Jun 29, 2023
Protocols for "Characterizing the diversity of enteric neurons using Dopamine Transporter (DAT)-Cre reporter mice".
- sherilyn.recinto1,2,
- Shobina Premachandran1,2,
- Sriparna Mukherjee3,2,
- Adam Macdonald1,2
- 1Dept. of Neuroscience and Neurology, Montreal Neurological Institute-Hospital, McGill University, Montreal, Quebec;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815;
- 3Dept. of Pharmacology and Physiology, Dept. of Neurosciences, Université de Montreal, Montreal, Quebec
Collection Citation: sherilyn.recinto, Shobina Premachandran, Sriparna Mukherjee, Adam Macdonald 2023. Protocols for "Characterizing the diversity of enteric neurons using Dopamine Transporter (DAT)-Cre reporter mice".. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4q792vo5/v1
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: June 29, 2023
Last Modified: May 31, 2024
Collection Integer ID: 84265
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP 000525
Abstract
Dopaminergic neurons (DA) are the predominant cell type in the midbrain that synthesize dopamine, a neurotransmitter implicated in a number of behavioural processes, including motor function, the reward pathway, and satiety. In diseases affecting these neurons, such as in Parkinson’s disease (PD), there is growing evidence that the gut-brain axis and selective vulnerability of DA neurons plays a crucial role in disease. Most investigations relating to DA neurons in the gut rely on immunoreactivity to tyrosine hydroxylase (TH) - a rate-limiting enzyme in the production of dopamine. However, the reliability of TH staining as a marker of DA neurons has been questioned in recent years. Our aim is to perform a comprehensive characterization of DA neurons in the gut using a well-accepted reporter mouse line, expressing a fluorescent protein under the dopamine transporter promoter (DAT). Our findings confirm a unique localisation of DA neurons in the gut, and also unveil that there are discrete subtypes of DA neurons in the gut, which we characterised using both immunofluorescence and single cell transcriptomics. We observed distinct subtypes of DAT neurons expressing co-transmitters and modulators; some of them likely co-releasing acetylcholine, and a smaller population likely releasing nitric oxide; while others were positive for a slew of canonical DA markers (Vmat2, Girk2, Foxa2). Given the clear heterogeneity of DA gut neurons, further investigation is warranted to define their functional signatures and discover their inherent biological differences that put these cells at risk for neurodegeneration.
Files
Dissociation of myenteric plexus of adult mice for single-cell RNAseq.docx
Immunofluorescence for staining fixed-frozen tissues on slide.docx
10XChromium single cell 3'RNAseq preparation_protocol.docx
RNAscope_FixedFrozedn_colons.docx