Name: real-time fluorescent measurement of synaptic functions in models of amyotrophic lateral sclerosis
Uploaded: 2021-07-16T14:30:00
Description: Watch this Scientific Journal Video about Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis at JoVE.com

We would like to acknowledge the present and former members of the Jefferson Weinberg ALS Center for critical feedback and suggestions for optimizing these techniques and their analyses. This work was supported by funding from the NIH (RF1-AG057882-01 and R21-NS0103118 to D.T), the NINDS (R56-NS092572 and R01-NS109150 to P.P), the Muscular Dystrophy Association (D.T.), the Robert Packard Center for ALS Research (D.T.), the Family Strong 4 ALS foundation and the Farber Family Foundation (B.K.J., K.K, and P.P).

All animal procedures performed in this study were approved by the Institutional Animal Care and Use Committee of Jefferson University.
1. Primary culture of neurons from embryonic rat cortex
NOTE: Primary cortical neurons are isolated from E17.5 rat embryos as previously described57,58. No strain bias appears to exist with the success of this culturing protocol. This method is described briefly below. The pre...

Three steps common to both methods described are of crucial importance for experimental success and quantifiable outcomes. First, preparation of fresh aCSF before each round of experiments is essential, following the attached instructions. Failure to do so may prevent appropriate neuronal depolarization. A sample of untreated control neurons should constantly be tested before stimulation of any experimental groups to ensure proper cellular depolarization and provide a benchmark for positive results obtained in that imagi...

Modeling amyotrophic lateral sclerosis (ALS) in the laboratory is made uniquely challenging due to the overwhelmingly sporadic nature of over 80% of cases1, coupled with the vast number of genetic mutations known to be disease-causative2. Despite this, all cases of ALS share the unifying feature that before outright neuronal degeneration, there is dysfunctional communication between presynaptic motor neurons and postsynaptic muscle cells3,4. Clinically, as patients lose connectivity of the remaining upper and lower motor neurons, they present with features of neuronal hyper- and hypoexcitability throughout the disease5,6,7,8,9, reflecting complex underlying molecular changes to these synapses, which we, as ALS researchers, seek to understand.
Multiple transgenic models have illustrated that deterioration and disorganization of the neuromuscular junction occur with the expression of ALS-causative genetic mutations, including SOD110, FUS11,12, C9orf7213,14,15,16, and TDP4317,18,19 through morphological assessments, including evaluation of synaptic boutons, spine densities, and pre/postsynaptic organization. Mechanistically, since the landmark papers of Cole, Hodgkin, and Huxley in the 1930s, it has also been possible to evaluate synaptic responses through electrophysiological techniques in either in vitro cell culture or tissue slice preparations20. Through these strategies, many models of ALS have demonstrated synaptic transmission deficits. For example, a mutant variant of TDP43 causes enhanced firing frequency and decreases action potential threshold in NSC-34 (spinal cord x neuroblastoma hybrid cell line 34) motor-neuron-like cells21. This same variant also causes dysfunctional synaptic transmission at the neuromuscular junction (NMJ) before the onset of behavioral motor deficits in a mouse model22. It was previously showed that mutant FUS expression results in reduced synaptic transmission at the NMJ in a drosophila model of FUS-ALS before locomotor defects11. A recent report using induced pluripotent stem cells derived from C9orf72-expansion carriers revealed a reduction in the readily releasable pool of synaptic vesicles23. Altogether, these studies and others highlight the importance of building a more comprehensive understanding of the mechanisms underlying synaptic signaling in disease-relevant models of ALS. This will be pivotal in understanding the pathobiology of ALS and developing potential therapeutic targets for patients.
Methods of current and voltage clamping cells have been invaluable in determining membrane properties such as conductance, resting membrane potential, and quantal content of individual synapses20,24. However, one of the significant limitations of electrophysiology is that it is technically challenging and only provides insights from a single neuron at a time. Live-cell confocal microscopy, coupled with specific fluorescent probes, offers the opportunity to investigate the synaptic transmission of neurons in a spatiotemporal manner25,26,27. Although not a direct measure of neuronal excitability, this fluorescence approach can provide a relative measurement of two molecular correlations of synaptic function: synaptic vesicle release and calcium transients at synaptic terminals.
When an action potential reaches the presynaptic terminal region of neurons, calcium transients are triggered, facilitating the transition from an electrical signal to the process of neurotransmitter release28. Voltage-gated calcium channels localized to these areas tightly regulate calcium signaling to modulate the kinetics of neurotransmitter release29. The first reported fluorescence-based recordings of calcium transients were performed using either the dual-wavelength indicator Fura-2 AM or the single wavelength dye Fluo-3 AM30,31,32. While these dyes offered great new insight at the time, they suffer from several limitations such as non-specific compartmentalization within cells, active or passive dye loss from labeled cells, photobleaching, and toxicity if imaged over extended periods of time33. In the past decade, genetically encoded calcium indicators have become the workhorses for imaging various forms of neuronal activity. These indicators combine a modified fluorescent protein with a calcium chelator protein that rapidly switches fluorescence intensity after the binding of Ca2+ ions34. The application of these new indicators is vast, allowing for much easier visualization of intracellular calcium transients both in in vitro and in vivo settings. One family of these genetically encoded reporters, known as GCaMP, are now broadly utilized. These indicators contain a C-terminal calmodulin domain, followed by green fluorescent protein (GFP), and are capped by an N-terminal calmodulin-binding region35,36. Calcium-binding to the calmodulin domain triggers an interaction with the calmodulin-binding region, resulting in a conformational change in the overall protein structure and a substantial increase in the fluorescence of the GFP moiety35,36. Over the years, this family of reporters has undergone several evolutions to enable distinct readouts for particular calcium transients with specific kinetics (slow, medium, and fast), each with slightly different properties37,38. Here, the usage of the reporter GcaMP6 has been highlighted, which has been previously shown to detect single action potentials and dendritic calcium transients in neurons both in vivo and in vitro37.
Calcium transients in the presynaptic region trigger synaptic vesicle fusion events, causing neurotransmitter release into the synapse and initiation of signaling events in the postsynaptic cell28,39. Synaptic vesicles are both rapidly released and recycled, as the cell homeostatically maintains a stable cell membrane surface area and readily releasable pool of fusion capable membrane-bound vesicles40. The styryl dye used here has an affinity toward lipid membranes and specifically changes its emission properties based on the ordering of the surrounding lipid environment41,42. Thus, it is an ideal tool for labeling recycling synaptic vesicles and subsequent tracking of these vesicles as they are later released following neuronal stimulation41,42. The protocol that has been generated and optimized is an adaptation of the concepts described initially by Gaffield and colleagues, which allows us to visualize styryl dye-labeled synaptic vesicle puncta over time continuously41.
Here, two related fluorescence-based methodologies are described, reliably reporting specific cellular events involved in synaptic transmission. Protocols have been defined to probe the dynamics of depolarization-mediated presynaptic terminal calcium influx and synaptic vesicle exocytosis in cultured neurons. Here, methods and representative results are focused on using primary rodent cortical or motor neurons as the in vitro model system, as there are published studies using these cell types43,44. However, these methods are also applicable to differentiated human i3 cortical-like neurons45, as we have also had success with both protocols in presently ongoing experimentation in our laboratory. The general protocol is outlined in a stepwise linear format, shown in Figure 1. In brief, to study calcium dynamics in neurites, mature neurons are transfected with plasmid DNA to express the fluorescent reporter GCaMP6m under a Cytomegalovirus (CMV) promoter37,46. Transfected cells have a low level of basal green fluorescence, which increases in the presence of calcium. Regions of interest are specified to monitor fluorescence changes throughout our manipulation. This allows for highly spatially and temporally localized fluctuations in calcium to be measured37,46. For evaluating synaptic vesicle fusion and release, mature neurons are loaded with styryl dye incorporated into synaptic vesicle membranes as they are recycled, reformed, and reloaded with neurotransmitters in presynaptic cells41,42,43,47,48. The current dyes used for this purpose label synaptic vesicles along neurites and are used as a proxy for these regions in live-imaging experiments, as was shown by co-staining of styryl dye and synaptotagmin by Kraszewski and colleagues49. Included here are representative images of similar staining that have also been performed (Figure 2A). Previous investigators have extensively used such dyes to report synaptic vesicle dynamics at the neuromuscular junction and hippocampal neurons48,49,50,51,52,53,54,55,56. By selecting punctate regions of dye-loaded vesicles and by monitoring decreases in fluorescence intensity following vesicle release, functional synaptic transmission capacity and temporal dynamics of release can be studied following stimulation43. For both methods, a medium containing a high concentration of potassium chloride is employed to depolarize cells to mimic neuronal activity. Imaging parameters are specified to capture sub-second intervals spanning a baseline normalization followed by our stimulation capture period. Fluorescence measurements at each time point are determined, normalized to the background, and quantified over the experimental time period. Calcium-influx mediated GCaMP6m fluorescence increase or effective synaptic vesicle exocytosis styryl dye release fluorescence decrease can be detected through this strategy. Detailed methodological setup and parameters for these two protocols and a discussion on their advantages and limitations are described below.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62813/62813fig01.jpg" /> 
Figure 1:&#160;Visual rendering of overall general protocol process. (1) Isolate and culture primary rodent neurons in vitro to chosen maturation timepoint. (2) Introduce GCaMP DNA or styryl dye as reporters of synaptic activity. (3) Setup imaging paradigm using live-imaging equipped confocal microscope and associated software. Begin baseline recording period. (4) While cells are still undergoing live-image capture, stimulate neurons via high KCl bath perfusion. (5) Assess fluorescence intensity measurements over time to measure calcium transients or synaptic vesicle fusion. <a href="https://www.jove.com/files/ftp_upload/62813/62813fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20x air objective</td>
			<td>Nikon</td>
			<td></td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>40x oil immersion objective</td>
			<td>Nikon</td>
			<td></td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Scientific</td>
			<td>17504044</td>
			<td>Neuronal growth supplement</td>
		</tr>
		<tr>
			<td>BD Syringes without Needle, 50 mL</td>
			<td>Thermo Scientific</td>
			<td>13-689-8</td>
			<td>Part of gravity perfusion assembly</td>
		</tr>
		<tr>
			<td>Biosafety cell culture hood</td>
			<td>Baker</td>
			<td>SterilGARD III SG403A</td>
			<td>Asceptic cell culturing, transfection, and dye loading</td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol</td>
			<td>Millipore Sigma</td>
			<td>M3148</td>
			<td>For culturing and maintenance of neuronal cultures</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Millipore Sigma</td>
			<td>A9418</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Millipore Sigma</td>
			<td>223506</td>
			<td>Component of aCSF solutions</td>
		</tr>
		<tr>
			<td>Cell culture CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>13-998-123</td>
			<td>For culturing and maintenance of neurons</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>For neuronal culture preparation</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti +A1R core</td>
			<td>For fluorescence imaging</td>
		</tr>
		<tr>
			<td>CoolSNAP ES2&#160; CCD camera</td>
			<td>Photometrics</td>
			<td></td>
			<td>For image acquisition</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Millipore Sigma</td>
			<td>G8270</td>
			<td>Component of aCSF solutions</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Millipore Sigma</td>
			<td>D5025</td>
			<td>For neuronal culture preparation</td>
		</tr>
		<tr>
			<td>Female, timed-pregnancy Sprague Dawley rats</td>
			<td>Charles river</td>
			<td>400SASSD</td>
			<td>For preparing embryonic cortical and spinal motor neuron cultures</td>
		</tr>
		<tr>
			<td>FITC Filter cube</td>
			<td>Nikon</td>
			<td>77032509</td>
			<td>For imaging Gcamp calcium transients</td>
		</tr>
		<tr>
			<td>FM4-64 styryl dye</td>
			<td>Invitrogen</td>
			<td>T13320</td>
			<td>For imaging synaptic vesicle release</td>
		</tr>
		<tr>
			<td>Glass bottom petri dishes (Thickness #1.5)</td>
			<td>CellVis</td>
			<td>D35-10-1.5-N</td>
			<td>For growth of neurons on imaging-compatible culture dish</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Grainger</td>
			<td>52NK56</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Millipore Sigma</td>
			<td>H6648</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td>Component of aCSF solutions</td>
		</tr>
		<tr>
			<td>High KCl artifical cerebrospinal fluid (aCSF)</td>
			<td></td>
			<td></td>
			<td>For imaging. Please see recipes*</td>
		</tr>
		<tr>
			<td>horse serum</td>
			<td>Millipore Sigma</td>
			<td>H1138</td>
			<td>For culturing and maintenance of neurons</td>
		</tr>
		<tr>
			<td>Laminar flow dissection hood</td>
			<td>NUAIRE</td>
			<td>NU-301-630</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermo Scientific</td>
			<td>23017015</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Thermo Scientific</td>
			<td>11415064</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td>Thermo Scientific</td>
			<td>21083027</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Thermo Scientific</td>
			<td>25030149</td>
			<td>Neuronal culture supplement</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Thermo Scientific</td>
			<td>11668019</td>
			<td>For neuronal transfections</td>
		</tr>
		<tr>
			<td>Low KCl artifical cerebrospinal fluid (aCSF)</td>
			<td></td>
			<td></td>
			<td>For imaging. Please see recipes*</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Millipore Sigma</td>
			<td>208337</td>
			<td>Component of aCSF solutions</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Software for data analysis/normalization</td>
		</tr>
		<tr>
			<td>Nalgene&#160; Filter Units, 0.2 &#181;m PES</td>
			<td>Thermo Scientific</td>
			<td>565-0020</td>
			<td>Filter unit for aCSF solution</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Scientific</td>
			<td>21103049</td>
			<td>For culturing and maintenance of neuronal cultures</td>
		</tr>
		<tr>
			<td>NIS-Elements Advanced Research</td>
			<td>Nikon</td>
			<td></td>
			<td>Software for image capture and analysis</td>
		</tr>
		<tr>
			<td>Nunc 15 mL Conical tubes</td>
			<td>Thermo Scientific</td>
			<td>339650</td>
			<td>For preparing neuronal culture and buffer solutions</td>
		</tr>
		<tr>
			<td>Nunc 50 mL conical tubes</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td>For preparing neuronal culture and buffer solutions</td>
		</tr>
		<tr>
			<td>Optiprep</td>
			<td>Millipore Sigma</td>
			<td>D1556</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Millipore Sigma</td>
			<td>P4762</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Scientific</td>
			<td>15140122</td>
			<td>To prevent bacterial contamination of neuronal cultures</td>
		</tr>
		<tr>
			<td>Perfusion system</td>
			<td>Warner Instruments</td>
			<td>SF-77B</td>
			<td>For exchange of aCSF</td>
		</tr>
		<tr>
			<td>Perfusion tubing</td>
			<td>Cole-Parmer</td>
			<td>UX-30526-14</td>
			<td>Part of gravity perfusion assembly</td>
		</tr>
		<tr>
			<td>pGP-CMV-Gcamp6m plasmid</td>
			<td>Addgene</td>
			<td>40754</td>
			<td>For imaging calcium transients</td>
		</tr>
		<tr>
			<td>Poly-D-lysine hydrobromide</td>
			<td>Millipore Sigma</td>
			<td>P7886</td>
			<td>Coating agent for glass bottom petri dishes</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Millipore Sigma</td>
			<td>P3911</td>
			<td>Component of aCSF solutions</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Millipore Sigma</td>
			<td>S5761</td>
			<td>Component of aCSF solutions</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td>Component of aCSF solutions</td>
		</tr>
		<tr>
			<td>Stage Top Incubator</td>
			<td>Tokai Hit</td>
			<td></td>
			<td>For incubation of live neurons during imaging period</td>
		</tr>
		<tr>
			<td>TRITC Filter cube</td>
			<td>Nikon</td>
			<td>77032809</td>
			<td>For imaging FM4-64</td>
		</tr>
		<tr>
			<td>Trypsin Inhibitor</td>
			<td>Millipore Sigma</td>
			<td>T6414</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Scientific</td>
			<td>25200056</td>
			<td>For preparing neuronal cultures</td>
		</tr>
		<tr>
			<td>Vibration Isolation table</td>
			<td>New Port</td>
			<td>VIP320X2430-135520</td>
			<td>Table/stand for microscope</td>
		</tr>
	</tbody>
</table>

real-time fluorescent measurement of synaptic functions in models of amyotrophic lateral sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Following the successful implementation of the above protocol, representative results are shown for a typical styryl dye synaptic vesicle release experiment. Cultured rat primary cortical neurons were loaded with dye using the method described in section 6. The specificity of dye loading was determined by co-labeling with synaptic vesicle marker synaptophysin. A majority of styryl dye positive puncta are co-positive for this marker (Figure 2A). To determine whether the settings used for styr...

Watch this Scientific Journal Video about Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis at JoVE.com

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

These methods enable quick assessment as to whether an experimental manipulation, disease causative protein, or RNA can impact synaptic processes, and whether therapeutic compounds can restore function. These techniques provide a reliable readout of synaptic function from a group of neurons in a relatively short period of time compared to electrophysiology. This protocol can be applied to any disease of neuronal development or degeneration.This works well for primary rodent neuronal cultures, and for neurons derived from induced pluripotent stem cells. To begin, optimize the image acquisition settings using confocal imaging acquisition software. Keep excitation power exposure time, detector gain and frame rate constant across all samples.For time lapse imaging, select an aspect ratio of 512 by 512, and a frame rate of two images per second to minimize dye bleaching. Then select the fluorescence excitation, dichroic and emission filter combinations for GCaMP six M/GCaMP three with Fitzy and stearoyl dye with Trixi. To avoid Z drift during time lapse imaging, use the perfect focus feature of the confocal imaging acquisition software.Next, select the time tab in the image acquisition panel. For phase one, set interval 500 milliseconds and duration three to five minutes. And for phase two, set interval 500 milliseconds and duration, five minutes.Then to assemble the gravity perfusion apparatus for artificial cerebrospinal fluid or ACSF, load the high potassium chloride ACSF buffer into a 50 milliliter syringe at the top of the apparatus and set the flow rate to one milliliter per minute. Then, load a 35 milliliter glass dish containing neurons onto the confocal imaging stage with the end of the perfusion tubing placed at the dish edge and choose the field for imaging. 48 hours post transfection, incubate the primary cortical or motor neurons in low potassium chloride ACSF buffer for 10 minutes at 37 degrees Celsius.Then remove the buffer by aspiration and using a pipette, load the neurons in the dark on a glass bottom Petri dish filled with ACSF containing 50 millimolar potassium chloride and 10 Micro molar styryl dye. After five minutes, remove the loading solution and bath the neurons in low potassium chloride ACSF buffer at 37 degrees Celsius for 10 minutes to eliminate the nonspecific dye loading. Then place the dish onto the imaging stage of the inverted confocal microscope and observe the cells under a 20 times air objective or 40 times oil immersion objective.Excite the steel dye using a 546 nanometer laser and collect emission using a 570 to 620 nanometer bandpass filter. After selecting the imaging field and engaging perfect focus, take a single still image with bright field, Trixi and fluorescence marker channels to mark neuronal boundaries. Then initiate Run Now in the acquisition software and carry out the basal recording for three to five minutes to exclude variations in dye intensity.Right at the switch to phase two, trigger the on button for the perfusion system and constantly perfuse 50 millimolar potassium chloride to the neurons to facilitate dye unloading. Carry out the recordings for five minutes then trigger the off switch for the perfusion system. Save the experiment for data analysis later using the confocal software.48 hours post transfection with GCaMP six M, incubate the primary rodent cortical neurons with low potassium chloride ACSF for 15 minutes. Then mount the dish on the imaging platform and visualized GCaMP six M fluorescence using a Fitzy filter and a 20 times or 40 times objective. After selecting the imaging field and engaging perfect focus, take a single still image with brightfield, Fitzy, and fluorescence marker channels to mark neuronal boundaries.Next, initiate Run Now in the acquisition software and carry out the basal recording for three to five minutes. Then perfuse ACSF containing 50 millimolar potassium chloride to the neurons as demonstrated previously and record for five minutes. When the image acquisition stops, save the experiment and proceed to data analysis using the confocal software.For image analysis, open the time lapse images in the confocal software and align the images by clicking on Image, followed by processing, followed by align current document, then select Align to the first frame. Select regions of interest along the neurites using the ROI selection tool and also mark an ROI representing background fluorescence intensity. Next, initiate the measure function from the time measurement panel to measure raw fluorescence over time for the selected ROIs.After measurement export the raw fluorescence intensities to the spreadsheet software. Cultured rat primary cortical neurons loaded with styryl dye are shown here. The specificity of dye loading was determined by co labeling with synaptic vesicle marker synaptophysin and the majority of sterile dye positive puncta are co positive for this marker.The analysis of the raw intensity values over the entire imaging period reveals that synaptophysin and intensity remains constant while styryl dye intensity decreases following stimulation. For green fluorescent protein transfected control neurons, successful synaptic vesicle release resulted in the striking loss of dye fluorescence upon high potassium chloride depolarization. This representative video shows a neuron selectively unloading styryl dye in a new right region following stimulation.In contrast, in neurons transfected with a C nine ORF 72 linked to dye peptide repeat construct to GA 50, impaired synaptic transmission is represented by retained dye fluorescence even after high potassium chloride depolarization. Representative fluorescence images of cortical neurons transfected with GCaMP six M before and after potassium chloride depolarization are shown here. Increased fluorescence values and cortical neurons transfected with GCaMP six M indicate calcium entry into neurites following potassium chloride inducted depolarization.At the end of the baseline recording period, the neurons expressed GCaMP six M with a low fluorescence. A dramatic fluorescence increase is then observed at the start of stimulation. Make sure fluorescence baseline recordings for styryl dye and GCaMP are stable.Always use perfect focus to avoid image drift, which would make post image data processing challenging. Further culturing of neurons is not recommended, as dishes are exposed to air during profusion. However, immuno staining or extraction of protein or RNA can assess potential causes of synaptic alterations.

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The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...