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Reviews | Xiaohong Xu (Center for Excellence in Brain Science and Intelligent Technology, Chinese Academy of Sciences), Zhihua Gao (Zhejiang University), and Tian Xue (University of Science and Technology of China).
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 is stored in large dense-core vesicle, 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 dysregulation of OT/OTR signaling is closely related
to a variety of negative emotions and psychiatric diseases such as anxiety, autism and schizophrenia 。 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, Li Yulong's lab at Peking University published an online report entitled A genetically encoded sensor measures temporal oxytocin release from different neuronal in the journal Nature Biotechnology A research paper by compartments reporting on the novel genetically encoded oxytocin fluorescent probe GRABOT1.
0 and 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 molecule detection technology, has developed fluorescent probes for choline, monoamines, purines and lipid neurotransmitters/conditioners, the reported GRABOT1.
0 is the first neuropeptide fluorescent probe in the series, further expanding the GRAB fluorescent probe family
.
In this work, Li Yulong's lab used the GRAB probe strategy (GPCR-Activation Based Sensor) to graft the cyclically rearranged green fluorescent protein cpGFP to the third intracellular loop of the oxytocin receptor OTR.
Through the screening of OTRs derived from different species and the systematic optimization of peptide linkage, the best OT fluorescent probe GRABOT1.
0 (referred to as OT1.
0) was finally developed based on bovine oxytocin receptor (bovine OTR).
In in vitro cultured HEK293T cells and primary neurons, the OT1.
0 probe exhibited excellent membrane localization
.
The neuron-expressed OT1.
0 probe has a ~450% fluorescence signal response (ΔF/F0) and ~3 nM affinity (EC50) for exogenously added oxytocin (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: How OT1.
0 probes behave 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 paraventricular nucleus (PVN) of the hypothalamus could also be detected
by the OT1.
0 probe.
Interestingly, further pharmacological experiments have shown that the release of oxytocin in the soma and axon terminals is dependent on very different calcium channels: OT release in somal 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
.
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 had specific dynamic changes
in brain regions at different stages of mating behavior.
These findings further confirm that the OT1.
0 probe has high sensitivity, excellent signal-to-noise ratio, and fast kinetics
in vivo applications.
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 group has also recently developed a variety of neuropeptide GRAB fluorescent probes, including adrenocorticotropic hormone-releasing factor (CRF), somatostatin (SST), and cholecystokinin (CCK) , vasoactive intestinal peptide (VIP), neurotensin (NTS), and neuropeptide Y (NPY), et al.
bioRxiv, 2022)
。 In addition, the team of Tommaso Patriarchi of ETH Zurich in Switzerland and Daisuke Ino of Kanazawa University in Japan have also developed fluorescent probes OxLight1 (Duffet L et al.
, Nature Methods, 2022) and MTRIAOT for the detection of orexin and oxytocin, respectively （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
.
Beijing BigProfessor Li Yulong from the School of Life Sciences 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.
For more information on Li Yulong's laboratory work, see: http://yulonglilab.
org/
.
In addition, Li Yulong's laboratory recruits associate researchers, postdoctoral fellows and technicians from different discipline backgrounds, and the remuneration is excellent
.
Welcome aspiring young people who are interested in brain science to join!
Expert comment: Xu Xiaohong (senior researcher, Center for Excellence in Brain Science and Intelligent Technology, Chinese Academy of Sciences) Oxytocin It is an evolutionarily highly conserved neuropeptide involved in the regulation of a variety of important body functions, often colloquially referred to as "the love hormone" because of its role in parent-child behavior and gender relations
.
Here, Li Yulong's research group of Peking University reported a new genetically encoded oxytocin fluorescent probe, GRABOT1.
0, which greatly breaks through the limitations of previous microdialysis-based detection methods in terms of time, space and sensitivity, and realizes the real-time monitoring of
the dynamic release of oxytocin in behavior in different brain regions of awake animals 。 They found that the mechanism of release of oxytocin at the end of the axons and somal dendrites differed, and during the courtship of male mice, oxytocin was released through axons in the prefrontal lobe during the olfactory exploration phase, in the hypothalamic paraventricular nucleus during mating, and in the ejaculation phase in the prefrontal lobe and ventral tegmental area
.
More importantly, GRABOT1.
0 is a "sharp weapon" that breaks through the bottleneck of detecting the dynamic release of oxytocin in vivo, and opens up a new world for future cross-species research on the role of oxytocin in social behavior and social cognition
.
Expert comment: Gao Zhihua (professor of Zhejiang University School of Medicine): Oxytocin, which is composed of only nine amino acids, is a "magical" hormone
.
Unlike other hormones such as growth hormone and glucocorticoids, which are secreted directly by the glandular cells of endocrine organs, oxytocin is secreted
by endocrine neurons located in the hypothalamus.
These neurons can not only secrete oxytocin into the blood, reach distant target organs to regulate women's labor and lactation, but also release the emotional, cognitive, social and other behaviors
of oxytocin in the brain.
Studies have shown that oxytocin in the brain can significantly enhance the family bond between spouses, mothers and babies, so it is also known as "love hormone" and "maternal love hormone"
.
Although there are many studies on oxytocin, there has been a lack of good detection tools to dynamically observe the changes
in oxytocin in the brain in real time.
Li Yulong's laboratory at Peking University has developed a novel oxytocin fluorescent probe (OT1.
0)
using the GRAB probe strategy.
They cleverly grafted fluorescent proteins into bovine-derived oxytocin receptors, and then screened out fluorescent receptor probes that best responded to oxytocin through different combinations
.
The probe not only responds quickly to changes in oxytocin concentrations in vitro, but can also be used to record oxytocin changes
in the brains of living mice.
Using this probe, the research group successfully recorded the dynamic changes of oxytocin in different brain regions during the mating process of male mice, and based on the high temporal and spatial resolution of the probe, the relevant regulatory mechanism of oxytocin release patterns in different regions of oxytocin neurons was deeply explored, which provided a new perspective
for further understanding the precise regulation of oxytocin.
Li Yulong's laboratory focuses on the development of neurochemical molecular detection technology, and has developed a variety of fluorescent probes for different neurotransmitter types, which provide important tools and means
for analyzing the role of neurotransmitters in the brain in the regulation of mood, cognition and morphology 。 The newly developed genetically encoded oxytocin fluorescent probe has good spatiotemporal resolution and dynamic responsiveness, which provides a good research tool for further exploring the function of oxytocin in the brain, which will greatly promote and expand the development of
oxytocin-related research.
Expert comment: Xue Tian (professor at University of Science and Technology of China) oxytocin (OT), mainly by the paraventricular nucleus of the hypothalamus (PVN) and nonapeptide
secreted by oxytocinergic neurons within the supraoptic nucleus (SON).
It was first extracted
from animals a hundred years ago by British pharmacologist Sir Henry Hallett Dale.
At that time, it was found that it had the effect of promoting uterine contraction, and it was named oxytocin
.
With the revelation of OT function over the past century, in addition to the secretion of OT into the blood through the posterior pituitary gland to participate in the lactation function of childbirth, oxytocinergic neurons directly innervate many brain regions and OT can be directly released into the cerebrospinal fluid, and these OTs through nerve projection and into the cerebrospinal fluid play an important role
in promoting brain development, social interaction, finding a mate and maternal paternity.
Different from the convenient collection and detection of blood samples, it is very challenging
to monitor the spatiotemporal dynamics of OT in the brain.
With the wide application of advanced technologies such as fiber recording, in-vivo endoscopic imaging systems and in vivo two-photon calcium imaging in the field of neuroscience in recent years, the development of a fluorescent probe that can monitor OT release in vivo spatiotemporal can effectively decode the relationship between OT and dynamic behavior, which has become an important problem
to be solved in the field.
Recently, Nature Biotechnology reported that Professor Li Yulong's team of Peking University independently developed a bovine-derived OT receptor as the probe skeleton, grafting conformation-sensitive cpEGFP with it to form a genetically encoded oxytocin fluorescent probe with high specificity, high signal-to-noise ratio and high temporal resolution, GRAB OT1.
0
。 Further, this work analyzes the characteristics of OT at the time of release, that is, the OT released by axon terminals mainly relies on N-type voltage-sensitive calcium channels (N-type VGCC), and the release of OT in dendrites and somatosomes depends on L-type VGCC
.
This provides insight into the mechanism of neuropeptide release
.
Importantly, the probe allows scientists to analyze the spatial and temporal dynamics of OT in the brain in real time, giving the field powerful tools to analyze the dynamic
relationship between a range of neurophysiological processes and OT.
For example, this paper analyzes the three stages of mating, sniffing (OT release participation in PFC), mating (OT release participation in PVN), and ejaculation (VTA and PFC co-OT release participation).
The temporal and spatial sequence of OT participation in the process, directly visualizing the dynamic relationship
between OT and behavior.
Professor Li Yulong has independently developed and improved a large number of fluorescent probes of neurotransmitters since returning to China, which are currently widely used in the analysis of neural circuits and the study of the regulation mechanism of transmitter release in physiological and pathological states, and have been widely recognized by the international academic community as China's original brain science and technology, which has greatly accelerated the pace of
brain science research.
It is expected that they will develop more neuroscience tools in the future and light up the exploration
of brain science research.
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