Cellular and Metabolic Origins of Flavoprotein Autofluorescence in the Cerebellar Cortex in vivo

Abstract

Flavoprotein autofluorescence imaging, an intrinsic mitochondrial signal, has proven useful for monitoring neuronal activity. In the cerebellar cortex, parallel fiber stimulation evokes a beam-like response consisting of an initial, short-duration increase in fluorescence (on-beam light phase) followed by a longer duration decrease (on-beam dark phase). Also evoked are parasagittal bands of decreased fluorescence due to molecular layer inhibition. Previous work suggests that the on-beam light phase is due to oxidative metabolism in neurons. The present study further investigated the metabolic and cellular origins of the flavoprotein signal in vivo, testing the hypotheses that the dark phase is mediated by glia activation and the inhibitory bands reflect decreased flavoprotein oxidation and increased glycolysis in neurons. Blocking postsynaptic ionotropic and metabotropic glutamate receptors abolished the onbeam light phase and the parasagittal bands without altering the on-beam dark phase. Adding glutamate transporter blockers reduced the dark phase. Replacing glucose with lactate (or pyruvate) or adding lactate to the bathing media abolished the on-beam dark phase and reduced the inhibitory bands without affecting the light phase. Blocking monocarboxylate transporters eliminated the on-beam dark phase and increased the light phase. These results confirm that the on-beam light phase is due primarily to increased oxidative metabolism in neurons. They also show that the on-beam dark phase involves activation of glycolysis in glia resulting in the generation of lactate that is transferred to neurons. Oxidative savings in neurons contributes to the decrease in fluorescence characterizing the inhibitory bands. These findings provide strong in vivo support for the astrocyte–neuron lactate shuttle hypothesis.

Rapid Postnatal Expansion of Neural Networks Occurs in an Environment of Altered Neurovascular and Neurometabolic Coupling

“…3E). This limited response distribution is similar to the lateralized somatosensory-evoked neural activity previously observed in early postnatal mice and rats (Marcano-Reik and Blumberg, 2008;Quairiaux et al, 2011;McVea et al, 2012). In the P10 -P13 age group, the response initiates in the contralateral hindpaw region and then spreads quickly to the ipsilateral side.…”

“…The FAD results presented above demonstrate that the absence of functional hyperemia in the neonatal brain leads to locally depleted oxygen in the responding region. This observation could provide a possible mechanism for activity-linked angiogenesis, driven by the sequential neurally evoked localized hypoxic events observed here (Shweiki et al, 1992;Pugh and Ratcliffe, 2003). Equally, the lack of hyperemia to replenish supplies of both oxygen and glucose in the immature brain could impose an energetic limit on sustained neural activity; consistent with the similarities in timing between the early cessation of neural activity in younger animals and the onset of the dark phase of the FAD response.…”

Single-cell imaging tools for brain energy metabolism: a review

“…Fast fluctuations in the autofluorescence of brain tissue in acute slices and in vivo have revealed that metabolism can be as dynamic as intracellular calcium. [35][36][37][38][39] The extremely rapid response of NADH is consistent with its turnover time, which in brain cells is in the order of a few milliseconds. 40 However, the interpretation of autofluorescence data is limited by several factors, including uncertainty about the relative weight of free and protein-bound NADH, interference from NADPH, which in the cytosol is much more abundant, difficulties in assigning the signals to specific subcellular compartments, and the inherent ambiguity of concentration with respect to flux, compounded by the requirement for UV light, which is toxic and unavailable in standard confocal microscopes.…”

Activity-dependent Increases in Local Oxygen Consumption Correlate with Postsynaptic Currents in the Mouse CerebellumIn Vivo

“…CF stimulation evokes large rises in lactate that are coupled to rises in synaptic activity and spike rate (Caesar et al, 2008a). Thus, in the cerebellum preservation of normal function and restoration of ionic gradients is powered by both glycolysis and respiration (Reinert et al, 2011). Likewise, stimulation of parallel fiber beams increases respiration and aerobic glycolysis (Thomsen et al, 2009).…”