Spontaneous activity (SA;electrical activity independent of external stimulus) in the developing central nervous system is often recorded as waves of propagating electrical events in different brain structures. These waves are expressed only in a relatively short window of development, often occurring during a period of synaptogenesis;a change in the window of expression alters development of the relevant circuit. They have also been shown to influence neuron proliferation and phenotype, axon pathfinding and extension, and synaptic formation and maintenance. The waves of activity successively invade group of cells, allowing coincident firing between neighboring cells and mutual strengthening of developmental events. Modulation of this SA by changes in ion channels, propagation mechanisms or transmitter release would modify both the timing and the extent of SA, and would likely alter circuit development. In the embryonic brainstem, we have shown that SA originates within the developing serotonergic (5HT) hindbrain raphe;postnatally, 5HT neurons regulate of mood and behaviors such as depression and schizophrenia. 5HT-dependent waves propagate widely early in brainstem formation, at embryonic day (E) 11.5;soon after, waves begin to retract towards the pacemaker region, and disappear by E14.5. At specific stages, the waves propagate into the midbrain tegmentum, where they activate newly differentiating dopamine (DA) neurons;later in life these neurons mediate reward and addictive behaviors. This is an unusual example of SA in one brain region regulating neuronal excitability in another structure. We have shown that retraction of the 5HT-dependent waves is caused by upregulation of specific K channels, such that waves are unable to propagate into regions of the hindbrain in a defined spatiotemporal pattern. Because the initiator cells driving SA maintain their pacemaker role for the entire period of SA, we propose to use genetic labeling and optogenetic techniques to selectively silence or augment waves of SA, and ask how that modulation changes the development of serotonergic AND dopaminergic circuits. We have recently used specific 5HT genes to transgenically label live 5HT pacemaker neurons and identify the mechanisms by which they generate SA. We now propose to use the same genetic tags to introduce light- regulated ion channels that can silence (halorhodopsin) or stimulate (channelrhodopsin) the neurons. These experiments will be performed on cultured embryos, using controlled light stimulation during the culture period. We will be able to examine how modulation of SA changes the expression of the K channel that mediates wave retraction, and to characterize changes in 5HT and DA neuron number, position, axon extension and pathfinding. These experiments will demonstrate how modifications of neuronal excitability in the 5HT-dependent pacemaker population, such as might be induced during pregnancy by prescription or street drugs, can alter development of important behavior-regulating circuits.
During development of the brain, spontaneous electrical activity influences neuron number and morphology. Disruption or changes in this electrical activity, by endogenous disorders or by prescription drugs or drugs of abuse, will change the properties of developing neurons and their postnatal circuits, leading to neurodevelopmental and mental health disorders. We propose to characterize how brainstem activity modulation alters developing neurons that regulate addiction and mood disorders.