nGUDMAP Connectome Project
The primary purpose of the Nociceptive Genito-Urinary Developmental Anatomy (nGUDMAP) programme is to build a murine molecular anatomy atlas of the nociceptors (pain receptors) and associated cell types in pain processing of the urinary tract and pelvic region. In particular this will require the detailed description of the developmental origins, and the anatomical and biochemical heterogeneity of the murine nociceptors and associated cells. To do this, we propose to develop a spatiotemporal atlas of the developing neural connectome of the lower lumbar and pelvic region of the mouse during the critical developmental period when the neural connections are forming and differentiating into their innervatory and sensory fates. To capture the key events in the development of the neuronal system we propose to develop 3D atlases at a series of timepoints from mid-gestation through to birth. We plan to use Cre-lox technology to identify individual nerve fibres within the developing neural connectome. Central to identifying individual nerve fibres is our choice of Flox-STOP reporter systems. We propose to use the ‘Confetti’ (Snippert et al, 2010) Flox-STOP reporter systems to fluorescently label individual nerve fibres in the developing urogenital system. Confetti is a refined version of Brainbow 2.1 that was originally used to identify individual neurons in the adult brain (Livet et al, 2007). It is anticipated that the Confetti construct, that utilises a knock-in to the Rosa26 locus, may circumvent many of the pitfalls (random integration, copy number) associated with transgenic mice produced by microinjection.
Using Cre drivers, we propose to develop a spatiotemporal atlas of the developing nociceptive system. The voltage-gated sodium channel Nav1.8 is enriched in nociceptor populations and is expressed in a subset of sensory neurons of which >85% are nociceptors (Akopian et al., 1996; Djouhri et al., 2003). We propose to use the Nav1.8-Cre to induce stochastic expression of GFP, YFP, RFP, or CFP in Confetti mice and use this to identify individual nociceptor neurons and DRG ganglion cells in a timecourse through development. In addition, we plan to use TRPV1-Cre as an alternative method of labeling nociceptor populations. Numerous TRP (transient receptor potential) channels – a type of ion channel - are found in nociceptors and have an important role in thermosensation and thermoregulation. TRPV1-Cre is expressed in DRG and peptidergic sensory neurons (Cavanaugh et al., 2011) and is of wide interest to the nociceptor research community. We aim to use novel next-generation confocal technology to capture images of the full 3D connectome without distortion and histological processing artefacts arising from tissue sectioning. Recent advances in microscopy include the Mesolens (Saini 2012) which allows micron-resolution wide-field confocal imaging of objects up to 6mm wide and 3mm deep. The Edinburgh group is part of the Mesoscope consortium that is using the unique technology for biomedical research.
It is anticipated that a combination of Nav1.8-Cre/TRPV1-Cre drivers, the Confetti 4-colour reporter, and the Mesolens imaging system will enable us to develop a cellular-resolution spatiotemporal atlas of nociceptor distribution in the developing genitourinary system.
Akopian AN, Sivilotti L, Wood JN. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature. 1996 Jan 18;379(6562):257-62.
Cavanaugh DJ, Chesler AT, Bráz JM, Shah NM, Julius D, Basbaum AI. Restriction of transient receptor potential vanilloid-1 to the peptidergic subset of primary afferent neurons follows its developmental downregulation in nonpeptidergic neurons. J Neurosci. 2011 Jul 13;31(28):10119-27. doi: 10.1523/JNEUROSCI.1299-11.2011.
Djouhri L, Fang X, Okuse K, et al. The TTX-resistant sodium channel Nav1.8 (SNS/PN3): expression and correlation with membrane properties in rat nociceptive primary afferent neurons. J Physiol. 2003 Aug 1;550(Pt 3):739-52.
Livet J, Weissman TA, Kang H, et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature. 2007;450:56-62.
Saini A. New Lens Offers Scientist A Brighter Outlook. Science 2012; 335: 1562-1563
Snippert HJ, van der Flier LG, Sato T, et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell. 2010;143:134-144.
Grant number: 1U01DK101037