Modern functional imaging techniques of the brain measure local hemodynamic responses evoked by neuronal activity. Capillary pericytes recently were suggested to mediate neurovascular coupling in brain slices, but their role in vivo remains unexplored. We used two-photon microscopy to study in real time pericytes and the dynamic changes of capillary diameter and blood flow in the cortex of anesthetized mice, as well as in brain slices. The thromboxane A 2 analog, 9,11-dideoxy-9α,11α-methanoepoxy Prostaglandin F2α (U46619), induced constrictions in the vicinity of pericytes in a fraction of capillaries, whereas others dilated. The changes in vessel diameter resulted in changes in capillary red blood cell (RBC) flow. In contrast, during brief epochs of seizure activity elicited by local administration of the GABA A receptor antagonist, bicuculline, capillary RBC flow increased without pericyte-induced capillary diameter changes. Precapillary arterioles were the smallest vessels to dilate, together with penetrating and pial arterioles. Our results provide in vivo evidence that pericytes can modulate capillary blood flow in the brain, which may be important under pathological conditions. However, our data suggest that precapillary and penetrating arterioles, rather than pericytes in capillaries, are responsible for the blood flow increase induced by neural activity.cerebral blood flow | neurovascular coupling | cortical spreading depolarization | electrophysiology

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.

In the evolution of the cerebral cortex, the sophisticated organization in a steady state far away from thermodynamic equilibrium has produced the side effect of two fundamental pathological network events: ictal epileptic activity and spreading depolarization. Ictal epileptic activity describes the partial disruption, and spreading depolarization describes the near-complete disruption of the physiological double Gibbs–Donnan steady state. The occurrence of ictal epileptic activity in patients has been known for decades. Recently, unequivocal electrophysiological evidence has been found in patients that spreading depolarizations occur abundantly in stroke and brain trauma. The authors propose that the ion changes can be taken to estimate relative changes in Gibbs free energy from state to state. The calculations suggest that in transitions from the physiological state to ictal epileptic activity to spreading depolarization to death, the cortex releases Gibbs free energy in a stepwise fashion. Spreading depolarization thus appears as a twilight state close to death. Consistently, electrocorticographic recordings in the core of focal ischemia or after cardiac arrest display a smooth transition from the initial spreading depolarization component to the later ultraslow negative potential, which is assumed to reflect processes in cellular death.

Neurovascular coupling provides the basis for many functional neuroimaging techniques. Nitric oxide (NO), adenosine, cyclooxygenase, CYP450 epoxygenase, and potassium are involved in dilating arterioles during neuronal activation. We combined inhibition of NO synthase, cyclooxygenase, adenosine receptors, CYP450 epoxygenase, and inward rectifier potassium (Kir) channels to test whether these pathways could explain the blood flow response to neuronal activation. Cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO(2)) of the somatosensory cortex were measured during forepaw stimulation in 24 rats using a laser Doppler/spectroscopy probe through a cranial window. Combined inhibition reduced CBF responses by two-thirds, somatosensory evoked potentials and activation-induced CMRO(2) increases remained unchanged, and deoxy-hemoglobin (deoxy-Hb) response was abrogated. This shows that in the rat somatosensory cortex, one-third of the physiological blood flow increase is sufficient to prevent microcirculatory increase of deoxy-Hb concentration during neuronal activity. The large physiological CBF response is not necessary to support small changes in CMRO(2). We speculate that the CBF response safeguards substrate delivery during functional activation with a considerable 'safety factor'. Reduction of the CBF response in pathological states may abolish the BOLD-fMRI signal, without affecting underlying neuronal activity.

Recently, a universal, simple, and fail-safe mechanism has been proposed by which cerebral blood flow (CBF) might be coupled to oxygen metabolism during neuronal activation without the need for any tissue-based mechanism. According to this concept, vasodilation occurs by local erythrocytic release of nitric oxide or ATP wherever and whenever hemoglobin is deoxygenated, directly matching oxygen demand and supply in every tissue. For neurovascular coupling in the brain, we present experimental evidence challenging this view by applying an experimental regime operating without deoxy-hemoglobin. Hyperbaric hyperoxygenation (HBO) allowed us to prevent hemoglobin deoxygenation, as the oxygen that was physically dissolved in the tissue was sufficient to support oxidative metabolism. Regional CBF and regional cerebral blood oxygenation were measured using a cranial window preparation in anesthetized rats. Hemodynamic and neuronal responses to electrical forepaw stimulation or cortical spreading depression (CSD) were analyzed under normobaric normoxia and during HBO up to 4 ATA (standard atmospheres absolute). Inconsistent with the proposed mechanism, during HBO, CBF responses to functional activation or CSD were unchanged. Our results show that activation-induced CBF regulation in the brain does not operate through the release of vasoactive mediators on hemoglobin deoxygenation or through a tissue-based oxygen-sensing mechanism.