Stellate cells release NO and induce vasodilation of intraparenchymal blood vessels in rat cerebellar slices

Introduction

The tight coupling between neuronal activity and local perfusion, known as functional hyperemia, is central to normal brain function. In the cerebellum functional hyperemia depends almost exclusively on nitric oxide (NO), a potent vasodilator. The neuronal isoform of nitric oxide synthase (nNOS), a NO synthesizing enzyme, is expressed by different neuronal types in the cerebellum, among which stellate cells are suspected to be critical for neurovascular coupling.

Methods

To demonstrate this functional role, we applied the NO donor diethylamine NONOate (DEA-NONOate) in rat cerebellar acute slices. NO was detected by amperometry with a platinized carbon NO probe (poised at E = 650 mV vs Ag/AgCl) placed in the molecular layer. Cerebellar microvessel diameter changes were monitored by infrared videomicroscopy. Stellate cells characterization was achieved by patch-clamp recordings and single-cell reverse transcriptase-multiplex PCR (RT-mPCR), and their associations with responsive microvessels identified by confocal microscopy following biocytin labeling and immunodetection of blood vessel laminin.

Results

Bath application of DEA-NONOate (100 μM) produced NO flux (84.8 107 molecules/s) and vasodilation (86%) of intraparenchymal blood vessels. Similarly, neuronal stimulation with NMDA (100 μM) induced NO flux (99.8 106 molecules/s) and microvessel vasodilatations (42%) that were completely abolished by TTX (1 μM) or by L-NAME (1 mM), a NOS inhibitor. In patch clamp recordings the evoked firing of single stellate cells also induced NO flux (22.5 106 molecules/s) and dilatation (10%) of neighboring intraparenchymal microvessels. Molecular and morphological characterization of stimulated stellate cells by single cell RT-PCR and confocal microcopy revealed the expression nNOS mRNAs and the association of stellate cell neurites with responsive blood vessels.

Conclusions

These ex vivo results confirm a functional role of stellate cells in neurovascular coupling mediated by NO release. Further, these data demonstrate that single interneurons activation is sufficient to alter the tone of local microvessels, emphasizing their role in the regulation of local perfusion.