New Technologies to Investigate Neuropeptides at Scale

Abstract

Neuropeptides are some of the most elusive molecules to monitor in neuroscience. Detecting their release and spread in brain tissue requires the development and use of advanced technologies that enable specific neuropeptide measurements with high spatial and temporal resolution. This Viewpoint highlights some of the emerging tools and techniques that are already advancing our knowledge of neuropeptide physiology and discusses possible future developments.

We have come a long way since the 1969 when David de Wied coined the term neuropeptide (NPP) to identify a protein-based hormone acting as a neurotransmitter within the brain.¹ Over 100 individual neuropeptide species are present in the brain, and they are typically cotransmitted by the same neurons along with other neurotransmitters or modulators. The sheer heterogeneity and complexity of neuropeptide actions in the brain combined with their elusive nature present a formidable challenge to investigators. Yet, due to the fundamental modulatory roles they possess that can profoundly shape neural circuit function and animal behavior, they have been subjects of intense investigation since their initial identification. As new transformative optical tools and techniques are being developed that afford us the high spatial and temporal resolution power of light, we might just be at the cusp of a neuropeptide revolution, where one can for the first time shine new light on the hidden details of neuropeptide signals, which in a way could be considered the “dark matter” of the brain (i.e., insofar hidden to the eye but necessarily present).

NPPs are unlike fast neuromodulators. First, they are produced within large dense core vesicles by proteolytic cleavage of larger precursor proteins, where oftentimes a single precursor can give rise to several neuropeptide species acting through different targets. Another striking difference between neuropeptides and classical neuromodulators are their distinctive release mechanisms. While “fast” neurotransmitters and neuromodulators (i.e., glutamate, GABA, dopamine, etc.) are recycled back into release vesicles, neuropeptides, once released, are thought to continue diffusing into the extracellular milieu until they are degraded by the local action of proteases, resulting in their signals spreading thin over hundreds of micrometers away from the sites of release, akin to a long-range broadcast signal. This long-range diffusion and the corresponding gradual dissipation of the signal dilutes the NPP concentration so much that only their high-affinity cognate G-protein coupled receptors (GPCRs) expressed on the “receiving” cells can still detect them. The low active concentrations of NPPs in brain tissue thus pose a major obstacle for their detection.

Our ability to monitor neuropeptide actions in brain tissue is evolving. Traditionally, classical patch-clamp electrophysiology has been the gold standard for investigating NPP effects on neurons. This technique, albeit limited by very low throughput (i.e., recording one NPP and one neuron at a time), provides a simple and direct way to ascertain NPP action on a specified target cell type, simply by direct application onto the patched neuron of a set concentration of NPP. Microdialysis, on the other hand, succeeded in measuring neuropeptide fluctuations with high throughput (>10 peptides can be measured at a time in specific brain areas), but it provides only coarse temporal and spatial information. Fast scan voltammetry has been adapted to the detection of certain neuropeptide species (i.e., tyrosine-containing), such as met-enkephalin.² While exciting and useful, this technique still remains technically challenging to perform and may not be easy to repurpose for the detection of every single neuropeptide species. To be able to match the local release kinetics and concentrations of NPPs with the required sensitivity and specificity of detection and at the same time provide information on the spatial and temporal patterns of their release, three relatively new classes of technologies take the center stage, and they share the same molecular detector: a GPCR.

The first class of technologies discussed here that was deployed to detect a neuropeptide signal in vivo is called iTango2. This is a fully genetically encoded system relying on the expression of a GPCR and a β-arrestin construct each carrying half of a tobacco-etch virus protease. Upon light exposure and ligand-induced β-arrestin recruitment to the receptor, the protease recovers its function and cleaves a receptor-tethered transcription factor, which then translocates to the nucleus to initiate the downstream expression of a reporter or actuator gene. The first version of this system that has been deployed for the detection of endogenous neuropeptides has been directed toward oxytocin (OXTR-iTango2³) and could successfully mark a subpopulation of dopaminergic neurons in the mouse ventral tegmental area that receive oxytocin signals when mice socially interact. Main advantages of this technique are the ability to temporally confine neuropeptide detection to the period coincident with light exposure, as well as the cellular resolution of the readout. However, by being reliant on gene expression, this method loses the possibility of gathering any subcellular-level of information regarding neuropeptide dynamics and has an inherently slow readout that can only be obtained once the animal is sacrificed.

The second class of technologies are called CNiFERs (for cell-based neurotransmitter fluorescent engineered reporter). These are engineered cells that stably express a GPCR cognate to the neuropeptide of interest and a modified G-protein that allows them to transduce the NPP signal into an intracellular calcium fluctuation that is then detected via a FRET-based calcium sensor expressed in the same cells. These cells are meant to be microinjected at specified positions in brain tissue where they can be imaged over the course of days using two-photon microscopy. By utilizing an unmodified receptor as neuropeptide detector, CNiFERs leverage the highest detection affinity and specificity that can be found through natural sources. Building on this approach, researchers developed an all-optical platform for locally releasing and detecting neuropeptide signals in brain tissue called PACE.⁴ This breakthrough approach allowed them to obtain a rather precise estimate of functional neuropeptide signal spread from the area of release, and it even permitted determination of the contribution of an extracellular matrix component to the NPP diffusion rate, which moves the boundaries of our knowledge a step forward.

The third and most recent class of technologies are genetically encoded sensors based on the engineering of a circularly permuted green fluorescent protein (cpGFP) into a GPCR. The ligand-induced conformational activation of the GPCR directly triggers a rapid increase in cpGFP fluorescence that permits real-time monitoring of extracellular ligand concentrations. These tools can be expressed directly in brain tissue via adeno-associated viral delivery, thus facilitating long-term imaging of neuromodulator release. To date only one such sensor, named OxLight1,⁵ has been developed and validated for the detection of endogenous neuropeptide release. This sensor named OxLight1⁵ detects orexins (also known as hypocretins), two with very high sensitivity. These are highly conserved NPPs that play an important role in the maintenance of stable wakefulness, as well as feeding and motivation. Thanks to this tool it has been possible to precisely determine the release kinetics of these NPPs directly in awake behaving mice, revealing unexpectedly rapid changes of these signals, which ensued and decayed within a few seconds. Furthermore, in combination with two-photon imaging this tool revealed the presence of localized orexin signals in the upper cortical space of the mouse brain (i.e., Layer 2/3, somatosensory cortex). Given the great value and promise of this technology, it is essential to continue developing many more genetically encoded NPP sensors in an effort to match the large number of NPPs present in the brain.

While the exciting prospect of sensing NPPs with high spatiotemporal resolution is certainly appealing, it is important to keep in mind that all the approaches mentioned above can only provide correlative evidence of the presence of a NPP within a specific behavioral context and/or brain state. Causative evidence is still missing. It will be necessary for the field to move in the direction of researching and developing new molecular tools endowed with the fine activation modes and rapid kinetics of optogenetic tools but targeted toward specific NPP signaling pathways. A new generation of light-activatable neuropeptide receptors based on the concept of engineered chimeric opsin/GPCR constructs (originally known as OptoXRs), for example, could allow us to control the activation of NPP receptor signaling in a particular cell type in vivo. Yet, due to the much higher expression levels of these tools compared to endogenous receptors and the lack of a “reference signal” for what the endogenous NPP fluctuation actually looks like, this technology runs into the risk of producing NPP-looking stimuli not necessarily matching the more nuanced signaling events that endogenous receptors can actually trigger. Therefore, more suited to the causal investigation of NPP function would be tools capable of triggering endogenous NPP receptor activation or inactivation in response to illumination. Such tools would prove to be invaluable assets in combination with optical sensing technologies and could truly transform NPP research into something more tractable across scales.

Despite all the inherent difficulties involved in monitoring NPPs, the advent of the new molecular and cellular techniques described above comes now to the aid of microdialysis, voltammetry, and electrophysiology.

All these technologies should continue to be developed and should be deployed in concert wherever possible, so that they can benefit from their collective strengths and overcome their individual limitations. For example, a new format of PACE could be deployed by combining two-photon NPP release with NPP detection using a genetically encoded sensor. This would enable us to generate a more spatially detailed map of NPP signal spread and provide additional information on their extracellular kinetics. Furthermore, the development of red-shifted neuropeptide sensors could free up wavelength space in the UV-green range (405−510 nm) that could be leveraged upon to develop all optical assays for the screening and development of new tools for controlling endogenous NPP receptor activity.

Hopefully with this and the next-generation of tools at hand, we will be able to learn a great deal about the nature of neuropeptide signals in the brain. By monitoring and controlling when and where these signals are coming from and to whom they are directed, we will have a better chance at understanding their precise message and physiological function.