Developmental roles of spontaneous network activity during motor circuit assembly

Cells of the adult brain are organised into a vast array of networks that relay, process and respond to information from the external world. These networks are composed of neurons, specialized cells that carry electrical information. Neurons within networks communicate with one another by releasing neurotransmitters, small chemical messengers, across specialized junctions called synapses. By using a combination of electrical and chemical signalling, neuronal networks of the adult brain can process and respond to a wide spectrum of information from the environment. Until recently, it was believed that the embryonic brain does not generate activity before sensory systems develop. However, recent research has demonstrated that this is not the case. Immature neuronal networks spontaneously generate patterns of electrical activity in the absence of sensory input. This activity is unique to developing networks, bearing no resemblance to activities seen in the adult brain. Such spontaneous activity is observed in the brains of virtually all developing animals. Whilst it is known to be important for maturation of nervous tissue, its role during early development remains poorly described. We will address this problem by studying network activity during development of the zebrafish embryo. The zebrafish, a small freshwater cyprinid, is ideal for developmental studies because fertilization occurs externally so that steps during embryonic development can be easily monitored. In the zebrafish embryo, before sensory systems are established, immature neuronal networks generate spontaneous activity. It is our goal to establish precisely how such activity is generated and how it influences the early stages of development. Our work has two broad goals. The first is to understand how the immature network produces activity. To do this we will monitor electrical properties of developing embryonic neurons. We will examine how these neurons are able to spontaneously generate electrical activity in the absence of external input. Our second goal will be to determine developmental roles of early activity in the zebrafish embryo. We will use molecular genetic methods to disrupt electrical signals and determine how this influences network maturation. We will focus on the neuronal network that generates swimming as it can be used as a simple model network for the study of behaviour. In this way we will be able to examine how early network activity affects many aspects of development, from the growth of individual neurons through to the maturation of adult behaviours. Our work will cast new light on the importance of electrical activity in the embryonic nervous system and may provide important clues about how aberrant activity causes developmental disease.

The developing zebrafish spinal cord is an exceptionally tractable model for the study of in vivo neural network formation because it contains a simple motor circuit that drives a stereotyped form of swimming behaviour. During the first day of development, a small subset of early-born primary neurons are formed that generate spontaneous patterns of network activity. This activity, unique to the embryonic network, occurs prior to the formation of functional synaptic contacts. It is thought to be generated by intrinsic pacemaker properties of developing neurons which are synchronized through gap-junction hemichannels. This proposal seeks to explore how this early activity sculpts assembly of the spinal motor network. To do so, we will use a battery of electrophysiological, morphological and molecular genetic techniques. We will begin by characterizing the ontogeny of voltage gated ion channels on developing primary neurons across the first two days of life. We will use whole cell patch clamp electrophysiology combined with pharmacology to isolate Na+, K+ and Ca2+ conductances for study so that we can determine which specific conductances are present during early forms of network activity. We will next characterize the ion channels responsible for generation of pacemaker activity. To do this we will study the effects of ion channel antagonists on early network activity, monitored with the whole cell patch clamp technique. Finally we will perform a series of functional studies to establish the consequences of perturbing spontaneous network activity during early development. We will knock down ion channel subunits with antisense morpholino oligonucleotides and use immunohistochemical and electrophysiological analyses to determine the consequences for assembly of the spinal motor circuit.