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

Summary

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.

Abstract

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 tissue^1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates^4-6.

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 vivo^7-10. 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 Förster 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 α4 nAChR subunits (α4YFP), 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 α4YFP 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 α4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of α4YFP receptors in the medial perforant path of the hippocampus.

Protocol

1. Pump implantation

1. 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 μl/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.
2. 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 hydr....

Discussion

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 α4YFP gene product is expressed at WT levels, being under control of the same promoter.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
mini-osmotic pumps	Alzet	model 2002
saline	TEKnova, Inc.	S5819
(-)-nicotine hydrogen tartrate salt	Sigma-Aldrich	N5260
eye drops	Novartis AG	Tear-Gel
Vetbond glue	3M	1469SB
heparin sodium salt	Sigma-Aldrich	H4784
10x PBS	Invitrogen	70011
ketamine	Wyeth Animal Health	0856-4403-01
medatomidine hydrochloride	Pfizer Pharma GmbH	1950673
23G butterfly needle	BD Biosciences	367253
paraformaldehyde	Electron Microscopy Sciences	15710
plastic embedding mold	VWR international	18986-1
O.C.T. Mounting Compound	Tissue-Tek	4583
Mowiol 4-88	EMD Millipore	475904	pH 8.5