We use the central pattern generator (CPG) network of neurones found in invertebrates as a simple analogue of the behavioural switching neurone network found in humans. A particular focus of our work is on the voluntary control of respiratory function. Our aim is to understand this process at a developmental, functional and molecular level and to do this we use a combination of behavioural, physiological and cell-molecular biological techniques as described in more detail below.
Control of Respiration during Development
In humans, the generation of the respiratory rhythm arises from a group of pacemaker neurones in the pre-Bözinger complex of the Medulla Oblongata. During neonatal growth, this rhythm is generated and sustained by interactions between the pacemaker neurones and excitatory connections within the respiratory network. In adults, synaptic inhibition plays an increasingly important role in establishing the modulatory control of respiration.
We are using histofluorescence techniques to map the development of the CPG neurones involved in initiating, sustaining and terminating the respiratory cycle in the slug Arion rufus and the tropical snail Marisa cornuarietis.
We use animals at different developmental stages and train them using operant conditioning techniques to suppress their respiratory activity even under hypoxic conditions. The effect of this training on CPG neurone connectivity is characterised using histofluorescence.
In Vitro Reconstruction of the Respiratory Network
Whilst the neuronal organisation of invertebrates such as slugs and snails is relatively simple in comparison to humans, in vivo analysis is still challenging. Fortunately, studying the development of the CPG responsible for respiratory control is simplified considerably by the ability to reconstruct it in vitro. Previous work using other model gastropods has indicated that neurones of the CPG make the same type of synaptic connections with each other in vitro as they do in vivo. The aim of this project is to characterise how modulation of respiratory activity is encoded within the neurones that make up the CPG and to characterise the developmental influences on this process.
We are using a combination of electrophysiological and fluorescence microscopy techniques to characterise the connections made between different neuronal soma taken from the CPGs of animals at different developmental stages.
Maintenance of Inhibitory Synapses in the CPG
An interesting feature of the CPG-R found in many gastropods is the presence of two large neurones, LPeD1 (dopaminergic) and RPeD1 (serotonergic) which form a mutually inhibitory synapse with each other. Whilst it is known that LPeD1 is responsible for initiating the respiratory cycle, the function of RPeD1 is unclear but it probably acts as an ‘arousal level’ modulator of LPeD1. The signalling between these two neurones is complex, involving a number of adhesion complexes and cell membrane ligand-receptor proteins. The aim of this project is to characterise synapse-specific proteins in both A. rufus and M. cornuarietis and investigate their function in this inhibitory synapse.
We are using PCR-based methods to identify, sequence and clone a number of known synapse proteins and fluorescent tagging to characterise their activity in our in vitro soma cultures.