Simultaneous monitoring of tissue P2 and NADH fluorescence during synaptic stimulation and spreading depression reveals a transient dissociation between oxygen utilization and mitochondrial redox state in rat hippocampal slices

Nicotinamide adenine dinucleotide (NADH) imaging can be used to monitor neuronal activation and ascertain mitochondrial dysfunction, for example during hypoxia. During neuronal stimulation in vitro, NADH normally becomes more oxidized, indicating enhanced oxygen utilization. A subsequent NADH overshoot during activation or on recovery remains controversial and reflects either increased metabolic activity or limited oxygen availability. Tissue P2 measurements, obtained simultaneously with NADH imaging in area CA1 in hippocampal slices, reveal that during prolonged train stimulation (ST) in 95% O2, a persistent NADH oxidation is coupled with increased metabolic demand and oxygen utilization, for the duration of the stimulation. However, under conditions of either decreased oxygen supply (ST-50% O2) or enhanced metabolic demand (K+-induced spreading depression (K+-SD) 95% O2) the NADH oxidation is brief and the redox balance shifts early toward reduction, leading to a prolonged NADH overshoot. Yet, oxygen utilization remains elevated and is correlated with metabolic demand. Under these conditions, it appears that the rate of NAD+ reduction may transiently exceed oxidation, to maintain an adequate oxygen flux and ATP production. In contrast, during SD in 50% O2, the oxygen levels dropped to a point at which oxidative metabolism in the electron transport chain is limited and the rate of utilization declined

Coupling and uncoupling of activity-dependent increases of neuronal activity and blood flow in rat somatosensory cortex

Electrical stimulation of the infraorbital nerve was used to examine the coupling between neuronal activity and cerebral blood flow (CBF) in rat somatosensory cortex by laser Doppler flowmetry and extracellular recordings of field potentials.The relationship between field potential (FP) and CBF amplitudes was examined as a function of the stimulus intensity (0–2.0 mA) at fixed frequency (3 Hz). FP amplitudes up to 2.0–2.5 mV were unaccompanied by increases of CBF. Above this threshold, CBF and FP amplitudes increased proportionally.At fixed stimulus intensity of 1.5 mA, CBF increases were highly correlated to FP amplitudes at low frequencies of stimulation (< 2 Hz), but uncoupling was observed at stimulation frequencies of 2–5 Hz. The evoked responses were independent of stimulus duration (8–32 s).The first rise in CBF occurred within the first 0.2 s after onset of stimulation in the upper 0–250 μm of the cortex. Latencies were longer (1.0–1.2 s) in lower cortical layers in which CBF and FP amplitudes were larger.Local AMPA receptor blockade attenuated CBF and FP amplitudes proportionally.This study showed that activity-dependent increases in neuronal activity and CBF were linearly coupled under defined conditions, but neuronal activity was well developed before CBF started to increase. Consequently, the absence of a rise in CBF does not exclude the presence of significant neuronal activity. The CBF increase in upper cortical layers preceded the rise in lower layers suggesting that vessels close to or at the brain surface are the first to react to neuronal activity. The activity-dependent rise in CBF was explained by postsynaptic activity in glutamatergic neurons

Modification of activity-dependent increases in cerebellar blood flow by extracellular potassium in anaesthetized rats

The hypothesis that potassium ions mediate activity-dependent increases of cerebral blood flow was examined in rat cerebellar cortex using ion-selective microelectrodes and laser-Doppler flowmetry. Increases of cerebellar blood flow (CeBF) and extracellular potassium concentration ([K+]o) were evoked by stimulation of parallel fibres and climbing fibres, and by microinjection of KCl into the cortex.For parallel fibre stimulation, there was a maximal increase in [K+]o to 6.3 ± 0.5 mm and in CeBF of 122 ± 11%. Climbing fibre stimulation gave a maximal increase in [K+]o to 4.4 ± 0.2 mm and in CeBF of 157 ± 20%. This indicates different maxima for [K+]o and CeBF, dependent on the afferent system activated.[K+]o and CeBF responses evoked by parallel or climbing fibre stimulation increased rapidly at the onset of stimulation, but exhibited different time courses during the remainder of the stimulation period and during return to baseline.Microinjections of KCl into the cortex increased [K+]o to levels comparable to those evoked by parallel fibre stimulation. The corresponding CeBF increases were the same as, or smaller than, for parallel fibre stimulation, and much smaller than for climbing fibre stimulation. This suggests that mediators other than [K+]o are important for activity-dependent cerebral blood flow increases.The present study showed that increased [K+]o is involved in CeBF regulation in the parallel fibre system, but is of limited importance for CeBF regulation in the climbing fibre system. The hypothesis that K+ is a major mediator of activity-dependent blood flow increases is probably not generally applicable to all brain regions and all types of neuronal stimulation