Neurovascular relationships in hippocampal slices: Physiological and anatomical studies of mechanisms underlying flow-metabolism coupling in intraparenchymal microvessels

Experiments were carried out to investigate the functional and anatomical r elationships between neuronal elements and cerebral microvessels in 300-350 -mu m thick coronal hippocampal slices maintained at 33-35 degrees C, obtai ned from 150-200 g male Wistar rats. Cerebral arterioles (9-22 mu m in diam eter) were visualized in situ and pre-constricted by 22.0 +/- 6.6% by the a ddition of the thromboxane AZ agonist U46619 (75 nM), to the bathing medium . The glutamate agonist N-methyl-D-aspartate (0.01-1 mM) produced a dose-re lated increase in luminal diameter of pre-constricted vessels. In the prese nce of 4 mu M haemoglobin to scavenge nitric oxide from the extravascular e nvironment of the slice, the increase in diameter evoked by 0.1 mM N-methyl -D-aspartate was significantly reduced from 17.5 +/- 4.6% to 4.8 +/- 1.7% i ndicating that N-methyl-D-aspartate-induced vasodilatation of cerebral micr ovessels is mediated via a mechanism which involves neuronally-derived nitr ic oxide. In a parallel anatomical study, P-nicotinamide adenine dinucleoti de phosphate-dependent diaphorase staining was used to reveal the enzyme ni tric oxide synthase in vascular endothelium and neurons in slices. A small subpopulation (<11 cells per slice) of darkly-stained multipolar neurons, 2 1-32 mu m in diameter was observed to give rise to a dense network of fine diaphorase-reactive nerve fibres that ramified throughout the whole of the hippocampus and appeared to come into close apposition with arterioles. Mor phometric analysis of the relationship between cerebral microvessels, P-nic otinamide adenine dinucleotide phosphate, reduced form-dependent diaphorase -reactive neuronal elements and individual pyramidal layer neurons, identif ied by filling with biocytin, revealed that for a given point on a pyramida l layer neuron, the proximity of the nearest diaphorase-reactive nerve fibr e was less than 10 mu m, whilst the distance to the nearest arteriole (the smallest functional unit for controlling blood flow) was in excess of 70 mu m Such a distance would probably preclude diffusion of vasoactive metaboli tes in effective concentrations from the area of increased neuronal activit y. We therefore propose that the diaphorase-reactive nerve network constitu tes the functional link. It is possible that during periods of increased ne uronal activity, spillover of glutamate from synapses may activate the diap horase-reactive network. Release of nitric oxide from the network in the vi cinity of local cerebral arterioles may then produce relaxation of the vasc ular smooth muscle, enabling increased blood flow into the capillary networ k supplying the region of increased metabolic activity. This study has shown that the process whereby increases in neuronal activit y elicit a local change in cerebral blood flow remains functionally intact in hippocampal slice preparations. Nitric oxide of neuronal origin appears to be involved in mediating the coupling between neurons and cerebral arter ioles. Stereological analysis of the relationship between neuronal and vasc ular elements within hippocampal slices suggested that a small subpopulatio n of nitric oxide synthase-containing neurons which give rise to a diffuse network of fine nitric oxide synthase-containing nerve fibres that lie in c lose apposition to cerebral arterioles may provide the anatomical substrate for coupling of blood flow to metabolism. (C) 1999 IBRO. Published by Else vier Science Ltd.