Adaptation of Mechanoelectrical Transduction in Hair Cells of the Bullfrog’S Sacculus

The Journal of Neuroscience, September 1987, 7(9): 2821-2836 Adaptation of Mechanoelectrical Transduction in Hair Cells of the Bullfrog’s Sacculus FL A. Eatock,‘B2 D. P. Corey,‘131a and A. J. Hudspeth1.4.b ‘Division of Biology, California Institute of Technology, Pasadena, California 91125; ‘Eaton-Peabody Laboratory of Auditory Physiology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114; 3Neuroscience Group, Howard Hughes Medical Institute and Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts 02114; and 4Department of Physiology, University of California School of Medicine, San Francisco, California 94143 Adaptation in a vestibular organ, the bullfrog’s sacculus, was cess in the function of the frog’s sacculus as a very sensitive studied in viva and in vitro. In the in vivo experiments, the vibration detector is discussed. discharge of primary saccular neurons and the extracellular response of saccular hair cells were recorded during steps The term “sensory adaptation” is applied to a wide variety of of linear acceleration. The saccular neurons responded at phenomena. In a broad sense,it can be defined as any decrease the onset of the acceleration steps, then adapted fully within with time in a sensory responseto a constant input. Such re- 1 O-50 msec. The extracellular (microphonic) response of the sponsedecays occur via diverse mechanisms,including refrac- hair cells adapted with a similar time course, indicating that toriness in spike generation, decreasedsynaptic gain, changein the primary sources of the neural adaptation are peripheral the sensitivity of a transduction process,and reduction of the to the afferent synapse-in the hair cell, its mechanical in- input to receptor cells. Responsedecays during constant stimuli puts, or both. Evidence for hair cell adaptation was provided are often seenin afferent neurons innervating organsof the inner by 2 in vitro preparaiions: after excising the sacculus and ear, where they are usually attributed to decay of the mechanical removing the accessory structures, we recorded either the input to the sensory hair cells or to processesat the afferent extracellular hair cell response to displacement of the oto- synapse.In this paper we report a different kind of adaptation lithic membrane or the intracellular hair cell response to hair in an inner-ear organ: hair cells in the sacculusof the bullfrog bundle displacement. In both cases the response to a step adapt through a processthat resets the range of sensitivity of stimulus adapted. The adaptation involved a shift in the dis- the mechanoelectricaltransducer. placement-response curve along the displacement axis, so The sacculus(Figs. 1, 2) is an otolithic organ. The character- that the cell’s operating point was reset toward the static istic feature of such organs is the otoconial mass,an aggregate position of its hair bundle. This displacement shift occurred of calcareous crystals (otoconia) within the endolymphatic in response to both depolarizing and hyperpolarizing stimuli. chamber. An otolithic membrane couples the otoconial mass Its time course varied among cells, from tens to hundreds to the sensory epithelium, the macula, which comprises hair of milliseconds, and also varied with the concentration of cells and supporting cells. The hair bundles of the hair cells Ca*+ bathing the apical surfaces of the hair cells. Voltage- protrude from the endolymphatic surfaceof the epithelium and clamp experiments suggested that the displacement shift are connected by strands to the otolithic membrane. The hair does not depend simply on ion entry through the hair cell’s cells are innervated on their basolateralsurfaces by both afferent transduction channels and can occur at a fixed membrane and efferent nerve fibers. Electrophysiological studies in several potential. The possible role of the displacement-shift pro- speciesshow that otolithic afferents respond to linear acceler- ations (for a review, see Goldberg and Fernandez, 1975). A Received Oct. 27, 1986; revised Feb. 20, 1987; accepted Mar. 10, 1987. widely accepted model of the mechanical behavior of otolithic This research was supported by National Institutes of Health Grants NS- 13 154 organs is based on the observation that the otoconial massis and NS-20429 (to A.J.H.), NS-22059 (to D.P.C.), GM-02031 and GM-07616 (to denser than the surrounding fluid and can move within that the California Institute of Technology); by fellowships from the Natural Sciences and Engineering Research Council of Canada, the Medical Research Council of fluid (Young, 1969; Goldberg and Femandez, 1975). The model Canada, and the Gordon Ross Medical Foundation (to R.A.E.); and by grants proposes that when the animal undergoeslinear acceleration, from the William Randolnh Hearst Foundation. the Pew Memorial Trust. the Alfred P. Sloan Foundation, and the Howard Hughes Medical Institute. We thank the position of the otoconial masslags behind that of the sur- Richard Jacobs for excellent and good-natured technical assistance, John Maunsell rounding fluid and tissues. The lag deflects the hair bundles, and Andy Moiseff for computer assistance, and Wendy Smith for participating in which are coupled to the otoconial massby the otolithic mem- the experiments of Figures 8 and 11. We thank Aniruddha Das, Joe Howard, Walter Koroshetz, Villu Maricq, John Maunsell, Aline McKenzie, Erich Phillips, brane. Hair bundle deflection modulates the hair cell’s conduc- and Bill Roberts for comments on the manuscriot. tance; the resulting receptor current produces a receptor poten- Correspondence should be addressed to R. A. Eatock, Department ofPhysiology, tial (Hudspeth and Corey, 1977). University of Rochester Medical Center, Box 642, 601 Elmwood Avenue, Roch- ester, NY 14642. In this paper we show that the spike activity in primary sac- a Present address: Neuroscience Group, Howard Hughes Medical Institute and cular neurons adapts during a maintained linear acceleration. Department of Neurology, Massachusetts General Hospital, Boston, MA 02114. b Present address: Department of Physiology, University of California School Such adaptation could be due to any of several mechanisms of Medicine, San Francisco, CA 94 143-0444. (Fig. lB), including negative feedback mediated by efferents, Copyright 0 1987 Society for Neuroscience 0270-6474/87/092821-16$02.00/O spike refractoriness, synaptic adaptation, decay of the hair cell 2822 Eatock et al. Adaptation by Hair Cells A Reduction of . Saccular nerve mechanical input Extracellular Mechanical relaxation microphonic currents within hair bundle and potentials Activation of voltage- dependent conductances Receptor currents Fatigue of transmitter release Receptor potentials Postsynaptic desensitization Postsynaptic potentials Spike adaptation Afferent-nerve Endolymph action potentials Efferent activity Figure 1. A, Schematic cross section through the entire sacculus. The otoconial mass is coupled to hair bundles by the perforated otolithic membrane. One of the roughly 3000 hair cells in the saccular macula is indicated by a box. B, Higher-magnification diagram of a hair cell innervated by an afferent and an efferent fiber. The labels to the left and right indicate, respectively, the possible sites of adaptation in this organ and the different types of recording that were made in this study. receptor potential or receptor current, and relaxation of the of this region suggested that most of these recordings were from fibers, mechanical input to the hair cells. We examine these possibil- but that some may have been from cell bodies. Before surgery, the bullfrog was anesthetized by immersion for about ities by presenting data recorded from stagesantecedent to spike 30 min in 6 mM m-aminobenzoic acid ethyl ester methane sulfonate generation: the synaptic potentials in the afferent terminals, ex- (Tricaine; Calbiochem, San Diego, CA). During surgery and recording, tracellular hair cell responses(the microphonic current and mi- the animal’s skin was kept moist with a gauze pad soaked in the an- crophonic potential), and intracellular hair cell responses(the esthetic solution. It was often necessary to supplement the anesthesia receptor current and receptor potential). We show that the ad- with up to 100 ~1 of sodium pentobarbital (Nembutal; Abbott Labo- ratories, North Chicago, IL, 50 pm/liter) injected intraperitoneally in aptation involves a shift of the sensitive range of the mechano- aliquots no greater than 30 ~1. electrical transduction processin the hair cells. Some of these The saccular macula and the saccular nerve lie ventrally within the data have beenreported in preliminary form (Eatock et al., 1979; otic capsule and are exposed easily through the roof of the mouth. In Eatock and Hudspeth, 1981; Corey and Hudspeth, 1983a). over half of the experiments we used this ventral surgical approach and the frog remained supine during recording. To test whether saccular responses are affected by the animal’s position, we used a dorsal surgical Materials and Methods approach in 13 of the intrasaccular experiments and 9 of the nerve The experimental animals were American bullfrogs, Ram cutesbeiuna, experiments, and recorded with the animal prone. Because the results weighing 75-150 gm. Unless otherwise indicated, all surgery and ex- of these experiments did not differ from those obtained with the ventral periments were conducted at 20-24°C. approach, we have pooled the data. In vivo experiments. Anesthetized frogs were stimulated with linear Following

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