Pattern of synaptophysin immunoreactivity in the efferent nerve terminals of the guinea pig cochlea

Pattern of synaptophysin immunoreactivity in the efferent nerve terminals of the guinea pig cochlea

Neuroscience Letters 222 (1997) 199–203 Pattern of synaptophysin immunoreactivity in the efferent nerve terminals of the guinea pig cochlea S.A. Coun...

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Neuroscience Letters 222 (1997) 199–203

Pattern of synaptophysin immunoreactivity in the efferent nerve terminals of the guinea pig cochlea S.A. Counter a , b, B. Canlon b ,*, E. Borg b , c, H. Aldskogius d , e a

Neurology Department, Harvard University Biological Laboratories, Cambridge, MA 02138 USA Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden c ¨ rebro Medical Center Hospital, O ¨ rebro, Sweden Department of Medical Audiology, O d Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden e Uppsala University, Uppsala, Sweden

b

Received 15 November 1996; revised version received 2 January 1997; accepted 7 January 1997

Abstract The goal of the present study was to analyze the distribution of efferent 8th nerve synaptic endings in a surface preparation of the guinea pig cochlea using synaptophysin antibodies. Employing light and confocal microscopy synaptophysin immunoreactivity was found exclusively at the base of the outer hair cells (OHCs) and the inner hair cells (IHCs) axosomatic efferent synapses. Qualitative and quantitative differences were found between the OHCs and the IHCs immunoreactivity. Efferent nerve endings innervating IHCs were comparatively smaller, more numerous and densely packed. Efferent terminals demonstrated a longitudinal gradient for the IHCs and a longitudinal and radial gradient for the OHCs. Quantitative analysis of synaptophysin immunofluorescence demonstrated a higher percentage of efferent terminals innervating the IHCs and the OHCs in the mid and basal segments of the cochlea than in the apical regions. In addition, a radial gradient from the 1st to 3rd row of OHCs was evident. The results from the present study show that the analysis of synaptophysin immunoreactivity on cochlear surface preparations allows the efferent innervation to be determined throughout the entire cochlea. This technique allows for a rapid assessment of the normal cochlea as well as after cochlear insult.  1997 Elsevier Science Ireland Ltd. Keywords: Cochlea; Efferent innervation; Synaptophysin; Guinea pig

The cochlear efferent neurons originate from the superior olivary complex in the brainstem. The lateral and medial olivo-cochlear bundles innervate the inner (IHC) and the outer (OHC) hair cells of the cochlea, respectively [14]. Over 90% of the efferent terminals innervate the OHC while fewer than 10% synapse on the radial fibers associated with the IHC [12]. Using the monoclonal antibody SY 38, Gil-Loyzaga and Pujol [6] have shown that the Ca2 + binding glycoprotein, synaptophysin, an intrinsic membrane protein of the small synaptic vesicles, was present in the efferent presynaptic terminals of neurons innervating the inner and outer spiral bundles of the guinea pig. At the ultrastructural level, they found the immunoreactivity to be localized in the varicosities of efferent fibers at the * Corresponding author. Tel.: +46 8 7287248; e-mail: [email protected]

base of the outer hair cells, but not in the afferent fibers or supporting cells. As early as postnatal day 5 synaptophysin immunoreactivity can be localized in efferent fibers projecting to the OHC in the basal cochlear segments and in the apical turns by postnatal day 14. The efferent innervation to the IHCs can be detected by synaptophysin antibody labeling by the second postnatal week [8]. Neuroanatomical studies of the inner ear of several lower mammals, and of the human fetal cochlea, have shown a gradient efferent terminals at the OHCs from row 1 to row 3 (radial), and at the IHC and OHC from the basal to the apical portion of the cochlea (longitudinal) [2,9–12]. In this study, we investigated the organization, density and spatial innervation pattern of efferent 8th nerve endings serving the IHCs and the OHCs from the apex to the base of a surface preparation in the guinea pig cochlea.

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )1 3364-X

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Synaptophysin immunoreactivity in a surface preparation was used for a continuous, quantitative and qualitative analysis of the cochlear efferent innervation using fluorescence light and confocal microscopy. Four cochleae from normal hearing, healthy adult pigmented Hartley strain guinea pigs weighing 300–500 g were examined in this study. The animals were deeply anesthetized with sodium pentobarbital (Nembutal 40 mg/kg body weight) and perfused intracardially with body temperature physiological saline, followed by 4% paraformaldehyde with 14% saturated picric acid solution in 0.15 M phosphate buffer (pH 7.3–7.4, 4°C). The cochleae were removed and post fixed for 2 h, then stored in a refrigerated phosphate buffer solution containing 10% sucrose for 24 h at 4°C. The cochleae were removed from the temporal bone and placed in a phosphate-buffered saline (PBS) (pH 7.4) for 1–2 h. The bone surrounding the organ of Corti, the stria vascularis, the spiral ligament, and the tectorial membrane was dissected free. The organ of Corti was rinsed with PBS, 10% bovine serum albumin (BSA) and 3% Triton X-100 for 1 h 15 min, followed by two additional 10 min rinses in 0.3% Triton X-100. The organ of Corti was then placed in a solution containing

rabbit anti-synaptophysin (1:100), diluted in 1% BSA plus 0.3% Triton X-100 overnight. The preparations were then rinsed three times, 10 min each followed by TRITC-conjugated (anti-rabbit) IgG for 1 h, then rinsed three times, 10 min each. The organ of Corti was dissected into 1/2–3/ 4 segments and placed on a microscope slide in Citi-flour, and covered with a cover slip and sealed with non-fluorescent nail polish. The efferent terminal synapses associated with the IHCs and OHCs were examined throughout the surface preparation between 18 and 6 mm distance from the round window. Images were visualized with oil immersion epifluorescence microscopy (Zeiss Axioscope using 40 × and 100 × neofluor objectives) interfaced with an Optimas image analysis system (Optimas, Co., USA). The image was collected using a black and white high resolution Dage video camera (MTI CCD 72EX) and processed using the image analysis software. The software program sets the densitometric range from absolute blank (0) to darkest (255) which was used to quantify each 0.25 mm cochlear section from the computerized images. The values obtained from the density measurements are referred to as the mean gray area (MGA). The MGA is the

Fig. 1. Confocal image reconstruction from a Z-series of 0.5 mm optical sections (30–50 mm total thickness of the tissue and 60–100 sections total) showing anti-synaptophysin immunoreactivity in the efferent nerve endings innervating the radial fibers of the IHCs (upper) and the OHCs (lower) in the apical, middle and basal turns of the guinea pig cochlea. The surface preparations of efferent terminals shows a gradients of large, raspberry-like puncta around the infranuclear area of the OHC from apex to base and from rows 1 to 3. The efferent terminals under the IHCs are smaller and more densely packed from apex to base. Scale bar, 10 mm.

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struction of the cochlea specimens. Optical section of the surface preparations were performed by using 0.5 mm sections. The microscope was first focused on the fluorescing efferent fibers and then the focal plane was changed in the direction of the nucleus until the efferent fibers were no longer visible. This distance was usually between 15 and 25 mm. The focal plane was then returned to the fluorescing efferent terminals and the focal plane was changed in the direction below the terminals. This distance was often between 15 and 25 mm. As a result there was between 30 and 50 mm total thickness of the tissue that was analyzed. While the optical sectioning was 0.5 mm there was a total of 60 and 100 sections in total.

Fig. 2. Light microscopic (100 × ) tracings of anti-synaptophysin immunoreactive efferent neuron endings associated with identifiable OHCs in rows 1, 2, and 3 of a normal preparation, showing the cuticular plates (stars) and an orderly arrangement of associated efferent terminals tracked to their base. Scale bar, 10 mm.

value obtained by calculating the area under the density curve. Separate analysis was performed for each hair cell type. Ten neighboring hair cells from each 0.25 mm section were manually outlined and its density measured. The average number of IHCs in a 0.25 mm section is 25 and there are between 28–30 OHCs depending on the row to be analyzed. A cochleogram was then constructed showing the MGA versus the distance along the basilar membrane. A number of manual tracings were performed on the surface preparation in order to identify an efferent nerve terminal to its innervated outer hair cell. By focusing on the cuticular plate of an outer hair cell the cell membrane was followed by readjusting the focus until the base of the cell was in view together with the efferent nerve endings. This procedure allowed for a definite identification of an efferent nerve ending innervating a particular OHC. Fluorescence-labeled sections from the basal, middle and apical portions of the organ of Corti were viewed also through a confocal microscopy imaging system. Confocal microscopy provided three-dimensional microscopy with improved resolution of digitally recorded consecutive optical sections of the fluorescent specimens [3]. The photometric instrument for optical sectioning (PHOIBOS Sarastro 1000) employed a laser spot illumination system with two computer-controlled scanning mirrors which direct the image to a Nikon OPTIPHOT fluorescence microscope with an argon ion laser light source (described by Walle´n et al. [13]). The wavelength of the laser, the beam-splitter and filters were adjusted for optimal recon-

Fig. 3. MGA measures of the anti-synaptophysin immunoreactivity (in 48 data points per cochlea) in the IHCs and rows 1, 2 and 3 of the OHCs over the length (12 mm) of the specimen averaged for four normal guinea pig cochleae. The greater the MGA value the more intense is the immunoreactivity.

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Intense anti-synaptophysin immunoreactivity was localized in the efferent terminals around the basal pole of the IHCs from apex to base (Fig. 1, upper). These efferent nerve endings form synapses on the dendrites of the afferent radial fibers under the IHCs. The IHC immunofluorescent efferent endings were spherical in appearance, and considerably smaller than the terminals innervating the OHCs. The IHC efferents were densely packed around the base of the soma from the apical turns to the mid or basal regions where they were slightly more dispersed and slightly reduced in quantity. Distinct anti-synaptophysin immunoreactivity was found in the base of the OHCs but not in the soma of the OHCs from the apex to the base of the cochlea. The terminals form large clumps of spherical, raspberry-like puncta (Fig. 1, lower). In the extended confocal image of OHC rows 1, 2, and 3, there were more distinct immunoreactive terminals in the 1st row of the apical region with fewer terminals in the 2nd and 3rd rows. Efferents terminals projecting to the OHC rows 2 and 3 begin to increase in number and regularity in the mid cochlea turns, and were more dense and symmetrically organized in the basal turn. The confocal images revealed a gradation in size and number of the efferent nerve endings from the 1st to the 3rd row of the OHCs in the middle and basal turns. When analyzed under the light microscope (100 × ), the OHCs could be traced from the cuticular plate to the infranuclear area for identification of the associated efferents terminals. Fig. 2 shows the cuticular plates of OHCs 1, 2, and 3, and tracings of anti-synaptophysin immunoreactive efferent endings of fairly uniform in size and distribution, associated with identifiable OHCs in each of the three rows of a normal preparation. The advantage of this tracing technique is that it directly identifies an efferent synapse terminating on its associated OHC. This technique has obvious advantages of investigating pathological cochlea where efferent nerve endings and/or OHCs are injured. Quantitative analysis of the IHC efferent terminal density and spatial distribution revealed intense anti-synaptophysin immunoreactivity from apex to base. Fig. 3 shows the MGA values for the IHCs and OHCs for normal cochleae as a function of distance (mm) from the apex. The MGA values averaged from four individual cochleae in 0.25 mm divisions are shown. The MGA measures for the IHC were slightly lower in the apical region than the mid and basal areas in each immunostained cochlea. Integration of the area under the curve for the baseline to peak MGA values over the length of the cochlea in four cochleae gave integrated synaptophysin MGA values which indicate only slight differences in the level of IHC efferent innervation from the apical to the basal turn. The mid-cochlea segment of 4.25–8 mm showed a 29% increase in MGA values over the most apical segment (0.25–4 mm). The basal cochlea segment of 8.25–12 mm showed a 20% increase in MGA values over the most apical segment.

Quantitative analysis of the OHC efferent nerve endings reflected high anti-synaptophysin immunoreactivity in each of the cochleae segments. The MGA measures of the anti-synaptophysin immunoreactivity in the three rows of OHC were consistent with the light and confocal histological observations of higher efferent terminal density in the mid and basal cochlear regions than in the apical turns, and a radial gradient from the 1st to the 3rd row. Integration of the baseline to peak MGA values for 4 mm segments of the cochlea over the length of the specimen from 0.25 (apex) to 12.0 mm (base) in four cochleae showed MGA differences for each row of OHC. Longitudinally, the mid-cochlea segment of 4.25–8 mm showed a 16% increase in MGA values over the most apical segment (0.25–4 mm). At the basal end 8.25–12 mm there was an 8% increase in MGA values over the most apical segment. Radially, the MGA values of OHC row 2 indicated a 17.5% decrease in anti-synaptophysin immunofluorescent terminals, and OHC row 3 showed a 28% decrease in efferent terminal representation relative to OHC row 1 in the apical segments (0.25–4 mm). In the mid-cochlea, (4.25–8 mm), the integrated MGA values of OHC row 2 and OHC row 3 reflected a 5 and 11% decrease in efferent terminals, respectively in comparison with OHC row 1. In the basal segment of the cochlea (8.25– 12 mm), the integrated MGA values of OHCs row 1, 2 and 3 were essentially equal. OHC row 1 was slightly lower (2.5%) than OHC rows 2 and 3 which had the same MGA level. The observation of a progressive increase MGA antisynaptophysin immunoreactivity in the OHC from the apex to base along the cochlea reflects a higher density of efferent axosomatic synapses in the basal and mid segments. This finding is consistent with the observation that the OHC of mammals receive innervation from more than one efferent fiber [7]. The abundance of synaptophysin in the membrane of synaptic vesicles and the specificity of this protein for Ca2+ suggests an active role in synaptic transmission at the receptor cell (OHC) and afferent fiber (IHC) levels. The advantage of applying synaptophysin antibodies to the cochlea is that the total efferent innervation to the IHCs and OHCs is obtained. This is in contrast with other neurotransmitter studies [1,4] which have not found a similar organization of efferent cholinergic synapses on the guinea pig IHCs and OHCs. The efferent synapses in the guinea pig cochlea also contain several types of g-aminobutyric acid (GABA)-containing fibers [5]. However, the GABA containing fibers are more predominant in the more apical cochlear regions than the basal regions and therefore has limited use in analyzing the total efferent innervation in the cochlea. The findings of the present study are also consistent with the observation of an earlier developmental appearance, and possible biological significance of a more abundant efferent innervation in the basal compared to the apical region of the cochlea [6,8].

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The use of the surface preparation permitted a quantitative analysis (MGA) of the spatial organization and pattern of efferent nerve endings in the guinea pig cochlea. It is feasible only through the use of the surface preparation to analyze all of the efferent endings innervating the IHCs and OHCs. Another advantage to the quantitative analysis of the surface preparation is that it is rapid. The analysis of the entire cochlea requires approximately 4 h. In addition, the positive identification of the efferent nerve ending that innervates a particular outer hair cell by the ‘tracing’ method has obvious advantages, particularly for the study of pathological cochlea. The relationship between a damaged or missing OHC to the status of the efferent synapse can be determined precisely. This would afford important information regarding the rate of degeneration of the efferent synapse in relation to the degeneration of the hair cell. This study was supported by grants from the Swedish Council for Work Life Research (79-0800), Medical Research Council (09476), Stiftelsen Tysta Skolan and Karolinska Institute and Harvard University funds. We thank Dr. Reinhard Jahn for the synaptophysin antibodies and Dr. Peter Walle´n for use of the confocal microscope. We gratefully acknowledge the excellent technical assistance of Britt Mejer, Anette Fransson and Agneta Viberg. [1] Altschuler, R.A., Kachar, B., Rubio, J.A., Parakkal, M.H. and Fex, J., Immunocytochemical localization of choline acetyltransferaselike immunoreactivity in the guinea pig cochlea, Brain Res., 338 (1985) 1–11. [2] Anniko, M., Arnold, W. and Thornell, L.E., Localization of the integral membrane glycoprotein synaptophysin and the surface gly-

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