In-vivo two-photon imaging of the honey bee antennal lobe

Due to the honey bee's importance as a simple neural model, there is a great need for new functional imaging modalities. Herein we report on the use of two-photon microscopy for in-vivo functional and morphological imaging of the honey bee's olfactory system focusing on its primary centers, the antennal lobes (ALs). Our imaging platform allows for simultaneously obtaining both morphological measurements of the AL and in-vivo calcium recording of neural activities. By applying external odor stimuli to the bee's antennas, we were able to record the characteristic odor response maps. Compared to previous works where conventional fluorescence microscopy is used, our approach offers all the typical advantages of multi-photon imaging, providing substantial enhancement in both spatial and temporal resolutions while minimizing photo-damages and autofluorescence contribution with a four-fold improvement in the functional signal. Moreover, the multi-photon associated extended penetration depth allows for functional imaging within profound glomeruli.Comment: 3 pages, 3 figure

Dependence of sub-micron vaterite container release properties on pH and ionic strength of the surrounding solution

We report on the synthesis and characterization of porous monodisperse vaterite containers with controllable average sizes from 400 nm to 10 mu m. Possible release strategies of enclosed substances via recrystallization or by pH-change are presented. As a model experiment, a fluorescent marker was encapsulated and imaged by two-photon microscopy to monitor the dye release. The release process was found to be controllable via the immersion medium's properties. Release times can be further tuned by covering the containers with additional polymer layers, creating a flexible system with promising perspectives for pharmaceutical applications

Second-harmonic generation sensitivity to transmembrane potential in normal and tumor cells.

Second-harmonic generation (SHG) is emerging as a powerful tool for the optical measurement of transmembrane potential in live cells with high sensitivity and temporal resolution. Using a patch clamp, we characterize the sensitivity of the SHG signal to transmembrane potential for the RH 237 dye in various normal and tumor cell types. SHG sensitivity shows a significant dependence on the type of cell, ranging from 10 to 17% per 100 mV. Furthermore, in the samples studied, tumor cell lines display a higher sensitivity compared to normal cells. In particular, the SHG sensitivity increases in the cell line Balb/c3T3 by the transformation induced with SV40 infection of the cells. We also demonstrate that fluorescent labeling of the membrane with RH 237 at the concentration used for SHG measurements does not induce any measurable alteration in the electrophysiological properties of the cells investigated. Therefore, SHG is suitable for the investigation of outstanding questions in electrophysiology and neurobiology

A multimodal approach for tracing lateralization along the olfactory pathway in the honeybee through electrophysiological recordings, morpho-functional imaging, and behavioural studies

Recent studies have revealed asymmetries between the left and right sides of the brain in invertebrate species. Here we present a review of a series of recent studies from our labs, aimed at tracing asymmetries at different stages along the honeybee's (Apis mellifera) olfactory pathway. These include estimates of the number of sensilla present on the two antennae, obtained by scanning electron microscopy, as well as electroantennography recordings of the left and right antennal responses to odorants. We describe investigative studies of the antennal lobes, where multi-photon microscopy is used to search for possible morphological asymmetries between the two brain sides. Moreover, we report on recently published results obtained by two-photon calcium imaging for functional mapping of the antennal lobe aimed at comparing patterns of activity evoked by different odours. Finally, possible links to the results of behavioural tests, measuring asymmetries in single-sided olfactory memory recall, are discussed.Comment: 28 pages, 8 figure

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19