The work was supported by grants from the DGICYT PB9610087, PB82/0268 and Junta de Castilla y León, Consejería de Bienestar. CJ and LN are recipients of fellowships from Eusko Jaurlaritza and DGICYT, respectively.Peer Reviewe

Jiménez, Cristina

Núñez, Lucía

Digital.CSIC

De\'e/upmeillaillellrohi%gy 159SGLUTAMATE RECEPTORS IN THE DEVELOPING COCHLEAR GANGLIONCristina JIMENEZ and Lucia NUNEZInstituto de 8iologla y Genetica Molecular (18GM)Facuftad de Medicina. Universidad de Valladolid-CSICValladolid, SpainThe cochlear ganglion contains the primary afferent neurones of the vertebrate auditory system and it constitutes the first synaptic relayafter sound transduction. The developmental expression of voltage-activated ionic channels in chick cochlear neurones has been describedin some detail (Vaiverde et aI., 1992; Jimenez et aI., 1996), however, little is known about the expression of ligand operated channels, Mostevidenceindicatesthat glutamate is the neurotransmitter between transducing hair cells and the cochlear neurones (see Eybalin, 1993 fora review), We have studied the developmental pattern of expression of the response of cochiear neurones to giutamate by analyzingintracelluiar Ca" transients with microfiuorescence methods,Cochlear neurones were isolated from chick embryos between days 6 and 11 of incubation (E-6, E-11). For fiuorescence measurements,the cel!-coated coverslips were washed with standard medium containing (in mM) : NaCl, 140; KCI, 5; MgCl" 1; CaCi" 1; glucose, 10; sodium-HEPES, 10, pH, 7.4, and loaded with fura-2 by incubation with 5 ~M fura-2/AM at room temperature for 1 h. The value of intracellular freeCa" concentration was estimated from the ratio of the fiuorescence values excited at 340 and 380 nrn (Grynkiewicz et aI., 1985), Calibrationwas performed by comparison with fura-2 standards. For single-cell measurements, the coverslips coated with fura-2-loaded cells weremounted under a microscope (Nikon Oiaphot) in a chamber thermostated at 36'C and epi-iluminated alternately at 340 and 380 nm. Lightemitted above 520 nm was recorded by an extended ISIS-M camera (Photonic Science, Robertbridge, UK) and analysed using an AppliedImaging Magical image processor (Sunderland, UK). For routine expe~iments, four video frames of each wavelength were averaged byhardware, with an overall time resolution of about 3 see for each pair of images at alternate wavelengths.Cell depolarisation with high K' (50 mM) induced an increase inthe concentration of intracelluiar Ca" (Fig, 1, traces iabeHed K').The response consisted of a rapid and transient component thatreached a peak in about 15 sec, to relax to a steady-state valuethroughout the K'-pulse. This response was similar to thatdescribed in most neuronal populations and refiects most probablythe infiux of Ca" through voltage-dependent Ca"-channels(Nunez et aI., 1996). The early expression of type N and type LCa2+ -channels in cochlear neurones has been recentlydocumented by Jimenez et ai., (1996). The response to high K'was used to asses the viability of the neurones in the preparation.The experimentwas accepted only if at least 50% of the neuronesresponded to high K'. The fraction of ceHs responding to high K',between developmental stages E-6 and E-11 was from 80 to 85%.This fraction did not change for the different developmentaistages, indicating that cell viability was not dependent on the stageselecte.i for the study. The increase in intracellular Ca2+concentr,'tion induced by high K' was between 250 and 400 nMand did not show a particular trend for the different developmentalstages.Alter testing responses to high-K', cells were superfused witha solution containing 100 ~M glutamate and 10 ~M glycine. Theresponse elidted by glutamate was similar to that evoked by K' butcf smaller ampl~ude. The fraction of cells responding to both, highK' "nd glutamate was between 40 and 80% (62f7%, n;91, in E-6and 6O:t5%, n=46, in E-11). The increase in the concentration ofCa2+ in~'Jced by glutamate was about 200-300 nM and was similarthroughol..:t development. The response to glutamate was presentas soon as c-6 in the development of cochlear neurones.Glutamate receptors in the mature cochiear neurones are of the NMDA type (Yamaguchi & Ohmori, 1990). In order to make a preliminarycharactenzation of early occurnng glutamate receptors, ceHs were isolated from E-7 and E-8 embryos and challenged with a 100 ~M NMOAsoiution, At both stages the majority of neurones responding to K' and glutamate, responded also to NMDA Average values were 78f6%(n;4) for E-7and 93f2% (n;4) for E-8. Therefore, early glutamate receptors were, like in mature neurones, of the NMOA type.Neurotrophic factors are known to allow survival and differentiation of target neurones, within the cochlear neurones pool. The NGF-familyof neurotrophlns modulate the expres510n of intermediate filament molecules (San Jose et al., 1996) and Ca" ionic channels (Jimenez et aI.,1096). We decIded to study whether neurotrophlns were also able to modulate glutamate receptor expression. Cells were isolated from E-7cOG~learganglions and cuHured for 48 h in the presence of 2 nplml NT-3. Neurones cuHured w~h NT-3 developed typical neuriies, whereasmost of control neurones did not survive, Responses to high K and glutamate were analyzed as described above, The fraction of neuronescultured vvth NT-3 that responded to high K' in these experiments was 94f2%, n;117. From these cells, 9H3%, n;106, responded alsoto glutamate. These figures are higher than the m;""mum v~iues measured from freshly isolated neurones (see above). Cells rescued by NT-3,therefore, were excitable, responded to hlgh-K vvth Ca transients, and also expressed functional glutamate receptors.5001 min400200300100oFigure 1. Increase of [Ca21induced by depolarization with high-K+ (SO mM)solution. The trace shown is the average of 88 neurons present in the samemicroscope field.1000800:>:~"600"1 min~;':""'NO 4002.2000NMDA GW'"160S f)e~'eI()p1l1e/lt{l1 nellrfJbi%gyThe results show the early expression of glutamate receptors indeveloping cochlear neurones. These receptors are of the NMDAtype and they are rescued, along with cell survival, by theneurotrophin NT-3. It is not yet possible to establish the preciserelation betvveen NT -3 and the functional expression of glutamatereceptors. NT-3 may regulate directly the expression of glutamatereceptors but it may be also possible that the effect is not specificon receptor expression regulation but secondary to cell survival.The phenotype of cochlear neurones may be highly specified atthe stage when they adquire their dependence of neurotrophinsand the action of NT-3 could be only to allow survival and, as aconsequence, the development of their differentiation program.At stage E-7, when we first detect glutamate receptors,connections betvveen hair cells and cochlear neurones do not yetexist. We face a situation where the expression of the physiologicalneurotransmitter anticipates the establishment of the actualsynaptic communication. This is interesting because it suggeststhat glutamate may have a role in the innervation of the cochlea,which is not synaptically mediated. During recent years it has beenshow that glutamate displays pleiotropic responses, including.trophic and plastic effects on developing neurones (McDonald &Johnston, 1990). Our results suggests that this may be the casein cochlear neurones. We propose that both glutamate and NT-3may act as target-derived signals and interact to regulate thesurvival and differentiation of cochlear neurones.Figure 2. Response of cochlear neurons to sequential stimulation withNMDA (1 ()() ~M), L-glutamate (1()() ~M) and high-K- (50 mM) solutions.The trace shown is the average of 47 single neurons present in the samemicroscope field and selected by responding to all the three stimuli.ReferencesEybalin, M. (1993). Neurotransmitters and neuromodulators of Ihe mammalian cochlea. Physiol. Rev. 73: 309-373.Jimenez,C., Giraldez, F., Represa, J. and Garcia-Diaz, F. (1996). Calcium currents in dissociated cochlear neurons from the chick embryo and their modification by neurotrophin-3. Neuroscience. In press.McDonald, J.W.and Johnston, M.V.(1990). Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res. Rev.15: 41-70.Nunez, l., Sanchez, A., Fonteriz, R.I. and Garcia-Sancho, J. (1996). Mechanisms for synchronous calcium oscillations in cultured rat cerebellar neurons. Eur. J. Neurosci. 8:192.201.San Jose, I., Vazquez, E., Garcia-Ataref, N., Rodriguez, F., Vega, JA. and Represa, J. (1996). Expression of the cytoskeletal protein MAP-5 and his regulation by neurolrophin-3 in the inner ear sensory neurons. Anatomy and Embryology. In press.Valverde, MA, Sheppard, D.N., Represa, J. and Girt!ddez, F. (1992). Development of Na' and K' currents in cochlear ganglion of the chick embryo. Neuroscience. 51: 621-630.Acknowledgements: The work was supported by grants from the DGICYT PB9610087, PB82/0268 and Junta de Castilla y Le6n, Consejeriade Bienestar. CJ and LN are recipients of fellowships from Eusko Jaurlaritza and DGICYT, respectively. We like to thank Javier GARCiA-SANCHO & Fernando GIRALDEZ for helpful discussions and encouragement.

Glutamate receptors in the developing cochlear ganglion.

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