Brainstem and spinal cord expression of novel and existing neuronal connexins determined using transgenic reporter mice.

The brain contains millions of nerve cells (neurones), generally organised into discrete collections to perform specific functions. These neurones communicate with selected partners to make such functions possible. Until recently the only way that brain cells were thought to communicate with one another was through chemical synapses where an electrical impulse in one neurone releases a chemical which diffuses across a small space to set up an electrical signal in another neurone. However, it has recently emerged that neurones can communicate by channels which allow two cells to be directly coupled / gap junctions. These gap junctions allow the passage of electrical signals and some other cellular metabolites directly between the cells. This direct connection permits fast transfer of information between the cells and allows groups of neurones to become synchronised in their electrical activity. Such synchrony is now seen as important for determining many brain functions. The gap junctions are made up of different proteins, some of which are a family called connexins. There are many connexins, but only two of these have been known to be present in neurones within the brain, Cx36 and Cx45. However, their distribution is not known in the brainstem and spinal cord, areas of the nervous system directly affecting breathing, blood pressure, heart rate, pain sensation and movement etc. In addition, our preliminary data shows that a new connexin, Cx30.2, is present in many neurones in these regions. We therefore propose to investigate the distribution of these connexins in the brainstem and spinal cord. Unfortunately, two conventional approaches to determining connexin distribution have proven unreliable, with many false answers. We will therefore base our studies on a new, validated approach where mice have been genetically altered to produce a marker within the cells that make each individual connexin. By a simple reaction we can see in tissue sections the cells where the connexins are made. Since the brainstem and spinal cord contain neurones with different functions we will work out which cell types express the particular connexins using additional anatomical and physiological methods. We will also cross breed mice with different types of markers to see if one cell can produce more than one connexin. This is important as the presence of different connexins in a single cell may influence how the particular gap junction works. These studies will therefore provide new data about the distribution, cell types and co-localisation of the neuronal connexins. Since our preliminary studies have suggested the presence of connexins in neuronal groups not previously known to express gap junctions, full analysis is likely to reveal novel populations for future studies. The identification of such neuronal groups with the capabilities of synchronising their activity will provide new areas of understanding of how these brain regions function.

Many functions underpinned by the brainstem and spinal cord are reflected by synchronous, often rhythmic, electrical discharge in the underlying neuronal circuits. Such activity patterns frequently result from electrical coupling of neurones via gap junctions. However, the pattern and types of gap junction proteins expressed in these areas remains largely to be identified. Unfortunately, even in intensely studied regions, conventional methods to localise expression or protein distribution of gap junctions have proven unreliable. This project will use transgenic mice to examine the expression of the neuronal gap junction proteins from the connexin family in the brainstem and spinal cord. In these mice the expression of reporter protein such as GFP or beta-galactosidase is controlled by the endogenous connexin reporter, enabling visualisation of expressing neurones. Preliminary data shows expression of Cx36, Cx45 and a novel connexin, Cx30.2. In the first stage we shall fully determine the expression of each connexin in the brainstem and spinal cord. We shall next determine the phenotype of the connexin expressing neurones by staining reporter labelled neurones with antibodies to marker proteins such as calcium binding proteins, or by neuronal tracing. Expression in GABA or glutamatergic neurones will be determined using in situ hybridisation and by breeding connexin mice with other strains expressing reporters in these cell types. Finally, we shall cross breed mice from connexin strains using different reporter proteins and co-localise these in tissue sections to reveal co-expression. These experiments will reveal the expression pattern of Cx30.2, Cx36 and Cx45 in spinal cord and brainstem neurones, identify the cell types expressing the connexins and the patterns of co-expression. Such data will are essential to fully understanding the determinants of neuronal activity in these regions and in turn the critical important functions that they control.