ELSEVIER Neuroscience Letters 175 (1994) 141-144

NHROSClENCE LETTERS

Cytochrome oxidase histochemistry in Scarpa's ganglion after hemilabyrinthectomy

Golda Anne Kevetter*, Adrian A. Perachio

Departments of Anatomy and Neuroscience, Physiology and Biophysics, and Otolaryngology, ENT Research, MRB 2.104 J-63, University of Texas Medical Branch; Galveston, TX 77555-1063, USA

Received 10 January 1994; Revised version received 15 April 1994; Accepted 3 May 1994

Abstract Cytochrome oxidase histochemistry was studied in neurons in the vestibular ganglion in gerbils two weeks after hemilabyrinthec-

tomy. This study measured the staining density in ganglion ceils on both the lesioned and non-lesioned side of the brainstem. Cytochrome oxidase staining was significantly reduced in ganglion cells ipsilateral to the lesion. This decrease may have been related to the concomitant loss of spontaneous discharge and reduced energy demand for oxidative metabolism.

Key words: Labyrinthectomy; Vestibular compensation; Plasticity; Cytochrome oxidase

Unilateral destruction of the labyrinth results in a va- riety of postural and oculomotor deficits [23]. Static symptoms are encountered when the head is stationary. Such manifestations include spontaneous nystagmus and a head tilt toward the lesioned ear. Dynamic symptoms are seen during head movements and include a reduction in the gain of the vestibuloocular reflex (VOR). Most of these behavioral symptoms recover in a process referred to as vestibular compensation. This process appears to rely on the re-establishment of symmetry in the activity of neurons in the vestibular nuclear complex (VNC) (e.g. [3,7,8,12,28,32]).

Type I neurons are defined as central neurons whose firing properties during angular acceleration resemble those of the vestibular nerve on the same side [25]. Imme- diately following hemilabyrinthectomy, type I neurons in the medial vestibular nucleus (MVN) ipsilateral to the lesion exhibit little or no spontaneous activity and reduced responses to horizontal head rotation [4,18,21,22,27]. During compensation type I neurons begin to exhibit spontaneous activity and increased dy- namic responses; however, the number of detectable cells of this class recorded on the side of the lesion is lower

* C0rrespgnding author: Fax: (1) (409) 772-5894.

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than in labyrinth-intact preparations [18,22]. Within the vestibular nuclei, cellular glucose metabolism, as esti- mated by the 2-deoxyglucose method, is reduced imme- diately after hemilabyrinthectomy on the side of the le- sion, with some recovery over time [13,14,15]. However, a persistent asymmetry remained in the MVN 21 days post-lesion [14].

There is little evidence that the vestibular afferents play a role in compensation; however, they do not degen- erate after peripheral lesions [1,6,24]. Moreover, afferent discharge can be induced both acutely and following compensation by the application of electrical current across the ganglion [18]. This suggests that the viable cells should exhibit reduced metabolic processes since the energy demand associated with the relatively high rates of afferent discharge in labyrinth-intact animals (average firing rate = 88 impulses/s for gerbil canal afferents [19]) is obviated. The oxidative enzyme cytochrome oxidase (CO) has often been used as an indicator that is related to discharge activity in neurons [30,31]. The density of histochemical staining expressed appears to depend on both the activity of the Na+,K+-ATPase activity within the neuron and presynaptic activity due to interactions between neurons [11,16,30].

The present study was designed to determine whether bilateral differences in cytochrome oxidase activity could

142 G.A. Kevetter, A.A. Perachio/Neuroscience Letters 175 (1994) 141 144

be detected in neurons in the vestibular ganglion in ger- bils after hemilabyrinthectomy. A preliminary report of a portion of this work has been presented previously [20].

Adult Mongolian gerbils (Merlones unguiculatus) were anesthetized with Brevital (60 mg/kg i.p.). The right bulla was opened and a hemilabyrinthectomy was performed by aspiration of the sensory end organs of the vestibular labyrinth. Fourteen days later, animals were re-anesthe- tized with an overdose of urethane. Animals were per- fused with 4% paraformaldehyde and 0.1% glutaralde- hyde in 0.1 M phosphate buffer (pH 7.4), the brains were removed, and 30/2m frozen sections were cut through the cerebellum, brainstem, and Scarpa's ganglia. Sections were reacted for CO histochemistry [30] with a procedure modified by Mawe and Gershon [16]. They were preincu- bated for 10 min in 10% sucrose, 27.4% cobalt chloride, 4% DMSO in 0.05 M Tris buffer, and then incubated in a solution containing 50 mg DAB, 6.4 mg cytochrome c (Sigma type III), 5 g sucrose, and 0.2 ml DMSO in 100 ml of 0.1 M phosphate buffer.

Measures for the density of the CO reaction product were made with a computer-aided densitometry pro- gram. The amount of transmittance of light through the selected area was measured. A sample of randomly cho- sen 50 individual primary afferent neurons on the intact side and 50 ganglion cells on the hemilabyrinthectomized side was obtained for each gerbil. Only ganglion cells with visible nuclei in the section were assessed. The non- stained nucleus was excluded from the optical density measure for each cell. Adjacent neurons in which the cytoplasm overlapped were also excluded.

A linear transform was made on each transmittance measurement in order to eliminate processing variables that could exist between animals. This transform was 1-transmittance measurement/mean transmittance for 100 cells/animal. Independent t-tests were run between the left and right sides of each animal for both the ob- served data and the transformed data to verify that the transformation had not changed the relationship of the numbers. The t- and P-values (P < 0.05) were identical for both sets of data. A 3-way analysis of variance with repeated values was run on the variables: treatment, an- imal, and side to analyze the effect of the lesion. There was a significant difference between the sides of the ex- perimental animals, but not between the sides of the control animals. Subsequently, a simple 2-way analysis of variance was performed analyzing the effect of the lesion on the staining of ganglion cells.

In control animals, CO staining was present in neu- rons in both divisions of Scarpa's ganglia bilaterally. The cytoplasm was densely stained and the nucleus was un- stained. The staining was approximately the same (P > 0.05) on both sides (Fig. 1).

Following hemilabyrinthectomy, all animals exhibited behavioral signs of postural compensation characteristic of this species [17]. A head tilt toward the lesioned side

was the only sign remaining after two weeks. Differences in the density of CO staining were found between the right (lesioned side) and left Scarpa's ganglia (Fig. 1). Two weeks after the lesion, ganglion cells on the injured side stained less densely than those that innervated the intact labyrinth (Fig. 2). There was a significant differ- ence (P < 0.01) in CO density between sides in the ani- mals with lesions (Fig. 1).

We have described a significant decrease in the activity of the mitochondrial enzyme CO within Scarpa's gan- glion neurons two weeks following hemilabyrinthec- tomy. It had been hypothesized that the remaining nerve might be an important factor in the recovery after hemil- abyrinthectomy [2,26]. However, the behavioral recov- ery after labyrinthectomy and section of the vestibular nerve central to the ganglion do not differ significantly in time course or amount of recovery [2]. Therefore, if the surviving afferent nerve serves any function, it is not required for vestibular compensation.

Vestibular afferents are spontaneously active when the hair cells they innervate are viable [29]. Extraction of the sensory neuroepithelia results in an immediate cessation of afferent discharge activity [10,26]. However, the affer- ent neurons remain responsive to extracellularly applied galvanic stimulation [18], and the number of cells in Scarpa's ganglia remains undiminished for 8-12 weeks post-lesion [1] and many cells survive more than 3 years

C y t o c h r o m e Oxidase Dens i t y in Gang l ion Ceils

160

~ 140 ~ ~ .~ left

"-~ 120 ~, right

10o

"~ 40

o 1 2 3 4 5 6

control experimental Animal N u m b e r

Fig. 1. Bar graphs comparing the density of cytochrome oxidase stain- ing in the ganglion cells on the intact (left) side with those of the right side (ipsilateral to the lesion). The density of staining is proportional to the intensity of light transmitted through a designated area. There- fore, the higher the number, the lighter the staining, and the lower the metabolic activity. The mean and standard error of the mean are shown for each side. Animals 1, 2 and 3 are controls. Animals 4, 5 and 6 had hemi|abyrinthectomies on the right side. There is a significant difference between sides for animals 4, 5 and 6.

G.A. Kevetter, A.A. Perachio/Neuroscience Letters 175 (1994) 141-144 143

Fig. 2. Photomicrographs of Scarpa's ganglion. A hemilabyrinthectomy was performed on this gerbil two weeks prior to sacrifice. A: Scarpa's ganglion on the non-lesioned side. B: Scarpa's ganglion on the side ipsilateral to the lesion. These micrographs are taken from the same section with the same intensity of light. Bar = 70/lm.

after lesion [6,24]. Lacking spontaneous activity, it is reasonable to expect that the energy demands of the afferents, as reflected by CO activity, would be signifi- cantly reduced. Lesions of the cochlea or the retina result in reduction in CO staining in neurons in the associated central sensory pathways [9,30,31].

Since ablation of the sensory neuroepithelium does not result in the death of Scarpa's ganglion neurons, the reduction in CO staining for afferent cell bodies on the injured side may simply reflect the loss of spontaneous discharge consequent to the lesion. The remaining stain- able enzyme may therefore be involved in the oxidative metabolism that maintains the integrity of the deaffer- ented neurons. The loss of afferent drive is accompanied by an immediate silencing or reduction of post-synaptic activity in second-order vestibular nuclei neurons. We have observed reduced CO staining following hemilaby- rinthectomy in the MVN [20], suggesting a comparable relationship in central vestibular neurons between neu- ronal activity and CO.

A mechanism that could account for the incomplete functional recovery of second-order vestibular neurons may involve neurotrophic factors. A nerve growth factor receptor (p75 NGFR) has been found by immunocytochem- ical labeling to be distributed in specific zones within the cuneate and gracile nuclei in a pattern that closely corre- sponds to that of CO activity [5]. Those regions contain concentrations of second-order somatosensory relay neurons. The loss of vestibular afferent activity may re- sult in reduction of a comparable trophic factor needed to maintain either the pre- or post-synaptic neuronal elements in the vestibular afferent pathway.

The authors would like to thank James Koppe for assistance with the statistics in this study, Dr. Ramptin Kassir for help in the initial experiments, and Heather McMullin for her technical assistance. We thank

Drs. Robert Leonard and Richard Puzdrowski for read- ing earlier versions of this manuscript. This work was supported in part by NIDCD/NIH DC00052 and Deaf- ness Research Foundation (G.A.K.) and NASA, NAG2- 26 (A.A.P.).

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