Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and mediates diverse functions including sensory and motor processing, as well as learning and memory. The energeticdemandofthisexcitatoryactivityismetbyalocalizedincreaseinbloodflow.Althoughthisincrease inbloodflowisimportanttosupporttheenergydemandsofneuraltissueandrepresentsthebasisofthesignal monitoredwithfunctionalmagneticresonanceimaging(fMRI),themechanism(s)thatunderliethiseffectremain unresolved. Unlike other classical neurotransmitters, that are directly recycled into the presynaptic nerve terminal, most glutamate is cleared into astrocytes. This clearance is mediated by two Na+-dependent transporters, called GLT-1 and GLAST (or EAAT2 and EAAT1, respectively). These transporters are almost exclusivelyexpressedbyastrocytesandenrichedonfineastrocyteprocessesnearsynapsesandonastrocyte endfeet. We have recently shown that glutamate transporters, Na+/Ca2+ exchangers, and mitochondria are functionally coupled to one another in astrocyte processes. We provide a strong scientific premise for the hypothesisthatincreasesinbloodflowuponneuronalactivationareduetoglutamatetransportintoastrocytes. InstudiesproposedinSpecificAim1,wewilluse2-photonimagingcombinedwithpharmacologicandgenetic manipulations to test the hypothesis that glutamate transport and Na+/Ca2+ exchange increase calcium in astrocyte endfeet and that this increase in calcium is necessary for stimulus-evoked increases in arteriole diameterinvivo.Normally,excitatoryactivitycausesanincreaseinbloodflow,butundersomecircumstances, theresponsebecomesinverted.
In SpecificAim2, wewilltestthehypothesisthatpreventingmitochondriafrom dockinginastrocyteprocesses/endfeetresultsinexaggeratedstimulus-evokedcalciumsignalinginendfeetand inversionoftheneurovascularresponse.Afterastroke,decreasesinbloodflowextendbeyondtheoccluded vessel.
In Specific Aim 3, we will test the hypothesis that focal ischemia results ina lossof mitochondria from astrocyte processes, exaggerated stimulus-evoked calcium signaling in endfeet, and inversion of the neurovascular response in the penumbra These studies will define a novel mechanism by which neuronal activitycausesanincreaseinneuronalbloodflow,willdefineanovelmechanismbywhichthisresponseinverts, andadeterminehowthesephenomenacontributetodysregulatedbloodflowobservedafterstroke.
Glutamate is the predominant excitatory neurotransmitter in the mammalian CNS. After release, it is cleared into neighboring cells, called astrocytes. The goals of this proposal are to determine if this clearance into astrocytes contributes to increases in blood flow observed with neuronal activity, to determine if a loss of mitochondria from astrocytes results in an inversion of the blood flow response, and to determine how these phenomenacontributetothedecreasedbloodflowobservedafterstroke.
