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.

No abstract

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.

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.

We searched for an optimal voltage-sensitive dye for optical measurements of neural activity in the hippocampal slice by evaluating several merocyanine-rhodanine and oxonol dyes. The wavelength dependence (action spectra), pharmacological effects of staining, signal size, signal-to-noise ratio, and the utility of the dyes for long-term continuous recording were examined for four merocyanine-rhodanine dyes (NK2761, NK2776, NK3224 and NK3225), which had been reported to be optimal in embryonic nervous systems, and for two oxonol dyes (NK3630 (RH482) and NK3041 (RH155)), which have been among the most popular potentiometric probes for the hippocampal slice preparation. NK2761, NK3224 and NK3225 provided large signal-to-noise ratios, and proved to be useful for optical recordings lasting several hours. NK3630 was most suitable for long-term recording, although the signal-to-noise ratio was slightly inferior to that of the merocyanine-rhodanines. Using NK3630 (RH482) on the hippocampal slice preparation, we demonstrate here that long-term potentiation can be monitored stably for more than 8 hr.