366 Brain Research, 460 (1988) 36(~ 368 Elsevier BRE 23100

Putative in the rat at several ages

Douglas W. Hoffman 1, Kitty Lea Jones-King 1 and Richard A. Altschuler 2 INeurochemistry Laboratory. Departments of Psychiatry and Pharmacology, Dartmouth Medical School, Hanover, NH ( U. S. A. ) and 2Kresge Research Institute, University of Michigan, Ann Arbor, MI (U.S.A.) (Accepted 31 May 1988)

Key words: Cochlea; Aging; Presbycusis; Enkephalin; Dynorphin;

We have performed a longitudinal study of the content of the putative neurotransminer substances, enkephalin, dynorphin, and acetylcholine (ACh), in the cochlea of the Fischer 344 rat. It is the first study of transmitters in the rat cochlea over an extended time span. This study also provides biochemical verification of the presence of ACh in cochlear tissues. No change was seen in the cochlear content of these transmitter candidates up to 24 months of age.

In the past several years it has become clear that olivocochlear efferent fibers innervating the coch- there are anatomically and chemically distinct effer- lea 1-7'9~12'16"18-2°'23. These include enkephalins, dyn- ent systems innervating the cochlea; the lateral sys- orphins and neo-endorphins. Opioid receptors as tem, which terminates on eight nerve dendrites un- well have been reported in the cochlea m'2°. Enke- der inner hair cells, and the medial, which directly in- phalin- and dynorphin-like immunoreactivities have nervates outer hair cells 14'30~31. The lateral efferents also been seen in the same cells in the superior oli- of species studied to date are known to contain enke- vary region 1,7, as have opioid peptide-like and cho- phalins, dynorphins, calcitonin gene-retated peptide line acetyltransferase-like immunoreactivity 1,3, and and acetylcholine (ACh), while in the medial system enkephalin-like immunoreactivity and acetylcholin- only ACh has so far been identified as a putative esterase activity 5. transmitter substance i- ~2,1~.IX 2023.24. Acetylcholine and opioid peptides appear to serve A considerable body of evidence has been devel- as neurotransmitters or neuromodulators of the su- oped supporting a role for acetylcholine as the trans- perior olivary fibers in the cochlea. The manner in mitter of the efferent fibers arising in the superior oli- which they may interact with each other, and the rea- vary region which innervate the cochlea. Acetylcho- sons for their co-localization at these , have line has been identified by bioassay in one mamma- not yet been elucidated. In the course of studying lian cochlea 2s, and enzymes related to the synthesis these neuroactive substances we have determined and degradation of ACh have been similarly local- their levels in the rat cochlea at different stages in the ized by histochemical and biochemical means 1'3 life span. Fischer 344 rats at 3, 12 and 24 months of s.13.2~. Acetylcholine has also been shown to mimic in age were used, representing the mature adult, old certain respects electrical stimulation of the cochlear adult, and the extreme of the normal life span, re- efferents s. spectively. Both proenkephalin- and prodynorphin-derived The combined high-performance liquid chroma- peptides have been identified in cochlear tissues and tography-radioimmunoassay (HPLC-RIA) for en- fluids, and have been localized by immunocytochem- kephalins has been described in earlier publica- ical, histochemical and biochemical techniques to the tions 17"18. Whole were sonicated in 7.5 mM

Correspondence: D.W. Hoffman, Department of Psychiatry-HB7770, Dartmouth Medical School, Hanover, NH 03756, U.S.A.

0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division) 367 trifluoroacetic acid in 25% acetonitrile and centri- TABLE I fuged. Supernatants were either chromatographed Levels of putative substances at different ages for enkephalins and dynorphin, or lyophilized direct- in the rat cochlea ly in a vacuum centrifuge. Dynorphin B chromato- Values are mean _+ S.E.M., expressed in fmol/cochlea. graphed as a single sharp peak using the same HPLC n 3 Months 12 Months 24Months method as used for enkephalins. Dynorphin was as- sayed as dynorphin B (rimorphin) using a commer- Enkephalin 5 675 _+ 95 733 + 129 620 _+ 211 Dynorphin 5 54 _+ 6 63 + 4 52 + 7 cially available kit (Peninsula Labs). This antiserum Acetylcholine 5 1340 + 231 1290_+398 1497 + 435 does not cross-react with other dynorphins, endor- phins or dynorphins, and so results were the same whether or not samples were chromatographed prior rat a less desirable model for studying neural mecha- to assay. Acetylcholine was assayed using the radio- nisms of auditory aging 15. chemical method of McCaman and Stetzler 25 as mod- The functions of these olivocochlear fiber systems ified by Marchi et al. 26. are not clear at this time, in spite of much research, The results are summarized in Table I. These data but are known to involve some inhibitory activity demonstrate the presence of Met-enkephalin, dyn- (see review by Wiederhold32). Nieder and Nieder 27 orphin B and ACh in the rat cochlea. No significant reported that efferent stimulation increases the abili- changes in levels of these putative transmitter sub- ty to detect sound stimuli in a ltoisy background, by stances are seen at the different ages studied. increasing the signal:noise ratio. This discriminative Enkephalin-like, dynorphin-like and choline ace- ability rapidly declines with age, and is greatly im- tyltransferase-like immunoreactivities have pre- paired in neural presbycusis. It is well known that viously been reported to be co-localized in lateral oli- hair cells in many species decline in number or dem- vocochlear cell bodies in the rat 1. Although 3 classes onstrate damage with increasing age 15'22. However, of putative neurotransmitters are identified in the rat nothing is known to date about anatomical changes in cochlea in this study, no change in their levels with the olivocochlear efferent neurons in aged animals of age was seen. As auditory testing was not performed any species, although such information would be of on these animals, it is possible that a deficit in audito- great value in understanding the expression in the au- ry perception and cochlear transmitters may occur ditory system of the aging process. together at ages later than those studied. losses have been reported to increase in the rat coch- This work has been supported by grants to D.W.H. lea at even later ages 22. However, aging changes in from the National Institutes of Health (AG05499 and auditory perception have been reported in Fischer EY06180) and the National Science Foundation 344 rats with an average age of 25 months ~9, which is (BNS8646563). Cochleas were provided by Dr. Jef- the normal lifespan of these animals. Also, some pro- frey Chesky of the Gerontology Program, Sangamon nounced differences in the aging of the rat and hu- State University, with support from the National In- man cochleas have been noted, which may make the stitutes of Health to Dr. Chesky.

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