Name: multi-photon intracellular sodium imaging combined with uv-mediated focal uncaging of glutamate in ca1 pyramidal neurons
Uploaded: 2014-10-08T14:00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons at JoVE.com

This study was supported by a grant of the German Science Foundation (DFG, Ro2327/6-1) to C.R.R. The authors wish to thank S. Durry and C. Roderigo for expert technical assistance, and M. D&#252;bbert (Electronics Laboratory, Zoological Institute, University of Cologne, Germany) for help in implementing the Pockels cell controller and the HF switch.

This study was carried out in strict accordance with the institutional guidelines of the Heinrich Heine University D&#252;sseldorf, Germany, as well as the European Community Council Directive (86/609/EEC). All experiments were communicated to and approved by the Animal Welfare Office at the Animal Care and Use Facility of the Heinrich Heine University D&#252;sseldorf, Germany (institutional act number: O52/05). In accordance with the German Animal Welfare Act (Tierschutzgesetz, Articles 4 and 7), no formal additional approval for the postmortem removal of brain tissue was necessary.
For generation of acute slices, mice were anaesthetized with CO2 and quickly decapitated (following the recommendation of the European Commission published in: Euthanasia of experimental animals, Luxembourg: Office for Official Publications of the European Communities, 1997; ISBN 92&#8211;827-9694-9).
1. Preparation of Solutions
<ol>
	<li>Prepare artificial cerebrospinal fluid (ACSF) for dissection containing (in mM): 125 NaCl, 2.5 KCl, 0.5 CaCl2, 6 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose, bubbled with 95% O2 and 5% CO2, resulting in a pH of 7.4.</li>
	<li>Prepare ACSF for experiments containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose, bubbled with 95% O2 and 5% CO2, resulting in a pH of 7.4.</li>
	<li>Prepare intracellular solution (ICS) containing (in mM): 150 KMeSO3, 12.5 hydroxy ethyl piperazine ethane sulfonic acid (HEPES), 40 KCl, 5 NaCl, 1.25 ethylene glycol tetraacetic acid (EGTA), 5 Mg-ATP, 0.5 Na-GTP. Adjust pH to 7.3. Store aliquots of 1 ml at -20 &#176;C.</li>
	<li>Dilute sodium-binding benzofuran isophthalate (SBFI)-salt in double-distilled water and prepare aliquots of 5 &#181;l of a 10 mM stock solution.</li>
	<li>Thaw ICS and add SBFI stock solution at a final concentration of 1 mM. Vortex and micro-filtrate (0.22 &#181;m). Keep at 4 &#176;C until used in experiment. Do not refreeze and only use for one day.</li>
	<li>Prepare MNI glutamate stock solution of 50 mM by dissolving the compound in double-distilled water. Dilute MNI glutamate stock solution to a final concentration of 5 mM in normal ACSF. Keep at 4 &#176;C until used in experiment. Do not refreeze and only use for one day. Store aliquots, which are not immediately used in the experiment, at -20 &#176;C.</li>
</ol>
2. Dissection of Tissue
NOTE: The preparation of acute hippocampal slices of the rodent brain was described in detail earlier13,14. In brief, the following protocol was employed in the present study.
<ol>
	<li>After decapitation, rapidly remove the brain from the skull.</li>
	<li>Immediately place the brain in a petri dish with ice-cold dissection ACSF and dissect a hemisphere by performing a sagittal cut along the midline.</li>
	<li>Perform a second cut in the desired orientation as a blocking surface and attach this to the cutting stage of a vibratome with superglue.</li>
	<li>Take cutting chamber and cooling element (both kept at -20 &#176;C) of the vibratome out of the freezer and place cutting stage with brain section in the chamber. Then put cooling element in the chamber and submerge tissue in ice-cold dissection ACSF. To stabilize the preparation, one may want to counter the tissue block with agar gel.</li>
	<li>Cut 250 &#181;m thick, parasagittal slices of the hippocampus with the vibratome. Ensure that all ACS fluids are bubbled at all times.</li>
	<li>After slicing, keep tissue on a mesh in a beaker with the normal ACSF and incubate at 34 &#176;C for 30 min. Then keep at room temperature.</li>
</ol>
3. Preparation of Hardware
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/52038/52038fig1highres.jpg" width="500" /> 
Figure 1. Scheme showing light paths and experimental control of the rig consisting of multi-photon imaging, laser-scanning UV flash photolysis, and electrophysiology. The multi-photon beam (red) is produced by a pulsed, tunable infrared (IR) laser (TiSa). It passes a mechanical shutter, a Pockels&#8217; cell, and the IR detector (detection of beam intensity), all of which enable control of the laser power and its administration duration. The flip-in/flip-out optional laser diode is employed for basic alignment of the laser beam. The beam expander may be used in combination with objectives with an extremely wide back-focal plane. The remote-controlled ND filter wheel may be used in addition or instead of the Pockels&#8217; cell to control laser beam power. After passing the scan head, the pulsed IR-light is guided to the specimen. Emitted fluorescence light (light green) is collected either by the external or the internal photomultiplier detectors (PMTs). The external detectors are fully synchronized with the internal PMTs via a high frequency switch (PMT controller). The uncaging beam (light blue) is produced by a UV solid-state laser (DPSS UV-Laser). It is then directed to the galvo-driven scanning unit at the top-back of the epi-fluorescence condenser by a light guide. The precise positioning (chromatic aberration between imaging and uncaging beam) of the uncaging spot or area is enabled by the image export from the imaging software. The timing management for synchronizing the imaging, the electrophysiology, and the flash photolysis is electronically controlled. The transillumination detector (TL-PMT) is of need for documentation of the positioning of the pipettes within the tissue. The control units and software of the imaging system, which has been modified, is used to control and synchronize all other devices needed to run the system. System components labeled as &#8220;custom&#8221; were designed and build/adapted by the authors. Some components were adapted to meet the requirements of the custom-build multi-photon system and are labeled as &#8220;modified&#8221;. <a href="https://www.jove.com/files/ftp_upload/52038/52038fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Switch on components of the multi-photon system. Test and adjust infrared laser beam alignment.
	<ol>
		<li>Control beam positioning by flipping in the centering prism instead of an objective. If the beam is dislocated, re-adjust it by the mirrors in the periscope. Note that the centering plane of the prism must be at the same level as the back focal plane of the objective while imaging.</li>
		<li>Switch on the spectrometer and check for the multi-photon characteristics of the beam.</li>
		<li>Set parameters of the imaging software to the following values: Choose a frame size of 512 x 512 pixels for overview images of the cell (smaller clip boxes with a zoom factor of 2.5 for high frame rate and high spatial resolution, respectively). Set imaging speed to fast mode and scan mode to XYT. Z-stepping should be 1 &#181;m for overview stacks and 0.2 &#181;m for detailed stacks to match spatial sampling requirements for deconvolution performed later.</li>
	</ol>
	</li>
	<li>Adjust multi-photon laser beam (790 nm) intensity by altering settings of the Pockels&#8217; cell (final power under the objective: &#8776; 14 - 16 &#181;W) and the photo-multipliers (PMTs).</li>
	<li>Switch on and calibrate uncaging system by placing a fluorescent sample slide under the objective lens.
	<ol>
		<li>Using the binoculars, adjust the focus of the UV optics and spatially limit the UV laser spot to a size of 2 &#181;m in diameter at maximum by employing the focusing unit at the UGA scan head.</li>
		<li>Start the calibration routine of the uncaging unit control software.
		<ol>
			<li>Set the UV laser to several points within its scan range, while the fluorescence is captured with a CCD camera. By clicking on the UV laser spot at each point, adjust the positioning of the galvanic scan mirrors to correspond to a certain coordinate in the software.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52038/52038fig2highres.jpg" width="500" /> 
Figure 2. Detailed scheme illustrating the characteristics ofexcitation, emission and uncaging beam paths. The IR excitation beam (red) passes the beam combining dichroic mirror at the level to the filer turret in the epi-fluorescence condenser of the microscope and the objective to reach the specimen. The uncaging beam (blue) is delivered via a quartz optical light fiber to the collimation and beam focusing unit and then subsequently guided to the scanning mirrors in the scanning unit, passes a dichroic mirror to the beam combining dichroic mirror. Here it is combined with the excitation scanning beam. The emitted light from the specimen follows the path of the excitation scanning beam path in the opposite orientation towards the imaging scan head. The camera is used for visual control of the patch pipette and for positioning of the uncaging beam in combination with the confocal laser scanning microscope (not shown). The green light path represents an optional epi-fluorescence illumination which may be of use with other applications.
<ol start="3">
	<li>
	<ol start="2">
		<li>
		<ol start="2">
			<li>Use the flash photolysis system in the spot mode to gain focal applications. Ensure that the focal adjustment of the uncaging beam (average power underneath the objective: 0.55 mW) results in a spot size of 1.5 &#181;m in diameter (xy extension) as revealed at a fluorescent slide positioned at a z-level that corresponds to that of a tissue slice (not shown).</li>
		</ol>
		</li>
		<li>The accurate positioning of the uncaging spot is achieved by the import of an image of the imaging system via a network connection between uncaging- and imaging-computers.
		<ol>
			<li>Using a screen grabber software, continuously read out the frames of the imaging software (instead of the camera feed) and adjust the imaging and uncaging frames congruently. Export every 10th frame from the imaging software as a reference image into the flash unit to ensure proper adjustment during the entire experiment.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" fo:content-width="6.5in" src="/files/ftp_upload/52038/52038fig3highres.jpg" width="600" /> 
Figure 3. Adjustment of the uncaging spot: To precisely position the uncaging spot, a screen shot of the imaging software (yellow frame) is imported into the uncaging software (green frame). The left image within the yellow frame represents a Hi-Lo coded image (blue: black pixels, red: saturated pixels) of the entire CA1 pyramidal cell. The image is overlaid by the calibration grid of the uncaging software (green &#8220;X&#8221;s). To the right, a magnified part of the cell is shown with the overlaid uncaging spot (red cross). The yellow spot is the automatically overlaid image of the uncaging beam. <a href="https://www.jove.com/files/ftp_upload/52038/52038fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Turn on micromanipulators, electrophysiology components, and pressure application device for delivery of the caged compound to the target region.</li>
	<li>Install a focal pressure application device for local perfusion of caged compounds. This will reduce costs enormously as compared to bath perfusion of these substances. Adjust the holding pressure to the given atmospheric pressure to prevent a drag of ACSF into or a leakage of caged substances from the application pipette. 
	NOTE: The pressure application device hosts a highly precise and ultrafast micro-valve in the pipette holder. This enables to employ minimal application pressures and therefore reduces movement artifacts to a minimum during the local perfusion with the caged compound.</li>
	<li>Pull pipettes for whole-cell patch-clamp and local perfusion using fire-polished borosilicate glass capillaries. Pipettes should have a tip diameter of ~1 &#181;m and a resistance of R &#8776; 3 M&#8486; (values determined with K-MeSO3-based ICS).</li>
	<li>Place slice in the experimental bath and affix it with a grid (platinum frame 250 &#181;M thick, spanned with surgical filaments, o.d. 40 &#181;m) (Figure 4A, B). Use an inverted, tip-broken, and fire-polished Pasteur pipette (suction ball at the side of the broken tip) for slice transfer. Avoid bending and any other harsh handling of the slice.</li>
</ol>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/52038/52038fig4highres.jpg" width="500" /> 
Figure 4. Components of the experimental bath and positioning of the bath at the microscope stage. (A) The experimental bath consists of the bath chamber itself (blue square), which is surrounded by a magnetic metal ring. The tubing ensures continuous perfusion of the bath with saline (ACSF). The grid to hold down and fix the slice is put into the bath chamber. (B) Acute slice preparation positioned within the bath chamber. The slice is fixed by the threads of the grid. (C) Positioning and geometry of the experimental bath at the microscope stage during the experiments.
<ol start="8">
	<li>Place bath at the microscope stage and permanently perfuse slice with ACSF.</li>
</ol>
4. Whole-cell Patch-clamping
<ol>
	<li>Load patch pipette with ICS containing SBFI and load local perfusion pipette with caged compound. Attach pipettes to corresponding micromanipulators. Place reference electrode in bath. Make sure that you are grounded permanently to avoid damage to the head stage (c.f. Figure 4C).</li>
	<li>Lower both pipettes into bath and place them above the hippocampal CA1 region. Apply gentle pressure to patch pipette (+40 mbar) to avoid dilution of the ICS with ACSF.</li>
	<li>Compensate the offset potential of the patch pipette using the electrophysiology software.</li>
	<li>Approach a CA1 pyramidal cell with patch pipette employing IR-DIC video microscopy. Apply gentle suction until a Giga-seal is obtained. Choose a cell the soma of which is located 30 -70 &#181;m below the surface of the slice to ensure intact cell morphology on the one hand side and low scattering and attenuation of the uncaging beam on the other hand side.</li>
	<li>Compensate for fast capacity. Break membrane and open cell to gain whole-cell configuration.</li>
	<li>Compensate for slow capacity and series resistance.</li>
	<li>Allow the cell to be dialyzed with the SBFI-ICS for at least 30 min before starting the imaging experiments.</li>
</ol>
5. Multi-photon imaging and stimulation
<img alt="Figure 5" fo:content-width="6in" src="/files/ftp_upload/52038/52038fig5highres.jpg" width="600" /> 
Figure 5. Experimental protocol. To initialize an experiment, a trigger pulse (indicated by red sign (&#7464;)and the red line) is set to simultaneously start the imaging (blue), the electrophysiology (green), and the administration of the caged compound (yellow) at time point 0 sec. During this first period, the imaging beam should be entirely dimmed (light blue). After 4.5 sec (dashed line), the local perfusion of the caged compound is terminated and the imaging beam should be set to its working intensity for data acquisition (blue). 1.5 sec afterwards (time point 6 sec), a second trigger pulse is given (indicated by red sign (&#7464;) and the red line), which initializes the UV flash unit (duration of the flash: 300 msec; indicated by the yellow flash) and sets markers in the electrophysiology and the imaging protocol.
<ol>
	<li>Add tetrodotoxin (TTX, 500 nM) to ACSF to prevent activation of voltage-gated sodium channels and generation of action potentials.</li>
	<li>Visualize cellular morphology using multi-photon excitation and resulting SBFI fluorescence and choose a spiny dendrite for experiment. Zoom in for images at a higher resolution and place a clip box around the dendrite.</li>
	<li>Fill a standard patch pipette with 10 &#181;l ACSF containing caged glutamate. Connect the pipette to the pressure application system and attach it to the micromanipulator.</li>
	<li>Place pipette with caged compound near the dendrite (~30 &#181;m). Position the pipette to allow efficient local perfusion of the dendrite of choice. Adjust the uncaging laser: position the uncaging spot close (~1 - 2 &#181;m) to the structure of interest.</li>
	<li>Set regions of interest on the chosen dendrite and adjacent spines using imaging software.</li>
	<li>Approach the region of interest and focally inject the caged compound for several sec with low pressure (&#60;3 PSI). Start patch clamp and fluorescence recordings (at 790 nm multi-photon excitation) via trigger signal. 
	NOTE: During the local perfusion of the caged compound, the excitation beam is completely dimmed to prevent bleaching.</li>
	<li>Stop local perfusion of the caged compound. Increase intensity of the two-photon laser to enable efficient excitation of SBFI and apply a UV flash to initialize uncaging (uncaging duration 300 msec).</li>
	<li>Monitor changes in SBFI fluorescence. Stop recording after SBFI fluorescence has recovered back to baseline.</li>
</ol>
6. Pharmacology
<ol>
	<li>To test the involvement of ionotropic glutamate receptors in the generation of the currents and/or sodium signals evoked by UV flash photolysis of caged glutamate, employ receptor blockers.
	<ol>
		<li>Switch to ACSF containing the AMPA-receptor blocker cyano-nitroquinoxaline-dione (CNQX, 10 &#181;M) and the NMDA-receptor blocker amino-phosphonopentanoate (APV, 50 &#181;M) in addition to TTX (see above) and perfuse slice for at least 10 min.</li>
		<li>Repeat stimulation procedure (see 5.4 and 5.5.)</li>
	</ol>
	</li>
	<li>Reversibility of the inhibitors&#8217; effects
	<ol>
		<li>Switch back to normal ACSF containing only TTX.</li>
		<li>Perfuse slice for 20 min.</li>
		<li>Repeat stimulation procedure (see 5.4 and 5.5.).</li>
	</ol>
	</li>
</ol>
7. Morphology
<ol>
	<li>Record a XYZ-stack of the clip box-region set for the performed measurements. Ensure that this stack is oversampled spatially (at least 0.2 &#181;m per pixel) to enable optimal image deconvolution and to increase image quality and resolution.</li>
	<li>Record a XYZ-stack of the entire cell to assess cell morphology.</li>
	<li>Run deconvolution algorithm.</li>
</ol>

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The authors declare no competing interests. The authors received financial support enabling Open Access Publication by Rapp OptoElectronic (Wedel, Germany), which produces an instrument used in the video article. The company was neither involved in the experiments nor in the data handling nor in the manuscript writing.

The present study shows that SBFI is well suited for two-photon imaging of intracellular sodium transients in small cellular compartments. It has to be kept in mind, however, that the quantum efficiency of SBFI is rather low15 and relative changes in the sodium concentration are quite small with physiological activity. Thus, high-resolution measurement of Na+ transients in fine processes is a relatively tedious task, and binning or averaging of several trials may be necessary to obtain satisfactory signals. In addition, the kinetics of sodium transients are surprisingly slow, making it obligatory to record for extended time periods. This observation corresponds to those in earlier studies, where monoexponential decay of sodium transients in neuronal dendrites and in astrocytes was characterized by large decay time constants in the range of 10 sec at room temperature1,2,6. Sodium transients thus seem to exert a much slower time course and much larger decay time constants as compared to calcium transients16.
Set aside these drawbacks, sodium imaging proves to be a valuable tool for the investigation of physiological properties of neuronal subdomains. For example, sodium imaging can serve to monitor excitatory synaptic activity at or close to active synapses. Because sodium is essentially not buffered8,17, activity-induced sodium transients are linearly related to a wide range of synaptic glutamate release or exogenously applied glutamate. They hence represent direct and unbiased indicators of neuronal glutamatergic activity. Moreover, sodium indicator dyes exhibit high Kd&#8217;s (SBFI&#8217;s Kd is in the range of 25 mM1). Even at the relatively high intracellular dye concentrations used to achieve sufficient brightness (usually 0.5 - 1 mM), the high Kd&#8217;s imply that the dyes themselves do not act as buffers for sodium. Consequently, they do not distort the amplitude nor time course of sodium transients, which is always a concern when calcium-sensitive dyes are introduced into the cells18. It can thus be assumed that the detected signals represent a good measure of the &#8220;real&#8221; changes in intracellular sodium. Their slow time course then implies that the velocity for intracellular diffusion for sodium in neurons is much smaller than previously assumed19.
A critical requirement for the successful execution of experiments addressing the properties of excitatory synaptic transmission and sodium influx pathways into spines and dendrites is the induction of a fast and highly localized activation of postsynaptic structures. This can be obtained by fast and local application of glutamate or glutamate agonists in the direct vicinity of a spine or a dendrite by the flash photolysis of caged compounds. Flash photolysis does not mechanically damage the tissue, is free of movement artifacts and is well established for the investigation of related questions (e.g. local calcium signaling). The diameter of the uncaging spot was 1.5 &#181;m in the xy-plane. Uncaging close to a spiny dendrite induced sodium signals in both spines and the parent dendrite. Whereas the signals tended to be largest in spines closest to the uncaging spot, the amplitudes in the parent dendrite and spines further away were still rather similar to those in direct vicinity of the uncaging spot. Uncaging was performed for 300 msec during which inward currents increased in amplitude. Uncaged glutamate might have diffused in the tissue during the same time period, activating ionotropic glutamate receptors and sodium influx on dendritic regions and spines further away from the uncaging spot.
An intrinsic problem that occurs when using a UV laser for photolysis of caged compounds administered through the cell bathing solution is &#8216;inner filtering&#8217;. Because of the high absorption rate of the cage, UV light is attenuated strongly along the way from the objective to the stimulation site20,21. A feasible way to avoid this is local perfusion with the cage that is restricted only to the stimulation site, which furthermore decreases the amount of caged compound needed to a minimum. As compared to UV laser-scanning-mediated uncaging, near-infra-red two-photon uncaging22 is spatially more precise, mainly because the accuracy of the stimulation is increased in the z-axis. On the other hand, due to the small cross-section of commonly used cages for longer wavelengths as employed with two-photon excitation, either a high concentration of the caged compound or very high, potentially phototoxic light intensities are needed. Also, two-photon uncaging necessitates a much higher investment in the equipment; among others, an additional IR laser plus the required optical components, are needed.
Both SBFI and MNI-glutamate are excitable in the identical wavelength range. This may lead to an unintended interdependency of UV uncaging laser and SBFI or IR imaging laser and MNI-glutamate, resulting in bleaching of SBFI by the uncaging flash and /or a continuous uncaging of glutamate by the IR beam. However, as shown in Figure 6C, neither a UV flash by itself nor the multi photon imaging caused such reciprocal effects under our experimental conditions.
UV-flash systems similar to the one described here are used routinely in many other laboratories and can relatively easily be incorporated into any existing multi-photon imaging microscope. It offers the advantage that the laser beam for photolysis can be positioned freely in the field of view. In addition, the system enables a fast and automated repositioning of the laser beam and the release volume, respectively, in the field of view during an experiment. Our modification simplifies and improves the accurate positioning of the uncaging spot, so that the frames of the imaging software can serve to adjust imaging and uncaging frames congruently.
Taken together, whole-cell patch-clamp and multi-photon sodium imaging in dendrites and spines of central neurons, combined with a modified procedure for UV-light-induced uncaging of glutamate, allows reliable and focal activation of glutamate receptors in the tissue. It can thus serve to analyze the properties of excitatory synaptic transmission and of postsynaptic sodium signals in neurons in the intact tissue.

De senaste förbättringarna i lätta mikroskopiska tekniker såsom flera foton mikroskopi har gjort det möjligt att studera morfologiska och fysiologiska parametrar av hjärnceller i intakt vävnad med hög spatial och temporal upplösning. Kombinerat med elektrofysiologi är dessa tekniker nu allmänt används för att analysera aktivitetsrelaterade elektriska signaler på neuroner liksom samtidig kalciumsignaler i små subcellulära fack, nämligen i fina dendriter och Dendritutskotten. Förutom kalcium transienter, synaptisk aktivitet inducerar också natrium signaler i dendriter och ryggar, egenskaper som är i stort sett outforskade. Sådana signaler kan analyseras med två-foton-avbildning av intracellulärt natrium ([Na +] i) som gör det möjligt att on-line-mätning av [Na +] i-transienter under längre tidsperioder utan signifikant färgblekning eller fotoskador 1,2. För avbildning av [Na +] i- </sub&gt;, bara några kemiska indikator färgämnen är tillgängliga, t.ex. corona Grön eller Asante Natrium Grön 3,4. Det mest använda fluorescerande sond för Na + avbildning är natrium-bindande bensofuran isoftalat (SBFI). Det är en ratiometrisk, UV-glada färgämne som liknar den välkända kalciumkänsliga färgämnet fura-2 och har varit anställd för konventionell Na + avbildning i många celltyper (t.ex. 5,6). Det finns olika möjligheter spännande färgämnet och samla sin fluorescens. Om hög tidsupplösning (dvs. en hög bildbildhastighet) krävs i kombination med geografisk information, kan SBFI vara glada med en xenonlampa eller en hög effekt lysdiod (LED)-enhet och dess utsläpp detekteras med en hög hastighet laddning -kopplade enhet (CCD) kamera 7,8. För maximal rumslig upplösning djup i vävnaden, är multi-photon laser scanning microscopy metoden för val 9. Den relativt låga kvant efficieNCY av SBFI nödvändiggör relativt höga färgämneskoncentrationer (0,5 till 2 mM) och direkt lastning av den membran ogenomtränglig form av SBFI via en skarp mikroelektrod 10 eller fläckpipetten 1. Använda SBFI, tidigare arbete som utförs i akut vävnadsskivor av gnagare hippocampus visade aktivitetsrelaterade natrium transienter i dendriter och ryggar av CA1 pyramidala nervceller som främst orsakas av inflödet av natrium genom jonotropa NMDA-receptorer 1,2. För att studera egenskaperna hos sådana lokala natrium signaler i mer detalj är specifik aktivering av postsynaptiska receptorer genom tillämpning av receptoragonister en väl lämpad metod. För att efterlikna presynaptisk aktivitet och frigör sändare, bör ansökan vara relativt korta och fokuserade för att möjliggöra lokal stimulering. Detta är emellertid visar sig vara ganska utmanande i intakt vävnad. Lokalt tryck tillämpning av receptoragonister med en spetsig pipett möjliggör mycket bränn apkomplikation, men är värd den potentiella risken för att producera rörelse strukturen av intresse (t.ex. såsom en dendrit eller Dendritutskotten), och på så sätt hindrar högupplösta bilder. Lämpligheten jontofores av neuroaktiva ämnen beror på deras elektriska egenskaper och höga ström amplituder kan producera cellulära artefakter också. Ett sätt att kringgå dessa hinder är anställning av fotoaktiverade föreningar och deras flash fotolys. I grunden är två olika principer som används för fotoaktivering av bur ämnen: I) Wide-field flash fotolys 11 och II) focal uncaging använder skanningsmoduler i kombination med laser 12. Medan brett fält flash fotolys används för att aktivera större regioner av intresse, t ex en hel cell, är fokal uncaging utnyttjas för att specifikt stimulera små cellulära avdelningar. I föreliggande studie visar vi ett förfarande för hel-cell patch-clamp och multi-pHoton natrium avbildning i dendriter och ryggar av centrala neuroner, i kombination med ett modifierat förfarande för UV-ljusinducerad uncaging av glutamat, vilket möjliggör tillförlitlig och fokal aktivering av glutamatreceptorer i vävnad.

<table border="0" cellpadding="0" cellspacing="0" width="1138">
	<tbody>
		<tr height="33">
			<td height="33" style="height:33px;width:239px;">Hadrware/Software Component</td>
			<td style="width:231px;">Manufacturer</td>
			<td style="width:183px;">Type</td>
			<td style="width:487px;">Comment</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Borosilicate-glass capillaries</td>
			<td>Hilgenberg</td>
			<td>1405059</td>
			<td>75 mm x 2 mm, wall thickness 0.3 mm</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Camera</td>
			<td>Jenoptik</td>
			<td>ProgRes MF</td>
			<td>IR-DIC compatible camera</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Deconvolution software</td>
			<td>SVI</td>
			<td>Huygens Pofessional X11</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Fiber optic spectrometer</td>
			<td>Ocean Optics</td>
			<td>USB 4000</td>
			<td>Miniature fiber optic spectrometer</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Filter tubes</td>
			<td>VWR</td>
			<td>cenrifugal filter, 0.2 &#181;m</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Imaging software</td>
			<td>Olympus Europe</td>
			<td>Flowview Ver 5.0c, Tiempo</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">IR beam power controller</td>
			<td>Custom built</td>
			<td></td>
			<td>Laser beam intensity control + fast switch to zero on flyback via pockels cell</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">IR laser</td>
			<td>SpectraPhysics</td>
			<td>Mai-Tai VF-N1S</td>
			<td>fs-pulsed Ti:Sa tunable laser (710-990 nm); CAUTION</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">IR power monitor</td>
			<td>Custom built</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Micromanipulator</td>
			<td>Burleigh</td>
			<td>PCS 5000</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Microscope/Imaging System</td>
			<td>Olympus Europe</td>
			<td>BX51WI/FV300</td>
			<td>Both, the microscope and the imaging systems were strongly modified</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">ND filter wheel</td>
			<td>Newport</td>
			<td>50Q04AV.2</td>
			<td>UV fused silica, optical density 0.05-2.0</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Objective</td>
			<td>Nikon Instruments Europe</td>
			<td>NIR Apo 60x/1.0w</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Patch clamp amplifier</td>
			<td>HEKA</td>
			<td>EPC-10</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Pipette puller</td>
			<td>Narishige</td>
			<td>Model PP-830</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Pneumatic drug ejection system</td>
			<td>NPI Electronic</td>
			<td>PDES-DXH</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Pockels cell</td>
			<td>Conoptics</td>
			<td>350-80 LA-BK</td>
			<td>EOM, used as fast switch and for power adjustment</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Pockels cell driver</td>
			<td>Conoptics</td>
			<td>M302-RM</td>
			<td>High voltage differential amplifier</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Shutter</td>
			<td>Vincent Associates</td>
			<td>LS6ZM2</td>
			<td>ALMgF2-coated BeCu blades</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Shutter controller</td>
			<td>Custom built</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Shutter driver</td>
			<td>Vincent Associates</td>
			<td>Uniblitz VCM D1</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Slicer / Vibratome</td>
			<td>Thermo Scientific</td>
			<td>Microm HM 650 V</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Uncaging software</td>
			<td>Rapp OptoElectronic</td>
			<td>UGA40 1.1.2.6 + NetCam</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">UV laser&#160;</td>
			<td>Rapp OptoElectronic</td>
			<td>DL-355/10</td>
			<td>cw DPSSL Laser, 355 nm; 10 mW; CAUTION</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">UV laser scanning system</td>
			<td>Rapp OptoElectronic</td>
			<td>UGA 40</td>
			<td>Galvanometric scanning system</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Name of Compound</td>
			<td>Catalog Number</td>
			<td></td>
			<td>Comments/Description</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">CNQX</td>
			<td>Sigma Aldrich</td>
			<td>C-127</td>
			<td>AMPA receptor antagonist; CAUTION</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">DL-AP5</td>
			<td>Alfa Aesar</td>
			<td>J64210</td>
			<td>NMDA receptor antagonist; CAUTION</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">SBFI K+ Salt</td>
			<td>Teflabs</td>
			<td>OO32</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">TTX</td>
			<td>Ascent Scientific</td>
			<td>Asc-055</td>
			<td>Inhibitor of voltage-dependent Na+ channels; CAUTION</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">NMI-caged-L-Glutamate</td>
			<td>Torcis</td>
			<td>1490</td>
			<td>Caged glutamatereleased upon UV absobtion</td>
		</tr>
	</tbody>
</table>

multi-photon intracellular sodium imaging combined with uv-mediated focal uncaging of glutamate in ca1 pyramidal neurons

Fler photon fluorescensmikroskopi har möjliggjort analys av morfologiska och fysiologiska parametrar hos hjärnceller i intakt vävnad med hög rumslig och temporal upplösning. I kombination med elektrofysiologi, är det allmänt används för att studera aktivitetsrelaterade kalciumsignaler i små subcellulära fack såsom dendriter och Dendritutskotten. Förutom kalcium transienter, synaptisk aktivitet inducerar också postsynaptiska natrium-signaler, vars egenskaper är endast marginellt förstås. Här beskriver vi en metod för kombinerad hel-cell patch-clamp och multifoton natrium avbildning i cellulära mikro domäner centrala neuroner. Vidare introducerar vi ett modifierat förfarande för ultraviolett (UV) -ljus-inducerad uncaging av glutamat, vilket möjliggör tillförlitlig och fokal aktivering av glutamatreceptorer i vävnad. För detta ändamål var helcells-inspelningar utförs på Cornu Ammonis delfältet 1 (CA1) pyramidala neuroner i akuta vävnadsskivor avmus hippocampus. Nervceller fylldes med natrium-känsliga fluorescerande färgämne SBFI genom patch-pipett, och multi-foton-excitation av SBFI gjorde det möjligt för visualisering av dendriter och angränsande ryggar. Att etablera UV-inducerad fokal uncaging har flera parametrar, bland annat ljusintensitet, volym påverkas av UV uncaging trålen, positionering av strålen samt koncentration av bur föreningen testas och optimeras. Våra resultat visar att lokal perfusion med bur glutamat (MNI-glutamat) och dess bränn UV-uncaging resultat i aktiv strömmar och natrium transienter i dendriter och ryggar. Tidsförlopp och amplitud både inåt strömmar och natrium-signaler korrelerar med den tid som uncaging pulsen. Vidare visar våra resultat att intracellulära natriumsignaler blockeras i närvaro av blockerare för jonotropa glutamatreceptorer, vilket visar att de förmedlas av natrium tillströmning även om denna väg. Sammanfattningsvis ger vår metod ett pålitligt verktyg förundersökning av intracellulära natrium signaler inducerade av fokal receptoraktivering i intakt hjärnvävnad.

In the present study, we demonstrate a procedure for multi-photon microscopy of cellular sodium dynamics with the sodium-dependent fluorescent dye SBFI in CA1 pyramidal neurons of acute mouse hippocampal tissue slices. Moreover, we show how to combine this imaging technique with laser-scanning-based uncaging of neuro-active compounds (e.g. caged glutamate) and its precise targeting to cellular micro-domains.
Loading neurons with SBFI through the patch-pipette enabled the visualization of the entire cell including fine dendrites and adjacent spines by employing multi-photon excitation (Figure 6A, B, D and Figure 7A).
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/52038/52038fig6highres.jpg" width="500" /> 
Figure 6. Sodium signals and synaptic currents induced by flash photolysis of caged glutamate. (A) Maximal projection image of a CA1 pyramidal neuron loaded with SBFI via the patch pipette (PP). The box indicates the area shown enlarged in B. LP indicates the position and orientation of the pipette for local perfusion of caged glutamate. (B) High power maximum projection of a stack of optical sections of a dendrite with adjacent dendritic spines. The stacks of optical sections underwent z-alignment and deconvolution. The red cross indicates the target region of the uncaging beam. The orange dotted line delineates the region of interest from which the fluorescence emission was recorded. The box indicates the area shown enlarged in D. (C) Left: Sodium signal (upper row) and somatic inward current (lower row) induced by flash photolysis of caged glutamate (indicated by yellow flash). The red line represents a fit of the experimental data. The grey area represents the period in which the uncaging flash (300 msec) obstructed the imaging of SBFI fluorescence. Right: Without pre-perfusion with caged glutamate, the same UV-flash neither evoked a change in SBFI emission, nor an inward current. (D) Left: High power image of the dendrite and adjacent spines as delineated in B. Alongside, the same image with inverted grey values. The dotted lines indicate the regions of interest in which the fluorescence emission was recorded. The red cross indicates the localization of the uncaging beam. Right: Sodium transients induced by UV-flash photolysis of caged glutamate. Blue trace: sodium signals in the dendrite; red trace: averaged response from three spines adjacent to the uncaging spot; green trace: averaged response from three distant spines. <a href="https://www.jove.com/files/ftp_upload/52038/52038fig6highres.jpg" target="_blank">Please click here for a larger version of this figure.</a>
After local perfusion with caged glutamate, applying a UV-flash close to a dendrite resulted in a transient decrease in fluorescence emission of SBFI, reflecting an increase in the intracellular sodium concentration (Figure 6C, D and Figure 7B). At the same time, an inward current was recorded at the soma (Figure 6C and Figure 7B). Increasing the duration of the UV flash resulted in increasing amplitudes of both the elicited inward currents and the sodium signals (data not shown), indicating that the system was well within its dynamic range and that neither uncaging nor cellular responses were saturated. Application of a UV-flash of identical or longer duration to slices which had not been pre-perfused with caged glutamate, never elicited changes in SBFI fluorescence nor inward currents, indicating that these signals are due to the uncaging of glutamate (Figure 6C). Moreover, these results show that no interdependency of imaging- and uncaging components is observed under our experimental conditions. Sodium signals could also be detected in dendritic spines (Figure 6D). While we did not attempt to achieve stimulation of a single spine only with glutamate uncaging, peak amplitudes tended to be slightly higher in spines close to the uncaging spot, whereas spines further away showed virtually identical fluorescence changes as the parent dendrite (Figure 6D).
Finally, we studied the pathway for sodium influx into dendrites and spines in response to uncaging of glutamate. To this end, we employed CNQX and APV, which are selective blockers for the sodium-permeable, ionotropic glutamate receptors of the AMPA- and NMDA-subtype, respectively. Our results show that glutamate-induced intracellular sodium signals and the elicited somatic currents were omitted in the presence of these blockers (Figure 7B). Upon wash-out of the blockers, the signals are regained. This demonstrates that uncaging of glutamate activates ionotropic glutamate receptors on CA1 pyramidal neurons, which mediate the influx of sodium into dendrites and spines, resulting in intracellular sodium transients and inward currents.
<img alt="Figure 7" fo:content-width="6in" src="/files/ftp_upload/52038/52038fig7highres.jpg" width="600" /> 
Figure 7. Pharmacological profile of evoked sodium signals and inward currents. (A) SBFI-filled CA1 pyramidal neuron with patch pipette (PP) attached to the soma. (B) Left: Sodium transient and somatic current induced by photo-activation of caged glutamate in a dendrite and attached spines. Center: Perfusion with the ionotropic glutamate receptor blockers CNQX and APV inhibits both the sodium signal and the inward current induced by uncaging. Right: Upon wash-out of the blockers, the signals are restored. The red line represents a fit of the experimental data. The grey area represents the period in which the uncaging flash (300 msec) obstructed the imaging of SBFI fluorescence. <a href="https://www.jove.com/files/ftp_upload/52038/52038fig7highres.jpg" target="_blank">Please click here for a larger version of this figure.</a>

Watch this Scientific Journal Video about Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons at JoVE.com

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

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Optisk avbildning av nervceller i Crab Stomatogastric Ganglion med spänningskänsliga Färgämnen

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

En metod för High Fidelity Optogenetic kontroll av enskilda pyramidala nervceller<em&gt; In vivo</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

Intracellulär inspelning, Sensorisk Field Mapping, och odling identifierade nervceller i Leech, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

Snabb Micro-iontophoresis av glutamat och GABA: Ett användbart verktyg för att undersöka Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

Funktionell Förhör av Adult Hypothalamic neurogenes med Focal Radiological Hämning

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Lukt Nervceller erhållits genom Nasal Biopsi Kombinerad med Laser-Capture Microdissection: En Potentiell Approach att studera behandlingssvar hos psykiska störningar

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

3-D Imaging och analys av nervceller Infekterade<em&gt; In Vivo</em&gt; Med<em&gt; Toxoplasma gondii</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Kombinerade optogenetic och Freeze-fraktur Replica immunomärkning att granska Input specifika Arrangemang av glutamatreceptorer i mus Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

Proportionerlig kalcium avbildning av enskilda nervceller i beter sig Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Två-foton avbildning av kortikala nervceller hos neonatala möss

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...