Optical recording of oscillatory activity in the absence of external Ca2+ in the embryonic chick olfactory bulb.

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.

Functional development of olfactory nerve-related neural circuits in the embryonic chick forebrain revealed by voltage-sensitive dye imaging.

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.

Prenatal exposure to nicotine disrupts synaptic network formation by inhibiting spontaneous correlated wave activity.

Correlated spontaneous activity propagating over a wide region of the central nervous system is expressed during a specific period of embryonic development. We previously demonstrated using an optical imaging technique with a voltage-sensitive dye that this wave-like activity, which we referred to as the depolarization wave, is fundamentally involved in the early process of synaptic network formation. We found that the in ovo application of bicuculline/strychnine or d-tubocurarine, which blocked the neurotransmitters mediating the wave, significantly reduced functional synaptic expression in the brainstem sensory nucleus. This result, particularly for d-tubocurarine, an antagonist of nicotinic acetylcholine receptors, suggested that prenatal nicotine exposure associated with maternal smoking affects the development of neural circuit formation by interfering with the correlated wave. In the present study, we tested this hypothesis by examining the effects of nicotine on the correlated activity and assessing the chronic action of nicotine in ovo on functional synaptic expression along the vagal sensory pathway. In ovo observations of chick embryo behavior and electrical recording using in vitro preparations showed that the application of nicotine transiently increased embryonic movements and electrical bursts associated with the wave, but subsequently inhibited these activities, suggesting that the dominant action of the drug was to inhibit the wave. Optical imaging with the voltage-sensitive dye showed that the chronic exposure to nicotine in ovo markedly reduced functional synaptic expression in the higher-order sensory nucleus of the vagus nerve, the parabrachial nucleus. The results suggest that prenatal nicotine exposure disrupts the initial formation of the neural circuitry by inhibiting correlated spontaneous wave activity.

Voltage-sensitive dye recording of glossopharyngeal nerve-related synaptic networks in the embryonic mouse brainstem.

The glossopharyngeal nerve (N.IX) transfers motor and sensory information related to visceral and somatic functions, such as salivary secretion, gustation and the control of blood pressure. N.IX-related neural circuits are indispensable for these essential functions. Compared with the strenuous analysis of morphogenesis, we are only just starting to elucidate the functiogenesis of these neural circuits during ontogenesis. In the present study, we applied voltage-sensitive dye recording to the embryonic mouse brainstem, and examined the functional development of the N.IX-related neural circuits. First, we optically identified the motor nucleus (the inferior salivatory nucleus (ISN)) and the first-order sensory nucleus (the nucleus of the tractus solitarius (NTS)). We also succeeded in recording optical responses in the second/higher-order sensory nuclei via the NTS, including the parabrachial nucleus. Second, we pursued neuronal excitability and the onset of synaptic function in the N.IX-related nuclei. The neurons in the ISN were excitable at least at E11, and functional synaptic transmission in the NTS was first expressed at E12. In the second/higher-order sensory nuclei, synaptic function emerged at around E12-13. Third, by mapping optical responses to N.IX and vagus nerve (N.X) stimulation, we showed that the distribution patterns of neural activity in the NTS were different between the N.IX and the N.X from the early stage of ontogenesis. We discuss N.IX-related neural circuit formation in the brainstem, in comparison with our previous results obtained from chick and rat embryos.

Optical analysis of functional development of the facial motor nucleus in the embryonic rat brainstem.

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.

Developmental roles of the spontaneous depolarization wave in synaptic network formation in the embryonic brainstem.

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.

Intraoperative intrinsic optical imaging of human somatosensory cortex during neurosurgical operations.

Intrinsic optical imaging as developed by Grinvald et al. is a powerful technique for monitoring neural function in the in vivo central nervous system. The advent of this dye-free imaging has also enabled us to monitor human brain function during neurosurgical operations. We briefly describe our own experience in functional mapping of the human somatosensory cortex, carried out using intraoperative optical imaging. The maps obtained demonstrate new additional evidence of a hierarchy for sensory response patterns in the human primary somatosensory cortex.

Functiogenesis of the embryonic central nervous system revealed by optical recording with a voltage-sensitive dye.

Clarification of the functiogenesis of the embryonic central nervous system (CNS) has long been problematic, because conventional electrophysiological techniques have several limitations. First, early embryonic neurons are small and fragile, and the application of microelectrodes is challenging. Second, the simultaneous monitoring of electrical activity from multiple sites is limited, and as a consequence, spatiotemporal response patterns of neural networks cannot be assessed. We have applied multiple-site optical recording with a voltage-sensitive dye to the embryonic CNS and paved a new way to analyze the functiogenesis of the CNS. In this review, we discuss key points of optical recording in the embryonic CNS and introduce recent progress in optical investigations on the embryonic CNS with special emphasis on the development of the chick olfactory system. The studies clearly demonstrate the usefulness of voltage-sensitive dye recording as a powerful tool for elucidating the functional organization of the vertebrate embryonic CNS.

Development of Spontaneous Activity in the Avian Hindbrain.

Spontaneous activity in the developing central nervous system occurs before the brain responds to external sensory inputs, and appears in the hindbrain and spinal cord as rhythmic electrical discharges of cranial and spinal nerves. This spontaneous activity recruits a large population of neurons and propagates like a wave over a wide region of the central nervous system. Here, we review spontaneous activity in the chick hindbrain by focusing on this large-scale synchronized activity. Asynchronous activity that is expressed earlier than the above mentioned synchronized activity and activity originating in midline serotonergic neurons are also briefly mentioned.

Development of synaptic networks in the mouse vagal pathway revealed by optical mapping with a voltage-sensitive dye.

The central issue in developmental neuroscience is when and how neural synaptic networks are established and become functional within the central nervous system (CNS). Investigations of the neural network organization have been hampered because conventional electrophysiological means have some technical limitations. In this study, the multiple-site optical recording technique with a voltage-sensitive dye was employed to survey the developmental organization of the vagal system in the mouse embryo. Stimulation of the vagus nerve in E11-E14 mouse embryos elicited optical responses in areas corresponding to the vagal sensory and motor nuclei. Postsynaptic responses in the first-order sensory nucleus, the nucleus of the tractus solitarius (NTS), were identified from E11, suggesting that sensory information becomes transferred to the brain at this stage. In addition to the NTS, optical responses were identified in the rostral and contralateral brainstem regions, which corresponded to second/higher order nuclei of the vagus nerve including the parabrachial nucleus (PBN). Postsynaptic responses in the second/higher-order nuclei were detected from E12, suggesting that polysynaptic networks were functional at this stage. We discuss the results of our optical mapping, comparing them with previous findings obtained in the chick and rat embryos, and suggest some fundamental principles in the functional organization of synaptic networks in the embryonic brain.

Oscillations in the embryonic chick olfactory bulb: initial expression and development revealed by optical imaging with a voltage-sensitive dye.

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.

Monitoring Population Membrane Potential Signals During Development of the Vertebrate Nervous System.

The functional organization of the vertebrate central nervous system (CNS) during the early phase of development has long been unclear, because conventional electrophysiological means have several technical limitations. First, early embryonic neurons are small and fragile, and the application of microelectrodes is often difficult. Second, the simultaneous recording of electrical activity from multiple sites is limited, and as a consequence, response patterns of neural networks cannot be assessed. Optical recording techniques with voltage-sensitive dyes have overcome these obstacles and provided a new approach to the analysis of the functional development/organization of the CNS. In this review, we provide detailed information concerning the recording of optical signals in the embryonic nervous system. After outlining methodological considerations, we present examples of recent progress in optical studies on the embryonic nervous system with special emphasis on two topics. The first is the study of how synapse networks form in specific neuronal circuits. The second is the study of non-specific correlated wave activity, which is considered to play a fundamental role in neural development. These studies clearly demonstrate the utility of fast voltage-sensitive dye imaging as a powerful tool for elucidating the functional organization of the vertebrate embryonic CNS.

Voltage-sensitive dye imaging during functional development of the embryonic nervous system: a brief review with special thanks to Professor Larry Cohen.

Investigating the developmental organization of the embryonic nervous system is one of the major challenges in the field of neuroscience. Despite their significance, functional studies on the vertebrate embryonic central nervous system (CNS) have been hampered by the technical limitations associated with conventional electrophysiological methods. The advent of optical techniques using voltage-sensitive dyes, which were developed by Dr. Cohen and his colleagues, has enabled electrical activity in living cells to be monitored noninvasively and also facilitated the simultaneous recording of neural responses from multiple regions. Using optical recording techniques, it is now possible to follow the functional organization of the embryonic CNS and image the spatiotemporal dynamics involved in the formation of this neural network. We herein briefly reviewed optical studies on the embryonic CNS with a special emphasis on methodological considerations and the study of neuronal circuit formation, which demonstrates the utility of fast voltage-sensitive dye imaging as a powerful tool for elucidating the functional organization of the embryonic CNS.

Positive feedback of NR2B-containing NMDA receptor activity is the initial step toward visual imprinting: a model for juvenile learning.

Imprinting in chicks is a good model for elucidating the processes underlying neural plasticity changes during juvenile learning. We recently reported that neural activation of a telencephalic region, the core region of the hyperpallium densocellulare (HDCo), was critical for success of visual imprinting, and that N-Methyl-D-aspartic (NMDA) receptors containing the NR2B subunit (NR2B/NR1) in this region were essential for imprinting. Using electrophysiological and multiple-site optical imaging techniques with acute brain slices, we found that long-term potentiation (LTP) and enhancement of NR2B/NR1 currents in HDCo neurons were induced in imprinted chicks. Enhancement of NR2B/NR1 currents as well as an increase in surface NR2B expression occurred even following a brief training that was too weak to induce LTP or imprinting behavior. This means that NR2B/NR1 activation is the initial step of learning, well before the activation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors which induces LTP. We also showed that knockdown of NR2B/NR1 inhibited imprinting, and inversely, increasing the surface NR2B expression by treatment with a casein kinase 2 inhibitor successfully reduced training time required for imprinting. These results suggest that imprinting stimuli activate post-synaptic NR2B/NR1 in HDCo cells, increase NR2B/NR1 signaling through up-regulation of its expression, and induce LTP and memory acquisition. The study investigated the neural mechanism underlying juvenile learning. In the initial stage of chick imprinting, NMDA receptors containing the NMDA receptor subunit 2B (NR2B) are activated, surface expression of NR2B/NR1 (NMDA receptor subunit 1) is up-regulated, and consequently long-term potentiation is induced in the telencephalic neurons. We suggest that the positive feedback in the NR2B/NR1 activation is a unique process of juvenile learning, exhibiting rapid memory acquisition.

Optical survey of initial expression of synaptic function in the embryonic chick trigeminal sensory nucleus.

We examined the initial expression of synaptic function in the embryonic chick trigeminal nucleus using voltage-sensitive dye recording. Brainstem preparations with three trigeminal nerve afferents, the ophthalmic nerve (N.V1), maxillary nerve (N.V2) and mandibular nerve (N.V3), were dissected from 5.5- to 6.5-day-old chick embryos. In our previous study [Sato et al., 1999], we detected slow signals corresponding to glutamatergic excitatory postsynaptic potentials and identified the principal sensory nucleus of the trigeminal nerve (Pr5), spinal sensory nucleus of the trigeminal nerve (Sp5) and trigeminal motor nucleus. In this study, we examined the effects of removing Mg(2+) from the physiological solution, which enhanced N-methyl-d-aspartate receptor function in the sensory nuclei. In 6.5-day-old (St 29) embryos, the slow signal was observed in Pr5 and Sp5 only when N.V1 was stimulated, whereas it appeared in Mg(2+)-free solution with every nerve stimulation. In 6-day-old (St 28) embryos, the slow signal was observed in Sp5 with N.V1 stimulation, and the appearance of synaptic function in Mg(2+)-free solution varied, depending on the nerves and preparations used. In 5.5-day-old (St 27) embryos, synaptic function was not detected even when external Mg(2+) was removed. These results indicate that the initial expression of synaptic function in the trigeminal system occurs earlier than previously considered, and that the developmental organization of synaptic function differs among the three trigeminal nerves and between the two sensory nuclei.

Evaluation of voltage-sensitive fluorescence dyes for monitoring neuronal activity in the embryonic central nervous system.

Using an optical imaging technique with voltage-sensitive dyes (VSDs), we investigated the functional organization and architecture of the central nervous system (CNS) during embryogenesis. In the embryonic nervous system, a merocyanine-rhodanine dye, NK2761, has proved to be the most useful absorption dye for detecting neuronal activity because of its high signal-to-noise ratio (S/N), low toxicity and small dye bleaching. In the present study, we evaluated the suitability of fluorescence VSDs for optical recording in the embryonic CNS. We screened eight styryl (hemicyanine) dyes in isolated brainstem-spinal cord preparations from 7-day-old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recording system. The signal size, S/N, photobleaching, effects of perfusion and recovery of neural responses after staining were compared. We also evaluated optical responses with various magnifications. Although the S/N was lower than with the absorption dye, clear optical responses were detected with several fluorescence dyes, including di-2-ANEPEQ, di-4-ANEPPS, di-3-ANEPPDHQ, di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA and di-2-ANEPPTEA. Di-2-ANEPEQ showed the largest S/N, whereas its photobleaching was faster and the recovery of neural responses after staining was slower. Di-4-ANEPPS and di-3-ANEPPDHQ also exhibited a large S/N but required a relatively long time for recovery of neural activity. Di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA and di-2-ANEPPTEA showed smaller S/Ns than di-2-ANEPEQ, di-4-ANEPPS and di-3-ANEPPDHQ; but the recovery of neural responses after staining was faster. This study demonstrates the potential utility of these styryl dyes in optical monitoring of voltage changes in the embryonic CNS.

Large-scale synchronized activity in the embryonic brainstem and spinal cord.

In the developing central nervous system, spontaneous activity appears well before the brain responds to external sensory inputs. One of the earliest activities is observed in the hindbrain and spinal cord, which is detected as rhythmic electrical discharges of cranial and spinal motoneurons or oscillations of Ca(2+)- and voltage-related optical signals. Shortly after the initial expression, the spontaneous activity appearing in the hindbrain and spinal cord exhibits a large-scale correlated wave that propagates over a wide region of the central nervous system, maximally extending to the lumbosacral cord and to the forebrain. In this review, we describe several aspects of this synchronized activity by focusing on the basic properties, development, origin, propagation pattern, pharmacological characteristics, and possible mechanisms underlying the generation of the activity. These profiles differ from those of the respiratory and locomotion pattern generators observed in the mature brainstem and spinal cord, suggesting that the wave is primordial activity that appears during a specific period of embryonic development and plays some important roles in the development of the central nervous system.

Optical imaging of the spontaneous depolarization wave in the mouse embryo: origins and pharmacological nature.

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.

Neural basis of imprinting behavior in chicks.

Newly hatched chicks memorize the characteristics of the first moving object they encounter, and subsequently show a preference for it. This "imprinting" behavior is an example of infant learning and is elicited by visual and/or auditory cues. Visual information of imprinting stimuli in chicks is first processed in the visual Wulst (VW), a telencephalic area corresponding to the mammalian visual cortex, congregates in the core region of the hyperpallium densocellulare (HDCo) cells, and transmitted to the intermediate medial mesopallium (IMM), a region similar to the mammalian association cortex. The imprinting memory is stored in the IMM, and activities of IMM neurons are altered by imprinting. Imprinting also induces functional and structural plastic changes of neurons in the circuit that links the VW and the IMM. Of these neurons, the activity of the HDCo cells is strongly influenced by imprinting. Expression and modulation of NR2B subunit-containing N-methyl-D-aspartate (NMDA) receptors in the HDCo cells are crucial for plastic changes in this circuit as well as the process of visual imprinting. Thus, elucidation of cellular and molecular mechanisms underlying the plastic changes that occurred in the HDCo cells may provide useful knowledge about infant learning.

Optical survey of vagus nerve-related neuronal circuits in the embryonic rat brainstem.

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.