0306.4522,‘92 $5.00 + 0.00

.Yrur-rw,~w~eVol 51. No. 3, pp. 621 -630. 1992 Printed 111Great Britain

DEVELOPMENT COCHLEAR M. A.

Per~amon Press Lid

t

OF Na+GANGLION

VALVERDE,*

AND K+-CURRENTS IN THE OF THE CHICK EMBRYO

D. N. SHEPPARD.*

*Departamento de Bioquimica y Biologia Morfolbgicas. Facultad de Medicina.

1992 IBRO

J. REPRESA~

and F.

GIRALDEZ*$

Molecular y Fisiologia and i‘Departamento Universidad de Valladolid, 47005.Valladolid.

de Ciencias Spain

development of Na + - and K + -currents m the primary afferent neurons of the cochlear ganglion was studied using the patch-clamp technique. Cells were dissociated between days 6 and I7 of development and membrane currents recorded within the following 24 h. Outward currents were the first to appear between days 6 and 7 of embryonic development and their magnitude increased throughout development from 200 pA on day 7 to 900 pA on days 14- 16. Threshold for activation decreased by 20 mV between days 8 and 14. Outward currents were absent when Cs + replaced K + in the pipette and were partially blocked by external tetraethylammonium. Outward currents contained at least three components: (i) a non-inactivating outward current, similar to the delayed-rectifier. predominating in mature neurons: (ii) a slowly inactivating current (tau about 200 ms). most evident in early and intermediate stages (days 7 IO); and (iii) a rapidly inactivating outward current (tau about 20ms) similar to the A-current (I,) described in other neurons. which was distinctly expressed in mature neurons. Sodium currents were identified as fast transient inward currents, sensitive to tetrodotoxin and extracellular Na’ -removal. The) appeared later than K + -currents and increased in size from about 100 pA between days 9 I I to 600 pA by days l3- 16. The development of membrane currents in cochlear ganglion neurons corresponded to delined stages of the innervation pattern of the chick cochlea [Whitehead and Merest (1985) h’euro.vci~nce 14, 255 -2761. These currents could be functionally related to the establishment of synaptic connections between transducing cells and primary afferent neurons. Abstract-The

The cochlear ganglion contains the primary afferent neurons of the vertebrate auditory system. Cochlear (acoustic) neurons are bipolar. with a central process that synapses either in the cochlear or tangential nucleus of the brainstem and a peripheral process that terminates on the sensory epithelium of basilar papilla of the inner ear. Morphological studies have shown that there are at least two types of cochlear ganglion neurons. About 95% are type I, which are large. myelinated and bipolar with a round nucleus. The remaining 5% are Type II, which are less than half the sizz, unmyelinated and pseudo-monopolar with a lobulated nucleus.“’ The cochlear and vestibular ganglia develop embryonic days 2-3 (E2-3) as a single unit, the cochlcovestibular ganglion (CVG). The CVG is a mixture of cells with different embryological origins, the majority of the neurons arise by migration from the otic vesicle (large-sized cells) while all support. satellite and Schwann cells are derived from the cephalic neural crest (small-sized cells).’ As development proceeds (E4&5) the CVG splits into distinct cochlear and vestibular ganglia. Evcntually ganglion cells develop peripheral processes and the innervation of the sensory epithelium begins

around E6 in the chick, by which time most ganglionic cells have performed their terminal mitosis.” It proceeds in parallel with hair cell maturation to form the first synaptic contacts by E8-9 and the mature synapsis by El 5-l 7.‘” The electrophysiological properties of fully developed chick cochlear ganglion neurons have recently been studied by Yamaguchi and Ohmoriz5 but no information is yet available on the ontogeny of these properties. The development of membrane excitability has been studied in several neuronal systems and the sequence of appearance of different ionic currents varies from neuron to neuron.7.‘x.” This might be related to their functions throughout development and/or to how the specific cellular connections emerge. The aim of this work was to describe the development of membrane ionic currents in isolated chick cochlear ganglion neurons, using the patch-clamp technique. The results show the early appearance of inactivating outward currents followed by Na ’ currents and fast transient outward currents (A-currents). This shapes a distinct pattern of expression of voltage-dependent currents whose significance will be discussed in connection with the innervation pattern.

:To whom

correspondence should be addressed. PBS, Ca’+- and Mg2+-free phosphatebuffered saline; CR, chick Ringer; CVG. cochleovestibular ganglion; E, embryonic day; EGTA, 5-ethyleneglycol-bis(fl-aminoethyl ether)N,N,N’,N’-tetra-acetic acid; PBS, phosphate-buffered saline; PBSBSA. PBS supplemented with bovine serum albumin.

Ahhrwicr/ionv: CMF

EXPERIMENTAL

PROCEDURES

Cochlear ganglia were isolated from the membranous labyrinths of chick embryos ranging in age from Eh to El 7. Embryos were removed from the egg, placed in Hank’s 621

622

U. :\

\‘hl.vI.KI)t. C’, r,/

balanced salt solution and staged. The cephahc region was split by a mid-sagittal section and the brain removed to expose the petrous portion of the anlage of the temporal bone. The labyrinth was then isolated and the otic capsule of the cochlear duct opened longitudinally in order to dissect the cochlear part of the cochieo-vestibular ganglion. The dissection is illustrated in Fig. IA. Dissected ganglia were washed in Ca’ ’ and Mg’ ’ -free phosphate buffered saline (CMF-~PBS) and incubated in CMF-PBS containing 0.5% disoase for 30 min at 37’C with gentle agitation. Isolated cells w&e released by gentle trituration and were washed twice in Hank’s balanced salt solution supplemented with 20% fetal calf serum. The dissociated cell suspension was plated onto polylysine collagen coated glass coverslips and enriched for neurons by differential attachment.“ Dissociated neurons were maintained at 37’C and used for electrophysiological recording within 24 h of isolation. Solutims

The composition of the standard pipette solution was (in mM): 10 NaCl; 150 KCl; 10 HEPES; 5 EGTA; 15 KOH, pH 7.2. Neurons were bathed in a Na ‘-rich physiological solution (chick Ringer, CR) (in mM): 160 NaCl; 5 KCl; 1 MgCl,; 2.5 CaCI,; 10 HEPES; 17 glucose, pH 7.4. In Na ’ replaced by choline. free solutions, Na+ was isotonically

Neurons plated onto polylysme ~collagen coaled glass c‘o\~_: slips were placed inside a chamber of volume 0.5 cm.’ I or electrophysiological recordings the culture medium \\:I, substituted by CR and the chamber transferred lo the stage of an inverted microscope. Neurons were virwcd using phase-contrast optics at a total magnification of * 400. The bath was perfused with test solutions flowing under graver) and waste removed by a vacuum pump. All solutions were filtered through 0.2 pm Millipore filters and experiments conducted at room temperature.

Patch-pipettes were fabricated from thin-walled borosillcate (hard) glass capillary tubing of outside diameter I .5 mm (Clark Electromedical Ltd. U.K.) using a two-stage vertical pipette puller (pp-83 Narishige. Japan). The tips of pipettes were polished using a microforge before back-filling with intracellular solution. Patch-pipettes used for whole-cell recording had resistances of 1 4 MR when filled with K ’ rich intracellular solution. Whole-cell currents were recorded according to the method of Hamill e/ ul..’ using a Biologic RK 300 patch-clamp amplifier (Biologic. France). Seals were achieved by applying light suction to patchpipettes pressed gently against neurons. Formation of a satisfactory seal was observed as a large increase in

B

Fig. 1. (A) Isolation of the cochlear ganglion. (a) Lateral view of the inner ear primordium isolated from a 12-day-old chick embryo. Cochlear (C) and vestibular (V) portions of the membranous labyrinth are discernible across the otic capsule. (b) Dorsal view of a microdissected cochlea, showing both cochlear ganglion and cochlear duct (arrowheads). (c) Isolated cochlear ganglion following separation from the mediodorsal wall of the cochlear duct. Scale bar = 200 pm. (B) Immunocytochemical recognition of neurofilament proteins in developing cochlear cells. Phase-contrast (upper photograph) and fluorescence (lower) images of dissociated cells from a ltday cochlear ganglion of chick embryo are shown. Cells are stained after electrophysiological recording with an anti- 160,000 mol. wt neurofilament antibody and processed for immunocytochemistry as described in Experimental Procedures. Both large placode-derived neurons (arrows) and small crest-derived support cells (arrowheads) were observed. Note that only large cells show positive neurofilament immunoreactivity.

Ionic currents

in developing

resistance as measured by the current response to small voltage pulses. Seals of lo-40 CR were routinely obtained. Following appropriate compensation of the fast component of the capacitive current further suction was applied to destroy the membrane patch and achieve the whole-cell recording configuration. The series capacitance was then adjusted to compensate for this additional (whole cell) capacitive current, Cells were clamped at a holding potential (typically --80 mV) and membrane currents in response to depolarizing and hyperpolarizing voltage steps measured. The established sign convention was used throughout. That is, ionic currents produed by positive charge moving from intra- to extracellular solutions (anions moving in the opposite direction) are shown as positive currents. The bath electrode consisted of a Ag:AgCl pellet connected to the bathing solution via an agar bridge filled with CR. Liquidjunction potentials following bath solution changes were determined and the currenttvoltage relationship corrected for the measured offset.

A microcomputer and two suites of programmes written by J. Dempster (Department of Physiology and Pharmacology. University of Strathclyde, Glasgow, U.K.) were used for pulse generation, data acquisition and analysis. Voltage pulse protocols were applied to the pulse input of the patch-clamp amplifier following digital to analogue conversion using a CED 1401 laboratory interface (Cambridge Electronic Design Ltd, U.K.). The signal from the patch-clamp amplifier was simultaneously viewed on a storage oscilloscope and recorded together with triggering pulses using a digital tape recorder (DTR-1200 Biologic, France) for subsequent analysis. Replayed records were analysed “off line”. The signal was filtered at 0.5-5 kHz using a variable g-pole Bessel filter (Frequency Devices Inc., U.S.A.) and digitized at I-IO kHz using the CED 1401 laboratory interface. Data shown have not been capacitanceand leak-subtracted unless indicated. Results are expressed as means + S.E.M. of rr observations.

Cells on coverslips were fixed for 2 h at 4 C in 4% (w/v) pamformaldehyde in phosphate-buffered saline (PBS). pH 7. and washed three times with PBS supplemented with bovine serum albumin (PBSBSA) for IOmin at room temperature. The coverslips were treated with 0.5% Triton X- 100 in PBS-BSA for IO min, rinsed with PBS-BSA and incubated for I2 h at 4’C with a rabbit antiserum

cochlear

623

neurons

against 160,000 mol. wt neurofilament, diluted I : 100 in PBSBSA. Controls were treated with pre-immune rabbit serum diluted in I : 100 in PBS-BSA. The coverslips were then rinsed in PBSBSA and incubated with a fluorescein isothiocyanate-labelled goat anti-rabbit IgG (Gibco), diluted 1:50 in PBS-BSA for 2 h at room temperature. The coverslips were then mounted for photography with DAKO-glycerol (Fig. I B). Morphometrical measurements of neuronal surface were done on coverslips at seven. 10, 12 and 16 days of development. Three squared fields were chosen at random and IO cells were measured on each.

RESULTS The results that follow were obtained from 107 cells, dissociated from the chick embryo cochlear ganglion. The earliest stage explored was E6, which corresponds to the end of the mitotic period and the beginning of the invasion of the sensory targets. The latest cells examined were large, 15-20-pm diameter neurons of E17. exhibiting the adult mosaic of membrane currents described by Yamaguchi and Ohmori.‘s Currents were recorded from cell cultures enriched in neurons which were recognized by their characteristic shape and size.“‘,” Large cells that fitted placode-derived neuron relative size and Kiang’s description of type I ganglion neurons’” were chosen for electrophysiological recording while remaining small-sized cells (support neural-crest-derived cells) were systematically discarded. Immunocytochemical recognition of 160,000 mol. wt neurofilament proteins in dissociated cells were performed in selected coverslips in order to further assess the neuronal phenotype of large-sized recorded cells. A dissociated culture containing large type I neurons, which were positively labelled with 160,000 mol. wt neurofilament antibody throughout the entire period under analysis, as opposed to small non-neuronal cells which were devoid of immunofluorescence is shown in Fig. 1. Changes in the resting membrane potential (open circles) and input resistance (filled circles) recorded throughout development are illustrated in Fig. 2.

65 z

60

*;o

55

5 ‘d a

5o

,D g

45

II E

40

s H

35 4

6

6

10

12

14

16

18

Days of Development Fig. 2. Relationship days of development.

between membrane potential (open circles) and input resistance (filled circles) with Values are means i S.E.M. n = 8821. The lines through the data have been drawn by eye.

624

M. A. VALVEKDE YI ul E 10

_A -43mv

E 16

OmV

$“--7-

u

-

_60mv&

20 rns

Fig. 3. Development of membrane excitablity of cochlear neurones. Current clamp records from isolated

cochlear neurons at the stage of development indicated by the day. Positive and negative square current pulses of 20 pA from a zero-current baseline were applied and membrane potential recorded. The numbers to the left of each record indicate the zero-current potential, hence an estimate of the resting membrane potential.

Membrane potential rose gradually from -40 + 5mVonE7(n = 15)to -61+3mV(n = 12)onE16, whilst the input resistance fell between two- to threefold over the same period. The input resistance corresponds to the slope resistance calculated from the current-voltage relationship at very negative values, from -80 to - 120 mV. Changes in the input resistance, calculated in this way, are taken to reflect changes in cell size, occurring between E6 and E16, with which they show good correspondence.6*” For the period under study, actual measurements of cell size carried out on coverslips used for

l=lt60

-6O_ 50

recordings gave the following values (in pm2): 116 f 16; 257 + 33; 365 + 30 and 466 f 26 (n = 30 cells) for E7, ElO, El2 and E16, respectively. Representative current clamp records at three different developmental stages of development are shown in Fig. 3. Depolarizing and hyperpolarizing current pulses were in all cases 20 pA and the zero-current membrane potential is indicated at the left. E8 neurons showed strong rectification with sometimes a small spike activity. By ElO, spikes were clearly developed but small. At E14, regenerative activity with large overshooting spikes was clearly developed.

Ez+6o

-0o.w

PA

El4 E IO

Fig. 4. General pattern of membrane currents recorded in isolated cochlear ganglion neurons throughout development. Cells were clamped at a holding potential of -8OmV and voltage steps applied as illustrated. Calibration of records labeled El0 and El4 are Iike E7.5. Note the presence of inward transients, poorly resolved at this time-scale. Cells were bathed in a normal Ringer and patch-pipettes contained the Ca*+-free K+-rich intracellular solution. Records were performed as described in Experimental Procedures.

Ionic currents

in developing

cochlear

neurons

potential

(mV)

625

200 PA

c

Membrane -80

-120

0

-40

80

40

25

Fig. 5. Voitag~-activated currents. (A) Na+ currents recorded in an El6 cochlear ganglion neuron in response to the illustrated voltage protocol are shown. (B) Inhibition of Na‘ currents by I PM tetrodotoxin recorded in an E8.5 cell (bottom) or bath substitution of Na + ions in an El6 cell (top). Holding and test potentials were -80 and -22 mV. respectively, for both cells. Traces recorded in TTX or Na + -free-CR are indicated by arrows. Note the change in calibration bars. (C) Steady-state properties of Na + -currents. Current-voltage relationships of voltage-activated Na + -currents recorded in an E8.5 cell (filled circles) and El6 cell (open circles). Peak Na +-current values were plotted against the command potential. Holding potential was -80 mV.

Neurons recorded during these late stages showed a characteristic inflexion or “hump” in the falling phase of the action potential and also a distinct afterhyper-polarization which could not be detected in earlier stages of developemcnt. The general pattern of membrane currents observed throughout development is illustrated in Fig. 4. Membrane current families generated by similar voltageclamp protocols are displayed. Cells were routinely clamped at - 80 mV and command potentials between - 100 and + 60 mV in 20-mV steps were applied. Cells sampled on Eb showed very small linear or weakly outwardly rectifying currents. From E7 onwards, how-

Table

1. Nat

and K+ currents

Days of development E6-8 E9-12 El3-17

I,,,: 0 (0) 0 (0) 40 (95)

ever, outward currents were systematically present and they typically inactivated with time (E7.5 in Fig. 4). These currents increased in size throughout development and by E8 were accompanied by fast transient inward currents that also increased in magnitude over the following days. The typical current pattern of such intermediate developmental stage is illustrated by the El0 neuron shown in Fig. 4. Inward current transients, poorly resolved at the illustrated time-scale. correspond to sodium currents (see below). By El2-14. the basic pattern of currents did not differ strikingly from that of the adult.‘s The proportion of cells expressing each current type at different periods of development

throughout Iiii,) 36 (78) 17 (89) 19 (45)

development I,’ 38 (82) 16 (84) 42 (100)

of cochlear INn

22 (49) 18 (95) 42 (100)

Number

neurons of cells 46 19 42

Figures are number of cells recorded at each period and figures in brackets are percentage of occurrences. Current types identified (see text and Ref. 20) are &. fast transient outward current, I,,,. slow transient outward current, I,,. steady-state outward current and INa, sodium current.

626

M. A. 1200

-

1000

-

800

-

600

-

400

-

200

-

2 5 -

01

6

8

VALVERDE

10

et ul.

12

14

16

18

Days of Development Fig. 6. Relationship between membrane currents and days of development. Values are means 4 S.E.M.. n = 8-12, for Na-currents (triangles) and n = 8-20 for K-currents (circles). is given in Table 1. What follows describes in more detail the composition and developmental changes in these currents.

absolute current size to allow direct comparison (see below). Threshold for activation was in both cases -40 mV, and maximal current amplitude was reached at about - 20 mV. The extrapolated reversal potential was between +60 and f80 mV, which is in agreement with the value of the Na+ equilibrium potential (ENa= + 70 mV) calculated for the Na + concentrations in the pipette and bath solutions. Steady-state inactivation of the Na + -current also remained unchanged throughout development (results not shown). The absolute magnitude of Na +current, however, was very small before El 1 but increased steeply by E12-14. Peak Na +-current values are shown in Fig. 6 (triangles), where it can be seen that values were below 200 pA until E14-16 whereupon they increased to about 600pA. This reflected only in part the increase in cell membrane area due to the increase in cell size that occurs between days E6 and E16. When

Sodium currents

Depolarizing pulses in fully developed neurons generated fast transient inward currents with a threshold of activation of about -4OmV (Fig. 5). Activation of these currents was fast, occurring between 0.1 and 1 ms as it was their inactivation. Figure 5B shows that inward currents were blocked by 1 pm tetrodotoxin and suppressed by bathing the cells in a Na+-free Ringer solution. This indicates that these currents are generated by the activation of voltage-dependent Na +-channels ubiquitously present in excitable cells. Figure SC compares the current-voltage plots of Na +-currents recorded from young (filled circles) and mature (open circles) neurons. Plots were normalized for the difference in

A

C +50

-60

100

25

-90

-60

Membrone

200 PA

-30

potential

0

30

60

(mV)

2ms

Fig. 7. Voltage-activated outward currents. (A) Outward currents recorded in an El6 cochlear ganglion neuron in response to the ihustrated voltage protocol are shown. The cell was bathed in Na+-free CR and the patch-pipette contained the Ca ‘+-free K + -rich intracellular solution. (B) Membrane currents recorded in an El6 cochlear ganglion neuron bathed in CR using a CsCl-rich pipette filling solution. Voltage protocols as shown in A. (C) Peak current amplitude of E6-8 neuron (filled circles, n = 4) and El416 neurons (open circles, n = 6) were plotted against the command potential.

Ionic currents

Fig. El5 CR. the

in developing

cochlear

neurons

X. Slowly-inactivating outward currents. (A) Families of outward currents recorded in E8 (top) and (bottom) cells in response to the voltage protocol shown in the inset. Cells were bathed in Na’ -fret Note the change in scale of the calibration bars. (B) Fraction of total outward current representing slowly inactivating outward current plotted versus days of development. Values are means k S.E.M. of 11= 4~ 12 observations.

of Na +-current were referred to the membrane resistance and normalized to the maximal amplitude. Na ’ -channel density rose three-fold bctwecn EIO-12 and El4-16. For fully differentiated neurons with diameters of I5 pm, the corresponding value of current density was 88 x IO ’ A’crn-‘. Finally. inward currents were always observed in association with outward currents and never in isolation or before E7. values

input

A typical current family generated in Na ’ -free Ringer in an El6 neuron, is illustrated in Fig. 7A. Under these conditions only outwardly rectifying currents remained upon depolarization. These currents are seen to be activated at potentials positive

Fig. 0. Fast inactivating outward currents. Membrane currents recorded in an El6 cochlear ganglion neuron in response to the pre-pulse protocol illustrated in the inset. The asterisk indicates the current recorded after - 50 mV prepulse. Cell bathed in chick Ringer. Computer subtraction of currents shown above reveal the fast transient outward current component in isolation.

to -50 mV with a time-to-peak of between I and 2 ms. They incompletely relaxed to a tinal steadystate value. These currents were suppressed when intracellular K + was replaced by Cs + (Fig. 7B). and with the exception of the initial transient (see below). these currents were blocked by extracellular exposure to 20 mM tctraethylammonium (more than 60% inhibition in I2 cells tested), indicating that they were K +-currents. CurrentPvoltage plots for outward currents recorded in early (filled circles) and late (open circles) developmental stages are shown in Fig. 7C. The activation threshold was about -50 mV in young neurons but it was shifted to the left by about 20mV in mature neurons. The steady-state activation profile. however. was similar and typical of K+-currents. Like I,,,. the absolute magnitude of outward currents increased in size with development (Fig. 6. circles). Outward currents wet-c about 200 pA on E7 and reached I nA by E16. When referred to the input rcsistancc these values revealed a constant increase in channel density between E6 and E12. The corresponding current density for a typical day I6 cell of diameter IS Elm was 135 x IO- ’ A’cm I. Nine out of thirty-foul cells. recorded in CR, between E6 and E8.5 showed outward currents in the absence of detectable inward currents. K +-currents, therefore. precede Na ’ currents. The composition of the outward currents. however. was not simple and, furthermore, it changed throughout development. A slowly inactivating component was prominent in early and intermediate cells. E7- IO (Fig. XA. upper current family). This component was apparent at potentials positive to -20 mV. This current was less evident at later stages of development. The contribution of the slowly decaying current to the total outward current recorded throughout development is illustrated in Fig. 8B.

638

M. A

Fast transient outward currents. A-currenr

L’AI.VlXlX

t!y.w

Another distinct component of the outward current generated upon depolarization was a very fast activating-inactivating current, which resembled the A-current first described in molluscan neurons and subsequently in many other excitable cells.‘3 This current could be isolated by applying short (25550 ms) depolarizing pre-pulses. One such experiment is shown in Fig. 9, in a mature, El6 cell, where it can be observed that a depolarizing pulse to + 50 mV fails to activate the fast inactivating outward current when the membrane potential was held at - 50 mV (trace labelled with an asterisk) instead of - 100 mV. Note that the non-inactivating current shows much slower activation kinetics, reaching a plateau in about 10 ms. The subtracted record is shown in the lower part of Fig. 9 and illustrates the speed of activation and inactivation of the fast transient outward current, which was completed in about 10 ms. These currents were sometimes hidden behind the initial inward current transient. They were resistant to extracellular exposure to 20 mM tetraethylammonium and, moreover, they were sometimes revealed only after blocking large non-inactivating outward currents with tetraethylammonium. Finally, it is worth mentioning that the functional expression of this fast transient outward current appeared to be dependent on the substrate used for the cultures. Polylysine-coated coverslips seemed to dramatically reduce the occurrence of this current, as compared with collagen or collagen plus polylysinecoated coverslips. In parallel batches of cells between El 5 and El 7, fast decaying currents were recorded in 15 out of 17 neurons plated onto collagen-polylysine coverslips but only in four out of 18 in polylysine alone. This might explain the absence of evidence for transient outward currents in cochlear neurons reported in previous studies.‘5

DISCUSSION

The aim of this study was to trace the development of Na + - and K +-currents in the cochlear ganglion of the chick embryo. The results show a distinct pattern of expression of Na+- and K +-currents throughout development. The earliest currents recorded were outward, most likely K+-currents, that appeared at E6. Inward currents developed later and were voltage-activated Na + -currents, selectively blocked by tetrodotoxin. Na +-currents were never recorded in isolation and they were very small before El2 whereupon they sharply increased in density to reach the values of late embryonic life.25 A similar sequence consisting of voltage-dependent outward currents preceding the development of inward currents has been observed in Drosophila flight muscle,“~‘* grasshopper neurons’ and avian neural crest cells.’ Na+-currents are most probably responsible for the development of the rising phase of the action poten-

(‘I u/

tial present in current-clamp recordings at mtermcdrate stages (E8-~10) which was distinctly observed b! E14, when spike overshoot was close to the expected value of ENa (approx. + 70 mV). The “hump” in the declining phase of the action potential. seen at these late stages most probably reflects the expression ol Cal ’ -currents, the study of which is out of the scope of the present report. Outward currents contained at least three components which predominate differently throughout development: (i) a slowly activating, noninactivating current; (ii) a slowly inactivating outward current (tau > 200 ms at + 50 mv); and (iii) a rapidly inactivating outward current (tau < 20 ms at +50 mV). A detailed kinetic characterization of these currents is reported in the accompanying paper.‘” The co-existence of different types of K ’ -currents within a single cell has been consistently observed in several adult neurons. The non-inactivating component closely rcsembles the conventional delayed rectifier. Ik. present in most excitable cellsY,‘h which is believed to contribute mainly to action potential repolarization. The slowly inactivating outward current described here also shows strong similarities with K ’ -currents recorded in other cell types including embryonic dorsal root ganglion neurons.‘b,” The fast-activating outward current resembles the A-current (I,,) originally characterized in molluscan neurons by Connor and Stevens’ and Neher.” The population of neurons sampled in this work was homogeneous in terms of their electrical properties, most cells behaving similarly at a given stage of development. This is consistent with the morphology of the cells and with an homogeneous receptor population, since membrane properties of primary afferents are known to differ for different receptor types.’ Inactivating K +-currents are thought to modulate cell excitability by transiently hyperpolarizing the membrane.’ Such a mechanism can operate to control repetitive tiring and the efficacy of synaptic transmission.” Although little is known about the functional properties of the synapsis between hair cells and cochlear fibres, it is possible that inactivating currents play a role in sound transmission through the regulation of synaptic efficacy. In addition, synaptic efficiency could be regulating the development of the synaptic contacts. At the normal resting potential of mature neurons (approx. -6OmV) inactivating currents will be inactivated so that their contribution to the control of cell excitability should not appear until the cell is hyperpolarized, either as a consequence of the triggering of an action potential or by the effect of inhibitory neurotransmitters (see also Ref. 20). Finally, a correspondence can be traced between the development of membrane currents in cochlear neurons reported here and the innervation pattern of the chick cochlea described by Whitehead and Morest.24 On E&7, the peripheral endings of the cochlear neurons invade the otocyst. A fraction of the

629

Ionic currents in developing cochlear neurons

cells in the ganglion concomitantly begin to show voltage-dependent currents, primarily outward currents. Early synaptic contacts are established near E&9, in association with continued hair cell differentiation. This period is associated with the appearance of Na’ -currents, of small amplitude, although the dominating current types are inactivating outward currents. This picture continues through El0 and El2 which correspond to the intermediate stages of synaptogenesis. By El4-15, the mature synaptic structure of the cochlear receptor develops. Hair cells and synaptic endings are evident and this coincides with the adult mosaic of current types with fully developed Na +- and K + -currents found in mature neurons. It is interesting to note that whilst cochlear potentials can be evoked in chick embryos by EIO-1 I. central responses cannot be recorded until day 11 ,I4 Following El 5, central responses become more synchronized and shorter in duration. Embryonic days 14-15 are also a transitional period during which behavioural responses evoked by auditory stimulation can be first elicited.‘J Further changes in membrane properties may cxplain the increase in the maximum discharge rate after El 7.” In making these correlations, however, it

should be remembered that the experiments reported in the present paper were done on dissociated neurons whose properties are taken to reflect those occurring in z+vo. This assumption requires confirmation. The possibility exists that culture procedures select a particular subtype of neurone or that they induce or suppress currents, which are risks inherent to the technique.

CONCLUSlONS

The expression of Na ’ and K +-currents in embryonic chick cochlear ganglion neurons follows a precise temporal sequence. Predominant current types are associated in time with defined stages of the development of synaptic contacts between hair cells and primary afferent neurons. Current work is devoted to further studying this relationship. Acknor~/edXm2ents-This work was supported hy grants from the DGICYT (PB86/0357) and FIS (91’0361). D. N. Sheppard is the recipient of a Royal Society European Science Exchange postdoctoral fellowship and M. A. Valverde is a postgraduate student of the DGICYT. We thank J. Dempster (Strathclyde) for providing some of the software used in this work and Dr F. V. Sephlvcda and Gerard Mintenig for constructive criticism.

REFERENCES

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