Anthony Renda was supported by a University of Victoria Graduate Fellowship Award. This research was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant, a NARSAD Young investigator Award (to R.N.), a Victoria Foundation - Myre and Winifred Sim Fund, a Canadian Foundation for Innovation grant, a British Columbia Knowledge Development Fund and a Natural Sciences and Engineering Research Council of Canada Research Tools and Instrumentation Grant. We thank Jillian McKay, Christina Barnes, Ariel Sullivan, Jennifer MacDonald and Daniel Morgado for excellent mouse husbandry.

1. Pump implantation
<ol>
	<li>Before pump implantation, fill and prepare the Alzet mini-osmotic pumps (Alzet, Model 2002, Cupertino, USA) being careful not to introduce air bubbles. This model of mini-osmotic pump delivers solution at a rate of 0.5 &mu;l/hr for 14 days. Ensure sterile conditions. Weigh empty and filled pumps. At the conclusion of experiment (10 days after implantation), the remaining liquid in the pump can be removed with a syringe and needle and weighed to calculate the volume pumped.</li>
	<li>Pumps with control solution contain saline (0.9% w/v, Teknova, S5819, Hollister, USA). To prepare nicotine solution, a 1 M stock solution of (-)-nicotine hydrogen tartrate salt (Sigma, cat# N5260) is diluted in saline (0.9% w/v) and sterilized by filtering through a 0.22 &mu;m syringe-end filter. We have previously administered nicotine at 0.4 and 2 mg/kg/hr (calculated as free base of nicotine) for 10 days.</li>
	<li>Prepare three saline baths (three 10 cm saline filled petri dishes). Once the pump has been filled and capped, wash pump thoroughly in each successive saline bath to remove any traces of the drug on the outer shell. Immerse filled pumps in saline solution for storage until surgery, keeping the control and nicotine pumps in separate saline containers.</li>
	<li>To encourage a healthy recovery and reduce the risk of post-surgical infection, have clean cages prepared for long-term recovery.</li>
	<li>For short-term recovery immediately following surgery, prepare a cage containing a heating pad and a heat lamp.</li>
	<li>To examine chronic nicotine effects we have 5 to 6 homozygous &alpha;4YFP knock-in mice (2-3 months old) in each group (control or nicotine). The &alpha;4YFP knock-in mouse line have been backcrossed for 10 generations to the C57BL/6J mouse strain. We make sure the age and sex of all mice are the same for the study and that all surgeries are performed on the same day to minimize variability. To prevent hypothermia to the mice during and after surgery, equip surgical table with a heating pad covered in sterile surgical drape.</li>
	<li>Induce the &alpha;4YFP knock-in mouse with 3 L/min oxygen and 3% isoflurane and then maintain anesthesia at 2.5 L/min oxygen and 1% isoflurane. We prefer isoflurane anesthesia because on completion of the surgery isoflurane clears the system rapidly and the mice are conscious and mobile within ~2 min. Apply eye drops immediately (Tear-Gel, Novartis) to avoid corneal damage.</li>
	<li>Pumps are implanted subcutaneously through a skin incision on the back of the neck and the pump is pushed caudally down the dorsal aspect of the back. Wipe the area on the back between the forelimbs with 95% ethanol to matte the fur. Pinch the skin with 0.8mm Graefe forceps (Fine Science Tools, 11050-10) and make a 1 cm central lateral cut using iris scissors (Fine Science Tools, 14060-10).</li>
	<li>To create a subcutaneous space for the pump, insert standard pattern scissors (Fine Science Tools, 14101-14) into the incision and push them carefully toward the caudal end of the animal.</li>
	<li>Using 1.0 mm Graefe forceps (Fine Science Tools, 11650-10), remove the pump from saline. Hold the incision open using forceps and insert the pump into the incision with the osmotic pump cap facing the caudal end of the animal and push the pump to the caudal end.</li>
	<li>Pinch wound shut using 0.8mm forceps and apply an adequate amount of Vetbond (3M, cat# 1469SB) glue. Hold until wound is sealed.</li>
	<li>Remove the animal from isoflurane mask, inject with the analgesic, meloxicam (0.1 mg/kg s.c.), and place into a short-term recovery cage until conscious and mobile. Then place in a long-term recovery cage with food and water available ad libitum.</li>
</ol>
2. &alpha;4YFP knock-in mouse fixation by intracardiac perfusion
<ol>
	<li>Make solutions one day prior to procedure and leave at 4&deg; C. To minimize variability all mice will be perfused on the same day and with the same batch of solutions.</li>
	<li>Perform perfusion fixations in a well ventilated area. Flush line of peristatic pump (Masterflex Easy Load, 7518-00; Masterflex Pump Controller, 60648) with ddH2O.</li>
	<li>Add ~0.0015g of heparin (Sigma, cat# H4784), an anticoagulant, to 20 ml PBS pH 7.6 (Invitrogen, cat# 70011).</li>
	<li>Anesthetize &alpha;4YFP knock-in mouse by injecting intramuscularly a mixture of ketamine (25 mg/kg, Wyeth Animal Health) and medatomidine hydrochloride (0.25 mg/kg, Pfizer) into the hind leg muscle and immediately place the animal back into its home cage.</li>
	<li>Pin animal to a styrofoam lid inserted into a metal tray. Wipe thorax with 95% ethanol. Pinch skin with adson forceps (Fine Science Tools, 91106-12) and snip open the thoracic cavity with iris scissors. Clamp the ribcage using an ultra fine hemostat (Fine Science Tools, 13021-12) and expose the heart.</li>
	<li>Begin pumping PBS at 4 ml/min and insert a 23G butterfly needle (Becton Dickinson, 367253) into the left ventricle of the animal. Immediately snip the right atrium to allow blood and perfusate to escape.</li>
	<li>Perfuse 20 ml of PBS (pH 7.6), then 30 ml of 4% paraformaldehyde (pH 7.6, diluted with PBS from a 16% PFA stock, Electron Microscopy Sciences, cat# 15710), then 20ml of 5% sucrose (pH 7.6) . We found that over fixation increases autofluorescence. Perfusion with 5% sucrose rinses residual PFA from the brain, which lowers autofluorescence.</li>
	<li>Remove brain and store in 30% sucrose for 3 days.</li>
	<li>To freeze brains for coronal sectioning, cut off the cerebellum with a razor blade and place brain in a plastic embedding mold (VWR, cat# 18986-1) rostral side up and submerge in O.C.T. Mounting Compound (Tissue-Tek, cat# 4583). Freeze in dry ice and store at -20 &deg;C before slicing.</li>
	<li>Section brains (30 &mu;m thick) on a cryostat and then transfer to coated slides. Store the brain sections at -20&deg;C in slide boxes containing one anhydrous calcium sulphate stone. The slide box should be in a zip lock sealed bag to avoid moisture and subsequent freezer burn when stored at -20&deg;C.</li>
</ol>
3. Imaging fluorescent nAChRs using spectral confocal microscopy
<ol>
	<li>Ensure slides have minimal exposure to any light source to minimize photobleaching.</li>
	<li>Coverslip the brain section slides with a mounting medium that does not inhibit fluorescent protein fluorescence. We have good success with Mowiol 4-88 (pH 8.5 in Tris-HCl and glycerol, EMD-Calbiochem, cat# 475904), which hardens after a few hours. Make sure the Mowiol equilibrates to room temperature before cover slipping in order to avoid air bubbles. Do not use nail polish when cover slipping as it will quench YFP fluorescence.</li>
	<li>Images are acquired using a Nikon C1si spectral confocal microscope system. Details on the method of spectral confocal imaging and linear unmixing are provided elsewhere 13-15. The rationale for using a spectral confocal microscope is that fixed brain tissue has inherent autofluorescence. Spectral confocal imaging on the Nikon C1si uses an array of 32 photomultiplier tube detectors that sample a specified range of wavelengths of fluorescent emitted light, refracted spatially into its different wavelengths via a grating dispersive element just like a prism refracting white light into a rainbow of colors16. What is collected is a lambda stack of images -- images collected at different wavelengths of light so that an emission spectrum is collected for each pixel of a lambda stack of images. Since YFP and tissue autofluorescence each have characteristic spectral signatures, the lambda stack can be deconvolved using a linear unmixing algebraic algorithm into separate YFP and autofluorescent signals (Fig. 2). Thus, very accurate quantification of YFP fluorescence can be determined even in tissue with significant levels of autofluorescence.</li>
	<li>Various settings can be adjusted to optimize image quality and fluorescence collection efficiency. We will report settings we typically use but these settings can be adjusted depending on the sample and the confocal microscope. To accurately quantify changes in &alpha;4YFP subunit expression with chronic nicotine we ensure that the grey scale level intensity of the signals of all pixels are below saturating value (&lt; 4095 for 12 bit grey scale). Moreover, we accommodate for potential increase in signal due to receptor upregulation by adjusting the confocal settings so that pixels are around one third of saturating value (~1300-1400) or less. Once the settings are optimized, the settings are maintained identical for all imaging sessions and samples.</li>
	<li>We use the following settings for imaging &alpha;4YFP using a 60X oil CFI Plan Apo VC objective (1.40 N.A., 0.13 mm working distance): 488 nm laser line at 15% maximal transmission of a 40 mW Argon laser, spectral detector gain at 220, spectral range imaged from 496.5 nm to 656.5 nm at 5 nm resolution, 512 x 512 pixels over a 50 &mu;m x 50 &mu;m area, a medium pinhole size (60 &mu;m diam), a 4.08 pixel dwell time, averaging over two scans and 12 bits of grey scale.</li>
</ol>
4. Linear unmixing of spectral confocal images and image analysis
<ol>
	<li>To perform linear spectral unmixing on a sample image taken with a spectral confocal microscope, one must first acquire a reference spectrum for YFP and a reference spectrum for tissue autofluorescence.</li>
	<li>An important requisite for any reference spectrum is to obtain a high signal to noise fluorescence spectrum imaged with the same laser line used for imaging of your samples. Although the 514 nm laser line of Argon has a higher excitation efficiency to excite YFP we prefer to image YFP with the 488 nm laser line because there is better separation from the peak emission of YFP and the 488 nm line is less likely to distort the peak and one can obtain the entire YFP emission spectrum including the rise to peak. We image soluble YFP transfected in a cell line so that a strong YFP signal is obtained and the resultant YFP spectrum is stored in our library of reference spectra. Transfected &alpha;4YFP over expressed in cell lines may also be used since it will also provide a high signal to background spectrum.</li>
	<li>To obtain a reference spectrum of autofluorescence we image autofluorescence from a wildtype mouse brain section from the various brain regions that we intend to image from the &alpha;4YFP mouse and obtain their corresponding spectra. We use the same laser line, 488 nm, and image using nearly identical parameters, although we may alter the detector gain, laser intensity or perform average imaging to maximize signal to noise.</li>
	<li>After obtaining a spectral confocal image from a brain region of the &alpha;4YFP mouse, the image is then deconvolved into its YFP and autofluorescence signals by applying a linear spectral unmixing algorithm using the reference spectrum of YFP and the reference spectrum of wildtype mouse autofluorescence from the same brain region.</li>
	<li>The unmixed &alpha;4YFP image can then be opened in an image analysis software such as ImageJ (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) and then the mean pixel intensity is calculated for the region of interest. A plugin for ImageJ called "loci_tools.jar" (<a href="http://www.loci.wisc.edu/bio-formats/imagej" target="_blank">http://www.loci.wisc.edu/bio-formats/imagej</a>) can be used to import Nikon ics/ids confocal files.</li>
	<li>We repeat the same linear spectral unmixing and analysis to wildtype mice in the same brain region to obtain a background residual value.</li>
	<li>Then one obtains the corrected mean &alpha;4YFP intensity by subtracting the background residual value (4.6) from the mean uncorrected &alpha;4YFP value (4.5).</li>
</ol>
5. Representative Results
We show a representative true color projection of a lambda stack of images of the medial habenula from a homozygous &alpha;4YFP mouse (Fig. 3A) taken with a spectral confocal microscope. We also show the emission spectrum from a region of interest containing &alpha;4YFP positive neurons from the same lambda stack image (Fig. 3B). A distinct emission peak is evident at ~527 nm, which is the peak fluorescence emission of YFP. The region neighbouring the medial habenula shows an emission spectrum lacking a spectral peak at 527 nm, indicating the absence of &alpha;4YFP nAChR subunits. Following linear spectral unmixing using reference spectra of YFP and mouse brain autofluoresence with significant overlap of emission (Fig. 4), separation of YFP and autofluorescent signal is possible yielding an &alpha;4YFP unmixed image, an autofluorescent unmixed image and a remainder channel. Clear localization of &alpha;4YFP fluorescence can be identified in the tightly packed soma of the medial habenula (Fig. 4).
In the hippocampus &alpha;4YFP is localized mainly in the medial perforant path, the tempero-ammonic path and the alveus12. These are all glutamatergic innervation of the hippocampus. We examined the effects of chronic nicotine on &alpha;4YFP expression in the hippocampal perforant path (Fig. 5). Chronic administration of nicotine (2 mg/kg/hr for 10 days) resulted in a significant increase (69 &plusmn; 14%) in &alpha;4YFP fluorescence from control saline treated mice to chronic nicotine treated mice (p = 0.001, Wilcoxon rank sum test) (Fig. 5).
<img alt="Figure 1" src="/files/ftp_upload/3516/3516fig1.jpg" /> 
Figure 1. Flow chart showing procedure to image changes in &alpha;4YFP nAChR subunits with chronic nicotine. Mini-osmotic pumps are filled with either saline or nicotine and implanted subcutaneously in &alpha;4YFP homozygous mice. After 10 days mice are perfused and fixed with 4% paraformaldehyde and the mouse brains are sectioned (30 &mu;m thick) on slides. The brain section is imaged on a spectral confocal microscope (Nikon C1si) and spectrally unmixed into YFP and autofluorescent images. Then the &alpha;4YFP images are analyzed further with ImageJ software.
<img alt="Figure 2" src="/files/ftp_upload/3516/3516fig2.jpg" /> 
Figure 2. A schematic diagram of a lambda stack imaged from a spectral confocal microscope and linearly unmixed into its spectral components. (A) A lambda stack of images is collected. (B) A lambda stack consists of images collected at different wavelengths of light so that an emission spectrum is collected for each pixel across the entire stack. (C) Since YFP and tissue autofluorescence each have characteristic spectral signatures, the lambda stack can be deconvolved using a linear unmixing algebraic algorithm into separate YFP and autofluorescent signals. Thus, very accurate quantification of YFP fluorescence can be determined even in tissue with high levels of autofluorescence.
<img alt="Figure 3" src="/files/ftp_upload/3516/3516fig3.jpg" /> 
Figure 3. Spectral confocal image of a brain region expressing &alpha;4YFP nAChRs. (A) A true color projection of a lambda stack of images of the medial habenula from an &alpha;4YFP mouse taken with a Nikon C1si spectral confocal microscope. (B) Plots of spectra from a region of interest, which includes &alpha;4YFP containing neurons (green), and a region of interest outside of the medial habenula (red).
<img alt="Figure 4" src="/files/ftp_upload/3516/3516fig4.jpg" /> 
Figure 4. Linear spectral unmixing of the medial habenula. (A) Images of unmixed &alpha;4YFP and (B) autofluorescence following linear spectral unmixing. (C) Reference spectra of YFP (green triangles) and autofluorescence (yellow circles) used for spectral unmixing.
<img alt="Figure 5" src="/files/ftp_upload/3516/3516fig5.jpg" /> 
Figure 5. Upregulation of &alpha;4 nAChRs in the hippocampus of &alpha;4YFP knock-in mice exposed to chronic nicotine. (A) A tiled montage of &alpha;4YFP fluorescence from the hippocampus. The two dashed selection areas are the approximate locations on the inferior limb of the perforant path of the hippocampus where analyses were performed for each mouse. (B) &alpha;4YFP fluorescence was significantly higher in the perforant path of mice exposed to chronic nicotine than chronic saline (*, p = 0.001, Wilcoxon rank sum test). Results represent mean &plusmn; SEM from n = 20 measurements for both saline and chronic nicotine treated mice (5 mice for each treatment group).
<img alt="Figure 6" src="/files/ftp_upload/3516/3516fig6.jpg" /> 
Figure 6. Better depth expression of &alpha;4YFP as compared to antibody labeling. X-Z orthogonal views of &alpha;4YFP fluorescence (A) and VGlut2 antibody with Cy5 as a secondary label (B). (C) Plots showing greater fluorescence signal intensity degradation over depth for antibody staining (black squares) as compared to &alpha;4YFP (open circles).

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta2-subunit-containing+nicotinic+acetylcholine+receptors+are+critical+for+dopamine-dependent+locomotor+activation+following+repeated+nicotine+administration.">Beta2-subunit-containing nicotinic acetylcholine receptors are critical for dopamine-dependent locomotor activation following repeated nicotine administration.</a> Neuropharmacology. 47, 132-139 (2004).</li><li>Robinson, S. F., Marks, M. J., Collins, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbred+mouse+strains+vary+in+oral+self-selection+of+nicotine.">Inbred mouse strains vary in oral self-selection of nicotine.</a> Psychopharmacology (Berl). 124, 332-339 (1996).</li><li>Sparks, J. A., Pauly, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+continuous+oral+nicotine+administration+on+brain+nicotinic+receptors+and+responsiveness+to+nicotine+in+C57Bl/6+mice.">Effects of continuous oral nicotine administration on brain nicotinic receptors and responsiveness to nicotine in C57Bl/6 mice.</a> Psychopharmacology (Berl). , 141-145 (1999).</li><li>Rahman, S., Zhang, J., Engleman, E. A., Corrigall, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroadaptive+changes+in+the+mesoaccumbens+dopamine+system+after+chronic+nicotine+self-administration:+a+microdialysis+study.">Neuroadaptive changes in the mesoaccumbens dopamine system after chronic nicotine self-administration: a microdialysis study.</a> Neuroscience. 129, 415-4124 (2004).</li><li>Picciotto, M. R., Zoli, M., Rimondini, R., Lena, C., Marubio, L. M., Pich, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+containing+the+Î²2+subunit+are+involved+in+the+reinforcing+properties+of+nicotine.">Acetylcholine receptors containing the Î²2 subunit are involved in the reinforcing properties of nicotine.</a> Nature. 391, 173-177 (1998).</li><li>Fowler, C. D., Lu, Q., Johnson, P. M., Marks, M. J., Kenny, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habenular+&#945;5+nicotinic+receptor+subunit+signalling+controls+nicotine+intake.">Habenular &#945;5 nicotinic receptor subunit signalling controls nicotine intake.</a> Nature. 471, 597-601 (2011).</li><li>Maskos, U., Molles, B. E., Pons, S., Besson, M., Guiard, B. P., Guilloux, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+reinforcement+and+cognition+restored+by+targeted+expression+of+nicotinic+receptors.">Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors.</a> Nature. 436, 103-107 (2005).</li><li>Matta, S. G., Balfour, D. J., Benowitz, N. L., Boyd, R. T., Buccafusco, J. J., Caggiula, A. R., Craig, C. R., Collins, A. C., Damaj, M. I., Donny, E. C., Gardiner, P. S., Grady, S. R., Heberlein, U., Leonard, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+on+nicotine+dose+selection+for+in+vivo+research.">Guidelines on nicotine dose selection for in vivo research.</a> Psychopharmacology. 190, 269-319 (2007).</li><li>Lang, T., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+protein+clusters+at+nanoscale+resolution:+more+than+pretty+pictures.">Membrane protein clusters at nanoscale resolution: more than pretty pictures.</a> Physiology (Bethesda). 25, 116-1124 (2010).</li></ol>

We have nothing to disclose.

The use of a fluorescent receptor in a knock-in mouse model to determine quantity and localization of a specific ion channel provides a number of advantages. In contrast to proteins such as actin, which is ubiquitously expressed in all cells, ion channels are present in far fewer numbers and their expression varies between neuronal subtypes making accurate analysis via traditional immunohistochemical techniques challenging. The &alpha;4YFP gene product is expressed at WT levels, being under control of the same promoter...

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>mini-osmotic pumps</td>
 <td>Alzet</td>
 <td>model 2002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>saline</td>
 <td>Teknova</td>
 <td>S5819</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>(-)-nicotine hydrogen tartrate salt</td>
 <td>Sigma </td>
 <td>N5260</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>eye drops</td>
 <td>Novartis</td>
 <td>Tear-Gel</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vetbond glue</td>
 <td>3M</td>
 <td>1469SB</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>heparin sodium salt</td>
 <td>Sigma </td>
 <td>H4784</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10x PBS</td>
 <td>Invitrogen</td>
 <td>70011</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ketamine</td>
 <td>Wyeth Animal Health</td>
 <td>0856-4403-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>medatomidine hydrochloride</td>
 <td>Pfizer</td>
 <td>1950673</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>23G butterfly needle</td>
 <td>Becton Dickinson</td>
 <td>367253</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>paraformaldehyde</td>
 <td>Electron Microscopy Sciences</td>
 <td>15710</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>plastic embedding mold</td>
 <td>VWR</td>
 <td>18986-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>O.C.T. Mounting Compound</td>
 <td>Tissue-Tek</td>
 <td>4583</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mowiol 4-88</td>
 <td>EMD-Calbiochem</td>
 <td>475904</td>
 <td>pH 8.5</td>
 </tr>
</table>

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.

Watch this Scientific Journal Video about Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration at JoVE.com

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

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 ...

spectral-confocal-imaging-fluorescently-tagged-nicotinic-receptors

Research

JoVE Journal

Neuroscience

13.4K Views.  University of Victoria. 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.

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

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

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 ...

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 ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior are identified in freely moving animals, this three-fold subdivision becomes too simplistic. During wakefulness, ensembles of global and local cortical activities, together with peripheral parameters such as pupillary diameter and sympathovagal balance, define various degrees of arousal. It remains unclear the extent to which sleep also forms a continuum of brain states-within which the degree of resilience to sensory stimuli and arousability, and perhaps other sleep functions, vary gradually-and how peripheral physiological states co-vary. Research advancing the methods to monitor multiple parameters during sleep, as well as attributing to constellations of these functional attributes, is central to refining our understanding of sleep as a multifunctional process during which many beneficial effects must be executed. Identifying novel parameters characterizing sleep states will open opportunities for novel diagnostic avenues in sleep disorders.
We present a procedure to describe dynamic variations of mouse non-REM sleep states via the combined monitoring and analysis of electroencephalogram (EEG)/electrocorticogram (ECoG), electromyogram (EMG), and electrocardiogram (ECG) signals using standard polysomnographic recording techniques. Using this approach, we found that mouse non-REM sleep is organized into cycles of coordinated neural and cardiac oscillations that generate successive 25-s intervals of high and low fragility to external stimuli. Therefore, central and autonomic nervous systems are coordinated to form behaviorally distinct sleep states during consolidated non-REM sleep. We present surgical manipulations for polysomnographic (i.e., EEG/EMG combined with ECG) monitoring to track these cycles in the freely sleeping mouse, the analysis to quantify their dynamics, and the acoustic stimulation protocols to assess their role in the likelihood of waking up. Our approach has already been extended to human sleep and promises to unravel common organizing principles of non-REM sleep states in mammals.

Here, we present experimental and analytical procedures to describe the temporal dynamics of the neural and cardiac variables of non-REM sleep in mice, which modulate sleep responsiveness to acoustic stimuli.

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

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.

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 ...