Departments of 1Surgery (Otolaryngology-Head and Neck Surgery) and 2Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Brichta, Alan M. and Jay M. Goldberg. Morphological Identification of Physiologically Characterized Afferents Innervating the Turtle Posterior Crista. J. Neurophysiol. 83: 1202-1223, 2000. The turtle posterior crista consists of two hemicristae. Each hemicrista extends from the planum semilunatum to the nonsensory torus and includes a central zone (CZ) surrounded by a peripheral zone (PZ). Type I and type II hair cells are found in the CZ and are innervated by calyx, dimorphic and bouton afferents. Only type II hair cells and bouton fibers are found in the PZ. Units were intraaxonally labeled in a half-head preparation. Bouton (B) units could be near the planum (BP), near the torus (BT), or in midportions of a hemicrista, including the PZ and CZ. Discharge properties of B units vary with longitudinal position in a hemicrista but not with morphological features of their peripheral terminations. BP units are regularly discharging and have small gains and small phase leads re angular head velocity. BT units are irregular and have large gains and large phase leads. BM units have intermediate properties. Calyx (C) and dimorphic (D) units have similar discharge properties and were placed into a single calyx-bearing (CD) category. While having an irregular discharge resembling BT units, CD units have gains and phases similar to those of BM units. Rather than any single discharge property, it is the relation between discharge regularity and either gain or phase that makes CD units distinctive. Multivariate statistical formulas were developed to infer a unit's morphological class (B or CD) and longitudinal position solely from its discharge properties. To verify the use of the formulas, discharge properties were compared for units recorded intraaxonally or extracellularly in the half-head or extracellularly in intact animals. Most B units have background rates of 10-30 spikes/s. The CD category was separated into CD-high and CD-low units with background rates above or below 5 spikes/s, respectively. CD-low units have lower gains and phases and are located nearer the planum than CD-high units. In their response dynamics over a frequency range from 0.01-3 Hz, BP units conform to an overdamped torsion-pendulum model. Other units show departures from the model, including high-frequency gain increases and phase leads. The longitudinal gradient in the physiology of turtle B units resembles a similar gradient in the anamniote crista. In many respects, turtle CD units have discharge properties resembling those of calyx-bearing units in the mammalian central zone.
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INTRODUCTION |
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As was established by early studies of
silver-stained material (Lorente de Nó 1926;
Poljak 1927
) and since confirmed by modern neuroanatomical techniques (Fernández et al. 1988
,
1995
), afferents innervating the cristae differ in their axon
diameters, terminal morphology, and the zones of the neuroepithelium
they supply. When it became evident that fibers also differed in their
discharge properties (Baird et al. 1988
; Boyle
and Highstein 1990
; Goldberg and Fernández
1971
; Honrubia et al. 1989
; Lysakowski et
al. 1995
; Myers and Lewis 1990
), the question
arose as to the relation between the morphology and physiology of
individual afferents. Taking advantage of the fact that fiber diameter
was correlated with terminal morphology and crista location, the first
attempts to study this question characterized the discharge properties
of thick, medium-sized and thin axons. Axon caliber was estimated by measuring conduction velocities (Goldberg and Fernández
1977
; Lysakowski et al. 1995
; Yagi et al.
1977
) or by labeling axons (Honrubia et al.
1989
). More recently, intraaxonal labeling methods have been
used to visualize not only the parent axons but also the peripheral
terminations of physiologically characterized fibers. Such studies have
now been done in fish (Boyle et al. 1991
), frogs (Myers and Lewis 1990
), and mammals (Baird et al.
1988
).
In anamniotes (fish and amphibians), only type II hair cells are found
in the cristae and other vestibular organs (Lysakowski 1996; Wersäll and Bagger-Sjöbäck
1974
). Afferents in these animals, nevertheless, have diverse
morphological and physiological properties related to their
longitudinal position in the neuroepithelium. In describing the
results, it is useful to recall that the crista is saddle-shaped, a
narrow isthmus region at the center of the organ giving way to a
broader region near either planum semilunatum. The innervation near the
planum consists of thin axons terminating in relatively simple arbors,
whereas fibers supplying the isthmus have thicker axons with more
robust arbors (Boyle et al. 1991
; Honrubia et al.
1989
; Myers and Lewis 1990
). By correlating
terminal morphology and physiology (Boyle et al. 1991
;
Myers and Lewis 1990
), it was found that the planum
fibers are regularly discharging and have small gains and small phase
leads re angular head velocity; in contrast, many of the isthmus fibers
are irregularly discharging with large gains and large phase leads. A
similar conclusion arises from a correlation between fiber size and
physiological properties (Honrubia et al. 1989
).
A different organization is seen in the mammalian crista. Both type I
and type II hair cells are found throughout the neuroepithelium (Fernández et al. 1995; Lindeman
1969
; Lysakowski and Goldberg 1997
). Based on
the size, shape, and spacing of hair cells (Fernández et
al. 1995
; Lindeman 1969
; Lysakowski and
Goldberg 1997
), on the morphology of their synaptic inputs
(Lysakowski and Goldberg 1997
), and on the branching
patterns of the afferents (Fernández et al. 1988
,
1995
), the neuroepithelium has been divided into three
concentrically arranged zones. There is a central zone, occupying the
apex of the crista except near the planum, successively surrounded by
an intermediate and a peripheral zone. Extracellular labeling has been
used to describe the afferent innervation (Fernández et
al. 1988
, 1995
). Calyx units innervate type I hair cells in the
central zone; bouton units supply type II hair cells in the peripheral
zone; and dimorphic units provide a mixed innervation to both kinds of
hair cells throughout the neuroepithelium.
The discharge characteristics of the various kinds of mammalian
afferents have been determined by intraaxonal labeling (Baird et
al. 1988). Calyx and bouton fibers are each relatively
homogeneous in their behavior. In contrast, the physiological
properties of dimorphic units vary with the zones in which they
terminate. Dimorphic units in the central zone are irregularly
discharging, and their gains and phase leads are large; in comparison,
dimorphic units in the peripheral zone have a regular discharge, small
gains, and small phase leads. Calyx units, although resembling central dimorphic units in their irregular discharge and large phase leads, have considerably smaller gains. Because of their thin axons, bouton
afferents have been difficult to impale and label (Baird et al.
1988
). Fortunately, such afferents can be recognized by their
distinctively slow conduction velocities (Goldberg and
Fernández 1977
; Lysakowski et al. 1995
;
Yagi et al. 1977
). Bouton units so identified resemble
peripheral dimorphic units in discharge regularity, gain, and phase.
The results for the peripheral zone emphasize the concentric
organization of the mammalian crista because labeled units in this zone
have similar discharge properties whether they are located at the apex
of the organ near the planum or at the base of the organ, near the
planum, or near the isthmus (Baird et al. 1988
).
The turtle posterior crista shares features with the cristae of both
mammals and anamniotes. As illustrated in Fig.
1, the turtle crista consists of two
triangular-shaped hemicristae. Each hemicrista extends from the planum
to a nonsensory torus found at the isthmus. Within each hemicrista,
there is a central zone and a surrounding peripheral zone. Type I hair
cells are confined to the central zone, which also contains a smaller
number of type II hair cells (Brichta and Peterson 1994;
Jørgensen 1974
; Lysakowski 1996
). The
type I hair cells are innervated by calyx and dimorphic fibers; the
type II hair cells, by dimorphic and bouton fibers (Brichta and
Peterson 1994
). Only type II hair cells and bouton fibers are
found in the peripheral zone (Brichta and Peterson 1994
;
Jørgensen 1974
; Lysakowski 1996
).
Similar to the longitudinal gradient in axon size and terminal arbors
described in anamniotes (Boyle et al. 1991
;
Honrubia et al. 1989
; Myers and Lewis
1990
), bouton fibers ending near the planum have thin axons and
sparse terminal arbors, whereas those terminating in the remainder of the organ, including the portion near the nonsensory torus, have thicker axons and can have more robust arbors (Brichta and
Peterson 1994
). Bouton fibers in midportions of the hemicrista
are similar in their morphology whether they innervate the central or
peripheral zones.
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The purpose of the present study was to determine the discharge
properties of the various afferent groups in the turtle posterior crista. A specific hypothesis was that in their physiology turtle bouton fibers would show a longitudinal gradient similar to that described in anamniotes (Boyle et al. 1991;
Honrubia et al. 1989
; Myers and Lewis
1990
), whereas calyx-bearing units in the turtle central zone
would resemble the corresponding units of the mammalian central zone
(Baird et al. 1988
; Lysakowski et al.
1995
). The hypothesis led to three predictions: 1)
bouton units near the planum would be regularly discharging and have
low gains and phases; those near the torus would be irregular and have
high gains and phases; and those in midportions of the hemicrista would
have intermediate properties whether they innervated the central or peripheral zones. 2) Turtle calyx-bearing units should
resemble one another in their discharge regularity and phase, but calyx units should have distinctively lower gains than dimorphic units. And
3) the gains and phases of turtle calyx-bearing units
should fall between those of bouton units located near the planum and near the isthmus. This last prediction was based on a comparison of
bouton units in the anamniote crista with centrally located calyx and
dimorphic units in the mammalian crista.
The discharge properties of turtle afferents were studied in a half-head preparation. Intraaxonal labeling was used to relate the physiology of individual afferents with the morphology and locations of their terminal trees. Impaled units were tested with rotations at a single sinusoidal frequency (0.3 Hz), chosen because preliminary studies indicated that variations between units in gain and phase were largest there. Even with this limited testing, bouton and calyx-bearing afferents were distinctive. On the other hand, calyx and dimorphic units were so similar that they had to be placed in a single calyx-bearing class. For either bouton or calyx-bearing units, discharge properties varied with longitudinal position in a hemicrista. Data from labeled units were used to develop statistical formulas from which the morphological classes and longitudinal positions of extracellularly recorded units could be inferred.
To verify the use of the statistical formulas, we first compared the
discharge properties in units recorded intraaxonally or extracellularly
in the half-head or extracellularly in intact animals. The formulas
were then used in the half-head to relate the inferred morphological
class and longitudinal position of each extracellularly recorded unit
with its other physiological properties. One such property, the
response dynamics over a broad frequency range, is considered in this
paper. Other properties are studied in the next paper (Brichta
and Goldberg 2000).
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METHODS |
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Preparation and recording
We used red-eared turtles [Pseudemys (Trachemys) scripta
elegans] of both sexes. Animals weighed 200-400 g and had
carapace lengths of 11-14 cm. Experiments were done at room
temperature (21-23°C). On the basis of a previously published
procedure (Crawford and Fettiplace 1980), the animal was
decapitated, the head was split in the midsagittal plane, and the two
half-heads were placed in a turtle Ringer solution. One half-brain was
blocked at the levels of the trigeminal nerve rostrally and the
glossopharyngeal nerve caudally. To expose the dorsal surface of the
posterior division of the VIIIth nerve, including the fibers
innervating the posterior crista, we pivoted the brain stem 90° about
the VIIth and VIIIth nerves. After removal from Ringer solution, the half-head was placed on its lateral surface in a recording chamber and
a moist gas mixture (95% O2-5%
CO2) was passed continually over the tissue. The
chamber was bolted to the superstructure of a rotating device whose
motion was controlled by a velocity servomechanism (Inland 823, Pittsburgh, PA).
To gauge the influence of in vitro conditions, we also studied intact
animals anesthetized with pentobarbital sodium (10 mg/kg im). The skin,
hyoid cartilage, and soft tissue under the mandible were reflected to
expose the tongue, glottis, and trachea. The animal was then intubated
and respired with compressed air. Artificial respiration mimicked
natural breathing patterns with nonventilatory periods followed by
breathing episodes (Burggren 1975; Frankel et al.
1969
). To accomplish this, we built a three-phase respirator (Hasan 1986
). A breathing episode consisted of 10 breaths, each including an 8-s expiration followed by a 5-s inspiration
with the tidal volume adjusted to 15 ml/kg. After the 10th inspiration, there was a 10-min apneic period. The procedure resulted in a heart
rate of 30-40/min and an expired pCO2 of 3-4%.
Analysis of blood drawn from the femoral artery and measured at 37°C
gave a pH of 7.35-7.45, a p02 of 15-30%, and a
pCO2 of 4-8%, values that are close to normal
(Frankel et al. 1969
).
In intact animals, both divisions of the eighth nerve were exposed by drilling through the hard palate with a dental burr. Care was taken not to disturb the blood supply in and around the nerve. The animal was placed in a supine position on the superstructure with the head clamped inside a recording chamber.
In both the half-head and intact preparations, recording
microelectrodes were advanced by a screw-micrometer drive attached to
the top of the chamber. For extracellular recordings, microelectrodes were filled with 3 M NaCl (20-40 M impedance). Intraaxonal labeling was done with beveled microelectrodes containing 4% biocytin
(Molecular Probes, Eugene OR) in 0.5 M KCl and 0.05 M Tris (pH = 7.4; 40-80 M
impedance). Recordings were made from the posterior
division of the VIIIth nerve proximal to its ganglion. Unless otherwise stated, data are from the half-head preparation.
Physiological testing
The posterior division of the VIIIth nerve supplies five organs.
Fibers innervating the posterior crista and papilla neglecta responded
to head rotations, while those supplying the other three organs did not
(see RESULTS). Rotation-sensitive units were characterized as follows. The head was kept in a fixed position relative to the
horizontal plane of rotation. For all units, a 5-s sample of background
discharge was recorded, as was the response to a 0.3-Hz sinusoidal head
rotation. In some extracellularly recorded units, responses to
sinusoidal rotations at frequencies ranging from 0.01 to 3 Hz and
spaced one-half decade apart were studied. Maximal head velocities were
320 deg/s for 0.01 and 0.03 Hz, 160 deg/s for 0.1 Hz, 80 deg/s for 0.3 Hz, 40 deg/s for 1.0 Hz, and 20 deg/s for 3.0 Hz; the number of cycles
ranged from 4 at 0.01 Hz to 128 at 3.0 Hz. Phase histograms were viewed
on-line to ensure that each unit was tested with rotation amplitudes in
its linear range. Linearity was judged by a lack of harmonic distortion
and, in spontaneously active units, an absence of inhibitory silencing. When in doubt, we continued halving the stimulus intensity until there
was no consistent change in response gain or phase. In some especially
sensitive fibers, this required testing at velocities approaching 1 deg/s. We used Fourier analysis to extract the fundamental component of
the response. A similar analysis was done on the servo's
table-velocity (tachometer) signal. Gains were obtained as the ratio of
the response amplitude (in spikes/s) to the table-velocity amplitude
(in deg/s). For the phase (in degrees), the table-velocity phase was
subtracted from the response phase; positive phases correspond to the
response leading table velocity. The effective posterior-canal plane
was delineated in a previous paper (Brichta and Goldberg
1998a). In the half-head preparation, the effective plane
deviated 45° from the rotation plane and the gains stated in the
paper should be multiplied by
to get their maximal values.
For intact animals, the effective canal plane was 60° from the
rotation plane, leading to a correction factor of 2.
The coefficient of variation (cv) normalized to a standard mean
interval provides a measure of discharge regularity (Goldberg et
al. 1984). As a standard interval, we chose 50 ms because it corresponds to the average background rate of
20 spikes/s found for
a large population of nonsilent units from the turtle posterior crista
(Fig. 11). To convert the cv of a steady-state sample to its normalized
value, cv*, we used a power-law regression,
cv(
) = a(
) · cv*b(
), relating the cv to the mean
interval,
. cv* can be viewed as a
parameter that varies from unit to unit but does not vary with
(
). In contrast,
a(
) and
b(
) vary with
in an manner identical for all units.
Values of a(
) and
b(
) were obtained by fitting
data from 28 posterior-canal units, where sinusoidal head rotations
were used to vary
between 20 and 100 ms.
The details of the calculations are presented elsewhere
(Goldberg et al. 1984
). Data were selected near the
peaks and troughs of responses, where discharge was nearly stationary.
Relations for nine individual units are shown in Fig.
2A, along with the relations for constant values of cv* between 0.1 and 1.0. Whenever possible, the
cv* of a unit was calculated from its background discharge. When the
background
fell outside the normalization
range (Fig. 2B), we used the rotation responses of the unit,
selecting near-stationary data with a
as
close as possible to 50 ms.
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Galvanic sensitivity of individual units was tested in three half-head
preparations. A chlorided silver wire was placed near the posterior
ampullary nerve as it exited the ampulla, and a second chlorided silver
wire was placed in the nasal cavity. Polarity is stated as that of the
ampullary electrode. The resting discharge measured in the immediately
preceding 5-s period was subtracted from the discharge rate averaged
over the last 2.5 s of a 5-s 50-µA cathodal (excitatory) current
step. Data were fit by a power-law relation,
ij = ai(cv*ij)b,
between the galvanic sensitivity (
ij) and
cv*ij, where the subscripts refer to the
jth unit in the ith animal. To estimate the
sensitivity factor for each animal (ai),
as well as the exponent (b) for all animals, an analysis of
covariance was run between the logarithmic transforms of
ij and cv*ij. It was
verified that there was no statistically significant difference between
the exponents from different animals. To eliminate interanimal
differences in sensitivity, which are likely to reflect electrode
placement and other technical factors, we calculated a normalized
galvanic sensitivity,
*ij =
ij/ai.
Intracellular labeling
After physiological testing, impaled axons were injected
iontophoretically with biocytin. Currents were 5 nA alternating every 500 ms between anodal and cathodal pulses. These were interrupted every
30 s to measure the resting potential and the size of the action
potential. Injections, which were started only if the resting potential
was more negative than 25 mV and the action potential exceeded 5 mV,
continued for a total of 10 min or until the resting potential fell
below
10 mV or the action potential fell below 1 mV. Only one
posterior-canal afferent was injected in each preparation. Two to 12 h later the tissue was fixed in 0.1 M phosphate buffer (pH 7.4)
containing 2.5% paraformaldehyde and 2.5% glutaraldehyde. The skull
was removed and the remaining block, consisting of the labyrinth, the
VIIIth nerve and the attached brain stem, was handled en
toto. The block was placed in a phosphate-buffered 30% sucrose solution until it sank and was then embedded in 12% gelatin and cut
into 40-µm frozen serial sections. Biocytin labeling was demonstrated by treating sections with an avidin-biotinylated horseradish peroxidase procedure (ABC kit, Vector Laboratories, Burlingame, CA) with diaminobenzidine (DAB) as the chromogen. Sections were rinsed, mounted,
dehydrated, and cover-slipped.
Material was examined with a ×100 oil-immersion, planapochromatic objective under bright-field illumination. Peripheral arborizations of labeled afferents were reconstructed from serial sections with the aid of a drawing tube (total magnification ×1,650). In most cases, the posterior crista was sectioned so that the entire terminal field of a labeled unit was contained in one to three sections. A terminal field was judged to be complete when it was darkly labeled and each of its processes ended either as a terminal bouton or as a calyx ending.
The approximate center of the terminal field was taken as the location
of the unit. To determine the average diameter of the parent axon
immediately below the neuroepithelium, we measured the area of the
axon's silhouette over a length >100 µm and then divided by the
length (Liberman and Oliver 1984). For bouton units, an
irregular polygon was drawn that included all of the terminal branches;
an approximate terminal-field diameter was calculated as the square
root of the polygon's area. Other morphological features of bouton
units
total bouton area, mean bouton area, and number of terminal
branches
were measured as described by Brichta and Peterson
(1994)
. For calyx and dimorphic units, the numbers of calyx and
bouton endings, when present, were counted separately, as was the
number of type I hair cells enclosed by each calyx ending. Calyx
endings were called "simple" if they innervated a single hair cell
or "complex" if they innervated two or more hair cells.
Discriminant analysis
To develop an empirical formula distinguishing calyx-bearing
(CD) and bouton (B) units, we used discriminant analysis. Three variables-x1 = log10(cv*), x2 = log10(gain), and
x3 = phase-were measured for every
labeled fiber; the gain (in spikes · s1/deg · s
1) and
phase (in degrees) came from the responses to 0.3-Hz sinusoidal head
rotations. Because the covariance matrices for CD and B units were
unequal, a quadratic (rather than a linear) discriminant function was
calculated (Morrison 1990
). The discriminant score, g(x), is a function of the vector,
x = (x1,
x2, x3). The coefficients defining
g(x) were chosen so that units with
g(x) > 0 and g(x) < 0 were assigned, respectively, to the B and CD groups (see Fig.
7C).
The probability of misclassification is indicated by the proportion of known (labeled) units that were assigned to the wrong category. A more accurate procedure is provided by the "jack-knife" method. Here, a unit is removed from the sample, a revised discriminant function is calculated and used to classify the removed unit. The procedure is repeated for each unit and leads to a second estimate of the proportion of misclassified units. Because each removed unit does not contribute to the function used to classify it, the jack-knife simulates the procedure used when a new unit is classified.
The larger the magnitude of g(x), the more
reliable the classification. The probability that an individual case
would be misclassified is related to the normalized score,
z = g(x)/s, where
s is the pooled intragroup SD obtained from labeled B and CD
units. We calculated the mean normalized scores,
CD/s and
B/s. To simplify matters,
the two means were adjusted so they were symmetrically disposed about
zero, i.e.,
CD =
(|
CD| + |
B|)/2s and
B = (|
CD| + |
B|)/2s. We assumed
that the z scores were distributed normally for either
morphological class, in which case the conditional probabilities,
p(z/CD) = N(z
CD) and
p(z/B) = N(z
B), where N is the
standardized normal probability density function. In the rest of the
derivation, we use standard relations between the joint and conditional
probabilities for two events, x and y:
p(x, y) = p(y/x) p(x) = p(x/y) p(y).
The unconditional probability, p(z) = p(z, CD) + p(z, B) = p(z/CD) p(CD) + p(z/B) p(B). p(B) and
p(CD) can be taken from the relative proportions of B and CD
units in a particular sample. When z < 0, a unit will be
assigned to the CD group. The misclassification probability is the
conditional probability that the unit actually belongs to the B group,
i.e.,
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Other statistical procedures were run in SYSTAT for the Macintosh. Unless otherwise stated, means are presented ±SE.
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RESULTS |
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Organs innervated by rotation-sensitive units
Units encountered in the posterior division of the VIIIth nerve responded to sound, vibration, head tilt, a combination of vibration and head tilt, or head rotation. To determine which of these units innervated the posterior crista, we labeled 86 fibers. None of the auditory (n = 6), vibratory (n = 2), tilt-sensitive (n = 6), or tilt-plus-vibration-sensitive (n = 14) fibers was traced to the posterior crista or the papilla neglecta. Fifty-eight rotation-sensitive units were labeled, of which 54 innervated the posterior crista and 4 supplied the papilla neglecta.
As described elsewhere (Brichta and Goldberg 1998a),
units innervating the posterior crista encoded between angular velocity and angular acceleration, whereas papilla neglecta units encoded between angular acceleration and angular jerk. The difference in coding
properties was reflected in the phases of the response to 0.3-Hz
sinusoidal head rotations, which ranged from 5 to 91° in labeled
posterior-crista fibers and between 125 and 146° in labeled
papilla-neglecta fibers. On the basis of these ranges, we assigned an
extracellular unit to the posterior crista if its 0.3-Hz phase was
90° and to the papilla neglecta if it was
110°.
The properties of the papilla-neglecta units, which made up
<10% of our extracellular sample, already have been described (Brichta and Goldberg 1998a). This and the companion
paper (Brichta and Goldberg 2000
) will only consider
posterior-crista units.
Intraaxonally labeled units
MORPHOLOGY OF LABELED UNITS. Of the 54 labeled posterior- crista units, 23 were bouton units, 11 were calyx units, and 18 were dimorphic units. In addition, two calyx-bearing units were too faintly labeled to be assigned to the calyx or dimorphic categories. Terminal fields were labeled incompletely in five bouton units, and in one bouton unit, the terminal field was completely labeled but a labeled axon could not be found.
The locations of all labeled units in the neuroepithelium are shown in Fig. 3A. A large proportion (37/54 = 69%) of the units were located in the medial hemicrista. As expected, calyx-bearing units were only found in the central zone. Three bouton units were also centrally located. The remaining 20 bouton units were distributed in the peripheral zone with a higher concentration near the torus than near the planum or in midportions of the crista. Terminal fields are illustrated by photomontages in Fig. 4 and by drawings in Fig. 5.
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BIAS IN THE INTRAAXONAL SAMPLE.
Large axons are easier to impale than small axons. This source of bias
was evaluated in Fig. 6 by comparing
diameters of the 54 intraaxonally labeled axons with 105 extracellularly labeled axons from a previous study (Brichta and
Peterson 1994). As expected, the intraaxonal sample is missing
the smallest axons seen in the extracellular sample and has
proportionately more axons with diameters
2 µm. Similar biases are
seen for bouton (Fig. 6B) and calyx-bearing axons (Fig.
6C).
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PHYSIOLOGY OF LABELED FIBERS.
Figure 7 plots the gains and phases for
0.3-Hz sinusoidal head rotations versus cv* for the 54 labeled units.
Units are separated by their morphological classes as well as by their
background rates (high-rate, 5 spikes/s; low-rate, < 5 spikes/s). C
and D units were placed into a single CD class because, as is described in the following text, the two kinds of units had similar discharge properties. Combining the terminology based on morphology with that
based on background rates, we have B-high, B-low, CD-high, and CD-low
categories.
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DISCRIMINATION BETWEEN BOUTON AND CALYX-BEARING UNITS.
B and CD units cannot be distinguished by any of the individual
variables plotted in Fig. 7. CD units are irregular and have relatively
small gains and phases. But they are neither the most irregular units
nor do they have the smallest gains or phases. Rather it is the
relationship between cv* and either gain or phase that makes CD units
distinctive. In particular, CD units have larger cv*s than do B units
with comparable values of gain and phase. Equivalently, when cv*s are
equated, CD units have smaller gains and phases. To exploit this
observation, we did a quadratic discriminant analysis involving three
variablesx1 = log10 (cv*), x2 = log10 (gain), and
x3 = phase. Because of differences in
gain and phase related to background discharge, a separate analysis was
done for high-rate and low-rate groups. Since three-dimensional discriminant functions can be difficult to visualize, results are
illustrated with the two-dimensional functions obtained by eliminating
x2 or
x3.
DISCHARGE PROPERTIES AND LONGITUDINAL POSITION.
In Fig. 8, cv*, gain and phase are
plotted versus normalized longitudinal position, .
= 0 corresponds to the torus;
= 1, to the planum. Gain (Fig.
8B) and phase (Fig. 8C) are both strongly related
to
; because the relations are statistically indistinguishable for
the B and CD groups, a single regression line is drawn for all units in
each plot. In contrast, the relations between cv* and
differed for
B and CD units and separate regression lines are drawn in Fig.
8A for the two groups. The latter difference is not
surprising as it forms the basis for the discriminant analysis described in the preceding section.
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DISCHARGE PROPERTIES AND OTHER MORPHOLOGICAL FEATURES. Each of the labeled units was characterized by several morphological features besides its longitudinal position. To investigate whether any of these other features was related to the unit's discharge properties, forward stepwise multiple regression was used separately on B and CD units. Dependent variables included the background rate as well as the three discharge properties considered in Fig. 8.
For B units, the morphological features considered included longitudinal position, axon diameter, number of boutons, total bouton area, mean bouton area, number of terminal branches, and terminal-field diameter (Brichta and Peterson 1994Relation between the discharge properties from various samples
In this section, we compare the physiology of the intraaxonally labeled and extracellularly recorded units from the half-head. The comparison is needed to determine if the multivariate equations, which are based on data from the intraaxonal sample, can be used to specify the morphological class and location of extracellular units. In addition, extracellular samples from the half-head and from intact animals are compared to gauge the effects of in vitro conditions on afferent discharge.
INTRAAXONAL VERSUS EXTRACELLULAR SAMPLES, HALF-HEAD. To be included in the extracellular sample, a unit had to be characterized in terms of its background discharge, its discharge regularity, and the gain and phase of its response to 0.3-Hz sinusoidal head rotations. The extracellular sample from the half-head comprised 567 units. Several other units, possibly as many as 5-10% of the sample, had to be discarded because they were silent at rest and were so insensitive that we were unable to drive them to rates needed to calculate a cv* or to determine their gains and phases. We labeled two such "very insensitive" units. They were C units with complex calyx endings.
The assignment of extracellular units is illustrated in Fig. 10, A and B, which shows the relations between gain and cv* and between phase and cv* for extracellular units identified as B or CD by discriminant functions. Comparisons with the intraaxonal sample (Fig. 7, A and B) showed that the relations for B units from the two samples were statistically indistinguishable. Because of the larger size of the extracellular sample, it provided more precise estimates of the regression coefficients. A power-law regression, gain = a(cv*)b, for extracellular B units gave a = 41.6 ± 3.5 spikes · s
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IN VITRO VERSUS IN VIVO EXTRACELLULAR SAMPLES. Forty units were obtained in eight intact turtles. The in vivo sample was classified by the discriminant analysis into B-high (n = 17), CD-high (n = 14), and CD-low (n = 9) units (Fig. 10, C and D); none of the units were B-low. There were no statistically significant differences between the intact and half-head preparations in the mean values of background rate, cv*, gain, or phase for B-high, CD-high, or CD-low units. Nor were there significant differences in the relations between cv* and gain or phase for B-high units from the two preparations. This was so even when gains were corrected for the angles between the plane of the posterior canal and the rotation plane (see METHODS). "Very insensitive" units, presumably of the CD-low variety, were found in vivo. The results suggest that in vitro conditions did not result in a deterioration of vestibular transduction.
Discharge properties of extracellular units from the half-head
Because of its larger size, the extracellular sample provides a more accurate picture of physiology than does the intraaxonal sample. In addition, the extracellular sample is likely to be less biased in recording from thin axons. In the following sections, discharge properties from the extracellular sample are described.
BACKGROUND DISCHARGE. The mean value for B units is 20.0 ± 0.6 spikes/s and that for CD units is 16.7 ± 0.9 spikes/s. Although the mean rates for the two groups are similar, the distributions differ (Fig. 11). Rates for B units are almost symmetrically distributed about a mode just above 20 spikes/s. The CD distribution, in contrast, is positively skewed with many of its units having no background discharge. Silent units make up a larger proportion of CD units (42/279, 15.1%) than of B units (7/288, 2.4%).
|
LONGITUDINAL POSITION. Multiple-regression equations were used to predict the normalized longitudinal positions of extracellular B and CD units (Fig. 12A). The distribution for B units has two peaks, one at 0.3 and the other near 1.0. The trough between 0.4 and 0.8, which corresponds to the position of the central zone, may be explained by the small size of the peripheral zone in this range and the large proportion of CD units in the central zone (Fig. 1). Consistent with this interpretation, it is in this range that CD units are placed (Fig. 12A).
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DISCHARGE REGULARITY. B units can be regular or irregular, whereas almost all CD units are irregular (Fig. 12C). There is a small, but statistically significant difference in mean values of cv* for CD-high and CD-low units (t test, P < 0.001; Fig. 12D and Table 3).
GAIN AND PHASE.
There are two peaks in the gain (Fig. 12E) and phase (Fig.
12G) distributions for extracellular B units. The gain peaks
are at 0.5 and 10-20 spikes · s1/deg · s
1, the
phase peaks, at 0-10° and 50-60°. The separate peaks are related
to longitudinal position. This can be seen in Table 3 in which the B
units are divided into BT, BM, and BP categories. BT units have an
average gain >50 times that of BP units. There is a large difference
in phase for the two groups. BM units have intermediate values of gain
and phase. The differences are not a statistical artifact of the fact
that gains and phases were used in calculating the presumed locations.
Large regional differences still were observed when gains or phases
were removed from the location calculation.
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GALVANIC SENSITIVITY.
Responses to externally applied 50-µA currents are illustrated in
Fig. 14, A and B,
for two units recorded consecutively from one animal. Cathodal currents
increase discharge and anodal currents decrease it. The top unit (Fig.
14A) is irregular (cv* = 0.65), whereas the bottom unit is
regular (cv* = 0.13). For the irregular unit, cathodal currents result
in a response of 55 spikes/s, whereas anodal currents abolish
firing. Smaller,
2.5 spikes/s responses are seen in the cathodal and
anodal responses of the regular unit.
|
RESPONSE DYNAMICS. Sinusoidal head rotations were presented in half-decade steps over the frequency range, 0.01-3 Hz. Rotation amplitude was adjusted to stay within the linear range of each unit. Bode plots are shown for individual BP, BT, CD-high, and CD-low units (Fig. 15, A-G). Of the CD-low units, only those with some background discharge could be studied because silent units did not respond to the maximal rotation amplitudes possible at the higher frequencies. Mean gains and phases for the other groups are shown in Fig. 15, D and H.
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DISCUSSION |
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Functional organization of the turtle posterior crista
Previous studies suggested that there were similar spatial
gradients in the morphology of bouton afferents innervating the cristae
of turtles (Brichta and Peterson 1994), frogs
(Honrubia et al. 1989
; Myers and Lewis
1990
), and fish (Boyle et al. 1991
). On the
basis of this similarity, we hypothesized that turtle B fibers would
show longitudinal gradients in afferent discharge properties resembling
those found in anamniotes (Boyle et al. 1991
;
Honrubia et al. 1989
; Myers and Lewis
1990
). The hypothesis was confirmed. In the turtle crista,
bouton (BP) units terminating near the planum have a more regular
discharge, smaller rotational gains, and smaller rotational phases than
do bouton (BT) fibers ending near the torus. BM units, supplying
midportions of the hemicrista, are intermediate in all three respects.
Furthermore BM units had similar discharge properties whether they
innervated the central (CZ) or peripheral zones (PZ). A similar
functional organization is seen in the anterior and horizontal cristae
of turtles (Igic and Brichta 1997
; unpublished data).
We also predicted that in their physiology CD units, restricted to the
turtle CZ, would resemble the comparable units in the mammalian CZ. The
latter prediction was only partly confirmed. As is the case for
centrally located mammalian calyx (C) and dimorphic (D) units
(Baird et al. 1988; Lysakowski et al.
1995
), turtle CD units are irregularly discharging and have
gains and phase leads that are intermediate between those of turtle BP
and BT units. Despite the basic similarity between calyx-bearing CZ
units in the mammals and turtles, there are two obvious differences. First, in mammals, centrally located C and D units differ in their rotational phases and gains, with the C units having slightly larger
phases and considerably lower gains. This may be contrasted with the
situation in the turtle where C and D units have similar gains and
phases. We shall return to this topic in a later section (see
Determinants of response gain). Second, turtle CD units can be divided into those with low and high background rates. In contrast, almost all mammalian afferents have an appreciable background rate. In
the turtle, CD-low units have considerably lower rotational gains than
CD-high units. Low rotational gains may help to extend the range of
angular head velocities that can be linearly encoded (Baird et
al. 1988
; Brichta and Goldberg 2000
). In this
respect, CD-low units in turtles may serve the same function as do C
units in mammals.
A comparison of the crista in anamniotes, turtles, and mammals has led
to speculations concerning the evolution of the vertebrate crista
(Goldberg and Brichta 1998). In particular, it has been suggested that the presumed transformation of the crista from a
longitudinal organization in anamniotes (Boyle et al.
1991
; Honrubia et al. 1989
; Myers and
Lewis 1990
) to a partly concentric organization in turtles is
related to the presence of type I hair cells in the turtle CZ
(Brichta and Peterson 1994
; Jørgensen
1974
; Lysakowski 1996
) and that the presumed
transformation to a fully concentric organization in mammals is related
to the distribution of type I hair cells throughout the mammalian
neuroepithelium (Fernández et al. 1995
;
Lindeman 1969
; Lysakowski and Goldberg 1997
). Although the proposed scheme provides a useful
framework, there are indications that it may be overly simplified. One
such indication is the early finding in the guitarfish of a transverse variation in afferent physiology (O'Leary and Dunn
1976
;O'Leary et al. 1974
).
Relation between morphology and physiology
In the turtle crista, the physiology of a bouton afferent is
related to its longitudinal position. Longitudinal gradients in
afferent morphology were described previously in an extracellular labeling study (Brichta and Peterson 1994) but were less
evident in the present sample of intraaxonally labeled fibers. Some of the differences between the two studies can be ascribed to a bias in
intraaxonal labeling toward larger diameter fibers. Possibly reflecting
such a bias, there was little difference in the morphology of
intraaxonally labeled BP, BT, and BM units. Despite the similarity in
their morphology, the three subclasses of intraaxonally labeled B units
differed in their physiology. This suggests that morphological features
other than longitudinal position are unimportant in determining discharge properties. The conclusion was confirmed by a statistical analysis of the structural correlates of the cv*, gain, and phase of B
units. Each of these discharge properties was related to longitudinal
position, but not to axon diameter, number of boutons, or terminal
branching patterns. A similar result held for CD units. All CD units
were irregularly discharging, and their cv*s were unrelated to any
measured morphological variable, including longitudinal position. In
addition, the background discharge, gain, and phase were related to
longitudinal position but not to axon diameter, the number of type I
hair cells, or the number of bouton endings.
Perhaps the strongest evidence that discharge properties are unrelated
to branching patterns comes from a comparison of B and CD units.
Despite their distinctive terminal trees, CD units cannot be
distinguished from B units by any single physiological variable,
including gain, phase, or cv*. The gain and phase of CD units overlap
those of BM units, whereas the cv* of CD and BT units resemble each
other. In a previous study of intraaxonally labeled fibers in the
lizard horizontal crista, Schessel et al. (1991)
suggested that C units in lizards had a distinctively irregular discharge, but only one B unit was labeled and the cv's of C and D
units were not corrected for differences in mean interval. In any case,
our study indicates that discharge regularity, by itself, cannot be
used to classify units into B, C, and D groups. Furthermore our results
do not support the suggestion, made by Boyle et al. (1991)
, that response dynamics and dendritic branching patterns are causally related.
Determinants of response gain
There is a 200-fold variation in the gains of bouton afferents,
with the gain at 0.3 Hz (g0.3 Hz in
spikes · s1/deg · s
1) varying from 0.2 in the most regularly
discharging BP units to 40 in the most irregularly discharging BT
units. The results can be summarized by a power-law relation between
g0.3 Hz and cv* (Fig.
16A). On the basis of a
previous analysis done in mammals (Baird et al. 1988
;
Goldberg et al. 1990
), it can be supposed that there are
at least two factors contributing to the relation. The first is the
sensitivity of the postsynaptic spike encoder, which as measured by
galvanic sensitivity (
*) increases with cv* (Goldberg et al.
1984
; Smith and Goldberg 1986
). The
second is the influence of response dynamics. As we have shown
elsewhere (Brichta and Goldberg 1998a
), gain
increases with sinusoidal frequency (f)
according to a formula, gain
(f/f0)
/90,
where f0 is a reference frequency and
is
the average phase lead (in degrees) over the frequency interval from
f0 to f. Because of the
semilogarithmic relation between
and cv*, response dynamics will
also contribute to the power-law relation between gain and cv*.
|
To estimate the relative contributions of the two factors, we started
with the power-law relation for B units, g0.3
Hz = 41.6 cv*2.66 (Fig. 16A). To
eliminate the influence of encoder sensitivity, we divided
g0.3 Hz by the power law, * = 0.47 cv*0.50 (Fig. 14C, - - -), to give
g*0.3 Hz = 88.5 cv*2.16
(Fig. 16B). This left an exponent, b = 2.16, to be explained by response dynamics. The relation between
(f/f0)
/90
and cv* is closely fit by a power-law whose exponent increases with the
ratio, (f/f0). By
substituting different values of the ratio, we found that the
desired exponent of 2.16 was obtained when
(f/f0) = 125 or f0 = 0.0024 Hz. The result was
a flat relation between g*0.0024 Hz and
cv* (Fig. 16C). A 200-fold variation in gain corresponds
to a 7.4-fold variation in cv*. The analysis suggests that of the
7.42.65 = 200 × variation in
g0.3 Hz, response dynamics makes a larger contribution (7.42.16 = 75X) than does encoder
sensitivity (7.40.50 = 2.7X).
A similar analysis was done for CD-high and CD-low units. The
value of g0.3 Hz for each CD unit
(Fig. 16A) was divided by * = 1.30cv*0.50 to give g*0.3
Hz (Fig. 16B); the leading coefficient in the
power law for
* was increased to reflect the finding that galvanic sensitivity was 2.75 times higher in CD than in B units. Because the
Bode plots for CD-high units paralleled those for BM units (Fig. 15,
D and H), we used the value of
f/f0 = 125 gotten from B
units to calculate g*0.0024 Hz = g*0.3 Hz/125(
/90) for CD
units (Fig. 16C).
Mean values (in spikes · s1/deg · s-1) of g*0.0024 Hz for the
various extracellular groups were: 0.141 ± 0.006 (B), 0.039 ± 0.002 (CD-high), and 0.016 ± 0.002 (CD-low). Values for the
three groups are in the ratio, 1:0.28:0.11.
As was done in mammals (Baird et al. 1988;
Goldberg et al. 1990
; Lysakowski et al.
1995
), we wished to estimate the relative contributions of type
I hair cells (wI) and bouton endings
(wB) to
g*f0,
f0 = 0.0024 Hz.
g*f0 may be taken as a measure of the total synaptic input to all of an afferent's endings
independent of encoder sensitivity and response dynamics. Encoder
sensitivity and response dynamics are eliminated because the former
reflects the channel properties of the nerve terminal (Smith and
Goldberg 1986
), whereas the latter is likely to reflect early
stages in transduction (Highstein et al. 1996
). As such, these two factors presumably are unrelated to the types and numbers of
endings. wI was computed as the ratio
between the mean values of
g*f0 and of the
number of type I hair cells for 11 C units. Similarly,
wB was taken as the ratio between the mean values of g*f0 and of
the number of bouton endings for 20 B units. The weighting coefficients
(in spikes · s
1/deg · s
1)
were wI = 0.0084 ± 0.0020, wB = 0.0014 ± 0.0002 and
wI:wB = 6.0 ± 1.7.
wI and wB
were used to obtain predictions of
g*f0 for individual
B, C, and D units. As can be seen in Fig.
17, the agreement between actual and
predicted values is only fair as judged by the correlation between the
two variables (r = 0.42, df = 47, P < 0.01). In addition, the correlation within
each group is poor. In calculating predicted responses, we took into
account encoder sensitivity, response dynamics, and the numbers of type I hair cells and bouton endings. The poor correlations imply that there
is considerable intragroup variability in the weighting coefficients,
and, hence, that factors other than the three considered are important
in determining rotational gains. A similar conclusion was reached in
our studies in the chinchilla (Baird et al. 1988; Goldberg et al. 1990
).
|
Type II inputs to dimorphic units
The present results differ from those obtained in the chinchilla
crista concerning the inputs from type II hair cells to D units. In the
chinchilla, g*f0 was
fivefold larger in D, as compared with C, units (Baird et al.
1988). Because both kinds of units contacted similar numbers of
type I hair cells, it was presumed that the larger values of
g*f0 for D units could be
explained by the additional bouton inputs they received. A linear
model, g*f0 = nIwI + nBwB, was
applied to C and D units to obtain a
wI:wB
ratio of 3:1. In the present study, we found that turtle C and D units
had comparable values of
g*f0, a finding that is
most easily explained were there a negligible type II input to turtle D
units. The conclusion was verified when the same linear model was
applied to turtle C and D units, giving an estimate of
wI identical to that stated in the
preceding text (wI = 0.0084 ± 0.0020), but a near-zero estimate of
wB (
0.0017 ± 0.0013). The
latter value may be contrasted with wB = 0.0014 ± 0.0002 obtained from turtle B units.
There are two differences between the turtle and chinchilla studies. The first concerns rotational frequencies. Turtle units were tested at 0.3 Hz; chinchilla units at 2 Hz. This is probably an unimportant difference. In particular, the Bode plots of Fig. 15, C and G, show that there is a high correlation between the gains at 0.3 and 3 Hz, implying that results would have been similar had we tested at the higher frequency. The second difference concerns f0, the frequency at which dynamic effects on gain are assumed comparable for the various unit groups. In the chinchilla, response phases converged at f0 = 0.2 Hz, whereas in the turtle f0 was more than a 100-fold lower. The very low value of f0 in turtles reflects the large range of phases seen in turtle B units at 0.3 Hz. It seems unlikely that the value of f0, used in comparing B and CD units, also should be used when C and D units are compared. In fact, a regression between g*0.3 Hz and response phase suggests that the response dynamics of turtle C and D units would converge between 0.1 and 0.15 Hz. Using this higher value of f0 resulted in an increase in the relative value of wB, albeit one that was still not statistically distinguishable from zero.
There is no obvious functional advantage of a D unit making contact
with type II hair cells and, yet, not receiving a significant input
from them. At the same time, any of several mechanisms could be
responsible for an ineffective input. So for example, a significant fraction of the bouton endings in the chinchilla crista lack afferent synapses (Lysakowski and Goldberg 1997), and the same
conceivably could be true for the bouton endings of turtle D afferents.
A second possibility is suggested by a recent survey of outwardly rectifying basolateral conductances in turtle crista hair cells (Brichta and Goldberg 1998b
; Brichta et al.
1998
). Many type II hair cells, including almost all those from
the PZ and many of those from the CZ, have small, rapidly activating
outward conductances. In contrast, a substantial proportion of type II
hair cells from the CZ have larger, slowly activating conductances. The
larger size of the conductances would lead to small receptor
potentials, which in turn would make synaptic transmission from the
hair cell less effective. Because the values of
g*f0 are similar for B
units in the CZ and PZ, it might be supposed that hair cells with
larger, slowly activating conductances preferentially synapse with D
units. A third explanation can be offered. It is possible that the
enlarged surface area of calyx endings would lower the input impedance
of the postsynaptic terminal and, hence, the gains of both calyx and
dimorphic units (Baird et al. 1988
; Goldberg 1996
). There were several reasons why the mechanism was thought not to operate in mammals (Goldberg 1996
;
Lysakowski et al. 1995
). Evidence on this point is
lacking in turtles. Fourth, there is a suggestion that differences in
the response properties of calyx and dimorphic units in mammals may be
related to differences in the intracellular machinery of the two kinds
of afferents, rather than to differences in their synaptic inputs from
type I and type II hair cells. In particular, calyx units in mammals
are immunoreactive for the calcium-binding protein, calretinin, whereas
dimorphic units are not (Desmadryl and Dechesne 1992
).
It is unclear whether calyx and dimorphic units in the turtle differ in
this way (Monk and Peterson 1995
).
Response dynamics
In the present paper, we developed statistical procedures to infer
the morphological type and location of an afferent solely from its
discharge properties. This indirect approach was used to characterize
the response dynamics of several groups of extracellularly recorded
units. BP units had the simplest response dynamics, which could be
approximated by a first-order torsion pendulum model. The same is also
true of regularly discharging afferents in other species (Baird
et al. 1988; Boyle and Highstein 1990
;
Goldberg and Fernández 1971
;
Honrubia et al. 1989
). Because a torsion pendulum
parallels the expected macromechanics of the semicircular canals
(Oman et al. 1987
; Steinhausen 1931
), it
is customary to presume that the transduction mechanisms following
macromechanics do not greatly alter the response dynamics of such afferents.
Less regularly discharging afferents show high-frequency deviations
from the torsion-pendulum model, and these can sometimes be described
by a fractional (sk) operator,
k > 0, which introduces a fixed phase
lead, k · 90°, and a gain enhancement,
(f/f0)k,
as frequency increases from f0 to
f (Baird et al. 1988; Boyle and
Highstein 1990
; Honrubia et al. 1989
;
Schneider and Anderson 1973
). An
sk operator describes the deviations seen
in our CD and in many of our BM units but not in our BT units. The
latter units show a high-frequency decline in phase. A similar decline
also was seen in afferents innervating the turtle papilla neglecta
(Brichta and Goldberg 1998a
) but not in acceleration
afferents in the toadfish (Boyle and Highstein 1990
).
The cellular mechanisms responsible for the high-frequency phase
decline are unknown.
Afferent responses to externally applied currents have been used to
study the etiology of response dynamics. Currents have been applied to
afferents by way of the perilymph (Ezure et al. 1983;
Goldberg et al. 1982
) or to hair cells by way of the
endolymph (Highstein et al. 1996
). The results imply
that interunit differences in high-frequency response dynamics do not
reflect later stages in transduction, including voltage-sensitive
basolateral currents, the various stages of synaptic transmission, and
the conversion of postsynaptic currents to spike frequency. Studies of
solitary hair cells harvested from distinct regions of the turtle
posterior crista are consistent with the conclusion (Brichta and
Goldberg 1998b
; Brichta et al. 1998
). In
particular, type II hair cells supplying BP and BT units show almost
identical voltage responses to intracellularly injected sinusoidal
currents in the frequency range of vestibular transduction. Moreover,
responses are in phase with injected current. The conclusion may not
extend to type I hair cells or to some central type II hair cells.
Because of their slow kinetics, the basolateral currents obtained from
these hair cells could contribute a significant fraction of the modest
phase leads seen in CD units.
Classification methods
Multivariate statistical procedures have been used to classify
neurons based on anatomical (Brichta and Peterson 1994;
Matesz et al. 1995
; Moschovakis et al.
1988
) or physiological criteria (Frank et al.
1988
; Leem et al. 1993
; Pennartz et al.
1998
). In the present study, a different approach was used. We
first placed intraaxonally labeled neurons into morphological classes
and then used a discriminant analysis to show that the classified
neurons also could be distinguished by their physiological properties. The approach was used by Mason (1997)
to determine that
serotonergic neurons in the ventromedial medulla could be distinguished
by their slow and steady background discharge. Similarly, calyx
afferents innervating the chinchilla crista were distinctive in having
an irregular discharge coupled with a small rotational gain
(Baird et al. 1988
). In the latter case, the
classification rule defining calyx afferents was so simple that it was
arrived at without the help of multivariate statistics.
There are two motives for determining the physiological properties
distinguishing morphologically defined classes. The first motive can be
expressed by two questions of theoretical interest: what is the minimal
set of physiological properties that can distinguish two or more
morphological classes? Does the minimal set represent one or more
physiological mechanisms? There is no a priori reason why a single
property should not suffice as a minimal set. In fact, the discriminant
score defines a single property as a linear or quadratic combination of
the measuring physiological variables. At the same time, the
combination need not represent a single mechanism. This would appear to
be the case for vestibular afferents, whether from mammals
(Baird et al. 1988) or turtles (the present paper). To
see this for turtle afferents, we need only consider the fact it is the
relation between discharge regularity and either gain or phase that
makes CD units distinctive. Discharge regularity (cv*) is likely to
reflect postsynaptic mechanisms (Goldberg et al. 1984
;
Smith and Goldberg 1985
). The ratio between gain and cv*
is an estimate of the total synaptic input to the afferent and, as
such, reflects presynaptic mechanisms (see Determinants of
response gain). Response dynamics, including the phase at an individual frequency, is also likely to be determined presynaptically (see Response dynamics).
The second motive is more pragmatic. Data from intraaxonally labeled units can be used to develop statistical formulas that infer the morphological class and location of an extracellularly recorded unit from its physiological properties. In the present paper, the applicability of the formulas to extracellular units was confirmed by comparing the discharge properties of extracellular and intraaxonal samples. The indirect determination of the morphology and location of an extracellularly recorded afferent has several advantages. First, in our hands, only one labeled unit can be identified unambiguously in a single preparation, whereas many extracellular units can be recorded. Because the extracellular sample can be much larger than the labeled sample, a more reliable picture of morphophysiological relations emerges. Second, an extracellular sample is less biased because units with thin axons are recorded more readily extracellularly than intraaxonally. Third, although it is relatively easy to impale units, it remains a challenge to maintain intracellular contact for the long periods required to complete some physiological protocols.
Like all statistical predictions, the morphological classification and predicted location of an extracellularly recorded unit are subject to error. Based on the intraaxonally labeled sample, the probability of misclassification was estimated to be <10% and the error in predicting the location was 5-10% of the entire length of a hemicrista. As the method is used to study more discharge properties, several of them may prove useful in supplementing the three variables used in the original discriminant scheme. Consider, for example, the Bode plots shown in Fig. 15. In most BT units, phase continues to decline above 0.3 Hz (Fig. 15F). This is not the case for most CD units (Fig. 15G). In only 2/28 CD-high units, were there high-frequency phase declines overlapping those of BT units. From their z scores near 0.5, the units had a large misclassification probability (Fig. 7D, P > 0.2), consistent with their being misclassified.
The present classification scheme requires the collection of a sample
of background activity and the response to a single sinusoidal rotation
at a frequency of 0.3 Hz. Typically, the classification protocol can be
completed in 1 min. A similar situation occurs in mammals where the
testing of crista units is confined to 2 Hz (Baird et al.
1988; Lysakowski et al. 1995
). The
classification error might be reduced by including several other
variables in the discriminant analysis, but this would require a more
complicated testing protocol. Our preference has been to keep the
protocol simple and look for potentially misclassified units when
studying a new discharge property. Potentially misclassified units are indicated by their having an atypical response in the new paradigm when
compared with other, similarly classified units, together with a large
misclassification probability as indicated by a near-zero discriminant
score based on the original variables.
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ACKNOWLEDGMENTS |
---|
Drs. R. A. Eatock and A. Lysakowski made helpful comments on the manuscript. J. Joos and K. Dempsey helped in the preparation of histological material.
This research was supported by National Institute on Deafness and Other Communication Disorders Grant DC-02508 (J. M. Goldberg, principal investigator).
Present address of A. M. Brichta: Discipline of Anatomy, Medicine and Health Sciences, University of Newcastle, Callaghan, NSW 2308 Australia.
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FOOTNOTES |
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Address for reprint requests: J. M. Goldberg, Dept. of Neurobiology, Pharmacology and Physiology, University of Chicago, 947 E. 58th St., Chicago, IL 60637.
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 22 April 1999; accepted in final form 11 October 1999.
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REFERENCES |
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