UC San Diego

Specification of neurotransmitters is a crucial and fundamental aspect of development. It allows the establishment of functional connections at synapses, which ensures normal functioning of the entire nervous system. Establishing the appropriate expression pattern of neurotransmitters in different populations of neurons is a critical challenge for neural development. It is a complicated process that involves multiple mechanisms, including intrinsic genetic coding, early neuronal activity and environmental factors. Embryonic Xenopus spinal neurons generate spontaneous transient elevations of intracellular calcium. Calcium activity regulates early neuronal differentiation by regulating neurotransmitter phenotype choice, but the mechanisms by which this activity is transduced to achieve these changes are unclear. We developed a novel practical method to manipulate activity in single neurons in vivo. Although we found that suppression of spike activity globally in developing spinal neurons increased the incidence of glutamatergic neurons and reduced the incidence of GABAergic neurons, we didn't find the same change when spike activity was suppressed in single isolated neurons. Our results indicate that GABAergic/glutamatergic selection is regulated by the level of activity in surrounding neurons. We found that activity-dependent brain-derived factor (BDNF) modulated GABAergic/ glutamatergic switching, mimicking the effect of activity enhancement. Abolition of BDNF function by blocking its tyrosine kinase B (TrkB) receptor using K252a generated the opposite neurotransmitter switch, mimicking the effect of activity suppression. Simultaneous manipulation of BDNF function and suppression of Ca²⁺ activity phenocopied the effect on neurotransmitter phenotype of BDNF alone, indicating that BDNF works downstream of Ca²⁺ activity in regulating neurotransmitter specification. We propose that activity from neighboring neurons regulates the expression and release of BDNF, which then activates the TrkB signaling cascade leading to several physiological or genetic pathways that determine neurotransmitter specification. This mechanism provides a basis for early activity-dependent regulation of neurotransmitter phenotype in developing neurons. In addition to the role of Ca²⁺ activity, we wanted to determine whether there is a target effect on neurotransmitter specification. For this purpose we developed a neuron-muscle co-culture system. We found that muscle contact refines transmitter expression in cultured neurons by reducing expression of the non-cholinergic transmitters, GABA, glycine and glutamate, while having no effect on the incidence of choline acetyltransferase expression. The results indicate that muscle, as a neuronal target, plays important role in regulating transmitter specification