The sense of smell, its signalling pathways, and the dichotomy of cilia and microvilli in olfactory sensory cells

Smell is often regarded as an ancillary perception in primates, who seem so dominated by their sense of vision. In this paper, we will portray some aspects of the significance of olfaction to human life and speculate on what evolutionary factors contribute to keeping it alive. We then outline the functional architecture of olfactory sensory neurons and their signal transduction pathways, which are the primary detectors that render olfactory perception possible. Throughout the phylogenetic tree, olfactory neurons, at their apical tip, are either decorated with cilia or with microvilli. The significance of this dichotomy is unknown. It is generally assumed that mammalian olfactory neurons are of the ciliary type only. The existance of so-called olfactory microvillar cells in mammals, however, is well documented, but their nature remains unclear and their function orphaned. This paper discusses the possibility, that in the main olfactory epithelium of mammals ciliated and microvillar sensory cells exist concurrently. We review evidence related to this hypothesis and ask, what function olfactory microvillar cells might have and what signalling mechanisms they use

A new protocol for whole-brain biodistribution analysis of AAVs by tissue clearing, light-sheet microscopy and semi-automated spatial quantification

© The Author(s), under exclusive licence to Springer Nature Limited 2022Recombinant adeno-associated virus (rAAV) has become one of the most promising gene delivery systems for both in vitro and in vivo applications. However, a key challenge is the lack of suitable imaging technologies to evaluate delivery, biodistribution and tropism of rAAVs and efficiently monitor disease amelioration promoted by AAV-based therapies at a whole-organ level with single-cell resolution. Therefore, we aimed to establish a new pipeline for the biodistribution analysis of natural and new variants of AAVs at a whole-brain level by tissue clearing and light-sheet fluorescence microscopy (LSFM). To test this platform, neonatal C57BL/6 mice were intravenously injected with rAAV9 encoding EGFP and, after sacrifice, brains were processed by standard immunohistochemistry and a recently released aqueous-based clearing procedure. This clearing technique required no dedicated equipment and rendered highly cleared brains, while simultaneously preserving endogenous fluorescence. Moreover, three-dimensional imaging by LSFM allowed the quantitative analysis of EGFP at a whole-brain level, as well as the reconstruction of Purkinje cells for the retrieval of valuable morphological information inaccessible by standard immunohistochemistry. In conclusion, the pipeline herein described takes the AAVs to a new level when coupled to LSFM, proving its worth as a bioimaging tool in tropism and gene therapy studies.This work was funded by the ERDF through the Regional Operational Program Center 2020, Competitiveness Factors Operational Program (COMPETE 2020) and National Funds through FCT (Foundation for Science and Technology): Imagene (PTDC/BBB-NAN/0932/2014 | POCI-01-0145-FEDER-016807), MODELPOLYQ 2.O (CENTRO-01-0145-FEDER-181258), MJDEDIT (CENTRO-01-0145-FEDER-181266), BDFORMJD (CENTRO-01-0145-FEDER-181240), CENTRO-01-0246-FEDER-000010 (Multidisciplinary Institute of Ageing in Coimbra), BrainHealth2020 projects (CENTRO-01-0145-FEDER-000008), UID/NEU/04539/2019, UIDB/04539/2020, UIDP/04539/2020, LA/P/0058/2020, PPBI (POCI-01-0145-FEDER-022122), ViraVector (CENTRO-01-0145-FEDER-022095), CortaCAGs (PTDC/NEU-NMC/0084/2014 | POCI-01-0145-FEDER-016719), SpreadSilencing (POCI-01-0145-FEDER-029716), CancelStem (POCI-01-0145-FEDER-016390), POCI-01-0145-FEDER-030737, POCI-01-0145-FEDER-032309, as well as SynSpread, ESMI and ModelPolyQ under the EU Joint Program – Neurodegenerative Disease Research (JPND), the last two co-funded by the European Union H2020 program, GA No.643417; by National Ataxia Foundation (USA), the American Portuguese Biomedical Research Fund (APBRF) and the Richard Chin and Lily Lock Machado-Joseph Disease Research Fund. MML was supported by a PhD fellowship from FCT (2021.05776.BD).info:eu-repo/semantics/publishedVersio