The widespread use of Glutamate (Glu) as the major excitatory neurotransmitter (NT) in the mammalian brain
is both critical for normal physiology and a source of predicaments: a) As seen in stroke and a range of
neurodegenerative diseases, any disruption in Glu clearance causes its accumulation, leading to over-
excitation of postsynaptic cells and excitotoxic neurodegeneration. b) The use of the same NT in so many
adjacent synapses can cause signaling to “bleed over” between neuronal circuits, and the loss of processing
fidelity. An idealized view of the brain describes synapses as well insulated from each other, enveloped by glia
that expresses high levels of Glu clearing transporters (GluTs). However, a more realistic examination reveals
that some particularly-important brain areas (e.g., hippocampus) show severely deficient glial isolation, with
estimated 2/3 of released Glu seeping out of the original synapse. How sufficient Glu clearance is achieved in
glia-deficient brain areas remains unclear. To overcome the limitation of current techniques we will study Glu
clearance in the glia-deficient synaptic hub of the C. elegans nerve ring. We are aided by the availability of
information on the precise identification of individual neurons, the exact location of their synapses, the circuits
that they participate in, and the sensory inputs and behavioral outputs of these circuits. Together with animal
transparency and the wide availability of optogenetic tools, this is an ideal system to study Glu clearance
without perturbing interstitial fluids. In our recent studies we have discovered that specific synapses fall into
watershed territories of Glu clearance, and that synapses might be affected by the agitation of body fluids. We
therefore propose a novel concept, where Glu clearance in a glia-deficient synaptic hub can be robust enough
to allow functional synaptic isolation. Such robust clearance depends on division of labor between proximal
and distal GluTs, and is facilitated by agitation and perfusion of interstitial fluids. To provide further support to
this model we will use genetically-encoded florescent Ca2+ reporters (GCaMP) to follow synaptic activity and
assign additional synapses and circuits to GluT drainage territories; we will stimulate one circuit and record
responses from an adjacent one to detect spillover; we will use genetically-encoded fluorescent detectors to
study the flow of Glu in the interstitial space; we will study the effect of paralysis on neuronal responses and
Glu flow; We will correlated the differences between the structure of proximal and distal GluTs to potential
differences transport in affinity and capacity. These studies will provide novel insights to mechanisms of robust
Glu clearance in the absence of glia, and highlight the significance of agitation of interstitial fluids in synaptic
areas that are deficient in glia insulation, a feature shared between nematodes and some areas of the
mammalian brain. These insights will aid in the design of future therapeutic interventions to prevent
excitotoxicity (seen in stroke and a range of neurodegenerative diseases), and highlight the significance of
vascular pulsatility in CNS physiology.