ANR Blanc - BrainVasc Project 2011-2015

The relationship between blood flow and neuronal activity

It has been known for more than a century that the tight coupling between neuronal activity and regional cerebral blood flow (rCBF) is essential to the normal brain function (Roy and Sherrington, 1890). The increase in neuronal activity is associated with an increase of local perfusion, known as functional hyperemia. In most neuroimaging studies, activation of the brain with cognitive tasks or sensory stimuli results in a local functional hyperemia. The measure of local hyperemia is usually done by functional Magnetic Resonance Imaging (fMRI) a widely used technique that measures haemodynamic changes. In fMRI, the blood-oxygen-level-dependent (BOLD) signal resulting from the paramagnetic properties of deoxyhaemoglobin in red blood cells is measured with precise spacial and temporal resolution (Logothetis, 2008). The change in BOLD signal appears in the second following brain activation. In the last ten years, fMRI has become the method of choice in human studies although the precise mechanisms involved in the changes of local blood flow are still poorly understood. How activation of the brain transmutes in functional hyperemia is an important question whose answer could shed light on several pathology such as Alzheimer’s disease, migraine and stroke, neurovascular coupling is known to be impaired (Zlokovic, 2005).

How activation of the brain transmutes in functional hyperemia

Precise mechanisms involved in functional hyperemia are still poorly understood. At present, an intense debate among neuroscientists exists with two hypotheses, the “metabolic” and the “neurogenic” (Estrada and DeFelipe, 1998; Hamel, 2006). The metabolic hypothesis assumes a causal link between neuronal energy demand and the regulation of local cerebral blood flow. The general assumption, which is supported by PET findings showing comparable functional increases in blood flow and glucose uptake (Raichle and Mintun, 2006), is that CBF is coupled to regional glucose utilization, which in turn is directly related to neuronal activity (Magistretti, 2006). Excitatory neuronal activity releases glutamate which activates glia through metabotropic glutamate receptors. The activation of glial cells will induce at the same time an increase in the diameter of nearby blood vessels and increase of glucose uptake. In this metabolic hypothesis, the activity-dependent regulation of local CBF is a feedback mechanism that does not anticipate possible demand.

In contrast with this metabolic feedback hypothesis, the neurogenic hypothesis (Estrada and DeFelipe, 1998; Hamel, 2006) describes a feedforward mechanism where the hyperemia evoked by cerebral activation is linked to neuronal synaptic signalling rather than to the metabolic needs of the tissue. The neurogenic hypothesis was highlighted recently by Sirotin and Das (2009) who have found a large haemodynamic signal that could deliver arterial blood to the somatosensory cortex in anticipation of an increase of local neuronal activity. In the present project, we wish to highlight the importance of a network of subclasses of interneurons in controlling local blood flow. Our working hypothesis is more on the neurogenic side of the current dispute. In the neurovascular unit composed of neurons, glia and muscles we propose that interneurons containing vasoactive neuropeptides are key elements in guiding the blood vessel regulation.

Description and role of interneurons in the neocortex

Our laboratory has particularly described the intrinsic properties of neocortical interneurons. We have analysed their electrophysiological properties together with the detailed description of their morpholog. In addition, the expression of several molecular markers has been explored by combining these experiments with immunocytochemistry or with the single cell RT-PCR method pioneered in our laboratory (Lambolez et al. 1992). These approaches gave rise to a large dataset, from which emerged the description of distinct interneuron populations according to their electrophysiological, morphological and molecular characteristics. The last classification according to the Petilla 2005 nomenclature (Ascoli et al., 2008; Karagiannis et al., 2009) described in the somato-sensory cortex four classes of neocortical interneurons 1) the Fast spiking (FS) expressing the calcium binding protein parvalbumin (PV), 2) the Martinotti cells expressing somatostatin (SOM) and NPY, 3) the VIP bipolar and 4) the neurogliaform expressing NPY and NOS.

In 2004, our laboratory reported that activation of a single interneuron by patch-clamp was able and enough to induce changesin the diameter of the blood vessels localized in the vicinity of the stimulated neuron (see picture below from Cauli et al., 2004; Rancillac et al., 2006). The strongest evidence for the direct involvement of interneurons controlling blood vessels was obtained with three classes of interneurons co-expressing neuropeptides with GABA. VIP bipolar interneurons inducevasodilatation. SOM Martinotti and NPY-neurogliaform interneurons induce vasoconstriction. In the 2004 paper we described also that the neurogliaform interneurons express NOS and could induce vasodilation by release of NO. If the release of NO and NPY by these neurons is spatially or temporally differentially regulated, neurogliaform interneurons could play a key and essential role in local blood flow regulation. Morevover we have shown that neurogliaform interneurons are preferentially located in the close vicinity of blood vessels (Cauli et al., 2004).

VIP and neurogliaform interneurons have recently been studied in details by our group (Vucurovic et al., 2010). They have in common their embryonic origin: they are both generated around embryonic day 14.5 in the caudal ganglionic eminence. They both express the ionotropic 5-HT3 receptor that is not expressed in other neurons. We have obtained many additional experimental evidences for the involvement of these VIP and neurogliaform interneurons in the control of local blood vessels tonus, including experiments with optogenetics.

Why are we proposing that the local net of interneurons is regulating local blood flow ? Our current works on brain slices give us confidence that interneurons are indeed involved in blood flow control. Although we should stress that these “ex vivo” conditions are far away from an in vivo situation. Indeed in a slice preparation, the blood vessels have open ends and therefore lack intraluminal blood flow and pressure. In vascular research it is of paramount importance to work with pressurised blood vessels. In order to take into account such requirements, our brain slices are perfused with an optimal concentration of thromboxane agonist to induce a small contraction mimicking arterial muscle tone. In any case, brain slice preparations were very useful to get the seminal observation that interneurons control blood vessels diameter. Brain slices are of course lacking most of the three dimensional connectivity and therefore in this project we should delineate the roles of the various interneurons in intact brain.

Deciphering the neuronal circuitry controlling local blood flow in the cerebral cortex of rodents with optogenetics

The objective of the present proposal is to understand the functional role of specific subset of cortical interneuron in the neurovascular coupling. The physiological model used is the whisker to barrel sensory cortex in the mouse. In the present proposal we wish to study ensemble of similar interneurons in their original environment. Such an approach of studying an ensemble of similar interneurons intermingled a variety of other cells in the cortex of a living animal is highly original. It will take profit of the recent availability of transgenic Cre-driver mouse line expressing recombinase in a restricted cell population. This will be coupled with the use of recombinase-dependent viral vectors containing specific expression cassette for the new optogenetic tools.

Optogenetic transgenes are actuators and transduce optical signals into physiological signals; they make cellular function controllable. The actuators used in this proposal are ChETA an improved channelrhodopsin for activation and Archeorhodopsin (Arch) for inhibition.

The proposal will particularly study three classes of interneurons: the bipolar adapting VIP, the multipolar (neurogliaform) adapting NPY and the somatostatin (SOM) Martinotti interneurons expressing also NPY. In 2004, our laboratory reported that activation of a single interneuron was able to induce changes in the diameter of the blood vessels localized in the vicinity of the stimulated neuron. Since then we have obtained many additional experimental evidences for the involvement of interneurons in the control of local blood vessels tonus. Some of these unpublished experiments are displayed in this proposal. In brief in a slice preparation, pharmacological stimulation of subset of interneurons induces dilatation or contraction of penetrating arterioles, recorded in infrared videomicroscopy. Similar experiments have already been performed using optogenetics tools. Light stimulation of a slice preparation containing VIP interneurons expressing ChETA in VIP driver Cre-mice induces vasodilations.

To demonstrate the role of VIP interneurons in local blood flow control, the optogenetic silencer Arch will be used. Optic fibers will be implanted in the area where stimulation of whiskers increases in local blood flow. Then VIP interneurons will be silenced by light stimulation of Arch. In these conditions we expect that whisker stimulation would no more induce local change in blood perfusion. The same type of experiments will be repeated by targeting the other classes of interneurons as SOM Martinotti and NPY neurogliaform.

This study will define the role of interneurons in the control of vascular tone by combining ex vivo and in vivo experiments and contribute to a better understanding of the cellular and molecular basis of neurovascular coupling. In addition, this will give a new perspective to the interpretation of functional imaging. Finally this project could have an impact on the treatment of certain pathologies such as Alzheimer disease or migraine where neuronal and vascular deficits overlap.

Optogenetic stimulation of PV-expressing neurons elicits vasoconstriction of penetrating arterioles in the cortex

In the brain, neuronal activation triggers a local increase of cerebral blood flow (CBF) that is controlled bythe neurogliovascular unit composed of terminals of neurons, astrocytes and blood vessel muscles. It is generally accepted that the regulation is adjusted to local metabolic demand by local circuits. Today experimental data led us to realize that the regulatory mechanisms are more complex. Indeed, how to explain that a stimulus of the whisker pad in rodents is associated with local increase in the corresponding barrel field but also with decreases in the surrounding deeper area and in the opposite barrel field.

By combining both in vitro studies in acute slice and in vivo imaging in anesthetized rats, we demonstrated that neurovascular coupling is a process through which neuronal activity leads to increases of local CBF inthe activated areabut also to decreases in other brain regions. In order to explain these observations, we propose that a neuronal system within the brain is devoted to the control of local brain blood flow.

Expression of ChETA-EYFP in the cerebral cortex of mice. A. High resolution mosaic images of around 100 individual frames obtained from a 50 µm thick coronal section of PV::Cre mouse cerebral cortex after 14 days of rAAV infection. Level of expression in somatosensory cortex was controlled by fluorescence microscopy (EYFP filter). The red star indicates the injection site. TH: thalamus, fi: fimbria of hippocampus, vhc: ventral hippocampal commissure, CC: corpus callosum, GP: globus pallidus, IC: internal capsule, CPu: caudate putamen, GP: globus pallidus, S1FL: primary somatosensory forelimb cortex, S1BF: primary somatosensory barrel field cortex, S2: secondary somatosensory cortex, S1ULp: primary somatosensory upper limb cortex. B. Diagram of a sagittal brain slice showing the injection site in the S1BF of the transgenic mouse brain. C. The cell-type specificity of the viral expression was assayed using immunocytochemistry with an anti-parvalbumin antibody coupled to Alexa Fluor 568 (in red). EYFP expression was found to be largely restricted to parvalbumin expressing neurons (white arrow) in layer 2/3 of PV::Cre mouse somatosensory cortex.

Our optogenetic experiments revealed that neurons expressing the calcium binding protein parvalbumin could control local blood flow. We demonstrated that ChR2-based photostimulation of these cells give rise to an effective contraction of penetrating arterioles. These results therefore support the neurogenic hypothesis of a complex distributed nervous system controlling the CBF.

Vasoconstriction induced by optogenetic stimulation of PV expressing neurons. A. Infrared microscopy images of a blood vessel in the somatosensory cortex showing changes of the luminal diameter after light stimulation of PV-interneurons. Images of the microvessels were acquired every 15 s after a control period of 5 min. Arrows indicate region of high vascular reactivity.Note the accumulation of red bloodcells on both sides of the arrows in the right panel. B. Mean vascular constriction ± SEM (n = 5) induced by 2 min stimulation at 20 Hz (473 nm, 5 ms pulse width, 35 mW/mm²) in the layer II/III of the somatosensory cortex.

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