Whole-brain wiring diagram of oxytocin system in adult mice

Quantitative comparison between Oxt output and Oxtr expression showed no significant correlation across the whole brain.

“Surprisingly, quantitative comparison between Oxt output and Oxtr expression showed no significant correlation across the whole brain, suggesting abundant indirect Oxt signaling in Oxtr-expressing areas.”

• Also stated in: § Introduction · plain

  “we found lack of quantitative correlation between Oxt output and Oxtr expression across the whole brain”

Oxt neurons in the PVH broadly project to nine functional circuits that control cognition, brain state, and somatic visceral response.

“Oxt neurons in the paraventricular hypothalamus (PVH) broadly project to nine functional circuits that control cognition, brain state, and somatic visceral response.”

• Also stated in: § Abstract · plain

  “We found Oxt connections with nine functional circuits controlling cognition, brain state, and somatic visceral response.”

The cerebrum (cerebral nuclei and cerebral cortex) receives more sparse Oxt projection.

“In contrast, the cerebrum (the cerebral nuclei and cerebral cortex) that works as high cognitive controller receives more sparse projection (Fig. 2E).”

Robust Oxt expression in the TU area was not colocalized with vasopressin staining.

“Importantly, we confirmed the robust Oxt expression in the TU area that was not colocalized with vasopressin staining (Fig. 1I).”

PVH regions contain the highest density of Oxt neurons (~39%, 742 out of total 1899 cells) followed by the tuberal nucleus (TU), SO, and other areas.

“The PVH regions (PVH, descending division of PVH, anterior, intermediate, and subparaventricular zone) contain the highest density of Oxt neurons (∼39%, 742 out of total 1899 cells) followed by the tuberal nucleus (TU), SO, and other areas (Table 1).”

The largely overlooked TU area contains almost as high density of Oxt neurons as the PVH area.

“Notably, the largely overlooked TU area contains almost as high density of Oxt neurons as the PVH area (Fig. 1E).”

The somatic visceral module includes pain, sensory motor, and body physiology/metabolism circuits.

“The somatic visceral module includes pain, sensory motor, and body physiology/metabolism circuits (Fig. 2E).”

Dense Oxt projection occurs largely onto the hindbrain (DMX, PB), midbrain (SNc), and hypothalamus (MPN).

“Dense projection occurs largely onto the hindbrain (e.g., the dorsal motor nucleus of the vagus nerve; DMX, the PB), the midbrain (the substantia nigra compacta; SNc), and the hypothalamus (the medial preoptic nucleus; MPN) to directly modulate motor output and sensory input (Fig. 2E).”

The estimated number of Oxt neurons in the PVH with immunostaining was 1095 cells out of total 3149 cells (~35%).

“For example, the estimated number of Oxt neurons in the PVH with the immunostaining was 1095 cells out of total 3149 cells (∼35%), which is higher than our transgenic based estimate mostly likely because of sensitive labeling based on antibody detection.”

There is no significant correlation between relative Oxt projection and relative Oxtr density across the whole brain and major brain areas, except for the thalamus and the medulla.

“When we examined whether relative projection of Oxt neurons is correlated with relative Oxtr density across the entire brain, we found no significant correlation across the whole brain and major brain areas, except for the thalamus and the medulla (Fig. 3C).”

• AssumptionRests on an assumption: The quantitative relationship between Oxt projection and Oxtr expression across the whole brain is currently lacking.

SO and AN Oxt neurons project to a small subset of PVH-Oxt efferent areas.

“Overall, the PVH neurons showed the broadest projection pattern followed by the SO and the AN, which project to a small subset of PVH-Oxt efferent areas (Fig. 2D,E; Movie 2).”

Oxt neurons in the SO and AN have limited central projection to a small subset of the nine circuits.

“In contrast, Oxt neurons in the supraoptic (SO) and accessory (AN) nuclei have limited central projection to a small subset of the nine circuits.”

PVH-Oxt neurons project to these areas at varying degrees.

“PVH-Oxt neurons project to these areas at varying degrees.”

PVH Oxt neurons show the broadest projection pattern among the four anatomic areas (PVH, SO, AN, TU).

“Overall, the PVH neurons showed the broadest projection pattern followed by the SO and the AN, which project to a small subset of PVH-Oxt efferent areas (Fig. 2D,E; Movie 2).”

In the PVH, 76% of tdTomato labeled cells were Oxt immuno positive (1733 out of 2277 cells).

“In contrast, 76% of tdTomato labeled cells were Oxt immuno positive (1733 out of 2277 cells) in the PVH.”

Oxt neuronal expression is in four hypothalamic domains including under-characterized Oxt neurons in the tuberal nucleus (TU).

“We use a flatmap to describe Oxt neuronal expression in four hypothalamic domains including under-characterized Oxt neurons in the tuberal nucleus (TU).”

• Also stated in: § Quantitative density mapping of Oxt neurons reveals four clusters in the adult mouse brain · boosted

  “Oxt density heatmap on the hypothalamic flatmap clearly shows four clusters: (1) the PVH; (2) the SO; (3) accessory nuclei (AN); (4) the TU area”

Oxt neurons in both the PVH and SO receive similar monosynaptic inputs from a subset of the nine circuits mainly in the thalamic, hypothalamic, and cerebral nuclei areas.

“Unlike output, Oxt neurons in both the PVH and SO receive similar monosynaptic inputs from a subset of the nine circuits mainly in the thalamic, hypothalamic, and cerebral nuclei areas.”

Each circuit is composed of multiple brain regions from the hindbrain, midbrain, thalamus, hypothalamus, cerebral nuclei, and cerebral cortex that process low-to-high order information.

“Each circuit is composed of multiple brain regions from the hindbrain, midbrain, thalamus, hypothalamus, cerebral nuclei, and cerebral cortex that process low-to-high order information.”

Nine functional circuits receive long-range projection from Oxt neurons in three different injection areas (PVH, SO, AN).

“Nine functional circuits that receive long-range projection from Oxt neurons in the three different injection area 1–3.”

Oxt fibers with thick varicosities are frequently found at the lateral, third, and fourth ventricle surface.

“Indeed, we frequently found Oxt fibers with thick varicosities at the lateral, third, and fourth ventricle surface (Fig. 3D).”

Almost all Oxt immuno positive neurons (97%, 1733 out of 1790 cells, n = 4 animals) were labeled by tdTomato from Oxt-Cre;Ai14 mice.

“We confirmed that almost all Oxt immuno positive neurons (97%, 1733 out of 1790 cells, n = 4 animals) were labeled by tdTomato from Oxt-Cre;Ai14 mice (Extended Data Fig. 1-1).”

Smaller portions of tdTomato cells in the SO (44%, 654 out of 1508 cells) and the MEA (8%, 31 out of 375 cells) retain active Oxt expression.

“Smaller portions of tdTomato cells in the SO (44%, 654 out of 1508 cells) and the MEA (8%, 31 out of 375 cells) retain active Oxt expression (Extended Data Fig. 1-1).”

TU-Oxt neurons do not show any long-range projections.

“The TU-Oxt neurons did not show any long-range projections.”

Many basal ganglia circuit components including CP, GP, STN, and SN receive Oxt projection.

“For instance, many basal ganglia circuit components including the caudate putamen (CP), globus pallidus (GP), subthalamic nucleus (STN), and substantia nigra (SN) receive Oxt projection to modulate motor function (see sensory motor regulation in Fig. 2E).”

The cerebral cortex provides little to no input to the Oxt neurons.

“Moreover, the cerebral cortex provides little to no input to the Oxt neurons, further supporting very weak direct interaction between cerebral cortical areas and Oxt neurons (Fig. 4D; Movie 4).”

Mid-level circuits including the diencephalon (thalamus, hypothalamus), midbrain, and cerebral nuclei provide major input to inform action of Oxt neurons.

“In contrast, mid-level circuits including the diencephalon (the thalamus, hypothalamus), the midbrain, and the cerebral nuclei, provide major input to inform action of Oxt neurons, providing anatomic substrate to perform an integrative role (Fig. 5).”

All brain regions providing inputs to Oxt neurons also receive output from Oxt neurons except the triangular nucleus of septum (TRS).

“All brain regions providing inputs to the Oxt neurons also receive output from the Oxt neurons except the triangular nucleus of septum (TRS), creating reciprocal connections with afferent areas (Figs. 2E, 4D).”

In addition to well-described Oxt neurons in PVH, SO, and AN, there is another major population in the TU area in the hypothalamus.

“In addition to well-described Oxt neurons in the PVH, SO, and AN, we described another major population in the TU area in the hypothalamus (Jirikowski, 2019).”

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SO-Oxt neurons show input from relatively more lateral parts of the brain compared with PVH-Oxt neurons.

“When monosynaptic input from the PVH-Oxt and SO-Oxt neurons are compared, SO-Oxt neurons show input from relatively more lateral parts of the brain (Fig. 4C, arrows).”

Each circuit consists of a set of brain regions processing different behavioral aspects.

“Each circuit consists of a set of brain regions processing different behavioral aspects.”

Oxt neuronal cell counts remain similar across male, virgin female and lactating female mice.

“Oxt neuronal cell counts remain similar across male, virgin female and lactating female mice, which is consistent with an earlier study in rats (Knobloch et al., 2012).”

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Oxt neurons from the PVH mainly receive inputs from the thalamus, hypothalamus, and cerebral nuclei.

“Overall, Oxt neurons from the PVH mainly receive inputs from the thalamus, hypothalamus, and cerebral nuclei (Fig. 4C).”

• AssumptionRests on an assumption: Oxt neurons integrate external stimuli and internal state.

SO-Oxt neurons receive overall similar input compared with PVH-Oxt neurons.

“SO-Oxt neurons receive overall similar input compared with the PVH-Oxt neurons (Fig. 4D).”

PVH-Oxt neurons project broadly to all nine identified functional circuits throughout the brain.

“PVH-Oxt neurons project broadly to all nine identified functional circuits throughout the brain, indicating that Oxt neurons can modulate information processing at different level of circuits with overall stronger influence in the midbrain and hindbrain circuits (Fig. 5).”

Oxt neurons receive little input from the hindbrain despite strong output to the same area.

“Noticeably, Oxt neurons received little input from hindbrain despite strong output to the same area, suggesting that Oxt neurons provide largely unilateral output to the hindbrain (Figs. 2E, 4D).”

The broad afferent pattern of SO-Oxt neurons is in sharp contrast to their very sparse efferent projection to the brain.

“The broad afferent pattern is in sharp contrast to the very sparse efferent projection of SO-Oxt neurons to the brain (Figs. 2E, 4D).”

TU-Oxt neurons have almost no central projection.

“Our anterograde tracing showed that these neurons have almost no central projection, suggesting their contribution to brain information processing is limited.”

Oxt neurons project to sensory-motor and pain circuits from the hindbrain and midbrain to cerebral cortex and cerebral nuclei.

“For example, we found that Oxt neurons project to sensory-motor and pain circuits from the hindbrain and midbrain to cerebral cortex and cerebral nuclei.”

Long-range processes from Oxt neurons contact the ventricle surface.

“Indeed, we found that long-range processes from Oxt neurons contact the ventricle surface, suggesting potential release of Oxt into the CSF via long-range processes.”

Brain regions with abundant Oxt projection without Oxtr expression exist, such as sensory related hindbrain areas.

“Another noteworthy Oxt-Oxtr discrepancy is brain regions with abundant Oxt projection without Oxtr expression such as sensory related hindbrain areas.”

Almost all afferent brain regions to PVH-Oxt neurons also receive efferent projections.

“Particularly, almost all afferent brain regions to PVH-Oxt neurons also receive efferent projections, suggesting strong reciprocal control of target regions by PVH-Oxt neurons except the hindbrain for unidirectional output.”

The majority of monosynaptic inputs to Oxt neurons are from the cerebral nuclei, thalamus, hypothalamus, and midbrain with little input from the hindbrain.

“Our Oxt monosynaptic input maps showed that the majority of inputs are from the cerebral nuclei, thalamus, hypothalamus, and midbrain with little input from the hindbrain.”

The analysis revealed no significant quantitative correlation between Oxt and Oxtr across entire brain regions.

“Despite a few areas with high levels of both Oxtr and Oxt projection (e.g., the paraventricular thalamus), our analysis revealed no significant quantitative correlation between Oxt and Oxtr across entire brain regions.”

The actions of Oxt are mainly mediated by a single subtype of the Oxt receptor (Oxtr) expressed in distinct brain regions as well as peripheral tissues.

“The actions of Oxt are mainly mediated by a single subtype of the Oxt receptor (Oxtr) expressed in distinct brain regions as well as peripheral tissues (Gimpl and Fahrenholz, 2001; Grinevich et al., 2016; Newmaster et al., 2020).”

• cites Gimpl and Fahrenholz, 2001Not checked yet

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• cites Grinevich et al., 2016Not checked yet

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• cites Newmaster et al., 2020Not checked yet

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Dense Oxt projections to hindbrain, midbrain, and hypothalamic areas directly modulate motor output and sensory input.

“Dense projection occurs largely onto the hindbrain (e.g., the dorsal motor nucleus of the vagus nerve; DMX, the PB), the midbrain (the substantia nigra compacta; SNc), and the hypothalamus (the medial preoptic nucleus; MPN) to directly modulate motor output and sensory input (Fig. 2E).”

The comprehensive projectome analysis uncovers anatomic substrates to explain pleiotropic effects of Oxt neurons regulating diverse behavioral outcomes.

“Together, our comprehensive projectome analysis uncovers anatomic substrates to explain pleiotropic effect of Oxt neurons regulating diverse behavioral outcomes.”

SO and AN Oxt neurons can further modulate a subset of the nine functional circuits, albeit less powerfully.

“These data suggest that Oxt neurons in these two areas can further modulate a subset of the nine functional circuits, albeit less powerfully.”

There is a lack of quantitative and spatial correlation between Oxt projections and Oxtr expression in the mouse brain.

“Overall, our results highlight lack of quantitative and spatial correlation between Oxt projections and Oxtr expression in the mouse brain.”

• AssumptionRests on an assumption: The quantitative relationship between Oxt projection and Oxtr expression across the whole brain is currently lacking.

PVH-Oxt neurons serve as a central modulator to integrate external and internal information via largely reciprocal connection with the nine circuits.

“Our results suggest that PVH-Oxt neurons serve as a central modulator to integrate external and internal information via largely reciprocal connection with the nine circuits while the SO-Oxt neurons act mainly as unidirectional Oxt hormonal output.”

SO-Oxt neurons act mainly as unidirectional Oxt hormonal output.

“while the SO-Oxt neurons act mainly as unidirectional Oxt hormonal output.”

The lack of quantitative correlation between Oxt output and Oxtr expression across the whole brain suggests abundant indirect Oxt signaling in Oxtr-expressing areas.

“suggesting abundant indirect Oxt signaling in Oxtr-expressing areas.”

• Also stated in: § Introduction · hedged

  “suggesting abundant indirect Oxt signaling in Oxtr-expressing brain areas.”

Oxt projection to basal ganglia components CP, GP, STN, and SN modulates motor function.

“For instance, many basal ganglia circuit components including the caudate putamen (CP), globus pallidus (GP), subthalamic nucleus (STN), and substantia nigra (SN) receive Oxt projection to modulate motor function (see sensory motor regulation in Fig. 2E).”

Oxt signaling may transmit to the brain via the CSF route in addition to direct transmission in target areas.

“This further suggests that Oxt signaling may transmit to the brain via the CSF route in addition to direct transmission in target areas.”

PVH-Oxt neurons project to three functional modules that control the internal state, somatic visceral, and cognitive response.

“Based on known functions of each anatomic region, we found that PVH-Oxt neurons project to three functional modules that control the internal state, somatic visceral, and cognitive response.”

The higher immunostaining estimate compared with the transgenic based estimate is most likely because of sensitive labeling based on antibody detection.

“which is higher than our transgenic based estimate mostly likely because of sensitive labeling based on antibody detection.”

Oxt neurons undergo Oxt expression changes during neurodevelopmental processes.

“This result suggested that Oxt neurons undergo Oxt expression changes during neurodevelopmental processes (Madrigal and Jurado, 2021).”

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This provides anatomic substrate for Oxt neurons to perform an integrative role.

“In contrast, mid-level circuits including the diencephalon (the thalamus, hypothalamus), the midbrain, and the cerebral nuclei, provide major input to inform action of Oxt neurons, providing anatomic substrate to perform an integrative role (Fig. 5).”

Most Oxtr-expressing areas contain at least sparse Oxt projection.

“Although earlier studies mentioned Oxt-Oxtr discrepancy, recent studies showed that most Oxtr-expressing areas contain at least sparse Oxt projection (Knobloch et al., 2012; Grinevich et al., 2016; Mitre et al., 2016; Zhang et al., 2021).”

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• cites Grinevich et al., 2016Not checked yet

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• cites Mitre et al., 2016Not checked yet

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Secreted Oxt level per cell may change based on physiological conditions (e.g., lactation) without changing Oxt cell numbers in normal mice.

“This suggests that secreted Oxt level per cell may change based on physiological conditions (e.g., lactation) without changing Oxt cell numbers in normal mice, although reduction of Oxt neurons has been reported in animal models of neurodevelopmental disorders (Peñagarikano et al., 2015; Dai et al., 2018).”

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The projection maps with entire process labeling provide possible release sites of Oxt throughout the whole brain.

“Hence, our projection maps with entire process labeling provide possible release sites of Oxt throughout the whole brain.”

Oxt neurons provide largely unilateral output to the hindbrain.

“Noticeably, Oxt neurons received little input from hindbrain despite strong output to the same area, suggesting that Oxt neurons provide largely unilateral output to the hindbrain (Figs. 2E, 4D).”

An alternative rabies tracing approach with optimized G and split TVA gave near identical results.

“We found near identical results with this alternative virus approach (Extended Data Fig. 4-2; N = 2 animals, each for the PVH and SO).”

A quantitative mismatch exists between Oxt projection and Oxtr distribution pointing toward abundant non-synaptic Oxt signaling within the brain.

“A quantitative mismatch exists between Oxt projection and Oxtr distribution pointing toward abundant non-synaptic Oxt signaling within the brain.”

These provide anatomic evidence that Oxt neurons can finely modulate sensory motor processing throughout different circuit levels.

“These provide anatomic evidence that Oxt neurons can finely modulate sensory motor processing throughout different circuit levels.”

Oxt neurons in the PVH and SO receive similar input from a subset of brain areas that receive majority of input from hypothalamic areas followed by cerebral nuclei and thalamic areas.

“Collectively, we conclude that Oxt neurons in the PVH and the SO receive similar input from a subset of brain areas that receive majority of input from hypothalamic areas followed by cerebral nuclei and thalamic areas (Fig. 4D).”

• AssumptionRests on an assumption: Oxt neurons integrate external stimuli and internal state.

Nine functional circuits with reciprocal or unidirectional connection with Oxt neurons serve as anatomic entities to exert varied behavioral control.

“We also identify nine functional circuits with reciprocal or unidirectional connection with Oxt neurons that serve as anatomic entities to exert varied behavioral control.”

Many Oxtr-expressing neurons may receive Oxt signal non-synaptically via CSF.

“A previous study suggested that many Oxtr-expressing neurons may receive Oxt signal non-synaptically via CSF (Zheng et al., 2014).”

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There is very weak direct interaction between cerebral cortical areas and Oxt neurons.

“Moreover, the cerebral cortex provides little to no input to the Oxt neurons, further supporting very weak direct interaction between cerebral cortical areas and Oxt neurons (Fig. 4D; Movie 4).”

Extensive connections of Oxt neurons to somatic visceral, cognitive, and state control modules can help to fine-tune activity of different circuit components to generate enhanced response to socially salient stimuli.

“Extensive connections of Oxt neurons to somatic visceral, cognitive, and state control modules can help to fine-tune activity of different circuit components to generate enhanced response to socially salient stimuli.”

3D immunolabeling independently validated the existence of the extra TU Oxt population.

“Our 3D immunolabeling independently validated the existence of this extra population.”

The circuit map can help to understand neural entities of Oxt that modulate different behavioral aspects.

“Thus, our circuit map can help to understand neural entities of Oxt that modulate different behavioral aspects.”

Oxt neurons create reciprocal connections with afferent areas.

“All brain regions providing inputs to the Oxt neurons also receive output from the Oxt neurons except the triangular nucleus of septum (TRS), creating reciprocal connections with afferent areas (Figs. 2E, 4D).”

Injecting TVA in the PVH of adult C57 mice did not result in any infection.

“We also confirmed no leakiness of TVA labeling by injecting TVA in the PVH of adult C57 mice which did not result in any infection (Extended Data Fig. 4-1).”

SO-Oxt neurons mainly serve as peripheral hormonal output.

“This suggests that SO-Oxt neurons mainly serve as peripheral hormonal output.”

By mapping Oxt efferent processes and clustering brain regions based on known functions, the authors identified nine functional circuits where Oxt processes interact to modulate distinct behavioral circuits.

“By extensively mapping Oxt efferent processes and clustering brain regions based on known functions, we identified nine functional circuits where Oxt processes interact to modulate distinct behavioral circuits.”

Oxt circuits provide broad projections to modulate external and internal information throughout the entire brain circuit.

“Overall, Oxt circuits provide broad projections to modulate external and internal information throughout the entire brain circuit.”

The contribution of TU-Oxt neurons to brain information processing is limited.

“Our anterograde tracing showed that these neurons have almost no central projection, suggesting their contribution to brain information processing is limited.”

Abundant bidirectional connections with nine functional circuits suggest that PVH-Oxt neurons can be an allostatic tool to interactively orchestrate and facilitate social and nonsocial information processing based on external stimuli and internal state.

“Abundant bidirectional connections with nine functional circuits suggest that PVH-Oxt neurons can be an allostatic tool to interactively orchestrate and facilitate social and nonsocial information processing based on external stimuli and internal state (Quintana and Guastella, 2020).”

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The study provides an anatomic foundation to understand diverse functions based on Oxt neurons in the brain.

“In summary, our study provides an anatomic foundation to understand diverse functions based on Oxt neurons in the brain.”

Oxt can bind to another receptor to exert its effect.

“Although Oxtr is a main Oxtr, Oxt can bind to another receptor to exert its effect.”

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There is potential release of Oxt into the CSF via long-range processes.

“Indeed, we found that long-range processes from Oxt neurons contact the ventricle surface, suggesting potential release of Oxt into the CSF via long-range processes.”

Despite having similar afferent areas to SO-Oxt neurons, their limited central projection suggests that SO-Oxt neurons serve largely as unidirectional hormonal output to the periphery rather than reciprocal circuit modulator.

“In contrast, despite having similar afferent areas to SO-Oxt neurons, their limited central projection suggests that SO-Oxt neurons serve largely as unidirectional hormonal output to the periphery rather than reciprocal circuit modulator.”