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INTRODUCTION |
Alterations in sensory input
can induce plastic reorganizational changes in sensory maps of adult
mammals (Kaas 2000
). Postlesional reactive
synaptogenesis subsequent to unilateral section of N. VIII or of its
ramus anterior (RA) is well documented for frogs (Dieringer
1995
; Goto et al. 2000
), resembles the
lesion-induced reorganization of sensory maps after nerve injury in
mammals, and raises the issue of a reaction pattern common for
different sensory modalities and vertebrate species. "Vestibular
compensation," the partial behavioral normalization following N. VIII
section, is an excellent model for the study of adult subcortical
plasticity and its motor consequences. In vitro frog brain studies have
demonstrated a remarkable functional specificity of semicircular canal
afferent inputs (Straka et al. 1997
) and commissural
inhibitory postsynaptic potentials (IPSPs) from the contralateral
coplanar canals (Holler and Straka 2001
). In vivo
studies characterized the spatial response vectors of maculo- and
canal-ocular innervation patterns (Rohregger and Dieringer
1999
). Such detailed species-specific information about the
synaptic convergence patterns of a sensorimotor system provides a
platform for the analysis of postlesional reorganization and evaluation
of functional specificity. As in an earlier study, the afferent inputs
from the utricle, horizontal, and anterior vertical canals were
inactivated unilaterally by RA section (Goto et al.
2000
). Two months later the brain was isolated in vitro, and
second-order vestibular neurons (2°VN) were identified by the
presence of monosynaptic excitatory postsynaptic potentials (EPSPs) as
2°PC, 2°RA, or 2°RA + PC neurons after electrical stimulation of
the posterior vertical canal (PC) nerve or of the sectioned RA on the
operated side. Commissural EPSPs and IPSPs of identified 2°VN were
studied following separate stimulation of each of the canal nerve
branches on the intact side.
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METHODS |
Surgery was performed in deeply anesthetized grass frogs
(Rana temporaria; 0.1% MS-222). The optic capsule was
opened, and the RA of N. VIII was sectioned under visual control distal
to the entry of the saccular nerve branch. About 2 mo after the lesion, chronic RA frogs (n = 11) and control frogs
(n = 10) were anesthetized and perfused transcardially
with iced Ringer solution. The brain together with the attached N. VIII
and their branches to the labyrinthine end organs were removed and
prepared for in vitro experimentation (see Straka et al.
1997
). The PC and the RA nerve branches on the operated side
and each of the three canal nerve branches on the intact side were
electrically stimulated separately with short pulses via suction
electrodes. The characteristics of recording electrodes and the
limitations of stimulation intensities were as described by Goto
et al. (2000)
. Field potentials were searched in depth tracts
throughout the entire rostrocaudal extent of the vestibular nuclear
complex on the operated side. The responses of identified 2°VN were
analyzed provided their membrane potentials was at least
40 mV and
stable over the recording time. Single sweeps of the evoked afferent or
commissural responses were digitized, stored on a computer, averaged
15-20 times, and analyzed off-line. EPSPs and IPSPs from 2°VN were
analyzed after electronic subtraction of the extracellular field
potential recorded in the vicinity of the neuron. In most control or RA
frogs, the responses of about 20 neurons were recorded, except for two
individuals in which only 3 or 6 and one in which 38 neurons were
recorded. A possible bias of our results due to exceptional data from
individual animals was excluded by a Kruskal-Wallis test
(nonparametric ANOVA). For further statistical analysis, the
Mann-Whitney U test (unpaired parameters) was used. The mean
of the average numbers of EPSPs or IPSPs in each particular cell type
(i.e., 2°PC, 2°RA, or 2°RA + PC neurons) from
individual RA frogs were significantly different from controls except
for 2°PC neurons. The mean values for each recorded cell type from
the RA frog population were significantly different from the mean
values of controls as well except for 2°PC neurons.
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RESULTS |
Semicircular canal nerve stimulation evoked afferent field
potentials in the ipsilateral and commissural field potentials (Fig.
1A) in the contralateral
vestibular nuclear complex. Commissural field potentials consisted of
one (cN1) or more postsynaptic negativities. The
onset latency of cN1 was 8.3 ± 0.9 (SD) ms
(n = 24;
in Fig. 1A) and characterized
postsynaptic commissural responses with a disynaptic onset (
in Fig.
2, B-D). The amplitudes of
control cN1 potentials were small, and a clear
depth profile was absent (Fig. 1B). Commissural field
potentials recorded on the operated side of chronic RA frogs were much
larger than in controls (Fig. 1, A and B). These
canal nerve-evoked cN1 potentials exhibited a
depth profile with a first peak at a depth of about 0.5-0.6 mm (center
of the vestibular nuclear complex) and a secondary increase at a depth
below the ventral border of the vestibular nuclei (0.7 mm and deeper;
Fig. 1B).

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Fig. 1.
Commissural field potentials recorded in controls and on the operated
side of chronic ramus anterior (RA) frogs following separate electrical
stimulation of individual semicircular canal nerves on the
contralateral side. A: commissural field potentials
(cN1) following stimulation of the contralateral horizontal
semicircular canal nerve had much larger amplitudes on the operated
side of RA frogs than in controls. B: averaged
commissural field potentials recorded on the operated side of RA frogs
following stimulation of the anterior vertical (AC), posterior vertical
(PC), or horizontal canal (HC) nerve were larger than in controls.
- - - (A), the baseline, , onset of
stimulation; , the average ± SD of the
cN1 onset latency. Each record in A
represents the average of 20 responses.
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Fig. 2.
Commissural canal inputs converging on second-order vestibular neurons
(2°VN) in controls and in frogs 2 mo after RA nerve section.
A-D: monosynaptic excitatory postsynaptic potentials
(EPSPs) following ipsilateral posterior vertical canal (iPC) nerve
stimulation (A) identified a 2°PC neuron that received
disynaptic inhibitory postsynaptic potentials (IPSPs; B)
following contralateral anterior vertical canal (cAC) nerve stimulation
and oligosynaptic EPSPs (C and D)
following contralateral horizontal (cHC) or posterior vertical canal
(cPC) nerve stimulation. E and F: in
chronic RA frogs, the average number of commissural IPSPs per 2°VN
(E) was significantly smaller in 2°RA
(*P 0.01) and in 2°RA + PC neurons
(***P 0.0001) than in controls. The average
number of commissural EPSPs per 2°VN (F) of chronic RA
frogs was significantly increased in 2°RA (**P 0.001) and in 2°RA + PC neurons (***P 0.0001)
with respect to controls. Commissural inhibitory (E) and
excitatory (F) inputs of 2°PC neurons remained
unchanged (n.s., not significant). (A-D), stimulus onset; , mean ± SD of monosynaptic (A) or disynaptic
(B-D) response onset latencies. Each trace in
A-D represents an average of 20 single sweeps.
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Intracellular records were collected from 430 identified 2°VN. With
respect to controls, we encountered on the operated side of chronic RA
frogs fewer 2°RA, more 2°RA + PC, and about equal numbers of 2°PC
neurons (Table 1). The numerical shift
from 2°RA to 2°RA + PC neurons in chronic RA frogs confirmed our
earlier report on the expansion of afferent inputs from intact
fibers (e.g., from PC nerve) onto 2°RA neurons deprived
of an active vestibular nerve afferent input (Goto et al.
2000
). Canal nerve-evoked commissural inputs of 2°PC neurons
(Fig. 2A) recorded on the operated side of chronic RA frogs
consisted typically (25 of 28 neurons) of disynaptic IPSPs (Fig.
2B) from the nerve of the contralateral anterior vertical
canal, which is coplanar to the ipsilateral PC and di- or oligosynaptic
EPSPs (Fig. 2, C and D; 14 of 28 neurons) from
the nerves of the two remaining contralateral canals, which are
noncoplanar to the ipsilateral PC. Accordingly, the average number of
inhibitory commissural canal inputs per 2°PC neuron was close to one
(Fig. 2E) and that of excitatory commissural canal
inputs was close to two (Fig. 2F). A very similar
commissural input pattern was observed in 2°PC neurons of control
animals (Fig. 2, E and F).
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Table 1.
Percentage of 2°VN with monosynaptic afferent EPSPs following
ipsilateral stimulation of the RA of N. VIII or of the PC nerve
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In chronic RA frogs, the commissural canal inputs of 2°RA and of
2°RA + PC neurons were clearly different from those recorded in
controls. Commissural IPSPs were detected in fewer of these neurons and
commissural EPSPs were evoked from more contralateral canal nerves than
in controls (Table 2). Hence, the average
number of commissural IPSPs per identi-fied 2°RA or 2°RA + PC neuron was significantly lower than in controls (Fig.
2E). The average number of commissural EPSPs,
however, was significantly higher in 2°RA neurons and in 2°RA + PC
neurons than in controls (Fig. 2F). Thus more 2°RA
and 2°RA + PC neurons on the operated side of chronic RA frogs were
excited by commissural inputs following stimulation of each of the
three contralateral canal nerves than in controls (Table 2).
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Table 2.
Percentages of 2°VN, as presented in Table 1, exhibiting commissural
inhibitory or commissural excitatory responses
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DISCUSSION |
Canal nerve-evoked commissural field potentials increased
on the operated side in amplitude in part because commissural EPSPs had
increased and commissural IPSPs had decreased in number. This increase
in the amplitude of commissural field potentials was not restricted to
the region of the vestibular nuclei but included also the more
ventrally located reticular formation, which receives vestibular nerve
afferent fibers as well.
Lesion-induced cooperative changes in excitatory and inhibitory inputs
as reported here for afferent and commissural inputs of deprived 2°RA
neurons were observed also in the somatosensory system of monkeys
(Garraghty et al. 1991
) and in the visual system of cats
(Arckens et al. 2000
). After a vestibular nerve lesion, Calza et al. (1992)
and Yamanaka et al.
(2000)
observed a transient down-regulation of the inhibitory
GABAergic input of unidentified vestibular neurons in rats. An
increased cerebellar inhibition, however, was observed in addition
to an increased commissural excitation in frogs (Dieringer
1995
). A general downregulation of GABAergic inputs in deprived
2°VN is thus unlikely. Possibly, more efficient excitatory
commissural inputs from the intact side simply masked persisting
commissural inhibitory inputs. Such a masking of commissural inhibition
by convergent commissural excitation occurs in 2°VN of controls if
all three canal nerves are stimulated simultaneously (Holler and
Straka 2001
).
RA nerve-evoked responses recorded on the operated side were unaltered
when compared with those recorded on the intact side or in controls
(Goto et al. 2000
). Hence, the synapses of inactivated RA afferents were maintained but the missing afferent excitation was
replaced by cooperative changes in commissural excitation and
inhibition and by new, spatially inadequate afferent inputs. Reactivation of deprived 2°RA neurons might facilitate the survival of these neurons and reduce the postlesional asymmetry in bilateral resting activities. However, this reactivation did not restore but
altered the original spatial response specificity of deprived 2°RA
neurons. Their target neurons (e.g., extraocular motoneurons) will
therefore receive spatially inappropriate dynamic input signals. In
fact, the spatial response vectors of extraocular motoneurons were
altered in RA frogs, and a new response component, not present in
controls, emerged during linear vertical acceleration (M. Rohregger and N. Dieringer, unpublished data). Similar
functionally inappropriate subcortical and cortical reorganizations and
the emergence of undesired sensations and motor reactions were
described for other sensory or motor systems of adult mammals after
nerve injury (Kaas 2000
). As for the vestibular system
of frogs (Dieringer 1995
), multiple intracellular
mechanisms at multiple anatomical sites are involved in the
reactivation of deprived neurons in the monkey's somatosensory system
(Florence and Kaas 1995
) and in the cat's visual system
(Arckens et al. 1998
). These extensive similarities across sensory modalities and vertebrate species suggest the presence of a fundamental and common lesion-induced neural reaction pattern.
We gratefully acknowledge the support by Bundesministerium
für Bildung und Forschung-Förderschwerpunkt
"Neurotraumatologie" (Teilprojekt D3) and by Deutsche
Forschungsgemeinschaft (SFB 462; Teilprojekt B2). 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 the experiments was granted by Regierung von Oberbayern
(211-2531-98/99).
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).