Project 4

Bridging guidance cues and activity: how to lead a horse to water and make it drink.

The mature central nervous system is exquisitely complex. In the human brain, approximately 1012 cells become ordered within discrete regions, each subserving a variety of functions with specific connections both within and between them. Most connections are topographically organised and one of the greatest challenges in biology is to understand the rules underlying the establishment of such connectivity, and its re-establishment following neurotrauma.

The restoration of a functional topographic map involves the recapitulation of molecular events that occur during development (Beazley, Tennant et al. 1999). The first of these is the expression of guidance cues (Ephs and ephrins) that guide incoming axons to approximately correct locations within the target (Flanagan and Vanderhaeghen 1998). The second is the expression of molecules involved in strengthening functionally appropriate connections (NMDA and GABA receptors) to produce a fine-tuned functional map (activity-dependent mechanisms; (Cramer and Sur 1995)). The Ephs and ephrins lead the horse to water, but only the appropriate expression and function of neurotransmitter receptors will allow it to drink.

The relevance of this analogy in injury is that axons in the damaged brain or spinal cord can be encouraged to regenerate but do not reconnect with their target cells (water) and normal function (drinking) is not restored. The malfunction is thought to be primarily due to the abnormal expression of neurotransmitter receptors both on the axon terminals and on their targets. Specifically, the balance between inhibitory (GABA, glycine) and excitatory (NMDA, AMPA) receptors is not restored. Following denervation of the superior colliculus (SC), there is a significant upregulation of the GABA and NMDA neurotransmitter receptor (Houser, Lee et al. 1983; Janusonis and Fite 1997). The result is not only abnormal function, but also in some cases the development of neuropathic pain (Obata, Yamanaka et al. 2003; Bursztajn, Rutkowski et al. 2004; Jang, Kim et al. 2004; Karchewski, Bloechlinger et al. 2004).

There is considerable evidence that guidance cues and activity-dependent mechanisms interact. Ephs and ephrins have been implicated in synaptic plasticity, a phenomenon that is traditionally considered activity-dependent. In addition, recent experiments have demonstrated a direct interaction between guidance cues and activity-dependent mechanisms: EphB can directly regulate the function, structure and subcellular localisation of NMDA receptors in the hippocampus (Dalva, Takasu et al. 2000; Grunwald, Korte et al. 2001; Takasu, Dalva et al. 2002).

Here, we use normal development as a model to examine the physical and functional interactions between Eph receptors and excitatory and inhibitory neurotransmitter receptors.

Aims

1. To fully characterise the physical interactions between Eph receptors and excitatory and inhibitory neurotransmitter receptors during normal development
2. To assess the functional effects of Eph receptors on neurotransmitter channel properties during normal development

Significance

We will determine the role of guidance cues in establishing normal neurotransmission. The results will suggest novel targets for the treatment of dysfunctional connections that occur following injury. These include anatomically intact tracts that are silenced due to secondary damage as well as abnormal regenerate connections subserving neuropathic pain, increased tone and spasticity.

The honours project will address a selection of the aims listed below depending on the areas of interest to the student and taking time constraints into consideration.

Aim 1. To fully characterise the physical interactions between Eph receptors and excitatory and inhibitory neurotransmitter receptors in the developing mouse SC

Experiment 1a: Immunoprecipitation of SC proteins from P8 mice during the refinement of the retinocollicular projection will be used to determine the endogenous physical interactions between Eph and neurotransmitter receptors.

Proteins will be extracted and incubated with the primary antibody. To minimize false negatives, immunoprecipitation will be carried out in both directions. Antibodies to EphAs and EphBs are routinely used in our laboratory for immunohistochemistry and immunoprecipitation. Antibodies to neurotransmitter receptors have successfully been used for immunoprecipitation in mouse. Bound complexes will be captured using Trueblot agarose beads and analysed by western blotting. We will run aliquots of the same lysate and probe each of the resultant blots with a different antibody.

Experiment 1b: Immunohistochemistry on frozen sections of P8 mouse brain will be used to examine expression and localisation of Eph/ephrins and neurotransmitter receptors in vivo.

To identify whether complexes are located in RGC or collicular cells, we will label a small number of terminals by applying the anterograde tracer DiI to the retina using a picospritzer 48 hours prior to sacrifice. Mice will be perfused with 4% paraformaldehyde and brains cryosectioned to expose the labelled area. Primary antibodies will be applied overnight at 4oC and detected using fluorescent secondaries (controls: pre-immune serum and absence of primary antibody). Neuronal and glial markers will be applied to identify cell types in the colliculus. Localisation of target proteins in relation to RGC axon terminals and collicular glia and neurons will be assessed by confocal microscopy.

Aim 2: To assess the functional effects of Eph receptors on neurotransmitter receptors

Experiment 2a: Do ephrins inhibit or activate neurotransmitter receptor channels?

Whole cell patch recordings will be made from isolated mouse retinal ganglion cells in culture.

Cells will be voltage clamped and exposed to increasing concentrations of recombinant ephrin-A or ephrin-B proteins. The sensitivity of NMDA, GABA and AMPA receptors to ephrin-A and ephrin-B will be determined. Specifically changes in peak inward current in the presence of increasing concentrations of ephrin-A or ephrin-B will be determined. Increased current in the presence of ephrin will indicate activation, will decreased current will indicate inhibition. Data will be fit to a logistic equation using a nonlinear least-squares curve-fitting routine (GraphPad Prism) and the concentration of ephrin at which the current is half-maximally activated or inhibited will be calculated.

Experiment 2b. Do ephrins recruit neurotransmitter receptor channels to the membrane?

The effects of ephrin-A and ephrin-B on current density will be studied in the presence of a single concentration of ligand. Cell capacitance will be calculated for each cell and current density determined (pA/pF). The responses are acute and unlikely to involve changes in transcription or translation of receptor protein. Therefore, an increase in current density at a maximally stimulating concentration of ephrin may suggest the recruitment of functional receptors to the plasma membrane by ephrin or an ephrin-mediated signal.