Initial evidence that electrical excitability is both an early aspect of neuronal differentiation and a developmentally regulated property was obtained from recordings of action potentials in vivo. Subsequently, the analysis of the underlying voltage-dependent currents during early stages of embryogenesis was facilitated by investigation of dissociated neurons and muscle cells differentiating in culture. Calcium and potassium currents play a major role in the differentiation of the action potential of Xenopus spinal neurons, and calcium influx triggers specific features of neuronal differentiation. However, the extent to which differentiation of currents in vitro parallels that in vivo is uncertain. We have undertaken a study of in vivo differentiation of these macroscopic currents in Xenopus embryos. Spinal cords were isolated from embryos at several early stages of neurogenesis. Neurons in these isolated spinal cords were accessible to patch-clamp electrodes. Neuronal currents were recorded within 1 hr to assure that the characteristics of the currents resulted from developmental events occurring in vivo prior to the experiment. Whole-cell voltage-clamp recordings from neurons in these acutely isolated and intact embryonic spinal cords demonstrate that both the delayed-rectifier and inactivating potassium current and a low-voltage-activated calcium current mature in a manner closely parallel to that observed in culture. The results validate those from the culture system and indicate that the spinal cord is another region of the CNS accessible to cellular analysis in an intact preparation.

Neurons in the CNS generally receive inputs form multiple afferent sources. These afferent systems seldom all use the same neurotransmitter, so most central neurons are required to express multiple neurotransmitter receptors. This work addresses the issue of how multiple neurotransmitter receptors are regulated on the surface of individual neurons. We made whole-cell voltage-clamp recordings from identified chick sympathetic preganglionic neurons (SPNs) in dissociated cell cultures. The neurons were derived from stage 30-31 (7 d) chick embryos and were studied within the first week in vitro. We found that by 1 week in vitro, most SPNs responded to the application of GABA, glycine, and glutamate. The responses of SPNs to the amino acid neurotransmitters were similar to the responses of other CNS neurons to these 3 substances. SPNs became sensitive to these substances at different times in culture. At 1 d in vitro, most cells already responded to GABA, and about half of the cells also responded to glycine and kainate. In contrast, responses to NMDA and quisqualate were usually not seen until day 3-4 in vitro. Although there was a general trend for the amplitude of the responses of SPNs to each of the neurotransmitters to increase with time in vitro, there was an immense amount of cell-to-cell variability. By measuring the amplitudes of the responses of a series of SPNs to all 3 transmitters, we were able to test whether a common regulatory mechanism governed the level of responsiveness of SPNs to all 3 amino acid transmitters. We found no correlation between cells in the amplitudes of their responses to the 3 transmitters. Both the differences in time course of appearance of responsiveness and the lack of correlation in the amplitude of responses suggest that the multiple receptors on the surface of SPNs in vitro are independently regulated.

The influence of non-neuronal cells and interneurons on the morphological development of chick sympathetic preganglionic neurons (SPNs) and on the responsiveness of these neurons to the neurotransmitters GABA, glycine, and glutamate was studied. SPNs were retrogradely labeled with the fluorescent dyes dil and diO, then separated from spinal-cord non-neuronal cells and interneurons by fluorescence-activated cell sorting. SPNs were grown in culture, either alone or in coculture with non-neuronal cells alone, with interneurons alone, or with both of these cell types (control cultures). The responsiveness of SPNs to neurotransmitters was assessed by whole-cell recording, while cell morphology was assessed after intracellular staining with 6-carboxyfluorescein. Cell size and morphology were affected by non-neuronal cells. In the absence of non-neuronal cells, SPNs had smaller cell bodies and fewer major processes, whether or not interneurons were present. In contrast, responses to the 3 neurotransmitters were affected by both non-neuronal cells and interneurons, but in ways that differed slightly for each transmitter. In the absence of both non-neuronal cells and interneurons, responses to all 3 transmitters were much smaller than in control cultures, with responses to glutamate most profoundly affected. The addition of either non-neuronal cells or interneurons slightly increased the amplitude of SPN responses to glutamate, but the level of responsiveness with either cell type alone was much lower than for SPNs grown in the presence of both cell types. The addition of interneurons also slightly increased the responsiveness of SPNs to GABA, but non-neuronal cells alone had no significant effect on the responses of SPNs to GABA. Finally, the glycine responsiveness of SPNs was raised to control levels when either non-neuronal cells or interneurons were added. These experiments demonstrate that, though interneurons can have a significant inductive effect on the responses of SPNs to neurotransmitters, not all of the changes in neurotransmitter responsiveness can be related to the formation of functional synapses.

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