Flavoprotein Autofluorescence Imaging of Neuronal Activation in the Cerebellar Cortex In Vivo

Abstract

Autofluorescence has been used as an indirect measure of neuronal activity in isolated cell cultures and brain slices, but only to a limited extent in vivo. Intrinsic fluorescence signals reflect the coupling between neuronal activity and mitochondrial metabolism, and are caused by the oxidation/reduction of flavoproteins or nicotinamide adenine dinucleotide (NADH). The present study evaluated the existence and properties of these autofluorescence signals in the cerebellar cortex of the ketamine/xylazine anesthetized mouse in vivo. Surface stimulation of the unstained cerebellar cortex evoked a narrow, transverse beam of optical activity consisting of a large amplitude, short latency increase in fluorescence followed by a longer duration decrease. The optimal wavelengths for this autofluorescence signal were 420-490 nm for excitation and 515-570 nm for emission, consistent with a flavoprotein origin. The amplitude of the optical signal was linearly related to stimulation amplitude and frequency, and its duration was linearly related to the duration of stimulation. Blocking synaptic transmission demonstrated that a majority of the autofluorescence signal is attributed to activating the postsynaptic targets of the parallel fibers. Hypothesized to be the result of oxidation and subsequent reduction of flavoproteins, blocking mitochondrial respiration with sodium cyanide or inactivation of flavoproteins with diphenyleneiodonium substantially reduced the optical signal. This reduction in the autofluorescence signal was accomplished without altering the presynaptic and postsynaptic components of the electrophysiological response. Results from reflectance imaging and blocking nitric oxide synthase demonstrated that the epifluorescence signal is not the result of changes in hemoglobin oxygenation or blood flow. This flavoprotein autofluorescence signal thus provides a powerful tool to monitor neuronal activity in vivo and its relationship to mitochondrial metabolism.

Differences in O2 availability resolve the apparent discrepancies in metabolic intrinsic optical signals in vivo and in vitro

“…An increase in fluorescence (oxidation) (uncorrected fluorescence recording) outlasted the stimulation. Other studies have shown biphasic stimulation-evoked responses of flavoprotein fluorescence in rat sensory cortex [54] and in mouse cerebellar cortex [55]. During stimulation, fluorescence briefly increased, indicating oxidation, and then decreased.…”

“…Weber et al [54] consider it plausible that the apparent delayed decrease in light intensity was in fact caused by interference by Hb. However, Reinert et al [55] report evidence that the delayed decrease in FAD fluorescence (reduction) after the stimulation was due neither to changes in blood flow nor to Hb oxygenation.…”

Initial Phase of Neuropathic Pain within a Few Hours after Nerve Injury in Mice

“…However, observation of the sensory modality shift at the spinal cord level might serve as a straightforward method for investigating neuropathic pain. Activity-dependent fluorescence changes derived from mitochondrial flavoproteins have been used in vivo for visualizing brain activities (Shibuki et al, 2003;Reinert et al, 2004). Cortical fluorescence changes can be imaged through the intact skull without surgical damage to the brain in mice (Tohmi et al, 2006;Kubota et al, 2008;Kitaura et al, 2010).…”

NAD(P)H Fluorescence Transients after Synaptic Activity in Brain Slices: Predominant Role of Mitochondrial Function

“…The time courses of the NAD(P)H and flavoprotein responses were of opposite sign and had very similar spatial and temporal characteristics (Shuttleworth et al, 2003) as would be expected if the signals were of mitochondrial origin, as initial phases may arise primarily from NADH and flavin adenine dinucleotide (reduced form; FADH 2 ) oxidation in the respiratory transport chain, followed by regeneration in the trichloroacetic acid (TCA) cycle, where they share obligatory coupling. The activity of TCA cycle enzymes can be stimulated by Ca 2 + elevations (McCormack and Denton, 1993;Rutter and Rizzuto, 2000) and mitochondrial Ca 2 + increases have been implicated as key contributors to NAD(P)H transients after neuronal stimulation (Duchen, 1992;Duchen and Biscoe, 1992;Kann et al, 2003;Reinert et al, 2004;Schuchmann et al, 2001). However, a significant portion of evoked responses appear resistant to Ca 2 + removal (Kann et al, 2003;Reinert et al, 2004;Shuttleworth et al, 2003), emphasizing the role that other mechanisms (including ATP depletion because of Na + extrusion; Lewis and Schuette, 1975) may play as possible triggers for mitochondrial metabolism.…”