Postlesional Vestibular Reorganization in Frogs: Evidence for a Basic Reaction Pattern After Nerve Injury

Fumiyuki Goto, Hans Straka, and Norbert Dieringer

Department of Physiology, University of Munich, 80336 Munich, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Goto, Fumiyuki, Hans Straka, and Norbert Dieringer. Postlesional Vestibular Reorganization in Frogs: Evidence for a Basic Reaction Pattern After Nerve Injury. J. Neurophysiol. 85: 2643-2646, 2001. Nerve injury induces a reorganization of subcortical and cortical sensory or motor maps in mammals. A similar process, vestibular plasticity 2 mo after unilateral section of the ramus anterior of N. VIII was examined in this study in adult frogs. The brain was isolated with the branches of both N. VIII attached. Monosynaptic afferent responses were recorded in the vestibular nuclei on the operated side following ipsilateral electric stimulation either of the sectioned ramus anterior of N. VIII or of the intact posterior vertical canal nerve. Excitatory and inhibitory commissural responses were evoked by separate stimulation of each of the contralateral canal nerves in second-order vestibular neurons. The afferent and commissural responses of posterior vertical canal neurons recorded on the operated side were not altered. However, posterior canal-related afferent inputs had expanded onto part of the deprived ramus anterior neurons. Inhibitory commissural responses evoked from canal nerves on the intact side were detected in significantly fewer deprived ramus anterior neurons than in controls, but excitatory commissural inputs from the three contralateral canal nerves had expanded. This reactivation might facilitate the survival of deprived neurons and reduce the asymmetry in bilateral resting activities but implies a deterioration of the original spatial response tuning. Extensive similarities at the synaptic and network level were noted between this vestibular reorganization and the postlesional cortical and subcortical reorganization of sensory representations in mammals. We therefore suggest that nerve injury activates a fundamental neural reaction pattern that is common between sensory modalities and vertebrate species.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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.


    RESULTS
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INTRODUCTION
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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, black-down-triangle , 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). black-down-triangle  (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.

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

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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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.


    ACKNOWLEDGMENTS

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).


    FOOTNOTES

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).

Received 2 February 2001; accepted in final form 14 March 2001.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society