Martha Bosma - US grants

Understanding the mechanisms and roles of spontaneous electrical activity in brain development is key to overall understanding of how connections among individual nerve cells are created. These connections are required for the correct functioning of the adult brain. The hindbrain contains several populations of motor neurons that control muscles serving critical functions in speech, respiration, and facial expression. Dr. Bosma's laboratory has developed methods for identifying specific hindbrain motor neurons in the early mouse embryo, and is the first to record spontaneous activity from these identified soma during the developmental stages when spontaneous activity first appears. The ability to characterize the onset, properties, and developmental functions of these cells makes this an ideal preparation in which to understand the role that spontaneous activity plays in establishing functional connections in the central nervous system and in the peripheral musculature.

Because the PI's laboratory has developed unique methods to study spontaneous activity in this preparation, the PI has already established collaborations with laboratories in the US and Europe that wish to study these questions in chick and zebrafish hindbrain. Such collaborative experiments will help advance the understanding of the evolution and genetics of activity-dependent hindbrain development. Because the PI teaches undergraduate courses in neurobiology and developmental neurobiology, the research efforts will be easily integrated into undergraduate education. In addition to other undergraduates in the lab, the PI has already sponsored three undergraduate researchers to work on this project, including one currently in the lab who is the first author on a published paper on this work. Two of these students won the University of Washington's Franco Award for best undergraduate research paper. The PI is also involved in educational outreach at the K-12 and community college level. Basic ideas of brain function and development have been incorporated into grades 1-4 hands-on classroom exercises, and the PI has obtained funds to extend that outreach to disabled students in a local K-5 special education classroom. The PI sponsors a graduate student, whose salary is included in this proposal, who is on leave from a local community college, where she teaches biology.

Spontaneous electrical activity, taking the form of action potentials, in developing mammalian brain structures has been shown to be required for the correct circuit wiring and cellular development of those structures. However, the mechanisms of generation of spontaneous activity have not been elucidated in many of these structures; in those where the mechanism is known, spontaneous electrical activity is caused by a network of mutually excitable neurons that synchronize each others activity. Recently, it has been shown by this laboratory that within the early embryonic mouse hindbrain, a region that contains many autonomic and widespread modulatory neuronal networks, as well as facial motor control, that synchronized spontaneous activity is controlled by a discrete population of neurons, the newly differentiating midline serotonergic raphe. This grant proposes to examine the mechanisms by which the midline raphe neurons mediate this widespread synchronized activity using combined imaging of intracellular Ca2+ levels and electrical recordings of individual neurons; in addition, it will explore the developmental ramifications of manipulation of the activity by culturing the hindbrain under control and pharmacologically altered conditions. Understanding these mechanisms, and their modulation by altered drug conditions, will be crucial to understanding how an important brain structure develops.

The PI integrates her research results into her undergraduate teaching. Seven undergraduates participated in the previously funded award. Four of these seven undergraduate students won the University award for best undergraduate paper. Five of these undergraduates are listed as an author on at least one major publication from the lab. Dr. Bosma also participates in outreach at the K-12 and community college level and sponsors a teacher from a local community college to pursue a graduate degree The PI is actively involved in outreach for students from kindergarten through community college by developing and implementing several hands-on exercises. She has also recently obtained Howard Hughes funds to extend this outreach to K-5 students with disabilities in a local special education classroom. The PI also sponsors a PhD student who is on leave from community college biology teaching. This student is a tenured instructor at an inner city campus serving older and returning students who has taken a sabbatical, and who plans to return to her campus, better equipped to offer modern science education. Support of the present proposal will allow Dr. Bosma to continue and expand these activities.

In the developing brain, spontaneous activity (not driven by external input) is critical for appropriate circuit formation. These experiments will investigate the mechanisms by which serotonergic neurons in the embryonic mouse hindbrain begin and cease expression of spontaneous activity. These neurons drive waves of electrical activity across the entire hindbrain, which is important in specifying axonal projections both of the serotonergic neurons, and in follower neurons. Previous work has shown that pharmacological blockade of the serotonin receptor prevents the onset of the spontaneous activity. The current goals, using physiological and anatomical techniques, are elucidating the mechanism(s) governing the onset of activity and the role of serotonin receptor signaling in that onset. In addition, the mechanism(s) that dictate the retraction of activity to the driver cells and cessation over 3 developmental days will be determined. This work characterizes important processes in the regulation of spontaneous activity which directs correct network formation. Thus, it examines the basic mechanisms by which neurons find and maintain correct innervation patterns during development. The lab has had many undergraduates and a number of high school students participate in the research project, to the point of publication. This includes a week-long workshop for disabled high school students as part of a summer program, where the students are able to use physiological techniques and observe spontaneous activity.

DESCRIPTION (provided by applicant): 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.