Name: probing nicotinic acetylcholine receptor function in mouse brain slices via laser flash photolysis of photoactivatable nicotine
Uploaded: 2019-01-25T13:00:00
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>Instruments, Consumables, and Miscellaneous Chemicals</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>
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			<td>Alexa FluorTM 488 Hydrazide</td>
			<td>ThermoFisher</td>
			<td>A10436</td>
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		<tr>
			<td>Alexa FluorTM 568 Hydrazide</td>
			<td>ThermoFisher</td>
			<td>A10437</td>
			<td>red fill dye</td>
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			<td>6-carboxy-AF594 (Alexa Fluor 594)</td>
			<td>Janelia Research Campus</td>
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			<td>red fill dye</td>
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			<td>QX 314 chloride</td>
			<td>Tocris</td>
			<td>2313</td>
			<td>voltage-gated sodium channel blocker</td>
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		<tr>
			<td>Power Meter&#160;</td>
			<td>ThorLabs</td>
			<td>S120C</td>
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			<td>Chemicals for Solutions</td>
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			<td>N-Methyl-D-glucamine</td>
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			<td>M2004</td>
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			<td>Potassium chloride</td>
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			<td>P3911</td>
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			<td>H3375</td>
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			<td>D-(+)-Glucose</td>
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			<td>G5767</td>
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			<td>(+)-Sodium L-ascorbate</td>
			<td>Sigma</td>
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			<td>P2256</td>
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			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>230391</td>
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			<td>Sigma</td>
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			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
			<td>Sigma</td>
			<td>E3889</td>
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		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate magnesium salt</td>
			<td>Sigma</td>
			<td>A9187</td>
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		<tr>
			<td>Guanosine 5&#8242;-triphosphate sodium salt hydrate</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td></td>
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			<td>Name</td>
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			<td>Components of 2-Photon Microscope</td>
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			<td>&#160;Ultima Laser Scanner for Olympus BX51 Microscope</td>
			<td>Bruker Nano, Inc.</td>
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			<td>imaging software and galvos</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; Imaging X-Y galvanometers</td>
			<td>Cambridge Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160; Mai Tai HP1040</td>
			<td>Spectra-Physics</td>
			<td></td>
			<td>Tuneable IR laser</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;Pockels cell M350-80-02-BK with M302RM Driver</td>
			<td>Conoptics, Inc.</td>
			<td></td>
			<td>for IR laser attenuation</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Integrating Sphere Photodiode Power Sensor</td>
			<td>Thorlabs, Inc</td>
			<td></td>
			<td>laser power pick-off photodiode</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; Uncaging X-Y galvanometers</td>
			<td>Cambridge Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160; Helios 2-Line Laser Launch</td>
			<td>Bruker Nano, Inc.</td>
			<td></td>
			<td>uncaging laser components</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; OBIS LX/LS 405 nm (100 mW)</td>
			<td>Coherent, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; OBIS LX/LS 473 nm (75 mW)</td>
			<td>Coherent, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;Point-Photoactivation / Fiber Input Module for Limo Sidecar - Uncaging</td>
			<td>Bruker Nano, Inc.</td>
			<td></td>
			<td></td>
		</tr>
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			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Upright Microscope</td>
			<td>&#160;Olympus</td>
			<td>BX51WIF</td>
			<td>Upright microscope chasis</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; Objective: Olympus M Plan FL 10x; NA 0.3 WD 11 mm</td>
			<td>&#160;Olympus</td>
			<td></td>
			<td>10x objective</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; Objective: Olympus M Plan Fluorite 60x/1.0 WD=2mm NIR</td>
			<td>&#160;Olympus</td>
			<td></td>
			<td>60x water-dipping objective</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 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>&#160;&#160;&#160;&#160;&#160;&#160;&#160; 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>&#160;&#160;&#160;&#160;&#160;&#160;&#160; 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>&#160;&#160;&#160; B&#38;W CCD camera; Watec, 0.5in B/W CCD</td>
			<td>Watec Co., LTD.</td>
			<td></td>
			<td>CCD camera for patch clamp recording</td>
		</tr>
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			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>External Detectors - Dual Reflected Emission - Olympus Upright (Multi-Alkali, GaAsP)</td>
			<td>Bruker Nano, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; Multi-alkali side-on PMT</td>
			<td>Hamamatsu</td>
			<td>R3896</td>
			<td>red channel PMT</td>
		</tr>
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			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160; 595/50m</td>
			<td>Chroma Technologies</td>
			<td></td>
			<td>red channel emission filter</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 565lpxr</td>
			<td>Chroma Technologies</td>
			<td></td>
			<td>dichroic beam splitter</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; GaAsP end-on PMT</td>
			<td>Hamamatsu</td>
			<td>7422PA-40</td>
			<td>green channel PMT</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160; 525/70m</td>
			<td>Chroma Technologies</td>
			<td></td>
			<td>green channel emission filter</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; High-Speed Shutter for Hamamatsu H7422 PMT</td>
			<td>Vincent Associates / Bruker</td>
			<td>517329</td>
			<td>PMT shutter mount</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dodt Gradient Contrast Transmission Detection Module</td>
			<td>Bruker Nano, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 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/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/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/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/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 ...

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