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1Department of Physiology, Tokyo Medical and Dental University, Graduate School and Faculty of Medicine; and 2Department of Anatomy, Tokyo Women's Medical University, School of Medicine, Tokyo, Japan
Submitted 13 January 2005; accepted in final form 27 April 2005
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ABSTRACT |
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-glycyrrhetinic acid. In the immunohistochemical study, significant immunoreactivity of connexin26 and connexin32 was also observed. Wave activity detected with a voltage-sensitive dye was accompanied by a Ca2+-wave, indicating that it not only provides electrical synchrony but also biochemical signals associated with [Ca2+]i elevation. These characteristics of the wave activity are similar to those of the depolarization wave reported in the chick embryo, suggesting that the large-scale depolarization wave is globally generated across different species. |
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INTRODUCTION |
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; Champagnat and Fortin 1997; Feller 1999; O'Donovan 1999; Roerig and Feller 2000). Although the roles of this activity during ontogenesis remain unresolved, one traditional view is that it contributes to the activity-dependent developmental organization of neuronal circuits with specific functions (Goodman and Shatz 1993; Hanse et al. 1997; Katz and Shatz 1996; Sur and Leamey 2001). For example, in the developing mammalian cortex, the synchronized activity of neurons in locally correlated areas, termed neuronal domains, has been suggested to lead to a columnar unit of interconnected neurons in the adult brain (Yuste et al. 1992). In slice preparations including the retina and lateral geniculate nucleus, the bursting activity of retinal ganglion cells is transmitted to the lateral geniculate nucleus, suggesting a role for retinal inputs in the regulation of visual pathway formation (Mooney et al. 1996).
A recent finding of a novel form of correlated wave activity in the embryonic chick CNS suggested another possibility concerning the role of the correlated activity during embryogenesis. This activity was recorded using a multiple-site optical recording technique with a fast voltage-sensitive dye as widely spreading depolarizing optical signals and was termed the depolarization wave (Momose-Sato et al. 2001b). One novel phenomenon of the depolarization wave is that it travels over a wide region of the CNS including the medulla, spinal cord, pons, cerebellum, midbrain, and telencephalon (Mochida et al. 2001; Momose-Sato et al. 2001b, 2003a). Another outstanding feature is that it is nonspecifically triggered by various types of cranial and spinal sensory inputs as well as endogenous spontaneous activity (Mochida et al. 2001; Momose-Sato et al. 2003b). These depolarization wave profiles suggest that it may not serve as a simple regulator of specific neuronal circuit formation but might play a more global role in CNS development (Momose-Sato et al. 2003b).
An important question is whether correlated activity similar to the depolarization wave is generated in other species, especially in mammals. The aim of the present study was to examine whether widely propagating wave activity can be observed in the embryonic rat CNS and if so, then to examine the characteristics of the wave activity including spatiotemporal patterns and pharmacological natures for comparison with the chick depolarization wave. Preliminary results have appeared in abstract form (Momose-Sato and Sato 2004; Sato et al. 2004).
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METHODS |
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The experiments were carried out in accordance with the guidelines of 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. Wistar rats at 15–17 days gestation (E15–E17; Saitama Experimental Animals Supply, Saitama, Japan) were used. Females were caged with males in the evening and were checked for sperm positivity the next morning, and this day was termed E0. Pregnant rats were anesthetized with ether, and their fetuses were then surgically removed. To investigate the spatial distribution pattern of evoked waves of activity, three types of semi-intact preparations were used: Type-A, brain stem preparations that included the medulla, cervical spinal cord, pons, cerebellum with or without the midbrain (Fig. 1A); Type-B, whole brain preparations that included the diencephalon and telencephalon in addition to Type-A preparation; Type-C, spinal cord preparations that included the medulla and spinal cord. Most experiments were carried out using E16 brain stem (Type-A) preparations. The number of tested preparations and the incidence of wave activity are summarized in Table 1. The preparations were kept in a bathing solution of (in mM) 149 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 10 glucose, and 10 Tris-HCl buffer (pH 7.4), and the solution was equilibrated with oxygen. In some experiments, we used artificial cerebrospinal fluid (ACSF) that contained (in mM) 124 NaCl, 5 KCl, 1 MgSO4, 2.5 CaCl2, 1.25 NaH2PO4, 22 NaHCO3, and 10 glucose, equilibrated with a mixture of 95% O2-5% CO2 (pH 7.4) and obtained similar results. The pia mater was carefully removed in the bathing solution under a dissection microscope, and the preparation was next stained by incubating it for 10–20 min in the solution containing 0.2–0.4 mg/ml of the voltage-sensitive merocyanine-rhodanine dye NK2761 (Hayashibara Biochemical Laboratories/Kankoh-Shikiso Kenkyusho, Okayama, Japan) (Momose-Sato et al. 1995). After staining, the preparation was attached to the silicone bottom of a recording chamber with the ventral or dorsal side facing up by pinning it down with tungsten wires. The preparation was continuously perfused with the bathing solution at 2–3 ml/min at 26–30°C. Wave activity was evoked by applying square current pulses to the upper cervical cord 2–3 mm caudal to the obex using a concentric bipolar tungsten electrode at 200 µA/1 ms or to the cranial nerves with a glass micro-suction electrode at 8 µA/5 ms.
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For the pharmacological experiments, DL-2-amino-5-phosphonovaleric acid (APV), strychnine, bicuculline, picrotoxin, tetrodotoxin, octanol, and 18-glycyrrhetinic acid were acquired from Sigma Chemical (St. Louis, MO), 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) was obtained from Research Biochemicals International (Natic, MA), and d-tubocurarine and atropine were acquired from Wako Pure Chemical Industries(Osaka, Japan).
Optical recording using a voltage-sensitive dye
The methods used for the multiple-site optical recording of electrical activity in embryonic nervous systems are described elsewhere (for reviews, see Kamino 1990; Momose-Sato et al. 2001a). In brief, bright-field illumination was provided by a 300 W tungsten-halogen lamp (Type JC-24V/300W, Kondo Philips, Tokyo, Japan) driven by a stable DC power supply, and incident light was collimated and rendered quasi-monochromatic with an interference filter with a transmission maximum at 703 ± 12 nm (half-width; Asahi Spectra, Tokyo, Japan). The objective (Plan Apo, x4, 0.2 NA) and photographic eyepiece (x2.5) projected a real image of the preparation onto a 34 x 34-element silicon photodiode matrix array mounted on a microscope. Changes in transmitted light intensity through the preparation were detected with the photodiode array, and were recorded with a 1,020-site optical recording system that was constructed in our laboratory. Each pixel (element) of the array detected light transmitted by a square region (116 x 116 µm2 using x10 magnification) of the preparation. The time resolution of the system was 
1 ms (1,024 frames/1,000 ms). The recordings were made in single sweeps at 10- to 15-min intervals.
The fractional change in dye absorption 
A/Ar is equal to –I/(Ibefore staining – Iafter staining), where I is the light intensity transmitted through the preparation (Ross et al. 1977). In E16 preparations (n = 3), we compared Ibefore staining and Iafter staining by measuring the light that reached the detectors before and after the preparation was stained for 10 min with 0.4 mg/ml NK2761. Iafter staining/Ibefore staining averaged 34% in the pons, 36% in the rostral medulla, 38% in the caudal medulla, and 34% in the cervical spinal cord. To stain the preparation well, the dye was allowed to diffuse from the ventral and dorsal sides of the brain before the preparation was pinned to the chamber. Thus the measurement of Ibefore staining and Iafter staining from the same position was technically difficult. Because variations in Iafter staining/Ibefore staining between the regions were relatively small, we measured Iafter staining and I and expressed the optical signal as I/Iafter staining. Color-coded representation used for the construction of a spatiotemporal activity map was carried out with "NeuroPlex" software (RedShirtImaging LLC, Fairfield, CT), and that for the maximum signal amplitude was performed using "Transform" software (Fortner Research LLC, Sterling, VA). The color code for each figure was linearly distributed between the minimum and maximum values of I/I.
Ca2+ fluorescence imaging
For the detection of changes in intracellular Ca2+ concentration ([Ca2+]i), we used the Ca2+ indicator Ca green-1 AM (Molecular Probes, Eugene, OR). The preparation was stained by incubating it for 60 min in HEPES-buffered (HEPES-NaOH, pH 7.4, 10 mM) Ringer solution containing 20 µg/ml Ca green-1 AM with 0.5% dimethylsulfoxide (DMSO) and 0.005% cremophor. It was then perfused with dye-free Ringer solution for 60 min before recording. The preparation chamber was mounted on an inverted microscope (Axiovert 135TV, Carl Zeiss, Göttingen, Germany), and a 75-W Xenon lamp provided epi-illumination of the field. The excitation light was passed through a heat filter and a band-pass filter (450–490 nm) to the preparation, and the emitted light was then passed through a dichroic mirror (510 nm) and a band-pass filter (515–565 nm). The objective (Plan NEOFLUAR, x5, 0.15 NA) projected a real image of the preparation (magnification: x5) onto a cooled CCD camera system (PXL 3700, Photometrics, Tucson, AZ) mounted on the microscope, where the camera head contained a half-masked 512 x 512 pixel CCD chip operated in the frame-transfer mode and the system had a 12-bit dynamic range. Groups of 2 x 2 pixels were binned together, and images were recorded with a frame interval of 500 ms.
Electrical recording
Wave activity was electrically monitored by recording the population activity of vagal motoneurons with a suction electrode applied to the cut end of the vagus nerve root. Signals were amplified with filters set at 0.08 Hz and 1 kHz and digitally recorded at 4 kHz with an A/D converter (MacLab/8S, ADInstruments, Castle Hill, Australia).
Immunohistochemistry
Ten fetal (E16) rats were used. Their brains and spinal cords were removed and immersed in cold (4°C) fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4), for 2–3 h, and the tissues were then cryoprotected in 10 mM PB containing 30% sucrose. They were finally directly embedded in Tissue-Tek/O.C.T. (Sakura Finetechnical, Tokyo, Japan) and rapidly frozen in liquid nitrogen. Tissues from the liver of adult rats (n = 2) were used as a positive control. Horizontal sections of the flattened brains and spinal cords were cut at a thickness of 6–8 µm using a cryostat, air-dried, and processed following the labeled streptavidin-biotin (LSAB) method using a Dako LSAB2 kit (DakoCytomation, Kyoto, Japan). The sections were washed three times for 10 min each in 10 mM phosphate-buffered saline (PBS, pH 7.4), and then endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 30 min at room temperature. After washing in PBS, the sections were blocked for 30 min in 10% normal goat serum (Vector Laboratories, Burlingame, CA), followed by incubation overnight at 4°C with the primary antibodies against connexins. In the rat embryo, connexins26, -32, -43, -45 are expressed in the cortex and/or midbrain floor (Leung et al. 2002; Nadarajah et al. 1997), although no study has been carried out on the lower brain stem. Thus we examined the immunoreactivity of these connexins in the brain stem region, in which the optical signals were most prominent. We used 1/200 diluted rabbit polyclonal anti-mouse connexin26 antibody (Chemicon International, Temecula, CA), 1/100 diluted mouse monoclonal anti-connexin32 antibody (Zymed Laboratoriez, South Sara Francisco, CA), 1/100 diluted rabbit anti-connexin43 antibody (Zymed Laboratoriez), and 1/100 diluted rabbit polyclonal anti-connexin45 antibody (Chemicon International). The sections were then washed again three times in PBS, incubated with streptavidin conjugated to horseradish peroxidase (DakoCytomation) for 10 min at room temperature, and reacted for 10 min in 50 mM Tris-HCl buffer (pH 7.4) containing 0.02% 3,3'-diaminobenzidine (DAB) and 0.005% hydrogen peroxide. Control sections were treated using the same immunohistochemical protocol except for incubation with primary antibodies. No immunostaining was seen in the control sections. After counterstaining with hematoxylin, the sections were dehydrated in a graded alcohol series, cleared with xylene, and coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ). Photomicrographs were then taken using a microscope (Nikon ECLIPSE E600, Tokyo) equipped with a Nikon DXM digital camera system at a resolution of 3,600 x 2,880 pixels. The captured images were processed using Adobe Photoshop software (Adobe Systems) and were printed at a resolution of 300 dpi.
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RESULTS |
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In the present study, we first examined whether correlated wave activity was evoked by direct stimulation of the upper cervical cord, which had been shown in the chick embryo to induce depolarization waves (Momose-Sato et al. 2003a). Figure 1B shows an example of the optical recordings obtained from an E16 brain stem preparation. When we applied stimuli to the upper cervical cord, depolarizing optical signals were evoked throughout the medulla, pons, and cerebellar primordium. Optical signals were not detected at 630 nm (Fig. 1C), where the membrane potential-related absorption change of NK2761 is absent (Momose-Sato et al. 1995), indicating that the obtained signals were voltage-dependent absorption changes in the dye and not intrinsic signals or mechanical artifacts. Similar results were obtained from 24 brain stem preparations dissected from E15 to E16 embryos, although the responses in the cerebellum were significant (I/I 
2.0 x 10–4) only in five E16 preparations (Tables 1 and 2).
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; Obaid et al. 1985; Orbach et al. 1985). Thus if the action potentials were asynchronous between neighboring neurons or originated from a small active membrane area, it is possible that they were not observed as clear spike-like signals.
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; Weissman et al. 2004), suggesting that the wave in the present study is different from previously reported glial waves. We tested the oxonol dye RH482 (NK3630), which is relatively insensitive to glial cell membrane potentials in the skate cerebellum (Konnerth et al.1987), and observed similar wave activity, although this does not exclude the possibility of a glial cell contribution to the optical signal because the cell specificity of RH482 has not been proven in the embryonic rat brain. It was interesting to determine to what extent the waves were propagated within the embryonic brain. Figure 3 shows the propagation of the optical signals in the whole brain (Fig. 3A) and intact spinal cord (Fig. 3B) dissected from E16 to E17 embryos. When we applied stimuli to the upper cervical cord, optical signals were propagated rostrally to the medulla, pons and midbrain (Fig. 3A). Small signals were also observed in the thalamus and part of the cerebrum. In a caudal direction, the wave was propagated to the lumbosacral region (Fig. 3B). These results show that the depolarizing wave activity is widely distributed in the embryonic CNS (Table 2).
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Next, we examined whether the wave activity shown in Figs. 1 and 2 was elicited by other natural triggers such as synaptic inputs from the sensory nerves. Figures 4 and 5 show that this was the case. In Fig. 4A, two patterns of optical responses obtained by stimulation of the maxillary branch of the trigeminal nerve (N. V2) are shown. In the upper images, trigeminal nerve stimulation evoked pre- and postsynaptic responses in a restricted region corresponding to the trigeminal nuclear complex (Momose-Sato et al. 2004), whereas in the lower images trigeminal nerve stimulation at the same intensity elicited the wide propagation of optical signals following the nuclear response. The former pattern was often observed shortly after dissection of the preparation or after repetitive stimuli, whereas the latter pattern was usually observed after the preparation was fully recovered. In Fig. 4B, enlargements of the wave-related optical signals detected from four regions shown in Fig. 4C are presented. Similar results were obtained via maxillary nerve stimulation from 9 of 10 preparations dissected from E15 to E16 embryos (Table 1).
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). When we stimulated the ophthalmic nerve in the Mg2+-free solution, in which an N-methyl-D-aspartate (NMDA) receptor-mediated component of the EPSP was potentiated (Momose-Sato et al. 2004), the wave appeared in every preparation (Table 1). In Tables 2–4, we compared the spatiotemporal characteristics of the waves evoked by upper cervical cord stimulation and cranial nerve stimulation. The spatial extent of the wave activity (Table 2) and the time course of the wave-related optical signal (Table 3) were similar in every preparation although the onset latency of the signal (Table 4) showed some differences between stimulations: the onset latency was more variable with cranial nerve stimulation than with direct stimulation of the cervical cord. A comparison of the onset latency of the signal between different regions (Table 4, bottom) showed that the delay was most variable between the cranial sensory nucleus and the medulla, and that propagation of the wave to the pons and cerebellum was not largely different between stimulations.
Similarities in the characteristics of the wave-related optical signals between different nerves suggests that although each nerve stimulation first activated a specific set of neural structures within the sensory nucleus, there was a nonspecific spread of depolarization from the nucleus that led to global activation of the brain. Another possibility is that the net effects of secondary and tertiary synaptic links led to widespread depolarization with each nerve stimulation. Figure 6 suggests that the spatiotemporal characteristics of the wave activity were different from those of the second/higher-ordered synaptic responses evoked by cranial nerve stimulation as seen by the multiple-site optical recordings of maxillary nerve responses detected in the E16 medulla. In Fig. 6A, left (1-s recording), optical signals related to the EPSP were identified in the lateral region corresponding to the trigeminal nuclear complex as shown by the red asterisk in the right short-time recording, and wave-related optical signals were observed over the entire medulla. The expanded time base of the same recording (Fig. 6A, right: 125-ms recording) revealed that there was another population of neural responses with a short latency on the contralateral side as indicated by the dotted circle. Comparison of the onset latency of the signals from three different regions, the trigeminal nuclear complex (red asterisk), the contralateral short-latency response area (green asterisk), and the region with wave activity alone (blue asterisk; Fig. 6B) showed that there was a small delay of 10–20 ms between the first-ordered EPSP in the trigeminal nuclear complex (red asterisk) and the contralateral response (green asterisk). The onset latency of the wave-related optical signal (blue asterisk) was long and highly variable as indicated in Table 4. Contour line maps of the contralateral responses (Fig. 6C, red lines) and wave activity (Fig. 6C, blue lines) exhibited different patterns with no correlation between the amplitudes of the two responses (Fig. 6D, correlation coefficient = 0.333); in one position (Fig. 6C, top right trace) the contralateral response (arrowhead) was detected with the small wave-related optical signal (asterisk), whereas in another (Fig. 6C, bottom right trace) no contralateral response was identified despite the large wave-related optical signal. Furthermore, wave activity was detected from preparations in which contralateral responses were not clearly identified.
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Ca2+ waves associated with the depolarizing wave activity
One question we asked was whether the widely propagating depolarizing wave activity detected with a voltage-sensitive dye could afford information limited to the electrogenic interaction of cells or provide biochemical signaling such as via Ca2+ through the network. We thus examined whether the spreading depolarization was accompanied by a change in the intracellular Ca2+ concentration ([Ca2+]i) using a Ca2+-imaging technique and the fluorescent Ca2+-indicator Ca green-1 AM.
Figure 7 shows an example of Ca2+-imaging obtained via stimulation of the N. V2. When we stimulated the nerve, Ca2+ responses were first observed in the trigeminal nuclear complex (1st frame), followed by an elevation in [Ca2+]i over a wide region of the medulla, spinal cord and pons. Ca2+ responses exhibited a multiple-peak pattern with peak locations in the rostral medulla, lateral and medial regions of the pons, and spinal cord just caudal to the obex. This spatial distribution pattern was similar to that of the spreading depolarizing optical signals (Fig. 2). These results suggest that the wave activity identified in the present experiment provides not only electrical synchrony but also biochemical signals through the network distributed over a wide region of the CNS.
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We next performed pharmacological examinations to study neural network mechanisms underlying the propagation of the wave. Experiments were performed using E16 brain stem preparations with triggering wave activity by upper cervical cord stimulation.
The wave activity was eliminated by Cd2+ (10 µM; n = 7 preparations; Figs. 8A and 9A), Ca2+-free solution (n = 2; data not shown) and tetrodotoxin (TTX; 1 µM; n = 2; data not shown), suggesting that activity in chemical synaptic networks was involved. To assess which neurotransmitters mediate the wave activity, we applied antagonists to neurotransmitter receptors. The wave activity was markedly inhibited by the application of glutamate receptor antagonists. In the presence of the 
-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainite receptor antagonist CNQX (5 µM; n = 4) or the NMDA receptor antagonist APV (200 µM; n = 5), the wave-related optical signals were significantly reduced in amplitude (Figs. 8B and 9A). The wave activity was also inhibited when the nicotinic acetylcholine receptor antagonist d-tubocurarine (dTc; 100 µM; n = 4) was applied, although the muscarinic acetylcholine receptor antagonist atropine (10 µM; n = 4) did not significantly affect it (Figs. 8C and 9A).
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; Ben-Ari 2002). In embryonic rat brain stem preparations, application of the glycine receptor antagonist strychnine (20 µM; n = 4) or GABAA receptor antagonists bicuculline (200 µM; n = 6) and picrotoxin (200 µM; n = 4) reversibly inhibited the wave activity (Figs. 8D and 9A). These results show that GABA as well as glycine play an excitatory role in the embryonic rat brain by mediating the propagation of wave activity. In the preceding experiments, the wave activity was not completely eliminated by a single receptor antagonist (Fig. 9A). Thus we examined the effects of combined applications of the drugs (Fig. 9B). The blockade of glutamate receptors by APV together with CNQX strongly suppressed the wave activity but did not eliminate it. Also, addition of the acetylcholine receptor antagonists atropine and dTc further reduced the wave-related optical signals. However, it was not until the addition of strychnine and bicuculline that the wave activity was completely eliminated. In the presence of a mixture of glutamatergic, cholinergic, glycinergic, and GABAergic blockers, the optical signals were completely eliminated among all tested preparations in the pons, and for 8 of 11 preparations in the medulla (Fig. 9C). These results indicate that the wave activity in the embryonic rat brain is mediated by multiple neurotransmitters including glutamate, acetylcholine, glycine, and GABA.
We previously reported that the depolarization wave in the chick embryo is blocked by gap junction blockers such as octanol, 18-glycyrrhetinic acid and carbenoxolone, in addition to chemical synaptic antagonists (Momose-Sato et al. 2003a). To compare the pharmacological characteristics of the wave activity with those of the chick depolarization wave, we examined the effects of these blockers. When we applied octanol to the bathing solution (1 mM; n = 4), the wave-related optical signals were reversibly suppressed within 10–20 min (Fig. 9D). Similar results were obtained with 18-glycyrrhetinic acid (100 µM; n = 4), although the degree of suppression was relatively mild (Fig. 9D).
Connexin immunohistochemistry
A gap junction is formed by two hemichannels (connexons), each of which is a hexamer composed of connexin (Cx) protein (Evans and Martin 2002). We performed an experiment involving connexin immunohistochemistry in the embryonic rat brain (Fig. 10).
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DISCUSSION |
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In the mammalian CNS, using electrophysiological methods or Ca2+-imaging techniques, it was found that some activities travel widely over distances of a few millimeters or more (Garaschuk et al. 2000; Hanson and Landmesser 2003; Leinekugel et al. 1998; Nakayama et al. 1999; Peinado 2000; Ren and Greer 2003). However, these activities were essentially described within specific structures that become part of functional ensembles such as the cerebral cortex, limbic system, and medulla-spinal cord. The wave activity optically identified in the present study showed wide propagation of membrane depolarization and [Ca2+]i elevation, which has not been previously described for the mammalian CNS. The only comparable phenomenon noted in past studies is the depolarization wave in the chick embryo, which was also recorded using the optical recording technique with voltage-sensitive dyes (Mochida et al. 2001; Momose-Sato et al. 2001b, 2003a,Momose-Sato et al. 2003b). Between the wave activity in the rat embryo and the depolarization wave in the chick embryo, there are many similarities (summarized in Table 5). These include the extent of wave propagation, although responses in the cerebellum were smaller in the rat embryo, the waveform of the optical signal, the conduction velocity, the trigger sources, association with an increase in [Ca2+]i, and sensitivity to multiple blockers of chemical neurotransmitters and gap junctions. These similarities suggest that the large-scale depolarization wave is globally generated in different species. In preliminary experiments, we also detected similar depolarizing wave activity in mouse embryos.
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; Hanson and Landmesser 2003; Ren and Greer 2003). In the rat spinal cord, spontaneous electrical activity travels throughout the cord during these developmental stages (Nakayama et al. 1999; Ren and Greer 2003). It is important to clarify the relationship between the optically identified depolarization wave and the electrically recorded spontaneous activities. Although we preliminarily observed spontaneous optical signals that spread like a wave, detailed analyses could not be performed in the present study because of technical problems associated with our optical recording system. Resolving this issue will be a major challenge in future.
We examined network mechanisms responsible for wave propagation via pharmacological manipulation. Propagation of the wave-related optical signals was dependent on multiple neurotransmitters such as glutamate, acetylcholine, GABA, and glycine and was most sensitive to the NMDA and GABAA receptor antagonists. In the mouse spinal cord between E15 and E17, it has been suggested that functional inhibitory connections mediated by glycine are in the process of developing (Whelan 2003). In the E16 rat brain stem preparation, the effects of GABA and glycine receptor antagonists on the wave activity were inhibitory in every case.
In addition to chemical synaptic antagonists, wave activity was inhibited by gap junction blockers such as octanol and 18-glycyrrhetinic acid. Recently, several investigations reported using octanol and glycyrrhetinic acids (18-glycyrrhetinic acid, 18-glycyrrhetinic acid, and carbenoxolone) that gap junction function is necessary in addition to chemical synaptic transmission in mediating correlated neuronal activity in the embryonic retina (Catsicas et al. 1998; Wong et al. 1998) and spinal cord (Hanson and Landmesser 2003; Milner and Landmesser 1999). In the embryonic mouse spinal cord, gap junctions are necessary for the propagation of local episodes of activity throughout the cord but not for the local episode itself evoked by antidromic motoneuron activation (Hanson and Landmesser 2003). Another study on the neonatal mouse retina showed that spatially restricted retinal waves are triggered by local, synaptically mediated depolarization and that widely propagating waves induced by L-type Ca2+-channel activation depend on gap junction function (Singer et al. 2001). These studies suggest that the synergistic activities of gap junctions and chemical synapses might be necessary for the long-distance propagation of correlated activity in the developing CNS.
Lipophilic compounds such as octanol and heptanol have been widely used as gap junction inhibitors (Spray and Bennett 1985). Glycyrrhetinic acid and its derivatives such as carbenoxolone have been developed as more specific inhibitors of gap junctions (Davidson and Baumgarten 1988). However, nonspecific effects of these reagents have also been suggested (Deutsch et al. 1995; Rouach et al. 2003; Todorovic and Lingle 1998; Vessey et al. 2004), and thus it is of concern that clear interpretations of results gained using these blockers are not forthcoming. Although our morphological observation of connexin immunoreactivity suggests that gap junctional intercellular communication systems are generated in the embryonic rat brain, the hypothesis that these systems contribute to the propagation of wave activity will require careful confirmation in future.
In our immunohistochemical study, we observed significant imuunoreactivity of connexins26 and 32 in the E16 rat brain stem. These connexins are expressed in several types of neural cells in the brain. In the postnatal and adult rodent brain, expression of the connexin26-protein was previously reported in leptomeningeal cells, ependymal cells, astrocytes, and neurons (Dermietzel et al. 1989; Nadarajah et al. 1997; Nagy et al. 2001; Solomon et al. 2001). Some neurons and oligodendrocytes express connexin32 in the postnatal and adult rat brain (Dermietzel et al. 1989; Kunzelmann et al. 1997; Nadarajah et al. 1996). Unfortunately, we could not identify specific cell-type connexin immunoreactivity in the present study. The expression of connexins is developmentally regulated in the rat brain. In the midbrain floor, connexins26 and 32 are highly expressed during the embryonic period with peaks at E14 and E16, respectively. Thereafter they significantly decrease toward birth (Leung et al. 2002). Throughout the entire cortex, connexin26 expression also occurs during the embryonic stages (Nadarajah et al. 1997). However, in the rat striatum and cerebral cortex, connexin32 shows no distinct activity throughout the prenatal and neonatal periods but gradually increases during the postnatal stages (Dermietzel et al. 1989; Nadarajah et al. 1997). Our results showed for the first time the dense distribution of immunoreactive spots of both connexins at the levels of the pons and medulla in E16 rat embryos. The punctate labeling pattern of connexins26 and 32 showed that the immunoreactive spots were concentrated on the plasma membrane as plaques, suggesting that these connexins organize fully functional gap junction plaques (Yeager et al. 1998).
What might be the functional significance of the wave activity in the embryonic rat CNS? The wave activity identified in the present study provides evidence of the existence of two kinds of information that travel through the network, one being electrical signals characterized by membrane depolarization and the other being biochemical signals accompanied by [Ca2+]i elevation. It was previously suggested that transient elevation in [Ca2+]i is important in some aspects of growth and differentiation including proliferation (LoTurco et al. 1995), migration (Komuro and Rakic 1996), neuronal differentiation, and the expression of transmitter phenotypes (Gu and Spitzer 1997), axonal pathfinding (Kater et al. 1988), and dendritic growth and patterning (Wong and Ghosh 2002). In the brain stem and spinal cord in which the wave activity was most prominent in the present study, neurogenesis occurs in most regions by E15, although in several brain stem nuclei such as the cochlear nucleus, cell production continues beyond E16 referring the day of sperm positivity as E0 (Altman and Bayer 1982, 1984). Thus the role of the wave activity is not limited to the control of early neuronal proliferation/differentiation and probably involves the regulation of later development such as synaptogenesis. Although activity-dependent processes are required for the proper formation of neuronal circuitry (Goodman and Shatz 1993; Hanse et al. 1997; Katz and Shatz 1996; Sur and Leamey 2001), the profile of large-scale, nonspecific wave activity propagation across anatomical boundaries suggests that this activity may not serve as a simple regulator of specific neuronal circuit formation either.
One possible role of the wave activity is that it transmits electrical and biochemical signals that occur in any restricted region of the nervous system throughout the CNS via a widely distributed network. This would allow neurons/neural precursor cells involved in the network to be activated at a higher rate than expected for local, poorly developed specific neuronal circuits and then facilitate the processes responsible for neural development via its nurturing effects.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: Y. Momose-Sato, Dept. of Physiology, Tokyo Medical and Dental University, Graduate School and Faculty of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan (E-mail: yoko.phy2{at}tmd.ac.jp)
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) and 10 positions in the lateral pons (
) of an E16 preparation, and the blockers were applied during the period indicated by the horizontal lines. In the presence of a mixture of glutamatergic, cholinergic, glycinergic, and GABAergic blockers, the signals were eliminated. Atr, atropine; Str, strychnine; Bic, bicuculline; PTX, picrotoxin. C: normalized amplitudes of the wave-related optical signals with a mixed application of blockers. The optical signals were eliminated in the pons in all tested preparations (n = 7 using bicuculline and n = 2 using picrotoxin). In the medulla, the optical signals were eliminated in 5 of 8 preparations using bicuculline and 3 of 3 preparations using picrotoxin. D: effects of gap junction blockers on the wave activity. Normalized signal amplitudes of the wave activity with the application of octanol (1 mM: 10–20 min application) and 18