 |
INTRODUCTION |
Adult vestibular plasticity can be achieved by different
experimental approaches, most prominently by a peripheral lesion, e.g.,
unilateral labyrinthectomy or neurectomy. After a peripheral section of
N. VIII the ganglion cells survive and electric stimulation of the
sectioned nerve branch evokes monosynaptic field potentials in the
vestibular nuclei similar to those recorded in control animals even two
months after the lesion (Kunkel and Dieringer 1994
). Postoperatively, several neural changes were
documented with various methods in different species (Dieringer
1995
; Smith and Curthoys 1989
). However,
detailed studies concerning the spatial or functional specificity of
these rearrangements within the central vestibular system akin to the
functional reorganization of somatosensory maps at cortical and
subcortical levels (O'Leary et al. 1994
) are so far
absent. To investigate a possible expansion of signals from intact
vestibular afferent fibers onto second-order vestibular neurons
(2°VN) with silenced vestibular afferent inputs, we sectioned the
frog's anterior branch of N. VIII on one side. Thereby, the afferent
inputs from utricle, horizontal, and anterior vertical semicircular
canals were eliminated, whereas the afferent inputs from saccule,
lagena, and posterior vertical semicircular canal, and from the
auditory organs, remained intact. Two months after this lesion we
recorded from 2°VN and studied the convergence of monosynaptic inputs
from different vestibular nerve branches. Convergence of afferent
signals from different canals is rare in controls, since most (about
90%) of the 2°VN receive their monosynaptic vestibular excitation
from only one of the three ipsilateral semicircular canal nerves in
pigeon (Wilson and Felpel 1972
), cat (Kasahara
and Uchino 1974
), and frog (Straka et al. 1997
).
 |
METHODS |
In deeply anesthetized grass frogs (Rana
temporaria; 0.1% MS-222) the otic capsule was opened, and the
ramus anterior (RA) of N. VIII was sectioned under visual control
distal to the entry of the saccular nerve branch. Two months after this
lesion chronic animals were reanesthetized and perfused transcardially
with iced Ringer solution. The brain together with the attached N. VIII and their branches to the labyrinthine endorgans were removed and
prepared for in vitro experimentation (see Straka et al.
1997
). The posterior vertical semicircular canal (PC) and the
RA nerve branches were electrically stimulated on either side with
short pulses via suction electrodes. Extra- and intracellular records in the vestibular nuclei ipsilateral to the side of stimulation were
obtained with glass pipettes (2 M sodium chloride: 1-3 M
; or 2 M
potassium acetate and 0.3 M potassium chloride: 90-120 M
). The
stimulus intensity was limited to a value five times above the
threshold of the first postsynaptic negativity
(N1) in the evoked field potential (Precht
et al. 1974
). This threshold was determined at the beginning of
each experiment, and absolute values (between 2 and 3.5 µA) were
similar between different experiments. Field potentials were searched
in depth tracts with bilaterally symmetrical coordinates throughout the
entire rostro-caudal extent of the vestibular nuclear complex. Our
observations were restricted to monosynaptic excitatory postsynaptic
potentials (EPSPs) in 2°VN with membrane potentials of at least
40
mV and to crossed early responses in the abducens nerves. About equal
numbers of neurons were recorded on a given side of the brain stem per
animal. Single sweeps of the evoked responses were digitized, stored on a computer, averaged 15-20 times, and analyzed off-line. EPSPs from
2°VN were analyzed after electronic subtraction of the extracellular field potential recorded in the vicinity of the neuron. Abducens nerve
responses evoked by contralateral PC nerve stimulation were analyzed by
calculating the area covered by the positive response over the first 20 ms (in mV × ms). Statistical differences were calculated
according to the Mann-Whitney U test (unpaired parameters) or to the Wilcoxan signed rank test (paired parameters).
 |
RESULTS |
Field potentials in the vestibular nuclei evoked by
stimulation of the ipsilateral PC or RA nerve consisted of a
presynaptic (N0) and one
(N1) or more postsynaptic negativities (Fig.
1A) (Precht et al.
1974
). The latencies of N1 responses were
shorter following RA nerve stimulation (2.64 ± 0.30 ms; mean ± SD, n = 27) than following PC nerve
stimulation (3.41 ± 0.46 ms; n = 27) because of a
shorter distance between the stimulation and the recording site
(Straka et al. 1997
). These latencies characterized monosynaptic EPSPs in intracellular records (shaded bars in Fig. 2). PC nerve evoked
N1 potentials on the operated side were
significantly larger in amplitude (Fig. 1, A and
B) than PC nerve evoked N1 potentials
recorded at corresponding sites on the intact side (Fig. 1,
A and B). Following RA nerve stimulation, very
similar amplitudes of N1 potentials and depth
profiles were recorded in controls and on either side of operated
frogs.

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Fig. 1.
Posterior canal (PC) nerve evoked afferent field potentials in the
vestibular nuclei. A: PC nerve evoked pre-
(N0) and postsynaptic (N1) field potentials
recorded 65 days after the transection of the ramus anterior of N. VIII. B: depth profiles of PC nerve evoked
N1 potentials of controls (n = 6) and
of operated frogs recorded on the intact (n = 6) or
on the operated side (n = 6). Traces in
A are the averages from 20-24 single sweeps and were
recorded 0.7 mm caudal to the caudal end of the entry of N. VIII at a
depth of 0.5 mm. The shaded vertical bar in A shows the
average ± SDs of the N1 onset latencies.
|
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Fig. 2.
Convergence of afferent inputs from different vestibular endorgans in
intact and operated frogs. A-C: monosynaptic response
components were evoked in some 2nd-order (2°) vestibular neurons only
after stimulation of the posterior canal (PC) nerve (A),
in others only after stimulation of the ramus anterior (RA) of N. VIII
(B) and in a 3rd group after stimulation of the PC as
well as of the RA nerve (C). D: the
percentages of these 2°PC neurons, of 2°RA neurons and of 2°RA + PC neurons differed in part significantly between controls and operated
frogs as between the intact and the operated side of operated frogs
(#P 0.0001; *P 0.01). Closed arrow heads in A-C for stimulus onset
and shaded vertical bars for the average ± SDs of monosynaptic
onset latencies following PC or RA nerve stimulation. Each trace in
A-C represents an average of 30 single sweeps.
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Intracellular records were collected from more than 700 identified 2°VN (Table 1). On the
operated side of chronic frogs, significantly more 2°VN received
monosynaptic EPSPs following PC nerve stimulation than on the intact
side or in controls (Table 1). The number of 2°VN that responded with
monosynaptic EPSPs after RA nerve stimulation was larger than that
after PC nerve stimulation, but similar values were encountered on
either side of operated frogs and in controls (Table 1). According to
the convergence of afferent canal inputs on 2°VN, three subgroups of
2°VN were differentiated: monosynaptic EPSPs in 2°PC neurons were
evoked by PC but not by RA nerve stimulation (Fig. 2A),
monosynaptic EPSPs in 2°RA neurons were evoked by RA but not by PC
nerve stimulation (Fig. 2B), and monosynaptic EPSPs in
2°RA + PC neurons were evoked by RA as well as by PC nerve
stimulation (Fig. 2C). The percentage of 2°RA + PC neurons
was significantly increased on the operated side (Fig. 2D)
when compared with data from controls or from the intact side of
operated frogs. Interestingly, the percentage of 2°RA neurons was
significantly reduced on the same side (Fig. 2D). The
decrease in the number of 2°RA neurons on the operated side (about
24%) corresponded to the increase in the number of 2°RA + PC neurons
(about 21%) on the same side.
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Table 1.
Percentage of 2°VN with monosynaptic EPSPs following stimulation of
the ramus anterior (RA) of N. VIII or following stimulation of the PC
nerve
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|
Long-lasting excitatory abducens nerve responses were recorded on
either side with suction electrodes following stimulation of the
contralateral PC nerve. The onset latency of responses on the intact
side (5.88 ± 0.48 ms; n = 6) was significantly
shorter (P
0.05) than on the operated side
(6.67 ± 0.50 ms; n = 6) or in controls (7.53 ± 1.27 ms; n = 8). Abducens responses (area of
the 1st 20 ms) recorded on the intact side were significantly (P
0.05) larger (1.40 ± 0.19 mV × ms; n = 6) than the corresponding response components
recorded on the operated side (0.79 ± 0.21 mV × ms;
n = 6) or in controls (0.62 ± 0.40 mV × ms;
n = 8).
 |
DISCUSSION |
The significant increase in the amplitude of the PC nerve
evoked N1 field potentials on the operated side
corresponds with the increased number of 2°VN that exhibited
monosynaptic EPSPs following PC nerve stimulation on this side.
Apparently, PC nerve afferent inputs expanded on the operated side onto
some of those 2°VN that were vestibularly deprived after RA nerve
section. Consistent with this interpretation is the fact that the
percentage of true 2°PC neurons, i.e., of 2°VN with a monosynaptic
vestibular afferent input exclusively from the PC nerve, had not
changed in operated frogs. Rather, the number of 2°RA + PC neurons
increased, and the number of 2°RA neurons decreased on the operated
side by about the same amount. This shift in the relative size of these
two subpopulations strongly supports the notion of an expansion of PC
nerve afferent inputs onto vestibularly deprived ipsilateral 2°VN. A
direct consequence of such an expansion may be seen in the increased
responsiveness of the abducens nerve on the intact side after PC nerve
stimulation on the operated side. More efficient excitatory inputs and
an earlier recruitment of action potentials could explain shorter onset
latencies as observed for abducens nerve responses on the intact side.
Functional reorganization of somatosensory maps after nerve injury
includes an expansion of signals from intact peripheral afferents into
the territory of deprived afferents and an activation of neurons that
had not responded to this input before the lesion (O'Leary et
al. 1994
). As for the somatosensory system, axonal or dendritic
sprouting of intact afferent fibers and the formation of new synaptic
contacts or a postoperative increase in the efficacy of already
existing but silent terminals are possible mechanisms for the expansion
of vestibular signals on the operated side. Activity-dependent
interaxonal competition between vestibular afferent fibers for
functional synaptic contacts, as discussed for lesion-induced reactive
synaptogenesis in the somatosensory system (O'Leary et al.
1994
), is a possible trigger for these changes. Accordingly,
the terminals of PC nerve afferent fibers were expected to have an
activity-related competitive advantage compared with injured RA nerve
afferent fibers with the result that functionally new synaptic contacts
emerged. However, the expansion of PC nerve afferent signals on the
operated side was neither paralleled by an obvious reduction in the
number of 2°RA neurons nor by a decrease in the amplitude of RA nerve
evoked field potentials on this side. Both results speak against a
degeneration of the axotomized afferent fibers and for the survival of
their synaptic contacts. A similar observation was made after
unilateral labyrinthectomy (Kunkel and Dieringer 1994
).
The same competitive mechanism, assumed to be responsible for the
expansion of vestibular afferent signals after RA nerve section, might
also account for the expansion of ascending spinal projections and for
the amplification of vestibular commissural signals in frogs after
unilateral labyrinthectomy (see Dieringer 1995
). In
fact, preliminary data from this study indicate that the synaptic
efficacy of commissural fibers terminating on the operated side was
increased two months after a RA nerve section as well. The time course
of this increase after RA nerve section is so far unknown but should be
as delayed in its onset as it is after labyrinthectomy (Kunkel
and Dieringer 1994
), provided both increases share a common mechanism.
We gratefully acknowledge the support by Bundesministerium
für Bildung und Forschung, Förderschwerpunkt
"Neurotraumatologie" (Teilprojekt D3) and by the
Friedrich-Baur-Stiftung 44/95. F. Goto was supported by
Graduierten-Kolleg "Sensorische Interaktion in biologischen und
technischen Systemen." The National Institutes of Health
"Principles of Laboratory Animal Care" were followed, and
permission for these experiments was granted by Regierung von
Oberbayern (211-2531-31/95).
Address for reprint requests: N. Dieringer, Dept. of
Physiology, University of Munich, Pettenkoferstr. 12, 80336 Munich,
Germany (E-mail: dieringer{at}phyl.med.uni-muenchen.de).
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.