Nonsynaptic Neurotransmitter Effects on Developing Spinal Cord Circuitry

Lead Research Organisation: University of Leicester
Department Name: Biology

Abstract

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.

Technical Summary

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.

Publications

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Description 1) We have found two distinct roles for GABA signalling in the zebrafish CNS. First, perturbation of GABA synthesis with AMOs targeted against GAD67b, the predominant GAD isozyme of the spinal cord, profoundly affects the onset of spinal glutamatergic transmission. GAD67b knockdown fish develop precocious glutamatergic synapses that can be detected with electrophysiological methods several hours prior to those seen in control fish. Second, knockdown of GAD67a causes marked effect perturbation of fore and midbrain development: Abrogation of this enzyme disrupts neuronal proliferation in these regions, dramatically affecting neuronal architecture. Taken together, our findings provide in vivo evidence for developmental roles of GABAergic systems in vertebrate nervous tissue.



2) We have found that DA has a key role in regulating the earliest forms of spinal network activity observed in the zebrafish embryo. We have shown that DA signalling strongly inhibits "spontaneous network activity" (SNA), an immature form of network activity that propagates between electrically coupled spinal neurons prior to the formation of synapses. Addition of DA during this period silences SNA, an effect which can be occluded by the D4 receptor antagonist clozapine. Systematic patch clamp analysis of spinal neuron responses to DA revealed that this signalling molecule acts by specifically modulating ionic conductances of descending spinal cord interneurons. We have shown that DA enhances a leak potassium conductance and suppresses a persistent sodium conductance in these cells, causing network hyperpolarisation and cessation of spontaneous activity. To examine the functional role of this immature form of network activity, we exposed early stage zebrafish to the DA neurotoxin 6-OH-DA. This drug perturbed maturation of spinal network activity, suggesting that DA may have key roles in regulating the maturation of more mature forms of network output during spinal development.



3) We have examined the functional roles of NO signalling during spinal cord maturation. We have found that pharmacological and antisense perturbation of spinal NOS1 expression has a dramatic impact on the growth of nascent primary motoneurons. Loss of NOS1 activity dramatically increases motor axon arborisation whilst exogenous NO has the opposite effect. These findings demonstrate that NO is a critical regulator of spinal motoneuron maturation during early development.
Exploitation Route This work may help to instruct strategies for treatment of developmental brain disorders. Our work suggests that developmental perturbations in GABA/NO signalling can have a marked impact on nervous system development, which may have relevance to the mechanisms underpinning or treatment of neurodevelopmental disorders.
Sectors Pharmaceuticals and Medical Biotechnology,Other

 
Description Invited speaker, University of Bristol 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Talk title: "Development of the zebrafish locomotor network"

no actual impacts realised to date
Year(s) Of Engagement Activity 2008