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We investigated the functional organization of the glossopharyngeal and vagal motor nuclei during embryogenesis using multiple-site optical recording with a fast voltage-sensitive dye. Intact brain stem preparations with glossopharyngeal and vagus nerves were dissected from 4- to 8-day-old chick embryos. Electrical responses evoked by glossopharyngeal/vagus nerve stimulation were optically recorded from many loci of the stained preparations. In 4- to 6-day-old preparations, action potential-related fast spikelike signals were detected from the nucleus of the glossopharyngeal nerve and the dorsal motor nucleus of the vagus nerve. Contour line maps of the signal amplitude showed multiple-peak patterns, suggesting that the neurons and/or their activity were not uniformly distributed within the nuclei at early developmental stages. As development proceeded from 4 to 6 days, the peaks fused with each other and the number of peaks decreased gradually. In most 7- and 8-day-old preparations, only a single peak was identified in the nuclei, and the distribution of the signal amplitude formed a layered pattern surrounding the peak-signal area. These results suggest that functional organization of the motor nuclei in the embryonic hindbrain changes dynamically with development, resulting in a rearrangement of functional nuclear cores from multiple-peaks to a single peak.

Multiple‐site optical recordings with NK2761, a voltage‐sensitive absorption dye, were applied to the embryonic chick olfactory system, and the functional development of olfactory nerve (N.I)‐related neural circuits was examined in the forebrain. The stimulation of the N. I elicited neural responses in N.I‐olfactory bulb (OB)‐forebrain preparations at the embryonic 8–12 day (E8‐E12) stages. At the E11 stage, we functionally identified two circuits projecting from the OB to the forebrain. The first circuit passed through the ventral side of the forebrain and spread in the dorso‐caudal direction, whereas the second circuit passed through the dorsal side to the first circuit. Pharmacological experiments showed that N‐methyl‐D‐aspartate (NMDA) receptor function was more significant for the transfer of sensory information in these circuits. The functional development of N.I‐related circuits was investigated, and the results obtained revealed that the ventral circuit was generated earlier than the dorsal circuit. Neural responses in the ventral circuit were detected from the E9 stage in normal physiological solution and the E8 stage in Mg2+‐free solution, which activated NMDA receptor function. At the E10 stage, neural responses in the dorsal circuit were clearly recognised in addition to ventral responses. We attempted to identify possible candidates for relay nuclei in the forebrain by comparing contour line maps of the optical signal amplitude with previously reported neuroanatomical data. The results suggest that N.I‐related neural circuits from the periphery to the subpallium functionally mature earlier than those to the pallium during ontogenesis. [ABSTRACT FROM AUTHOR]

One of the earliest activities expressed within the developing central nervous system is a widely propagating wave-like activity, which we referred to as the depolarization wave. Despite considerable consensus concerning the global features of the activity, its physiological role is yet to be clarified. The depolarization wave is expressed during a specific period of functional synaptogenesis, and this developmental profile has led to the hypothesis that the wave plays some roles in synaptic network organization. In the present study, we tested this hypothesis by inhibiting the depolarization wave in ovo and examining its effects on the development of functional synapses in vagus nerve-related brainstem nuclei of the chick embryo. Chronic inhibition of the depolarization wave had no significant effect on the developmental time course, amplitude, and spatial distribution of monosynaptic excitatory postsynaptic potentials in the first-order nuclei of the vagal sensory pathway (the nucleus of the tractus solitarius (NTS) and the contralateral non-NTS region), but reduced polysynaptic responses in the higher-order nucleus (the parabrachial nucleus). These results suggest that the depolarization wave plays an important role in the initial process of functional synaptic expression in the brainstem, especially in the higher-order nucleus of the cranial sensory pathway. [ABSTRACT FROM AUTHOR]

• We optically detected oscillatory activity in the embryonic chick olfactory bulb. • Olfactory nerve stimulation induced the oscillation following the EPSP. • The short-term perfusion of a Ca2+-free solution blocked the EPSP and oscillation. • The long-term perfusion of a Ca2+-free solution newly induced oscillatory activity. • The patterns of the oscillatory activity are different between with and without Ca2+. We applied 464/1020-site optical recording systems with a voltage-sensitive dye (NK2761) to the embryonic chick olfactory system and detected oscillatory activity in the olfactory bulb (OB) in the absence of synaptic transmission. In embryonic day 8–10 (E8-E10) chick olfactory nerve (N.I)-OB-forebrain preparations, the removal of Ca2+ from the external solution completely blocked the glutamatergic excitatory postsynaptic potential (EPSP) from the N.I to the OB as well as oscillations following the EPSP. However, a novel type of oscillatory activity was detected in the OB with the long-term perfusion of a Ca2+-free solution. The characteristics of oscillatory activity in the Ca2+-free solution differed from those in normal physiological solution. The present results suggest the existence of a neural communication system in the absence of synaptic transmission at the early stage of embryonic development. [ABSTRACT FROM AUTHOR]

In a previous study, we applied a multiple-site optical recording technique with a voltage-sensitive dye to the embryonic chick olfactory system and showed that functional synaptic transmission in the olfactory bulb was expressed at embryonic 6-7-day stages. It is known that oscillations, i.e. stereotyped sinusoidal neural activity, appear in the olfactory system of various species. The focus of the present study is to determine whether the oscillation is also generated in the embryonic chick olfactory bulb and, if this is the case, when the oscillation appears and how its profiles change during embryogenesis. At the early stages of development (embryonic 6- to 8-day stages), postsynaptic response-related optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted for a few seconds. At embryonic 9-day stage, the optical signal became multi-phasic, and the oscillatory event was detected in some preparations. The oscillation was restricted to the distal half of the olfactory bulb. As development proceeded, the incidence and duration of the oscillation gradually increased, and the waveform became complicated. In some cases at embryonic 12-day stage, the oscillation lasted for nearly a minute. The frequency of the oscillation increased slightly with development, but it remained in the range of theta oscillation during the 9- to 12-day stages. We discuss the ontogenetic dynamics of the oscillation and the significance of this activity in the developing olfactory bulb. [ABSTRACT FROM AUTHOR]

Abstract: Using voltage-sensitive dye recording, we traced the ontogenetic expression of neural excitability related to the glossopharyngeal nerve (N. IX) and the vagus nerve (N. X) in the embryonic chick brainstem. At the 3.5-day embryonic stage, by averaging optical signals, we succeeded in recording very small action potential-related optical responses (ΔI/I <10−4) induced by N. IX stimulation in the nucleus of the glossopharyngeal nerve (motor nucleus). This suggests that glossopharyngeal excitability in the motor nucleus is first generated no later than this developmental stage. On the other hand, action potential-related optical responses induced by N. X stimulation were first detected at the 4-day embryonic stage. Comparison with morphogenesis indicated that glossopharyngeal and vagal motoneurons acquire their excitability and send their axons to the periphery soon after they leave the proliferative pool. [Copyright &y& Elsevier]

Multiple-site optical recording of neural activity was performed in the nucleus of the tractus solitarius (NTS) of the chick embryo with stimulation of the glossopharyngeal nerve (N. IX) and vagus nerve (N. X). We measured the amplitudes of the optical signals related to glutamate-mediated excitatory postsynaptic responses, and calculated the ratio of the signal evoked by simultaneous N. IX/N. X stimulation to the signal obtained after mathematical summation of the individual N. IX and N. X responses. The ratio was significantly lower than 100% in the rostral region of the NTS, in which postsynaptic responses were elicited by both N. IX and N. X stimulations. This result means that there is a convergence of visceral inputs via the N. IX and N. X in the embryonic chick NTS. The existence of the convergence suggests that the NTS performs complex integration of information from multiple sensory inputs from the early stages of embryogenesis. [Copyright &y& Elsevier]

We investigated the developmental pattern of functional synaptogenesis in the chick visual pathway using a multiple-site optical recording method. Responses to optic nerve stimulation were recorded from the diencephalon and mesencephalon of the chick embryo. The first excitatory postsynaptic responses to optic nerve stimulation appeared in the contralateral diencephalon at Hamburger-Hamilton stage 27, which corresponds to an incubation day 5.5 (E5.5). At more developed stages, the optical signals evoked by optic nerve stimulation spread to several different regions, including the tectum and extra-tectal visual nuclei. We constructed maps of neural activity in the diencephalon and mesencephalon at different stages to investigate the spatio-temporal patterns of functional development in the chick visual system. The maps revealed that distinct postsynaptic response areas in the extra-tectal regions showed different onsets of activity, suggesting that the corresponding visual nuclei exhibit different time courses of functional synaptogenesis. We also identified the onset and location of the first functional synaptic connection in the optic tectum, which had been a point of controversy in earlier studies. In the tectal region, the action potential and the excitatory postsynaptic potential first appeared at E8, although these signals were recognized in the tecto/tegmental region at E7. The response area expanded with retinotectal fibre elongation, and reached the area centralis at E9. These results show that the onset of synaptic function in the tectum occurs 2-3 days earlier than was previously reported. [ABSTRACT FROM AUTHOR]

Abstract Using a multiple-site optical recording technique with a voltage-sensitive dye, we found that widely spreading depolarization waves were evoked by dorsal root stimulation in embryonic chick spinal cords. Spatiotemporal maps of the depolarization waves showed that the signals were mainly distributed in the ventral half of the slice, with the highest activity in the ventrolateral area. The propagation velocity of the waves was estimated to be in the order of mm/s. Depolarization waves were evoked in the ventral root-cut preparation, but not in the dorsal root-cut preparation, suggesting that the wave was triggered by synaptic inputs from the primary afferents, and that activation of the motoneurons was not essential for wave generation. In intact spinal cord–brain preparations, the depolarization wave propagated rostrally and caudally for a distance of several spinal segments in normal Ringer's solution. In a Mg2+-free solution, the amplitude and extent of the signals were markedly enhanced, and the depolarization wave triggered in the cervical spinal cord propagated to the brainstem and the cerebellum. The depolarization wave demonstrated here had many similarities with the vagus nerve-evoked depolarization wave reported previously. The results suggest that functional cell-to-cell communication systems mediated by the depolarization wave are widely generated in the embryonic central nervous system, and could play a role in large-scale coactivation of the neurons in the spinal cord and brain. [ABSTRACT FROM AUTHOR]

Abstract We examined embryonic expression of postsynaptic potentials in stages 26–31 (E5 to E7) chick spinal cord slices. Slow optical signals related to the postsynaptic potentials which were evoked by electrical stimulation of afferent fibers were identified in the dorsal grey matter and the ventral motoneuronal area. In cervical spinal cord (C13) preparations, the dorsal slow signal appeared from stage 28 (E6), whilst the ventral slow signal was recognized from stage 29. At stages 26 and 27 (E5), no slow signal was observed in either the dorsal or ventral regions. On the other hand, in lumbosacral spinal cord (LS5) preparations, the dorsal, as well as ventral, slow signals appeared from stage 29; at stage 28 no slow signal was detected in the dorsal or ventral regions. These results suggest that there are differences in the ontogenetic expression of synaptic functions between the dorsal and ventral regions, and between the cervical and lumbosacral spinal cords. In embryos older than stage 29, removal of Mg2+ from the bathing solution markedly enhanced the amplitude and incidence of the ventral slow signal. In addition, in C13 preparations at stage 28, removal of Mg2+ elicited small slow signals in the ventral region in which no synaptic response was evoked in normal Ringer's solution. The slow signals induced in the Mg2+-free solution were blocked by 2-amino-5-phosphonovaleric acid (APV), showing that they are attributable to N-methyl- d-aspartate (NMDA) receptors. These results suggest that functional synaptic connections via polysynaptic pathways are already generated on motoneurons, but are suppressed by a Mg2+ block on the NMDA receptors at developmental stages when synaptic transmission from the primary afferents to the dorsal interneurons is initially expressed in the dorsal region. [ABSTRACT FROM AUTHOR]

We traced developmental changes in the ventro-dorsal distribution pattern of glossopharyngeal nerve (N. IX) responses by applying an optical sectioning method to thick slice preparations dissected from E4 to E8 chick embryos. We identified the motor and sensory nuclei related to the glossopharyngeal nerve in the rostral and caudal focal planes, respectively. In the E4 and E5 preparations, the motoneuronal responses appeared on the central part of the stimulated side of the brainstem. As development proceeded to E6, the response area became localized on the dorsal region. The change in the ventro-dorsal distribution pattern was similar to that observed in the vagus nerve-related nuclei, suggesting that there might be an essential process underlying functional organization of the brainstem nuclei. [Copyright &y& Elsevier]