It is accepted that glucose is the primary energy support of the brain, but what is contentious is the form of glucose that neural elements use: glucose or glucose-derived metabolites? Neurones contribute 80% of brain metabolic rate, but astrocytes consume up to 60% of brain glucose. This dichotomy is resolved if we accept that glucose is converted to a conduit, lactate, within astrocytes that is released into the interstitial space for neuronal metabolism. A broad conclusion is that astrocytes are primarily glycolytic whereas neurones are primarily oxidative. We demonstrate that lactate is released from the mouse optic nerve (MON), and that stimulus evoked activity leads to elevations in [K+]o and [lac-]o. The logarithmic relationship between [K+]o and [lac-]o and the stimulatory effects of barium in the absence of elevated [K+] supports the notion that it is the astrocyte membrane potential that dictates lactate release. All procedures were carried out in accordance with the Animals (Scientific Procedures) Act 1986 under appropriate authority of project and personal licenses. Adult male CD-1 mice were killed by cervical dislocation and decapitated. Optic nerves were dissected, placed in a perfusion chamber, superfused with aerated aCSF containing 10 mM glucose. The stimulus evoked compound action potential was evoked with supra-maximal stimuli. Lactate biosensors (sarissa.co.uk) recorded [lac-]o at the glial pia boundary and K+ sensitive microelectrodes measure interstitial [K+]. We sought to establish the relationship between [lac-]o and [K+]o by altering aCSF [K+]. aCSF containing increasing [K+] resulted in elevations in [lac-]o compared to aCSF containing 3 mM. The relationship between aCSF [K+] and [lac-]o was logarithmic. We used the K+ channel blocker barium to depolarize astrocytes, causing a sustained [lac-]o elevation. We quantified the relationship between stimulus intensity and [lac-]o by imposing 2-minute periods of stimulus ranging in intensity from 2 to 60 Hz, which produced stimulus frequency dependent increases in [lac-]o and [K+]o, which peaked at about 50 Hz. Stimulating the nerve with high intensity bursts of action potentials evoked increases in [lac-]o that attenuated with subsequent identical stimuli. Our data provides convincing evidence of an intimate link between interstitial [K+] and lactate release from astrocytes in MON. The relationship provides a means by which neural elements can signal to astrocytes their metabolic requirements. All neural elements release K+ in response to increased firing in a frequency dependent manner, and astrocytes are exquisitely sensitive to interstitial [K+], their membrane potential responding in a Nernstian manner to fluctuations in [K+]o. Thus astrocytes are uniquely equipped to respond rapidly to activity induced fluctuations of [K+]o and release lactate for use by neural elements as substrate.