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

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 neuron^1,2. 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 perspective^3,4,5. 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 sites^6,7,8,9,10.

Because it is known that astroglia play, in general, a major role in the pathogenesis of neurodegenerative disease^11,12 and, in particular, a role in the maintenance and plasticity of glutamatergic synapses^13,14,15,16, 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 (see^17,18,19,20 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 size^21,22. 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'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 synapses^25,26.

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 iGlu_u was expressed in corticostriatal pyramidal neurons. It was developed by Katalin Török²⁷ 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, iGlu_u 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 iGlu_u was determined in a stopped-flow device, which rendered a Tau_off value of 2.1 ms at 20 °C, but 0.68 ms when extrapolated to a temperature of 34 °C²⁷. Single Schaffer collateral terminals probed at 34 °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.

Protocol

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 iGlu_u for Expression in Corticostriatal Axons

1. Use the pipette puller (one step mode) to prepare borosilicate glass pipettes for the injection of the virus. After pulling, break the pipette manually to obtain a tip diameter of 30–50 µm. Autoclave the pipette and surgical instruments, including the drill for opening the skull.
2. Store the virus AAV9-CaMKIIa.iGlu_u.WPRE-hGH (7.5 x 10¹³gc/mL) at -80 °C in 10 µL aliquots. If injections are performed shortly after virus production (within 6 months), keep at 5 °C. Before surgery, take out the vial and maintain it at room temperature.
3. Fill the glass pipette and remove any bubbles.
4. Anesthetize the animal with an intraperitoneal injection of a solution containing 87.5 mg/kg ketamine and 12.5 mg/kg xylazine. Subcutaneously inject 0.25% bupivacain (8 mg/kg) for additional pain relief. Check the depth of anesthesia by monitoring the muscle tone and observing the absence of pain-induced reflexes.
5. Shave the skin on the head and sterilize it with 70% alcohol. Fit the mouse into the stereotaxic frame.
6. Use a scalpel to remove the skin and a high speed (38,000 rpm) drill to make a 1.2 mm hole in the bone above the motor cortex.
7. Mount the syringe with the attached injection pipette in the holder of a precision manipulator. Insert the vertically oriented pipette into the cortex at 4 different sites. The injection coordinates are, with respect to bregma (in mm): anterior 1.5, lateral 1.56, 1.8, 2.04, 2.28. The depth with respect to dura mater is (in mm): 1.5–1.7.
8. Using the injection system, inject 0.3 µL per site of the undiluted virus solution with a velocity of 0.05 µL/min. After each injection, leave the pipette in place for 1 min before withdrawing it slowly (1 mm/min).
9. Finish by closing the surgical wound with a nylon suture.
10. Leave the mouse for 0.5 to 1 h on a heating pad in a clean cage before returning it to its original cage.
11. Maintain the mouse on a 12 h day-night cycle for 6 to 8 weeks before the preparation of acute brain slices.
    NOTE: To avoid immune reactions resulting in cell damage and synapse loss, injection of multiple viral constructs must be performed simultaneously or within 2–3 hours after the primary injection. The coordinates for iGlu_u injection were selected according to the Paxinos and Franklin²⁹ brain atlas. They correspond to the M1 motor cortex. Immunostaining of injected brains visualized numerous but mostly well isolated axons and axon varicosities in the ipsi- and contralateral striatum and in the contralateral M1 and S1 cortices.

2. Search for Glutamatergic Terminals Expressing the Glutamate Sensor iGlu_u

1. Calibration mode
   1. Prepare the sCMOS camera and the camera control software with the following settings. On the Readout page set Pixel readout rate: 560 MHz (= fastest readout), Sensitivity/Dynamic Range: bit, Spurious Noise Filter: Yes, Overlap Readout: Yes. On the Binning/ROI page, select Full screen.
   2. Prepare a glass slide containing a drop of 5 mg/mL Lucifer Yellow (LY) under a cover slip.
   3. Place the LY slide under a 63x objective, open the laser shutter and perform a serial acquisition using the following settings: Pixel Binning: 1x1, Trigger mode: Internal, Exposure time: Minimal (to be determined).
   4. Adjust the laser power to produce a fluorescence spot of 4 µm in diameter (the image should not contain saturated pixels).
   5. To perform a calibration of the laser positioning system, select the following settings in the laser positioning software: Spot-size diameter: 10, Scanning velocity: 43.200 kHz. Click the Start image acquisition button on the right panel. Set Runs: 0, Run delay: 0, and select the Run at TTL option on the Sequence page. Click the Calibrate button on the Calibration page and calibrate the laser control software according instructions shown in the top-left corner of the acquisition screen.
   6. If the setup uses independent software for camera and laser control, acquire screenshots from the camera acquisition window and send it to the laser control software for a re-calculation of the XY coordinates. If the software is installed on different computers, then use a video grabber to import the image into the laser control software. The laser control software will need the information on the XY scaling factors and offsets. For this purpose, determine the coordinates of the top-left, bottom-left and bottom-right corners of the image within the acquisition window of the laser control program. Calculate the scaling factors according to the following equations: X factor = (X bottom-right – X bottom-left)/2048, X offset = X bottom-left, Y factor = (Y top-left – Y bottom-left)/2048, Y offset = Y bottom-left.
   7. At the end of the calibration procedure, create a rectangular region of interest (ROI) of 267x460 pixels, move it to the center of the screen and click the Start sequence button.
   8. Return to the following camera control software settings: Binning: 2x2, Trigger mode: External exposure, Exposure time: Minimal (to be determined).
2. Autofluorescence correction mode
   NOTE: The resting level of [Glu] in the environment of active synaptic terminals is typically below 100 nM^{14,30,31,32,33,34}. Accordingly, any sensor of glutamate, especially a low affinity sensor like iGlu_u will be rather dim in the absence of synaptic glutamate release. Nevertheless, some iGlu_u fluorescence can even be detected at rest, but must be distinguished from the tissue autofluorescence. The 473 nm illumination elicits both iGlu_u fluorescence and autofluorescence (Figure 1A–C). The latter occupies a wide range of wavelengths, while iGlu_u fluorescence is limited to 480–580 nm (with a maximum at 510 nm). The correction for autofluorescence is based on the acquisition of two images with different high-pass filters.
   1. Prepare brain slices in advance as described elsewhere³⁵. Keep bran slices ready.
   2. Transfer the slices into the recording chamber, submerging them into oxygenized artificial cerebrospinalfluid (ACSF) containing 125 mM NaCl, 3 mM KCl, 1.25 mM NaH₂PO₄, 25 mM NaHCO₃, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose (pH 7.3, 303 mOsm/L), supplemented with 0.5 mM sodium pyruvate, 2.8 mM sodium ascorbate and 0.005 mM glutathione. Use a flow rate of 1–2 mL/min. Keep the bath temperature at 28–30 °C.
   3. Locate the dorsal striatum under a 20x water immersion objective. Fix the slices with a nylon grid on a platinum harp to minimize the tissue movement. Switch to the 63x /NA 1.0 water immersion objective. Select filters reflecting light at 473 nm (dichroic mirror) and passing light with wavelengths >510 nm (emission filter).
   4. Synchronize illumination, stimulation and image acquisition using an AD/DA board with the respective control software. Set the trigger program to control acquisition with a laser exposure time of 180 ms and an image acquisition time of 160 ms. Use the laser positioning device to send the laser beam to a predetermined number of points and define the settings for Scanning velocity and Spot size.
   5. Acquire an image of both autofluorescence and iGlu_u-positive structures using a high pass filter at 510 nm ("yellow image").
   6. Acquire an image with autofluorescence alone using a high-pass filter at 600 nm ("red image").
   7. Scale the red and yellow images, using the mean intensities of the 10 brightest and the 10 darkest pixels to define the range. Perform a subtraction "yellow minus red image" and rescale the subtracted image to generate a standard 8-bit tif file for convenient visualization of the bouton of interest (Figure 1D). It contains the bright pixels from the iGlu_u-positive structures, grey pixels from the background and dark pixels from structures with autofluorescence.
      NOTE: With the given equipment the mean resting fluorescence (F) will be below 700 A.U.
3. Bouton search mode
   NOTE: iGlu_u-positive pixels may belong to functionally different elements of the axonal tree, such as axon branches of different order, sites of bifurcation, varicosities after vesicle depletion or fully active varicosities. However, it is almost impossible to identify functional synaptic terminals just by visual inspection. Therefore, each glutamatergic synaptic terminal needs identification by its responsiveness to electrical depolarization. Sites that do not respond to stimulation have to be discarded. The physiological means to induce glutamate release from corticostriatal axons is to elicit an action potential. This can either be achieved by using a channel rhodopsin of appropriate spectral characteristics or by electrical stimulation of an axon visualized by iGlu_u itself. To avoid accidental opsin activation, we preferred the latter approastep
   1. Use the micropipette puller (in a four-step mode) to produce stimulation pipettes from borosilicate glass capillaries. The internal tip diameter should be about 1 µm. When filled with ACSF, the electrode resistance should be about 10 MΩ.
   2. To induce the action potential-dependent release of glutamate from a set of synaptic boutons attached to the same axon, use 63x magnification, the 510 nm emission filter and the subtracted image to place a glass stimulation electrode next to a fluorescent varicosity. Avoid the proximity of additional axons.
   3. Turn on the stimulator to deliver depolarizing current pulses to the stimulation pipette. Use current intensities around 2 µA (no more than 10 µA).
   4. Turn on the multi-channel bath application system where one channel delivers the standard bath solution and the other channels deliver the necessary blockers of ion channels, transporters or membrane receptors. Control the flow at the site of recording and then switch to the tetrodotoxin (TTX) channel (bath solution plus 1 µM TTX). After 2–3 min, stimulate the bouton of interest again, but now in the absence of action potential generation. The release is directly due to calcium influx through voltage-depended calcium channels.
   5. By turning the intensity key on the stimulator, adjust the stimulation current to obtain responses similar to those elicited via action potentials. Typically, the stimulation current would be around 6 µA for a 0.5 ms depolarization.
   6. For basic testing of the preparation, apply 0.5 mM CdCl₂. The presence of this calcium channel blocker glutamate release completely prevents the synaptic glutamate release thereby validating the calcium dependence of the directly evoked glutamate signal.
      NOTE: The following general recommendation may help to increase the success rate of single synapse experiments in the striatum. To select glutamatergic synaptic terminals with intact release machinery, a varicosity should: (i) Have a smooth spindle-like shape; (ii) Not be associated with an axon bifurcation; (iii) Be brighter than other structures on the "yellow image"; (iv) Reside in the striatal neuropil rather than in the fiber tracts; (v) Reside on a very thin axon branch; (vi) Not reside within the deeper parts of the slice.

3. Visualization of Glutamate Release and Clearance

1. Recording mode
   NOTE: After the subtraction of the autofluorescence (step 2.2) and a few initial tests for responsiveness of the bouton of interest (Step 2.3), data acquisition can begin. The responses to electrical activation of glutamate release from single axon terminals can be observed in the image directly (Figure 2), without applying further analysis tools. However, it proved to be very convenient to immediately extract some basic indicators of synapse performance (Figure 3). This information is needed to make decisions on subsequent course of experiment, such as selection of particular terminal types, rapid assessment of HD-related alterations, the number and frequency of trials or drugs to be applied with the superfusion system. It might also be necessary to deal with eventually appearing artifacts. First, the standard settings for data recording is described.
   1. Using the microscope XY drives, place the tested iGlu_u-positive bouton close to the viewfield center. Stop the image acquisition with the Abort acquisition button. On the last acquired picture, determine the XY position of the resting bouton center by clicking on it with the left mouse button. The XY coordinates of the set cursor will be shown on the bottom status panel of the acquisition window.
   2. Using the calibration data (step 2.1.6), calculate the coordinates of the site where the laser beam should be sent for the excitation of the iGlu_u fluorescence. Use the following equations: X laser = X camera * X factor + X offset, Y laser = Y offset – Y camera * Y factor. While performing this recalculation, pay attention to the vertical or horizontal flip settings of the camera.
   3. Create a one-point sequence in the laser control software using the calculated coordinates. For this purpose, select Point in the Add to sequence box on the Sequence page of the laser control software. Select 10 µs for the delay to trigger onset and 180 ms for the laser pulse time. Move the mouse to the calculated coordinates and click left button.
   4. Select the following settings in the laser control software: Runs: 0, Run delay: 0, Sequence: Run at TTL. Then click Start sequence.
   5. In the camera control software, select the following settings: On the Binning/ROI page, set Image Area: Custom, Pixel Binning: 2x2, Height: 20, Width: 20, Left: X-coordinate of the resting iGlu_u-positive spot minus 10 px, Bottom: Y-coordinate of the resting iGlu_u-positive spot minus 10 px, Acquisition Mode: Kinetic Series, Kinetic Series Length: 400, Exposure Time: 0.0003744s (minimal value). With such settings, the acquisition rate will be 2.48 kHz.
   6. Select Trigger mode: External. Click Take signal in the camera control software. Initiate the experimental protocol laid down for the trigger device.
   7. Implement the experimental protocol Trial with the following time line: 0 ms – start trial, 1 ms – start laser illumination, 20 ms – start image acquisition with camera, 70 ms – start electrical stimulation 1, 120 – start electrical stimulation 2, 181 ms – end trial (laser and camera off). Thus, during one trial the camera acquires 400 frames with a frequency of 2.48 kHz. See steps 2.3.3 and 2.3.5. for details on electrical stimulation.
   8. To allow for sufficient recovery of presynaptic vesicle pools, apply the protocol Trial with a repetition frequency of 0.1 Hz or lower.
2. Off-line construction of the glutamate transient and rapid assessment of glutamate release and clearance for the identification of pathological synapses
   1. Turn on the evaluation routines. Here, we use an in-house-written software SynBout v. 3.2. (author: Anton Dvorzhak). The following steps are needed to construct a ΔF/F transient from the pixels with elevated iGlu_u fluorescence as in the video of Figure 2.
   2. To determinate the bouton size, calculate the mean and standard deviation (SD) of the ROI fluorescence intensity at rest (F), before the onset of stimulation. Determine and box the area occupied by pixels with F>mean + 3 SD (Figure 3A). Determine a virtual diameter (in µm) assuming a circular form of the supra-threshold area.
   3. Determine ΔF as the difference between the peak intensity value and F. Plot the stimulating current and ΔF/F against time (Figure 3B). Calculate the SD of ΔF/F at rest (before the onset of stimulation) and the "Peak amplitude". Use pixel with peak amplitude more than 3 SD of ΔF/F to perform monoexponential fitting for the decay from peak. Determine the time constant of decay TauD of ΔF/F.
   4. To estimate the "Maximal amplitude" at a given synapse, select the pixel with the highest ΔF/F value. It is typically located within or next to the boundaries of the resting bouton iGlu_u fluorescence. The "Maximal amplitude" would be the best indicator of the glutamate load presented to the clearance machinery of a single synapse.
   5. To estimate the spatial extension of the iGlu_u signal, determine the diameter of the area of all supra-threshold pixels combined to form a virtual circle. The respective diameter is called "Spread". The term "Peak spread" then refers to the peak value of the averaged spread transient (Figure 3C, difference between dotted red lines).
3. Corrections for possible artifacts
   NOTE: Electrical depolarization is associated with an outward flow of the intra-pipette solution. This may result in a small tissue displacement. To discriminate between stimulation-induced changes of iGlu_u and out-of-focus shifts of the image, one could use the following approach to identify and to eliminate displacement artifacts (Figure 4).
   1. Analyze the spatial characteristics of the suprathreshold pixels derived from a ROI with signs of displacement (Figure 4F–H).
   2. Find pixels outside the initially determined boundaries of the bouton at rest (Figure 4F–H, boxed in red).
   3. Identify eventually existing negative pixel intensity values (Figure 4I, J). Any ΔF/F in the negative direction with an amplitude larger than the pre-stimulation mean ± 3 SD should be regarded as artifact, and the record should be discarded.
   4. To avoid out-of-focus artifacts, place the stimulation electrode on the antero-lateral side of the synaptic bouton, use biphasic electrical stimulation and minimize the stimulation intensity/duration.
      NOTE: Out-of-focus artifacts can also be derived from the camera. If the latter is not optimally fixed to the microscope it can produce oscillations, most likely due to small vibrations of the camera cooling system. Such vibrations create a "yin and yang" pattern in the ΔF/F images rotating with a frequency of 50 Hz. The vibrations can be mathematically subtracted from the fluorescence signal, but the final quality of the measurements would then critically depend on the recording time.
   5. Fix the camera to the microscope such that vibrations are absent. If the latter remain, place a rubber gasket between the camera and microscope adapter.
      NOTE: One cannot neglect the possibility that the striatal neuropil around the synapse of interest contains glutamatergic varicosities that do not express iGlu_u and therefore remain invisible. In the case of their co-activation (more likely in the absence of TTX) the transients obtained from the boutons of interest might be affected by spill-over. The same applies to iGlu_u-expressing terminals that were out of focus. A characteristic phenomenon would in this case be that fluorescence transients originate from a much wider area. As this response is eliminated by TTX, one can interpret it as an unspecific response induced by unwarranted multi-fiber activation.
   6. To correct for this unspecific multi-fiber response, follow the procedure outlined in Figure 5. Use the fluorescence signal of pixels at the viewfield boundaries. To minimize background response, minimize stimulation intensity and duration or use TTX.

Results

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 varico...

Discussion

The experiments concern a question of general interest — 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 (>1 year) mice. Taking advantage of the improved kinetic characteristics of the recently introduced glutamate sensor iGlu_u the experiments illuminate the relationship between synaptic glutamate release and uptake in a way that has not been possible before.

Materials

List of materials used in this article

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