Kamm, G. B. | Search bioRxiv

bioRxiv2024

Chemigenetic far-red labels and Ca2+ indicators optimized for photoacoustic imaging.

Photoacoustic imaging is an emerging modality with significant promise for biomedical applications such as neuroimaging, owing to its capability to capture large fields of view, deep inside complex scattering tissue. However, the widespread adoption of this technique has been hindered by a lack of suitable molecular reporters for this modality. In this work, we introduce chemigenetic labels and calcium sensors specifically tailored for photoacoustic imaging, using a combination of synthetic dyes and HaloTag-based self-labelling proteins. We rationally design and engineer far-red "acoustogenic" dyes, showing high photoacoustic turn-ons upon binding to HaloTag, and develop a suite of tunable calcium indicators based on these scaffolds. These first-generation photoacoustic reporters show excellent performance in tissue-mimicking phantoms, with the best variants outperforming existing sensors in terms of signal intensity, sensitivity and photostability. We demonstrate the application of these ligands for labelling HaloTag-expressing neurons in mouse brain tissue, producing strong, specifically targeted photoacoustic signal, and provide a first example of in vivo labelling with these chemigenetic photoacoustic probes. Together, this work establishes a new approach for the design of photoacoustic reporters, paving the way towards deep tissue functional imaging.

biochemistry↗

bioRxiv2023

Diverging roles of TRPV1 and TRPM2 in warm-temperature detection

The accurate perception of innocuous temperatures, particularly those experienced as pleasantly warm, is essential for achieving thermal comfort and maintaining thermoregulatory balance. Warm-sensitive neurons (WSN) innervating the skin play a central role in non-painful warmth detection. The TRP ion channels TRPV1 and TRPM2 have been suggested as sensors of warm temperature in WSNs. However, the precise contribution of these channels to the process of warmth detection is not fully understood. A significant challenge in analysing WSNs lies in their scarcity: fewer than 10 % of sensory neurons in the rodent dorsal root ganglion (DRG) respond to innocuous warm temperatures. In this study, we examined >20,000 cultured mouse DRG neurons using calcium imaging and discovered distinct contributions of TRPV1 and TRPM2 to warm-temperature sensitivity. TRPV1, and to a lesser extent TRPM2, affect the abundance of WSNs, with TRPV1 mediating the rapid, dynamic response to warmth and TRPM2 subtly affecting the population response of WSNs. By carefully tracking animal movement in a whole-body thermal preference paradigm, we observe that these cellular differences correlate with nuanced thermal behaviours. Utilizing a drift-diffusion model to quantitatively analyse the decision-making process of animals exposed to different environmental temperatures, we found that: TRPV1 deletion primarily impairs the precision of evidence accumulation, whereas TRPM2 deletion significantly increases the total duration of exposure to warmer environments that are avoided by wildtype mice. Our findings provide valuable insights into the distinct molecular responses to warmth stimuli, and underpin the subtle aspects of thermal decision-making when encountering minor temperature variations.

neuroscience↗