Name: single synapse indicators of glutamate release and uptake in acute brain slices from normal and huntington mice
Uploaded: 2020-03-11T12:30:00
Description: Watch this Scientific Journal Video about Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice at JoVE.com

This work was supported by CHDI (A-12467), the German Research Foundation (Exc 257/1 and DFG Project-ID 327654276 &#8211; SFB 1315) and intramural Research Funds of the Charit&#233;. We thank K. T&#246;r&#246;k, St. George&#39;s, University of London, and N. Helassa, University of Liverpool, for the iGluu plasmid and many helpful discussions. D. Betances and A. Sch&#246;nherr provided excellent technical assistance.

All work has been carried out in accordance with the EU Directive 2010/63/EU for animal experiments and was registered at the Berlin Office of Health Protection and Technical Safety (G0233/14 and G0218/17).
NOTE: Recordings from Q175 wild-type (WT) and heterozygotes (HETs) can be performed at any age and sex. Here we studied males and females at an age of 51 to 76 weeks.
1. Injection of the Glutamate Sensor iGluu for Expression in Corticos...

The experiments concern a question of general interest &#8212; synapse independency and its possible loss in the course of neurodegeneration, and we describe a new approach to identify affected synapses in acute brain slices from aged (&#62;1 year) mice. Taking advantage of the improved kinetic characteristics of the recently introduced glutamate sensor iGluu the experiments illuminate the relationship between synaptic glutamate release and uptake in a way that has not been possible before.
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The relative impact of each synaptic terminal belonging to a &#34;unitary connection&#34; (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-overlapping territories may contribute a horizontal perspective that is based on local mechanisms of signal exchange, integration and synchronization at synaptic sites6,7,8,9,10.
Because it is known that astroglia play, in general, a major role in the pathogenesis of neurodegenerative disease11,12 and, in particular, a role in the maintenance and plasticity of glutamatergic synapses13,14,15,16, it is conceivable that alterations in synaptic performance evolve in accordance with the state of astrocytes in the shared target area of afferent fibers with diverse origin. To further explore the target-/astroglia-derived local regulatory mechanisms in health and disease, it is necessary to evaluate individual synapses. The present approach was worked out to estimate the range of functional glutamate release and clearance indicators and to define criteria that may be used to identify dysfunctional (or recovered) synapses in brain areas most closely related to movement initiation (i.e., first of all in the motor cortex and dorsal striatum).
The striatum lacks intrinsic glutamatergic neurons. Therefore, it is relatively easy to identify glutamatergic afferents of extrastriatal origin. The latter mostly originate in the medial thalamus and in the cerebral cortex (see17,18,19,20 for more). Corticostriatal synapses are formed by the axons of pyramidal neurons localized in cortical layers 2/3 and 5. The respective axons form bilateral intra-telencephalic (IT) connections or ipsilateral connections via a fiber system that more caudally constitutes the pyramidal tract (PT). It has further been suggested that IT- and PT-type terminals differ in their release characteristics and size21,22. In view of these data, one could also expect some differences in the handling of glutamate.
The striatum is the most affected brain area in Huntington&#39;s disease (HD)5. Human HD is a severe genetically inherited neurodegenerative disorder. The Q175 mouse model offers an opportunity to investigate the cellular basis of the hypokinetic-rigid form of HD, a state that has much in common with parkinsonism. Starting at an age of about 1 year, homozygote Q175 mice (HOM) exhibit signs of hypokinesia, as revealed by measuring the time spent without movement in an open field23. The present experiments with heterozygote Q175 mice (HET) confirmed the previous motor deficits observed in HOM and, in addition, showed that the observed motor deficits were accompanied by a reduced level of the astrocytic excitatory amino acid transporter 2 protein (EAAT2) in the immediate vicinity of corticostriatal synaptic terminals24. It has therefore been hypothesized that a deficit in astrocytic glutamate uptake could lead to dysfunction or even loss of respective synapses25,26.
Here, we describe a new approach that allows one to evaluate single synapse glutamate clearance relative to the amount of the released neurotransmitter. The new glutamate sensor iGluu was expressed in corticostriatal pyramidal neurons. It was developed by Katalin T&#246;r&#246;k27 and represents a modification of the previously introduced high-affinity but slow glutamate sensor iGluSnFR28. Both sensors are derivatives of the enhanced green fluorescent protein (EGFP). For spectral and kinetic characteristics, see Helassa et al.27. Briefly, iGluu is a low-affinity sensor with rapid de-activation kinetics and therefore particularly well suited to study glutamate clearance at glutamate-releasing synaptic terminals. The dissociation time constant of iGluu was determined in a stopped-flow device, which rendered a Tauoff value of 2.1 ms at 20 &#176;C, but 0.68 ms when extrapolated to a temperature of 34 &#176;C27. Single Schaffer collateral terminals probed at 34 &#176;C with spiral laser scanning in the CA1 region of organotypic hippocampal cultures under a 2-photon microscope exhibited a mean time constant of decay of 2.7 ms.

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	<tbody>
		<tr>
			<td>Stereo microsope</td>
			<td>WPI</td>
			<td>PZMIII</td>
			<td>Precision Stereo Zoom Binocular Microscope</td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Stoelting</td>
			<td>51500D</td>
			<td>Digital Lab New Standard stereotaxic frame</td>
		</tr>
		<tr>
			<td>High speed drill equipment</td>
			<td>Stoelting</td>
			<td>514439V</td>
			<td>Foredom K1070 cromoter Kit</td>
		</tr>
		<tr>
			<td>Injection system</td>
			<td>Stoelting</td>
			<td>53311</td>
			<td>Quintessential Stereotaxic Injector (QSI)</td>
		</tr>
		<tr>
			<td>Hamilton syringe 5 &#181;l</td>
			<td>Hamilton</td>
			<td>87930</td>
			<td>75RN Syr (26s/51/2)</td>
		</tr>
		<tr>
			<td>Laser positioning system</td>
			<td>Rapp OptoElectronic</td>
			<td>UGA-40</td>
			<td>UGA-40</td>
		</tr>
		<tr>
			<td>Blue laser for iGluu excitation</td>
			<td>Rapp OptoElectronic</td>
			<td>DL-473-020-S</td>
			<td>473 nm laser</td>
		</tr>
		<tr>
			<td>Dichroic mirror for 473 nm</td>
			<td>Rapp OptoElectronic</td>
			<td>ROE TB-355-405-473</td>
			<td>Dichroic</td>
		</tr>
		<tr>
			<td>1P upright microscope</td>
			<td>Carl Zeiss</td>
			<td>000000-1066-600</td>
			<td>Axioskop 2 FS Plus</td>
		</tr>
		<tr>
			<td>Objective 63x/1.0</td>
			<td>Carl Zeiss</td>
			<td>421480-9900</td>
			<td>W Plan-Apochromat</td>
		</tr>
		<tr>
			<td>4x objective</td>
			<td>Carl Zeiss</td>
			<td>44-00-20</td>
			<td>Achroplan 4x/0,10</td>
		</tr>
		<tr>
			<td>Dichroic mirror for iGluu</td>
			<td>Omega optical</td>
			<td>XF2030</td>
			<td></td>
		</tr>
		<tr>
			<td>Emission filter for iGluu</td>
			<td>Omega optical</td>
			<td>XF3086</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Omega optical</td>
			<td>QMAX_DI580LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Emission filter for autofluorescence subtr.</td>
			<td>Omega optical</td>
			<td>QMAX EM600-650</td>
			<td></td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>ZYLA4.2PCL10</td>
			<td>ZYLA 4.2MP Plus</td>
		</tr>
		<tr>
			<td>Acqusition software</td>
			<td>Andor</td>
			<td>4.30.30034.0</td>
			<td>Solis</td>
		</tr>
		<tr>
			<td>AD/DA converter</td>
			<td>HEKA Elektronik</td>
			<td>895035</td>
			<td>InstruTECH LIH8+8</td>
		</tr>
		<tr>
			<td>Aquisition software</td>
			<td>HEKA Elektronik</td>
			<td>895153</td>
			<td>TIDA5.25</td>
		</tr>
		<tr>
			<td>Electrode positioning system</td>
			<td>Sutter Instrument</td>
			<td>MPC-200</td>
			<td>Micromanipulator</td>
		</tr>
		<tr>
			<td>Electrical stimulator</td>
			<td>Charite workshops</td>
			<td>STIM-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicer</td>
			<td>Leica</td>
			<td>VT1200 S</td>
			<td>Vibrotome</td>
		</tr>
		<tr>
			<td>Brown/Flaming-type puller</td>
			<td>Sutter Instr</td>
			<td>SU-P1000</td>
			<td>P-1000</td>
		</tr>
		<tr>
			<td>Glass tubes for injection pipettes</td>
			<td>WPI</td>
			<td>1B100F3</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tubes forstimulation pipettes</td>
			<td>WPI</td>
			<td>R100-F3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Abcam</td>
			<td>ab120054</td>
			<td>TTX</td>
		</tr>
		<tr>
			<td>iGluu plasmid</td>
			<td>Addgene</td>
			<td>106122</td>
			<td>pCI-syn-iGluu</td>
		</tr>
		<tr>
			<td>Q175 mice</td>
			<td>Jackson Lab</td>
			<td>27410</td>
			<td>Z-Q175-KI</td>
		</tr>
	</tbody>
</table>

single synapse indicators of glutamate release and uptake in acute brain slices from normal and huntington mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;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 iGluu, 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.

Identification of two types of corticostriatal glutamatergic varicosities 
IT and PT afferents originate in layer 2/3 and 5, respectively, and exhibit differential ramification and termination patterns in the ipsilateral and contralateral (IT terminals only) striatum. Still little is known about the properties of glutamate release and clearance under repetitive activation conditions as observed during the initiation of movements, but it is well documented that the respective glutamate-releasing vari...

Watch this Scientific Journal Video about Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice at JoVE.com

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

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 iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

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