Nonsynaptic Neurotransmitter Effects on Developing Spinal Cord Circuitry

In the adult brain, nerve cells communicate by secreting neurotransmitters, small chemical messengers, at specialized junctions between cells called synapses. When neurotransmitter is secreted, it crosses the synapse and interacts with the apposing nerve cell which converts this chemical message into electrical information. In this way, information can rapidly spread between the 100 billion nerve cells of our brain so that complex computational tasks can be performed that allow us to receive, process and react to information from the world around us. Traditionally, it was believed that in the developing brain, before synaptic junctions form, neurotransmitters were not required for nerve cell signalling. However, we now know that this is not the case. Developing nerve cells secrete neurotransmitter before they establish synaptic contacts and a range of studies show profound neurotransmitter effects on the growth and maturation of developing nervous tissue. Whilst this work has provided important clues about how immature brain cells communicate, few whole organism studies have been undertaken. We will address this problem by studying neurotransmission 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 studied. In addition, zebrafish embryos are transparent which means that we can visually identify and monitor nerve cells as they develop within a living embryo. Our work has three broad aims. The first is to understand whether neurotransmitters affect the excitability of developing nerve cells before synaptic junctions form. We will use specialized techniques to record electrical activity in immature nerve cells following exposure to neurotransmitters. This will allow us to define when cells first become responsive to chemical signalling and what kind of neurotransmitters are involved in these responses. Our second aim will be to determine if embryonic nerve cells use neurotransmitter to talk to one another in the absence of synaptic junctions. We will apply chemicals to block responses to secreted neurotransmitters and measure how this affects electrical activity of developing nerve cells. This will allow us to determine when nerve cells of the developing nervous system begin to communicate. Our final aim is to determine the role for immature neurotransmission in the zebrafish embryo. We will use molecular genetic methods to disrupt neurotransmitter signalling from the onset of development and determine how this affects assembly of the nervous system. We will focus on the nerve cell 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 disruptions in transmitter signalling impact on the many aspects of nervous development, from the growth and maturation of individual cells through to the activity of nerve cell networks and the generation of behaviour. Our work will cast new light on the importance of embryonic neurotransmission and may provide important clues about how imbalances in neurotransmitter activity cause developmental disease.

The developing zebrafish spinal cord is an exceptionally tractable model for the study of in vivo neural network formation. Containing a simple motor circuit that drives a stereotyped form of swimming behaviour, the spinal cord can be used to study the processes governing assembly and operation vertebrate networks for behaviour. The current proposal seeks to capitalize on the strengths of this model to explore noncanonical neurotransmission during embryonic development. The project will use a combination of electrophysiological, morphological and molecular genetic techniques to achieve our goals. We will begin by determining the temporal expression patterns of ligand gated ion channels on developing spinal neurons at stages spanning neuronal birth through to synaptogenesis. Using whole cell patch clamp electrophysiology we will record membrane current responses to a range of ligand gated ion channel agonists. We will determine which precociously expressed ligand gated ion channels are endogenously activated by secreted neurotransmitters. Using whole cell patch clamping we will monitor membrane current responses to blockade of various ligand gated ion channels. Finally we will perform a detailed series of functional studies to establish the role of noncanonical signalling during assembly of the spinal swimming circuit. We will knock down receptor subunits with antisense morpholino oligonucleotides and use immunohistochemical and electrophysiological analyses to determine the consequences to spinal proliferation, differentiation, synaptogenesis, axon pathfinding and neural network formation.