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Properties of neurons from the rat medial vestibular nucleus in microexplant culture
Neuroscience Letters 338 (2003) 45–48 www.elsevier.com/locate/neulet
Properties of neurons from the rat medial vestibular nucleus in microexplant culture Marle`ne Genlaina, Denis Nonclercqb, Guy Laurentb, Ge´rard Toubeaub, Emile Godauxa,*, Laurence Risa a
Laboratory of Neurosciences, University of Mons-Hainaut, Place du Parc 20, B-7000 MONS, Belgium b Laboratory of Histology, University of Mons-Hainaut, Place du Parc 20, B-7000 MONS, Belgium Received 12 2002; received in revised form 18 November 2002; accepted 19 November 2002
Abstract This study is a first step in an attempt to identify the factors which determine and maintain the electrophysiological phenotype(s) of mature neurons of the medial vestibular nucleus (MVN). We cultured MVN microexplants obtained from slices of the brainstem of newborn rats, using a hollow punching needle. The electrophysiological maturation of the neurons was followed by analyzing their responses to 1 s steps of current of increasing amplitude. The maximal number of spikes that was generated in response to such stimuli increased dramatically over time in vitro. However, even after 28 days in vitro, it did not exceed about 60 spikes/s. At this stage of culture, the input – output properties of the spike generator of the MVN neurons were similar to those observed in brainstem slices of newborn rats, but clearly inferior to those of adult neurons which can generate sustained firing up to 150– 200 spikes/s. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vestibular nucleus; Primary culture; Whole-cell recording; Neuronal development; Immunochemistry; Rat
The vestibular system senses the movement and position of the head in space, and uses this information to stabilize gaze, control posture, and register the orientation of the body. The vestibular signals are generated in the labyrinth, which contains two different types of sensory detectors: the three semicircular canals register angular acceleration of the head in space, the two otolith organs (utricule and saccule) detect linear accelerations. The vestibular signals are relayed mainly at the level of the superior, medial, inferior and lateral vestibular nuclei, each detector having its particular distribution among these nuclei [1]. In mammals, many neurons of the medial vestibular nucleus (MVN) present a spontaneous activity [13], which enables them to work in frequency modulation [19]. This property is favored by the intrinsic membrane properties of the MVN neurons. These cells are able to respond to 1 s steps of intracellularly-injected current by sustained firing, the maximum of which can reach values up to 150 –200 spikes/s [4,8,16,17]. However, such an appropriate electrophysiological phenotype is not complete at birth and is * Corresponding author. Tel.: þ 32-65-373570; fax: þ 32-65-373573. E-mail address: [email protected] (E. Godaux).
achieved only after a few weeks of postnatal development [7,10]. Although primary culture techniques have been used to study the physiological properties of the neurons of the vestibular ganglion (Scarpa’s ganglion) [2,9,11,12], they have not yet been applied to neurons of the vestibular nuclei. Here, in a first step to study the factors which determine and maintain the electrophysiological phenotype of the MVN mature neurons, we developed a culture preparation of MVN neurons and analyzed their electrophysiological maturation for 28 days in vitro. Rat pups (from 1 to 3 days postnatal) were anesthetized with ether and rapidly decapitated. The entire brain was removed. Then, the part formed by the cerebellum and the medulla oblongata was excised with a knife blade and subsequently cut in transverse slices (350 mm) using a vibratome. From two slices from each brainstem, four samples of the vestibular nuclei were removed using a hollow punching needle with an internal diameter of 0.5 mm (Fine Science Tools, Germany) (Fig. 1A). Microexplants were separately plated on round glass coverslips coated with laminin [14] placed in 4-well multidishes (Nunc, Denmark). The growth medium was
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 9 4 0 ( 0 2 ) 0 1 3 5 9 - 9
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M. Genlain et al. / Neuroscience Letters 338 (2003) 45–48
Fig. 1. Changes in the number and in the appearance of cultured MVN neurons with time in vitro. (A) Photograph of a brainstem slice from a newborn rat showing the location of MVN sampling. DCN, dorsal cochlear nucleus. (B) Mean number (^ SD) of surviving neurons around a single microexplant as a function of DIV. (C–F) Changes in the morphology of cultured cells with time. The dendritic tree of the neurons developed and ramified progressively over time. The neurons were specifically immunolabeled with anti-MAP2-ab and anti-NeuN.
Neurobasal (Invitrogen, United Kingdom) containing 125 mM Glutamax (Invitrogen) and 2% B27 (Invitrogen), and supplemented with penicillin (100 U/ml) and streptomycin (100 mg/ml). The microexplants were cultured at 37 8C in a humidified 5% CO2 incubator. Half of the culture medium was changed every 2 days. After 4 days of culture, the microexplants were treated for 24 h with 1 mg/ml 50 -fluoro20 -deoxyuridine (Sigma, US) and 1 mg/ml uridine (Sigma, US) in order to avoid overgrowth of cultures by glial cells. Current stimulation and recording of the membrane potential were obtained using whole-cell technique [6] and an Axoclamp 2B system (Axon Instruments, United States). All electrophysiological experiments were carried out at 32 8C. The external bath solution containing (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2.6H20, 24 glucose and 10 HEPES had an osmolarity of 300 – 310 mOsm and its pH was adjusted to 7.4 with NaOH. Patch electrodes prepared from borosilicate tubing (Hilgenberg, Germany) had a resistance of about 5 MV. The solution in the pipette contained (in mM) 130 K gluconate, 4 NaCl, 1 EGTA, 10 HEPES, 0.3 GTP and 5 Mg2 ATP. Its pH was adjusted to 7.2 with KOH, whereas its osmolarity was adjusted to 290 mOsm with sucrose. Junction potential was corrected after the pipette entered the bath solution. Astrocytes were detected by using a rabbit polyclonal
anti-glial fibrillary acidic protein antibody (anti-GFAP 1/1000; Dakopatts, Denmark). Neurons were identified with a mouse monoclonal anti-microtubule-associated protein 2 (MAP2-ab 1/1000; Chemicon, USA) mixed with a mouse monoclonal anti-neuronal nuclei (anti-NeuN 1/500; Chemicon, USA). The statistical significance of a change in a variable with time was assessed by pooling the data week by week and performing a Kruskall –Wallis test [5]. On a laminin-coated substratum, both neurons and astrocytes migrated outwards from the microexplant. At DIV 3 (3rd day in vitro after plating), the glia tended to form a sheet of flattened cells beneath scattered neurons. From that time, because of the presence of antimetabolites which prevented glial overgrowth, this general pattern was conserved until our latest observations after 28 DIV. The number of neurons surrounding a single microexplant was 89 ^ 28 (mean ^ SD) after 3 DIV. Afterwards it decreased progressively (Kruskall –Wallis test, P , 0:001) (Fig. 1B). After 28 DIV, only 7 ^ 4 neurons remained present in the neighborhood of a microexplant. In contrast to the growing scarcity of the cell population, the surviving neurons appeared to flourish more and more. Their dendritic tree which was scanty after 3 DIV (Fig. 1C), developed progressively over time (Fig. 1D – F) to become large, multipolar and ramified after 21 DIV (Fig. 1F) and later. The cultured neurons were not spontaneously active at any time. Their intrinsic excitability was assessed by submitting the cells to 1 s steps of increasing amplitude. Using such stimuli in slices of adult animals, it was possible to induce sustained firing of action potentials in every vestibular neuron [4,8,16,17]. This was not the case in culture, although all the neurons were electrically excitable. Three electrophysiological phenotypes were observed. In the first, the greatest response obtained from a step was a single or a few action potentials at the beginning of the depolarization ( phasic neurons) (Fig. 2A). In the second type, the neuron fired a single action potential when threshold was reached, whereas increasing the stimulus led to a sustained discharge (tonic neurons) (Fig. 2B, top trace). In the third type, the neuron responded at threshold by a burst of action potentials (Fig. 2C, top trace), whereas it produced regular trains of spikes throughout the current pulse as the latter was increased (burst-tonic neurons) (Fig. 2C, middle trace). As shown in Fig. 2D, the ratios between the three categories changed over time in culture. The less responsive phenotype (phasic neurons) which corresponded to about half of the cell population examined during the first week, was absent among the neurons recorded in the fourth week. Conversely, the most responsive phenotype (bursttonic neurons) which was not yet present during the first week, characterized half of the cells observed in the fourth week. Interestingly, the MVN neurons recorded in brainstem slices from mature animals are either tonic or bursttonic neurons, but never phasic cells [4,8,16,17].
M. Genlain et al. / Neuroscience Letters 338 (2003) 45–48
Fig. 2. Changes in the intrinsic excitability of cultured MVN neurons with time in vitro. (A–C) Examples of the three types of responses evoked by intracellular injection of current in cultured MVN neurons. In each panel, membrane potential is plotted as a function of time in response to intracellular depolarization with current steps (displayed at the very bottom of each panel). The best response obtained with a phasic neuron (A) is one (in this case) or a few action potentials. A tonic neuron (B) can respond by sustained firing for some current intensities (B, top trace) although it can no longer sustain firing throughout the step at higher current intensities (B, bottom trace). At threshold, a burst-tonic neuron responds by a burst of action potentials occurring on the ridge of a transient hump which develops in the beginning of the depolarization (C, top trace). At higher current intensities (C, middle and bottom traces), the behavior of such a neuron resembles that of a tonic cell. (D) Changes in the ratios between the three types of neurons with time in vitro. Notice the progressive disappearance of the phasic cells and the progressive appearance of the burst-tonic neurons. (E) Maximal number of spikes observed in response to a 1 s step of current as a function of days in vitro. The solid line plots the least squares fit of this relationship. Triangles correspond to phasic neurons whereas circles and points correspond to tonic and burst-tonic cells, respectively.
At some level of current, MVN tonic and burst-tonic neurons could no longer sustain firing throughout the step (Fig. 2B,C, bottom traces). The maximum number of spikes generated by the cultured neurons in response to a 1 s step of current increased over time in culture (P , 0:001; Fig. 2E) from 9.1 ^ 8.6 spikes/s (mean ^ SD) (n ¼ 15) in cells recorded during the first week to 40.6 ^ 12.3 spikes/s (n ¼ 10) during the fourth week. Maturation changes were also observed in the characteristics of the individual action potential. The spike threshold determined in this study as the intersection between the linear, rapidly rising phase of the action potential and the gradual depolarizing phase preceding the action potential (Fig. 3A), tended to decrease over time in culture for the whole population of recorded neurons (P , 0:001; Fig. 3B). The width of the spike, measured at its threshold level, decreased over time in culture (P , 0:001; Fig. 3A,C). The amplitude of the action potential, measured from its threshold level to its peak, also changed with culture age (P , 0:001; Fig. 3D). However, the afterhyperpolarization (AHP) amplitude
47
(Fig. 3E), did not change significantly over time (P ¼ 0:53; Fig 3F). Moreover, action potentials were followed by a single AHP and never by a double one, whatever the age of the culture. From a qualitative point of view, it is worth remembering that the phasic phenotype has never been observed in MVN neurons studied in slices either in adult or newborn rats. When MVN neurons of a postnatal pup were cultured, we observed that an initial regression of maturation occurred. About half of the neurons studied during the first week were poorly excitable (phasic neurons). However, 4 weeks after subsequent maturation, the observed phenotypes were the same as those of the adult animal (tonic and burst-tonic). However, quantitatively, this maturation remained incomplete. Although adult MVN neurons can fire faster than 150 spikes/s in response to a broad range of current amplitudes, we observed that cultured MVN neurons could not discharge faster than 60 spikes/s, even after 28 DIV. This limit is similar to that shown by the MVN neurons from newborn rats, when they are tested in slices. Contrary to MVN neurons from adult rodents, cultured neurons remain unable to fire faster than 60/s. Analysis of the bottom of Fig. 2B and Fig. 2C – which shows a typical behavior of cultured MVN neurons – suggests that it is the repolarization capability subsequent to the spike which is deficient. As a result, the imposed depolarization is sustained and provokes inactivation of sodium channels [3]. One possible explanation is that Kv3 channels, a subfamily of high-threshold Kþ channels [15] which are
Fig. 3. Changes in action potential properties of cultured MVN neurons with time in vitro. (A) Membrane potential is plotted as a function of time for two examples of action potentials recorded in one neuron after 5 DIV and in another one after 18 DIV. The interrupted line indicates the threshold level. (B) Threshold is plotted versus culture age. Symbols are as defined in Fig. 2. (C) Width of action potential (measured at threshold level) versus DIV. (D) Amplitude of action potential (measured from threshold level to the peak) versus time in culture. (E) Example of a train of action potentials elicited by a step of current showing how the peak amplitude of the AHP was measured. AHP was measured on the response to the lowest level of a 1 s step of current which was able to elicit action potentials. It was measured after the first action potential, between the threshold level and the nadir subsequent to the spike. (F) AHP peak amplitude is plotted as function of days in vitro.
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M. Genlain et al. / Neuroscience Letters 338 (2003) 45–48
expressed at high concentrations in the adult MVN (unpublished observations quoted in Ref. [10]), are not expressed in cultured neurons. The spontaneous activity in this culture preparation is by far lower than that recorded in the MVN neurons in slices from very young animals. As Siebler et al. [18] have demonstrated in another system that the development of such an activity is dependent upon cell-to-cell contacts, the low density at plating and the cell loss during time in culture could be two of the factors responsible for this deficit. Finally, MVN cells recorded in slices from adult rats and guinea pigs are classified as either ‘type A’ showing a single deep post-spike afterhyperpolarization (AHP), or ‘type B’, showing an early fast AHP followed by a delayed slow AHP [8,17]. Even after 4 weeks in culture, no neuron presents a double AHP. This means either that only type A neurons are thriving in culture, or, more likely, that additional channels, needed to determine the specialization between A and B cells, are not yet expressed. In conclusion, MVN neurons from newborn rats cultured under the conditions described here, after an initial regression, undergo an electrophysiological maturation. However, this remains incomplete. After 28 DIV, this maturation is similar to that achieved by the MVN neurons in vivo at birth, but remains incomplete as compared to that achieved in adult animals. Besides, the progressive maturation observed here contrasts with the progressive decrease in the number of surviving neurons.
Acknowledgements We thank C. Busson for secretarial assistance and realization of the figures. This research was supported by the Belgian National Fund for Scientific Research (NFSR) and the Queen Elisabeth Fund for Medical Research. G.L. and L.R. are Senior Research Associate and Scientific Research Worker of the NFSR, respectively.
References [1] J.A. Bu¨ttner-Ennever, A review of otolith pathways to brainstem and cerebellum, Ann. N.Y. Acad. Sci. 871 (1999) 51 –64. [2] C. Chabbert, J.M. Chambard, A. Sans, G. Desmadryl, Voltage-
activated sodium currents in acutely isolated mouse vestibular neurones, NeuroReport 8 (1997) 1253–1256. [3] A. Erisir, D. Lau, B. Rudy, C.S. Leonard, Function of specific Kþ channels in sustained high-frequency firing of fast-spiking neocortical interneurons, J. Neurophysiol. 82 (1999) 2476–2489. [4] J.P. Gallagher, K.D. Phelan, P. Shinnick-Gallagher, Modulation of excitatory transmission at the rat medial vestibular nucleus synapse, Ann. N.Y. Acad. Sci. 656 (1992) 630– 644. [5] S.A. Glantz, Primer of Biostatistics, Mc Graw-Hill, New York, 1992, pp. 344–353. [6] O.P. Hamill, A. Marty, B. Neher, B. Sakmann, F.J. Sigworth, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pflu¨gers Arch. 391 (1981) 85– 100. [7] A.R. Johnston, M.B. Dutia, Postnatal development of spontaneous tonic activity in mouse medial vestibular nucleus neurones, Neurosci. Lett. 219 (1996) 17–20. [8] A.R. Johnston, N.K. Macleod, M.B. Dutia, Ionic conductances contributing to spike repolarization and after-potentials in rat medial vestibular nucleus neurones, J. Physiol. 481 (1994) 61–77. [9] P.P. Lefebvre, T.R. van de Water, J. Represa, W. Liu, P. Bernd, S. Modlin, G. Moonen, M.B. Mayer, Temporal pattern of nerve growth factor (NGF) binding in vivo and the in vitro effects of NGF on cultures of developing auditory and vestibular neurons, Acta Otolaryngol. 111 (1991) 304– 311. [10] G.J. Murphy, S. du Lac, Postnatal development of spike generation in rat medial vestibular nucleus neurons, J. Neurophysiol. 85 (2001) 1899–1906. [11] D. Rabejac, G. Devau, J. Raymond, AMPA receptors in cultured vestibular ganglion neurons: detection and activation, Eur. J. Neurosci. 9 (1997) 221 –228. [12] D. Rabejac, J. Raymond, C. Dechesne, Characterization of different neuron populations in mouse statoacoustic ganglion cultures, Brain Res. 652 (1994) 249– 256. [13] L. Ris, C. de Waele, M. Serafin, P.-P. Vidal, E. Godaux, Neuronal activity in the ipsilateral vestibular nucleus following unilateral labyrinthectomy in the alert guinea pig, J. Neurophysiol. 74 (5) (1995) 2087–2099. [14] B. Rogister, G. Moonen, Microexplant cultures of the cerebellum, in: S. Fedoroff, A. Richardson (Eds.), Protocols for Neural Cell Culture, Humana Press, Totowa, 2001, pp. 39–48. [15] B. Rudy, C.J. McBain, Kv3 channels: voltage-gated Kþ channels designed for high frequency repetitive firing, TINS 24(9) (2001) 517 –526. [16] C. Sekirnjak, S. du Lac, Intrinsic firing dynamics of vestibular nucleus neurons, J. Neurosci. 22 (6) (2002) 2083–2095. [17] M. Serafin, C. de Waele, A. Khateb, P.-P. Vidal, M. Mu¨hlethaler, Medial vestibular nucleus in the guinea pig: I: intrinsic membrane properties in brainstem slices, Exp. Brain Res. 84 (1991) 417– 425. [18] M. Siebler, H. Ko¨ller, C.C. Stichel, H.W. Mu¨ller, H.J. Freund, Spontaneous activity and recurrent inhibition in cultured hippocampal networks, Synapse 14 (1993) 206 –213. [19] V.J. Wilson, G. Melvill-Jones, Mammalian Vestibular Physiology, Plenum Press, New York, 1979.
Properties of neurons from the rat medial vestibular nucleus in microexplant culture Marle`ne Genlaina, Denis Nonclercqb, Guy Laurentb, Ge´rard Toubeaub, Emile Godauxa,*, Laurence Risa a
Laboratory of Neurosciences, University of Mons-Hainaut, Place du Parc 20, B-7000 MONS, Belgium b Laboratory of Histology, University of Mons-Hainaut, Place du Parc 20, B-7000 MONS, Belgium Received 12 2002; received in revised form 18 November 2002; accepted 19 November 2002
Abstract This study is a first step in an attempt to identify the factors which determine and maintain the electrophysiological phenotype(s) of mature neurons of the medial vestibular nucleus (MVN). We cultured MVN microexplants obtained from slices of the brainstem of newborn rats, using a hollow punching needle. The electrophysiological maturation of the neurons was followed by analyzing their responses to 1 s steps of current of increasing amplitude. The maximal number of spikes that was generated in response to such stimuli increased dramatically over time in vitro. However, even after 28 days in vitro, it did not exceed about 60 spikes/s. At this stage of culture, the input – output properties of the spike generator of the MVN neurons were similar to those observed in brainstem slices of newborn rats, but clearly inferior to those of adult neurons which can generate sustained firing up to 150– 200 spikes/s. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vestibular nucleus; Primary culture; Whole-cell recording; Neuronal development; Immunochemistry; Rat
The vestibular system senses the movement and position of the head in space, and uses this information to stabilize gaze, control posture, and register the orientation of the body. The vestibular signals are generated in the labyrinth, which contains two different types of sensory detectors: the three semicircular canals register angular acceleration of the head in space, the two otolith organs (utricule and saccule) detect linear accelerations. The vestibular signals are relayed mainly at the level of the superior, medial, inferior and lateral vestibular nuclei, each detector having its particular distribution among these nuclei [1]. In mammals, many neurons of the medial vestibular nucleus (MVN) present a spontaneous activity [13], which enables them to work in frequency modulation [19]. This property is favored by the intrinsic membrane properties of the MVN neurons. These cells are able to respond to 1 s steps of intracellularly-injected current by sustained firing, the maximum of which can reach values up to 150 –200 spikes/s [4,8,16,17]. However, such an appropriate electrophysiological phenotype is not complete at birth and is * Corresponding author. Tel.: þ 32-65-373570; fax: þ 32-65-373573. E-mail address: [email protected] (E. Godaux).
achieved only after a few weeks of postnatal development [7,10]. Although primary culture techniques have been used to study the physiological properties of the neurons of the vestibular ganglion (Scarpa’s ganglion) [2,9,11,12], they have not yet been applied to neurons of the vestibular nuclei. Here, in a first step to study the factors which determine and maintain the electrophysiological phenotype of the MVN mature neurons, we developed a culture preparation of MVN neurons and analyzed their electrophysiological maturation for 28 days in vitro. Rat pups (from 1 to 3 days postnatal) were anesthetized with ether and rapidly decapitated. The entire brain was removed. Then, the part formed by the cerebellum and the medulla oblongata was excised with a knife blade and subsequently cut in transverse slices (350 mm) using a vibratome. From two slices from each brainstem, four samples of the vestibular nuclei were removed using a hollow punching needle with an internal diameter of 0.5 mm (Fine Science Tools, Germany) (Fig. 1A). Microexplants were separately plated on round glass coverslips coated with laminin [14] placed in 4-well multidishes (Nunc, Denmark). The growth medium was
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 9 4 0 ( 0 2 ) 0 1 3 5 9 - 9
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M. Genlain et al. / Neuroscience Letters 338 (2003) 45–48
Fig. 1. Changes in the number and in the appearance of cultured MVN neurons with time in vitro. (A) Photograph of a brainstem slice from a newborn rat showing the location of MVN sampling. DCN, dorsal cochlear nucleus. (B) Mean number (^ SD) of surviving neurons around a single microexplant as a function of DIV. (C–F) Changes in the morphology of cultured cells with time. The dendritic tree of the neurons developed and ramified progressively over time. The neurons were specifically immunolabeled with anti-MAP2-ab and anti-NeuN.
Neurobasal (Invitrogen, United Kingdom) containing 125 mM Glutamax (Invitrogen) and 2% B27 (Invitrogen), and supplemented with penicillin (100 U/ml) and streptomycin (100 mg/ml). The microexplants were cultured at 37 8C in a humidified 5% CO2 incubator. Half of the culture medium was changed every 2 days. After 4 days of culture, the microexplants were treated for 24 h with 1 mg/ml 50 -fluoro20 -deoxyuridine (Sigma, US) and 1 mg/ml uridine (Sigma, US) in order to avoid overgrowth of cultures by glial cells. Current stimulation and recording of the membrane potential were obtained using whole-cell technique [6] and an Axoclamp 2B system (Axon Instruments, United States). All electrophysiological experiments were carried out at 32 8C. The external bath solution containing (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2.6H20, 24 glucose and 10 HEPES had an osmolarity of 300 – 310 mOsm and its pH was adjusted to 7.4 with NaOH. Patch electrodes prepared from borosilicate tubing (Hilgenberg, Germany) had a resistance of about 5 MV. The solution in the pipette contained (in mM) 130 K gluconate, 4 NaCl, 1 EGTA, 10 HEPES, 0.3 GTP and 5 Mg2 ATP. Its pH was adjusted to 7.2 with KOH, whereas its osmolarity was adjusted to 290 mOsm with sucrose. Junction potential was corrected after the pipette entered the bath solution. Astrocytes were detected by using a rabbit polyclonal
anti-glial fibrillary acidic protein antibody (anti-GFAP 1/1000; Dakopatts, Denmark). Neurons were identified with a mouse monoclonal anti-microtubule-associated protein 2 (MAP2-ab 1/1000; Chemicon, USA) mixed with a mouse monoclonal anti-neuronal nuclei (anti-NeuN 1/500; Chemicon, USA). The statistical significance of a change in a variable with time was assessed by pooling the data week by week and performing a Kruskall –Wallis test [5]. On a laminin-coated substratum, both neurons and astrocytes migrated outwards from the microexplant. At DIV 3 (3rd day in vitro after plating), the glia tended to form a sheet of flattened cells beneath scattered neurons. From that time, because of the presence of antimetabolites which prevented glial overgrowth, this general pattern was conserved until our latest observations after 28 DIV. The number of neurons surrounding a single microexplant was 89 ^ 28 (mean ^ SD) after 3 DIV. Afterwards it decreased progressively (Kruskall –Wallis test, P , 0:001) (Fig. 1B). After 28 DIV, only 7 ^ 4 neurons remained present in the neighborhood of a microexplant. In contrast to the growing scarcity of the cell population, the surviving neurons appeared to flourish more and more. Their dendritic tree which was scanty after 3 DIV (Fig. 1C), developed progressively over time (Fig. 1D – F) to become large, multipolar and ramified after 21 DIV (Fig. 1F) and later. The cultured neurons were not spontaneously active at any time. Their intrinsic excitability was assessed by submitting the cells to 1 s steps of increasing amplitude. Using such stimuli in slices of adult animals, it was possible to induce sustained firing of action potentials in every vestibular neuron [4,8,16,17]. This was not the case in culture, although all the neurons were electrically excitable. Three electrophysiological phenotypes were observed. In the first, the greatest response obtained from a step was a single or a few action potentials at the beginning of the depolarization ( phasic neurons) (Fig. 2A). In the second type, the neuron fired a single action potential when threshold was reached, whereas increasing the stimulus led to a sustained discharge (tonic neurons) (Fig. 2B, top trace). In the third type, the neuron responded at threshold by a burst of action potentials (Fig. 2C, top trace), whereas it produced regular trains of spikes throughout the current pulse as the latter was increased (burst-tonic neurons) (Fig. 2C, middle trace). As shown in Fig. 2D, the ratios between the three categories changed over time in culture. The less responsive phenotype (phasic neurons) which corresponded to about half of the cell population examined during the first week, was absent among the neurons recorded in the fourth week. Conversely, the most responsive phenotype (bursttonic neurons) which was not yet present during the first week, characterized half of the cells observed in the fourth week. Interestingly, the MVN neurons recorded in brainstem slices from mature animals are either tonic or bursttonic neurons, but never phasic cells [4,8,16,17].
M. Genlain et al. / Neuroscience Letters 338 (2003) 45–48
Fig. 2. Changes in the intrinsic excitability of cultured MVN neurons with time in vitro. (A–C) Examples of the three types of responses evoked by intracellular injection of current in cultured MVN neurons. In each panel, membrane potential is plotted as a function of time in response to intracellular depolarization with current steps (displayed at the very bottom of each panel). The best response obtained with a phasic neuron (A) is one (in this case) or a few action potentials. A tonic neuron (B) can respond by sustained firing for some current intensities (B, top trace) although it can no longer sustain firing throughout the step at higher current intensities (B, bottom trace). At threshold, a burst-tonic neuron responds by a burst of action potentials occurring on the ridge of a transient hump which develops in the beginning of the depolarization (C, top trace). At higher current intensities (C, middle and bottom traces), the behavior of such a neuron resembles that of a tonic cell. (D) Changes in the ratios between the three types of neurons with time in vitro. Notice the progressive disappearance of the phasic cells and the progressive appearance of the burst-tonic neurons. (E) Maximal number of spikes observed in response to a 1 s step of current as a function of days in vitro. The solid line plots the least squares fit of this relationship. Triangles correspond to phasic neurons whereas circles and points correspond to tonic and burst-tonic cells, respectively.
At some level of current, MVN tonic and burst-tonic neurons could no longer sustain firing throughout the step (Fig. 2B,C, bottom traces). The maximum number of spikes generated by the cultured neurons in response to a 1 s step of current increased over time in culture (P , 0:001; Fig. 2E) from 9.1 ^ 8.6 spikes/s (mean ^ SD) (n ¼ 15) in cells recorded during the first week to 40.6 ^ 12.3 spikes/s (n ¼ 10) during the fourth week. Maturation changes were also observed in the characteristics of the individual action potential. The spike threshold determined in this study as the intersection between the linear, rapidly rising phase of the action potential and the gradual depolarizing phase preceding the action potential (Fig. 3A), tended to decrease over time in culture for the whole population of recorded neurons (P , 0:001; Fig. 3B). The width of the spike, measured at its threshold level, decreased over time in culture (P , 0:001; Fig. 3A,C). The amplitude of the action potential, measured from its threshold level to its peak, also changed with culture age (P , 0:001; Fig. 3D). However, the afterhyperpolarization (AHP) amplitude
47
(Fig. 3E), did not change significantly over time (P ¼ 0:53; Fig 3F). Moreover, action potentials were followed by a single AHP and never by a double one, whatever the age of the culture. From a qualitative point of view, it is worth remembering that the phasic phenotype has never been observed in MVN neurons studied in slices either in adult or newborn rats. When MVN neurons of a postnatal pup were cultured, we observed that an initial regression of maturation occurred. About half of the neurons studied during the first week were poorly excitable (phasic neurons). However, 4 weeks after subsequent maturation, the observed phenotypes were the same as those of the adult animal (tonic and burst-tonic). However, quantitatively, this maturation remained incomplete. Although adult MVN neurons can fire faster than 150 spikes/s in response to a broad range of current amplitudes, we observed that cultured MVN neurons could not discharge faster than 60 spikes/s, even after 28 DIV. This limit is similar to that shown by the MVN neurons from newborn rats, when they are tested in slices. Contrary to MVN neurons from adult rodents, cultured neurons remain unable to fire faster than 60/s. Analysis of the bottom of Fig. 2B and Fig. 2C – which shows a typical behavior of cultured MVN neurons – suggests that it is the repolarization capability subsequent to the spike which is deficient. As a result, the imposed depolarization is sustained and provokes inactivation of sodium channels [3]. One possible explanation is that Kv3 channels, a subfamily of high-threshold Kþ channels [15] which are
Fig. 3. Changes in action potential properties of cultured MVN neurons with time in vitro. (A) Membrane potential is plotted as a function of time for two examples of action potentials recorded in one neuron after 5 DIV and in another one after 18 DIV. The interrupted line indicates the threshold level. (B) Threshold is plotted versus culture age. Symbols are as defined in Fig. 2. (C) Width of action potential (measured at threshold level) versus DIV. (D) Amplitude of action potential (measured from threshold level to the peak) versus time in culture. (E) Example of a train of action potentials elicited by a step of current showing how the peak amplitude of the AHP was measured. AHP was measured on the response to the lowest level of a 1 s step of current which was able to elicit action potentials. It was measured after the first action potential, between the threshold level and the nadir subsequent to the spike. (F) AHP peak amplitude is plotted as function of days in vitro.
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expressed at high concentrations in the adult MVN (unpublished observations quoted in Ref. [10]), are not expressed in cultured neurons. The spontaneous activity in this culture preparation is by far lower than that recorded in the MVN neurons in slices from very young animals. As Siebler et al. [18] have demonstrated in another system that the development of such an activity is dependent upon cell-to-cell contacts, the low density at plating and the cell loss during time in culture could be two of the factors responsible for this deficit. Finally, MVN cells recorded in slices from adult rats and guinea pigs are classified as either ‘type A’ showing a single deep post-spike afterhyperpolarization (AHP), or ‘type B’, showing an early fast AHP followed by a delayed slow AHP [8,17]. Even after 4 weeks in culture, no neuron presents a double AHP. This means either that only type A neurons are thriving in culture, or, more likely, that additional channels, needed to determine the specialization between A and B cells, are not yet expressed. In conclusion, MVN neurons from newborn rats cultured under the conditions described here, after an initial regression, undergo an electrophysiological maturation. However, this remains incomplete. After 28 DIV, this maturation is similar to that achieved by the MVN neurons in vivo at birth, but remains incomplete as compared to that achieved in adult animals. Besides, the progressive maturation observed here contrasts with the progressive decrease in the number of surviving neurons.
Acknowledgements We thank C. Busson for secretarial assistance and realization of the figures. This research was supported by the Belgian National Fund for Scientific Research (NFSR) and the Queen Elisabeth Fund for Medical Research. G.L. and L.R. are Senior Research Associate and Scientific Research Worker of the NFSR, respectively.
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