361

Journal of Physiology (1991), 439, pp. 361-381 With 14 figures Printed in Great Britain

OPTICAL DETERMINATION OF IMPULSE CONDUCTION VELOCITY DURING DEVELOPMENT OF EMBRYONIC CHICK CERVICAL VAGUS NERVE BUNDLES

BY TETSURO SAKAI, HITOSHI KOMURO*, YUSUKE KATOH, HIROSHI SASAKIt, YOKO MOMOSE-SATO AND KOHTARO KAMINOt From the Department of Physiology and the tDepartment of Anatomy, Tokyo Medical and Dental University School of Medicine, Bunkyo-ku, Tokyo 113, Japan

(Received 28 August 1990) SUMMARY

1. Employing an optical method for multiple-site simultaneous recording of electrical activity, we have determined the conduction velocity in cervical vagus nerve bundles isolated from 5- to 21-day-old chick embryos, and investigated its developmental changes. 2. The preparations were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761), and action potential- (impulse-) related optical signals were elicited by brief stimuli applied to the end of the vagus nerve bundle with a suction electrode. Optical signals were recorded simultaneously from many contiguous regions using a 12 x 12-element photodiode array. 3. The optical signals spread with small delay from the site of stimulation. From the relationship between the delay and distance from the current-applying electrode, conduction velocities were estimated in each tested preparation: the conduction velocity was very small and increased monotonically from about 0-1 m s-1 at 5 days embryonic age to about 0-4 m s-' by hatching. The increase in the conduction velocity was closely related to a developmental increase in the diameter of the vagus nerve bundle. 4. In addition, we have examined the spread of electrotonic potentials. The space constant was very small (200-450 ,um) and increased as development proceeded. 5. Compound optical action signals having two distinct components were also recorded. They often appeared to be concentrated in the preparations from 8- to 12day-old embryos. The conduction velocity of the second component was slower than that of the first. We suggest that appearance of the second component reflects degeneration of a subset of axons resulting from 'neural cell death' during the development of the vagus nerve. Present address: Department of Neurobiology, Yale University School of Medicine, New Haven, CT 06510, USA. t To whom correspondence should be sent. *

362

T. SAKAI AND OTHERS INTRODUCTION

The answers to many questions in developmental neurobiology require an understanding of the generation of neuronal functions during development. However, analyses of early functional development in nervous systems have been difficult because the neurons and processes of interest are relatively inaccessible: the electrophysiological examination with microelectrodes of neurons and processes, which provides the most direct test of their membrane potential activity, is often difficult because of the small size and fragility of young embryonic cells. Therefore, in comparison with morphological studies, functional studies on the developing nervous systems are rare (e.g. Jacobson, 1979; Harris, 1981; Spitzer, 1981). Voltage-sensitive dyes permit non-invasive optical monitoring of cellular electrical events in systems where the use of microelectrodes is either inconvenient or technically impossible (for reviews see Cohen & Salzberg, 1978; Freedman & Laris, 1981; Salzberg, 1989). Furthermore, optical techniques can facilitate the simultaneous recording of cellular electrical events from many adjacent sites in a preparation (for reviews see Salzberg, 1983; Cohen & Lesher, 1986; Grinvald, Frostig, Lieke & Hildesheim, 1988; Kamino, 1990, 1991). We have introduced optical recording techniques to monitor electrical activity from early developing embryonic systems, such as hearts (Hirota, Kamino, Komuro, Sakai & Yada, 1985; Hirota, Kamino, Komuro & Sakai, 1987; Kamino, Komuro & Sakai, 1988; Kamino, Komuro, Sakai & Hirota, 1988; and for reviews see Kamino, Hirota & Komuro, 1989a; Kamino, 1990, 1991), ganglia (Sakai, Hirota, Komuro, Fujii & Kamino, 1985; Sakai, Komuro & Kamino, 1990), and brain stems (Kamino, Katoh, Komuro & Sato, 1989b; Kamino, Komuro, Sakai & Sato, 1990). We report here the determination of the conduction velocity of impulses during development of embryonic chick cervical vagus nerve bundles using a multiple-site optical recording method that employs a 144-element photodiode array and a voltage-sensitive dye. Although the conduction velocity is a rather 'old fashioned' parameter in modern physiology, it remains an important factor for analysing functional organization and architecture during the development of the nervous system. However, in developmental neurobiology and neurophysiology; investigations of the conduction velocity have been reported infrequently in recent times, primarily because of methodological limitations. Some of the results reported here have appeared in preliminary form (Sakai, Komuro, Sato, Katoh, Sasaki & Kamino, 1989). METHODS

Preparation8 Fertilized eggs of chicks (White Leghorn) were incubated in a forced-draft incubator (Type P-03, Showa Incubator Lab., Urawa, Japan) at a temperature of 37 °C and 60% humidity, and were turned once each hour. Cervical vagus nerve bundles were dissected from the 4- to 21-day-old chick embryos. The embryos older than 14 days were anaesthetized with ether. The isolated vagus nerve bundle was attached to the silicone (KE 106; Shinnetsu Chemical Co., Tokyo, Japan) bottom of a simple chamber by pinning it with tungsten wires. The preparation was kept in a bathing solution with the following composition (in mM): NaCl, 138; KCl, 54; CaCl2, 1-8; MgC12, 0.5; glucose, 10; and Tris-HCl buffer (pH 7-2), 10. The solution was equilibrated with oxygen. The

COND UCTION IN DEVELOPING VAGUS NERVE

363

sheath of connective tissue attached to the vagus nerve bundle was carefully removed in the bathing solution under a dissecting microscope. Potential-sensitive dye staining The isolated preparations were incubated for 15 min in the bathing solution to which was added 0-10-2 mg ml-' of a merocyanine-rhodanine dye (NK2761: Fujii, Hirota & Kamino, 1981; Kamino, Hirota & Fujii, 1981; Salzberg, Obaid, Senseman & Gainer, 1983), which was synthesized

*.......:....; [.....s. ;....

~~~~~~~~~~~~~~~~~~~~.

.................~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~......... ............. -.6

:~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.......... :::

H:''~~~~~~~~~~. 2'...............

..e':-' ....

::::..H..,:... > :....^,..

.

... ~~~~~~~~~~~~~..

. . . . ...... ^~~~~~~~~~~~~~~~~~~U

..W

.::::::::

M. ..

.

............

..

......

; =~ ~ ~ ~~~ .S

.:

.1

~~~Electrode :.

..

.:: .: .:

.: .at.the.image.plane of the objective Magnification.was.usually.x.25..The.transmitted.light.intensity

1-0mm Fig. 1. Photomontage of the view of a 9-day-old embryonic cervical vagus nerve bundle with a suction electrode. NG is the nodose ganglion. An example of the relative positions of the photodiode array is also shown: the simultaneous multiple-site optical recordings were made at several successive times by sliding the photodiode array longitudinally over several positions in the image of the vagus nerve, indicated by squares. .....mtedlgtitnst tteim g ln f h betv Magnificatio usually .5 by Nippon Kankoh Shikiso Kenkyusho Co., Okayama, Japan. (The nature of this dye has been previously described in detail: see Kamino et al. 1989a; Kamino, 1991.) After staining, the preparation was washed with several changes of normal bathing solution.

Electrical stimulation As illustrated in Fig. 1, the cut end of the vagus nerve bundle was drawn into a suction electrode fabricated from Terumo haematocrit tubing (VC-H075P; Terumo Co., Tokyo, Japan), which had been hand pulled to a fine tip (about 100 ,um in calibre) over a low-temperature flame.

Optical recording The optical recording system is basically similar to that described by Cohen & Lesher (1986), with slight modifications (Hirota et al. 1985; Kamino et al. 1989b). The preparation chamber was mounted on the stage of an Olympus Vanox microscope (Type AHB-L-1). Bright field illumination was provided by a 300 W tungsten-halogen lamp (Type JC-24V-300W, Kondo Sylvania Ltd., Tokyo, Japan) driven by a stable DC power supply (Model PAD 35-20 L, 0-35 V, 20 A, Kikusui Electronic Corp., Kawasaki, Japan). Incident light was made quasimonochromatic by a 702+13 nm interference filter (Type lF-W, Vacuum Optics Co. of Japan, Tokyo, Japan) placed between the light source and the preparation. A microscope objective (S plan Apo, 040 n.a.) and a photographic eyepiece formed a magnified real image of the preparation at the image plane.

364

T. SAKAI AND OTHERS

and photographic eyepiece was detected using a 12 x 12 square array of silicon photodiodes (MD-144-4PV; Centronic Ltd, Croydon). The image of the preparation was focused onto the photodetector array (see Fig. 1). The output of each detector in the diode array was passed to an amplifier, via a current-to-voltage converter. The amplified outputs from 127 elements of the detector were first recorded simultaneously on a 128-channel recording system (RP-890 series, NF Electronic Instruments, Yokohama, Japan), and then were passed to a computer (LSI-11/73 system, Digital Equipment Co., Tewksbury, MA, USA). The 128-channel data recording system is composed of a main processor (RP-891), eight I/O processors (RP-893), a 64 K word wave-memory (RP-892) and a videotape recorder. The program for the computer was written in assembly language (Macro-1) called by FORTRAN, under the RT-1 1 operating system (Version 5.0). All experiments were carried out at room temperature, 26-28 'C. RESULTS

Multiple-site optical recordings of conducted action potentials Figure 2 illustrates two examples of original recordings of optical signals elicited by electrical stimulations in the embryonic chick cervical vagus nerve bundles. One was obtained from the cervical vagus nerve bundle isolated from an 8-day-old chick embryo and another from one isolated from a 19-day-old embryo. They were stained with a merocyanine-rhodanine dye (NK2761). The 8-day-old embryo preparation was efferently stimulated by applying 60 ,uA, 50 ms, 1-0 Hz depolarizing square current pulses with a suction electrode, and the 19-day-old embryo preparation was efferently stimulated by applying 30 ,A, 50 ms, 1-0 Hz depolarizing square current pulses. The evoked optical signals were recorded simultaneously from several sites of the nerve bundle using a 12 x 12-element photodiode array with a 702+13 nm interference filter. Eight trials were averaged. Successively sliding the photodiode array on the image of the preparation, we carried the measurements out on five contiguous areas of the preparation (see Fig. 1). These optical signals varied with the wavelength of incident light, and they were eliminated at 610 nm where the voltage-

related optical signal disappears. This action spectrum indicates that the optical signals are indeed absorption changes originating only from changes in membrane potential (Komuro, Sakai, Hirota & Kamino, 1986). No signals were detected from unstained preparations. Accordingly, in the present experiment, voltage-dependent light-scattering changes (Cohen, Keynes & Landowne, 1972) were not considered. In the 8-day-old embryo nerve bundle, two rows (along the fibre bundle) of elements in the photodiode array recorded optical signals and in the 19-day-old embryo fibre bundle, four rows of the elements detected signals. The pattern of elements of the array corresponded to the diameter of the vagus nerve bundle. In the recording obtained from the 8-day-old embryo preparation, the magnitude of the optical signals was relatively large, in comparison with those of the 19-day-old embryo preparation. Each detector received light from many nerve fibres: signals are likely to represent the summed responses of simultaneously activated nerve fibres. We further assume that the linearity with membrane potential of the fractional change in transmitted light intensity found in other preparations (Cohen & Salzberg, 1978) holds for experiments in the embryonic vagus nerve bundle, and that the fractional signal size is proportional as well to the percentage of activated nerve fibres and to the amount of dye bound to the membrane (Orbach, Cohen & Grinvald, 1985). In the present experiments, current strengths which induced the maximum action

E_ _ _ _ ~ ~ -.r,- _

CONDUCTION IN DEVELOPING VAGUS NERVE

365

signals in each preparation were used. On the other hand, younger embryonic nerve bundles are histologically loose, so that the dye diffuses well into the tissue and binds well to the relatively large membrane area of the deep fibres. Therefore, it is not surprising that the younger the embryo, the larger the optical signals detected. 8-day-old embryo

1 2 3 4 5 6

E

--

r

D _-. _- __

.-.

G

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

-

.-

120_gm _

-_

--

_

-_--

. _

.11 J.l -01, -A. -k

-k -k A A A -k J\. JK

- --1- - -

--------

H

KKK)LkJJLJ

_KK

_, _1 _N

_k _~ _k _k _A

__________

----------1-2

6MA, 5 ms

19-day-old embryo

1 2 3 4 5 6 7 8 9 10 11 1213 14 1516 17 18 19 20 21 22 23242526 27 28 29 3031 3233 34 35 36 37 383940

A

--.

----

---

B ______---___________1----------

H-

.-

H

_._.__.ii,i i.E~~j ..__

KJ L

...

JLKJJILX KxXJX _

-.-

ms

._._

NAAAA

___,___--------_____

30 ,A, 5

4-

-

________

_

..

A

. ---.--

Ns.._

r- - - - ----

---

--9 -'% Ad 1n-3 ,Iv

T A

100 ms

Fig. 2. Two examples of optical recording of electrical spikes from embryonic chick cervical vagus nerve bundles. The upper recording was made in a nerve from an 8-dayold embryo, and the lower recording was made from a 19-day-old embryo. The 8-day-old embryonic nerve was efferently stimulated by applying depolarizing square-current pulses (6-0 IuA, 5 0 ms, 1 0 Hz) to the proximal cut end of the nerve bundle (right side of the recording), and the 19-day-old embryonic nerve was efferently stimulated by applying depolarizing square-current pulses (30 ,uA, 5 0 ms, 1 0 Hz) to the proximal cut end of the nerve bundle (left side of the recording). These optical recordings were obtained from x 25 magnified images of the preparation, so that one photodiode detected optical signals from a 56 x 56 ,m2 area of the preparation (assuming no light scattering or out-of-focus signals). Eight trials were averaged. The figure was constructed with five multiple-site simultaneous recordings using the 12 x 12-element photodiode array. Each group of signals surrounded with an outline corresponds to one recording with the photodiode array.

Effects of tetrodotoxin If the conducted optical signals reflect voltage-activated Na+-dependent action potentials, they should be blocked by the Na+ channel blocker tetrodotoxin (TTX). We therefore examined the effects of TTX on the evoked optical signals. One example is shown in Fig. 3. In this case, 40 ,uA, 50 ms, 1.0 Hz depolarizing square current pulses were applied to the vagus nerve bundle. When 20 jtM-TTX was applied, optical signals were completely blocked in the regions far from the currentapplying electrode. However, within, and close to the electrode, some optical signals remained. These residual signals gradually decreased in amplitude with distance from the electrode. Accordingly, this result suggests that the TTX-sensitive component is action potentials, and the TTX-insensitive component is due to the non-conducted electrotonic potential.

T. SAKAI AND OTHERS Figure 4 shows two examples of hyperpolarizing stimulation-induced optical signals. In Fig. 4A, the optical signals were obtained with hyperpolarizing 10 jPA, 5-0 ms current pulses. In the recording made in the normal solution, the optical signals are composed of a hyperpolarizing electrotonic potential-related component 366

Nodose Electrode ganglion

Vagus nerve

Brain stem

Control

JL JL J I K K 1- _N -

-

i- K K JL J

----

--

TTX

2.0 ,A, 5.0 ms

120 um 100 ms

Fig. 3. Effects of tetrodotoxin (TTX) on the optical spikes evoked by depolarizing stimulations with 2-0 ,uA, 5 0 ms, 1-0 Hz square-current pulses in a nerve from a 5-day-old embryo. TTX at 20 ,uM was added to the bathing solution. In the vagus nerve and brain stem, optical signals were completely blocked by TTX, but within and near the electrode (including the nodose ganglion), TTX-insensitive electrotonic potential-related optical signals remained. Other experimental conditions were the same as in Fig. 2.

and an anode-break excitation-related component. The anode-break excitationrelated component was completely blocked by 20 /tM-TTX, but the electrotonic potential-related component remained. The recording shown in Fig. 4B was made using a larger hyperpolarizing current (2-0 ,tA, 5-0 ms). In this recording, only electrotonic potential-related signals appeared, and they were not affected by TTX. Conduction velocity In Fig. 5, the time courses of evoked optical impulses detected from twelve different positions are compared. The signals were recorded from the 8-day-old (the left panel) and 19-day-old (the right panel) embryonic vagus nerve bundles. These recordings clearly show delays in the onset of the signals, which we presume to reflect the conduction time of the impulses. We have measured the delays (time to foot of the signals), and plotted them against the distance from the current-applying electrode. Four examples obtained from 5-, 8-, 12- and 19-day-old embryo vagus nerve bundles are shown in Fig. 6.

367 CONDUCTION IN DEVELOPING VAGUS NERVE The delay was related linearly to the distance. This linear relationship shows that the impulse conducts at a uniform rate longitudinally along the vagus nerve bundle. From the reciprocal of the slope of the straight line, the conduction velocity was obtained. In the preparations shown in Fig. 6, the conduction velocities were A Control

TTX

B Control

TTX

Electrode

120gm 100 ms

Fig. 4. Additional examples of the effects of TTX on the optical signals. A 5-day-old embryonic nerve was stimulated by applying 1l0 1tA, 5-0 ms, 1l0 Hz (A) and 2-0 1A, 5 0 ms, 10 Hz (B) hyperpolarizing square-current pulses. In A, both electrotonic hyperpolarizing signals and anode-break excitation signals were detected. The hyperpolarizing optical signals were not blocked by TTX (20 /tM) but the anode-break signals were completely blocked. In B, the hyperpolarizing electrotonic signals again remained in the presence of TTX. In the suction electrode, TTX-sensitive signals were observed. Other experimental conditions were the same as in Fig. 2.

estimated to be 0-114 m s-' for 5-day-old; 0 161 m s-' for 8-day-old; 0-281 m s-1 for 12-day-old; and 0-440 m s-1 for 19-day-old preparations. In comparison with adult C fibres (e.g. Brinley, 1980), these values are smaller (see Discussion), but of the same order of the value optically obtained in the unmyelinated parallel fibres in skate cerebellum reported by Konnerth, Obaid & Salzberg (1987).

Developmental changes in the conduction velocity In order to examine developmental changes in the conduction velocity in more detail, we measured the conduction velocities using over 100 preparations of 5- to 21day-old embryonic vagus nerve bundles. In Fig. 7, the measured conduction velocities are plotted against embryonic age. This graphic representation indicates that the conduction velocity increases linearly as the embryonic age proceeds: the conduction velocity increases about fourfold from 5- to 21-day-old preparations

T. SAKAI AND OTHERS

368

(corresponding to hatching time). The changes in the conduction velocity with the embryonic age increased monotonically, and no abrupt changes were observed during 5- to 21-day embryonic stages. Further, we examined differences in the conduction velocity between the right and left nerve bundles. However, no right-left differences were observed. Figure 7 8-day-old embryo

19-day-old embryo

F7

F40

F10

F36

F13

F33

F16

F30

F17

F26

F20

F23

F23

F20

F26

F16

F27

F13

F30

F10

F33

F6 F3

F36 6 IA

30 /A

10 ms

Fig. 5. Comparisons of the time course of optical signals detected from twelve different sites of the 8- and 19-day-old embryonic nerve bundles. These preparations and the experimental conditions were the same as those in Fig. 2.

includes data obtained from both the right (0) and left (0) vagus nerve bundles. We also examined the differences in the conduction velocity between afferent and efferent directions of stimulus to the vagus nerve bundles; there were no differences in the conduction velocity between the two directions (data not shown).

CONDUCTION IN DEVELOPING VAGUS NERVE

10.0

8.0

E 6.0

a 4.0

2.0

1000 Distance (pm) Fig. 6. The delay in the firing time (the foot) of the optical spike as a function of distance from the stimulus electrode. Data were obtained from 5- (O), 8- (O), 12- (A), and 19-dayold (V) embryonic vagus nerve bundles. The feet of the optical signals were measured by extrapolating the upstroke of the signal to the baseline.

0.5 r

80

0.4 0 o

E

0.3

0

o0o oo1

[-

0

0

0

CJ

0 ._

0

0.2

*

8

o 8

10 C

0

o

u

0.1-

0

o

a

Hatching 0

10 20 15 Embryonic age (days) Fig. 7. Conduction velocities plotted against embryonic age. Each point corresponds to one preparation. The data obtained from the right vagus nerve bundles are indicated by 0, and * indicates the data obtained from the left vagus nerve bundles. 5

369

T. SAKAI AND OTHERS

370 300 r

0

0 0 0

8

250 0

8

0 0 0 0 0

:,

200 1

Oo

-

0

a)

000

8

.0

o 8

0

80 8 8 00 o0

o8

150 1

0

0

a)

E 100

0 0

0

[-

8o

@8

8

0

50 -

Hatching 0

20 15 10 Embryonic age (days) Fig. 8. Diameters of the vagus nerve bundle plotted against embryonic age. Each point corresponds to one preparation. 5

0.5 r 0 0 0

0.4 FoRo

E

.000090

0.3H

0 0

0

0

o0o 8

8

0

0

*0

00 0

0

0

c

C-.)_

8

0

0.2k

0 ( 0 0 0 Zo 0

0 c

0

0

0-1k

6

00 0

8 0 0

I

250 200 300 100 150 Diameter of nerve bundle (jum) Fig. 9. Conduction velocities plotted against diameter of vagus nerve bundles. Data were obtained from 5- to 21-day-old embryonic vagus nerve bundles. Each point corresponds to one preparation.

0

50

CONDUCTION IN DEVELOPING VAGUS NERVE

37i1

Conduction velocity-bundle size relationship We measured the diameters of 4- to 21-day-old embryonic vagus nerve bundles under a light microscope. In Fig. 8, the diameters are plotted against embryonic age. The diameter increased dramatically as embryonic age proceeded. This graphical 1.0

0.8 ._Ln

.50) 0.6 0

._A

E ._

E 0-4 0

z

0-2

0

0

200

400

Distance

600

(gum)

Fig. 10. Relationship between the signal size related to hyperpolarizing electrotonic potentials and distance from current-applying electrode. Data were obtained from a 5and a 15-day-old embryonic vagus nerve. Optical signals were elicited by hyperpolarizing square-current pulses (4 0 ,1A, 5 0 ms, 1-0 Hz for the 5-day-old embryo preparation and 4-0 1sA, 5-0 ms, 1.0 Hz for the 15-day-old embryo preparation), under conditions in which anode-break excitations were not elicited. The signal size is normalized to the maximum value. In these preparations, the space constants (distance at signal size = 1/e) were estimated to be 228 ,tm for the 5-day-old embryo preparation and 408 ,um for the 15-dayold embryo preparation. Five-day-old embryo preparations are shown by *, 15-day old embryo preparations by 0.

feature is similar to that of the conduction velocity shown in Fig. 7. Thus, there seems to be a correlation between the conduction velocity and the diameter of the embryonic nerve bundle. In Fig. 9, the conduction velocities are plotted against the measured diameters of vagus nerve bundles. As expected, the conduction velocity is closely correlated with the diameter of the nerve bundle.

The spread of electrotonic potential As shown in Fig. 4, when hyperpolarizing square-current pulses were applied to the nerve bundle, the spread of optical changes related to electrotonic potential was clearly observed. In order to see the characteristics of the electrotonic potential

T. SAKAI AND OTHERS

372

spread in the embryonic vagus nerve bundles, we plotted the measured amplitude of the electrotonic optical changes as a function of the distance from the hyperpolarizing current-applying electrode. In Fig. 10, two typical examples of the plots constructed from 5- and 15-day-old embryonic vagus nerve bundles are shown. Electrotonic potential-related optical 500 0

8

0

0

0

400

_0

0

0

0

8 0 0 0

0

0

00 0

080

0

$300

0

0 0

0

~~~00

CoO C 0

o0

o 200 _ cn

100

5

10 15 Embryonic age (days)

20

Fig. 11. Space constants plotted against embryonic age. The data were obtained with hyperpolarizing stimulations (under the conditions in which anode-break excitations were not elicited), in 4- to 21-day-old embryonic vagus nerve bundles. Each point corresponds to one preparation.

signals spread longitudinally along the nerve bundle, and it decreased exponentially with distance. In these preparations, space constants were estimated to be 228 ,um for the 5-day-old embryo bundle and 408 ,um for the 15-day-old embryo bundle. Figure 11 shows the correlation between the measured space constant and the embryonic age. Although the data are scattered, the space constant appears to increase with embryonic age.

Compound action ignal8 In addition to signals with a single peak, more complex signals, displaying two components, were also recorded. An example of the original recording obtained from a 10-day-old embryonic vagus nerve bundle is shown in Fig. 12. In this recording, the first and second components become increasingly separated in time with distance from the current-applying electrode. As shown in Table 1, such two-component

CONDUCTION IN DEVELOPING VAGUS NERVE A A A_ At AQA_9 A_ Ah _\ A 4 s

1000 Distance (jm)

1500

373

_o A

200

100 ms

Fig. 12. Compound optical action signals with two components recorded from a 10-dayold embryonic vagus nerve bundle. The signals were evoked by applying 12 ,uA, 5 0 ms, 10 Hz depolarizing current pulses. Distance is measured from the end of the currentapplying suction electrode. Experimental conditions were the same as in Fig. 2. Circles, see Fig. 13. TABLE 1. Incidence of single and compound action signals at every embryonic age Embryonic age Single Compound (days) action signals action signals 5 3 None 6 5 None 7 7 None 1 8 5 9 3 3 10 2 8 11 3 3 12 2 7 13 8 None 14 8 None 15 5 None 16 5 None 2 17 None 18 6 None 19 7 None 20 8 None 21 5 None

action signals appeared exclusively in preparations obtained from 8- to 12-day-old embryos. In addition, the second signals were often eliminated with weaker stimulating currents: the threshold value of the strength of the stimulating current needed to produce the second component is larger than that for the first component (data not shown). Figure 13A shows enlargements of the five compound action signals shown in Fig. 12. The action signals recorded at different distances from the site of stimulation are arranged according to the distance. Diagonal straight lines were then drawn through the onsets of the first component and through the peaks of the first and second signals. From the slopes of these straight lines, the conduction velocity of each component was estimated: in this preparation, values of 025 m s-I for the first signal and 0 11 m s-' for the second signal were obtained. The conduction velocity of the second signal was about half as fast as that of the first signal. As stated above, in the present optical recording of impulses in the nerve bundle, one photodiode detected simultaneously the impulses from many nerve fibres. The

T. SAKAI AND OTHERS

374

optical signals are the algebraic sum of individual fibre action potentials. Therefore, by analogy to the compound action potential of a nerve trunk (Patton, 1982), one hypothesis may be formulated to explain the bimodal contour of the compound action signal: the groups of fibres may conduct impulses at different speeds so that AB bt 500 C

~~~~0.30

a

EC;

0 20000

1000

0

0 0

11

12

0

0

~~~~~~~Eo0.2

E

o

9

10

~~~~~~~0

0C

o

o

-

C~~~~~~~~~~~~~~~~0 CC ~~0 I

C

C

20OMs

0~~~~~~~~~~

~~~~~0

8

Embryonic age (days)

Fig. 13. A, Enlarged illustrations of five sampled compound optical action signals from Fig. 12 (labelled by circles). The optical traces are arranged according to their distance from the electrode. The diagonal straight line a was drawn through the onsets of the first component, line b was drawn through the peaks of the first component, and line c was drawn through the peaks of the second component: the slopes of these lines give the conduction velocities of the most rapidly conducting populations of fibres. B, conduction velocities of the first (0) and second (@) components plotted against embryonic age (days). Each point corresponds to one preparation.

arrival time at the detecting photodiode is different for impulses in different groups within the nerve bundle. On this assumption, we suggest that the first and second signals reflect the condition that impulses beginning together at the stimulation site become temporally dispersed as the conduction distance increases because they traverse fibres with different uniform conduction speeds. Also, this result suggests that two populations of fibres with different continuous spectra of conduction velocities are comprised in the developing vagus nerve bundle at 8- to 12-day-old embryonic ages (also see Discussion). In Fig. 13B, the conduction velocities of the first and second components are plotted against embryonic age. In contrast to the first component, the conduction velocity of the second component did not appear to change with embryonic age.

CONDIUCTION IN DEVELOPING VAGUS NERVE

375

DISCUSSION

In the present experiments, using the multiple-site optical recording method and a voltage-sensitive dye, we measured the conduction velocity of impulses in the embryonic nerve fibres, perhaps for the first time. Conventional electrophysiological methods for measurement of conduction velocity cannot usually provide simultaneous recording from more than two sites on the nerve fibre. Multiple-site optical recording allowed us to measure the firing times of impulses at many adjacent points along the nerve fibre, and we found that there are no regional differences in the conduction velocity in the embryonic vagus nerve fibre. The conduction velocity increased dramatically and monotonically with embryonic age (Fig. 7). In electrophysiology, it is well known that several factors determine the velocity of impulse conduction in excitable fibres (Hille, 1989). Of these, the following seems relevant to the impact of developmental stage on conduction velocity: (i) the larger the cross-sectional area of individual nerve fibres, and the higher the concentration of highly mobile intracellular ions, the greater the current flow for a given voltage: large diameter axons conduct faster; (ii) an increase in the number of available Na+ channels would increase conduction velocity; (iii) a reduction in the amount of depolarization required to reach threshold would increase conduction speed; and (iv) myelination allows rapid conduction with smaller fibre diameter. In the present experiments, the following results were obtained: (1) the crosssectional area of the embryonic vagus nerve bundle increased as embryonic age proceeded from 4-21 days (corresponding to the time of hatching) (Fig. 8). (2) The developmental changes (increases) in the cross-sectional area were parallel with those of the conduction velocity (Figs 7 and 8). (3) The conduction velocity was closely correlated with the cross-sectional area of the bundle (Fig. 9). As shown in Fig. 14, the bundle includes many nerve fibres with various diameters. Here, it is reasonable to assume that the diameter of the bundle depends on the average diameter and the number of nerve fibres in the bundle, and other tissues, such as fibroblasts and connective tissues. Of these components, the diameter of the nerve fibres is a parameter which is directly related to the conduction velocity. Accordingly, it is likely that the principal factor in the increase in the conduction velocity is the increased diameter of the nerve fibre as a function of developmental stage. Indeed, the electron micrographs show that the diameter of the nerve fibres in the bundle increased as development proceeded (Fig. 14). The vagus nerve bundle contains both motor and sensory nerve fibres having various diameters. Duclaux, Mei & Ranieri (1976) identified the C and B fibres in the vagus nerve of the adult cat. However, in the embryonic vagus nerve bundle, the conduction velocity is slower than that of the adult C fibre, of which the conduction speed is the slowest among nerve fibres. Therefore, it is difficult to classify the embryonic nerve fibres according to the criteria of conduction velocity which have been used for adult nerve fibres. In nerve fibres without a myelin sheath, the space constant is proportional to the square root of fibre diameter if the electrical properties of the cytoplasm and membrane are constant. In the present experiment, apparently, the space constant

376

T. SAKAI AND OTHERS

100im

Fig. 14. For legend see facing page.

CONDUCTION IN DEVELOPING VAGUS NERVE

377

increases with embryonic age. Nevertheless, in comparison with the developmental increase in the conduction velocity, the rate of increase in the space constant seems to be relatively small. This behaviour may be due to developmental changes in the specific resistance of the cytoplasm and/or the membrane. These factors may also

1 .0 pm

Fig. 14. Electron micrographs of the cross-sections of the vagus nerve bundles isolated from the 5- (A), 10- (B) and 21-day-old (just after hatching: C) embryos. In A and B the bundle involves many small fibres with various sizes. No myelinated fibres were observed. In B and C the diameter of the nerve fibres evidently increases and, in C, some thicker myelinated fibres are present among the unmyelinated small fibres. The formation of myelination seems to be heterogeneous. Isolated vagus nerve bundles were fixed by immersion in Karnovsky's phosphate-buffered fixative (pH 7 3) (Karnovsky, 1965) for 2 h at room temperature, and postfixed with Palade's osmium (Palade, 1952) for 1 h at 4 'C. They were stained with 1 % uranyl acetate buffered to pH 6-0 with acetate veronal buffer overnight, dehydrated and embedded in Epon. Ultra-thin sections were cut on a PorterBlum MT2 ultramicrotome with a diamond knife and stained with lead citrate before examination with a JEOL-IOOB electron microscope.

partly account for the developmental changes in the conduction velocity itself. Also, we cannot rule out the possibility of developmental changes in the resting potential and in the threshold, but at present it is very difficult to examine these. In earlier stages of development, the vagus nerve fibres in the bundle are all unmyelinated (A and B in Fig. 14). In adult myelinated nerve fibres (e.g. Hille, 1989), the myelin sheath is composed of many layers of closely packed membranes and the myelin sheath is a good insulator. On the other hand, as pointed out by Hille (1982), myelination is one of the functional landmarks in the development of the nervous system. Indeed, the photograph shown in Fig. 14C indicates that, even at the

378

T. SAKAI AND OTHERS

hatching time, the myelination is poor and heterogeneous. (However, we have not yet examined systematically the embryonic origin of myelination.) In addition, in myelinated fibres, only the nodes of Ranvier would be stained with the voltagesensitive dye (Shrager & Rubinstein, 1990), and the fractional area of the stained nodes of Ranvier is presumed to be relatively small. Accordingly, we feel that in the present experiments, the component originating at the node of Ranvier is also very small. In unmyelinated fibres, the conduction velocity is proportional to a factor between the square root and the first power of the diameter, whereas in myelinated fibres it is directly proportional to diameter. As Hille (1989) stated, it is not possible to generalize about how much myelination increases conduction velocity, as other factors play a role. Also, in the present experiments on developing embryonic nerve fibres, it is not possible to determine quantitatively the percentage of the contribution of myelination to the developmental changes in the conduction velocity. As shown in Figs 3 and 4, the action potential in the embryonic vagus nerve depends upon voltage-sensitive Na+ channels which are blocked by TTX. In comparison with adult unmyelinated fibres, the conduction velocity is small considering the diameter of the nerve fibre. It is estimated to be about 041 m s-1 for about 0.5 jtm diameter for 5-day-old embryonic nerve fibres. This result suggests that the conduction velocity in the embryonic nerve fibres may be limited by the small number of available Na+ channels, and that a developmental increase in the number of Na+ channels contributes to the increase in the conduction velocity during development of the nerve fibre. In the results described here, an observation of interest is the fact that the twocomponent action signals appear to be concentrated in 8- to 12-day-old embryonic vagus nerve bundles. (As shown in Fig. 13, both the first and second component signals were conductive. Therefore, the second component in the present experiment is different from the 'slow component' which is associated with glial depolarization due to K+ accumulation, found in the rat optic nerves stained with a styryl dye (RH 414) (Lev-Ram & Grinvald, 1986).) Thus the present result suggests that the population of fibres comprising the vagus nerve bundle is divided into two groups according to the conduction velocity of impulses at the 8- to 12-day-old embryonic ages and that one group of fibres disappears by 13 days embryonic age: separation of the compound action signal into two peaks occurs because of the bimodal distribution of fibre size or activity during the 8- to 12-day-old embryonic ages, and, by the 13th day, one group disappears. One possible explanation is that the two groups of fibres reflect a population of normally growing fibres (axons) and a population of poorly growing fibres (axons), and that the poorly growing fibres are terminated by a process of neural cell death. Thus, in the compound action signal, the first component is due to action potentials in normally growing fibres and the second component reflects action potentials in poorly growing fibres. Based on the idea that conduction velocity depends on the cross-sectional area of axons and that extracellular stimulation first activates the large axons in a nerve and then progressively activates smaller ones, the first component originates in a group of healthy axons with developmentally programmed increases in their diameters, and the second component originates from a group of feeble axons on the verge of

CONDUCTION IN DEVELOPING VAGUS NERVE 379 elimination by 'neural cell death' (e.g. Purves & Lichtman, 1985). Accordingly, we suggest that the embryonic ages of 8-12 days seem to be a very critical period in the process of functional organization of the embryonic chick vagus nerve. Although, in the electron micrographs shown in Fig. 14B, morphological events are not observed distinctly, it may be said that the finding of the two-component compound action signal in the embryonic vagus nerve has revealed the functional consequences of 'cell death' during development of the nervous system. The degeneration of the axons would be related to various factors, for example, axonal transport function and neurotrophic factors. Here, it may be worth noting that in further analysis of cellular/subcellular factors related to 'neural cell death', a focus on the 8- to 12-dayold embryonic chick vagus nerve might prove fruitful. We are most grateful to Larry Cohen and Brian Salzberg for their thoughtful reading of the manuscript and many pertinent comments. We also express thanks to Akihiko Hirota for his comments on the manuscript. This work was supported by grants from the Ministry of Education, Science and Culture of Japan, the Suzuken Memorial Foundation, the Brain Science Foundation of Japan, Kowa Life Science Foundation and the Inoue Foundation of Science. REFERENCES

BRINLEY, F. J. JR (1980). Excitation and conduction in nerve fibers. In Medical Physiology, vol. 1, ed. MOUNTCASTLE, V. B., pp. 46-81. C. V. Mosby Company, St Louis, MO, USA. COHEN, L. B., KEYNES, R. D. & LANDOWNE, D. (1972). Changes in light scattering that accompany the action potential in squid giant axons: potential-dependent components. Journal ofPhysiology 224, 701-725. COHEN, L. B. & LESHER, S. (1986). Optical monitoring of membrane potential: methods of multisite optical measurement. In Optical Methods in Cell Physiology, ed. DEWEER, P. & SALZBERG, B. M., pp. 71-99. Wiley-Interscience, New York, USA. COHEN, L. B. & SALZBERG, B. M. (1978). Optical measurement of membrane potential. Reviews of Physiology, Biochemistry and Pharmacology 83, 35-88. DUCLAUX, R., MEI, N. & RANIERI, F. (1976). Conduction velocity along the afferent vagal dendrites: a new type of fibre. Journal of Physiology 260, 487-495. FREEDMAN, J. C. & LARIS, P. C. (1981). Electrophysiology of cells and organelles: studies with optical potentiometric indicators. International Reviews of Cytology, suppl. 12, 177-246. Fujii, S., HIROTA, A. & KAMINO, K. (1981). Action potential synchrony in embryonic precontractile chick heart: optical monitoring with potentiometric dyes. Journal of Physiology 319, 529-541. GRINVALD, A., FROSTIG, R. D., LIEKE, E. & HILDESHEIM, R. (1988). Optical imaging of neuronal activity. Physiological Reviews 68, 1285-1366. HARRIS, W. A. (1981). Neural activity and development. Annual Review ofPhysiology 43, 689-7 10. HILLE, B. (1982). Membrane excitability: Action potential and ionic channels. In Physiology and Biophysics, vol. iv, ed. GORDON, A., HILLE, B., PATTON, H. & WOODBURY, F. W., pp. 68-100. W. B. Saunders Co. Philadelphia, NJ, USA. HILLE, B. (1989). Membrane excitability: Action potential propagation in axons. In Textbook of Physiology, vol. 1, ed. PATTON, H. D., FuCHS, A. F., HILLE, B., SCHER, A. M. & STEINER, R., pp. 49-79. W. B. Saunders Co. Philadelphia, NJ, USA. HIROTA, A., KAMINO, K., KOMURO, H. & SAKAI, T. (1987). Mapping of early development of electrical activity in the embryonic chick heart using multiple-site optical recording. Journal of Physiology 383, 711-728. HIROTA, A., KAMINO, K., KOMURO, H., SAKAI, T. & YADA, T. (1985). Early events in development of electrical activity and contraction in embryonic rat heart assessed by optical recording. Journal of Physiology 369, 209-227. JACOBSON, M. (1979). Developmental Neurobiology, second edn. Plenum Press, New York.

380

T. SAKAI AND OTHERS

KAMINO, K. (1990). Optical studies of early developing cardiac and neural activity using voltagesensitive dyes. Japanese Journal of Physiology 40, 443-461. KAMINO, K. (1991). Optical approaches to the ontogeny of electrical activity and related functional organization during early heart development. Physiological Reviews 71, 53-91. KAMINO, K., HIROTA, A. & Fujii, S. (1981). Localization of pacemaking activity in early embryonic heart monitored using voltage-sensitive dye. Nature 290, 595-597. KAMINO, K., HIROTA, A. & KOMURO, H. (1989a). Optical indications of electrical activity and excitation-contraction coupling in the early embryonic heart. Advances in Biophysics 25, 45-93. KAMINO, K., KATOH, Y., KOMURO, H. & SATO, K. (1989b). Multiple-site optical monitoring of neural activity evoked by vagus nerve stimulation in the embryonic chick brain stem. Journal of Physiology 409, 263-283. KAMINO, K., KOMURO, H. & SAKAI, T. (1988). Regional gradient of pacemaker activity in the early embryonic chick heart monitored by multisite optical recording. Journal of Physiology 402, 301-314. KAMINO, K., KOMURO, H., SAKAI, T. & HIROTA, A. (1988). Functional pacemaking area in the early embryonic chick heart assessed by simultaneous multiple-site optical recording of spontaneous action potentials. Journal of General Physiology 91, 573-591. KAMINO, K., KOMURO, H., SAKAI, T. & SATO, K. (1990). Optical assessment of spatially ordered patterns of neural response to vagal stimulation in the early embryonic chick brainstem. Neuroscience Research 8, 255-271. KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. Journal of Cell Biology 27, 137A. KAMINO, K. (1986). Conduction pattern of excitation in the KOMURO, H., SAKAI, T., HIROTA, A. &Y amphibian atrium assessed by multiple-site optical recording of action potentials. Japanese Journal of Physiology 36, 123-137. KONNERTH, A., OBAID, A. L. & SALZBERG, B. M. (1987). Optical recording of electrical activity from parallel fibres and other cell types in skate cerebellar slices in vitro. Journal of Physiology 393, 681-702. LEV-RAM, V. & GRINVALD, A. (1986). Ca2+- and K'-dependent communication between central nervous system myelinated axons and oligodendrocytes revealed by voltage-sensitive dyes. Proceedings of the National Academy of Sciences of the USA 83, 6651-6655. ORBACH, H. S., COHEN, L. B. & GRINVALD, A. (1985). Optical mapping of electrical activity in rat somatosensory and visual cortex. Journal of Neuroscience 5, 1886-1895. PALADE, G. E. (1952). A study of fixation for electron microscopy. Journal of Experimental Medicine 95, 285-298. PATTON, H. D. (1982). Special properties of nerve trunks and tracts. In Physiology and Biophysics, vol. iv, ed. ROCH, T. & PATTON, H. D., pp. 101-127. W. B. Saunders Co., Philadelphia, NJ, USA. PURVES, D. & LICHTMAN, W. (1985). Principles of Neural Development. Sinauer Associates, Sunderland, MA, USA. SAKAI, T., HIROTA, A., KOMURO, H., FuJii, S. & KAMINO, K. (1985). Optical recording of membrane potential responses from early embryonic chick ganglia using voltage-sensitive dyes. Developmental Brain Research 17, 39-51. SAKAI, T., KOMURO, H. & KAMINO, K. (1990). Optical monitoring of evoked electrical activity in the embryonic chick superior cervical ganglion using a voltage-sensitive dye. Neuroscience Research, suppl. 10, S39. SAKAI, T., KOMuRO, H., SATO, K., KATOH, Y., SASAKI, H. & KAMINO, K. (1989). Developmental changes in conduction velocity of excitation in the embryonic chick cervical vagus nerve bundle assessed by multiple-site optical recording of action potential. Neuroscience Research, suppl. 9, S55. SALZBERG, B. M. (1983). Optical recording of electrical activity in neurons using molecular probes. In Current Methods in Cellular Neurobiology, vol. 3, Electrophysiological Techniques, ed. BARKER, J. L. & McKELVEY, J., pp. 139-187. John Wiley & Sons Inc., New York. SALZBERG, B. M. (1989). Optical recording of voltage changes in nerve terminals and in fine neuronal processes. Annual Review of Physiology 51, 507-526.

CONDUCTION IN DEVELOPING VAGUS NERVE

381

SALZBERG, B. M., OBAID, A. L., SENSEMAN, D. M. & GAINER, H. (1983). Optical recording of action potentials from vertebrate nerve terminals using potentiometric probes provides evidence for sodium and calcium components. Nature 306, 36-40. SHRAGER, P. & RUBINSTEIN, C. T. (1990). Optical measurement of conduction in single demyelinated axons. Journal of General Physiology 95, 867-890. SPITZER, N. (1981). Development of membrane properties in vertebrates. Trend8 in Neuro8cience8 4, 169-172.