Single synapse indicators of impaired glutamate clearance derived from fast iGlu_u imaging of cortical afferents in the striatum of normal and Huntington (Q175) mice

Abstract

Changes in the balance between glutamate (Glu) release and uptake may stimulate synaptic reorganization and even synapse loss. In the case of neurodegeneration, a mismatch between astroglial Glu uptake and presynaptic Glu release could be detected if both parameters were assessed independently and at a single synapse level. This has now become possible due to a new imaging assay with the genetically encoded ultrafast Glu sensor iGlu u . We report findings from individual corticostriatal synapses in acute slices prepared from mice aged >1 year. Contrasting patterns of short-term plasticity and a size criterion identified 2 classes of terminals, presumably corresponding to the previously defined IT and PT synapses. The latter exhibited a higher degree of frequency potentiation/residual Glu accumulation and were selected for our first iGlu u single synapse study in Q175 mice, a model of Huntington's disease (HD). It was found that in HD the time constant of perisynaptic [Glu] decay (TauD, as indicator of uptake) and the peak iGlu u amplitude (as indicator of release) were prolonged and reduced, respectively. Treatment of WT preparations with the astrocytic Glu uptake blocker TFB-TBOA (100 nM) mimicked the TauD changes in homozygotes (HOM). Considering the largest TauD values encountered in WT, about 40% of PT terminals tested in Q175 heterozygotes (HET) can be classified as dysfunctional. Moreover, HD but not WT synapses exhibited a positive correlation between TauD and the peak amplitude of iGlu u . Finally, EAAT2 immunoreactivity was reduced next to corticostriatal terminals. Thus, astrocytic Glu transport remains a promising target for therapeutic intervention. SIGNIFICANCE STATEMENTAlterations in astrocytic Glu uptake can play a role in synaptic plasticity and neurodegeneration. Until now, sensitivity of synaptic responses to pharmacological transport block and the resulting activation of NMDA receptors were regarded as reliable evidence for a mismatch between synaptic uptake and release. But the latter parameters are interdependent. Using a new genetically encoded sensor to monitor [Glu] at individual corticostriatal synapses we can now quantify the time constant of perisynaptic [Glu] decay (as indicator of uptake) and the maximal [Glu] elevation next to the active zone (as indicator of Glu release). The results provide a positive answer to the hitherto unresolved question whether neurodegeneration (e.g. Huntington's disease) associates with a glutamate uptake deficit at tripartite excitatory synapses.

Increased and synchronous recruitment of release sites underlies hippocampal mossy fiber presynaptic potentiation

“…The average PPR of the cumulative amplitudes (PPR Cum ) was 1.45 ± 0.25 (Table S1, Figure 1G), a value that is close to the PPR for excitatory postsynaptic currents (EPSCs) recorded at 2 mM extracellular Ca 2+ (Chamberland et al, 2014). The cumulative amplitude reflects the total amount of released glutamate (Dvorzhak et al, 2019;Dvorzhak and Grantyn, 2020) and is negatively correlated with their PPR (Figure S1 A), thus reflecting an activity-dependent form of short-term plasticity. Neither the mean amplitude nor the active area correlated with their PPR (Figure S1 B, C).…”

“…Glutamate release from single hMFBs was visualized using the genetically encoded ultrafast glutamate sensor iGlu u (Helassa et al, 2018) that has been used for high-speed glutamate imaging before (Dürst et al, 2019;Dvorzhak et al, 2019;Dvorzhak and Grantyn, 2020).…”

Expression of a modified astrocytic glutamate transporter alleviates Huntington’s hypokinesia, promotes synaptic glutamate clearance and counteracts potentially adverse EAAT2 interactions

“…Here we present new results from rapid assessment and detailed analysis of self-initiated motor activity before and after viral treatment. The mice underwent two video-recorded tests: i) the open field test, OFT (Menalled et al, 2012;Rothe et al, 2015) and ii) the step-over latency test, SOLT (Dvorzhak et al, 2019). The latter was developed in our lab and could be regarded as a direct indicator of the time needed to initiate exploration in the open field.…”

“…As the striatum is devoid of glutamatergic neurons, iGlu u fluorescence of single corticostriatal presynaptic varicosities could well be resolved even under condition of blocked action potential generation. This helps the application of standardized tests at the synapses of choice (Dvorzhak et al, 2019). Two types of corticostriatal synapses were identified according to the size of iGlu u -positive varicosities at rest and their response dynamics during repetitive activation.…”

“…Most studies point to functional uncoupling rather than enhanced glutamatergic input to the striatum (Plotkin and Surmeier, 2015;Reiner and Deng, 2018;Veldman and Yang, 2018). Moreover, there is a tendency for a down-regulation of vGlut1 immunofluorescence (Rothe et al, 2015), a decrease in the number of vGluT1+ terminals (Deng et al, 2013) and a reduction of synaptic glutamate release from individual corticostriatal terminals in mice with symptomatic HD (Dvorzhak et al, 2019). On the postsynaptic side, corticostriatal synaptic transmission might be affected by insufficient supply with brain-derived neurotrophic factor (Plotkin et al, 2014) or reduced signal transfer from distal dendrites (Carrillo-Reid et al, 2019).…”