The rodent optic nerve is a model tissue for the physiological investigation of axonal–glial interactions in a typical CNS white matter tract. There is strong evidence that nerve transmission is maintained by a considerable degree of dynamic signalling between axons and glia through a variety of mechanisms, such as regulation of the ionic environment, energy metabolism and calcium signalling. This review focuses on the methods used to examine axonal and glial functions and interactions, primarily in the rodent optic nerve. Techniques encompass intracellular microelectrodes, sucrose- and grease-gap recordings of membrane potentials, suction electrode to measure compound action potentials, the use of ion-sensitive electrodes, patch clamping and imaging. An overview of the advantages and drawbacks of each technique is given and the application of each to the understanding glial and axonal physiology is briefly discussed.