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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., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

The multiple-site optical recording technique with a voltage-sensitive dye, NK2761, was used to survey the functional organization of neuronal networks related to the vagus nerve (N.X) in the E16-stage rat brainstem. When we stimulated N.X, in addition to the responses in the vagal sensory nucleus (nucleus of the tractus solitarius (NTS)) on the stimulated side, other response areas were bilaterally detected. Characteristics of the optical signals in these areas suggested that they correspond to neural activity in the second/higher-ordered nucleus of the vagal pathway. The first area was located at the level of the pons. Based upon morphological information, we suggest that this area corresponds to the parabrachial nucleus (PBN), which receives inputs from the NTS. The second area was located between the NTS and the PBN. We suggest that this area is the A5 noradrenergic group. These results suggest that the N.X-related neural networks are established similarly to the adult pattern from an early developmental stage., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

During the early development of the nervous system, synchronized activity is observed in a variety of structures, and is considered to play a fundamental role in neural development. One of the most striking examples of such activity is the depolarization wave reported in chick and rat embryos. In the accompanying paper (Momose-Sato et al., 2012), we have demonstrated that a depolarization wave is also present in the mouse embryo by showing large-scale optical waves, which spread remarkably over the central nervous system, including the spinal cord, hindbrain, cerebellum, midbrain, and forebrain. In the present study, we examined the pharmacological nature of the mouse depolarization wave and its developmental changes. We show here that two types of switching in pharmacological characteristics occur during development. One is that the depolarization wave is strongly dependent on nicotinic acetylcholine receptors during the early developmental stage [embryonic day (E)11-12], but is dominated by glutamate at the later stage (E13 onwards). The second is that γ-aminobutyric acid (GABA), which acts as an excitatory mediator of the depolarization wave during the early phase, becomes an inhibitory modulator by E14. These changes seemed to occur earlier in the hindbrain than in the spinal cord. Furthermore, we show that the second switch causes the loss of synchronization over the network, resulting in the disappearance of the depolarization wave and segregation of the activity into discrete regions of the medulla and spinal cord. We suggest that pharmacological switching is a possible mechanism underlying replacement of the primordial correlated network by a mature neuronal circuit., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

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 (DeltaI/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.

We examined the intracellular Ca2+ ([Ca2+]i) elevation evoked by GABA in an 8-day embryonic chick brainstem slice using a Ca imaging technique with Ca green-1 AM. When small quantities of GABA were pressure-ejected on the surface of the slice, the [Ca2+]i elevation was clearly detected. The GABA-induced [Ca2+]i elevation was eliminated in a Ca2+-free solution, whereas the previously reported GABA-induced light-scattering change was independent of extracellular Ca2+. Although, micro-application of glycine or glutamate also induced [Ca2+]i elevation, these changes were smaller than that by GABA. These results suggest that the GABA-induced [Ca2+]i elevation is due to Ca2+ entry resulting from membrane depolarization and may play an important role in the development of the central nervous system (CNS).

Multiple-site optical recording with a fast voltage-sensitive dye, absorption dye NK2761, was used to study the developmental organization of functional synaptic networks in the vagal pathway. Glutamatergic excitatory postsynaptic potentials (EPSPs) evoked by vagus nerve stimulation was first detected from the nucleus of the tractus solitarius (NTS) at embryonic day 7 (E7) in chick embryos and E15 in rat embryos, when morphological differentiation of pre- and postsynaptic neurons is incomplete. When extracellular Mg2+ was removed, small EPSPs were elicited at E6 in chick embryos and E14 in rat embryos. These results suggest that synaptic function mediated by N-methyl-D-aspartate (NMDA) receptors is latently generated 1 day before the expression of glutamatergic EPSP. Functional synapses related to the glossophyaryngeal nerve appear to be generated at the same time as the vagus nerve, but their spatial distribution was different from that of the vagus nerve. We further investigated the development of second synaptic pathways from the NTS to higher centers, and found that neuronal circuits from the NTS are already generated when the primary afferents form functional synapses with NTS neurons.

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.

Facial motor neurons of the rat embryo are first generated in rhombomere 4 and then migrate in the caudo‐ventral direction. This migration forms a unique axonal trajectory called the genu, a loop of facial motor axons around the abducens nucleus. It is still unclear when and how this unique structure is functionally established during ontogenesis. Using voltage‐sensitive dye (VSD) recording and the DiI staining method, we identified neural responses evoked by facial nerve (N.VII) stimulation and examined developmental processes of the facial motor nucleus in E12‐E17 rat brainstems. We identified two types of fast spike‐like signals; a long‐duration signal, which corresponded to the action potential in the N.VII soma, and a short‐duration signal, which reflected the action potential in the N.VII axons. The long‐duration signal was detected as early as E13, suggesting that the N.VII motor neuron is already excitable at the beginning of cell migration. The response area of the long‐duration signal extended caudally at E13‐E14, and shifted in a ventral direction at E15. At E16‐E17, the long‐duration signal was concentrated in the caudo‐ventral area, which was comparable to the location of the facial motor nucleus in the adult rat brainstem. These results demonstrate that developmental processes of cell migration and nuclear organization can be visualized and identified functionally with the VSD recording. We discuss the results by comparing functiogenesis and morphogenesis of the N.VII pathway. Appling voltage‐sensitive dye recording and the DiI staining method to the embryonic rat brainstem, we examined functiogenesis/morphogenesis of the facial motor system. Stimulation of the facial nerve elicited a long‐duration signal, which reflected the activity in the soma, and a short‐duration signal, which was associated with the axonal pathway. By examining the spatiotemporal patterns of these signals, we elucidated the developmental dynamics and functional organization of the facial motor nucleus. [ABSTRACT FROM AUTHOR]

Highlights: [•] We examined the initial expression of synaptic function in the embryonic chick CNS. [•] We applied voltage-sensitive dye imaging to the N.V-brainstem preparation. [•] We optically identified N.V-related sensory and motor nuclei in the brainstem. [•] Synaptic function in the sensory nuclei emerged earlier than previously considered. [•] Development of synaptic function differs among nerves and between sensory nuclei. [ABSTRACT FROM AUTHOR]

Spontaneous embryonic movements, called embryonic motility, are produced by correlated spontaneous activity in the cranial and spinal nerves, which is driven by brainstem and spinal networks. Using optical imaging with a voltage-sensitive dye, we revealed previously in the chick and rat embryos that this correlated activity is a widely propagating wave of neural depolarization, which we termed the depolarization wave. One important consideration is whether a depolarization wave with similar characteristics occurs in other species, especially in different mammals. Here, we provide evidence for the existence of the depolarization wave in the mouse embryo by summarizing spatiotemporal characteristics and pharmacological natures of the widely propagating wave activity. The findings show that a synchronized wave with common characteristics is expressed in different species, suggesting its fundamental roles in neural development. [ABSTRACT FROM AUTHOR]

Abstract: To regulate the autonomic function, the vagus nerve transfers various sensory information from peripheral organs, and appropriate motor reflexes are produced in the neural circuit. The functional development of the vagal pathway during the early phase of embryonic development has long been unclear. Optical recording with voltage-sensitive dyes has provided a new approach to the analysis of the functional development of the embryonic central nervous system. In this review, we present recent progress in optical studies on the vagal pathway in the embryonic chick and rat brainstems. The topics include how neural excitability is initially expressed in the motor and sensory nuclei [e.g. the dorsal motor nucleus of the vagus nerve (DMNV) and the nucleus of the tractus solitarius (NTS)] and how synapse networks are formed in the primary and higher-ordered sensory nuclei [e.g. the parabrachial nucleus (PBN)]. We also refer to the functional development of the glossopharyngeal nuclei and compare the developmental steps with those of the vagal nuclei. [Copyright &y& Elsevier]

Abstract: We examined the intracellular Ca2+ ([Ca2+]i) elevation evoked by GABA in an 8-day embryonic chick brainstem slice using a Ca imaging technique with Ca green-1 AM. When small quantities of GABA were pressure-ejected on the surface of the slice, the [Ca2+]i elevation was clearly detected. The GABA-induced [Ca2+]i elevation was eliminated in a Ca2+-free solution, whereas the previously reported GABA-induced light-scattering change was independent of extracellular Ca2+. Although, micro-application of glycine or glutamate also induced [Ca2+]i elevation, these changes were smaller than that by GABA. These results suggest that the GABA-induced [Ca2+]i elevation is due to Ca2+ entry resulting from membrane depolarization and may play an important role in the development of the central nervous system (CNS). [Copyright &y& Elsevier]

The multiple-site optical recording technique with a voltage-sensitive dye, NK2761, was used to survey functional organization of neural circuits related to the vagus nerve in the embryonic chick brainstem. When we stimulated the vagus nerve, in addition to the responses in the vagal sensory nucleus (nucleus of the tractus solitarius (NTS)) and motor nucleus (dorsal motor nucleus of the vagus nerve (DMNV)) on the stimulated side, another response area was discriminated at the level of the pons/rostral medulla on the contralateral side. Characteristics of the contralateral optical signals suggested that they correspond to the neural activity in the second/higher-ordered nucleus of the vagal pathway, possibly the parabrachial nucleus, which receives inputs from the NTS. Blockade of non-N-methyl- d-aspartate (NMDA) receptors abolished the responses on the contralateral side, together with the postsynaptic firing in the NTS, suggesting the significance of non-NMDA receptor function in sensory information transfer via the NTS. The responses on the contralateral side were first detected from the 7-day-old embryonic stage, when the glutamatergic excitatory postsynaptic potentials were first expressed in the NTS. The results suggest 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. [ABSTRACT FROM AUTHOR]

We examined the functional organization of the rat trigeminal nuclear complex and its developmental dynamics using a multiple-site optical recording technique. Brainstem preparations were dissected from embryonic day 12 (E12)-E16 rat embryos, and stimulation was applied individually to the three branches of the trigeminal nerve (V1-V3). The action potential activity of presynaptic fibers was detected from E13, and the glutamate-mediated postsynaptic response was significantly observed from E15 on. At E14, the evoked signals usually consisted of only the action potential-related fast component. However, when extracellular Mg2+ was removed, a significant DL-2-amino-5-phosphonovaleric acid-sensitive slow component appeared. These results suggest that postsynaptic function mediated by NMDA receptors is latently generated as early as E14. The response area of the three branches of the trigeminal nerve showed some functional somatotopic organization, with the ophthalmic (V1) nerve area medially located and the mandibular (V3) nerve area laterally located. The center of the trigeminal nuclear complex in which the activity of neurons and synaptic function was greatest shifted caudally with development, suggesting that the functional architecture of the trigeminal nuclear complex is not fixed but changes dynamically during embryogenesis. By electron microscopy, we could not observe clear correlations between functional data and morphological information; when we surveyed E16 preparations, we could not identify typical synaptic structures between the 1,1′-dioctyldecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-labeled trigeminal nerve terminals and the neurons in the trigeminal nuclear complex. This implies that postsynaptic function in the trigeminal nuclear complex is generated before the appearance of the morphological structure of conventional synapses. [ABSTRACT FROM AUTHOR]

Functional organization of the brain stem vestibulocochlear nuclei during embryogenesis was investigated using a multiple-site optical recording technique with a fast voltage-sensitive dye. Brain stem slices with the cochlear and/or vestibular nerves attached were dissected from 6- to 8-day-old (E6-E8) chick embryos. Electrical responses evoked by cochlear or vestibular nerve stimulation were optically recorded simultaneously from many loci of the preparations. In E7 and E8 preparations, we identified two components of the optical response with cochlear or vestibular nerve stimulation; one was a fast spike-like signal related to the action potential, and the other was a slow signal related to the glutamate-mediated excitatory postsynaptic potential. The location of the cochlear nerve response area was mainly located on the dorsolateral region, while that of the vestibular nerve was deviated ventromedially. At E6, cochlear nerve stimulation evoked only the fast spike-like signals in normal Ringer solution. However, when we removed Mg2+ from the extracellular solution, significant slow signals were elicited in the E6 preparation. The present results demonstrated that in the chick vestibulocochlear nuclei, functional synapses are already generated by the E7 embryonic stage and that postsynaptic activity related to N-methyl-d-aspartate receptors emerges latently, at least in the cochlear nerve-related nucleus, at the E6 embryonic stage. This chronological sequence of the emergence of postsynaptic function is different from that reported previously (E10-E11), suggesting that the developmental origin of sensory information transfer in the auditory pathway is much earlier than has been anticipated. [ABSTRACT FROM AUTHOR]