The fluorescent false neurotransmitter. FFN 511 is the fluorescent monoamine that can act as a substrate for the synaptic physical monoamine transporter when applied to brain slices. FN 511 is taken up by dopaminergic synaptic vesicles via the monoamine transporter and can be used to visualize transmitter release directly from presynaptic terminals of mono Americ neurons such as those in the S stratum transmitter release from terminals is visible in the slice as a reduction in punctate fluorescent signals.
In this video, 250 micrometer thick coronal sal slices are prepared using a vibrato stained with FFN 511 and imaged using a multiphoton laser microscope. Detaining experiments are shown which reveal a frequency dependent heterogeneity of iCal release. Hi, I am HU from David s Lab in the Department of Neurology at Columbia University.
Hi, I'm Minerva also from the Soldier Lab. Today we'll show you a procedure for labeling dopamine terminals in brain slices with fluorescence false neurotransmitters. We use this procedure in our laboratory to Study dopamine release.
So let's get Started. Before preparing acute sal slices, one must oxygenate the artificial cerebral spinal fluid A CSF and chill it with ice For at least 15 minutes prior to the brain extraction, keep the A CSF ice cold and oxygenated During the dissection, extract the whole brain from the mouse and mounted on the vibram tray, placed on the vibram using crazy glue. Brain extraction and mounting must be done within two minutes.
Fill the tray with chilled oxygenated A CSF cut coronal straight or brain slices at 250 micrometer thickness between plus 1.54 to plus naught. Point six two three hole slices will be obtained, which can be cut into six half slices with a needle. Incubate the brain slices in oxygenated A CSF at room temperature for at least one hour while the brain slices are being incubated.
In oxygenated A CSF, prepare the 10 micromolar FFN 511 loading solution by adding 10 millimolar FFN 511 stock solution to the A CSF. After that, prepare a solution of a hundred micromolar A-D-V-A-S-E-P seven in A CSF oxygenate both solutions for at least 15 minutes prior to loading into the slices individually. Incubate each brain slice with FFN 511 loading solution for 30 minutes at room temperature.
Next, remove the di bound to extracellular tissue by incubating the loaded slice in oxygenated 100 micromolar advers, seven A CSF for 30 minutes at room temperature slices are now ready for imaging FFN 511 Staining will be imaged in the slices using a prairie Ultima Multiphoton laser scanning microscope. Place the FFN 511 loaded slice in the recording chamber and super fused with oxygenated A CSF at a flow rate of one to two milliliters per minute. Then place a platinum harp with nylon strings on top of the slice to minimize movement.
During the experiment to further minimize movement, allow the slices to stabilize for at least 10 minutes in the chamber prior to imaging. Visualize and locate the dorsal stri AUM with brightfield luminescence under a 10 x water immersion objective place and position the bipolar twisted tungsten electrodes in this region for performing a stimulation experiment. The idle imaging area is located within 300 micrometers.
In between the two tips of the stimulating bipolar electrode place this region in the center of the visual field image FFN 511 labeled Sal terminals under a 63 x water immersion ultraviolet objective FFN 511 is excited at 760 nanometers with a mi tai laser from spectral physics. Optimal fluorescence of SAL terminals is seen through a band pass filter capture images in 12 bit format with 75 by 75 micrometer regions of interest at 512 by 512 pixel resolution. An example image with Sal dopamine terminals labeled with FFN 511 is shown here.
The FFN 511 label can be distained by either high concentrations of potassium chloride amphetamine sulfate or by electrical stimulation in the potassium chloride detaining experiment when high potassium A CSF solution is applied to the brain slice the FFN 511 label disdains within four minutes. Record XYZT images to track FFN 511 detaining over time for FFN 511 staining by electrical stimulation. Obtain a control time series image of the Zack prior to stimulation for at least five minutes to ensure minimal movement of the slice and no photo bleaching by the laser following acquisition of the control images.
Continue the XYZT imaging while starting the stimulation at frequencies of one four or 20 hertz stimuli are applied to the striatum locally by an isof lex stimulus isolator triggered by a master eight pulse generator using by polar electrodes. This video shows the disdain of FFN 511 during exocytosis in a sal brain slice. Preparation when FFN 511 loading into the SAL is complete abundant labeling in the striatum sparse labeling in the cortex and no labeling in the corpus callosum are observed.
The selective labeling of dopamine terminals can be distained by high potassium chloride as shown in these representative images before potassium chloride and two minutes after the application of 70 millimolar potassium chloride. A CSF detaining of FFN 511 labeling can also be accomplished by amphetamine. And this image shows FFN 511 labeling in the striatum before amphetamine.
While this image shows the striatum 20 minutes after detaining with 20 micromolar amphetamine detaining of FFN 511 labeling by local electrical stimulation is illustrated in these images that show FFN 511 labeling in the stratum before stimulation and at 217 seconds and 514 seconds after the start of stimulation at four hertz. This graph of normalized fluorescence intensity over time shows that detaining of FFN 511 is calcium and frequency dependent controls represented by the unfilled triangles receive no stimulation. Detaining with cadmium chloride represented by unfilled circles was identical to unstimulated controls.
The disdaining curves for each stimulation frequency represented by the colored circles were fitted by a single exponential decay function and half-life values calculated as tau by Naut 0.693. We have just shown you how to load a fluorescent force neurotransmitter FFN five 11 into dopamine terminals in the acute al slice and how to release it by high potassium and evoke the stimulation. This is the first mean developed to examine neurotransmitter release optically at the level of individual synaptic terminals.
When Doing this procedure, it's important to remember to keep the slices very healthy and be conscious of loading time, bleaching background and movement artifacts, slices, display good FFN five 11 loading and imaging up to four hours after slice preparation. So that's it. Thanks for watching and good luck with your experiments.
