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NEUROVASCULAR COUPLING IN THE CEREBELLAR GRANULAR LAYER

Giuseppe Gagliano1*, Lisa Mapelli1, 2*, Teresa Soda1, 2, Umberto Laforenza3, Francesco Moccia4 and Egidio D‘Angelo1, 5*
  • 1 University of Pavia, Dept. of Brain and Behavioral Sciences, Italy
  • 2 Museo Storico della Fisica e Centro Studi e Ricerche Enrico Fermi, Italy
  • 3 University of Pavia, Dept. of Molecular Medicine, Italy
  • 4 University of Pavia, Dept. of Biology and Biotechnology "L.Spallanzani", Italy
  • 5 Brain Connectivity Center, C. Mondino National Neurological Institute, IRCCS, Italy

INTRODUCTION AND METHODS The tight coupling between neuronal activity and cerebral blood flow (CBF) is called neurovascular coupling (NVC). This phenomenon controls blood vessel diameter to ensure the proper supply of oxygen and nutrients to the brain and contributes to generate the BOLD (blood-oxygenation-level-dependent) signals in functional magnetic resonance imaging (fMRI).The NVC has been investigated in several brain regions, but its neuronal drive and biochemical pathways in the cerebellum are still unclear. In particular, attention has been mostly given to the cerebellar molecular layer components, as parallel fibers (Bouvier, 2016), local interneurons (Akgoren, 1994), and Purkinje cells (where NVC was found dissociated from spiking activity, Thomsen, 2004). This may be due to the inability of these cells to release nitric oxide (NO), a well known vasoactive agent. Surprisingly enough, there is no information about the role of the granular layer in this phenomenon, even though granule cells (GrCs): i) are the most abundant brain neurons and the most energy consuming elements in the cerebellum (Howarth, 2014), ii) show a high expression of NMDA receptors (NMDARs) (Monaghan and Anderson, 1991) and of the neural isoform of nitric oxide synthase (nNOS) (Southam, 1992), and iii) produce and release NO following high frequency mossy fibers (MFs) stimulation (Maffei, 2003). NO is also implicated in long-term synaptic plasticity at the MF-GrC connection in the cerebellar granular layer (D'Angelo, 2014). Therefore, the granular layer is particularly suitable for the study of NVC mechanisms and it was then compelling to investigate its role in cerebellar NVC. At first, we described the vascular organization of the rat cerebellar cortex in immunostained slices. Secondly, we investigated whether and how synaptic activity was coupled to vascular motility in the granular layer, by combining bright-field microscopy an NO-related imaging techniques. We focused our attention on MF-GrC synapses (the cerebellar input stage) and on capillaries, since these vessels are able to change their lumen diameter earlier than upstream arterioles, in response to neuronal activity and following pericytes activation (Hall, 2014). RESULTS In immunostained cerebellar slices, the molecular layer showed a more regular vascular architecture compared to the granular layer, with arterioles originating from the surface of the lamella, penetrating deep into the layer and giving off capillaries (Fig.1A). In the granular layer, these capillaries (inner diameter 4.23±0.29 μm, n=26) are surrounded by GrCs and are in close contact with pericytes (Fig. 1B, arrow). In slices treated with the thromboxane A2 agonist (U46619; 75nM) to restore the vascular tone, MFs stimulation (35s at 50Hz, 15V) induced a rapid initial vasodilation followed by a slower increase in lumen diameter at pericytes location (10.89±2.35%, n=13; p=0.00006) (Fig. 2). The vasodilation was converted into vasoconstriction in the presence of NMDARs and NOS inhibitors (respectively 100μM D-APV+50μM 7-ClKyn; -5.9±1.8%, n=7; p=0.02 and 200μM L-NAME; -9.0±1.4%, n=9; p=0.000004) (Fig. 3A-B) and partially turned into vasoconstriction in presence of guanylyl cyclase (sGC) inhibitor (10μM ODQ; -3.6±0.9%; n=10; p=0.005) (Fig. 3C). Moreover, L-NAME perfusion alone reduced capillary diameter (-55.7±2.8%; n=8; p=0.004) (Fig. 3b inset), reflecting a tonic NO release by vascular endothelial cells. In the presence of TTX (4μM), the stimulation failed to cause changes in capillary diameter confirming the synaptic drive of this process (Fig. 2). Therefore, MF-GrC synapses activation, and NMDARs, NOS and sGC recruitments are necessary to increase the local CBF. To further investigate this pathways, slices were pre-incubated with 12 μM of the NO production-related fluorescent dye DAF-FM, The granular layer responded to MFs stimulation with a fluorescence peak (0.029±0.003ΔF/F0) (Fig. 4A) which was absent in the presence of NMDARs and NOS inhibitors (respectively 100μM D-APV+50μM 7-ClKyn; -0.007±0.006ΔF/F0*ms; n=6 and 200μM L-NAME; 0.001±0.003 ΔF/F0*ms; n=7) (Fig. 4B, top). Moreover, in control condition the integral of the fluorescence signal showed a trend to increase, suggesting a residual NO production in the granular layer, (Fig. 4A, red trace) that was no longer observed in the presence of NMDARs and NOS blockers (Fig 4B, bottom). Moreover, the distance from the stimulating electrode where fluorescence peaks were observed was comparable to the distance where vessels showed the vasoactive response (158±10μm vs 191±10μm; n=20 and n=50, respectively). Interestingly, the fluorescence signal kinetics and the linear fitting of the vessel dilation rate showed a similar trend (p=0.37), allowing to speculate that the kinetics of vessel dilation might be dictated by the rate of NO production. Besides promoting vasodilation, NO released by granular layer neurons could block the synthesis of the vasoconstrictor 20-HETE (Attwell, 2010). MFs stimulation during the perfusion of 20-HETE synthesis inhibitor (1μM HET-0016) and of metabotropic glutamate receptors (mGluRs) inhibitors (500μM MCPG+300 μM CPPG) respectively reduced (-4.2±1.9%; n=9; p<0.007) (Fig. 5A) and abolished (1.1 ± 1.5%; n=15; p=0.9) (Fig. 5B) the vasoconstriction shown in the presence of L-NAME. These data support the conclusion that mGluRs drove the synthesis of 20-HETE during synaptic transmission at the MF-GrC relay. DISCUSSION AND CONCLUSIONS Herein, we demonstrated that synaptic activation of the cerebellar granular layer caused a NMDARs/NOS-dependent capillary vasodilation that required cGMP synthesis by sGC, presumably in pericytes. Our results are also compatible with a role of glial cells in mediating the vasoconstriction through a mGluRs-dependent mechanism leading to 20-HETE synthesis. Actually, in pericytes 20-HETE is synthesized from the arachidonic acid released after mGluRs activation on astrocytes (Sweeney, 2016). Therefore, synaptic activity and glutamate release could activate two different competing signaling pathways (promoting either vasorelaxation or vasoconstriction), both involved in the CBF control. In conclusion, GrCs are likely to play a pivotal role in the NVC in the cerebellar circuit. These cells are also known to elaborate and store complex spatio-temporal patterns at the cerebellar input stage (D’Angelo and De Zeeuw, 2009). Taken together, these evidences strongly stand for a huge role of GrCs in the coordination of computational and metabolic functions in the cerebellar network. Notably, since the granular layer regulation of local microvessels caliber might contribute to change the cerebellar BOLD signals (Diedrichsen, 2010), our results may help cerebellar fMRI data analysis, besides shedding new light on the mechanisms of NVC in this brain structure.

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Acknowledgements

This work was supported by: European Union grant Human Brain Project (HBP-604102) to ED and Fermi grant [13(14)] to ED and LM. The authors declare no competing financial interests.

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Keywords: neurovascular coupling, Cerebellum, granule cells, Capillaries, Glutamate, NMDA receptor, Nitric Oxide

Conference: The Cerebellum inside out: cells, circuits and functions , ERICE (Trapani), Italy, 1 Dec - 5 Dec, 2016.

Presentation Type: poster

Topic: Cellular & Molecular Neuroscience

Citation: Gagliano G, Mapelli L, Soda T, Laforenza U, Moccia F and D‘Angelo E (2019). NEUROVASCULAR COUPLING IN THE CEREBELLAR GRANULAR LAYER. Conference Abstract: The Cerebellum inside out: cells, circuits and functions . doi: 10.3389/conf.fncel.2017.37.000025

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Received: 29 Nov 2016; Published Online: 25 Jan 2019.

* Correspondence:
Mr. Giuseppe Gagliano, University of Pavia, Dept. of Brain and Behavioral Sciences, Pavia, Italy, giuseppe.gagliano02@universitadipavia.it
Dr. Lisa Mapelli, University of Pavia, Dept. of Brain and Behavioral Sciences, Pavia, Italy, lisa.mapelli@unipv.it
Prof. Egidio D‘Angelo, University of Pavia, Dept. of Brain and Behavioral Sciences, Pavia, Italy, egidiougo.dangelo@unipv.it