Oxytocin (OT), a neuropeptide synthesized in the hypothalamus, is a key regulator of diverse physiological processes in peripheral tissues and the central nervous system. While OT's peripheral roles in childbirth and lactation are well-established, its central actions as a neuromodulator are less understood. OT neurons project to various brain regions, modulating functions such as sensory processing, feeding behavior, social cognition, and emotion. Dysfunctional OT signaling is implicated in neurodevelopmental disorders (e.g., autism, schizophrenia) and brain aging. The timescale of OT's effects is highly variable, ranging from seconds to potentially much longer durations, suggesting dynamic fluctuations depending on behavioral state, stimuli, and physiological condition. Exogenous OT administration, initially considered a therapeutic avenue for psychiatric disorders due to its mood-enhancing effects in humans, has shown controversial efficacy. The extent to which peripherally administered OT reaches the brain remains unclear, highlighting the urgent need for reliable methods to detect brain OT dynamics in real-time. Current techniques like microdialysis and reporter gene-based assays have limitations, notably in temporal resolution. Recent advancements in genetically encoded fluorescent sensors, particularly GPCR-based sensors utilizing fluorescent proteins replacing amino acids in the intracellular loop (IL3), have shown promise for real-time neurotransmitter and neuromodulator detection. This study leverages this technology to create a highly sensitive and specific OT sensor for in vivo applications.

Existing methods for measuring oxytocin, such as microdialysis and iTango reporter gene assays, suffer from limitations in temporal resolution. Microdialysis, while offering some insight into extracellular OT, lacks the rapid sampling rate needed to capture the dynamic fluctuations hypothesized to underlie its diverse functions. Reporter gene-based assays, like iTango, offer improvements, but are still not able to provide the real-time resolution that would allow a more complete understanding of oxytocin's role in the brain. Studies on the effects of exogenous OT administration on behavior have shown inconsistent results. Some studies have suggested that intranasal administration, while somewhat problematic, may increase brain OT levels; however, other studies question this finding, suggesting that the effects of exogenous OT on behavior are far more complex and context-dependent. This highlights the need for improved methods to understand the relationship between brain OT levels and behavior. The development of sensitive, real-time fluorescent sensors offers the potential to overcome these limitations and provide unprecedented insights into the complex dynamics of OT signaling in the brain.

The researchers developed a novel fluorescent oxytocin sensor, MTRIA^OT, based on a medaka OT receptor (meOTR) and a circularly permutated green fluorescent protein (cpGFP) module (MTRIA). A key aspect of the methodology involved a multi-step screening process to optimize the sensor's properties. This included a selection of the best OTR scaffold for membrane localization, followed by optimization of the cpGFP insertion site in the IL3 of the meOTR, and subsequent refinement of linker regions, TM-to-loop regions, and residues within cpGFP itself. This screening yielded the MTRIA^OT sensor, characterized by a significant fluorescence response upon OT stimulation. Ligand specificity was assessed against OT analogs (isotocin, vasopressin orthologs, inotocin, nematocin), confirming high selectivity for OT. The sensor's functional properties were thoroughly investigated, including its coupling to downstream signaling pathways (Gα proteins, β-arrestin), long-term fluorescence stability, kinetic parameters, and pH sensitivity. These characterizations were performed in both HEK293T cells and cultured rat hippocampal neurons, demonstrating the sensor's robust performance in different cellular contexts. In vivo validation utilized adeno-associated viruses (AAVs) to express MTRIA^OT in the anterior olfactory nucleus (AON) of mice, a brain region with high OT receptor expression. Fiber photometry was then used to measure fluorescence changes in response to various stimuli. These stimuli included intracerebroventricular (ICV) injections of OT, peripheral OT administration (intranasal, intraperitoneal), optogenetic stimulation of OT neurons in the paraventricular nucleus (PVN), and behavioral manipulations (social interaction, acute stress, food deprivation). Furthermore, the impact of general anesthetics (dexmedetomidine/butorphanol/midazolam, isoflurane) and aging on OT dynamics were also investigated using fiber photometry.

The development of MTRIA^OT provided a sensitive and specific tool for real-time measurement of extracellular OT dynamics in the brain. In vivo experiments demonstrated that MTRIA^OT successfully detected artificially evoked OT signals following ICV OT injection, with a clear dose-dependent response (significant increases at 0.2 µg and above). However, peripheral OT administration (intranasal or intraperitoneal at 20 µg) failed to elicit detectable OT increases in the AON, suggesting limited brain penetration of peripherally applied OT. Optogenetic stimulation of OT neurons in the PVN using ChRmine-mSca resulted in dose-dependent increases in AON MTRIA^OT fluorescence, confirming the sensor's ability to detect endogenous OT release. Real-time recordings in freely behaving mice revealed an ultradian OT rhythm (OT oscillation) characterized by transient increases at approximately 2-hour intervals, absent in control experiments, and confirmed as dependent on OT release from PVN neurons. The sensor's rapid kinetics also allowed detection of faster OT responses. Social interaction with a conspecific mouse induced a gradual increase in AON MTRIA^OT fluorescence (rise time constant ~1 min), while acute stress (tail lift) elicited a rapid response (~6 s to peak). Anesthetic drugs (mix-anes, isoflurane) significantly suppressed AON OT levels, while food deprivation disrupted OT oscillations, leading to a pattern termed 'OT turbulence'. Finally, aging was associated with a decreased frequency of OT oscillations, although amplitudes remained unchanged.

This study demonstrates that the novel MTRIA^OT sensor successfully measures real-time oxytocin dynamics in the brain in vivo. The findings highlight the highly dynamic and context-dependent nature of oxytocin signaling. The failure to detect significant AON OT increases following peripheral OT administration challenges the notion of effective brain penetration via intranasal or intraperitoneal routes. The observed ultradian OT rhythm (OT oscillation) represents a novel finding, demanding further investigation into its underlying mechanisms and physiological significance. The disruption of OT oscillations by food deprivation suggests a potential link between OT and appetite regulation. The effect of anesthesia and aging on OT dynamics emphasizes the importance of carefully controlling for experimental conditions in studies involving oxytocin. The findings may help to explain the inconsistent results of human clinical trials involving exogenous OT administration by suggesting that participant-dependent and context-dependent factors can significantly affect brain oxytocin levels.

This study successfully developed and validated a novel fluorescent sensor, MTRIA^OT, enabling real-time measurement of extracellular oxytocin dynamics in the brain. The in vivo application of MTRIA^OT revealed previously unknown aspects of oxytocin's dynamic regulation, including an ultradian rhythm and sensitivity to anesthesia, food deprivation, and aging. Future research should focus on quantitative OT measurements using advanced sensor technologies, further elucidating the regulatory mechanisms and functional significance of OT oscillations and their association with various physiological processes and behaviors.

While MTRIA^OT provides unprecedented temporal resolution, the current study primarily focuses on the AON and PVN. Further investigations across other brain regions are necessary to gain a comprehensive understanding of OT dynamics. The study primarily uses female mice. Investigating potential sex differences in OT dynamics is crucial. Although the sensor shows high specificity, it is important to note that interactions with other closely related peptides might affect interpretation, and further investigation to minimize crosstalk is needed. The current study does not directly measure absolute OT concentrations. The development of next-generation sensors capable of quantitative measurement would improve the comparability of results across various experimental conditions.