acting on the cupula contains both a
viscous and an inertial component, a fluid or boundary layer occurs around the
cupula that ultimately influences the mechanical coupling of water and the
neuromast (van Netten, 1991
).
The morphology of pored canals creates a high-pass filter that attenuates low
frequencies (Denton and Gray,
1988
). In contrast, the underlying compliant skin of non-pored
canals may function as a low-pass filter that reduces high frequency
stimulation due to the physical constraints of skin movement. However, the
mechanics of tissue distortion produced by tactile or hydrodynamic flow are
likely complex and dependent on a number of factors such as the mechanical
properties of the skin and canal. Variations in frequencyresponse
properties between canal and superficial neuromasts of teleosts result from
factors such as different stiffness coupling between cupula and hair cells,
cupular geometry and canal dimensions (Denton and Gray,
1988
,
1989
; van Netten and Kroese,
1989; van Netten and Khanna,
1994
). Thus, these characteristics may also contribute to the
response differences observed in the pored and non-pored canals of the
stingray and warrant further investigation.
The Atlantic stingray feeds almost exclusively on small benthic
invertebrates, which they excavate from the sand substrate and contain in a
feeding depression beneath the body (Cook,
1994; Bradley,
1996
; Maruska and Tricas,
1998
). Motile as well as sedentary animals can produce
hydrodynamic flows and potential stimuli near the best frequency of the
stingray lateral line canal system. For example, many zooplankton generate
swimming vibrations of 550 Hz atconstant swimming speeds of 1015
cm s1 (Montgomery,
1989
), and bivalves generate hydrodynamic flow velocities of
614 cm s1 (Price
and Schiebe, 1978
; LaBarbera,
1981
). These values translate to accelerations in the cm
s2 range, which are well within the range of sensitivities
seen at the primary afferent level in the stingray. Hydrodynamic flow fields
generated by excavated prey can be detected by the pored section of the
ventral hyomandibular canal and the stingray can move its body to position the
non-pored canals, snout and, finally, the mouth directly above the prey for
localization and final capture. Several meso- to bathypelagic fishes (e.g.
Anoplogaster spp.) also have canal systems covered by thin, soft
membranes without any regular system of pores. Studies indicate this
morphology increases low-frequency sensitivity of the system and similar to
the stingray, may facilitate foraging on small fishes, squid and crustaceans
in low light environments (Denton and
Gray, 1988
).
Mechanotactile sensitivity
The mechanotactile hypothesis of lateral line function states that ventral
non-pored canals function as tactile receptors to facilitate prey localization
and capture (Maruska and Tricas,
1998). The present study confirms the prediction that primary
afferents in the non-pored ventral canals respond as velocity detectors driven
by movement of the skin. These afferents have receptive fields of 0.254
cm2 on the skin surface, which is equivalent to or greater than the
surface area of their small prey. Previous studies show that elasmobranch
cutaneous tactile receptors respond to skin depressions of 20 µm
(Murray, 1961
), which is
equivalent to a velocity of 1256 µm s1 at 10 Hz. The
present study shows that primary afferents from non-pored canals respond to
skin motion velocities as low as 63 µm s1 at 10 Hz. Thus
the mechanotactile lateral line system appears to provide a twentyfold or
greater sensitivity to tactile velocity stimuli and could increase the
stingray's foraging efficiency on small benthic prey.
In order to assess possible advantages of Vnp canals for prey detection, we compared frequency responses to hydrodynamic and tactile stimuli among Vnp and Dp canal units, but because of time constraints were unable to compare responses to tactile stimuli between Vnp and Vp units. Without this control we can only assert that the non-pored lateral line system is specialized for tactile stimulation compared to pored canals. However, in addition to enhanced tactile sensitivity to prey, there may be other advantages for a non-pored lateral line system, which include the following: (1) Hydrodynamic stimuli from emergent and infaunal invertebrates (e.g. amphipods, polychaetes, echinoderms) are minimal or do not adequately stimulate the pored canal system. (2) Non-pored canals do not vent local lateral line fluid motion, thus they could enhance sensitivity to tactile stimuli along a greater length of the canal. This would be dependent upon tactile stimulus velocities and pore separation. (3) Development of a non-pored lateral line increases sensitivity to velocity and low frequency stimuli. (4) A non-pored system reduces intrusion of sediments (e.g. sand) through skin pores that can interfere with hydrodynamic stimulation. (5) The absence of canal pores would reduce self-generated hydrodynamic noise during excavation and manipulation of prey. This could result in an enhanced signal-to-noise ratio in primary afferent neurons.
Only a few studies examine the physiology of the lateral line in
elasmobranchs, especially with regard to biological function of the system.
Sand (1937) demonstrated that
a constant flow in the ventral non-pored hyomandibular canal of the skate,
Raja spp., increased primary afferent discharges, and that touch of
the skin was also an effective stimulus for the lateral line in those species.
However, any natural tactile or hydrodynamic stimulus should only cause a
transient low volume movement of canal fluid and therefore the suprathreshold
constant flows used in those experiments were not biologically relevant
stimuli. Recordings in the medial octavolateralis nucleus (primary lateral
line processing center in the hindbrain) in the thornback ray show responses
to peak-to-peak (PTP) displacements of 0.02 µm (Bleckmann et al.,
1987
,
1989
), which is considerably
lower than that observed for primary afferents in the present study. This
discrepancy may be explained by several factors. First, the lowest stimulus
amplitude used for frequencyresponse analyses in the present study was
approximately 0.5 µm PTP at the skin surface, but many primary afferents in
the stingray responded to displacements much lower than 0.5 µm (K.P.M.,
personal observation). Second, the increased sensitivity in the central
nervous system results from high signal-to-noise ratios of principal cells in
the hindbrain due to convergence of many primary afferents onto a single
secondary cell (Montgomery,
1984
; Bleckmann and Bullock,
1989
; Tricas and New,
1998
). In addition, behavioral detection thresholds of sensory
stimuli are often much lower than neurophysiological thresholds due to
summation and sensory integration. Thus, in its natural environment the
stingray may respond to water movements at thresholds much lower than
indicated by their primary afferent responses.
Hair cell orientations
Maximum sensitivity to fluid flow in canals results from the orientation of
hair cells and their kinocilia parallel to the main canal axis. While canals
of some chondrichthyan species show these axial hair cell orientations
(Hama and Yamada, 1977;
Peach and Rouse, 2000
), others
have proportions of hair cells oriented nearly perpendicular to the canal axis
(Roberts, 1969
;
Roberts and Ryan, 1971
;
Ekstrom von Lubitz, 1981
). The
majority of hair cells (8694%) within all stingray canals were oriented
within 45° of the longitudinal canal axis. However, all neuromasts also
showed some hair cells that were oriented perpendicular to the canal axis
(614%), which may broaden the sensitivity to tactile stimulation of the
skin adjacent to the canal. Nevertheless, we found no difference in hair cell
orientations among Dp, Vp and Vnp canals. Thus these results did not support
our prediction that non-pored canals have a greater proportion of orthogonally
oriented hair cells compared to pored canals. One function of the non-pored
canals may be to facilitate movement of the body so that prey is passed along
the canal axis towards the mouth (Maruska
and Tricas, 1998
), so the most effective hair cell orientation
would be parallel to the canal axis as observed. The mechanotactile mechanism
of action for ventral non-pored canals is also supported by the 0.254
cm2 receptive field, large canal diameter that covers a greater
area of underlying skin surface, and more compliant dermal skin layers
compared to dorsal canals (Maruska and
Tricas, 1998
; Maruska,
2001
). Collectively, this morphological evidence is consistent
with the function of tactile receptors for the ventral non-pored canals of
batoids, but the performance consequences of non-axial hair cells in pored
canals remains to be determined.
In summary, our results show that the pored hyomandibular canals on the dorsal surface of the stingray differ in terms of primary afferent-response properties from the non-pored hyomandibular canals on the ventral surface. Primary afferents from dorsal pored canals respond as hydrodynamic acceleration detectors of transient water disturbances that may be caused by predators, conspecifics or prey. Ventral non-pored canals are sensitive to small movements of the skin and primary afferents encode the velocity of fluid induced in the canal by these stimuli. These results support the main predictions of the mechanotactile hypothesis and demonstrate a physiological basis for lateral line-mediated prey detection in this and possibly other elasmobranch species.
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References |
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Bleckmann, H. and Bullock, T. H. (1989). Central nervous physiology of the lateral line, with special reference to cartilaginous fishes. In The Mechanosensory Lateral Line Neurobiology and Evolution (ed. S. Coombs, P. Görner and H. Münz), pp. 387-408. New York: Springer-Verlag.
Bleckmann, H. and Münz, H. (1990). Physiology of lateral-line mechanoreceptors in a teleost with highly branched, multiple lateral lines. Brain Behav. Evol. 35,240 -250.[CrossRef][Medline]
Bleckmann, H., Breithaupt, T., Blickhan, R. and Tautz, J. (1991). The time course and frequency content of hydrodynamic events caused by moving fish, frogs, and crustaceans. J. Comp. Physiol. A 168,749 -757.[Medline]
Bleckmann, H., Bullock, T. H. and Jorgensen, J. M. (1987). The lateral line mechanoreceptive mesencephalic, diencephalic, and telencephalic regions in the thornback ray, Platyrhinoidis triseriata (Elasmobranchii). J. Comp. Physiol. A 161,67 -84.[Medline]
Bleckmann, H., Weiss, O. and Bullock, T. H. (1989). Physiology of lateral line mechanoreceptive regions in the elasmobranch brain. J. Comp. Physiol. A 164,459 -474.[Medline]
Boord, R. L. and Campbell, C. B. G. (1977). Structural and functional organization of the lateral line system of sharks. Amer. Zool. 17,431 -441.
Boyle, R. and Highstein, S. M. (1990). Resting discharge and response dynamics of horizontal semicircular canal afferents of the toadfish, Opsanus tau. J. Neurosci. 10,1557 -1569.[Abstract]
Bradley, J. L. (1996). Prey energy content and selection, habitat use and daily ration of the Atlantic stingray, Dasyatis sabina. MS thesis, Florida Institute of Technology, USA.
Chu, Y. T. and Wen, M. C. (1979). Monograph of Fishes of China: A Study of the Lateral-Line Canal System and that of Lorenzini Ampullae and Tubules of Elasmobranchiate Fishes of China. Shanghai, China: Science and Technology Press.
Cook, D. A. (1994). Temporal patterns of food habits of the Atlantic stingray, Dasyatis sabina (LeSeur, 1824), from the Banana River Lagoon, Florida. MS thesis, Florida Institute of Technology, USA.
Coombs, S. and Janssen, J. (1989). Peripheral processing by the lateral line system of the mottled sculpin (Cottus bairdi). In The Mechanosensory Lateral Line Neurobiology and Evolution (ed. S. Coombs, P. Görner and H. Münz), pp. 299-319. New York: Springer-Verlag.
Coombs, S. and Janssen, J. (1990a). Behavioral and neurophysiological assessment of lateral line sensitivity in the mottled sculpin, Cottus bairdi. J. Comp. Physiol. A 167,557 -567.[Medline]
Coombs, S. and Janssen, J. (1990b). Water flow detection by the mechanosensory lateral line. In Comparative Perception Volume II: Complex Signals (ed. C. Stebbins and M. A. Berkley), pp. 89-123. New York: Wiley and Sons, Inc.
Coombs, S. and Montgomery, J. C. (1992). Fibers innervating different parts of the lateral line system of the Antarctic fish, Trematomus bernacchii, have similar neural responses despite large variations in peripheral morphology. Brain Behav. Evol. 40,217 -233.[Medline]
Coombs, S. and Montgomery, J. C. (1999). The enigmatic lateral line system. In Comparative Hearing: Fish and Amphibians (ed. R. R. Fay and A. N. Popper), pp.319 -362. New York: Springer-Verlag.
Denton, E. J. and Gray, J. A. B. (1988). Mechanical factors in the excitation of the lateral line of fishes. In Sensory Biology of Aquatic Animals (ed. J. Atema, R. R. Fay, A. N. Popper and W. N. Tavolga), pp. 595-593. Berlin Heidelberg New York: Springer-Verlag.
Denton, E. J. and Gray, J. A. B. (1989). Some observations on the forces acting on neuromasts in fish lateral line canals. In The Mechanosensory Lateral Line Neurobiology and Evolution (ed. S. Coombs, P. Görner and H. Münz), pp.229 -246. New York: Springer-Verlag.
Ekstrom von Lubitz, D. K. J. (1981). Ultrastructure of the lateral-line sense organs of the ratfish, Chimaera monstrosa. Cell Tiss. Res. 215,651 -665.[Medline]
Ewart, J. C. and Mitchell, H. C. (1892). On the lateral sense organs of elasmobranchs. II. The sensory canals of the common skate (Raja batis). Trans. R. Soc. Edinburgh 37, 87-105.
Flock, A. (1965). Electron microscopic and electrophysiological studies on the lateral line canal organ. Acta Otolaryngol. Suppl. S199,7 -90.
Goldberg, J. M., Smith, C. E. and Fernandez, C.
(1984). Relation between discharge regularity and responses to
externally applied galvanic currents in vestibular nerve afferents of the
squirrel monkey. J. Neurophysiol.
51,1236
-1256.
Hama, K. and Yamada, Y. (1977). Fine structure of the ordinary lateral line organ II. The lateral line canal organ of the spotted shark, Mustelus manazo. Cell Tiss. Res. 176, 23-36.[Medline]
Johnson, S. E. (1917). Structure and development of the sense organs of the lateral canal system of selachians (Mustelus canis and Squalus acanthias). J. Comp. Neurol. 28,1 -74.
Kalmijn, A. J. (1989). Functional evolution of lateral line and inner-ear sensory systems. In The Mechanosensory Lateral Line Neurobiology and Evolution (ed. S. Coombs, P. Görner and H. Münz), pp. 187-215. New York: Springer-Verlag.
Kroese, A. B. A., Van der Zalm, J. M. and Van den Bercken, J. (1978). Frequency response of the lateral-line organ of Xenopus laevis. Pflüg. Archiv. 375,167 -175.[Medline]
Kroese, A. B. and Schellart, N. A. M. (1992).
Velocity- and acceleration-sensitive units in the trunk lateral line of the
trout. J. Neurophysiol.
68,2212
-2221.
LaBarbera, M. (1981). Water flow patterns in and around three species of articulate brachiopods. J. Exp. Mar. Biol. Ecol. 55,185 -206.[CrossRef]
Lowrance, C. (2000). The development of the mechanosensory and electrosensory lateral line: a model of sensitivity and resolution. MS thesis, Florida Institute of Technology, USA.
Maruska, K. P. (2001). Morphology of the mechanosensory lateral line system in elasmobranch fishes: ecological and behavioral considerations. Environ. Biol. Fish. 60, 47-75.[CrossRef]
Maruska, K. P. and Tricas, T. C. (1998). Morphology of the mechanosensory lateral line system in the Atlantic stingray, Dasyatis sabina: the mechanotactile hypothesis. J. Morph. 238,1 -22.[CrossRef]
Montgomery, J. C. (1984). Frequency response characteristics of primary and secondary neurons in the electrosensory system of the thornback ray. Comp. Biochem. Physiol. 79A,189 -195.
Montgomery, J. C. (1989). Lateral line detection of planktonic prey. In The Mechanosensory Lateral Line Neurobiology and Evolution (ed. S. Coombs, P. Görner and H. Münz), pp. 561-574. New York: Springer-Verlag.
Montgomery, J. C. and Skipworth, E. (1997). Detection of weak water jets by the short-tailed stingray Dasyatis brevicaudata (Pisces: Dasyatidae). Copeia 1997,881 -883.
Montgomery, J. C., Baker, C. F. and Carton, A. G. (1997). The lateral line can mediate rheotaxis in fish. Nature 389,960 -963.[CrossRef]
Münz, H. (1985). Single unit activity in the peripheral lateral line system of the cichlid fish Sarotherodon niloticus L. J. Comp. Physiol. A 157,555 -568.
Münz, H. (1989). Functional organization of the lateral line periphery. In The Mechanosensory Lateral Line Neurobiology and Evolution (ed. S. Coombs, P. Görner and H. Münz), pp. 285-297. New York: Springer-Verlag.
Murray, R. W. (1961). The initiation of cutaneous nerve impulses in elasmobranch fishes. J. Physiol. 159,546 -570.[Medline]
Peach, M. B. (2001). The dorso-lateral pit organs of the Port Jackson shark contribute sensory information for rheotaxis. J. Fish Biol. 59,696 -704.[CrossRef]
Peach, M. B. and Rouse, G. W. (2000). The morphology of the pit organs and lateral line canal neuromasts of Mustelus antarcticus (Chondrichthyes: Triakidae). J. Mar. Biol. Assn UK 80,155 -162.[CrossRef]
Price, R. E. and Schiebe, M. A. (1978). Measurements of velocity from excurrent siphons of freshwater clams. Nautilus 92,67 -69.
Roberts, B. L. (1969). Mechanoreceptors and the behavior of elasmobranch fishes with special reference to the acoustico-lateralis system. In Sensory Biology of Sharks, Skates, and Rays (ed. E. S. Hodgson and R. W. Mathewson), pp.331 -390. Arlington, Virginia: Office/Naval Research.
Roberts, B. L. (1972). Activity of lateral-line organs in swimming dogfish. J. Exp. Biol. 56,105 -118.
Roberts, B. L. and Ryan, K. P. (1971). The fine structure of the lateral-line sense organs of dogfish. Proc. R. Soc. Lond. B 179,157 -169.
Sand, A. (1937). The mechanism of the lateral sense organs of fishes. Proc. R. Soc. B 123,472 -495.
Späth, M. and Schweickert, W. (1977). The effect of metacaine (MS-222) on the activity of the efferent and afferent nerves in the teleost lateral-line system. Naunyn-Schmiedebergs Arch. Pharmakol. 297,9 -16.[Medline]
Tricas, T. C. and Highstein, S. M. (1991). Action of the octavolateralis efferent system upon the lateral line of free-swimming toadfish, Opsanus tau. J. Comp. Physiol. A 169,25 -37.[Medline]
Tricas, T. C. and New, J. G. (1998). Sensitivity and response dynamics of elasmobranch electrosensory primary afferent neurons to near threshold fields. J. Comp. Physiol. A 182,89 -101.[Medline]
van Netten, S. M. (1991). Hydrodynamics of the excitation of the cupula in the fish canal lateral line. J. Acoust. Soc. Amer. 89,310 -319.
van Netten, S. M. and Khanna, S. M. (1994). Stiffness changes of the cupula associated with the mechanics of hair cells in the fish lateral line. Proc. Nat. Acad. Sci. 91,1549 -1553.[Abstract]
van Netten, S. M. and Kroese, A. B. A. (1989a). Hair cell mechanics controls the dynamic behavior of the lateral line cupula. In Cochlear Mechanisms: Structure, Function and Models (ed. J. P. Wilson and D. T. Kemp), pp. 47-55. New York: Plenum.
van Netten, S. M. and Kroese, A. B. A. (1989b). Dynamic behavior and micromechanical properties of the cupula. In The Mechanosensory Lateral Line Neurobiology and Evolution (ed. S. Coombs, P. Görner and H. Münz), pp.247 -263. New York: Springer-Verlag.
Wubbels, R. J. (1992). Afferent response of a head canal neuromast of the ruff (Acerina cernua) lateral line. Comp. Biochem. Physiol. 102A,19 -26.