Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Anton Dvorzhak; Rosemarie Grantyn

doi:10.3791/60113

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Method Article

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

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DOI:

10.3791/60113

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March 11th, 2020

Anton Dvorzhak¹ , Rosemarie Grantyn¹

¹Synaptic Dysfunction Group, Neuroscience Research Center, Charité - University Medicine

Summary

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGlu_u for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Abstract

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington's disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGlu_u, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

Introduction

The relative impact of each synaptic terminal belonging to a "unitary connection" (i.e., the connection between 2 nerve cells) is typically assessed by its influence on the initial segment of the postsynaptic neuron¹^,². Somatic and/or dendritic recordings from postsynaptic neurons represent the most common and, until now, also the most productive means to clarify information processing under a top-down or vertical perspective³^,⁴^,⁵. However, the presence of astrocytes with their discrete and (in rodents) non-ov....