Nature Biotechnology Yulong Li's lab has developed a novel oxytocin fluorescent probe to reveal the spatiotemporal dynamics of neuropeptides

Oxytocin (OT) is a nine-amino acid neuropeptide that plays an integral role
in the endocrine and central nervous systems of mammals.
Oxytocin is synthesized primarily by oxytocinergic neurons located in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) and stored in large dense-core vesicles , LDCV
).
On the one hand, oxytocin is released into the blood circulation through the posterior pituitary gland, regulating childbirth and lactation behavior; On the other hand, it is released into the brain through somal dendrites and axon terminals projected to other brain regions, regulating important social behaviors
such as mate union, mother-infant relationship and social cognition.
OT functions by activating the oxytocin receptor (OTR) of the G protein-coupled receptor (GPCR) family, and the imbalance of OT/OTR signaling and anxiety, A variety of negative emotions such as autism and schizophrenia are closely related
to mental illness.
Although oxytocin is involved in many of the above physiological and pathological processes and plays important regulatory functions, the spatiotemporal dynamics and molecular mechanism of its release in the body are still unclear, and the main technical bottleneck is the lack of sensitive, specific, non-invasive, and high spatiotemporal resolution probes to track its dynamic changes
.

On January 2, 2023, the lab of Li Yulong of Peking University published an online publication in the journal Nature Biotechnology A genetically encoded sensor measures temporal oxytocin release from different neuronal compartments, a research paper reporting novel genetically encoded oxytocin fluorescent probesGRABOT1.
0and its application in live animals; Combined with the new tools, this article delves into the molecular mechanisms
of oxytocin release.
Li Yulong's laboratory has been committed to the systematic development of neurochemical molecular detection technology, and has successively developed fluorescent probes for choline, monoamines, purines and lipid neurotransmitters/conditioners, which was reportedGRABOT1.
0It is the first neuropeptide fluorescent probe in the series, further expanding the GRAB fluorescent probe family
.

In this work, Li's lab used the GRAB probe strategy (GPC R-ActivationBased Sensor) to graft the cyclically rearranged green fluorescent protein cpGFP to the oxytocin receptor OTR The third intracellular loop, through the screening of OTRs from different species sources and systematic optimization of linkage peptides, finally developed the best performing OT fluorescent probe based on bovine oxytocin receptor (bovine OTR).
GRABOT1.
0(OT1.
0 for short).

In in vitro cultured HEK293T cells and primary neurons, the OT1.
0 probe exhibited excellent membrane localization
.
Neuron-expressed OT1.
0 probes have ~450% fluorescence signal response to exogenously added oxytocin (ΔF/F0) and affinity of ~3 nM (EC50(Figure 1).

In addition, the OT1.
0 probe can respond to changes in extracellular OT concentration on sub-second (~0.
5s) time scales without activating the downstream signaling pathway of GPCR, so there is no significant effect
on the normal physiological activities of cells.

Figure 1: Performance of the OT1.
0 probe on HEK293T cells and primary cultured neurons

So can OT1.
0 be used to detect endogenous oxytocin release? The authors used AAV virus to express the OT1.
0 probe in the ventral dorsal area (VTA) of the midbrain (VTA) projected by oxytocinergic neuronal axons, and successfully recorded axonal OT release
induced by electrical stimulation in acute brain slices of mice.
In addition, the authors found that oxytocin released by the soma dendrites of the hypothalamic paraventricular nucleus (PVN) could also be detected
by OT1.
0 probes.
Interestingly, further pharmacological experiments have shown that oxytocin release in the soma and axon terminals is dependent on very different calcium channels: OT release in soma dendrites is mainly mediated by L-type voltage-gated calcium channels, while its release at axonal terminals is mainly mediated by N-type voltage-gated calcium channels (Figure 2).

Figure 2: OT1.
0 probe detects endogenous oxytocin release in mouse acute brain slices

The prefrontal cortex (PFC), a key brain region for social behavior, expresses oxytocin receptors and receives axonal projection from OT neurons in PVN
.
To investigate whether the OT1.
0 probe could be used to report the dynamics of oxytocin in the brain of living animals, the authors first specifically expressed the optogenetic tool ChrimsonR in OT neurons and the OT1.
0 probe in PFCs
.
Using fiber-optic recording technology, the authors successfully detected oxytocin release
caused by activation of OT neurons in freely active mice.
Further using the OT1.
0 probe to record the levels of extracellular oxytocin in each brain region during mating in male mice, the authors found that OT signals have specific dynamic changes in brain regions at different stages of mating behavior (Figure 3).

These findings further confirm that the OT1.
0 probe has high sensitivity, excellent signal-to-noise ratio, and fast kinetics
in vivo applications.

Figure 3: The OT1.
0 probe reports oxytocin signaling in the brain of freely active mice during optogenetic activation and mating behavior

In summary, in this work, the authors developed a novel genetically encoded oxytocin fluorescent probe, which achieved high spatiotemporal resolution recording of extracellular oxytocin in vitro and in vivo, and reported the similarities and differences in the molecular mechanisms of oxytocin in the two release modes of neuronal axon terminals and somal dendrites, which provided a new reference
for understanding the complexity of neuropeptide function.

It is worth mentioning that Li Yulong's research group has recently developed a variety of neuropeptide GRAB fluorescent probes, including adrenocorticotropin-releasing factor (CRF), somatostatin (SST), cholecystokinin (CCK), and vasoactive intestinal peptide (VIP).
Neurotensin (NTS) and neuropeptide Y (NPY) et al.
(Wang H et al.
, bioRxiv, 2022).

In addition, Tommaso Patriarchi's team at ETH Zurich in Switzerland and Daisuke Ino team at Kanazawa University in Japan have also developed fluorescent probes OxLight1 for the detection of orexin and oxytocin, respectively (Duffet L et al.
, Nature Methods, 2022).
) andMTRIAOT（Ino D et al.
, Nature Methods, 2022）
。 It is believed that the development of these new imaging tools will advance researchers to a deeper understanding of the function and regulation of neuropeptide signaling under physiological and pathological conditions at the molecular and neural circuit level
.

Professor Li Yulong from the School of Life Sciences, Peking University is the corresponding author of this paper; Qian Tongrui, a doctoral student from the School of Life Sciences of Peking University, Huan Wang, a doctoral graduate, and Dr.
Peng Wang from Beijing Chaoyang Hospital affiliated to Capital Medical University are co-first authors of this paper.
Geng Lan and Wang Lei, doctoral students of Peking University, made important contributions
to the paper.
The work was coordinated by the laboratory of Minmin Luo of the Beijing Center for Brain Science and Brain-like Research, the Dayu Lin laboratory of New York University, the Ron Stooop laboratory of the University of Lausanne in Switzerland, and the laboratory of Valery Grinevich of the University of Heidelberg in Germany, and the State Key Laboratory of Membrane Biology of Peking University and Peking University Tsinghua Life Science Joint Center, National Natural Science Foundation of China, Beijing Municipal Science and Technology Commission, Shenzhen-Hong Kong Brain Science Innovation Institute, Peak Fund and other institutions and funds strongly supported
.

For more details on Li Yulong's laboratory work, see: http://yulonglilab.
org/
.

Original link: