Cardiac optogenetics: shining light on signaling pathways.

In the early 2000s, the field of neuroscience experienced a groundbreaking transformation with the advent of optogenetics. This innovative technique harnesses the properties of naturally occurring and genetically engineered rhodopsins to confer light sensitivity upon target cells. The remarkable spatiotemporal precision offered by optogenetics has provided researchers with unprecedented opportunities to dissect cellular physiology, leading to an entirely new level of investigation. Initially revolutionizing neuroscience, optogenetics quickly piqued the interest of the wider scientific community, and optogenetic applications were expanded to cardiovascular research. Over the past decade, researchers have employed various optical tools to observe, regulate, and steer the membrane potential of excitable cells in the heart. Despite these advancements, achieving control over specific signaling pathways within the heart has remained an elusive goal. Here, we review the optogenetic tools suitable to control cardiac signaling pathways with a focus on GPCR signaling, and delineate potential applications for studying these pathways, both in healthy and diseased hearts. By shedding light on these exciting developments, we hope to contribute to the ongoing progress in basic cardiac research to facilitate the discovery of novel therapeutic possibilities for treating cardiovascular pathologies.

Functional optimization of light-activatable Opto-GPCRs: Illuminating the importance of the proximal C-terminus in G-protein specificity.

Introduction: G-protein coupled receptors (GPCRs) are the largest family of human receptors that transmit signals from natural ligands and pharmaceutical drugs into essentially every physiological process. One main characteristic of G-protein coupled receptors is their ability to specifically couple with different families of G-proteins, thereby triggering specific downstream signaling pathways. While an abundance of structural information is available on G-protein coupled receptorn interactions with G-proteins, little is known about the G-protein coupled receptor domains functionally mediating G-protein specificity, in particular the proximal C-terminus, the structure which cannot be predicted with high confidentiality due to its flexibility. Methods: In this study, we exploited OptoGPCR chimeras between lightgated G-protein coupled receptors (opsins) and ligand-gated G-protein coupled receptors to systematically investigate the involvement of the C-terminus steering G-protein specificity. We employed rhodopsin-beta2-adrenoceptor and melanopsin-mGluR6 chimeras in second messenger assays and developed structural models of the chimeras. Results: We discovered a dominant role of the proximal C-terminus, dictating G-protein selectivity in the melanopsin-mGluR6 chimera, whereas it is the intracellular loop 3, which steers G-protein tropism in the rhodopsin-beta2-adrenoceptor. From the functional results and structural predictions, melanopsin and mGluR6 use a different mechanism to bovine rhodopsin and b2AR to couple to a selective G-protein. Discussion: Collectively, this work adds knowledge to the G-protein coupled receptor domains mediating G-protein selectivity, ultimately paving the way to optogenetically elicited specific G-protein signaling on demand.

The PV2 cluster of parvalbumin neurons in the murine periaqueductal gray: connections and gene expression.

The PV2 (Celio 1990), a cluster of parvalbumin-positive neurons located in the ventromedial region of the distal periaqueductal gray (PAG) has not been previously described as its own entity, leading us to study its extent, connections, and gene expression. It is an oval, bilateral, elongated cluster composed of approximately 475 parvalbumin-expressing neurons in a single mouse hemisphere. In its anterior portion it impinges upon the paratrochlear nucleus (Par4) and in its distal portion it is harbored in the posterodorsal raphe nucleus (PDR). It is known to receive inputs from the orbitofrontal cortex and from the parvafox nucleus in the ventrolateral hypothalamus. Using anterograde tracing methods in parvalbumin-Cre mice, the main projections of the PV2 cluster innervate the supraoculomotor periaqueductal gray (Su3) of the PAG, the parvafox nucleus of the lateral hypothalamus, the gemini nuclei of the posterior hypothalamus, the septal regions, and the diagonal band in the forebrain, as well as various nuclei within the reticular formation in the midbrain and brainstem. Within the brainstem, projections were discrete, but involved areas implicated in autonomic control. The PV2 cluster expressed various peptides and receptors, including the receptor for Adcyap1, a peptide secreted by one of its main afferences, namely, the parvafox nucleus. The expression of GAD1 and GAD2 in the region of the PV2, the presence of Vgat-1 in a subpopulation of PV2-neurons as well as the coexistence of GAD67 immunoreactivity with parvalbumin in terminal endings indicates the inhibitory nature of a subpopulation of PV2-neurons. The PV2 cluster may be part of a feedback controlling the activity of the hypothalamic parvafox and the Su3 nuclei in the periaqueductal gray.