Differential involvement of projection neurons during emergence of spontaneous activity in the developing avian hindbrain.

To better characterize the emergence of spontaneous neuronal activity in the developing hindbrain, spontaneous activity was recorded optically from defined projection neuron populations in isolated preparations of the brain stem of the chicken embryo. Ipsilaterally projecting reticulospinal (RS) neurons and several groups of vestibuloocular (VO) neurons were labeled retrogradely with Calcium Green-1 dextran amine and spontaneous calcium transients were recorded using a charge-coupled-device camera mounted on a fluorescence microscope. Simultaneous extracellular recordings were made from one of the trigeminal motor nerves (nV) to register the occurrence of spontaneous synchronous bursts of activity. Two types of spontaneous activity were observed: synchronous events (SEs), which occurred in register with spontaneous bursts in nV once every few minutes and were tetrodotoxin (TTX) dependent, and asynchronous events (AEs), which occurred in the intervals between SEs and were TTX resistant. AEs occurred developmentally before SEs and were in general smaller and more variable in amplitude than SEs. SEs appeared at the same stage as nV bursts early on embryonic day 4, first in RS neurons and then in VO neurons. All RS neurons participated equally in SEs from the outset, whereas different subpopulations of VO neurons participated differentially, both in terms of the proportion of neurons that exhibited SEs, the fidelity with which the SEs in individual neurons followed the nV bursts, and the developmental stage at which SEs appeared and matured. The results show that spontaneous activity is expressed heterogeneously among hindbrain projection neuron populations, suggesting its differential involvement in the formation of different functional neuronal circuits.

Origin of the earliest correlated neuronal activity in the chick embryo revealed by optical imaging with voltage-sensitive dyes.

Spontaneous correlated neuronal activity during early development spreads like a wave by recruiting a large number of neurons, and is considered to play a fundamental role in neural development. One important and as yet unresolved question is where the activity originates, especially at the earliest stage of wave expression. In other words, which part of the brain differentiates first as a source of the correlated activity, and how does it change as development proceeds? We assessed this issue by examining the spatiotemporal patterns of the depolarization wave, the optically identified primordial correlated activity, using the optical imaging technique with voltage-sensitive dyes. We surveyed the region responsible for the induction of the evoked and spontaneous depolarization waves in chick embryos, and traced its developmental changes. The results showed that the wave initially originated in a restricted area near the obex and was generated by multiple regions at later stages. We suggest that the upper cervical cord/lower medulla near the obex is the kernel that differentiates first as the source of the correlated activity, and that regional and temporal differences in neuronal excitability might underlie the developmental profile of wave generation in early chick embryos.

Switching of the transmitters that mediate hindbrain correlated activity in the chick embryo.

Widely propagating correlated neuronal activity is a hallmark of the developing nervous system. The activity is usually mediated by multiple transmitters, and the contribution of gap junctions has also been suggested in several systems. In some structures, such as the retina and spinal cord, it has been shown that the dominant transmitter mediating the correlated wave switches from acetylcholine to glutamate during development, although the functional significance of this phenomenon has not been clarified. An important question is whether such a transmitter switch occurs in other systems, especially in the brain. In the present study, we demonstrate that the major transmitter mediating correlated wave activity in the embryonic chick hindbrain changes from acetylcholine/gamma-aminobutyric acid (GABA)/glycine to glutamate/GABA as development proceeds. The results show for the first time that the dominant transmitter switches from acetylcholine to glutamate in a region other than the retina and spinal cord. This finding sheds more light on the role of nicotinic acetylcholine receptors in the generation of correlated wave activity, which is considered to regulate the development of the nervous system.

Development of projection-specific interneurons and projection neurons in the embryonic mouse and rat spinal cord.

Interneurons and projection neurons in the lumbar spinal cord of mouse and rat embryos were labeled retrogradely with fluorescent dextran amines from a distance of one segment from the segment of origin [lumbar segment (L) 2]. Six classes with specific axonal projections (ipsilateral ascending, descending, and bifurcating, and commissural ascending, descending, and bifurcating) were identified by differential labeling in both species and followed from embryonic day (E)12 to birth in the mouse. Neurons with shorter projections (intrasegmental interneurons) were not studied. We show that the four nonbifurcating neuron classes occupy characteristic, partially overlapping domains in the transverse plane, indicating a systematic pattern of migration and settlement related to axon trajectories. The number of neurons in each of the nonbifurcating classes increased steadily during development. Bifurcating neurons represented a minor fraction of the total throughout development and had relatively scattered positions within the ipsilateral and commissural neuron domains. Combination of retrograde tracing and immunohistochemistry for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) showed that none of the spinal neurons in the six projection-specific classes was GABA positive, suggesting that all GABA-positive spinal neurons, including previously described GABA-positive commissural neurons, are unlikely to have projections exceeding one or two segments in either direction.

Optical analysis of depolarization waves in the embryonic brain: a dual network of gap junctions and chemical synapses.

Correlated neuronal activity plays a fundamental role in the development of the CNS. Using a multiple-site optical recording technique with a voltage-sensitive dye, we previously described a novel type of depolarization wave that was evoked by cranial or spinal nerve stimulation and spread widely over the whole brain region in the chick embryo. We have now investigated developmental expression and neuronal network mechanisms of this depolarization wave by applying direct stimulation to the brain stem or upper cervical cord of E5-E11 embryos, which elicited wave activity similar to that evoked by nerve stimulation. Spatial distribution patterns of the depolarization wave changed dynamically with development, and this change appeared to be related to the regional differences in neuronal differentiation. The depolarization wave was completely eliminated by application of either gap junction blockers or an N-methyl-D-aspartate (NMDA)-receptor antagonist, indicating that functions of both gap junctions and NMDA receptors are indispensable for wave propagation. A possible interpretation of the results is that dual networks of gap junctions and chemical synaptic coupling mediate large-scale depolarization waves in the developing chick CNS.

Optical imaging of intrinsic signals induced by peripheral nerve stimulation in the in vivo rat spinal cord.

We examined neural response patterns evoked by peripheral nerve stimulation in in vivo rat spinal cords using an intrinsic optical imaging technique to monitor neural activity. Adult rats were anesthetized by urethane, and laminectomy was performed between C5 and Th1 to expose the dorsal surface of the cervical spinal cord. The median, ulnar, and radial nerves were dissected, and bipolar electrodes were implanted in the forelimb. Changes in optical reflectance were recorded from the dorsal cervical spinal cord in response to simultaneous stimulation of the median and ulnar nerves using a differential video acquisition system. In the region of the cervical spinal cord, intrinsic optical signals were detected between C5 and Th1 at wavelengths of 605, 630, 730, 750, and 850 nm: the image with the largest signal intensity and highest contrast was obtained at 605 nm. The signal intensity and response area expanded with an increase in the stimulation intensity and varied with the depth of the focal plane of the macroscope. The intrinsic optical response was mostly eliminated by Cd(2+), suggesting that the detected signals were mainly mediated by postsynaptic mechanisms activated by sensory nerve fibers. Furthermore, we succeeded in imaging neural activity evoked by individual peripheral nerve stimulation. We found that the response areas related to each peripheral nerve exhibited different spatial distribution patterns and that there were animal-to-animal variations in the evoked neural responses in the spinal cord. The results obtained in this study confirmed that intrinsic optical imaging is a very useful technique for acquiring fine functional maps of the in vivo spinal cord.

Developmental organization of the glossopharyngeal nucleus in the embryonic chick brainstem slice as revealed by optical sectioning recording.

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.

Optical approaches to functional organization of glossopharyngeal and vagal motor nuclei in the embryonic chick hindbrain.

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.

Intraoperative intrinsic optical imaging of neuronal activity from subdivisions of the human primary somatosensory cortex.

We performed intrinsic optical imaging of neuronal activity induced by peripheral stimulation from the human primary somatosensory cortex during brain tumor surgery for 11 patients. After craniotomy and dura reflection, the cortical surface was illuminated with a xenon light through an operating microscope. The reflected light passed through a bandpass filter, and we acquired functional images using an intrinsic optical imaging system. Electrical stimulation of the median nerve, or the first and fifth digits, induced biphasic intrinsic optical signals which consisted of a decrease in light reflectance followed by an increase. The decrease in light reflectance was imaged, and we identified a neural response area within the crown of the postcentral gyrus. In experiments on first and fifth digit stimulation, we identified optical responses in separated areas within the crown of the postcentral gyrus, i.e. near the central sulcus and near the postcentral sulcus. In the former response area, separate representations of the two fingers were observed, whereas in the latter response area, the two fingers were represented in the same region. A similar somatotopic representation was observed with electrical stimulation of the first and third branches of the trigeminal nerve. These results seem to support the hypothesis of hierarchical organization in the human primary somatosensory cortex.