Development of vagal afferent projections circumflex to the obex in the embryonic chick brainstem visualized with voltage-sensitive dye recording

Neuroscience 148 (2007) 140 –150

DEVELOPMENT OF VAGAL AFFERENT PROJECTIONS CIRCUMFLEX TO THE OBEX IN THE EMBRYONIC CHICK BRAINSTEM VISUALIZED WITH VOLTAGE-SENSITIVE DYE RECORDING Y. MOMOSE-SATO,a,b M. KINOSHITAa AND K. SATOa*

gli et al., 2006). In order to understand the integration of the visceral information in the brainstem, one of the fundamental questions is how and when the neural circuits are established in the brainstem. Although many anatomical and molecular genetic investigations have been made so far on brainstem neural circuits (for reviews see Friauf and Lohmann, 1999; Glover, 2000; Rubel and Fritzsch, 2002), electrophysiological studies of neural network organization have been limited because of the technical difficulty of monitoring neural activity from small and fragile embryonic neurons (for reviews see Fortin et al., 2000; Borday et al., 2004, 2005). Voltage-sensitive dye recording has made it possible to monitor transmembrane voltage changes from living cells that are inaccessible to conventional electrophysiological means. Furthermore, the introduction of multi-element photodiode arrays has provided powerful tools for monitoring the spatio-temporal patterns of neural activity from a variety of invertebrate and vertebrate central nervous systems (for reviews see Cohen and Salzberg, 1978; Salzberg, 1983; Grinvald et al., 1988; Ebner and Chen, 1995; Baker et al., 2005). In our previous studies, we established the usefulness of the voltage-sensitive dye recording for monitoring electrical activity from the embryonic nervous systems (for reviews see Momose-Sato et al., 2001, 2002), and we investigated the development of neural functions related to various cranial nerves, including the glossopharyngeal and vagus nerve. First, we examined spatio-temporal patterns of neural activity evoked by glossopharyngeal/vagal stimulation in embryonic brainstems, and obtained three-dimensional profiles of neural responses in the visceral motor and sensory nuclei (Komuro et al., 1991; Momose-Sato et al., 1991, 1994, 1999; Sato et al., 1995, 1998, 2002). In the sensory nucleus of the vagus and glossopharyngeal nerves (nucleus of the tractus solitarius: NTS), (1) the optical signals were composed of fast and slow components, which corresponded to the action potential and the glutamatergic excitatory postsynaptic potential (EPSP), respectively; (2) the slow signal easily fatigued with repetitive stimuli, and the initial and later phases of the slow signal were mainly attributable to non-NMDA (N-methyl-D-aspartate) and NMDA receptors, respectively; (3) in normal Ringer’s solution, the postsynaptic responses were first expressed in the NTS at embryonic day 7 in the chick embryo and embryonic day 15 in the rat embryo. Second, we surveyed optical responses from a wider region of the chick brainstem, and identified the second/

a

Department of Physiology, Tokyo Medical and Dental University, Graduate School and Faculty of Medicine, 1-5-45 Yushima, Bunkyoku, Tokyo 113-8519, Japan

b

Department of Health and Nutrition, Kanto Gakuin University, College of Human and Environmental Studies, 1-50-1 Mutsuura-higashi, Kanazawa-ku, Yokohama 236-8501, Japan

Abstract—Using voltage-sensitive dye recording, we surveyed neural responses related to the vagus nerve in the embryonic chick brainstem. In our previous studies, we identified four vagus nerve–related response areas in the brainstem. On the stimulated side, they included (1) the nucleus of the tractus solitarius (NTS: the primary sensory nucleus) and (2) the dorsal motor nucleus of the vagus nerve (DMNV), whereas on the contralateral side, they corresponded to (3) the parabrachial nucleus (PBN: the second/higher-ordered nucleus) and (4) the medullary non-NTS region. In the present study, in addition to these areas, we identified another response area circumflex to the obex. The intensity of the optical signal in the response area was much smaller than that in the NTS/DMNV, and the spatio-temporal pattern could be discerned after signal averaging. The conduction rate to the response area was slower than that to the other four areas. Ontogenetically, the response area was distributed on the stimulated side at the 6-day embryonic stage, and it spread into the contralateral side in 7- and 8-day embryonic stages. These distribution patterns were consistent with projection patterns of vagal afferent fibers stained with a fluorescent tracer, suggesting that the response area included a primary sensory nucleus. In comparison with the functional development of the other four response areas, we traced the functional organization of vagus nerve–related nuclei in the embryonic brainstem. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: voltage-sensitive dye recording, chick embryo, vagus nerve, brainstem, area postrema, development.

In the CNS, the brainstem is a pivotal region to integrate various sensory information from somatic/visceral organs and to produce motor reflexes. In the brainstem, the autonomic nervous system has extensive networks that are indispensable for regulating visceral functions, including cardiovascular responses, respiratory activity and gastric function (Spyer, 1982; Dampney, 1994; Saper, 1995; Trava*Corresponding author. Tel: ⫹81-3-5803-5157; fax: ⫹81-3-5803-0118. E-mail address: [email protected] (K. Sato). Abbreviations: DiI, 1,1=-dioctadecyl-3,3,3=,3=- tetramethylindocarbocyanine perchlorate; DMNV, the dorsal motor nucleus of the vagus nerve; EPSP, excitatory postsynaptic potential; NA, nucleus ambiguus; NMDA, N-methyl-D-aspartate; NTS, the nucleus of the tractus solitarius; PBN, the parabrachial nucleus.

0306-4522/07$30.00⫹0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.05.032

140

Y. Momose-Sato et al. / Neuroscience 148 (2007) 140 –150

higher-ordered nucleus, possibly corresponding to the parabrachial nucleus (PBN), at the pons/rostral medulla on the contralateral side (Sato et al., 2004). Furthermore, we found a primary vagal projection to the contralateral nonNTS region in the embryonic chick brainstem (MomoseSato and Sato, 2005). During these studies, we noticed that very small optical responses were induced by vagal nerve stimulation in the area circumflex to the obex. In the present study, we examined the development of the small vagal responses in the embryonic chick brainstem with voltage-sensitive dye recording and optical signal averaging.

EXPERIMENTAL PROCEDURES Preparations Experiments were carried out in accordance with the guidelines of the US National Institutes of Health and Tokyo Medical and Dental University for the care and use of laboratory animals. All efforts were made to minimize the number of animals used and their suffering, and all of the experiments were done in Tokyo Medical and Dental University. Fertilized eggs of White Leghorn chickens (Saitama Experimental Animals Supply Co. Ltd., Saitama, Japan) were incubated for 6 – 8 days in a forced-draft incubator (type P-008, Showa Incubator Laboratory, Urawa, Japan) at 37 °C and 60% humidity, and were turned once each hour. In the present experiments, the day on which incubation was started was termed day 0: day 6 corresponds to the Hamburger-Hamilton stages (Hamburger and Hamilton, 1951) 28 –29, day 7 to 30 –32, and day 8 to 33–34. The embryos were decapitated, and intact brainstem preparations with the vagus nerve fibers attached were dissected from the embryos. For slice preparations, 1200 –1300 ␮m thick slices were made from the isolated brainstem at the level of the vagus nerve root. The meningeal tissue was carefully removed in a bathing solution that contained (in mM) NaCl, 138; KCl, 5.4; CaCl2, 1.8; MgCl2, 0.5; glucose, 10; and Tris–HCl buffer (pH 7.3), 10. The solution was equilibrated with 100% oxygen. Each preparation was stained by incubating it for 20 min in a solution containing 0.2 mg/ml of the voltage-sensitive merocyanine-rhodanine dye, NK2761 (Hayashibara Biochemical Laboratories Inc./ Kankoh-Shikiso Kenkyusho, Okayama, Japan: Kamino et al., 1981; Salzberg et al., 1983; Momose-Sato et al., 1995), and the excess (unbound) dye was washed away with a dye-free solution before recording. After staining with the dye, the preparation was attached to the silicone (KE 106LTV; Shin-etsu Chemical Co., Tokyo, Japan) bottom of a simple chamber with the ventral side up for the intact preparation or the caudal side up for the slice preparation.

Electrical stimulation The cut end of the vagus nerve was drawn into a glass microsuction electrode (about 100 ␮m internal diameter). Positive depolarizing square current pulses at 7– 8 ␮A/5 ms, which evoked maximum responses, were applied to the right vagus nerve. For single sweep recordings, a single stimulus was applied to the nerve. When the signals were averaged, 16 –50 stimuli were delivered at 1 Hz.

Optical recording The optical recording system which we used was similar to that previously described (Momose-Sato et al., 2001). In brief, light from a 300 W tungsten-halogen lamp (Type JC-24V/300W, Kondo Philips Ltd., Tokyo, Japan) was collimated, rendered quasi-monochromatic with a heat filter and an interference filter with a transmission maximum at 703⫾15 nm (Asahi Spectra Co., Tokyo,

141

Japan), and focused onto the preparation. An objective (Plan Apo, 10⫻, 0.45 numerical aperture) and a photographic eyepiece projected a real image of the preparation (magnification 25⫻) onto a multi-element silicon photodiode matrix array mounted on an upright microscope. We used a 128-site optical recording system with a 12⫻12-element silicon photodiode array which was constructed in our laboratory (for reviews see Kamino, 1991; Momose-Sato et al., 2001). The time resolution of the system was 1 ms at 1000 frames/s. The recordings were usually made with a time interval of 10 –15 min. To see the small fast component of the optical signal, 16 –50 recordings were averaged off-line. Optical measurements were carried out in a still chamber without continuous perfusion with the bathing solution at 26 –30 °C. For short time recording of optical signals evoked by vagal nerve stimulation, there was no difference in the signal intensity between with and without continuous perfusion and between the bathing solution at 26 –30 °C and at 35–37 °C. The fractional change in dye absorption ⌬A/Ar is equal to ⫺⌬I/(Ib⫺Ia), where I is the light intensity transmitted through the preparation and Ib, Ia are the intensity before and after staining, respectively (Ross et al., 1977). When we recorded vagal responses, we stained the preparation before pinning it to the chamber to allow the dye to diffuse into the tissue. Thus, the measurement of Ib and Ia from the same position was technically difficult. We compared Ib and Ia by measuring the light that reached the detectors before and after the preparation was stained for 20 min with 0.2 mg/ml NK2761 on the stage of the microscope (Momose-Sato and Sato, 2006). Under this condition, Ia/Ib averaged 64% in the 8-day-old intact medulla with only a small variation between regions (n⫽3 preparations). In slice preparations, although the central part of the slice was less intensely stained than the peripheral region, Ia/Ib was relatively constant within the vagal response area (dorsal region): in 8-day-old slices (n⫽3), Ia/Ib averaged 42% in the dorsomedial region and 40% in the dorsolateral region. Since regional variations in Ia/Ib in the vagal response area were small, we measured Ia and ⌬I, and expressed the optical signal as ⌬I/Ia.

DiI labeling The DiI labeling method that we used was essentially similar to that described by Godement et al. (1987). Brainstems with the vagus nerve attached were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. A small crystal of the fluorescent neuronal tracer, 1,1=-dioctadecyl-3,3,3=,3=-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes, Eugene, OR, USA), was placed in the cut ends of the vagus nerve. Preparations in which DiI had been placed were stored in 4% paraformaldehyde for 2– 8 weeks at room temperature. The brainstem dissected free from surrounding tissue was embedded in 3% gelatin, and was sectioned in the horizontal (coronal) plane at 50 ␮m on a Vibratome (microslicer DTK-2000, Dosaka EM, Kyoto, Japan). Wetmounted sections were examined with an epifluorescence microscope (FLUOPHOT, Nikon Co., Tokyo, Japan) equipped with a rhodamine filter set at excitation 520 –550 nm and emission ⬎570 nm using a 575 nm dichroic mirror.

RESULTS Optical signals induced by vagal nerve stimulation in the medulla Fig. 1A shows an example of multiple-site optical recordings of neural activity induced by vagal nerve stimulation in a 7-day-old embryonic chick brainstem intact preparation. The thickness (light-path from the dorsal surface to the

142

Y. Momose-Sato et al. / Neuroscience 148 (2007) 140 –150

Fig. 1. (A) An example of multiple-site optical recordings of neural responses to vagal nerve stimulation in a 7-day-old embryonic chick brainstem. The preparation was stained with a merocyanine–rhodanine dye (NK2761), and optical signals were evoked by applying a brief square current pulse (7 ␮A/5 ms) to the right vagus nerve bundle with a microsuction electrode. The evoked optical signals were detected simultaneously from the ventral side of the brainstem using a 12⫻12 element photodiode array, and 50 trials are averaged. Simultaneous 128-site optical recordings were made in seven areas of the preparation by moving the photodiode array over the image of the preparation. The direction of the arrow on the lower right side indicates an increase in transmitted light intensity (a decrease in absorption) and the length of the arrow represents the stated value of the fractional change. After averaging, spike-like signals (⌬I /I⬍10⫺3) in the caudal medulla became prominent. In this recording, two response areas were observed; one was located at the level of the vagus nerve on the stimulated side, and the other in the caudal medulla. The former area includes the NTS (sensory nucleus) [lateral region] and the DMNV (motor nucleus) [medial region] (Momose-Sato et al., 1991). Optical signals detected from three different positions (marked a, b and c) are enlarged in B. (B) Enlargement of optical signals to compare the timing of their peaks. The signals were detected from three different positions (a– c) shown in the left panel. In position a (which corresponds to the level of the root of the vagus nerve), the optical signal has two peaks, labeled 1 and 2. The first peak (1) of the optical signal in position a and the peak (marked by an asterisk) in position b (which corresponds to NTS/DMNV) are observed earlier than the second peak (2) in position a and the peak (marked by two asterisks) in position c.

ventral surface) of these preparations was 300 –1000 ␮m, and they were translucent. Thus, we could detect neural voltage responses as changes in transmitted light intensity. The evoked optical signals were recorded using a

12⫻12 element photodiode array, and 50 trials were averaged to detect smaller optical signals. After the averaging, a new response area was discernable in the caudal medulla in addition to response areas

Y. Momose-Sato et al. / Neuroscience 148 (2007) 140 –150

identified at the level of the root of the vagus nerve on the stimulated (ipsilateral) side. As we reported previously (Momose-Sato et al., 1991), the ipsilateral response area corresponds to the NTS (sensory nucleus) and the dorsal motor nucleus of the vagus nerve (DMNV; motor nucleus). In this paper, we will not focus on the responses in the NTS and DMNV. Fig. 1B shows enlargements of optical signals detected in positions a– c in Fig. 1A. By averaging 50 trials, very small optical signals (⌬I /I⬍10⫺3) were clearly visible. The averaging also revealed that optical signals in the lateral region at the level of the root of the vagus nerve had two peaks (Fig. 1Ba), whereas signals in the other regions had a single peak (Fig. 1Bb, c). The peak of the optical signals in the region corresponding to NTS/DMNV (Fig. 1Bb: marked by an asterisk) was observed later than the first peak detected near the root of the vagus nerve (Fig. 1Ba, marked 1) and earlier than the second one (Fig. 1Ba, marked 2). On the other hand, the second peak was observed earlier than the single peak in the caudal medulla (Fig. 1Bc, marked by two asterisks). These results suggest that the first peak detected near the root of the vagus nerve was medially conducted to the NTS/DMNV, and that the second peak was caudally conducted. Propagation patterns of optical signals and its developmental change To determine the propagation patterns of optical signals in each developmental stage, we made time sequential conduction maps (Fig. 2). The gray shadows in each map indicate areas in which spike-like signals (⌬I /I ⬎ 2⫻10⫺4) were observed within 5-ms time windows. In every embryonic stage, optical signals first propagated medially to regions corresponding to NTS and DMNV (the second maps in each stage: less than 10 ms after the first maps) from the root of the vagus nerve, and this propagation shows the conduction pattern of the first peak at the root of the vagus nerve (Fig. 1Ba, marked 1). Thereafter, optical signals were observed in the caudal medulla (the third or later maps in each stage: more than 10 ms from the first maps), and these signals indicate the conduction pattern of the second peak at the root of the vagus nerve (Fig. 1Ba, marked 2). The second peak appeared to be caudally conducted in the lateral area from the root of the vagus nerve, and then medially conducted toward the area around the obex. Although it was difficult to quantitatively evaluate the effective distance from the position of stimulation, the conduction velocities were estimated to be approximately 0.11– 0.12 m/s for the first peak signal and 0.09 – 0.11 m/s for the second peak signal. As development proceeded from the 6- to 8-day embryonic stage, the response areas in the caudal medulla expanded into the contralateral side to the stimulation. At the 6-day embryonic stage, the response area in the caudal medulla existed only on the stimulated side (Fig. 2A). On the other hand, at the 7-day embryonic stage, the response area in the caudal medulla spread into the contralateral side (Fig. 2B). The contralateral response further

143

expanded from the obex area to more rostral area at the 8-day embryonic stage (Fig. 2C). Spatial distribution patterns of the optical signals As shown in Figs. 1 and 2, using differences in the timing of signal peaks, optical signals induced by vagal nerve stimulation can be divided into two populations: (1) the population of the first peak signals and (2) that of the second peak signals. To trace developmental changes in the spatial distribution pattern of these two populations of the optical signal, we measured the amplitude of the signals and constructed contour line maps (Fig. 3). These maps revealed the following characteristics of the optical responses: (1) at the 6- to 8-day embryonic stages, the optical signals were distributed in layered patterns with the signal size decreased peripherally; (2) the distribution of the first peak signal (Fig. 3, black lines) corresponded to the NTS and DMNV and the pattern was consistent with our previous work (Momose-Sato et al., 1991); (3) the distribution area of the second peak signal (Fig. 3, red lines) expanded through the caudal side to the obex as development proceeded from the 6- to the 8-day embryonic stage; (4) at the 6-day embryonic stage, the second peak signals were distributed on the stimulated side (Fig. 3A), whereas at the 7- and 8-day embryonic stages, they spread into the contralateral side (Fig. 3B, C); and (5) the intensity of the second peak signal was largest at the position just caudal to the obex. For further examination of the distribution pattern of the second peak signal in the dorso-ventral plane, we recorded optical signals in 8-day-old slice preparations, and constructed contour line maps (Fig. 4). In a slice preparation that was coronally cut at the cephalic level to the obex (Fig. 4A), the second peak signals were distributed in the ipsilateral dorso-lateral region, and were never distributed in the contralateral side. On the other hand, in a slice preparation that was cut at the caudal level to the obex (Fig. 4B), the second peak signals were distributed in the dorsal region not only on the ipsilateral side but also on the contralateral side. These results confirm that the distribution area of the second peak signal expanded through the caudal side to the obex. Comparison with morphology To compare the spatial distribution patterns of the second peak signals with morphological structures, we investigated the distributions of vagal afferent fibers in the caudal medulla by tract tracing with a carbocyanine dye, DiI (Fig. 5). In the area around the obex, we could not observe retrogradely stained motoneuron somas, suggesting that the stained fibers were the orthodromically stained vagal afferent nerve fibers. Developmentally, at the 6-day embryonic stage, the vagal afferent projections did not cross the midline (Fig. 5A). However, at the 7-day embryonic stage, the projections crossed the midline on the caudal side to the obex (Fig. 5B). In the 8-day embryonic preparation, the projections extended toward the contralateral cephalic region via the caudal side to the obex (Fig. 5C). These distribution

144

Y. Momose-Sato et al. / Neuroscience 148 (2007) 140 –150

Fig. 2. Time lapse mapping of optical signals induced by vagal nerve stimulation in 6- to 8-day-old embryonic preparations. In each developmental stage, the gray shadow in the first map (map 1) means an area in which spike-like signals were observed within 5 ms after the optical signal first appeared in the brainstem. Maps 2– 6 show areas in which spike-like signals were observed within 5 ms after they were observed in the previous map. In every embryonic stage, optical signals were first conducted to regions corresponding to NTS and DMNV (map 2) from the root of the vagus nerve, and thereafter, optical signals were observed in the caudal medulla (maps 3– 6).

patterns of the vagal afferent fibers are well consistent with those of the optical signals (Fig. 3).

DISCUSSION In the present study, using optical recording with a fast voltage-sensitive dye, we succeeded in visualizing vagus

nerve–related neural responses in the newly identified area around the obex in the embryonic chick brainstem. This response area was not discernable in our previous studies because of the 10 times smaller signal intensity, and it was visible after signal averaging. Comparing the present results with those obtained in our previous studies (Momose-Sato et al., 1991; Sato et al., 2004; for reviews

Y. Momose-Sato et al. / Neuroscience 148 (2007) 140 –150

145

Fig. 4. Contour-line maps of the amplitude of the second peak signal evoked by vagal nerve stimulation. Data were obtained from two different 8-day-old slice preparations after 50 times averaging of the optical signals. The cut lines of slices A and B are shown in the lower figure. Slice A was cut at the cephalic level to the obex; the second peak signals were distributed in the ipsilateral dorso-lateral region. Slice B was cut at the caudal level to the obex; the second peak signals were distributed in the dorsal region not only in the ipsilateral side but also in the contralateral side. Numerals on the contour lines indicate the fractional change multiplied by 105.

see Momose-Sato et al., 2001; Momose-Sato and Sato, 2006), we traced the functional development of the vagal network in the embryonic chick brainstem. Optical detection of vagal afferent projections circumflex to the obex in the brainstem

Fig. 3. Contour-line maps of the amplitude of the optical signal evoked by vagal nerve stimulation in 6- to 8-day-old preparations. The stimulation was applied to the right vagus nerve (N. X), and the maximum amplitude of the fast spike-like signals was measured after 50 times averaging of the optical signals. The preparations are the same as those in Fig. 2. In each figure, contour lines for the first and second peaks in Fig. 1B are illustrated in black and red, respectively. The first peaks (shown in black) appeared at the timings of map 2 in the 6- and 7-day-old preparations and of maps 2–3 in the 8-day-old preparation.

Vagal nerve stimulation evoked neural responses in two separate areas in the 7-day-old embryonic brainstem: the ipsilateral area at the level of the vagus nerve (corresponding to the NTS and DMNV) and the area around the obex (Fig. 1A). Functional development of the NTS/DMNV has

The second peaks (shown in red) appeared at the timings of map 3 in the 6-day-old preparation, maps 3– 4 in the 7-day-old preparation and maps 4 – 6 in the 8-day-old preparation. Numerals on the contour lines indicate the fractional change multiplied by 105.

146

Y. Momose-Sato et al. / Neuroscience 148 (2007) 140 –150

Fig. 5. Morphological development of circumferential fibers caudal to the obex of the vagal pathway. Photographs were obtained from horizontal (coronal) sections of 6- to 8-day-old intact preparations. The position of the photograph in the 6-day-old brainstem is illustrated in the upper right figure. A carbocyanine dye (DiI) was applied to the right vagus nerve (left side of the figure), and nerve fiber projections were observed with an epifluorescence microscope.

already been reported (Momose-Sato et al., 1991; for reviews see Momose-Sato et al., 2001; Momose-Sato and Sato, 2006). The optical signal observed in the latter area has two major characteristics. Firstly, the size of the optical responses in the latter area was about 10 times smaller than that in the NTS and DMNV, and the entire response region was visible after 50 times averaging optical signals. Secondly, the conduction rate from the vagus nerve root to the latter area was slower than that to the NTS/DMNV (Figs. 1B and 2), and therefore these two areas can be

distinguished with differences in delays to the peak of the signal (Fig. 3). Under the assumption that neural pathways were linear from the stimulation site to the response areas, conduction velocities were estimated to be approximately 0.11– 0.12 m/s for the first peak signal and 0.09 – 0.11 m/s for the second peak signal. These conduction velocities are slower than that of the adult C fiber, whose conduction speed is the slowest among nerve fibers. Using voltagesensitive dye recording and electron microscopic observa-

Y. Momose-Sato et al. / Neuroscience 148 (2007) 140 –150

tion, Sakai et al. (1991) showed that, in the 6- to 8-day-old chick embryo, (1) the conduction velocity of the vagus nerve bundle was approximately 0.1– 0.2 m/s, (2) the conduction velocity was closely correlated with the crosssectional area of the bundle, and that (3) the vagus nerve bundle involved many small unmyelinated fibers with various sizes but no myelinated fibers. It was also shown that as development proceeded, the conduction velocity increased monotonically due to the increased diameter of the nerve fiber. The differences in nerve conduction velocities observed in the present study may also be due to differences in the diameters of unmyelinated nerve fibers, although we could not rule out contributions of differences in specific resistance of the cytoplasm and/or the membrane. Does the response area around the obex correspond to a motor nucleus or a sensory nucleus? The vagus nerve contains sensory (afferent) and motor (efferent) nerve fibers, and the stimulation applied to the vagus nerve was simultaneously orthodromic for the sensory nerve fibers and antidromic for the efferent fibers. In our previous study (Komuro et al., 1991; Momose-Sato et al., 1991), we showed that, in the NTS, vagal nerve stimulation evoked a fast spike-like signal followed by a long-lasting slow signal. The fast signal was completely blocked by TTX (20 ␮M), suggesting that the fast signal corresponds to a sodium-dependent action potential. On the other hand, the slow signal was blocked by kynurenic acid (1.2 mM), indicating that the slow signal corresponds to a glutamatergic EPSP. Other blockers of chemical transmission such as adrenaline, acetylcholine, GABA and glycine did not have significant effects on the slow signal, and it seems less likely that other transmitters are active in generating the slow signals. The EPSP-related slow signal was easily fatigued with repetitive stimuli (even with 0.1 Hz stimulation), and we needed to identify the slow signal without signal averaging. Since, in the present study, the size of the optical signal in the response area around the obex was very small and we averaged optical responses, we could not identify the EPSP-related slow signal. On the other hand, we could morphologically visualize the neural pathway to the response area around the obex by DiI staining (Fig. 5). No retrogradely stained motoneurons were observed in the response area, suggesting that the stained nerve fibers were mainly vagal afferent projections. This indicates that the optical signals observed in the response area around the obex contained neural responses of the orthodromically stimulated vagal afferent pathway. Comparison with anatomical information (Breazile, 1979; Kuenzel and Masson, 1988; Marín and Puelles, 1995; Cambronero and Puelles, 2000) indicated that it is most likely that the bilateral response area around the obex corresponds to the area postrema. Neurons in the area postrema are thought to be related to baroreflex and cardiovascular control (Undesser et al., 1985; Skoog and Mangiapane, 1988; Ferguson, 1991) and the vagal/glossopharyngeal nerve has been shown to send fiber projections to the area postrema (Altschuler et al., 1989). In

147

many species, including chicken, the area postrema appears as bilateral rounded eminences on either side of the fourth ventricle at its entrance to the central canal (Oldfield and McKinley, 1995). This anatomical information is consistent with the distribution patterns of the optical signals (Figs. 3 and 4). The area postrema is known to have afferent and efferent neural connections in the brain. In the rat, afferent neural connections to the area postrema mainly come from the hypothalamus, the PBN, the dorsal raphe, the A1 region of the ventrolateral medulla and the NTS (Torack et al., 1973; Vigier and Portalier, 1979; Hosoya and Matsushita, 1981; van der Kooy and Koda, 1983; Shapiro and Miselis, 1985; Larsen et al., 1991). On the other hand, the area postrema neurons send axons to the NTS, the DMNV, the caudal ventrolateral medulla, the dorsal aspect of the spinal trigeminal tract and nucleus, the paratrigeminal nucleus and the PBN (Torack et al., 1973; van der Kooy and Koda, 1983; Shapiro and Miselis, 1985). In the 8-day embryonic preparation (Fig. 3, the bottom panel), the optical response area around the obex extended toward the cephalic direction on the contralateral side. Although this response area may include neural responses of the internuclear connections related to the area postrema, we could not distinguish these responses in the present study. Another possible candidate that is included in the optical response area is the accessory nucleus (nucleus radicis spinalis nervi accessorii). The root of the vagus and accessory nerve could not be completely separated microsurgically, and there remains a possibility that the accessory nerve was partially stimulated with a suction electrode. Functional development of the vagus nerve–related neural pathway in the brainstem In our previous studies, using the voltage-sensitive dye recording technique, we identified several response areas in the embryonic chick brainstem with vagal nerve stimulation (Komuro et al., 1991; Momose-Sato et al., 1991, 1994; Sato et al., 2002, 2004; Momose-Sato and Sato, 2005). Fig. 6A shows a schematic representation of the vagus nerve–related optical response areas. The response areas are considered to include (1) the motor nucleus, (2) the primary sensory nuclei and (3) the second/ higher-ordered nucleus. We describe the functional development of these nuclei separately (Fig. 6B). The motor nucleus. The motor nucleus is the DMNV (the blue area in Fig. 6), and antidromic action potentials were first recorded in the DMNV from the 4-day-old embryonic stage (Momose-Sato et al., 1991). In mammals, another motor nucleus related to the vagus nerve, the nucleus ambiguus (NA), is distinguished, whereas in birds, the NA is not definitely determined (Cohen et al., 1970; Breazile, 1979; Kuenzel and Masson, 1988). In our optical studies, we could not identify any responses corresponding to the activity in the NA. The primary sensory nuclei. As sensory nuclei that receive direct inputs from the vagus nerve, we have identified three response areas: (i) the NTS (the green area in

148

Y. Momose-Sato et al. / Neuroscience 148 (2007) 140 –150

Fig. 6. (A) Schematic drawing of the vagal neural pathway in the embryonic chick brainstem. The left panel shows the cephalo-caudal distribution pattern of the vagal responses in the 8-day-old embryonic brainstem. The dorso-ventral distribution patterns in brainstem slices at the level of the vagus nerve root and at the obex are illustrated in the right panels. The yellow area around the obex was identified in this study. The NTS (the green area) and DMNV (the blue area) are cited from Momose-Sato et al. (1991). The contralateral cephalic response area (a red area), which appears to correspond to the PBN, is cited from Sato et al. (2002). The contralateral medullary non-NTS region (the pink area) is cited from Momose-Sato and Sato (2005). (B) Developmental sequences of emergence of the action potentials and EPSPs in each response area shown in A.

Fig. 6), (ii) the medullary non-NTS region (the pink area in Fig. 6) and (iii) the response area circumflex to the obex (possibly corresponding to the area postrema: the yellow area in Fig. 6). Although it is unclear which nucleus the medullary non-NTS region corresponds to, one possible candidate is nucleus intercalates, which has often been referred to as the dorsomedial nuclear group of the vagus nerve (Kuenzel and Masson, 1988). In the NTS, action potential-related spike-like signals evoked by vagus nerve stimulation were first detected from the 4-day embryonic stage (Momose-Sato et al., 1991), and in the medullary non-NTS region and the response area circumflex to the obex, the fast signals were discriminated at least at the

6-day embryonic stage (Momose-Sato and Sato, 2005). In the NTS and the medullary non-NTS region, the EPSPrelated slow signals were first detected from the same developmental stage, the 7-day embryonic stage, in normal Ringer’s solution (Momose-Sato et al., 1991; MomoseSato and Sato, 2005). In the present study, we could not identify slow signals in the response area circumflex to the obex because of the small sizes of the optical responses, and could not determine the emergence of synaptic function in this response area. The second/higher-ordered nucleus. Another response area (the red area in Fig. 6) was identified at the

Y. Momose-Sato et al. / Neuroscience 148 (2007) 140 –150

level of the pons/rostral medulla on the contralateral side, and it corresponded to the neural activity in the second/ higher-ordered nucleus of the vagal pathway, possibly the PBN (Sato et al., 2004). The PBN receives inputs from the NTS, and the responses on the contralateral side were first detected from the 7-day-old embryonic stage, when the glutamatergic EPSPs were first expressed in the NTS. This suggests that the synaptic pathway from the NTS to the contralateral nucleus is already generated when the primary vagal afferents make functional synapses on NTS neurons. From our optical studies concerning the vagus nerve– related pathway, we can conclude that (1) the fundamental vagal pathways in the embryonic chick brainstem are functionally generated by the 7-day embryonic stage, and that (2) despite the early formation, functional neuronal pathways related to the vagus nerve exhibit adult patterns from the earliest stages. It is known that higher centers of the vagal pathway exist in the CNS, such as the thalamus and the cortex (Saper, 1995), but we have not yet succeeded in recording vagus nerve–related neural activity outside the brainstem. By improving experimental methods, it will become possible to visualize whole activities in vagus nerve–related neural circuits and to elucidate the functional development of the circuits. Acknowledgments—We express our gratitude to Professor Hiroshi Sasaki (Tokyo Women’s Medical University) and Professor Joel C. Glover (University of Oslo) for discussion on anatomical data. This research was supported by grants from the MonbuKagaku-Sho of Japan and research funds from the Society for Research on Umami Taste and the Smoking Research Foundation. Masae Kinoshita was supported by the postdoctoral fellowship program of Japan Society for the Promotion of Science.

REFERENCES Altschuler SM, Bao X, Bieger D, Hopkins DA, Miselis RR (1989) Viscerotopic representation of the upper alimentary tract in the rat: Sensory gangli and nuclei of the solitary and spinal trigeminal tracts. J Comp Neurol 283:248 –268. Baker BJ, Kosmidis EK, Vucinic D, Falk CX, Cohen LB, Djurisic M, Zecevic D (2005) Imaging brain activity with voltage- and calciumsensitive dyes. Cell Mol Neurobiol 25:245–282. Borday C, Chatonnet F, Thoby-Brisson M, Champagnat J, Fortin G (2005) Neural tube patterning by Krox20 and emergence of a respiratory control. Resp Physiol Neurobiol 149:63–72. Borday C, Wrobel L, Fortin G, Champagnat J, Thaeron-Antono C, Thoby-Brisson M (2004) Developmental gene control of brainstem function: views from the embryo. Prog Biophys Mol Biol 84: 89 –106. Breazile JE (1979) Systema nervosum centrale. In: Nomina anatomica avium (Baumel JJ, King AS, Lucas AM, Breazile JE, Evans HE, eds), pp 417– 472. New York: Academic Press. Cambronero F, Puelles L (2000) Rostrocaudal nuclear relationships in the avian medulla oblongata: A fate map with quail chick chimeras. J Comp Neurol 427:522–545. Cohen LB, Salzberg BM (1978) Optical measurement of membrane potential. Rev Physiol Biochem Pharmacol 83:35– 88. Cohen DH, Schnall AM, MacDonald RL, Pitts LH (1970) Medullary cells of origin of vagal cardioinhibitory fibers in the pigeon. 1. Anatomical studies of peripheral vagus nerve and the dorsal motor nucleus. J Comp Neurol 140:299 –320.

149

Dampney RAL (1994) Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 74:323–364. Ebner TJ, Chen G (1995) Use of voltage-sensitive dyes and optical recordings in the central nervous system. Prog Neurobiol 46:463–506. Ferguson A (1991) The area postrema: A cardiovascular control center at the blood-brain interface? Can J Physiol Pharmacol 69:1026 –1034. Fortin G, del Toro ED, Abadie V, Guimaraes L, Foutz AS, DenavitSaubie M, Rouyer F, Champagnat J (2000) Genetic and developmental models for the neural control of breathing in vertebrates. Respir Physiol 122:247–257. Friauf E, Lohmann C (1999) Development of auditory brainstem circuitry activity-dependent and activity-independent processes. Cell Tissue Res 297:187–195. Glover JC (2000) Development of specific connectivity between premotor neurons and motoneurons in the brain stem and spinal cord. Physiol Rev 80:615– 647. Godement P, Vanselow J, Thanos S, Bonhoeffer F (1987) A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue. Development 101:697–713. Grinvald A, Frostig RD, Lieke E, Hildesheim R (1988) Optical imaging of neuronal activity. Physiol Rev 68:1285–1366. Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49 –92. Hosoya Y, Matsushita M (1981) A direct projection from the area postrema in the rat as demonstrated by the HRP, and autoradiographic methods. Brain Res 214:144 –149. Kamino K (1991) Optical approaches to ontogeny of electrical activity and related functional organization during early heart development. Physiol Rev 71:53–91. Kamino K, Hirota H, Fujii S (1981) Localization of pacemaking activity in early embryonic heart monitored using voltage-sensitive dye. Nature 290:595–597. Komuro H, Sakai T, Momose-Sato Y, Hirota A, Kamino K (1991) Optical detection of postsynaptic potentials evoked by vagal stimulation in the early embryonic chick brain stem slice. J Physiol (Lond) 442:631– 648. Kuenzel WJ, Masson M (1988) A stereotaxic atlas of the brain of the chick (Gallus domesticus), pp 1–166. Baltimore: Johns Hopkins University Press. Larsen PJ, Moller M, Mikkelsen JD (1991) Efferent projections from the periventricular and medial parvicellular subnuclei of the hypothalamic paraventricular nucleus to circumventricular organs of the rat: A Phaseolus vulgaris-leucoagglutinin (Pha-L) tracing study. J Comp Neurol 306:462– 479. Marín F, Puelles L (1995) Morphological fate of rhombomeres in quail/chick chimeras: A segmental analysis of hindbrain nuclei. Eur J Neurosci 7:1714 –1738. Momose-Sato Y, Sakai T, Hirota A, Sato K, Kamino K (1994) Optical mapping of early embryonic expressions of Mg2⫹-/APV-sensitive components of vagal glutaminergic EPSPs in the chick brainstem. J Neurosci 14:7572–7584. Momose-Sato Y, Sakai T, Komuro H, Hirota A, Kamino K (1991) Optical mapping of the early development of the response pattern to vagal stimulation in embryonic chick brain stem. J Physiol (Lond) 442:649 – 668. Momose-Sato Y, Sato K (2005) Primary vagal projection to the contralateral non-NTS region in the embryonic chick brainstem revealed by optical recording. J Membr Biol 208:183–191. Momose-Sato Y, Sato K (2006) Optical recording of vagal pathway formation in the embryonic brainstem. Auton Neurosci Basic Clin 126 –127:39 – 49. Momose-Sato Y, Sato K, Kamino K (1999) Optical identification of calcium-dependent action potentials transiently expressed in the embryonic rat brainstem. Neurosci 90:1293–1310.

150

Y. Momose-Sato et al. / Neuroscience 148 (2007) 140 –150

Momose-Sato Y, Sato K, Kamino K (2001) Optical approaches to embryonic development of neural functions in the brainstem. Prog Neurobiol 63:151–197. Momose-Sato Y, Sato K, Kamino K (2002) Application of voltagesensitive dyes to the embryonic central nervous system. In: Recent research developments in membrane biology, Vol. 1 (Fagan J, Davidson JN, Shimizu N, eds), pp 159–181. Kerara: Research Signpost. Momose-Sato Y, Sato K, Sakai T, Hirota A, Matsutani K, Kamino K (1995) Evaluation of optimal voltage-sensitive dyes for optical monitoring of embryonic neural activity. J Membr Biol 144:167–176. Oldfield BJ, McKinley MJ (1995) Circumventricular organs. In: The rat nervous system (Paxinos G, ed), pp 391– 403. San Diego: Academic Press. Ross WN, Salzberg BM, Cohen LB, Grinvald A, Davila HV, Waggoner AS, Wang CH (1977) Changes in absorption, fluorescence, dichroism, and birefringence in stained giant axons: optical measurement of membrane potential. J Membr Biol 33:141–183. Rubel EW, Fritzsch B (2002) Auditory system development: primary auditory neurons and their targets. Annu Rev Neurosci 25:51–101. Sakai T, Komuro H, Katoh Y, Sasaki H, Momose-Sato Y, Kamino K (1991) Optical determination of impulse conduction velocity during development of embryonic chick cervical vagus nerve bundles. J Physiol (Lond) 439:361–381. Salzberg BM (1983) Optical recording of electrical activity in neurons using molecular probes. In: Current methods in cellular neurobiology, Vol. 3, Electrophysiological techniques (Barber JL, ed), pp 139 –187. New York: John Wiley & Sons Inc. Salzberg BM, Obaid AL, Senseman DM, 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. Saper CB (1995) Central autonomic system. In: The rat nervous system (Paxinos G, ed), pp 107–135. San Diego: Academic Press. Sato K, Miyakawa N, Momose-Sato Y (2004) Optical survey of neural circuit formation in the embryonic chick vagal pathway. Eur J Neurosci 19:1217–1225.

Sato K, Mochida H, Yazawa I, Sasaki S, Momose-Sato Y (2002) Optical approaches to functional organization of glossopharyngeal and vagal motor nuclei in the embryonic chick hindbrain. J Neurophysiol 88:383–393. Sato K, Momose-Sato Y, Hirota A, Sakai T, Kamino K (1998) Optical mapping of neural responses in the embryonic rat brainstem with reference to the early functional organization of vagal nuclei. J Neurosci 18:1345–1362. Sato K, Momose-Sato Y, Sakai T, Hirota A, Kamino K (1995) Responses to glossopharyngeal stimulus in the early embryonic chick brainstem: Spatiotemporal patterns in three dimensions from repeated multiple-site optical recording of electrical activity. J Neurosci 15:2123–2140. Shapiro RE, Miselis RR (1985) The central neural connections of the area postrema of the rat. J Comp Neurol 234:344 –364. Skoog KM, Mangiapane ML (1988) Area postrema and cardiovascular regulation in rats. Am J Physiol 254:H965–H969. Spyer KM (1982) Central nervous integration of cardiovascular control. J Exp Biol 100:109 –128. Torack RM, Stranaham P, Hartman PK (1973) The role of norepinephrine in the function of the area postrema. I. Immunofluorescent localization of dopamine-beta-hydroxylase and electron microscopy. Brain Res 61:253–265. Travagli RA, Hermann GE, Browning KN, Rogers RC (2006) Brainstem circuits regulating gastric function. Annu Rev Physiol 68: 279 –305. Undesser KP, Hasser E, Haywood JR, Johnson AK, Bishop VS (1985) Interactions of vasopressin with the area postrema in arterial baroreflex function in conscious rabbits. Circ Res 56:410 – 417. van der Kooy D, Koda LY (1983) Organization of the projections of a circumventricular organ: The area postrema in the rat. J Comp Neurol 219:328 –338. Vigier D, Portalier P (1979) Efferent projections of the area postrema demonstrated by autoradiography. Arch Ital Biol 117: 308 –324.

(Accepted 23 May 2007) (Available online 16 July 2007)