Modulation of neurotransmitter release at central synapses: role of endocannabinoids and transporters

In the brain, most neurons (nerve cells) communicate with each other by releasing packets of a chemical, such as the amino acid glutamate, into the narrow gap between the neurons (the synapse). The glutamate passes across the gap and initiates a signal in the next cell. After release the glutamate is hoovered up and recycled to the nerve cell for re-use. Memory and learning occur as a result of alterations of this signalling at synapses. Synapses are densely packed so that glutamate released from one neuron can spread to neighbouring synapses that it was not supposed to signal to. This so-called synaptic crosstalk degrades the brain‘s information storage capacity. I have identified a detector of synaptic crosstalk, which makes neurons release another chemical called a cannabinoid (related to cannabis), which decreases synaptic crosstalk by reducing glutamate release. I am studying two fundamental mechanisms controlling glutamate release: (1) the cannabinoid system which reduces the number of packets of glutamate released; (2) the recycling of glutamate to releasing neurons which controls the amount of glutamate released from each packet. Understanding how glutamate release is controlled is essential to understand the normal function of the brain and what goes wrong in pathological conditions.

I will investigate two fundamental mechanisms controlling the amount of neurotransmitter glutamate released at CNS synapses:
(1) modulation by endocannabinoids of the number of vesicles of transmitter released, and its dependence on synaptic crosstalk mediated by glutamate spillover between synapses;
(2) modulation of the amount of glutamate released from each vesicle.

My model of investigation will be the cerebellar granule cell to Purkinje cell synapses where I recently started to investigate endocannabinoid signalling (Marcaggi & Attwell, 2005, Nature Neurosci. 8: 776-81). I will use patch-clamping and imaging of transfected cells and cells in cerebellar slices from mice expressing a Ca2+ indicator in granule cells.

(1) Suppression of presynaptic glutamate release by retrograde endocannabinoid signalling depends on glutamate spillover between adjacent synapses, which is detected by mGluR1 receptors. I will determine:
(a) which feature of the extracellular [glutamate] transient mGluR1 receptors respond to, in order to detect spillover;
(b) the spatial range over which endocannabinoids signal;
(c) the spatial pattern of synapse activation produced by stimulation which mimics the physiological situation, in order to understand how likely glutamate spillover evoked plasticity is to occur in vivo.
These experiments will significantly advance our understanding of the role of endocannabinoids in controlling the strength of glutamatergic synapses. This is fundamental if we wish to understand the effects of recreational use of cannabis, or to use cannabinoids (or related drugs) to treat diseases like multiple sclerosis or obesity.

(2) I will test the importance of the glutamate-glutamine recycling system for filling glutamate vesicles, by determining the route of recycling in presynaptic terminals loaded with fluorescent indicators and by blocking enzymes and transporters that are hypothesized to play crucial roles in the cycle.
On average, the amount of glutamate released at synapses equals the amount of the main energy substrate (glucose) consumed by the brain. The recycling of released glutamate is thus essential to avoid what would otherwise be a crippling energy loss from the brain (because lost glutamate would have to be made afresh from glucose). Characterizing the glutamate recycling pathway is essential to understand brain metabolism and hence to provide a basis for interpreting brain imaging (MRI or PET), which is based on detection of energetic demand in different regions of the brain.