Mechanisms of activity‐dependent increases in cerebral blood flow (CBF) were examined in rat cerebellar cortex using the laser Doppler flow technique and extracellular recordings of single unit activity and field potentials. Stimulation of the monosynaptic climbing fibre system evoked long‐lasting complex spikes in Purkinje cells, and extracellular field potentials with a characteristic profile that indicated contributions from both passive and active membrane mechanisms. The concomitant CBF increases were reproducible at fairly short intervals, and suggest that both synaptic activity and spikes may contribute to increased CBF. Stimulation of the disynaptic parallel fibre system inhibited the spiking activity in Purkinje cells, while the postsynaptic activity increased as indicated by the simultaneously recorded field potential. Nevertheless, CBF always increased. The inhibition of spike firing activity was partly dependent on GABAergic transmission, but may also relate to the intrinsic membrane properties of Purkinje cells. The CBF increases evoked by parallel or climbing fibre stimulation were highly correlated to the sum of neural activities, i.e. the negativity of field potentials multiplied by the stimulus frequency. This suggests a robust link between extracellular current flow and activity‐dependent increases in CBF. AMPA receptor blockade attenuated CBF increases and field potential amplitudes, while NMDA receptor antagonism did not. This is consistent with the idea that the CBF responses are of neuronal origin. This study has shown that activity‐dependent CBF increases evoked by stimulation of cerebellar parallel fibres are dependent on synaptic excitation, including excitation of inhibitory interneurones, whereas the net activity of Purkinje cells, the principal neurones of the cerebellar cortex, is unimportant for the vascular response. For the climbing fibre system, not only synaptic activity but also the generation of complex spikes from Purkinje cells contribute to the increases in CBF. The strong correlation between CBF and field potential amplitudes suggests that extracellular ion fluxes contribute to the coupling of brain activity to blood flow.

Functional neuroimaging relies on the robust coupling between neuronal activity, metabolism and cerebral blood flow (CBF), but the physiological basis of the neuroimaging signals is still poorly understood. We examined the mechanisms of activity-dependent changes in tissue oxygenation in relation to variations in CBF responses and postsynaptic activity in rat cerebellar cortex. To increase synaptic activity we stimulated the monosynaptic, glutamatergic climbing fibres that excite Purkinje cells via AMPA receptors. We used local field potentials to indicate synaptic activity, and recorded tissue oxygen partial pressure (P tiss,O 2 ) by polarographic microelectrodes, and CBF using laser-Doppler flowmetry. The disappearance rate of oxygen in the tissue increased linearly with synaptic activity. This indicated that, without a threshold, oxygen consumption increased as a linear function of synaptic activity. The reduction in P tiss,O 2 preceded the rise in CBF. The time integral (area) of the negative P tiss,O 2 response increased non-linearly showing saturation at high levels of synaptic activity, concomitant with a steep rise in CBF. This was accompanied by a positive change in P tiss,O 2 . Neuronal nitric oxide synthase inhibition enhanced the initial negative P tiss,O 2 response ('dip'), while attenuating the evoked CBF increase and positive P tiss,O 2 response equally. This indicates that increases in CBF counteract activity-induced reductions in P tiss,O 2 , and suggests the presence of a tissue oxygen reserve. The changes in P tiss,O 2 and CBF were strongly attenuated by AMPA receptor blockade. Our findings suggest an inverse relationship between negative P tiss,O 2 and CBF responses, and provide direct in vivo evidence for a tight coupling between activity in postsynaptic AMPA receptors and cerebellar oxygen consumption.

Neuronal activity is tightly coupled with brain energy metabolism. Numerous studies have suggested that lactate is equally important as an energy substrate for neurons as glucose. Lactate production is reportedly triggered by glutamate uptake, and independent of glutamate receptor activation. Here we show that climbing fibre stimulation of cerebellar Purkinje cells increased extracellular lactate by 30% within 30 s of stimulation, but not for briefer stimulation periods. To explore whether lactate production was controlled by pre-or postsynaptic events we silenced AMPA receptors with CNQX. This blocked all evoked rises in postsynaptic activity, blood flow, and glucose and oxygen consumption. CNQX also abolished rises in lactate concomitantly with marked reduction in postsynaptic currents. Rises in lactate were unaffected by inhibition of glycogen phosphorylase, suggesting that lactate production was independent of glycogen breakdown. Stimulated lactate production in cerebellum is derived directly from glucose uptake, and coupled to neuronal activity via AMPA receptor activation.

In a published paper we have provided two categories of information about the relationship between neuronal activity and functional increases in regional cerebellar blood flow (CeBF). First, we showed that activity-dependent increases in CeBF were not necessarily linked to increased spiking activity in the principal neurones of the region studied (Mathiesen et al. 1998). Second, a strong correlation was found between the maximal amplitude of the recorded field potentials and the maximal increase in CeBF (Mathiesen et al. 1998). The different time courses of the electrophysiological and vascular responses during activation precluded the existence of a simple relationship (Mathiesen et al. 1998): the CeBF response developed over tens of seconds while the electrophysiological response developed within milliseconds. The present study therefore examined the temporal correlation between the observed CeBF increases and neuronal activity. The hypothesis was that if the increases in CeBF were temporally coupled, albeit indirectly to neuronal activity, then it would be possible to model the time course of the vascular response by integrating the neuronal activity over time during all phases of the vascular response. The simplest mathematical approach to integrate neuronal activity over time was to obtain a running summation of the amplitudes of the evoked field potentials (runÓFP). The rat cerebellar cortex was used as a model since this brain region cannot generate the epilepsy that is common after stimulation of the cerebral cortex. The basic circuitry of the cerebellar cortex is organised around the Purkinje cells from which the final and only output from the cerebellar cortex originates (Fig. 1 and Eccles et al. 1967). The activity of Purkinje cells is influenced by two excitatory

Functional neuroimaging relies on the robust coupling between neuronal activity, metabolism and cerebral blood flow (CBF) to map the brain, but the physiological basis of the neuroimaging signals is still not well understood. Here we applied a pharmacological approach to separate spiking activity, synaptic activity, and the accompanying changes in CBF in rat cerebellar cortex. We report that tonic synaptic inhibition achieved by topical application of gamma-aminobutyric acid type A (GABAA) (muscimol) or GABAB (baclofen) receptor agonists abolished or reduced spontaneous Purkinje cell spiking activity without affecting basal CBF. The magnitude of CBF responses evoked by climbing fiber stimulation decreased gradually over time after exposure to muscimol, being more pronounced in the superficial than in the deep cortical layers. We provide direct evidence in favor of a laminar-specific regulation of CBF in deep cortical layers, independent of dilatation of surface vessels. With prolonged exposure to muscimol, activity-dependent CBF increments disappeared, despite preserved cerebrovascular reactivity to adenosine and preserved local field potentials (LFP). This dissociation of CBF and LFPs suggests that CBF responses are independent of extracellular synaptic currents that generate LFPs. Our work implies that neuronal and vascular signals evoked by glutamatergic pathways are sensitive to synaptic inhibition, and that local mechanisms independent of transmembrane synaptic currents adjust flow to synaptic activity in distinct cortical layers. Our results provide fundamental insights into the functional regulation of blood flow, showing important interference of GABAA receptors in translating excitatory input into blood flow responses.

Evoked neural activity correlates strongly with rises in cerebral metabolic rate of oxygen (CMRO 2 ) and cerebral blood flow (CBF). Activity-dependent rises in CMRO 2 fluctuate with ATP turnover due to ion pumping. In vitro studies suggest that increases in cytosolic Ca 2ϩ stimulate oxidative metabolism via mitochondrial signaling, but whether this also occurs in the intact brain is unknown. Here we applied a pharmacological approach to dissect the effects of ionic currents and cytosolic Ca 2ϩ rises of neuronal origin on activitydependent rises in CMRO 2 . We used two-photon microscopy and current source density analysis to study real-time Ca 2ϩ dynamics and transmembrane ionic currents in relation to CMRO 2 in the mouse cerebellar cortex in vivo. We report a direct correlation between CMRO 2 and summed (i.e., the sum of excitatory, negative currents during the whole stimulation period) field EPSCs (ΑfEPSCs) in Purkinje cells (PCs) in response to stimulation of the climbing fiber (CF) pathway. Blocking stimulus-evoked rises in cytosolic Ca 2ϩ in PCs with the P/Q-type channel blocker -agatoxin-IVA (-AGA), or the GABA A receptor agonist muscimol, did not lead to a time-locked reduction in CMRO 2 , and excitatory synaptic or action potential currents. During stimulation, neither -AGA or (-oxo)-bis-(transformatotetramine-ruthenium) (Ru360), a mitochondrial Ca 2ϩ uniporter inhibitor, affected the ratio of CMRO 2 to fEPSCs or evoked local field potentials. However, baseline CBF and CMRO 2 decreased gradually with Ru360. Our data suggest that in vivo activity-dependent rises in CMRO 2 are correlated with synaptic currents and postsynaptic spiking in PCs. Our study did not reveal a unique role of neuronal cytosolic Ca 2ϩ signals in controlling CMRO 2 increases during CF stimulation.

Functional neuroimaging in humans is used widely to study brain function in relation to human disease and cognition. The neural basis of neuroimaging signals is probably synaptic activity, but the effect of context, defined as the interaction between synaptic inhibition, excitation, and the electroresponsive properties of the targeted neurons, is not well understood. We examined here the effect of interaction of synaptic excitation and net inhibition on the relationship between electrical activity and vascular signals in the cerebellar cortex. We show that stimulation of the net inhibitory parallel fibers simultaneously with stimulation of the excitatory climbing fibers leads to a further rise in total local field potentials (LFP) and cerebral blood flow (CBF) amplitudes, not a decrease, as predicted from theoretical studies. However, the combined stimulation of the parallel and climbing fiber systems produced changes in CBF and LFP that were smaller than their algebraic sum evoked by separate stimulation of either system. This finding was independent of the starting condition, i.e., whether inhibition was superimposed on a state of excitation or vice versa. The attenuation of the increases in LFP and CBF amplitudes was similar, suggesting that synaptic activity and CBF were coupled under these conditions. The result might be explained by a relative neuronal refractoriness that relates to the intrinsic membrane properties of Purkinje cells, which determine the recovery time of these cells. Our work implies that neuronal and vascular signals are context-sensitive and that their amplitudes are modulated by the electroresponsive properties of the targeted neurons.

In the awake brain, the global metabolic rate of oxygen consumption is largely constant, while variations exist between regions dependent on the ongoing activity. This suggests that control mechanisms related to activity, that is, neuronal signaling, may redistribute metabolism in favor of active networks. This study examined the influence of gamma-aminobutyric acid (GABA) tone on local increases in cerebellar metabolic rate of oxygen (CeMR(O(2))) evoked by stimulation of the excitatory, glutamatergic climbing fiber-Purkinje cell synapse in rat cerebellum. In this network, the postsynaptic depolarization produced by synaptic excitation is preserved despite variations in GABAergic tone. Climbing fiber stimulation induced frequency-dependent increases in synaptic activity and CeMR(O(2)) under control conditions. Topical application of the GABA(A) receptor agonist muscimol blocked the increase in CeMR(O(2)) evoked by synaptic excitation concomitant with attenuation of cerebellar blood flow (CeBF) responses. The effect was reversed by the GABA(A) receptor antagonist bicuculline, which also reversed the effect of muscimol on synaptic activity and CeBF. Climbing fiber stimulation during bicuculline application alone produced a delayed undershoot in CeBF concomitant with a prolonged rise in CeMR(O(2)). The findings are consistent with the hypothesis that activity-dependent rises in CeBF and CeMR(O(2)) are controlled by a common feed-forward pathway and provide evidence for modification of cerebral blood flow and CMR(O(2)) by GABA.