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Postnatal developmental changes in the responses of mouse primary vestibular neurons to externally applied galvanic currents
Developmental Brain Research, 64 (1991) 137-143 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0165-3806/91/$03.50
137
BRESD 51378
Postnatal developmental changes in the responses of mouse primary vestibular neurons to externally applied galvanic currents Gilles Desmadryl INSERM U-254, Laboratoire de Neurophysiologie SensorieUe, U.S.T.L., Montpellier (France) (Accepted 13 August 1991) Key words: Vestibular system; Primary afferent neuron; Ontogenesis; Galvanic stimulation
The ontogenesis of vestibular primary neuron sensitivity to depolarisation produced by galvanic current stimulations was studied in mouse inner ear explants maintained in vitro. Cathodal galvanic stimulations, which elicit an increase of the discharge frequencies, are assumed to act on the spike initiation site by depolarizing the neuron. The responses of neurons to galvanic currents at various developmental stages were recorded. The pattern of responses reflected the sensitivities of the neurons to depolarization. At birth, about 75% of the vestibular neurons responded weakly to high intensity galvanic currents thus indicating that they were able to generate action potentials. However, the very low gain of the response to the stimulation revealed the immaturity of the neurons at the spike generation site. Between the day of birth and the ninth postnatal day, an increase in the gain of the responses was observed, indicating the enhancement of the sensitivity of the vestibular neurons to the galvanic currents. This increase in sensitivity was more pronounced from the fourth postnatal day. The response of the neurons to galvanic stimulation increased gradually during postnatal development without reaching a plateau at postnatal day 9 indicating that a further physiological maturation occurs after this stage. These results are consistent with the morphological maturation of the vestibular primary afferents and with previous studies showing that the physiological maturation parallels myelination of the afferent fibers. INTRODUCTION T h e physiology of the p r i m a r y vestibular neurons has been extensively studied in adult animals (for a review see ref. 12), and recently, different studies have correlated the physiological characteristics of the neurons with the m o r p h o l o g y of the hair cells afferentation inside the sensory epithelia 1'16'17. By contrast, the d e v e l o p m e n t of vestibular function in m a m m a l s has b e e n the subject of only a few studies dealing with the p r i m a r y vestibular afferents 5'1°'24 and the secondary vestibular neurons ~8. W i t h r e g a r d to the m a t u r a t i o n , analysis of p r i m a r y vestibular neurons, electrophysiological activities in rodents have b e e n p e r f o r m e d exclusively during the postnatal d e v e l o p m e n t . In the mouse, an increase of the frequency of s p o n t a n e o u s activities has b e e n r e p o r t e d both for the regular activities which a p p e a r e d at postnatal day 4, and for the irregular activities already present at birth 9'1°. In the rat, Curthoys 3'4 described the physiological d e v e l o p m e n t and m a t u r a t i o n of horizontal semicircular canal p r i m a r y vestibular neurons. The main functional changes which occurred during the first ten postnatal days were characterized by an increase of spontaneous activity and by the a p p e a r a n c e of regular activities. In this species, the responses of the vestibular
system to velocity trapezoid and sinusoidal angular acceleration stimulations showed an increase of the sensitivity of the r e c o r d e d neurons during these postnatal dev e l o p m e n t a l stages 5. The m a t u r a t i o n of these responses was hypothesized to be related to the m a t u r a t i o n of the gross m o r p h o l o g y of the vestibular system 5, the develo p m e n t of the sensory cells and synaptogenesis 21-23 as well as the d e v e l o p m e n t of the afferent innervation patterns 8. H o w e v e r , there is no information about the physiological d e v e l o p m e n t of the postsynaptic sensitivity of these neurons to depolarization. O n e way to study the m a t u r a t i o n of these mechanisms is to record intracellularly the synaptic functions. U n f o r t u n a t e l y such recordings, which can only be o b t a i n e d close to the epithelial synaptic zone, are difficult to realize, both in the adult animal and during the d e v e l o p m e n t . A n o t h e r a p p r o a c h is to study the physiological ontogenesis using externally applied galvanic currents. The galvanic constant current stimulations previously used to stimulate the vestibular system 1'2A1'14'15'16'19'2° are assumed to act at the spike generation site by depolarizing of hyperpolarizing the n e u r o n 14. The regularity of the discharge and the sensitivity of the neurons to polarization inputs which are in these e x p e r i m e n t a l conditions m i m i c k e d by the galvanic constant current stimulation, are governed by the
Correspondence: G. Desmadryl, INSERM U-254, Laboratoire de Neurophysiologie Sensorielle, USTL, C.P. 089, PI. E. Bataillon 34095 Montpellier Cedex 05, France. Fax: (33) 67 14 36 96.
138 postspike recovery mechanisms 13'L4'27. The postspike recoveries are determined by the magnitude and the time courses of the afterhyperpolarization ( A H P ) p h e n o m e n a following each spike. The A H P mechanisms reflect the
gl Itl qll . lg r.l
different m e m b r a n e properties of the different types of vestibular afferents, and in particular the presence of the
tO
ionic channels residing in the spike trigger site, exclud-
Fig. 1. Responses of a unit recorded at PD 4 to external galvanic current steps of -100 pA (A), -120 ~A (B) and -150 pA (C) applied for 5 s (calibration bars). The mean firing rates increased from a mean resting activity of 9.49 spikes.s-1 to 81.8 spikes.s-1 (A), 95.1 spikes.s-1 (B) and 138.4 spikes.s-1 (C). Note adaptation of the response during stimulation.
ing other various geometric factors (as the afferentation patterns, the diameter of the fiber or the myelination) TM 14,27
The purpose of the present experiment was to study
m
u
n
the ontogenesis of the sensitivity of the primary vestibular n e u r o n s to depolarization p h e n o m e n a which should occur during the m a t u r a t i o n of the synaptic activity, by means of recording the development of the evoked activity during galvanic current stimulations.
MATERIALS AND METHODS
Subjects Forty-seven explants isolated from inner ears of CBA × C57 mice, ranging in age from the day of birth, designated postnatal day (PD) 0 to PD 9, were used in this study. The explants, containing the vestibulocochlear system, the vestibular ganglion and a portion of the brainstem, were isolated and maintained for several hours in vitro as previously described 1°. They were placed in a recording chamber maintained at 35 °C and continuously perfused with a defined medium26 (124 mM NaC1, 5 mM KC1, 1.15 KH2PO4, 1.15 MgSOa.7H20, 2.5 mM CaCI2, 25 mM NaHCO3, 10 mM glucose). The recording medium was saturated with 95% O2 and 5% CO2.
Galvanic stimulation Electric currents were delivered between two electrodes made of insulated teflon silver wire (0.125 mm diameter). The extremities of the electrodes were terminated by a chlorided spherical tip (0:40.5 mm). One electrode was placed on the posterior canal membrane, close to the posterior ampulla, the other was placed on the cochlea. Stimuli ranging from 25 to 350/~A were delivered by a constant current generator (1 or 5 s, steps: 25, 50, 75, 100, 120, 160, 210, 255, 300 and 350 ktA). The stimuli were designated cathodal (--) or anodal (+) with respect to the polarity of the posterior canal electrode. Recordings Glass micropipettes filled with 3 M KCI (R -- 35-60 M)Q were inserted under visual control into the superior branch of the vestibular nerve at the level of the ganglion. At the first stages studied, some units were completely silent. Due to the difficulty in identifying these silent units, the vestibular afferent activity was recorded intracellularly and a galvanic stimulation test was systematically applied (-120/~A) when a DC potential inferior to 20 mV occurred while inserting the electrode. Single unit discharges were amplified, discriminated from the background noise and stored on-line on the hard disk of an AT Olivetti microcomputer. When a unit was identified, its spontaneous activity was first recorded for 10, 30 or 60 s, then constant galvanic currents were applied. Data analysis Spontaneous activities were averaged over periods of 10, 30 or 60 s. Spike trains were analysed on the computer and their mean frequencies were calculated. Individual recordings were pooled for each developmental stages due to difficulty of distinguishing the different pattern of activities (regular or irregular) at the first stages
of development. The analysis of the responses to the constant current stimulations was performed during the first second of the response. For purpose of statistical analysis, data were pooled and assigned to the following groups: PD 0-i, PD 2, PD 3-4, PD 5-6 and PD 7-9. The mean gain of the responses was calculated as the ratio of the change in the discharge rate between -25 and -350 #A, and the magnitude of the applied galvanic current. The gain of few individual units for which we could record at least three responses to different galvanic current steps, was expressed by the mean slope of the response increase. Statistical comparisons were based on Student's t-test.
RESULTS The responses of the primary vestibular afferents to galvanic stimulation were recorded from n e u r o n s located in the superior branch of the vestibular ganglion, at various stages of postnatal development. Cathodal constant currents always induced an increase in discharge rate while anodal currents decreased it. For the first stages of development the m e a n resting discharges of the primary vestibular afferents were low and the anodal stimulation almost always abolished the activities even with a low stimulation intensity. Consequently, only responses to cathodal stimulation were analysed. Typical responses to five seconds cathodal stimulation at different constant current steps are illustrated in Fig. 1. The firing rate increased rapidly at the beginning of the stimulation and the response sometimes presented an adaptation during the stimulation, as shown in this case. The magnitude of the response increased with the intensity of the stimulation and with the maturation of the neurons as illustrated, for some units at each developmental stages, in Fig. 2.
Resting discharge and response to constant current stimulations At PD 0-1, some units were totally silent. The resting activity of the others was characterized by a tow frequency discharging pattern (Table I). A t this stage, 16 out of 72 stimulated units did not respond to galvanic stimulation even at high intensities. A m o n g the responding units, a few presented a brief response during the
139
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Fig, 2. Examples of some responses of neurons recorded at different stages of development, for which at least 3 externally galvanic current steps were applied. At PD 0-1 and PD 2 some units do not show an increase of the response at high intensity of stimulation, indicating a possible saturation of the response. Note the appearance at PD 3-4 of units presenting steeper slopes than those of previous stages.
first t e n t h s of s e c o n d of t h e s t i m u l a t i o n , f o l l o w e d by a
s p o n s e of the v e s t i b u l a r n e u r o n s to - 1 2 0 / ~ A r a n g e d be-
silent p e r i o d . In c o n s e q u e n c e , the g a l v a n i c r e s p o n s e was
t w e e n 0 and 58.76 spikes.s -1 ( m e a n 24.67 spikes.s-I);
a n a l y s e d for units w h i c h r e s p o n d e d d u r i n g the w h o l e pe-
h o w e v e r , at this stage, s o m e n e u r o n s did n o t r e s p o n d to
r i o d of s t i m u l a t i o n . T h i s is illustrated by the r e s p o n s e of
- 1 2 0 A c o n s t a n t c u r r e n t s t i m u l a t i o n , but at h i g h e r inten-
the units to g a l v a n i c s t i m u l a t i o n
sities a r e s p o n s e was elicited.
at m e d i u m
strength
s t i m u l a t i o n of - 1 2 0 ~ A as s h o w n in T a b l e I. T h e re-
T h e resting discharge f r e q u e n c i e s of the n e u r o n s re-
TABLE I
Physiological properties of the vestibular neurons at the different stages studied First column: resting activity. Second column: examples of the response at medium constant current stimulation (-120 pA L Third column: gain of the response. Entries: minimum value, maximum value, mean ± S.E.M, Nine neurons did not respond at this intensity of stimulation, n, number units recorded; numbers in parentheses, number of explants.
Stages
Resting activity (spikes.s -1) Min
PD 0-1 (9) PD 2 (7) PD 3-4 (12) PD 5-6 (9) PD 7-9 (10)
Max
0.66
42.27
0.92
68.22
0.32
60.36
1.14
40.84
2.10
173.54
Evoked response to -120 pA (spikes.s -1) Mean
Min
Max
7.30 -+ 0.88 n=60 8.51 - 1.77 n=37 9.27 ± 1.34 n=81 10.95 ± 1.63 n = 37 40.98 +- 7.11 n=42
0.0"
58.76
9.90
48.05
10.14
129.53
15.35
127.76
29.73
193.19
Gain (spikes .s-I/#A)
Mean 24.67 n=28 30.35 n=13 42.69 n= 19 56.21 n = 18 66.98 n=22
± 4.45
0.1287
+- 3.35
0.1360
_+ 7,74
0.2474
- 7.35
0.2807
± 7.62
0.3434
140
150
TABLE
120
Gain of individual vestibular neuron responses galvanic stimulations
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9o
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-
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--+-+'11- 4 - - 1 I f lm~ -
0
50
100
t50
@00 250 300 350 uA Fig. 3. Relation between the mean responses recorded in the whole population of vestibular neurons and galvanic current steps for the five developmental stages. (Dashed lines, squares: PD 0, stars: PD 2, cross: PD 3-4; solid lines: squares: PD 5-6, stars: PD 7-9). The response augmentation elicited by increasing stimulus intensities is smaller for the first stages (PD 0-1 and PD 2) than for the oldest stages (PD 5-6 and PD 7-8).
corded at PD 2 remained low (Table I) and the difference to those recorded at PD 0-1 was not statistically significant (P > 0.5). By PD 2, the response to each constant current step persisted during the entire period of stimulation. From this stage on, all recorded neurons responded to galvanic stimulation of a -120 # A current. The frequency of the response to -120 ktA constant current increased continuously during maturation (Table I). At PD 3 - 4 and PD 5 - 6 the main spontaneous activity remained low (Table I) and the differences with those recorded at previous stages were not statistically significant (P > 0.2). At PD 7 - 9 the frequencies of resting discharges ranged between 2.10 and 173.54 spikes.s -1 (Table I). The spontaneous discharge rate was significantly higher than that recorded at previous stages (P < 0.001), but lower than the one previously reported in mouse 1° at PD 10 (P < 0.01).
0.4 "I O3
~0.2 r°r-i
a
r
~0.1
O-i
2 3-4 5-6 postnatal stages
7-9
Fig. 4. Gain of the response of the vestibular neurons at the different developmental stages. The gain is low at PD 0-1 and PD 2 and higher in the later stages.
1I
The minimum value and the maximum value of gain are given for each stage in the first column; n, number units recorded. The number of units assigned to 3 different groups according to their gains, is given in the three last columns. Significance levels for the differences in sensitivities: P > 0.5 between: PD 0-1/PD 2: PD 3-4/PD 7-9; PD 5-6/PD 7-9. P > 0.3 between: PD 3-4/5-6. P < 0.01 between: PD 0-1/PD 3-4; PD 2/PD 3-4; PD 0-1/5-6; PD 2/PD 5-6. P < 0.001 between: PD 0-1/PD 7-9; PD2/PD 7-9. Stages
Gain of the response Number of units (spikes. s-1/ltA ) Min
Max
below 0.1 0.1 to 0.2 Above 0,2 spikes spikes spikes •s-1/#A "S-l/~A "s-QIxA
PD 0-1
0.056
PD 2
0.048
PD 3-4
0.147
5/14 (35%) 4/20 (20%) 0/10
PD 5-6
0.116
PD 7-9
0.134
0.422 n=14 0.346 n=20 0.712 n=10 0.682 n=14 0.699 n=25
0/14 0/25
4/14 (28%) 11/20 (55%) 3/10 (30%) 4/14 (28%) 6/25 (24%)
5/14 (35%) 5/20 (25%) 7/10 (70%) 10/14 (71%) 19/25 (76%)
Development o f the response to constant current stimulations Data recorded at each developmental stage were pooled and plotted against the different current stimulation steps in Fig. 3. The response of the vestibular neurons increased with stimulus intensity. At PD 0-1, a small increase of the discharge rate occurred between -50 and -160 g A , and at higher constant current stimulations the responses seemed to reach a plateau suggesting the saturation of the response. At PD 2 and PD 3-4 the discharge frequencies for each current step were higher than those of the previous stages, but the increase in discharge rate evoked by the augmentation of the stimuli remained low. A t P D 5 - 6 and P D 7-9 discharge rates elicited by each current step were higher than those of the previous stages. At this stages, there was an almost linear relationship between the neural response and the stimulation intensity. The increase of the discharge rate elicited by galvanic stimulation during the early developmental stages was smaller than during older stages. This was confirmed by the analysis of the gain of the neural response during maturation (Table I, Fig. 4). The mean gain increased slowly during the two first postnatal stages and more quickly in the three last developmental stages (PD 3 - 4 to P D 7-9) without reaching saturation (Fig. 4) indicating a further increase of the response.
141
Development of the individual sensitivity of the vestibular neurons
Some insight of the individual sensitivity of the vestibular neurons to the galvanic stimulation could be obtained by the analysis of the gain of the responses in few units for which the responses to at least three current steps were recorded (Table II). At PD 0-1 and PD 2, there was a 7.5-fold range in the gain of the responses of individual units to the stimulation. At PD 0-1 the distribution of the gains was almost equal (Table II, 3 last columns), while 55% of the units recorded at PD 2 had gains ranging between 0.1 and 0.2 spikes.s-1//~A. The different gains of the neurons recorded in these two stages were not statistically significant (Table II). The individual gains of the recorded units increased at PD 3-4 and from this stage no units displayed individual gain below 0.1 spikes.s-1//~A, the proportion of units above 0.2 spikes.s-a//~A was around 70% (Table II). Difference in gains of neurons recorded at PD 3-4, 5-6 and 7-9 were not statistically significant, but higher than those of the previous stages (Table II).
(77%) responded to constant current stimulation, indicating that the neurons were able to respond to the depolarization elicited by galvanic simulation. However, at these stages, the more intense stimulation elicited a slight response with a very low gain. This implies an immaturity of the profiles of the recovery process following the action potentials. Since the postspike recovery phenomena are determined by the afterhyperpolarization (AHP) mechanisms x3AS'zv, AHPs at this stage would differ in magnitude and time course from those of the adult. AHPs reflect specific membrane properties, so the immaturity of these responses may reflect the immaturity of the vestibular primary neurons at the spike initiation site. These results are consistent with the low gain of the response of the vestibular neurons to natural stimuli during the first postnatal developmental stages reported in the rat 3'4, which is related to the vestibular hair cells immaturity. The limited sensitivity of the neuron at the initiation site level, and the small responses of the vestibular sensory cells may both contribute to the low gain of the responses of the vestibular system during the first stages of postnatal development.
DISCUSSION
Maturation of the responses Classification of the discharges At birth and during the two first postnatal days, the mean resting activities were similar to those previously described in the mouse w and comparable to those recorded in the rat 3'4. The increase of the spontaneous activity analysed in this study in the later stages is in accordance with previous works 3'4'1°. In the present study, the resting activities were not classified as regular or irregular discharging patterns, however some indications about the presence of different types of acitvities appeared as early as the fourth postnatal day. During maturation, the sensitivity of individual unit responses to constant current stimulations increased, and units with sensitivities below 0.1 spikes.-l/pA disappeared at PD 3-4, at stage when the first segregation between regular and irregular discharging units has been described w. So, the units with the higher galvanic sensitivity, which appeared at PD 3-4, may correspond to units to irregular units while the low sensitivity units correspond to the regular activities, as reported for the adult animal 13'14.
Response to galvanic stimulation at early stages At birth, some vestibular neurons were totally insensitive to the stimulus or presented only a slight response during the first tenths of second of the stimulation. This suggests that about one fourth of the vestibular primary afferents are completely immature. The main population
The sensitivity of neurons to constant current stimulation increased during development and, as early as the second postnatal day, the entire population of neurons responded. The increase in sensitivity of the vestibular neurons was sma!l between PD 0-1 and PD 2, and higher in the later stages. The progressive increase in sensitivity of the neurons to depolarization could be related to the structural maturation of the postsynaptic zone. This study shows that the last stage studied (PD 7-9) does not reflect the adult activities since the increase of sensivitity of the neurons did not reach a stable value at the oldest stages analysed. Presumably there will be an augmentation of the sensitivity of the primary vestibular neurons to depolarization after the 10 th postnatal day. This observation is in accordance with studies showing that the morphological adult stage is only reached during the fourth postnatal week 22'z3.
Correlations with the morphological neuronal development The maturation of the physiological properties of the neurons analysed in this study parallels the morphological development of the afferentation patterns of the hair cells, which occurred continuously during the 10 first postnatal days 8. The increase of the galvanic sensitivity, which arises at PD 3-4 is concomitant with the appearance of well differentiated hair cell afferentation patterns
142 inside the vestibular sensory epithelia s. The development of hair cells afferentation and the increase of the sensitivity of the neurons to depolarization, both contribute to the physiological maturation of the responses to natural stimuli described during postnatal maturation of the vestibular system 3'4. Some insight of the mechanisms underlying physiological maturation of the primary vestibular neurons are also brought by studies on the myelination of the neural peripheral processes. In the cat 25, myelinated fibers appeared at stages when regular units were first detected. Further myelination of the fibers paralleled the increase of the percentage of regular units 24. It has also been reported a linear relationship between the increase in diameter of the neuron processes and the number of myelin lamellae. In the mouse, the first regular activities were recorded at PD 41°, one day after the beginning of myelination of the preganglionic fibers 6. In consequence, although myelination appears not involved in postsynaptic physiological maturation, the myelination of the peripheral processes parallels the development of the sensitivity of the fibers to depolarization. Although the development of the sensitivity of the neurons is almost probably associated with biochemical
REFERENCES 1 Baird, R.A., Desmadryl, G., Fernandez, C. and Goldberg, J.M., The vestibular nerve of the Chinchilla. II. Relation between afferent response properties and peripheral innervation patterns in the semicircular canals, J. Neurophysiol., 60 (1988) 182-203. 2 Courjon, J.H., Precht, W. and Sirkin, D.W., Vestibular nerve and nuclei unit responses and eye movement responses to repetitive galvanic stimulation of the labyrinth in the rat, Exp. Brain Res., 66 (1987) 41-48. 3 Curthoys, I.S., The development of function of horizontal semicircular canal primary neurons in the rat, Brain Research, 167 (1979) 41-52. 4 Curthoys, I.S., Postnatal developmental changes in the response of rat primary horizontal semicircular canal neurons to sinusoidal angular accelerations, Exp. Brain Res., 47 (i982) 295-300. 5 Curthoys, I.S., The development of function of primary vestibular neurons. In R. Romand (Ed.), Development of Auditory and Vestibular Systems, Academic Press, New York, 1983, pp. 425-461. 6 Dechesne, C.J., Desmadryl, G. and Dem6mes, D., Myelination of the mouse vestibular ganglion, Acta Otolaryngol., 103 (1983) 18-23. 7 Dechesne, C.J., Sans, A. and Keller, A., Onset and development of neuron-specific enolase immunoreactivity in the peripheral vestibular system of the mouse, Neurosci. Lett., 61 (1985) 299-304. 8 Desmadryl, O. and Sans, A., The development of the innervation patterns in the mouse vestibular sensory epithelium, Dev. Brain Res., 52 (1990) 183-189. 9 Desmadryl, G., Raymond, J. and Sans, A., Technique d'enregistrement in vitro des neurones vestibulaires dans l'oreille interne lors du d6veloppement postnatal chez la Souris, C.R. Acad. Sci. Sdrie Ili (Paris), 298 (1984) 223-228. 10 Desmadryl, G., Raymond, J. and Sans, A., In vitro electro-
changes, studies using diverse markers have not detected specific modifications of the postsynaptic compartment of vestibular neurons during this postnatal period. Thus, the appearance of the neuronal marker, neuron specific enolase, which occurs as early as 15 days of gestation and parallels synaptogenesis 7, is unlikely associated with the emergence of physiological activity arising one week later. Conclusion
This study demonstrates that the development and the maturation of the sensitivity of the mouse vestibular neurons to depolarization occur throughout the first postnatal week. This process which is slow during the first postnatal days and quickens from the fourth postnatal day on, should be concomitant with the development of the hair cell synaptic activity. This functional ontogenesis is related to the maturation of the fibers around the trigger site, and may correspond quantitatively to the increase of the number of ionic channels in the spike trigger site and their qualitative maturation zs'29. Acknowledgements. The author is grateful to Dr A. Sans and to Dr C.J. Dechesne for critically reviewing the manuscript.
physiological study of spontaneous activity in neonatal mouse vestibular ganglion neurons during development, Dev. Brain Res., 25 (1986) 133-136. 11 Ezure, K., Cohen, M.S. and Wilson, V., Response of semicircular canal afferents to sinusoidal polarizing currents implications for the input-output properties of second-order neurons, J. NeurophysioL, 49 (1983) 639-648. 12 Goldberg, J.M. and Fernandez, C., The vestibular system. In: Handbook of Physiology. The Nervous System, Sect. 1, Vol. I11,
13 14
15
16
17 18 19
Chapt. 21, Am. Physiol. Soc., Bethesda MD, 1984, pp. 9771022. Goldberg, J.M., Fern,Sndez, C. and Smith, C.E., Responses of vestibular.nerve afferents in the squirrel monkey to externally applied galvanic current, Brain Research, 252 (1982) 256-160. Goldberg, LM., Smith, C.E. and Fernandez, C., Relation between discharge regularity and responses to externally applied galvanic currents in vestibular nerve afferents of the Squirrel Monkey, J. Neurophysiol., 51 (1984) 1236-1256. Goldberg, J.M., Desmadryl, G., Baird, R.A. and Fernandezi C., The vestibular nerve of the Chinchilla. IV. Discharge properties of utricular afferents, J. Neurophysiol., 63 (t990, a) 781790. Goldberg, J.M., Desmadryl, G., Baird, R.A. and Fernandez, C., The vestibular nerve of the Chinchilla. V. Relation between afferent discharge properties and peripheral innervation patterns in the utricular macula, J. NeurophysioL, 63 (1990, b) 791-804. Honrubia, V., Hoffman, L.E, Sitko, S. and Schwartz, I.R., Anatomic and physiological correlates in bullfrog vestibular nerve, J. Neurophysiol., 61 (1989) 688-701. Lannou, J., Preeht, W. and Cazin, L., The p~matal development of functional properties of central vestibular neurons in the rat, Brain Research, 175 (1979) 219-232. Lifschitz, W.S., Responses from first order neurons of the horizontal semicircular canal in the pigeon, Brain Research, 63 (1973) 43-57.
143 20 Lowenstein, O., The effect of galvanic polarization on the impulse discharge from sense ending in the isolated labyrinth in the thornback ray (Raja elavata), J. Physiol., 127 (1955) 104117. 21 Mbiene, J.P. and Sans, A., Differentiation and maturation of the sensory hair bundles in the fetal and postnatal vestibular receptors of the mouse a scanning electron microscopy study, J. Comp. Neurol., 254 (1986) 271-278. 22 Nordemar, H., Embryogenesis of the inner ear. II. The late differentiation of the mammalian crista ampullaris in vivo and in vitro, Acta Otolaryngol., 96 (1983) 1-8. 23 Nordemar, H., Postnatal development of the vestibular sensory epithelium in the mouse, Aeta Otolaryngol., 96 (1983) 447-456. 24 Romand, R. and Dauzat, M., Modification of spontaneous activity in primary vestibular neurons during development in the cat, Exp. Brain Res., 45 (1982) 265-268.
25 Romand, R., Sans, A., Romand, M.R. and Marty, R., The structural maturation of the stato-acoustic nerve in the cat, J. Comp. Neurol., 170 (1976) 1-15. 26 Schwartzkroin, P.A., to slice or not to slice. In G.A. Kerkut and H.V. Wheals (Eds.), Electrophysiology of Isolated Mammalian CNS preparation, Academic Press, New York, 1981, pp. 16-45. 27 Smith, C.E. and Goldberg, J.M., A stochastic after-hyperpolarization model of repetitive activity in vestibular afferents, Biol. Cybern., 54 (1986) 41-51. 28 Walton, K. and Fulton, B.P., Ionic mechanisms underlying the firing properties of rat neonatal motoneurons studied in vitro, Neuroscience, 19 (1986) 669-683. 29 Ziskind-Conhaim, L., Physiological and morphological changes in developing peripheral nerves of rat embryos, Dev. Brain Res., 42 (1988) 15-28.
137
BRESD 51378
Postnatal developmental changes in the responses of mouse primary vestibular neurons to externally applied galvanic currents Gilles Desmadryl INSERM U-254, Laboratoire de Neurophysiologie SensorieUe, U.S.T.L., Montpellier (France) (Accepted 13 August 1991) Key words: Vestibular system; Primary afferent neuron; Ontogenesis; Galvanic stimulation
The ontogenesis of vestibular primary neuron sensitivity to depolarisation produced by galvanic current stimulations was studied in mouse inner ear explants maintained in vitro. Cathodal galvanic stimulations, which elicit an increase of the discharge frequencies, are assumed to act on the spike initiation site by depolarizing the neuron. The responses of neurons to galvanic currents at various developmental stages were recorded. The pattern of responses reflected the sensitivities of the neurons to depolarization. At birth, about 75% of the vestibular neurons responded weakly to high intensity galvanic currents thus indicating that they were able to generate action potentials. However, the very low gain of the response to the stimulation revealed the immaturity of the neurons at the spike generation site. Between the day of birth and the ninth postnatal day, an increase in the gain of the responses was observed, indicating the enhancement of the sensitivity of the vestibular neurons to the galvanic currents. This increase in sensitivity was more pronounced from the fourth postnatal day. The response of the neurons to galvanic stimulation increased gradually during postnatal development without reaching a plateau at postnatal day 9 indicating that a further physiological maturation occurs after this stage. These results are consistent with the morphological maturation of the vestibular primary afferents and with previous studies showing that the physiological maturation parallels myelination of the afferent fibers. INTRODUCTION T h e physiology of the p r i m a r y vestibular neurons has been extensively studied in adult animals (for a review see ref. 12), and recently, different studies have correlated the physiological characteristics of the neurons with the m o r p h o l o g y of the hair cells afferentation inside the sensory epithelia 1'16'17. By contrast, the d e v e l o p m e n t of vestibular function in m a m m a l s has b e e n the subject of only a few studies dealing with the p r i m a r y vestibular afferents 5'1°'24 and the secondary vestibular neurons ~8. W i t h r e g a r d to the m a t u r a t i o n , analysis of p r i m a r y vestibular neurons, electrophysiological activities in rodents have b e e n p e r f o r m e d exclusively during the postnatal d e v e l o p m e n t . In the mouse, an increase of the frequency of s p o n t a n e o u s activities has b e e n r e p o r t e d both for the regular activities which a p p e a r e d at postnatal day 4, and for the irregular activities already present at birth 9'1°. In the rat, Curthoys 3'4 described the physiological d e v e l o p m e n t and m a t u r a t i o n of horizontal semicircular canal p r i m a r y vestibular neurons. The main functional changes which occurred during the first ten postnatal days were characterized by an increase of spontaneous activity and by the a p p e a r a n c e of regular activities. In this species, the responses of the vestibular
system to velocity trapezoid and sinusoidal angular acceleration stimulations showed an increase of the sensitivity of the r e c o r d e d neurons during these postnatal dev e l o p m e n t a l stages 5. The m a t u r a t i o n of these responses was hypothesized to be related to the m a t u r a t i o n of the gross m o r p h o l o g y of the vestibular system 5, the develo p m e n t of the sensory cells and synaptogenesis 21-23 as well as the d e v e l o p m e n t of the afferent innervation patterns 8. H o w e v e r , there is no information about the physiological d e v e l o p m e n t of the postsynaptic sensitivity of these neurons to depolarization. O n e way to study the m a t u r a t i o n of these mechanisms is to record intracellularly the synaptic functions. U n f o r t u n a t e l y such recordings, which can only be o b t a i n e d close to the epithelial synaptic zone, are difficult to realize, both in the adult animal and during the d e v e l o p m e n t . A n o t h e r a p p r o a c h is to study the physiological ontogenesis using externally applied galvanic currents. The galvanic constant current stimulations previously used to stimulate the vestibular system 1'2A1'14'15'16'19'2° are assumed to act at the spike generation site by depolarizing of hyperpolarizing the n e u r o n 14. The regularity of the discharge and the sensitivity of the neurons to polarization inputs which are in these e x p e r i m e n t a l conditions m i m i c k e d by the galvanic constant current stimulation, are governed by the
Correspondence: G. Desmadryl, INSERM U-254, Laboratoire de Neurophysiologie Sensorielle, USTL, C.P. 089, PI. E. Bataillon 34095 Montpellier Cedex 05, France. Fax: (33) 67 14 36 96.
138 postspike recovery mechanisms 13'L4'27. The postspike recoveries are determined by the magnitude and the time courses of the afterhyperpolarization ( A H P ) p h e n o m e n a following each spike. The A H P mechanisms reflect the
gl Itl qll . lg r.l
different m e m b r a n e properties of the different types of vestibular afferents, and in particular the presence of the
tO
ionic channels residing in the spike trigger site, exclud-
Fig. 1. Responses of a unit recorded at PD 4 to external galvanic current steps of -100 pA (A), -120 ~A (B) and -150 pA (C) applied for 5 s (calibration bars). The mean firing rates increased from a mean resting activity of 9.49 spikes.s-1 to 81.8 spikes.s-1 (A), 95.1 spikes.s-1 (B) and 138.4 spikes.s-1 (C). Note adaptation of the response during stimulation.
ing other various geometric factors (as the afferentation patterns, the diameter of the fiber or the myelination) TM 14,27
The purpose of the present experiment was to study
m
u
n
the ontogenesis of the sensitivity of the primary vestibular n e u r o n s to depolarization p h e n o m e n a which should occur during the m a t u r a t i o n of the synaptic activity, by means of recording the development of the evoked activity during galvanic current stimulations.
MATERIALS AND METHODS
Subjects Forty-seven explants isolated from inner ears of CBA × C57 mice, ranging in age from the day of birth, designated postnatal day (PD) 0 to PD 9, were used in this study. The explants, containing the vestibulocochlear system, the vestibular ganglion and a portion of the brainstem, were isolated and maintained for several hours in vitro as previously described 1°. They were placed in a recording chamber maintained at 35 °C and continuously perfused with a defined medium26 (124 mM NaC1, 5 mM KC1, 1.15 KH2PO4, 1.15 MgSOa.7H20, 2.5 mM CaCI2, 25 mM NaHCO3, 10 mM glucose). The recording medium was saturated with 95% O2 and 5% CO2.
Galvanic stimulation Electric currents were delivered between two electrodes made of insulated teflon silver wire (0.125 mm diameter). The extremities of the electrodes were terminated by a chlorided spherical tip (0:40.5 mm). One electrode was placed on the posterior canal membrane, close to the posterior ampulla, the other was placed on the cochlea. Stimuli ranging from 25 to 350/~A were delivered by a constant current generator (1 or 5 s, steps: 25, 50, 75, 100, 120, 160, 210, 255, 300 and 350 ktA). The stimuli were designated cathodal (--) or anodal (+) with respect to the polarity of the posterior canal electrode. Recordings Glass micropipettes filled with 3 M KCI (R -- 35-60 M)Q were inserted under visual control into the superior branch of the vestibular nerve at the level of the ganglion. At the first stages studied, some units were completely silent. Due to the difficulty in identifying these silent units, the vestibular afferent activity was recorded intracellularly and a galvanic stimulation test was systematically applied (-120/~A) when a DC potential inferior to 20 mV occurred while inserting the electrode. Single unit discharges were amplified, discriminated from the background noise and stored on-line on the hard disk of an AT Olivetti microcomputer. When a unit was identified, its spontaneous activity was first recorded for 10, 30 or 60 s, then constant galvanic currents were applied. Data analysis Spontaneous activities were averaged over periods of 10, 30 or 60 s. Spike trains were analysed on the computer and their mean frequencies were calculated. Individual recordings were pooled for each developmental stages due to difficulty of distinguishing the different pattern of activities (regular or irregular) at the first stages
of development. The analysis of the responses to the constant current stimulations was performed during the first second of the response. For purpose of statistical analysis, data were pooled and assigned to the following groups: PD 0-i, PD 2, PD 3-4, PD 5-6 and PD 7-9. The mean gain of the responses was calculated as the ratio of the change in the discharge rate between -25 and -350 #A, and the magnitude of the applied galvanic current. The gain of few individual units for which we could record at least three responses to different galvanic current steps, was expressed by the mean slope of the response increase. Statistical comparisons were based on Student's t-test.
RESULTS The responses of the primary vestibular afferents to galvanic stimulation were recorded from n e u r o n s located in the superior branch of the vestibular ganglion, at various stages of postnatal development. Cathodal constant currents always induced an increase in discharge rate while anodal currents decreased it. For the first stages of development the m e a n resting discharges of the primary vestibular afferents were low and the anodal stimulation almost always abolished the activities even with a low stimulation intensity. Consequently, only responses to cathodal stimulation were analysed. Typical responses to five seconds cathodal stimulation at different constant current steps are illustrated in Fig. 1. The firing rate increased rapidly at the beginning of the stimulation and the response sometimes presented an adaptation during the stimulation, as shown in this case. The magnitude of the response increased with the intensity of the stimulation and with the maturation of the neurons as illustrated, for some units at each developmental stages, in Fig. 2.
Resting discharge and response to constant current stimulations At PD 0-1, some units were totally silent. The resting activity of the others was characterized by a tow frequency discharging pattern (Table I). A t this stage, 16 out of 72 stimulated units did not respond to galvanic stimulation even at high intensities. A m o n g the responding units, a few presented a brief response during the
139
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200
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250 f
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. ~ 200.
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200
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0
300
100
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200
300
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Fig, 2. Examples of some responses of neurons recorded at different stages of development, for which at least 3 externally galvanic current steps were applied. At PD 0-1 and PD 2 some units do not show an increase of the response at high intensity of stimulation, indicating a possible saturation of the response. Note the appearance at PD 3-4 of units presenting steeper slopes than those of previous stages.
first t e n t h s of s e c o n d of t h e s t i m u l a t i o n , f o l l o w e d by a
s p o n s e of the v e s t i b u l a r n e u r o n s to - 1 2 0 / ~ A r a n g e d be-
silent p e r i o d . In c o n s e q u e n c e , the g a l v a n i c r e s p o n s e was
t w e e n 0 and 58.76 spikes.s -1 ( m e a n 24.67 spikes.s-I);
a n a l y s e d for units w h i c h r e s p o n d e d d u r i n g the w h o l e pe-
h o w e v e r , at this stage, s o m e n e u r o n s did n o t r e s p o n d to
r i o d of s t i m u l a t i o n . T h i s is illustrated by the r e s p o n s e of
- 1 2 0 A c o n s t a n t c u r r e n t s t i m u l a t i o n , but at h i g h e r inten-
the units to g a l v a n i c s t i m u l a t i o n
sities a r e s p o n s e was elicited.
at m e d i u m
strength
s t i m u l a t i o n of - 1 2 0 ~ A as s h o w n in T a b l e I. T h e re-
T h e resting discharge f r e q u e n c i e s of the n e u r o n s re-
TABLE I
Physiological properties of the vestibular neurons at the different stages studied First column: resting activity. Second column: examples of the response at medium constant current stimulation (-120 pA L Third column: gain of the response. Entries: minimum value, maximum value, mean ± S.E.M, Nine neurons did not respond at this intensity of stimulation, n, number units recorded; numbers in parentheses, number of explants.
Stages
Resting activity (spikes.s -1) Min
PD 0-1 (9) PD 2 (7) PD 3-4 (12) PD 5-6 (9) PD 7-9 (10)
Max
0.66
42.27
0.92
68.22
0.32
60.36
1.14
40.84
2.10
173.54
Evoked response to -120 pA (spikes.s -1) Mean
Min
Max
7.30 -+ 0.88 n=60 8.51 - 1.77 n=37 9.27 ± 1.34 n=81 10.95 ± 1.63 n = 37 40.98 +- 7.11 n=42
0.0"
58.76
9.90
48.05
10.14
129.53
15.35
127.76
29.73
193.19
Gain (spikes .s-I/#A)
Mean 24.67 n=28 30.35 n=13 42.69 n= 19 56.21 n = 18 66.98 n=22
± 4.45
0.1287
+- 3.35
0.1360
_+ 7,74
0.2474
- 7.35
0.2807
± 7.62
0.3434
140
150
TABLE
120
Gain of individual vestibular neuron responses galvanic stimulations
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9o
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-
--+-+'11- 4 - - 1 I f lm~ -
0
50
100
t50
@00 250 300 350 uA Fig. 3. Relation between the mean responses recorded in the whole population of vestibular neurons and galvanic current steps for the five developmental stages. (Dashed lines, squares: PD 0, stars: PD 2, cross: PD 3-4; solid lines: squares: PD 5-6, stars: PD 7-9). The response augmentation elicited by increasing stimulus intensities is smaller for the first stages (PD 0-1 and PD 2) than for the oldest stages (PD 5-6 and PD 7-8).
corded at PD 2 remained low (Table I) and the difference to those recorded at PD 0-1 was not statistically significant (P > 0.5). By PD 2, the response to each constant current step persisted during the entire period of stimulation. From this stage on, all recorded neurons responded to galvanic stimulation of a -120 # A current. The frequency of the response to -120 ktA constant current increased continuously during maturation (Table I). At PD 3 - 4 and PD 5 - 6 the main spontaneous activity remained low (Table I) and the differences with those recorded at previous stages were not statistically significant (P > 0.2). At PD 7 - 9 the frequencies of resting discharges ranged between 2.10 and 173.54 spikes.s -1 (Table I). The spontaneous discharge rate was significantly higher than that recorded at previous stages (P < 0.001), but lower than the one previously reported in mouse 1° at PD 10 (P < 0.01).
0.4 "I O3
~0.2 r°r-i
a
r
~0.1
O-i
2 3-4 5-6 postnatal stages
7-9
Fig. 4. Gain of the response of the vestibular neurons at the different developmental stages. The gain is low at PD 0-1 and PD 2 and higher in the later stages.
1I
The minimum value and the maximum value of gain are given for each stage in the first column; n, number units recorded. The number of units assigned to 3 different groups according to their gains, is given in the three last columns. Significance levels for the differences in sensitivities: P > 0.5 between: PD 0-1/PD 2: PD 3-4/PD 7-9; PD 5-6/PD 7-9. P > 0.3 between: PD 3-4/5-6. P < 0.01 between: PD 0-1/PD 3-4; PD 2/PD 3-4; PD 0-1/5-6; PD 2/PD 5-6. P < 0.001 between: PD 0-1/PD 7-9; PD2/PD 7-9. Stages
Gain of the response Number of units (spikes. s-1/ltA ) Min
Max
below 0.1 0.1 to 0.2 Above 0,2 spikes spikes spikes •s-1/#A "S-l/~A "s-QIxA
PD 0-1
0.056
PD 2
0.048
PD 3-4
0.147
5/14 (35%) 4/20 (20%) 0/10
PD 5-6
0.116
PD 7-9
0.134
0.422 n=14 0.346 n=20 0.712 n=10 0.682 n=14 0.699 n=25
0/14 0/25
4/14 (28%) 11/20 (55%) 3/10 (30%) 4/14 (28%) 6/25 (24%)
5/14 (35%) 5/20 (25%) 7/10 (70%) 10/14 (71%) 19/25 (76%)
Development o f the response to constant current stimulations Data recorded at each developmental stage were pooled and plotted against the different current stimulation steps in Fig. 3. The response of the vestibular neurons increased with stimulus intensity. At PD 0-1, a small increase of the discharge rate occurred between -50 and -160 g A , and at higher constant current stimulations the responses seemed to reach a plateau suggesting the saturation of the response. At PD 2 and PD 3-4 the discharge frequencies for each current step were higher than those of the previous stages, but the increase in discharge rate evoked by the augmentation of the stimuli remained low. A t P D 5 - 6 and P D 7-9 discharge rates elicited by each current step were higher than those of the previous stages. At this stages, there was an almost linear relationship between the neural response and the stimulation intensity. The increase of the discharge rate elicited by galvanic stimulation during the early developmental stages was smaller than during older stages. This was confirmed by the analysis of the gain of the neural response during maturation (Table I, Fig. 4). The mean gain increased slowly during the two first postnatal stages and more quickly in the three last developmental stages (PD 3 - 4 to P D 7-9) without reaching saturation (Fig. 4) indicating a further increase of the response.
141
Development of the individual sensitivity of the vestibular neurons
Some insight of the individual sensitivity of the vestibular neurons to the galvanic stimulation could be obtained by the analysis of the gain of the responses in few units for which the responses to at least three current steps were recorded (Table II). At PD 0-1 and PD 2, there was a 7.5-fold range in the gain of the responses of individual units to the stimulation. At PD 0-1 the distribution of the gains was almost equal (Table II, 3 last columns), while 55% of the units recorded at PD 2 had gains ranging between 0.1 and 0.2 spikes.s-1//~A. The different gains of the neurons recorded in these two stages were not statistically significant (Table II). The individual gains of the recorded units increased at PD 3-4 and from this stage no units displayed individual gain below 0.1 spikes.s-1//~A, the proportion of units above 0.2 spikes.s-a//~A was around 70% (Table II). Difference in gains of neurons recorded at PD 3-4, 5-6 and 7-9 were not statistically significant, but higher than those of the previous stages (Table II).
(77%) responded to constant current stimulation, indicating that the neurons were able to respond to the depolarization elicited by galvanic simulation. However, at these stages, the more intense stimulation elicited a slight response with a very low gain. This implies an immaturity of the profiles of the recovery process following the action potentials. Since the postspike recovery phenomena are determined by the afterhyperpolarization (AHP) mechanisms x3AS'zv, AHPs at this stage would differ in magnitude and time course from those of the adult. AHPs reflect specific membrane properties, so the immaturity of these responses may reflect the immaturity of the vestibular primary neurons at the spike initiation site. These results are consistent with the low gain of the response of the vestibular neurons to natural stimuli during the first postnatal developmental stages reported in the rat 3'4, which is related to the vestibular hair cells immaturity. The limited sensitivity of the neuron at the initiation site level, and the small responses of the vestibular sensory cells may both contribute to the low gain of the responses of the vestibular system during the first stages of postnatal development.
DISCUSSION
Maturation of the responses Classification of the discharges At birth and during the two first postnatal days, the mean resting activities were similar to those previously described in the mouse w and comparable to those recorded in the rat 3'4. The increase of the spontaneous activity analysed in this study in the later stages is in accordance with previous works 3'4'1°. In the present study, the resting activities were not classified as regular or irregular discharging patterns, however some indications about the presence of different types of acitvities appeared as early as the fourth postnatal day. During maturation, the sensitivity of individual unit responses to constant current stimulations increased, and units with sensitivities below 0.1 spikes.-l/pA disappeared at PD 3-4, at stage when the first segregation between regular and irregular discharging units has been described w. So, the units with the higher galvanic sensitivity, which appeared at PD 3-4, may correspond to units to irregular units while the low sensitivity units correspond to the regular activities, as reported for the adult animal 13'14.
Response to galvanic stimulation at early stages At birth, some vestibular neurons were totally insensitive to the stimulus or presented only a slight response during the first tenths of second of the stimulation. This suggests that about one fourth of the vestibular primary afferents are completely immature. The main population
The sensitivity of neurons to constant current stimulation increased during development and, as early as the second postnatal day, the entire population of neurons responded. The increase in sensitivity of the vestibular neurons was sma!l between PD 0-1 and PD 2, and higher in the later stages. The progressive increase in sensitivity of the neurons to depolarization could be related to the structural maturation of the postsynaptic zone. This study shows that the last stage studied (PD 7-9) does not reflect the adult activities since the increase of sensivitity of the neurons did not reach a stable value at the oldest stages analysed. Presumably there will be an augmentation of the sensitivity of the primary vestibular neurons to depolarization after the 10 th postnatal day. This observation is in accordance with studies showing that the morphological adult stage is only reached during the fourth postnatal week 22'z3.
Correlations with the morphological neuronal development The maturation of the physiological properties of the neurons analysed in this study parallels the morphological development of the afferentation patterns of the hair cells, which occurred continuously during the 10 first postnatal days 8. The increase of the galvanic sensitivity, which arises at PD 3-4 is concomitant with the appearance of well differentiated hair cell afferentation patterns
142 inside the vestibular sensory epithelia s. The development of hair cells afferentation and the increase of the sensitivity of the neurons to depolarization, both contribute to the physiological maturation of the responses to natural stimuli described during postnatal maturation of the vestibular system 3'4. Some insight of the mechanisms underlying physiological maturation of the primary vestibular neurons are also brought by studies on the myelination of the neural peripheral processes. In the cat 25, myelinated fibers appeared at stages when regular units were first detected. Further myelination of the fibers paralleled the increase of the percentage of regular units 24. It has also been reported a linear relationship between the increase in diameter of the neuron processes and the number of myelin lamellae. In the mouse, the first regular activities were recorded at PD 41°, one day after the beginning of myelination of the preganglionic fibers 6. In consequence, although myelination appears not involved in postsynaptic physiological maturation, the myelination of the peripheral processes parallels the development of the sensitivity of the fibers to depolarization. Although the development of the sensitivity of the neurons is almost probably associated with biochemical
REFERENCES 1 Baird, R.A., Desmadryl, G., Fernandez, C. and Goldberg, J.M., The vestibular nerve of the Chinchilla. II. Relation between afferent response properties and peripheral innervation patterns in the semicircular canals, J. Neurophysiol., 60 (1988) 182-203. 2 Courjon, J.H., Precht, W. and Sirkin, D.W., Vestibular nerve and nuclei unit responses and eye movement responses to repetitive galvanic stimulation of the labyrinth in the rat, Exp. Brain Res., 66 (1987) 41-48. 3 Curthoys, I.S., The development of function of horizontal semicircular canal primary neurons in the rat, Brain Research, 167 (1979) 41-52. 4 Curthoys, I.S., Postnatal developmental changes in the response of rat primary horizontal semicircular canal neurons to sinusoidal angular accelerations, Exp. Brain Res., 47 (i982) 295-300. 5 Curthoys, I.S., The development of function of primary vestibular neurons. In R. Romand (Ed.), Development of Auditory and Vestibular Systems, Academic Press, New York, 1983, pp. 425-461. 6 Dechesne, C.J., Desmadryl, G. and Dem6mes, D., Myelination of the mouse vestibular ganglion, Acta Otolaryngol., 103 (1983) 18-23. 7 Dechesne, C.J., Sans, A. and Keller, A., Onset and development of neuron-specific enolase immunoreactivity in the peripheral vestibular system of the mouse, Neurosci. Lett., 61 (1985) 299-304. 8 Desmadryl, O. and Sans, A., The development of the innervation patterns in the mouse vestibular sensory epithelium, Dev. Brain Res., 52 (1990) 183-189. 9 Desmadryl, G., Raymond, J. and Sans, A., Technique d'enregistrement in vitro des neurones vestibulaires dans l'oreille interne lors du d6veloppement postnatal chez la Souris, C.R. Acad. Sci. Sdrie Ili (Paris), 298 (1984) 223-228. 10 Desmadryl, G., Raymond, J. and Sans, A., In vitro electro-
changes, studies using diverse markers have not detected specific modifications of the postsynaptic compartment of vestibular neurons during this postnatal period. Thus, the appearance of the neuronal marker, neuron specific enolase, which occurs as early as 15 days of gestation and parallels synaptogenesis 7, is unlikely associated with the emergence of physiological activity arising one week later. Conclusion
This study demonstrates that the development and the maturation of the sensitivity of the mouse vestibular neurons to depolarization occur throughout the first postnatal week. This process which is slow during the first postnatal days and quickens from the fourth postnatal day on, should be concomitant with the development of the hair cell synaptic activity. This functional ontogenesis is related to the maturation of the fibers around the trigger site, and may correspond quantitatively to the increase of the number of ionic channels in the spike trigger site and their qualitative maturation zs'29. Acknowledgements. The author is grateful to Dr A. Sans and to Dr C.J. Dechesne for critically reviewing the manuscript.
physiological study of spontaneous activity in neonatal mouse vestibular ganglion neurons during development, Dev. Brain Res., 25 (1986) 133-136. 11 Ezure, K., Cohen, M.S. and Wilson, V., Response of semicircular canal afferents to sinusoidal polarizing currents implications for the input-output properties of second-order neurons, J. NeurophysioL, 49 (1983) 639-648. 12 Goldberg, J.M. and Fernandez, C., The vestibular system. In: Handbook of Physiology. The Nervous System, Sect. 1, Vol. I11,
13 14
15
16
17 18 19
Chapt. 21, Am. Physiol. Soc., Bethesda MD, 1984, pp. 9771022. Goldberg, J.M., Fern,Sndez, C. and Smith, C.E., Responses of vestibular.nerve afferents in the squirrel monkey to externally applied galvanic current, Brain Research, 252 (1982) 256-160. Goldberg, LM., Smith, C.E. and Fernandez, C., Relation between discharge regularity and responses to externally applied galvanic currents in vestibular nerve afferents of the Squirrel Monkey, J. Neurophysiol., 51 (1984) 1236-1256. Goldberg, J.M., Desmadryl, G., Baird, R.A. and Fernandezi C., The vestibular nerve of the Chinchilla. IV. Discharge properties of utricular afferents, J. Neurophysiol., 63 (t990, a) 781790. Goldberg, J.M., Desmadryl, G., Baird, R.A. and Fernandez, C., The vestibular nerve of the Chinchilla. V. Relation between afferent discharge properties and peripheral innervation patterns in the utricular macula, J. NeurophysioL, 63 (1990, b) 791-804. Honrubia, V., Hoffman, L.E, Sitko, S. and Schwartz, I.R., Anatomic and physiological correlates in bullfrog vestibular nerve, J. Neurophysiol., 61 (1989) 688-701. Lannou, J., Preeht, W. and Cazin, L., The p~matal development of functional properties of central vestibular neurons in the rat, Brain Research, 175 (1979) 219-232. Lifschitz, W.S., Responses from first order neurons of the horizontal semicircular canal in the pigeon, Brain Research, 63 (1973) 43-57.
143 20 Lowenstein, O., The effect of galvanic polarization on the impulse discharge from sense ending in the isolated labyrinth in the thornback ray (Raja elavata), J. Physiol., 127 (1955) 104117. 21 Mbiene, J.P. and Sans, A., Differentiation and maturation of the sensory hair bundles in the fetal and postnatal vestibular receptors of the mouse a scanning electron microscopy study, J. Comp. Neurol., 254 (1986) 271-278. 22 Nordemar, H., Embryogenesis of the inner ear. II. The late differentiation of the mammalian crista ampullaris in vivo and in vitro, Acta Otolaryngol., 96 (1983) 1-8. 23 Nordemar, H., Postnatal development of the vestibular sensory epithelium in the mouse, Aeta Otolaryngol., 96 (1983) 447-456. 24 Romand, R. and Dauzat, M., Modification of spontaneous activity in primary vestibular neurons during development in the cat, Exp. Brain Res., 45 (1982) 265-268.
25 Romand, R., Sans, A., Romand, M.R. and Marty, R., The structural maturation of the stato-acoustic nerve in the cat, J. Comp. Neurol., 170 (1976) 1-15. 26 Schwartzkroin, P.A., to slice or not to slice. In G.A. Kerkut and H.V. Wheals (Eds.), Electrophysiology of Isolated Mammalian CNS preparation, Academic Press, New York, 1981, pp. 16-45. 27 Smith, C.E. and Goldberg, J.M., A stochastic after-hyperpolarization model of repetitive activity in vestibular afferents, Biol. Cybern., 54 (1986) 41-51. 28 Walton, K. and Fulton, B.P., Ionic mechanisms underlying the firing properties of rat neonatal motoneurons studied in vitro, Neuroscience, 19 (1986) 669-683. 29 Ziskind-Conhaim, L., Physiological and morphological changes in developing peripheral nerves of rat embryos, Dev. Brain Res., 42 (1988) 15-28.