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...

<ol>
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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

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Neuroscience

13.4K ビュー.  University of Victoria. 私たちはより良いアプローチノックインおよびスペクトルを用いた受容体の蛍光タンパク質タグを含むアプローチの組み合わせを使用して、ニコチン中毒のメカニズムを理解するCNSニューロンの特定のサブタイプの細胞内領域内のニコチン性アセチルコリン受容体の変化を定量化する新しい技術を開発しました共焦点イメージング。