Neuropeptides produce robust effects on behavior across species, and recent research has benefited from advances in high-resolution techniques to investigate peptidergic transmission and expression throughout the brain in model systems. Neuropeptides exhibit distinct characteristics which includes their post-translational processing, release from dense core vesicles, and ability to activate G-protein-coupled receptors (GPCRs). These complex properties have driven the need for development of specialized tools that can sense neuropeptide expression, cell activity, and release. Current research has focused on isolating when and how neuropeptide transmission occurs, as well as the conditions in which neuropeptides directly mediate physiological and adaptive behavioral states. Here we describe the current technological landscape in which the field is operating to decode key questions regarding these dynamic neuromodulators.
Neuropeptides act as neuromodulators in the CNS, eliciting a wide variety of effects in many physiological processes as well as in behavioral and emotional states. Neuropeptides are specifically synthesized in neurons and stored in dense core vesicles (DCVs). They often act to modulate signaling and synaptic activity by binding to GPCRs and, in some cases, to neurotrophic tyrosine kinase receptors. Neuropeptide-induced GPCR activation occurs along many time scales.
Neuropeptide research has benefited from advances in techniques used to investigate their release, receptor targets, and coexpression with established cell-type markers. Novel fluid and tissue collection techniques have been used to finely measure evoked neuropeptide release, and slice electrophysiology has elucidated the circuit and synaptic architecture of neuropeptide and fast-acting neurotransmitter corelease. Providing granularity in cell-type identity and genetic expression, in situ hybridization and single-cell RNA sequencing have been instrumental in identifying neuropeptide expression patterns.
While single-cell transcriptomics has been able to identify the markers of peptidergic neurons, calcium imaging using fiber photometry allows for in vivo investigation of population dynamics of genetically encoded fluorescent calcium indicators, such as GCaMP6. The benefit of imaging a targeted population using fiber photometry lies in the diameter of the optical probes, with some fiber optics measuring as little as 50 μm in diameter. While this minimizes the tissue damage from implantation, it also restricts the field of view to a specific brain region.
Addressing questions about neuropeptide action – including localization, temporal effects, and network-wide distribution – requires tools with high spatiotemporal precision. Current tools such as fluorescent GPCR sensors may contribute to a more granular understanding of neuropeptide release physiology. In fact, GPCR sensors for small-molecule neurotransmitters, such as those for dopamine (dLight family) and norepinephrine (GRAB NE), have been used for monitoring release dynamics in vivo.
Photoactivatable tools have been a game changer in recent years for direct cell-type-specific manipulation of neural activity. Of the many different families of these tools, light-gated ion channels and light-driven ion pumps derived from microbial opsins have been widely used. Other photoactivatable tools – including photosensitive drugs and/or genetically modified GPCRs – can be harnessed for light-coupled control of GPCR dynamics. Photopharmacology uses these light-sensitive ligands to control endogenous receptor activity with high spatiotemporal precision.
During the past 20 years, important insights have been gleaned on how neuropeptide signaling affects synaptic architecture, signal transmission, and ultimately behavioral states in living animals. With that, a more refined understanding of how neuropeptides act to influence, sculpt, and shape brain activity is needed. Technological innovation in neural imaging techniques and cell transcriptomics has provided cell identification and observation of neural activity on a finer scale.
