Optical assessment of spatially ordered patterns of neural response to vagal stimulation in the early embryonic chick brainstem

255

Neuroscience Research, 8 (1990) 255-271 Elsevier Scientific Publishers Ireland Ltd. NEURES 00368

Research Reports

Optical assessment of spatially ordered patterns of neural response to vagal stimulation in the early embryonic chick brainstem Kohtaro Kamino, Hitoshi Komuro, Tetsuro Sakai and Katsushige Sato Department of Physiology, Tokyo Medical and Dental University School of Medicine, Tokyo (Japan) (Received 19 February 1990; Accepted 28 March 1990)

Key words: Optical assessment; Vagal response; Brainstem

SUMMARY Spatial dynamic patternings of electrical responses to vagal nerve stimulations in the embryonic chick brainstem preparation were assessed by means of simultaneous multiple-site optical recordings of electrical activity. The vagus/brainstem preparations were dissected from early 7-day-old chick embryos (165-172 h after incubation), and they were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). Application of depolarizing square current pulses to the vagus nerve fibers using a suction electrode evoked voltage-related optical (absorbance) signals that were recorded simultaneously from 127 contiguous sites in the whole brainstem preparation using a 12 x 12-element photodiode array. The optical signals evoked by the vagus nerve stimulation appeared to be concentrated longitudinally in the central region and in the lateral region ipsilateral to the site of brainstem stimulation. These response areas were orderly changed according to changes in the strength and in the duration of the stimulating current: the response area expanded as the strength or the duration of the stimulating current was increased. The size of the evoked optical signals also depended on the strength and on the duration of the stimulating current. We also measured critical (threshold) values of the strength and duration of the stimulating current to produce the optical responses, and we found that there was a regionally ordered distribution in the brainstem. In the experiments on transverse slices of the brainstem, the evoked optical responses were detected from limited areas near the dorsal surface of the stimulated side, and the response pattern depended on the strength and duration of the stimulating current in a manner similar to that in the whole bralnstem preparations. On the basis of the data obtained from these experiments, we have constructed 3 kinds of response maps for the embryonic brainstem, and the results suggest the functionally ordered arrangement of the neurons in the vagus-related area in the embryonic brainstem.

INTRODUCTION

Knowledge of physiological functions in the embryonic brainstem during the ontogenetic process would enhance our understanding of the functional organization of the central nervous control system(s) underlying autonomic functions such as circulation 31, respiration and others 24. However, although many morphological investigations have been devoted to the embryonic brainstem 1,2,32,33, physiological studies are rare 10. This is largely because conventional electrophysiological methods have been limited when apCorrespondence: K. Kamino, Department of Physiology, Tokyo Medical and Dental University School of Medicine, 1-5-45 Yushima Bunkyo-ku, Tokyo 113, Japan. Tel. 03-813-6111, ext. 3130. 0168-0102/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

256 plied to the early embryonic central nervous system. Optical recording methods using voltage-sensitive dyes 4,5,28 have been employed to monitor electrical activity from such early embryonic tissues as developing hearts 6-9, ganglia 27, and nerve fibers 19. Furthermore, the optical method provides a powerful tool for simultaneous recording of electrical activity from many areas of a preparation 3,11-13,16,18-20,23,28-30 In a previous paper 17, we demonstrated an application of the technique of multiple-site optical recording of transmembrane voltage to the embryonic chick brainstem and demonstrated the response area to vagal stimulation together with some of its electrophysiological properties. The experiments described in this article were undertaken to study the spatial patternings of the vagal response to various strengths and durations of stimulating currents in the embryonic chick brainstem, and we report here some novel findings concerning the response properties and the basic functional architecture of the embryonic brainstem (at the embryonic stage in which a synaptic response has not yet been generated). Some of these results have been communicated in preliminary form 2~ MATERIALSAND METHODS

Preparations In the present study, we have routinely used 7-day-old embryonic chick brainstems. Fertilized eggs of chicks (white Leghorn) were incubated usually for 165-172 h 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. Vagus-brainstem preparations were dissected from the 7-day-old embryos. The isolated vagus-brainstem preparation was attached dorsally 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): NaC1, 138; KC1, 5.4; CaC12, 1.8; MgC12, 0.5; glucose 10; and Tris-HC1 buffer (pH 7,2), 10. The solution was equilibrated with oxygen. The pia mater attached to the brainstem was carefully removed in the bathing solution under a dissecting microscope. Potential sensitive dye staining The isolated preparations were incubated for 15 rain in the bathing solution to which was added 0.1-0.2 mg/ml of a merocyanine-rhodanine dye (NK2761) 9,15,16,30,which was synthesized by Nippon Kankoh Shikiso Kenkyusho Co., Okayama, Japan. (The nature of this dye has been previously described: see Ref. 16). After staining, the preparation was washed with several changes of normal bathing solution. Electrical stimulation For preparations in which the vagus nerve was stimulated, the cut end of the nerve was drawn into a suction electrode fabricated from TERUMO-hematocrit tubing (VC-H075P; TERUMO Co., Tokyo, Japan), which had been hand-pulled to a fine tip (about 100 #m in caliber) over a low-temperature flame. Optical recording The optical recording system is basically similar to that described by Cohen and Lesher 3, with slight modifications 14'161 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 35-20L, 0-35V 20A, Kikusui Electronic Corp., Kawasald, Japan). Incident light was made quasimonochromatic by a 702 + 13 nm interference filter (Type 1F-W, Vacuum Optics Co. of Japan,

257 Tokyo, Japan) placed between the light source and the preparation. A microscope objective (S plan Apo, 0.40 n.a.) and a photographic eyepiece formed a magnified real image of the preparation at the image plane. Magnification was usually ×25. The transmitted light intensity at the image plane of the objective and photographic eyepiece was detected using a 12 × 12 square array of silicon photodiodes (MD-144-4PV; Centronic Ltd., Croydon, U.K.). The image of the preparation was focused onto the photodetector array. 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 (LSI11/73 system, Digital Equipment Co., Tewksbury, MA). The 128-channel data recording system is composed of a main processor (RP-891), eight I / O processors (RP-893), a 64K word wave-memory (RP-892) and a videotape recorder. The program for the computer was written in assembly language (Macro-ll) called by Fortran, under the RT-11 operating system (Version 5.0). All experiments were carried out at room temperature, 26-28 ° C. RESULTS

Evoked optical response in the embryonic brainstem The electrical activity in the embryonic bralnstem was evoked by applying a square current pulse to the attached vagus nerve fibers with a suction electrode. In the present experiment, the vagus nerve-brainstem preparation isolated from a 7-day-old chick embryo was used routinely. The preparation was stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). The evoked electrical responses were recorded as extrinsic absorption signals simultaneously from 127 adjacent loci using a 12 × 12-element photodiode array. Two kinds of experiments were carried out. First, the strength of the stimulating current was varied, and second, the duration of the stimulating current was varied.

Strength-dependence Figure 1 shows 3 examples of multiple-site optical recordings of electrical response evoked by different strengths of the stimulating current in the embryonic brainstem. A square pulse of current having a fixed duration of 5.0 ms was applied to the left vagus nerve fibers and the strength was varied. The simultaneous recordings from 127 adjacent loci were made in 4 areas by translating the photodiode array, in the order of Ill ~ II ~ IV ~ I, over the image of the brainstem. A sketch of the preparation as imaged onto the photodiode array is shown on the lower left corner in Figure 1. These measurements were carried out within less than 2 min, and the incident light was off except during the measuring period: dye bleaching usually was negligibly small. In each recording, stimulation was at 1 Hz and 8 trials were averaged. Magnification of the image was × 25, and a 702 + 13 nm interference filter was used. The objective was focused on the upper surface of the preparation. When the strength of the stimulating current was gradually increased, very small evoked optical signals were first detected with 1.0/~A current in a small lateral area near the root of the vagus nerve fibers (data not shown): the signal size was about 0.5 × 10 -4 expressed as the fractional change (AI/I) in transmitted intensity. No signals appeared in other areas (data not shown). The recording obtained with 2.0 #A current pulse stimulation is shown in Figure 1. In this recording, small optical signals appeared in a limited narrow central region, in addition to those in the lateral region, but the sizes of the signals were extremely small.

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Fig. 1. Multiple-site optical recording of electrical responses evoked by various strengths of vagus nerve stimulation in a 7-day-old embryonic chick bralnstem preparation. The duration of the stimulus current was fixed at 5.0 ms. The bralnstem preparation, to which the vagus nerve fibers were attached, was stained with a meroeyanlne-rhodanine dye (NK2761). The preparation was fixed to the bottom of the chamber with the dorsal side down. The figure was constructed using four 127-site simultaneous recordings from four areas on the left side (stimulated side) of the bralnstem, obtained with a 12 × 12-element photodiode array. In each recording, 8 trials were averaged. A 702 + 13 nm interference filter was used. Each group of detectors surrounded with an outline corresponds to one recording with the photodiode array. The photodiode array was positioned over a × 25 magnified image of the bralnstem, so that each trace represents signals detected by one photodiode from a 56 x 56 ~tm2 area of the preparation. The recordings were obtained with 2.0, 2.6 and 5.0/xA/5.0 ms depolarizing square current pulses applied to the left vagus nerve fibers with a suction electrode. On the left side, enlargements of the optical signals recorded from positions D-10, D-12, D-14, D-15, E-12, E-13, E-14 and E-15 (indicated by solid circles) are represented. In this figure and in Figs. 2 and 8, the traces are arranged so that their relative positions in the figures correspond to the relative positions of the area of the preparation imaged onto the detectors. The outputs of the individual detectors have been divided by the resting light intensity. The direction of the arrow in the lower right comer of the figure indicates a decrease in transmission (increase in absorbance) and the length of the arrow represents the stated value of the fractional change.

E n l a r g e m e n t s o f the s i g n a l s d e t e c t e d in p o s i t i o n s D - 1 0 , D - 1 2 , D - 1 4 , D - 1 5 , E-12, E-13, E - 1 4 a n d E-15 ( i n d i c a t e d b y solid circles) are s h o w n o n the left side: t h e s e signal s i z e s w e r e s m a l l e r t h a n 10 - 4 a n d the s i g n a l - t o - n o i s e ratio w a s a b o u t 1.0 or less. T h e s e signals w e r e c o m p l e t e l y e l i m i n a t e d w i t h b l u e - s h i f t e d i n c i d e n t light ( 6 3 0 n m ) , a n d s m a l l d e l a y s b e t w e e n firing o f the signals a n d s t i m u l a t i o n w e r e o b s e r v e d . W e s u p p o s e that t h e s e s i g n a l s w e r e d u e to c o n d u c t e d a c t i o n p o t e n t i a l s . T h e p a t t e r n o f r e s p o n s i v e areas w a s c o n s i s t e n t w i t h the result r e p o r t e d p r e v i o u s l y 17. T h e r e s p o n s e area in the central r e g i o n o f the b r a i n s t e m is referred to as the v a g u s n u c l e u s 17 In F i g u r e 1 the r e c o r d i n g s o b t a i n e d w i t h s t i m u l a t i o n s o f 2 . 6 / ~ A a n d 5.0 # A current p u l s e s are also s h o w n . A s c a n b e s e e n in t h e s e r e c o r d i n g s , the size o f the o p t i c a l signals

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Fig. 2. Multiple-site optical recording of vagal responses evoked by various stimulating currents in a 7-day-old embryonic brainstem. The recordings were obtained using 2 . 0 / t A / 7 . 5 ms, 2 . 0 / ~ A / 8 . 5 ms and 2 . 0 / ~ A / 1 1 . 0 ms depolarizing square current pulses applied to the left vagus nerve fibers with a suction electrode. The figure was constructed with three 127-site simultaneous recordings from three areas on the left side of the preparation. The inset in the lower left corner illustrates the location (I, II, III) of the image of the brainstem o n the photodiode array. Enlargement of the signals obtained from D-12, D-14, D-15, D-16, D-17, D-18, D-19 a n d D-20 (indicated by solid circles) are represented on the left side. Other experimental conditions are the same as in Fig. 1.

depended critically on the strength of the stimulating current. The size of the signals was dramatically increased as the strength of the current was increased, and the maximum size of the signals detected in the central area was estimated to be about 10 -3 with a 5.0 # A stimulating current pulse. The response area also expanded with increase in strength of the stimulating current. (The effects of reversal of the stimulus polarity have been previously demonstrated: see Ref. 17.)

Duration-dependence Generally, it is well known that pulse duration also is another parameter in electrical stimulation. Thus, a second experiment was carried out in which the duration of the stimulating current was varied. Because early embryonic brainstem preparations are relatively weak, in this experiment, other new preparations were used. The strength of the current pulse was fixed at 2.0/+A and the duration was varied in the range of 1.0-30.0 ms. Three examples of the original recordings are shown in Figure 2. The size of the signal and response area were also closely dependent on the duration of the stimulating current. When the duration was briefer than 3.5 ms, no signal appeared: very small signals were barely detectable in the central part of the brainstem with a 2.0 /+A/5.0 ms current pulse (data not shown). In Figure 2, the recording made with a 2.0 /+A/7.5 ms current pulse is shown. Enlargements of the signals detected in positions D-12, D-14, D-15, D-16, D-17, D-18, D-19 and D-20 (indicated by solid circles) are shown on the left side: the signal sizes were estimated to be around 10 -4 , with signal-to-noise ratios smaller than 1.0. In addition, in Figure 2, the recordings obtained by 2.0 /+A/8.5 ms and 2.0 / ~ A / l l . 0 ms current pulses are shown. As can be seen in these

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500 }2m Fig. 3. Contour maps of the optical signal size detected following 4 different stimulus strengths. The duration of the current was fixed at 5.0 ms. The regional differences in the size of the optical signal (expressed as a fractional change in transmitted intensity) is represented by contour lines. One contour interval represents an increment of 0 . 5 x 1 0 - 4 for A I / I <10 - 4 (drawn by dashed lines) and 10 - 4 for A I / I > 1 0 - 4 (drawn by solid lines): the numerals on the fines display the fractional change multiplied by 10 4. The maps were constructed with the recordings obtained using 1.8, 2.0, 2.4 and 6.0 # A / 5 . 0 ms, as labeled on each map. The preparation used for the original recordings and the relative location of the maps are the same as in Fig. 1.

recordings, the signal size and the response area gradually increased as the duration was lengthened. The size of the largest signal obtained under these conditions was greater than 10 -3, when the duration exceeded 11.0 ms. In such an experiment using a brief stimulus, the shapes of the optical signals were essentially similar, and they were completely blocked by tetrodotoxin (data not shown, but see Ref. 17). Therefore, it is likely that these optical signals do not include electrotonic response-related optical changes which are often observed with a stimulating current of relatively long duration. Mapping In order to examine the spatial distribution of the signal size and to evaluate the response area in more detail, we have measured the signal sizes, and we have made contour maps of the signals by means of an interpolation method. In Figure 3, 4 examples of the maps constructed using the recordings obtained with 1.8/~A, 2.0/~A, 2.4 /~A and 6.0 /~A current pulses (duration = 5.0 ms) are illustrated, and in Figure 4, the maps constructed using the recordings obtained with 5.5 ms, 6.5 ms, 8.5 ms a n d 10,0 ms current pulses (strength = 2.0/~A) are shown. Data were taken from the preparations used in the experiment illustrated in Figures 1 and 2. These maps give us more detailed information about regional patterns of evoked optical response under various conditions of vagus nerve stimulation. They include the following features: (1) the optical response evoked by very weak or brief current pulses first appeared to be concentrated in a very small area in the lateral region and consequently in the central part of the brainstem; (2) the response area expands radially with

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Critical values of stimulation for the optical response The results described above suggest that there exist critical values of the strength and the duration of the stimulating current to produce the optical responses. F r o m the viewpoint of functional architecture in the brainstem, it would be of interest to analyze their regional differences. In Figure 5A, the size of the evoked optical signal is plotted against the strength of the stimulating current (duration = 5.0 ms). These plots were made using signals detected from 8 positions: A-9, A-16, A-18, B-15, C-10, C-16, E-18 and 1-32 in the preparation shown in Figure 1. In this figure, there are differences in the critical values of the strength of the stimulating current for generation of the optical responses among the different positions: 1.9 # A for E-18, 2.0 # A for C-10, 2.2/~A for C-16 and B-15, 2.8/xA for 1-32, 3.5 # A for A-16 and 8.3 # A for A-18 were obtained. When stimuli of very small strength or duration were applied, the optical signals were not detectable because they were below noise level. Thus, these critical values were evaluated by extrapolation. After the strength of the stimulating current exceeded a critical value, the size of the optical signals

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increased exponentially to a plateau level. This behavior reflects the circumstance that one photodiode detected a sum of action potentials originating in many neurons, each possessing a different excitation threshold. Therefore, the critical value for each curve indicates the minimum current strength which is required to just elicit the electrical response in the neurons within the field detected by each photodiode. We have also plotted the size of the optical signals, against the duration of the stimulating current (in Fig. 5B). For this plot, the data were obtained from another different preparation. (Here, we note again that in comparison with adult brainstem preparations, early embryonic preparations are very weak, so that for every different experiment, we often used new preparations.) For the duration of the current, there was, again, a critical value necessary to elicit the optical response. In this graphic representation, 6 curves derived from the signals detected from 6 positions (A-2, A-13, B-24, C-2, D-12 and H - l ) in the brainstem preparation shown in Figure 2 are illustrated. When the current strength was fixed at 2.0/~A, and the duration was varied, different critical values to elicit excitation were obtained from individual positions: 5.0 ms for D-12, 8.5 ms for B-24, 9.0 ms for A-13, 10.0 ms for C-2 and 11.0 ms for H-1.

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Mapping We have also evaluated the critical values of the stimulating current strengths and durations for the evoked optical signs in all positions where these were detected, and we have mapped the regional distribution of the critical values. In Figures 6A and 7A, the numerical representations o f the critical values of the strength (in 6A) and the duration (in 7A) of the stimulating current are shown. The maps which were constructed using these data are illustrated in Figures 6B and 7B. In these maps, the impression is strong that critical values of both the strength and the duration of the current pulse to produce the excitation are arranged in graded contours towards the periphery, and that the distribution patterns for the strength and the duration are basically similar. Here, it is suggested that these maps correspond to the spatial distribution of sensitivity of neurons in the embryonic brainstem (see also Discussion). Transversely sectioned slice preparations The spatial resolution of the optical recording is somewhat limited for three-dimensional preparations 3,29, and accordingly, in the experiments described above, information

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Fig. 7. (A) Numerical representation of the critical values of the stimulus duration required to elicit an optical response. The data were obtained from the plots of the signal size versus the duration of the stimulating current. The current strength was fixed at 2.0 gA. Six samples of the plots are shown in Fig. 5B. The preparation for the original recording was the same as that in Fig. 2. (B) Contour maps of the critical values which were constructed from the data shown in Fig. 7A. One contour interval represents an increment of 0.5 or 1.0 m s as indicated on the lines. Other notations on the m a p are the same as in Fig. 6.

about the Z-axis distribution of the responses is lacking. Therefore, to compensate for this difficulty, we have carried out a series of experiments on transversely sectioned slice preparations from the brainstem. We prepared a slice preparation attached to the vagus nerve fibers by transversely sectioning a 7-day-old embryonic chick brainstem using the same method described previously 17. The thickness of the slice was about 1000 #m. As in the experiments on the intact preparation, we first applied various strengths of stimulating current, at constant duration, to the vagus nerve fibers. In Figure 8; two examples of original recordings are shown. These signals were evoked on the left side of the brainstem by stimulating with 2.0 #A, and 3.0 g A current pulses (duration = 5.0 ms) on the left vagus nerve fibers. Simultaneous 127-site recordings were made in 3 areas by moving the photodiode array on the image, in the order of I --, II ~ III as shown in the lower fight corner of the upper optical traces. The inset on the lower right illustrates the location of the image of the slice preparation on the photodiode array. Consistent with the results shown in Figure 1, both the signal size and the r e s ~ n s e area were strongly dependent on the strength of the stimulating current. S ~ optical signals were generated near the root of the vagus nerve fibers by 0.8 # A / 5 . 0 ms current pulses, and extremely small signals were first detected from one position (H-4) in response to a 1.4 gA current pulse near the dorsal surface of the stimulated side of the brainstem (data not shown). In Figure 8, the recording obtained using a 2,0 # A / 5 , 0 ms

265 2.0~A/S.0ms A

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Fig. 8. Multiple-site optical recordings of electrical responses evoked by various strengths of the stimulating current in a slice prepared from a 7-day-old embryonic brainstem. The slice was fixed to the bottom of the chamber with the cephalic side facing upward. Simultaneous multiple-site optical recordings with the 12 x 12element photodiode array were obtained from 3 areas in the left side of the slice. The depolarizing square current pulse was applied to the left vagus nerve fibers using a suction electrode. The recordings were made using 2.0 and 3.0 # A , and the duration of the stimulating current was fixed at 5.0 ms. In the lower left comer of the upper traces, enlargements of the signals obtained from the positions surrounded by a dotted line are shown, and in the lower fight comer in the upper traces, a schematic drawing of the slice and the relative positions of the diode array are illustrated. Other experimental conditions and notations are the same as in Fig. 1.

current pulse is shown. In this recording, small signals ( A I / I = 10 -4) were detected from several positions. The signals detected from the area surrounded by dotted lines are enlarged on the lower left comer of the upper traces. In Figure 8, the recordings made by 3.0 /~A current pulses (duration = 5.0 ms) are also shown. Both the signal size and the response area increased with the strength of the stimulating current. The duration of the stimulating current was also varied in a series of experiments (data not shown). In this experiment, the magnitude of the evoked optical signal was also related to the duration of the stimulating current: extremely small signals were first detected with a stimulating current of 2.5 ms duration (strength = 2.0 /~A) by a few

266 p h o t o d i o d e e l e m e n t s . T h e size a n d s p a t i a l e x t e n t i n c r e a s e d as the d u r a t i o n was l e n g t h e n e d to 10 ms.

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ms or 2.0 #A/2.5 ms current (data not shown), and located in the narrow area near the dorsal surface of the brainstem, and both the signal size and the response area expanded with increasing strength or duration of the stimulating current. Similarly to Figure 5, the signal size was plotted against the strength and the duration of the stimulating current, and we have evaluated the critical values for the strength and duration of the stimulating current. Subsequently, using these data, we have constructed maps of the spatial distribution of the critical values for the strength and the duration of the stimulating current to produce excitation in the transversely sectioned view. As shown in Figure 10, the patterns obtained for both strength and duration were basically similar to each other: the critical values are multiply layered and the area of maximum responsiveness favors the dorsal surface with the contour lines concentrated toward the dorsal side.

DISCUSSION

The results presented in this article show the spatial dynamic patterning of the vagal response in the embryonic brainstem for the first time, using the optical method for simultaneous multiple-site recording of electrical activity together with a voltage-sensitive

268 dye. We have provided 3 kinds of functional maps of vagal response in the 7-day-old embryonic chick brainstem. The first is the stimulus-strength-related response map, the second is the duration-related response map and the third is the map of the excitation threshold intensity and duration. These maps depict a fundamental feature of the functional organization of the embryonic brainstem. In a previous paper iv, we determined a basic pattern of the response area to vagal stimulation, in the 7-day-old embryonic brainstem, and we referred to the responsive region as the vagus nucleus, perhaps the nucleus dorsalis nervi vagi. The maps shown in this paper indicate that in the embryonic vagus nucleus, the spatial pattern of the response to vagal stimulation is adaptively modulated according to changes in the strength and the duration of the stimulating current. The change in the spatial pattern is accompanied by a change in the size of the evoked optical signal. In optical recording of electrical activity, the signal size is proportional to the percentage of activated cells and the magnitude of the membrane potential change in each activated cell within the field detected by one photodiode and depends also on the amount of dye bound to the membrane 3,16,26,28. It follows that the spread of the response area is related to an increase in the number of activated neurons produced by increasing the strength (or the duration) of the stimulus current. The number of neurons activated by vagus stimulation in the vagus nucleus is radially increased with the increase in the strength (or the duration) of the stimulus current. However, neither spatial variation in the density of inputs nor in the degree of staining can explain the maps of the vagal responses shown in Figures 3, 4, 6, 7, 9 and 10. Thus, it seems likely that the vagus nucleus is an orderly organized system which conforms to the conditions of various stimulations rather than a random assembly of neurons. Analyses of chronaxy and rheobase also support this idea (H. Komuro and K. Kamino, unpublished results). In the present experiment, there are many motor neurons and sensory neurons (nerve terminals) within the optically detected area, so that we detect both antidromic (for motor nerves) and orthodromic (for sensory nerves) activation in the vagus nucleus. Unfortunately, at the present stage at least, it is practically impossible to separate the motor and sensory nerve fibers contained in the vagus nerve bundle. In addition, the vagus nerve bundle is constructed of many unmyelinated nerve fibers with various thicknesses ~9 We have recently observed optical signals with the postsynaptic potential component in the later 7- to 8-day-old embryonic brainstem 22. However, no postsynaptic event was detected from the early 7-day-old embryonic brainstem preparations used in the present experiment. Thus, in the embryonic brainstem used in the present experiment, the functional property would be relatively simple. Thus, it is reasonable that we detected the optical signals from the motor and presynaptic sensory neurons and that no signal was detected from the postsynaptic neurons. In our optical method, generally, each element of the photodiode array monitored optical signals from many neurons and processes. As described above, the signal size is proportional to the magnitude of the action potential of each cell and to the number and size of electrically activated cells in the field detected by one photodiode element 16.26 Here, it is plausibly assumed that in the field detected by each element, the percentage of the space occupied by the sensory neurons (nerve terminals) is smaller than that of the motor neurons. It is thus postulated that the observed optical signals reflect mostly the action potential of the motor neurons and that the action potential component originating from the sensory neurons would be covered: it is likely that the optical signals recorded in the present experiment mostly correspond to the antidromic activation of the motor neurons.

269 Accordingly, although the present results must be interpreted cautiously, the third map of the threshold intensity and duration suggests a feature of the ordered organization of the neurons within the embryonic vagus nucleus: in this map, we are able to read, as a first approximation, (1) that the embryonic vagus nucleus has an ordered architecture determined by neurons having different degrees of responsiveness to vagal stimulation, and (2) that the responsiveness to vagal stimulation is the highest in the central core of the vagus nucleus and falls towards the edge. It is well known that extracellular stimulation first activates the largest axons in a nerve and then progressively activates smaller ones as the intensity increases. Thus, for the ordered distribution of the responsiveness (and the threshold intensity and duration), one possible explanation is that in the embryonic vagus nucleus the neurons are spatially arranged in a hierarchical order corresponding to t h e i r a x o n diameters. Although we anticipate that the present results would not be directly useful in understanding adult complete brainstem function, such a nature may be related to the embryonic origin of the functional architecture coupled with the morphogenesis of the neurons in the brainstem during early development. Nevertheless, the present experiment lacks data that require additional consideration: the variation of the response to stimuli could have been due to the position of the axons in the vagus nerve bundle. This could have been assayed by splitting the nerve into several components from or to various organs. However, in connection with this problem, we have not yet obtained complete results: more detailed experiments are required. As stated above, because the field of optical detection with one photodiode element was 56 x 56 # m 2, in the present experiment, we have not analyzed systematically the characteristics of individual neurons. However, quite recently we have been able to improve the two-dimensional spatial resolution to 2.7 x 2.7/~m 2. The size of cells in the embryonic brainstem is about 5.0 # m in diameter, so we feel that analysis of individual neuronal function might be possible. Effects of light-scattering on the spatial resolution pointed out by Orbach and Cohen 25 and London et al. 23 seem to be relatively small (see also Ref. 17). Possible reasons for this may be related to the small size of the neurons and to the low intrinsic absorbance and short optical path of the preparation. Nevertheless, resolution along the optic axis is one of the most serious difficulties in the use of optical recording methods, particularly in a complex tissue such as the embryonic brainstem. To determine the depth resolution, the size of the optical signal was measured when the preparation was moved both above and below the position of focus. The signal size was scarcely changed when the preparation was moved upward out of focus 1000/~m, but it was reduced by 50% when the preparation was moved downward out of focus by 500/~m, in the intact brainstem isolated from a 7-day-old embryo (K. Kamino, H. K o m u r o and T. Sakai, unpublished results). Salzberg et al. 29 reported that the signal size was reduced by 50% when the microscope stage was moved by + 300/~m from the level of focus in the supraesophageal ganglion isolated from a giant barnacle. The differences between our results and theirs may be due to differences in the thickness and the degree of transparency of the preparations, and to differences in the depth distribution of active cells. The 7-day-old embryonic chick brainstem is about 1000 /~m in thickness and has a relatively loose structure. In this report, we have focused on the 7-day-old chick brainstem in order to describe the basic characteristics of evoked responses in the embryonic vagus nucleus. We are now carrying out experiments on the ontogenesis and early development of the physiological events reported here, and on their role in the emerging functional organization of the brainstem.

270 ACKNOWLEDGEMENTS W e are m o s t g r a t e f u l to B r i a n S a l z b e r g for his critical r e a d i n g of the m a n u s c r i p t a n d useful d i s c u s s i o n s a n d to L a r r y C o h e n , A k i h i k o H i r o t a a n d Y o k o M o m o s e for their critical r e a d i n g of the m a n u s c r i p t a n d p e r t i n e n t c o m m e n t s . T h i s w o r k was s u p p o r t e d by g r a n t s f r o m the J a p a n e s e M i n i s t r y a n d E d u c a t i o n , S c i e n c e a n d C u l t u r e , f r o m the S u z u k e n M e m o r i a l F o u n d a t i o n , a n d f r o m the B r a i n S c i e n c e F o u n d a t i o n (Nos. 01480120, 01659504, 01639505) (to K . K . ) a n d b y R e s e a r c h A i d f r o m the I n o u e F o u n d a t i o n for S c i e n c e (to U.K.). REFERENCES 1 Altman, J. and Bayer, S.A., Development of the brain stem in the rat. I. Thymidine-radiographic study of the time of origin of neurons of the lower medulla, J. Comp. Neurol., 194 (1980) 1-35. 2 Altman, J. and Bayer, S.A., Development of the brain stem in the rat. II. Thymidine-radiographic study of the time of origin of neurons of the upper medulla, excluding the vestibular and auditory nuclei, J. Comp. Neurol., 194 (1980) 37-56. 3 Cohen, L.B. and Lesher, S., Optical monitoring of membrane potential: methods of multisite optical measurement. In P. De Weer and B.M. Salzberg (Eds.), Optical Methods in Cell Physiology, Wiley-Interscience, New York, 1986, pp. 71-99. 4 Cohen, L.B. and Salzberg, B.M., Optical measurement of membrane potential, Rev. Physiol. Biochem, Pharmacol., 85 (1978) 33-88. 5 Freedman, J.C. and Laris, P.C., Electrophysiology of cells and organdies: studies with optical potentiometric indicators, Int. Rev. Cytol., Suppl., 12 (1981) 177-246. 6 Fujii, S., Hirota, A. and Kamino, K., Optical signals from early embryonic chick heart stained with potential sensitive dyes: evidence for electrical activity, J. Physiol. (Lond.), 304 (1980) 503-518. 7 Fujii, S., Hirota, A. and Kamino, K., Optical recording of development of electrical activity in embryonic chick heart during early phases of cardiogenesis, J. Physiol. (Lond), 311 (1981) 147-160. 8 Fujii, S., Hirota, A. and Kamino, K., Optical indications of pace-maker potential and rhythm generation in early embryonic chick heart. J. Physiol. (Lond), 312 (1981) 253-263. 9 Fujii, S., Hirota, A. and Kamino, K., Action potential synchrony in embryonic precontractile chick heart: optical monitoring with potentiometric dyes, J. Physiol. (Lond.), 319 (1981) 529-541. 10 Gootman, P.M., Development of central autonomic regulation of cardiovascular function. In P.M. Gootman (Ed.), Developmental Neurobiology of the Autonomic Nervous System, Humana, Clifton, NJ, 1986, pp. 279-325. 11 Grinvald, A., Cohen, L.B., Lesher, S. and Boyle, M.B., Simultaneous optical monitoring of activity of many neurons in invertebrate ganglia using a 124-element photodiode array. J. Neurophysiol., 45 (1981) 829-840. 12 Grinvald, A., Frostig, R.D., Lieke, E. and Hildesheim, R., Optical imaging of neuronal activity, Physiol. Rev., 68 (1988) 1285-1366. 13 Grinvald, A., Manker, A. and Segal, M., Visualization of the spread of electrical activity in rat hippocampal slices by voltage-sensitive optical probes. J. Physiol. (Lond.), 333 (1982) 269-291. 14 Hirota, A., Kamino, K., Komuro, H. and Sakai, T., Early events in development of electrical activity and contraction in embryonic rat heart assessed by optical recording, J. Physiol. (Lond.), 369 (1985) 209-227. 15 Kamino, K., Hirota, A. and Fujii, S., Localization of pacemaking activity in early embryonic heart monitored using voltage-sensitive dye, Nature, 290 (1981) 595-597. 16 Kamino, K., Hirota, A. and Komuro, H., Optical indications of electrical activity and excitation-contraction coupling in the early embryonic heart, Ado. Biophys., 25 (1989) 45-93. 17 Kamino, K., Katoh, Y., Komuro, H. and Sato, K., Multiple-site optical monitoring of neural activity evoked by vagus nerve stimulation in the embryonic chick brain stem, J. Physiol. (Lond.), 409 (1989) 263-283. 18 Kamino, K., Komuro, H. and Sakai, T., Regional gradient of pacemaker activity in the early embryonic chick heart monitored by multisite optical recording. J. Physiol. (Lond), 402 (1988) 301-314. 19 Katoh, Y., Komuro, H., Sakai, T. and Kamino, K., Multiple-site optical recording of spreading electrical activity in the embryonic cervical vagus nerves using a voltage-sensitive dye (Abstract) J. Physiol. Soc. Jpn., 50 (1988) 448. 20 Komuro, H., Sakai, T., Hirota, A. and Kamino, K., Conduction pattern of excitation in the amphibian atrium assessed by multiple-site optical recording of action potentials, Jpn. J. Physiol., 36 (1986) 123-137.

271 21 Komuro, H., Sato, K., Sakai, T. and Kamino, K., Spatial pattern of neuronal responses to vagus nerve stimulation in the embryonic chick brain stem: multiple-site optical recording of electrical activity (Abstract), Neurosci. Res., 9 (1989) $47. 22 Komuro, H., Momose, Y., Sakai, T., Hirota, A. and Kamino, K., Optical recording of synaptic potential in the embryonic chick brain stem slice preparation using a voltage-sensitive dye (Abstract), Neurosci. Res., Suppl. 10 (1990) in press. 23 London, J.A., Zecevic, D. and Cohen, L.B., Simultaneous optical recording of activity from many neurons during feeding in Navanax, J. Neurosci., 7 (1987) 649-661. 24 Martin, J.H., Development as a guide to the regional anatomy of the brain. In E.R. Dandel and J.J. Schwartz (Eds.), Principles of Neural Science, 2rid edn., Elsevier, New York, 1985, pp. 248-258. 25 Orbach, H.S. and Cohen, L.B., Optical monitoring of activity from many areas of the in vitro and in vivo salamander olfactory bulb: a new method for studying functional organization in the vertebrate central nervous system, J. Neurosci., 3 (1983) 2251-2262. 26 Orbach, H.S., Cohen, L.B. and Grinvaid, A., Optical mapping of electrical activity in rat somatosensory and visual cortex, J. Neurosci., 5 (1985) 1886-1895. 27 Sakai, T., Hirota, A., Komuro, H., Fujii, S. and Kamino, K. Optical recording of membrane potential responses from early embryonic chick ganglia using voltage sensitive dyes, Dev. Brain Res., 17 (1985) 39-51. 28 Salzberg, B.M., Optical recording of electrical activity in neurons using molecular probes. In J.L. Barber (Ed.), Current Methods in Cellular Neurobiology, Wiley-Interscience, New York, 1983, pp. 139-187. 29 Salzberg, B.M., Grinvaid, A. Cohen, L.B., Davila, H.V. and Ross, W.N., Optical recording of neuronal activity in an invertebrate central nervous system: simultaneous monitoring of several neurons, J. Neurophysiol., 40 (1977) 1281-1291. 30 Salzberg, B.M., Obaid, A.L., Senseman, D.M. and Gainer, H., Optical recording of action potentials from vertebrate nerve terminals using potentiometric probes provides evidence for sodium and calcium components, Nature, 306 (1983) 36-40. 31 Spyer, K.M., Neural mechanisms involved in cardiovascular control during affective behaviour, Trends Neurosci., 12 (1989) 506-513. 32 Wright, L.L., Time of cell origin and cell death in the avian dorsal motor nucleus of the vagus, J. Comp. Neurol., 199 (1981) 125-132. 33 Young, S.R. and Rubel, E.W., Embryogenesis of arborization pattern and topography of individual axons in n. laminalis of the chicken brain stem, J. Comp. Neurol., 254 (1986) 425-459.