Name: probing nicotinic acetylcholine receptor function in mouse brain slices via laser flash photolysis of photoactivatable nicotine
Uploaded: 2019-01-25T13:00:00.000+00:00
Duration: 10 min 48 s
Description: Watch this Scientific Journal Video about Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine at JoVE.com

The authors thank laboratory members of the following Northwestern principal investigators: Ryan Drenan, D. James Surmeier, Yevgenia Kozorovitskiy, and Anis Contractor. This work was supported by the US National Institutes of Health (NIH) (grants DA035942 and DA040626 to R.M.D.), the PhRMA Foundation (fellowship to M.C.A.), and HHMI.

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D.L.W. serves as a paid consultant for Bruker Nano Fluorescence Microscopy.

The choice of PA-Nic application/delivery method is the most critical step in this localized photo-stimulation technique. The two methods, bath application and local perfusion, each offer distinct advantages and limitations. The choice is largely impacted by the nAChR functional expression level in the cell type of interest. It is often preferable to use bath application when functional expression levels are high, as bath application allows for a uniform probe concentration surrounding the recorded cell, facilitating data interpretation. Bath application also eliminates the need for a second perfusion pipette in the tissue, making the entire process easier. However, bath application of expensive compounds costs more per experiment.
Commonly, troubleshooting involves trying to understand why no nAChR activation is seen following flash photolysis. When working with a cell type that has not been previously studied with PA-Nic, the investigator should perform local puff-application of ACh or nicotine to determine whether sufficient receptors are functionally expressed5. To validate that the system is capable of detecting photolysis responses, control experiments should be done in medial habenula neurons that express large quantities of receptor30. In this brain area, PA-Nic bath application is possible, which is preferable for validation experiments. Only after performing these validation experiments should one move on to an unstudied cell type. If the experimental system has been validated and responses remain very small or undetectable, it may be warranted to increase the concentration of PA-Nic, increase the flash intensity or pulse duration, add a nAChR positive allosteric modulator to enhance nAChR activity6, or some combination of these.
Occasionally, uncaging responses are too large, with significant nAChR activation resulting in indirect voltage gated Na+ channel activation and unclamped inward currents due to poor space clamp. These artifacts, which completely obscure nAChR inward currents and make data interpretation impossible, can be eliminated by inclusion of QX-314 (2 mM) in the recording pipette. They may also be eliminated by reducing the concentration of PA-Nic or by reducing the flash intensity or pulse duration. In all visible light photo-stimulation experiments, care must be exercised when selecting stimulation sites to avoid unintended stimulation/photolysis above or below the desired focal plane. Additionally and when applicable, the laser power must always be titrated to reproduce physiological responses. It is especially important to be aware of z-axis photostimulation when working with caged ligands, as ligands that are activated above/below the focal spot may still diffuse and interact with the biological system (i.e., receptors) under study.
PA-Nic laser flash photolysis may not be appropriate for all investigators, as several limitations exist. The first is the relatively high cost of a suitable set-up. When working with intact brain slices, uncaging near small-diameter structures like dendrites requires a sophisticated visualization system such as a 2-photon microscope. Aside from the high cost of a Ti:sapphire, tunable IR pulsed laser for performing 2-photon microscopy, a dual-galvanometer system capable of independently positioning two laser beams further increases the system cost. Total system costs can be reduced by using a home-built system&#160;if the investigator has sufficient expertise and time to construct, troubleshoot, and maintain such a system. A second limitation often involves low nAChR functional expression, which can be partially mitigated by taking steps as mentioned above, but this may not guarantee success. Typically, if one cannot measure ligand-activated currents following puff-application of agonists, PA-Nic flash photolysis under voltage clamp may not yield acceptable results. A third limitation involves the intrinsic fluorescence of PA-Nic. PA-Nic absorbs ~405&#160;nm light and emits in a similar range as green fluorescent protein (GFP) or Alexa 4885. When PA-Nic concentrations exceed ~1 mM, this fluorescence property can make it challenging to simultaneously visualize neuronal structures. To mitigate this, it is critical to be able to easily control the flow of PA-Nic from the perfusion pipette. Periodically, the PA-Nic flow was stopped to allow fluorescent molecules to diffuse away. This permitted re-imaging of the neuron for checking the spot position of the uncaging beam. A fourth potential limitation to mention involves the use of 405 nm light for photolysis. Shorter wavelengths such as 405 nm are more prone to scattering in complex tissue such as a brain slice. Thus, at a given flash intensity and duration, uncaging response amplitudes and decay kinetics may be differentially affected by the depth of the uncaging focus within the slice. Conclusions about biological aspects of nAChRs should take this important caveat into account.
This localized laser flash photolysis technique has recently been used to uncover new details about nAChR neurobiology. For example, chronic nicotine exposure enhances perisomatic and dendritic nAChR function in medial habenula neurons5. It was also used to help demonstrate, for the first time, that ventral tegmental area glutamate neurons express functional nAChRs in their perisomatic and dendritic cellular compartments6. There are many potential future uses of this technique, and the approach could be applied to other key neuron types that are known to express nAChRs, such as cortical pyramidal neurons31 or interneurons in cerebral cortex32, striatum33, and hippocampus19. This technique could also be combined with pharmacology and/or nAChR gene editing34 to localize specific receptor subtypes to different neuronal compartments. The approach may be easily adapted to other coumarin-caged compounds, including, but not limited to, those developed in parallel with PA-Nic5. Finally, PA-Nic flash photolysis may one day be used in an awake/behaving animal to study nicotine&#39;s action in novel behavioral pharmacology paradigms.

Cholinergic signaling modulates numerous brain processes, including attentional control, volitional movement, and reward1,2. Drugs that enhance acetylcholine (ACh) transmission are used to treat cognitive impairment associated with Alzheimer&#39;s disease, implying an important role for cholinergic systems in cognition3. An improved understanding of cholinergic receptors and circuitry in healthy and diseased states could lead to better therapeutic approaches for several neurological diseases/disorders.
Nicotinic ACh receptors (nAChRs) are a family of ligand-gated ion channels that flux cations in response to endogenous ACh or exogenous nicotine from tobacco products. Given the fact that they were among the very first neurotransmitter receptors to be described4, nAChR pharmacology and location in muscle fibers is well-understood for muscle-type receptors. By contrast, comparatively little is known about the pharmacology and subcellular distribution of native nAChRs in the brain. This gap in knowledge was recently addressed by developing a novel chemical probe that allows for spatially restricted and rapid activation of nAChRs in brain tissue during cellular imaging and electrophysiological recording5. Here, the key methodological steps involved in this approach are described, with the overall goal of enhancing the ability to connect nAChR function with neuronal structure.
Photoactivatable nicotine (PA-Nic; chemical name: 1-[7-[bis(carboxymethyl)- amino]coumarin-4-yl]methyl-nicotine) can be photolyzed with ~405 nm laser flashes to efficiently release nicotine5,6. Prior to uncaging, PA-Nic is stable in solution and exhibits no untoward pharmacological or photochemical features5. After photolysis, released nicotine predictably activates nAChRs and the uncaging byproducts are pharmacologically inert5. A continuous-wave laser is used as the photolysis light source with an output power &#62;1 mW measured at the sample. When localized, targeted photo-stimulation is combined with the ability to locate cellular membranes with 2-photon laser scanning microscopy (2PLSM), and the two key advantages of this approach are fully realized: photolysis speed and spatial precision.
In most respects, photolysis of PA-Nic is superior to other methods of delivering nAChR ligands to receptors within brain slices. Such approaches include bath application7 and local drug delivery via a puffer pipette8. Whereas the former approach tends to over-emphasize the long-term effects of the applied drug, the latter approach can suffer from variability in response kinetics between trials and across cells. Neither of these alternative approaches can adequately distinguish receptor activities in different cellular locations from the same neuron. Optogenetically-activated release of ACh has been used for investigation of native nAChRs9,10,11, but it has not proven useful for mapping subcellular nAChR locations. Furthermore, most studies utilizing this approach have relied on a ChR2-expressing bacterial artificial chromosome transgenic mouse with abnormal cholinergic transmission12,13,14,15,16,17.
PA-Nic photolysis is not the only optical approach for studying cholinergic receptors. A caged carbachol was used to functionally map ACh receptor activities in cultured cells18 and brain slices19, but was not commercially available for comparative studies during the development of PA-Nic. A ruthenium bis(bipyridine)-nicotine complex (RuBi-nicotine) was reported to allow for nicotine uncaging20, but commercial preparations of RuBi-nicotine proved inferior to PA-Nic in a head-to-head comparison study5. It may be useful to repeat such comparative experiments with non-commercial, highly purified RuBi-nicotine, as its visible absorption could compliment PA-Nic&#39;s features for cholinergic studies. Finally, nAChRs have also been optically manipulated using a combination of photo-switchable ligands and genetically-modified receptors21. This approach is complementary to PA-Nic photolysis in brain tissue, with the ability/requirement of genetic targeting of the modified nAChR seen as both an advantage and a drawback.
Several key requirements of this approach should be noted. First, an appropriate visualization method is needed to accurately locate the neuronal membrane. Imaging with conventional epi-fluorescence microscopy may be sufficient when studying cultured cells, but for recording from neurons in brain slices or other thick tissue preparations, 2PLSM or confocal microscopy is a requirement. Second, a suitable method is needed to position the photolysis laser beam. This approach utilizes a dual-galvanometer scan head with two independent x-y mirrors for raster scanning of the imaging beam and point photoactivation using the uncaging laser beam22,23,24. Other, more limited solutions are possible, such as (1) a single-galvanometer scan head that alternatively raster scans the imaging beam and the uncaging beam, or (2) simply directing the uncaging beam to the center of the field of view such that the cell is brought to this position for flash photolysis.&#160;Third, a system is required for simultaneous electrophysiological recording if one wishes to collect physiological signals during experiments. The above requirements can be met with a suitable all-optical imaging technique, as recently described5. Below, a detailed protocol is included that describes the key steps of this approach.

<table><tbody><tr><td>&lt;strong&gt;Instruments, Consumables, and Miscellaneous Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Multiclamp 700B</td><td>Molecular Devices Corp.</td><td></td><td>Patch clamp amplifier</td></tr><tr><td>Pneumatic Picopump</td><td>World Precision Instruments</td><td>PV820</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument Co</td><td>P-97</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner Instruments</td><td>TC-324C</td><td></td></tr><tr><td>Vibrating blade microtome</td><td>Leica Biosystems</td><td>VT1200S</td><td></td></tr><tr><td>Ultrafree-MC Centrifugal Filter</td><td>MilliporeSigma</td><td>UFC30GV0S</td><td>internal solution filter</td></tr><tr><td>Borosilicate Glass Capillaries</td><td>World Precision Instruments</td><td>1B150F-4</td><td>patch and local application pipette</td></tr><tr><td>(-)-Nicotine hydrogen tartrate salt</td><td>Glentham</td><td>GL9693</td><td>nicotine salt</td></tr><tr><td>7-carboxymethylamino-4-methyl coumarin</td><td>Janelia Research Campus</td><td></td><td>PA-Nic by-product</td></tr><tr><td>1-[7-[bis(carboxymethyl)- amino]coumarin-4-yl]methyl-nicotine</td><td>Janelia Research Campus</td><td></td><td>PA-Nic</td></tr><tr><td>Euthasol (Pentobarbital Sodium and Phenytoin Sodium)</td><td>Virbac</td><td>ANADA #200-071</td><td></td></tr><tr><td>Alexa FluorTM 488 Hydrazide</td><td>ThermoFisher</td><td>A10436</td><td>green fill dye</td></tr><tr><td>Alexa FluorTM 568 Hydrazide</td><td>ThermoFisher</td><td>A10437</td><td>red fill dye</td></tr><tr><td>6-carboxy-AF594 (Alexa Fluor 594)</td><td>Janelia Research Campus</td><td></td><td>red fill dye</td></tr><tr><td>QX 314 chloride</td><td>Tocris</td><td>2313</td><td>voltage-gated sodium channel blocker</td></tr><tr><td>Power Meter&amp;nbsp;</td><td>ThorLabs</td><td>S120C</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals for Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>N-Methyl-D-glucamine</td><td>Sigma</td><td>M2004</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>Sodium phosphate monobasic monohydrate</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G5767</td><td></td></tr><tr><td>(+)-Sodium L-ascorbate</td><td>Sigma</td><td>A4034</td><td></td></tr><tr><td>Thiourea</td><td>Sigma</td><td>T8656</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>P2256</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>230391</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma</td><td>223506</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9625</td><td></td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-tetraacetic acid</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate magnesium salt</td><td>Sigma</td><td>A9187</td><td></td></tr><tr><td>Guanosine 5&amp;prime;-triphosphate sodium salt hydrate</td><td>Sigma</td><td>G8877</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Components of 2-Photon Microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;&amp;nbsp;Ultima Laser Scanner for Olympus BX51 Microscope&lt;/strong&gt;</td><td>Bruker Nano, Inc.</td><td></td><td>imaging software and galvos</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Imaging X-Y galvanometers</td><td>Cambridge Technology</td><td></td><td></td></tr><tr><td>&lt;strong&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Mai Tai HP1040&lt;/strong&gt;</td><td>Spectra-Physics</td><td></td><td>Tuneable IR laser</td></tr><tr><td>&lt;strong&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;Pockels cell M350-80-02-BK with M302RM Driver&lt;/strong&gt;</td><td>Conoptics, Inc.</td><td></td><td>for IR laser attenuation</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Integrating Sphere Photodiode Power Sensor</td><td>Thorlabs, Inc</td><td></td><td>laser power pick-off photodiode</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Uncaging X-Y galvanometers</td><td>Cambridge Technology</td><td></td><td></td></tr><tr><td>&lt;strong&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Helios 2-Line Laser Launch&lt;/strong&gt;</td><td>Bruker Nano, Inc.</td><td></td><td>uncaging laser components</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; OBIS LX/LS 405 nm (100 mW)</td><td>Coherent, Inc.</td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; OBIS LX/LS 473 nm (75 mW)</td><td>Coherent, Inc.</td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;Point-Photoactivation / Fiber Input Module for Limo Sidecar - Uncaging</td><td>Bruker Nano, Inc.</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Upright Microscope&lt;/strong&gt;</td><td>&amp;nbsp;Olympus</td><td>BX51WIF</td><td>Upright microscope chasis</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Objective: Olympus M Plan FL 10x; NA 0.3 WD 11 mm</td><td>&amp;nbsp;Olympus</td><td></td><td>10x objective</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Objective: Olympus M Plan Fluorite 60x/1.0 WD=2mm NIR</td><td>&amp;nbsp;Olympus</td><td></td><td>60x water-dipping objective</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; X-Cite 110, four-LED LLG coupled epi-fluorescence light source</td><td>Excelitas Technologies</td><td></td><td>LED Light Source</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Epi-Fluorescence Filter: ET-GFP (FITC/CY2) for Epi-Turret</td><td>Chroma Technologies</td><td></td><td>LED Filter for blue light excitation</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Epi-Fluorescence Filter: ET-DsRed (TRITC/CY3) for Epi-Turret</td><td>Chroma Technologies</td><td></td><td>LED Filter for green light excitation</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; B&amp;amp;W CCD camera; Watec, 0.5 in B/W CCD</td><td>Watec Co., LTD.</td><td></td><td>CCD camera for patch clamp recording</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;External Detectors - Dual Reflected Emission - Olympus Upright (Multi-Alkali, GaAsP)&lt;/strong&gt;</td><td>Bruker Nano, Inc.</td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Multi-alkali side-on PMT</td><td>Hamamatsu</td><td>R3896</td><td>red channel PMT</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 595/50m</td><td>Chroma Technologies</td><td></td><td>red channel emission filter</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 565lpxr</td><td>Chroma Technologies</td><td></td><td>dichroic beam splitter</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; GaAsP end-on PMT</td><td>Hamamatsu</td><td>7422PA-40</td><td>green channel PMT</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 525/70m</td><td>Chroma Technologies</td><td></td><td>green channel emission filter</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; High-Speed Shutter for Hamamatsu H7422 PMT</td><td>Vincent Associates / Bruker</td><td>517329</td><td>PMT shutter mount</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Dodt Gradient Contrast Transmission Detection Module&lt;/strong&gt;</td><td>Bruker Nano, Inc.</td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; Multi-alkali side-on PMT</td><td>Hamamatsu</td><td>R3896</td><td>Dodt PMT</td></tr></tbody></table>

probing nicotinic acetylcholine receptor function in mouse brain slices via laser flash photolysis of photoactivatable nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

For photolysis stimulation, the exposure dose (intensity and time), exposure location, and beam geometry are key variables. The system described in this article is capable of two different photostimulation beams, adjustable via moving a lens in/out of the photostimulation light path before the beam enters the galvanometer system. Without this lens, the photostimulation beam fills the entrance pupil of the 60x/1.0 NA water-dipping [60x WD] objective, producing a near-diffraction-limited, sub-&#181;m spot at the focal plane inside the sample. This is associated with photostimulation light with an hourglass shape, extending above and below the focal spot symmetric with the optical axis. With the lens inserted into the path, photostimulation laser light is focused into the entrance pupil of the objective lens and then exits as a pencil-like beam. This beam, which is expected to be ~10 &#181;m in diameter for a 60x objective, extends uniformly/vertically throughout the sample. In this mode, the light intensity at any given location within the stimulation spot will be ~1% of the near-diffraction-limited small spot intensity. Thus, higher laser powers are typically required when using ~10 &#181;m spot stimulation. For all experiments reported in this article, an hourglass-type photostimulation beam was used.
The delivered sample power can be plotted against the input voltage setting, after measuring the laser power at the sample using a power meter. These studies use a 60x WD objective with a working distance of 2 mm, but it is not submerged in water for power measurements to avoid potential damage to the detector element. When objectives with listed NA &#62; 0.95 are measured in air (without immersion fluid), there can be total internal reflection losses at the lens front face element due to the lower index (air). In this case, for a more accurate sample power measurement (to correct for total internal reflection losses), increase the measured power by the 1.0 NA objective (1.0/0.95)2 measured in air. Figure 1a shows a typical input/output plot for 405 nm and 473 nm visible lasers that are incorporated into the laser launch system in this study. These laser systems are ideal for photo-stimulation exposure dose control for the following reasons: (1) they are pre-calibrated to provide directly linear power output relative to input voltage (0-5 V), (2) they provide a silent shutter operation (with no laser output), and (3) they have rapid, sub-ms intensity pulse duration control (0.1 ms response). When using spot photo-stimulation with a laser/galvo system, routine calibration of MarkPoints spots is an essential task. Figure 1b (left panel) demonstrates a system that is out of calibration (desired point to photo-stimulate does not result in accurate stimulation of that point, as indicated by burn-hole location), with a return to accurate positioning of the spot after calibration (Figure 1b, right panel). 
PA-Nic is modestly fluorescent (emission peak at ~510 nm), exhibiting effective excitation between 350-450 nm (1-photon excitation) or 700-900 nm (2-photon excitation)5. To visualize PA-Nic during local application, PA-Nic (1 mM) was applied near brain tissue followed by simultaneous imaging of (1) brain tissue optical sectioned transmission context via Dodt contrast and (2) the emitted fluorescence from excitation (900 nm) of PA-Nic. PA-Nic 2PE fluorescence was easily detectable during pressure ejection from a local application pipette (Figure 2a). Nicotine and a monoalkylcoumarin, 7-carboxymethylamino-4-methyl coumarin, are the main photochemical products of the PA-Nic photolysis reaction5. Using the same imaging settings/parameters that were used for PA-Nic imaging, the tissue was imaged during delivery of either nicotine (1 mM) or 7-carboxymethylamino-4-methyl coumarin (1 mM). No fluorescent signal was detected (Figure 2a, middle and lower panels), demonstrating the specificity of the PA-Nic results. Finally, PA-Nic was applied within brain tissue and PA-Nic fluorescence emission was imaged (Figure 2b). This approach confirms that PA-Nic is present within 100-200 &#181;m of the local application pipette. Together, these data confirm that PA-Nic is effectively delivered to brain tissue via local application.
Electrophysiology recordings with simultaneous 2PLSM for visualization of cellular structures requires the investigator to balance considerations for both components of the experiment, and often a narrow time window (~20 min) is available for valid sample data acquisition from a patched cell. Without considering cellular visualization, it is best practice to begin recording as soon as possible after break-in because recording stability tends to decline with time. However, when imaging is a requirement, electrophysiological considerations must allow adequate time for fluorescence concentration increases in small/remote structures. This is exemplified by examining a dye concentration filling curve28, which is sometimes useful to derive when imaging a new cell type. Figure 3 shows several example neurons imaged as Z-stacks via 2PLSM and collapsed into a maximum intensity projection for presentation purposes. Figure 3a shows high-quality images where neuronal morphology appears to be complete, noise is minimized, and debris does not interfere with interpretation of cellular morphology. Figure 3b shows images of lower quality, owing to a lower signal-to-background ratio and substantial debris. This debris often appears as spherical pockets of intense fluorescence, arising from ejection of imaging dye from the patch pipette while approaching the cell. Notably, inclusion of 100 &#181;M PA-Nic in the bath (when performing bath application) tends to reduce the signal-to-background ratio and leads to sub-optimal image contrast. Alexa Fluor 568 or 594 is often quite useful in local application experiments as a finder dye or as a normalizing 2PE reference/normalization signal. An effective wavelength for two-photon excitation of these dyes is ~780 nm27, which allows simultaneous visualization of PA-Nic and identification of cellular compartments. This wavelength, however, does not fully avoid two-photon photolysis of PA-Nic5. Alexa Fluor 488 is advantageous in PA-Nic bath-application experiments; when excited with a suitable wavelength &#8805;900 nm, two-photon photolysis of PA-Nic5 can be avoided while still maintaining suitable visualization of cellular compartments.
Figure 4 shows example data for localized PA-Nic laser flash photolysis at MHb neurons in brain slices. Figure 4a (upper panels) shows an example of a &#34;reference&#34; image, which is a screen-capture of the last 2PLSM image taken before the MarkPoints protocol was run, overlaid with the photo-stimulation spot location. Figure 4a (lower image panels) shows a zoomed view of the photo-stimulation spot overlaid on the cellular morphology. The corresponding time-correlated electrophysiological response to PA-Nic photolysis is shown in the lower panels of Figure 4a. Previous work demonstrated that these currents are sensitive to nAChR antagonists5. Figure 4b shows representative data from different cells where single spot photolysis was performed at an interval of either 1 s or 10 s. Whereas a 10 s interval allowed sufficient recovery time for the baseline holding current, a shorter 1 s interval led to a gradual increase in the holding current as the protocol proceeded. The increasing current suggests that nicotine did not have enough time to diffuse away from the system with the 1 Hz interval29. Such temporal response analyses must be performed de novo on any new cell type being studied, as the neuropharmacology of nAChRs may differ between cell types.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58873/58873fig1.jpg" /> 
Figure 1: Photostimulation laser calibration. (a) Photo-stimulation laser power output. Power at the sample plane (through a 60x/1.0 NA water-dipping objective) was measured for 405 nm and 473 nm photo-stimulation lasers at the indicated output setting. (b) Photo-stimulation laser calibration. Screen capture images show the spatial relationship between the intended photo-stimulation spot and the corresponding location where photo-stimulation occurred (burn-hole) before (left) and after (right) running calibration in MarkPoints. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58873/58873fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58873/58873fig2.jpg" /> 
Figure 2: PA-Nic local application. (a) Detection of PA-Nic from a local application pipette. 1 mM PA-Nic, photolysis by-product, or nicotine were dissolved in ACSF, loaded into a local application pipette, and dispensed onto brain tissue during 2PLSM (900 nm excitation) imaging using the same imaging settings for each drug. Laser scanning Dodt contrast transmission image shows the tissue/pipette whereas a GaAsP cathode PMT was used to capture fluorescence emission. (b) PA-Nic (1 mM) was perfused into brain tissue and imaged via 2PLSM as in (a) to show the lateral spread of PA-Nic using its intrinsic fluorescence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58873/58873fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58873/58873fig3.jpg" /> 
Figure 3: Acquisition of 2-photon laser scanning microscopy images. (a) Optimal 2PLSM Z-stacks. Two examples of 2PLSM Z-stack maximum intensity projections are shown for MHb neurons with well-resolved dendrites and little to no interfering debris. (b) Sub-optimal 2PLSM Z-stacks. Two examples of 2PLSM Z-stack maximum intensity projections are shown for MHb neurons surrounded by debris (dye expelled from the pipette during cell approach). Such images are more difficult to interpret than images like those shown in (a). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58873/58873fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58873/58873fig4.jpg" /> 
Figure 4: Laser flash photolysis of PA-Nic. (a) MarkPoints reference images and inward currents evoked by PA-Nic photolysis. For one MHb neuron, raw reference images are shown for MarkPoints photo-stimulation trials at a single (indicated) cellular location. Note that for some photo-stimulation locations (the right-most image in this series), the dendritic structure is in focus but the soma and proximal dendrite are not. Below each reference image, the nicotine uncaging-evoked inward current is plotted. (b) Inter-stimulus intervals for PA-Nic photolysis. Exemplar recordings are shown for MHb neurons where nicotine was repeatedly uncaged at the same perisomatic location with an inter-stimulus interval of 1 s or 10 s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58873/58873fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine at JoVE.com

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

probing-nicotinic-acetylcholine-receptor-function-mouse-brain-slices

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.2K Views.  Northwestern University Feinberg School of Medicine. Deze techniek maakt gebruik van multi-photon laser-scanning microscopie in combinatie met laser-flash fotolyse van een fotoactiviteit nicotine molecuul. Het toestaan van nauwkeurige activering van nicotinic cholinerge receptoren. Deze techniek zorgt voor fijne spatiotemporale controle over nicotinische receptor agonistische toepassing, het verstrekken van een krachtig instrument voor de studie van receptor functionele expressie patronen en cholinerge synaptische of volumetransmissie.Ge...

Video: Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/58788/two-photon-imaging-microglial-processes-attraction-toward-atp-or'>Source: Etienne, F. et al. Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices.&#160;J. Vis. Exp. (2019)</a>This video demonstrates the use of two-photon microscopy to study the attraction of microglial processes toward an injected compound in brain slices. It provides an overview of preparing mouse brain slices, injecting a chemoattractant test compound, and imaging fluorescently labeled microglia to visualize their responses.

Source: Etienne, F. et al. Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices.&#160;J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Neuroimaging

Live Imaging & In Vivo Tracking

Imaging Nicotine-Induced Calcium Signaling along Ventral Hippocampal Axons in Synaptic Co-Cultures

Source: Zhong, C., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates how nicotine-mediated activation of nicotinic acetylcholine receptors in the mouse ventral hippocampus results in sustained calcium signaling along the axons.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Calcium Imaging

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Neurophysiology

Imaging and Optical Techniques

Analyzing the Glutamate Release and Clearance at a Single Neuron Synapse in a Mouse Brain Slice

Source: Dvorzhak, A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60113">Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice.</a> J. Vis. Exp. (2020)
This video demonstrates evaluating glutamate release and clearance at single corticostriatal synapses in a mouse brain slice. Electrical stimulation facilitates glutamate release at the synapse. The released glutamate alters sensor protein&#39;s fluorescence, which decays over time, indicating release and clearance efficiency.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE: Recordings from Q175 wild-type ...

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

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