Auditory stimulation alters the pattern of 2-deoxyglucose uptake in the inner ear

Brain Research, 234 (1982) 213-225

213

Elsevier Biomedical Press

A U D I T O R Y S T I M U L A T I O N ALTERS T H E P A T T E R N OF 2-DEOXYG L U C O S E U P T A K E IN T H E I N N E R EAR

ALLEN F. RYAN, PAUL GOODWIN, NIGEL K. WOOLF and FRANK SHARP Division of Otolaryngology, Departmentof Surgery and ( F.S.) Departmentof Neurosciences, University of California Medical School, San Diego, CA 92103 (U.S.A.)

(Accepted July 23rd, 1981) Key words: cochlea - - 2-deoxyglucose -- inner ear metabolism -- noise exposure - - frequency

selectivity

SUMMARY The 2-deoxy-D-glueose (2-DG) autoradiographic technique was adapted for application to the inner ear. The uptake of [14C]2-DG during silence was compared with that observed during exposure to wide band noise (WBN) or pule tones at an intensity level of 85 dB SPL. In silence, the highest levels of 2-DG uptake were observed in the spilal ligament, spiral prominence and stria vascularis, with approximately equal levels of uptake in each structure. The high levels of 2-DG uptake observed in the ligament and prominence are surprising, and suggest a more active role for these structures in cochlear function than has previously been suspected. Levels of uptake in the organ of Corti, spiral ganglion and VIIIth nerve were much lower, although well above background. During exposute to WBN, 2-DG uptake increased markedly in the VIIIth nerve, and spi~al ganglion throughout the cochlea, and in the organ of Corti in the lower basal turn. 2-DG uptake did not change significantly in the spiral ligament or stria vascularis. During pure tone exposure, increased 2-DG uptake was noted in localized regions of the VIIIth nerve and spiral ganglion.

INTRODUCTION The 2-deoxy-D-glucose (2-DG) autoradiographic method has been used to map functional activity in neural tissue at a variety of locations. In sensory systems, such mapping has been utilized to detect the activation of central structures in the visual 5,8,1°,27, olfactoryzl, vestibular z0 and auditory 18,22,2a,zg,88 systems. On the other hand, relatively little attention has been devoted to sensory receptors. Basinger et al. 2 demonstrated 2-DG uptake in photoreceptors and neurons of the goldfish retina 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press

214 which varied depending upon stimulus condition. The 2-DG method has not been applied to the inner ear. The cochlea should provide an excellent system in which to determine the relationship between 2-DG uptake and acoustically-evoked activity. The sensory and neural elements of the inner ear are organized longitudinally along the length of the basilar membrane, between 10 and 33 mm in common laboratory species. This provides far more spatial separation than is present in any central auditory structure. The various cochlear tissues are also locally separated, with the secretory epithelium of the stria vascularis located on the lateral cochlear wall, the sensory cells in the organ of Corti, and the cell bodies of the primary afferent fibers in the spiral ganglion, each separated from the other by distances of at least 100 /~m. The primary cochlear afferents display relatively uniform functional characteristics which have been extensively documented11,z5. However, the cochlea also presents unique difficulties for the utilization of diffusible tracers such as 2-DG. The tissues of the inner ear are surrounded by lalge fluid spaces and encased in a complex bony capsule, which makes frozen sectioning, as used in brain, impractical. Standard inner ear histological techniques, involving fixation and decalcification, would cause loss of the water-diffusible 2-DG and would probably alter its distribution pattern. We have previously demonstrated that cryogenic freezing followed by freezedrying of whole cochlear specimens leaves the tissues of the cochlea intact at the cellular and even subcellular level, and preserves the distribution of ions both in tissues and fluid residues3,16,17. It was hypothesized that freeze-dried cochlear specimens might be amenable to plastic embedding, as has been used to study [3H]galactose in the intestine2s, provided that the delicate cochlear tissues could withstand plastic infiltration following freeze-drying. The present investigation was designed to determine whether [14C]2-DG may be used to map functional activity in the cochlea, by combining cryogenic freezing, freeze-drying and plastic embedding.

MATERIALS AND METHODS Mongolian gerbils (Merionesunguiculatus)weighing from 45-85 g each were used as subjects. 2-Deoxy-D-l-[14C]glucose (57 mCi/mM, Amersham-Searle) was evapoated and reconstituted in a plasma substitute. Alert and unanesthetized gerbils were injected by cardiac puncture with 16.7 #Ci of 2-DG/100 g body weight. After injection, animals were caged or immobilized inside of a double-walled, sound-attenuated chamber (IAC) for one hour. 100/~t blood samples were obtained by cardiac puncture from some subjects at regular intervals following 2-DG injection. Control subjects were kept in silence. The external meati of some of these control animals were ligated under brief anesthesia (ketamine hydrochloride, 20 mg/kg) 24 h prior to administration of 2-DG. Experimental subjects were exposed to wide band noise (WBN) generated by a Grason-Stadler 2369 noise generator, or to

215 pure tones (0.75, 3.0 or 12.0 kHz) generated by a Wavetek 136 oscillator. WBN and pure tones at 0.75 and 3.0 kHz were amplified by a Mclntosh M50 amplifier and applied to a JBL 2482 midrange speaker coupled to an Altec 511B horn. The horn aperture was located 15 cm from the animal's head. Pure tones at 12.0 kHz were amplified by a Crown 060 amplifier and applied to a JBL 077 high-range radiator located 70 cm from the animal's head. Sound pressure levels were measured with a BriJel and Kjaer 2209 Sound Level Meter and 1613 Octave Band Filter, WBN by averaging the readings obtained at 2.0 and 4.0 kHz, pure tones on linear, and set at 85 dB SPL. Immediately following the one hour exposure or silent period, subjects were decapitated and the cochlea was rapidly dissected from the temporal bones. The whole, unbreached cochlea was then quenched in Freon 12 cooled to --159 °C in liquid nitrogen and freeze-dried at --40 °C for 72 h. The round and oval windows were opened. Most specimens were then vapor-stained for 10 min over a 4 % solution of OsO4 to fix tissue lipids followed by 15 min vapor-fixation with acrolein 18 to fix tissue proteins. The samples were then embedded in Spurr resin using only organic solvents. Cochleas were cut in midmodiolar section, either in half, or in ~ 200/zm sections, using a jeweler's saw mounted on a bone-cutting lathe and under continuous oil bath. The resultant sections were exposed on LKB Ultrofilm for 10-20 days and developed in Kodak D-19 (4 min) or Microdol-X (12 rain). Optical densities were measured on autoradiographs which had been exposed for 10 days and developed in D-19. A microdensitometer was used which consisted of a Leitz MPV photometer to measure light transmitted through the autoradiographs and magnified with a Leitz Ortholux microscope. The reading spot was 15/~m in diameter, and all readings were obtained at 40 × magnification. Locations of cochlear structures were determined from comparison of transmitted- and incident-light micrographs of each specimen with the corresponding autoradiograph. The densitometer was zeroed on the background of each autoradiograph before measurements were obtained. In order to standardize the optical density measurement procedure, the highest optical density measurement obtained from the image of a structure was taken as the reading for that structure. This procedure was found to produce the most consistent and repeatable optical density measurements. Blood samples were spun for 4 min in a Beckman Microfuge. Duplicate 10 #1 serum samples were added to 5 ml of scintillation fluid (Betablend, Weschem) and counted on a Beckman LS230 scintillation counter. [14C]toluene (New England Nuclear) was employed for external standardization. Serum glucose levels were determined from 10/zl samples on a Beckman Glucose Analyzer 2. Specific activity curves were calculated from serum data according to Sokoloff et al. 9-6. Tissue [14C] concentration [C;(T)] was calculated from the optical density of the tissue and a standard curve of optical densities versus nCi/g for a series of previously calibrated plastic standards. Plasma [14C] concentrations (C~) at each time point for each animal were divided by the plasma glucose concentration (Cp) and the integral of this curve calculated by the approximate method of rectangles. The following equation was used to perform the calculations: * Ci* (T)/o.[T Cp/Cp

216 with variable names as described by Sokoloff et al. 26 and the time T = 60 min in our experiments. In the absence of the kinetic constants necessary to quantify glucose utilization, the integral of the plasma specific activity curve provides a reasonable correction for differences between 2-DG dose and time course in plasma, as well as serum glucose competition, between different subjects. In a separate experiment to measure 2-DG uptake in cochlear per;lymph, an adult gerbil was anesthetized (20 mg/kg Nembutal, 35 mg/kg ketamine), and 16.7 #Ci/100 g of 2-DG administered by intracardiac injection. 0.5/~1 sampels of perilymph and CSF were obtained by round window and cisternal puncture, respectively, and 1.0 #1 serum samples from venous blood, for 50 min. The 2-DG content of all samples was determined from scintillation counts. RESULTS 2-DG in serum and perilymph Fig. 1 illustrates typical 2-DG and glucose levels in serum following intlacardiac injection of 2-DG in an unanesthetized gerbil. The highest 2-DG level was measured at 1 min post-injection and 2-DG then declined exponentially, as has been leported by other investigators with intravenous administration in the rat 26. Serum glucose remained relatively stable at 100 mg/ml, also similar to data obtained in the rat ~6. Fig. 2 illustrates the kinetics of 2-DG content in serum, CSF and perilymph after intracardiac injection of 2-DG in an anesthetized gerbil. The serum curve is similar to that of Fig. 1. In CSF, 2-DG level rose gradually, without reaching a pronounced peak, and then declined in a manner similar to that of the serum curve. Perilymph 2D G content was quite similar to that in CSF, although at a slightly lower level. In all

i~

SERUM[t4C]-2-OEOXYGLUCOSE

.2

~5 E

0

10

140I 0° 120 100

~" 8o

J ~ o--,~

0

20

30

40

50

GO

SERUMGLUCOSE o_.___./-o

lO 20 30 40 50 60 TIMEAFTERINJECTION (min)

Fig. 1. Kinetics of [a4C]2-deoxyglucoseand glucose in serum, following intracardiac injection of 2-DG in an unanesthetized gerbil.

217 lo

"~ 15 c

• Serum

,75 --

3

.50

&

25

o

10

2°

30

o

CSF

•

Pertlymph

,o

5'0

TIME AFTER INJECTION (rain)

Fig. 2. Comparison of the kinetics of [14C]deoxyglucose in serum, CSF and cochlear perilymph following intracardiac injection in an anesthetized gerbil. Serum obtained from venous blood is compared to CSF obtained by cisternal puncture and to perilymph obtained by round window puncture. CSF and perilymph 2-DG curves are similar, although the level of 2-DG in perilymph

consistentlyremains below that of CSF.

cases 2-DG content had declined well below maximum by 60 min post-injection, indicating that most of the radioactivity was cleared from serum, CSF and perilymph, and that most of the 2-DG was present in tissue at that time. Cochlear tissues The preservation of cochlear tissues after freeze-drying and plastic-embedding was a primary concern in this investigation. In unfixed tissue, preservation of all cochlear elements was excellent, with the exception of the organ of Corti. While the sensory epithelium was well preserved in about 80 ~ of cases, the organ of Corti was disrupted or even locally absent in the remaining 20 ~. Vapor fixation of tissue with acrolein and OsO4 appeared to enhance the stability of the organ of Corti. The epithelium was present in more than 90 ~ of cases and the most severe distortion observed was division of the organ at the level of the pillar cells into inner and outer halves. The typical preservation of tissue in a freeze-dried cochlea which has been vapor-fixed prior to embedding is illustrated in F;g. 3. All tissue elements are present. Artifacts are limited to a slight separation of the spiral ligament from the cochlear capsule, a break in Reissner's membrane, and a narrow split in the spiral ganglion. Distribution of 2-DG in silence The distribution of 2-DG which was observed during silence is illustrated in the autoradiograph of Fig. 4A. The highest level of uptake was observed in the lateral cochlear wall, including the spiral ligament, spiral prominence and stria vascularis. It should be noted that a uniform, high, grain density is present throughout the region of the lateral cochlear wall, with no preferential uptake in any one region. However, 2DG uptake in the lateral cochlear wall appears to be slightly higher in the lower basal turn than at other locations. The autoradiographic images associated with the organ of

218

Fig. 3. Plastic-embedded, freeze-dried inner ear. The figure illustrates the preservation of tissue in the lower middle turn. All of the cochlear structures are well preserved. The major artifacts present are a split between the spiral ligament (SL) and the cochlear capsule, a crack in the spiral ganglion (SG) and a break in Reissner's membrane (rm) at its point of attachment to the spiral ligament. Within the organ of Corti (OC), both inner (ihc) and outer (ohc) hair cells are clearly visible. SV, stria vascularis; SP, spiral prominence; tin, rectorial membrane; sl, spiral limbus. Corti, spiral ganglion and VIIIth nerve are much less dense than that of the lateral cochlear wall. Density of the organ of Corti image exhibited more variability than that of other cochlear structures. The data presented in the figure are typical of autoradiographs obtained from cochleas kept in silence. No difference in 2-DG distribution was noted between controls with or without ligation of the external meatus.

Distribution of 2-DG during WBN exposure The distribution of 2-DG which was observed dining exposme to 85 dB SPL WBN is illustrated in Fig. 4B. Relative to other cochlear structures, increased 2-DG uptake is visually apparent in the spiral ganglion, especially in the lower basal turn, and in the entire VIIIth nerve. N o increase is visually apparent in the organ of Corti. As in cochleas from animals kept in silence, 2-DG uptake in the lateral wall complex appeared to be maximal in the lower basal turn.

Distribution of 2-DG during pure tone exposure Autoradiographs obtained from cochleas after exposure to pure tones are

219

Fig. 4. Typical autoradiographs obtained from inner ears maintained in silence (A) or 85 dB WBN (B) for one hour after injection of [14C]2-DG. The light micrographs show the corresponding plastic sections, which were approximately 200/~m in thickness. The camera lucida drawings show the structures at the surface of each section which was apposed to the X-ray film. Autoradiographs exposed on LKB Ultrofilm 20 days and developed for 12 min in Kodak Microdol X. Note the missing segment of stria vascularis in the lower basal turn of (B), which appears as a gap in the autoradiographic image of the lateral wall structures. The stapedial artery was trimmed from the section in (/3) between exposure of the autoradiograph and photography, sv, stria vascularis; sl, spiral ligament; oC, organ of Corti; sg, spiral ganglion; sa, stapedial artery; ow, oval window.

illustrated in Fig. 5. The pure tone frequencies (0.75, 3.0 and 12.0 kHz) were chosen to correspond to lower apical, middle and basal turn locations exposed by our standard midmodiolar section, from the frequency/place map of Sokolich et al. ~5. A 0.75-kHz tone would be expected to excite the lower apical turn, 1.2 mm from the apex. As in the example in Fig. 5, exposure to this frequency at 85 dB characteristically produced increased 2-DG uptake in a diffuse area of the VIIIth nerve extending from apex to base and densest in the lateral portion. 2-DG uptake was also elevated in the spiral ganglion of the apical and lower middle turns. A 3.0-kHz tone would be expected to excite the lower middle turn, 4.7 mm from the apex. Increased 2-DG uptake is apparent in a discrete band in the medial portion of the VIIIth nerve, and in the spiral ganglion of the lower middle turn. A 12.0-kHz tone would be expected to excite the lower basal turn, 10.0 mm from apex. A discrete region of increased 2-DG uptake was characteristically observed in

220

750

HZ

3 kHz

12 kHz c~

g Fig. 5. Typical autoradiographs from inner ears exposed to pure tones at 3 frequencies. Spiral ganglion indicated by circles. Exposure frequencies chosen to correspond to the spiral ganglion location indicated by asterisk in each figure, based on the map of Sokolich et al.25. During exposure to a 750-Hz tone, 2-DG uptake is elevated diffuselythroughout the VIIIth nerve, with maximal uptake in the posterolateral portion (arrow). Uptake in the spiral ganglion was highest in the apical and upper middle turns. When the exposure frequency was 3.0 kHz, uptake in the nerve was highest in a sharplydefined band along the medial edge of the nerve (arrow) and in the ganglion of the lower middle turn. At 12.0kHz, 2-DG uptake was maximal in a discrete region of the extreme basal end of the nerve (arrow), and in the spiral ganglion of the lower basal turn. In each case, no localized increase in 2-DG uptake of the organ of Corti or lateral wall structures was apparent in the autoradiographs. the basal portion of the VIIIth nelve, as shown in Fig. 5. Increased 2-DG uptake is also apparent in the spiral ganglion of the lower basal turn. No localized increase in 2-DG content of the organ of Corti or of the lateral wall structures was noted in any of our pure tone subjects. 2-DG content of the organ of Corti was variable, and bore no relation to the pure tone stimuli.

Quantitation of 2-DG uptake in cochlear tissues Cochlear autoradiographs of control and W B N subjects were quantitated by optical density measurement. Fig. 6 illustrates the locations at which optical densities were measured in a typical cochlear autoradiograph. Optical densities were converted to tissue 2-DG levels with established [14C] and tissue 2-DG standards. Quantitation of glucose utilization in cochlear tissues, as has been employed in brain 26, could not be applied to our cochlear data. The relevant constants for glucose transport into and out of cochlear tissues are not available. Even the route taken by glucose into the organ of Corti has not been established with complete certainty, although it probably reaches the avascular epithelium via perilymph. In order to control for differences in the effective dose of 2-DG to which the cochlea was exposed, 2-DG content of cochlear structures was normalized by dividing the tissue concentration of [14C]2-DG by the integral of the plasmaspecific activity curve, as described in the Methods section, for 3 control and 3 WBN-exposed animals. The results of this analysis for the lower basal turn, the cochlear region in which 2-DG uptake tended to be maximal in all cochlear structures, are shown in Fig. 7. N o significant difference in 2-DG uptake in silence and in WBN was observed in the spiral ligament and stria vascularis, although there did appear to be some increase in the latter structure. A

221

Fig. 6. Locations at which optical density data were collected from a typical autoradiograph. Spiral ligament measurements were made just opposite the point at which the basilar membrane intersects the ligament. Stria vascularis densities were read from the center of the stria vascularis/spiral ligament image at the midpoint of the basal-to-apical extent of the stria. Organ of Corti and spiral ganglion measurements were made from the distinct image of each. VIIIth nerve measurements for each cochlear location were obtained from the midpoint of the closest part of the nerve, la, lower basal turn; Ib, upper basal turn; 2a, lower middle turn; 2b, upper middle turn; 3a, lower apical turn.

marginally significant (P < 0.05) increase in 2 - D G was observed in the organ of Corti, and a highly significant (P < 0.01) increase in the spiral ganglion and V I I I t h nerve. Quantitative analysis of optical densities from higher cochlear turns showed that WBN-induced increases in 2 - D G uptake were substantially higher in the lower basal turn than at more apical cochlear locations. While the increases were smaller more apically, they remained statistically significant (P < 0.05) in all cochlear turns for the spiral ganglion and the VIIIth nerve. This was not the case for the organ of Corti, which showed little or no increase in W B N subjects at cochlear locations higher than the lower base. Uptake of 2 - D G in the spiral ligament was unaffected by W B N exposure, while that of the stria vaseularis showed the same modest but non-significant increase, at all cochlear locations.

222

@

[]

WBN - 8 5 d B SPL

[]

Silence

o-

~_

.~A

t.a tt~

z~

t~

~-Q °V

~v

Zv~q

Fig. 7. Mean 2-DG levels in cochlear tissues of the lower basal turn from inner ears in silence and in 85 dB SPL WBN. Tissue levels have been normalized for each subject by the integral of the plasma specific activity curve. Each mean represents measurements from 6 cochleas, vertical bars show one standard deviation about each mean. Significant increases in 2-DG uptake were noted during WBN in the organ of Corti, spiral ganglion and the VIIIth nerve. DISCUSSION

The major observations of this investigation are that significant uptake of 2 - D G can be demonstrated autoradiographically in the cochlea, and that the pattern of this uptake changes as a result of exposure to sound. Uptake of 2 - D G increases in the spiral ganglion and the V I I I t h nerve during exposure to acoustic stimuli, with the location of increases dependent upon the frequency of exposure. 2 - D G uptake in the organ of Corti also increases during sound exposure, but only in the lower basal turn. Increased 2 - D G uptake in the spiral ganglion during sound exposure is consistent with previous reports of increased uptake in central auditory structures ls,22, 28,29,33. However, increased uptake in the VIIIth nerve represents a more unique observation. In general, myelinated nerve tracts do not increase 2 - D G uptake when stimulatedlO, 27. It should be noted that the VIIIth nerve is unusually vascular 1, suggesting the capacity for a high level of metabolic activity. Also, glutamate and aspartate have been suggested as possible neurotransmitters in primary auditory neurons 34. Increased 2 - D G uptake might thus be related to increased transmitter synthesis during acoustic stimulation. The patterns of increased 2 - D G uptake which occurred during exposure to pure tones are consistent with the tonotopic organization of primary auditory neurons m. It

223 is concluded that autoradiographic demonstration of 2-DG uptake can be used to map functional activity in the neural elements of the inner ear. Significant increases in 2-DG uptake of the organ of Corti were observed during sound exposure only in the lower basal turn. Either the glucose requirements of the more apical organ of Corti do not change during acoustic activation, or our methodology is not sensitive enough to detect changes which occur. Thalmann s2 demonstrated that after extended noise exposure there is no detectable change in ATP, glutamate, aspartate and other substances known to be involved in glucose metabolism, in the outer hair cell legion of the organ of Corti. The observations of Ishii et alp and Schnieder 19 further suggest that glycolytic metabolism may carry the metabolic load during acoustic stimulation in the organ of Corti. However, it should also be noted that glycolytic metabolism decreases in importance, and oxidative metabolism increases in importance, in the organ toward the cochlear baseg, 14. This metabolic gradient may explain why we observed significant WBN-induced increases in 2-DG uptake only in the lower basal turn. In any event, it is clear that with our p~esent methodology 2-DG uptake cannot be used to map the activation of the organ of Corti except perhaps in the high-frequency region of the cochlea. The pattern of 2-DG uptake in non-neural cochlear elements also provides significant information concerning their metabolic lole in cochlear function and auditory transduction. The high level of 2-DG uptake which we observed in stria vascularis, and the lack of a significant change in uptake during WBN exposure, are in agreement with our current understanding of the function of this structure. As the presumed source of the endocochlear resting potential 30 and of the endolymph 7,15, the metabolic requirements of the stria would be expected to be high at all times, and to be relatively unaffected by short-term activation of the auditory receptor. A high rate of 2-DG uptake is also consistent with the rich vascular supply of stria 1, as well as the high levels of metabolic enzymes 14,15 and substrates31, 32 which have been reported in this structure. The high level of 2-DG uptake which we observed in the spiral ligament and spiral prominence was not expected. This finding suggests that these structures play an active metabolic role in cochlear function. Quantitative data concerning metabolic enzymes and substrates are not available for the ligament and prominence. However, anatomical data suggest that at least the spiral prominence may exhibit specialized function 6,~2, and it has been hypothesized by some authors that the prominence acts in concert with the stria to produce the endocochlear potential and/or endolymph 4,12. The close relationship between the uptake of 2-DG in all of the structures of the lateral cochlear wall (see Fig. 4) would suggest that both the spiral ligament and spiral prominence may play an active role in the functions which have previously been attributed primarily to the stria vascularis. ACKNOWLEDGEMENTS

This research was supported by Grants 1K04 NS00176 and 5R01 NS14945 from the NIH/NINCDS. The technical contribution of Delores DiPietro in performing the

224 m i c r o d e n s i t o m e t r y a n d the assistance of R u t h Allen in typing the m a n u s c r i p t are gratefully acknowledged.

REFERENCES 1 Axelsson, A., The vascular anatomy of the cochlea in the guinea pig and man, Acta Otolaryngol., Suppl. 243 (1968). 2 Basinger, S. F., Gordon, W. and Lam, D. M. K., Differential labeling of retinal neurones by aH-2deoxyglucose Nature (Lond.), 280 (1979) 682-684. 3 Bone, R. C. and Ryan, A. F., Energy dispersive X-ray analysis of intracochlear ion shifts produced by anoxia, Laryngoscope, 90 (1980) 1169-1190. 4 Borghesan, E., On the function of the spiral prominence, Acta Otolaryngol., 63 (1967) 161-169. 5 Des Rosiers, M. H., Sakurada, O., Jehle, J., Shinohara, M., Kennedy, C. and Sokoloff, L., Functional plasticity in the immature striate cortex of the monkey shown by the [14C]deoxyglucose method, Science, 200 (1978) 447-449. 6 Duvall, A. J. and Sutherland, C. R., The ultrastructure of the extrasensory cells in the cochlear duct. In M. M. Paparella (Ed.), Biochemical Mechanisms in Hearing and Deafness, Charles C. Thomas, Springfield, IL., 1970, pp. 149-170. 7 Engstrom, H., Sjostrand, F. S. and Spoendlin, J., Feinstruktur der Stria vascularis beim Meerschweinchen, Pract. Otorhinolaryngol., 17 (1955) 69-84. 8 Hubel, D. H., Wiesel, T. N. and Stryker, M. P., Orientation columns in macaque monkey visual cortex demonstrated by the 2-deoxyglucose autoradiographic technique, Nature (Lond.), 269 (1977) 328-330. 9 Ishii, D., Takahashi, T. and Balogh, K., Glycogen in the inner ear after acoustic stimulation, Acta Otolaryngol., 67 (1969) 573-582. 10 Kennedy, C., Des Rosiers, M. H., Jehle, J. W., Reivich, M., Sharp, F. R. and Sokoloff, L., Mapping of functional neural pathways by autoradiographic survey of local metabolic rate with [14C]2-deoxyglucose, Science, 287 (1975) 850-853. 11 Kiang, N. Y.-C., Watanabe, T., Thomas, E. C. and Clark, L. F., Discharge Patterns of Single Fibers in the Cat's Auditory Nerve, Research Monograph 35, MIT Press, Cambridge, MA, 1965. 12 Kikuchi, K. and Hilding, D. A., The development of the stria vascularis in the mouse, Acta Otolaryngok, 62 (1966) 277-292. 13 Landis, W. J., Paine, M. C. and Glimcher, M. J., Use of acrolein vapor for the anhydrous preparation of bone tissue for electron microscopy, J. Ultrastruct. Res., 70 (1980) 171-180. 14 Matschinsky, F. M. and Thalmann, R., Energy metabolism of the cochlear duct. In M. M. Paparella (Ed.), Biochemical Mechanisms in Hearing and Deafness, Charles C. Thomas, Springfield, IL., 1970, pp. 137-147. 15 Nakai, Y., Histochemieal study of the stria vascularis in the inner ear by electron microscopy, Ann. Otolaryngol., 74 (1965) 326-339. 16 Ryan, A. F., Wickham, M. G. and Bone, R. C., Element content of intracochtear fluids, outer hair cells and stria vascularis as determined by energy-dispersive roentgen ray analysis, Otolaryngology, 87 (1979) 659-665. 17 Ryan, A. F., Wickham, M. G. and Bone, R. C., Studies of ion distribution in the inner ear: scanning electron microscopy and X-ray microanalysis of freeze-dried cochlear specimens, Hearing Res., 2 (1980) 1-20. 18 Scheich, H., Bonke, B. A., Bonke, D. and Langer, G., Functional organization of some auditory nuclei in the guinea fowl demonstrated by the 2-deoxyglucose technique, Cell 7~ss. Res., 204 (1979) 17-24. 19 Schnieder, E. A., Die Entstehung des Schalltraumas. Ein Beitrag iiber die Physiologie der Perilymphe, Thesis, Wurzburg, 1970. 20 Sharp, F. R., Rotation-induced increases of glucose uptake in rat vestibular nuclei and vestibulocerebellum, Brain Research, 110 (1976), 141-151. 21 Sharp, F. R., Kauer, J. S. and Shepherd, G. M., Laminar analysis of 2-deoxyglucose uptake in olfactory bulb and olfactory cortex of rabbit and rat, J. NeurophysioL, 40 (1977) 800-813. 22 Sharp, F. R., Ryan, A. F., Goodwin, P. and Woolf, N. K., Increasing intensities of wide band noise increase [14C]2-deoxyglucoseuptake in gerbil central auditory structures, Brain Research, in press.

225 23 Silverman, M. S., Hendrickson, A. E. and Clopton, B. M., Mapping of the tonotopic organization of the auditory system by uptake of radioactive metabolites, Neurosci. Abstr., 3 (1977) 11. 24 Sokolich, W. G., Hamernik, R. P., Zwislocki, J. J. and Schmeidt, R. A., Inferred response polarities of cochlear hair cells, J. Acoust. Soc. Amer., 59 (1976) 963-974. 25 Sokolich, W. G., Some Electrophysiological Evidence for a Polarity-Opposition Mechanism of Interaction between Inner and Outer Hair Ceils in the Cochlea, Ph.D. Thesis, Syracuse University, 1977. 26 Sokoloff, L., Reivich, M., Kennedy, C., DesRosiers, M. H., Patlak, C. S., Pettigrew, K. D., Kakaurda, O., Shinohara, M., The deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat, J. Neurochem., 28 (1977) 13-36. 27 Sokoloff, L., Relation between physiological function and energy metabolism in the central nervous system, J. Neurochem., 29 (1977) 13-26. 28 Stirling, C. E., and Kinter, W. B., High-resolution autoradiography of galactose-[SH]accumulation in rings of hamster intestine, J. Cell Biol., 35 (1967) 585-604. 29 Tanaguchi, I. and Saito, N., Plastic reorganization of the immature mouse studied by the [14C]deoxyglucose method, Proc. Jap. Acad., 54 Ser. B (1978) 496-499. 30 Tasaki, I. and Spyropolous, C. S., Stria vascularis as source of endocochlear potential, J. Neurophysiol., 22 (1959) 249-255. 31 Thalmann, I., Matschinksy, F. M. and Thalmann, R., Quantitative study of selected enzymes involved in energy metabolism of the cochlear duct, Ann. Otolaryngol., 79 (1970) 12-29. 32 Thalmann, R. R., Quantitative biochemical techniques for studying normal and noise-damaged ears. In D. Henderson, R. P. Hamernik, D. S. Dosanjh and J. H. Mills (Eds.), Effects of Noise on Hearing, Raven Press, New York, 1976, pp. 129-155. 33 Webster, W. R., Serviere, J., Batini, C. and LaPlante, J., Autoradiographic demonstration with [14C]deoxyglucose of frequency selectivity in the auditory system of cats under conditions of functional activity. Neurosci. Lett., 20 (1978) 43-48. 34 Wenthold, R. J., Glutamate and aspartate as neurotransmitters for the auditory nerve. In G. D. Chiana and G. L. Gessa, (Eds.), Glutamate as a Neural Transmitter, Advanc. Biochem. Psychopharmacol., Vol. 27, Raven Press, New York, 1981, pp. 69-78.