Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC57
Brain microcirculation is regulated by neuronal-derived nitric oxide: real time and in vivo demonstration
J. Laranjinha1, C. F. Lourenço2, E. Cadenas3, R. Radi4, R. M. Barbosa5
1. Center for Neuroscience and Cell Biology and Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal. 2. Center for Neuroscience and Cell Biology and Faculty of Pharmacy, University of Coimbra,, Coimbra, Portugal. 3. School of Pharmacy, University of Southern California, Los Angeles, California, United States. 4. Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Republica, Montevideo, Uruguay. 5. Center for Neuroscience and Cell Biology and Faculty of Pharmacy, University of Coimbra,, Coimbra, Portugal.
The neurovascular coupling (NVC) process establishes a functional bridge between active neurons and local blood flow, hence providing working neurons with energy substrates, glucose and oxygen. Although it is accepted that glutamate stimulus is a required trigger, the mechanistic details of such a process have remained controversial, largely because of experimental difficulties in addressing the problem in a real time, quantitative and dynamic fashion in vivo. Previous published evidences suggest that increased CBF occurs following glutamate NMDA receptor activation and that nitric oxide (NO) is a likely candidate to mediate NVC. Nitric oxide is produced upon glutamate-dependent neuronal nitric oxide synthase (nNOS) activation and is able to diffuse, generating a volume signaling that affects a large population of neighboring cells. Using a tri-component microsensor array with a versatile geometry and consisting of NO-selective microelectrode, an ejection pipette and a laser Doppler sensor inserted stereotaxically in the brain of anesthetized Wistar rat (via an intraperitoneal injection of urethane 1.25 g/kg) we show that a transient increase in NO production induced by an ejection 0.5 nmol glutamate (25 nL) during 1 s in a defined area of hippocampus, is followed, seconds later, by a transient change in cerebral blood flow (CBF). NO may peak at about 1 μM with a time rise of 22 ± 3 s and a total duration of 64 ± 4 s (15 individual experiments). The CBF increased 7 ± 2 s after stimulation reaching 122 ± 5% of the basal level after 62 ± 3 s and returning to basal levels after 216 ± 15 s. Blocking the NMDA receptor (MK-801 (1 mg/kg) elicited both an inhibition of NO production (73 ± 8%, p = 0.0017, n = 3) and a decrease of glutamate-induced CBF (74 ± 3%, p = 0.0008, n = 3). Similarly, the inhibition of the neuronal isoform of NOS by 7-NI (50 mg/Kg) induced an inhibition of both, NO and CBF responses to glutamate (62 ± 10 and 83 ± 6%, p = 0.0002 and p = 0.0025, respectively, n = 4). A local and transitory elevation of O2 tension was observed following the increase in CBF In summary, we have shown in vivo in rat brain and in a real-time and dynamic fashion that : 1) upon stimulation with glutamate, a transient increase in NO concentration is observed, 2) the dynamics of NO signal precedes that of CBF change which, in turn is followed by a transient increase in O2 tension, 3) both, NO, blood flow changes, may be pharmacologically modulated and are coupled in terms of time, space and amplitude. Thus, neurovascular coupling is mediated by nNOS-derived NO via a diffusional wireless connection between active glutamatergic neurons and blood vessels.
Where applicable, experiments conform with Society ethical requirements