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Published in final edited form as: Science. 2026 Feb 19;391(6787):800–806. doi: 10.1126/science.aea7140

Empathy and prosocial behavior powered by orexin-driven theta oscillations

Jae Gon Kim 1, Greg O Cron 1, Minsoo Kim 1, Aronee Hossain 1, Jin Hyung Lee 1,2,3,4,*
PMCID: PMC13005278  NIHMSID: NIHMS2152845  PMID: 41712718

Abstract

Empathy measured through observational fear in rodents has been associated with increased theta oscillations in the anterior cingulate cortex (ACC). However, upstream circuit mechanisms modulating these oscillations and the extent of the oscillations’ role in empathy-related behaviors remains elusive. Here, we found that in mice, ACC theta oscillations are involved in empathy-driven prosocial allogrooming. Moreover, orexinergic neurons are selectively activated in the ACC during observational fear and prosocial allogrooming, but only when the animals had prior fear experience. Real-time, gaze-dependent optogenetic inhibition of lateral hypothalamic orexinergic inputs to ACC suppressed theta power and reduced both behaviors. These findings show that hypothalamic orexinergic inputs drive ACC theta oscillations to modulate observational fear and prosocial behaviors, providing circuit-level insight into how affective empathy translates into prosocial action.

Many species, including humans, rely on group cohesion for survival and well-being. This social bonding provides evolutionary advantages through enhanced resource acquisition, predator defense, and collective problem-solving (1-3). To promote such critical social cohesion, prosocial behavior motivated by affective empathy—the ability to perceive and share negative emotional states of others—is essential (4-7). Rodents not only exhibit affective empathy behaviors including emotional contagion of fear, and pain from distressed conspecifics (8-11), but also engage in prosocial behaviors such as targeted helping or food-sharing (12-15). However, the mechanisms by which affective states drive prosocial actions are unknown.

Previous studies have implicated the anterior cingulate cortex (ACC) in both affective empathy and prosocial behavior, suggesting this region as a potential neural hub for connecting these processes. For instance, ACC projections to the basolateral amygdala facilitate observational fear learning—vicarious freezing response from distressed conspecifics—whereas ACC connections to the nucleus accumbens mediate emotional pain transfer through social interaction with pain-induced conspecifics (16-18). Additionally, neurons in the ACC play critical roles in prosocial allogrooming—consolation behavior measured by repeated licking toward distressed conspecifics (12, 19). Although these studies identify specific downstream circuits of ACC that modulate affective empathy, and implicate ACC in prosocial behavior, the extent of ACC involvement in affective empathy and prosocial behavior, its circuit mechanism, and common upstream targets that coordinate these processes, potentially linking them together, remain to be fully elucidated.

Recent work has shown that synchronized 5–7 Hz theta oscillations are critical for affective empathy during observational fear (16, 20). Given that the ACC is involved in both affective empathy and prosocial behavior, and that affective empathy is known to motivate prosocial behavior, we hypothesized that ACC theta oscillations may serve as a shared neural substrate underlying both behaviors. Furthermore, identifying upstream modulators of ACC theta oscillations could provide insight into how these oscillations coordinate affective empathy and prosocial behavior, potentially revealing novel targets for interventions in social disorders. Among potential upstream modulators, orexin (also known as hypocretin) has emerged as a particularly compelling candidate. This neuropeptide is produced by neurons in the lateral hypothalamus (LH) and regulates arousal, stress, and emotional processing by projecting to multiple brain regions (21-26), including the ACC (27-29). Orexin has been shown to causally promote hippocampal theta oscillations via septo-hippocampal GABAergic and cholinergic pathways (30-34). Additionally, orexin has been implicated in various social behaviors and responses to social challenges, including social interaction in chronic stress (35, 36), sociality and social novelty in social fear conditioning (37), and social impairments induced by early-life maternal separation stress (28). These results position orexin as an ideal candidate for coordinating empathetic responses. These converging lines of evidence raise the possibility that orexinergic inputs to the ACC modulate ACC theta activity to facilitate both affective empathy and prosocial behavior. Here, we tested this hypothesis using a combination of electrophysiological recordings, circuit-specific manipulations, and combined behavioral assays in a rodent model of affective empathy and prosocial paradigms.

Shared fear boosts empathy and prosociality

To investigate how affective empathy drives prosocial behavior, we combined observational fear (OF) and consolation assays (38), enabling simultaneous assessment of vicarious freezing and allogrooming within the same subjects. We performed two types of OF paradigms: a naive OF (16, 39, 40), in which observers had no prior fear experience, and an experience-dependent OF (Exp OF) (18, 41-43), in which observers underwent a prior fear experience induced by contextual fear conditioning (CFC; 0.5 mA, 2 s) one day before the OF and consolation assays. These behavioral paradigms allowed us to examine how shared fear experience influences both affective empathy and prosocial behavior (Fig. 1A, B).

Fig. 1. Shared experience enhances affective empathy and prosocial behavior.

Fig. 1.

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(A) Schematic of naive observational fear (naive OF) and consolation assays. OB, observer; DM, demonstrator. (B) Schematic of experience-dependent observational fear (Exp OF) assay following contextual fear conditioning (0.5 mA) in observers, and consolation assay. (C–G) OB behaviors during habituation and conditioning periods in OF. (C) Four different behavioral events selected for analysis (arrows indicate direction of OB’s gaze). (D, E) Percentage of freezing without gazing (D) or with gazing (E) quantified in 1-minute time bins. (F, G) Mean freezing percentage without gazing (F) or with gazing (G). (H–K) OB behaviors in consolation assay. (H) Raster plots of behavioral events during baseline and reunion. (I, J) Duration of self-grooming (I) and allogrooming (J) quantified in both groups. (K) Group comparison of cumulative reunion allogrooming duration. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. Statistical details are provided in table S1. Panels A–C were created with BioRender.com.

To determine whether observational fear is driven by socially relevant visual cues, we subdivided vicarious freezing into two categories: freezing with gazing—defined as the observer facing the demonstrator during freezing—and freezing without gazing. This classification enabled us to assess which vicarious freezing events were more indicative of affective states during observational fear. Both naive and Exp OF observers exhibited more freezing with gazing compared with freezing without gazing, with this effect markedly stronger in the Exp OF group (Fig. 1D to G and fig. S2A, C). These findings indicate that vicarious freezing is shaped by social visual information and further enhanced by shared prior experience, reinforcing the link between shared experience and affective empathy.

Given that shared fear experience enhanced vicarious freezing, we next examined whether this enhancement extends to prosocial comforting behaviors during the subsequent consolation assay (Fig. 1H-K). Exp OF groups showed increase in self-grooming and allogrooming during the reunion period (post-OF) compared with baseline (pre-OF). Exp OF observers exhibited greater allogrooming than naive OF observers (Fig. 1J, K), whereas reunion self-grooming did not differ between groups (Fig. 1I). Thus, shared experience selectively promotes prosocial allogrooming without altering self-directed emotional regulation. This increase in allogrooming was specifically directed towards distressed demonstrators (fig. S3). When the demonstrator was replaced with either a non-social object (wood-block) or a non-stressed littermate during the reunion period, allogrooming no longer increased (fig. S3F), confirming that it reflects genuine consolation rather than a non-specific social or stress-buffering response.

We next tested how excessively strong fear memories might influence observational fear and allogrooming (fig. S1). To test this, Exp OF observers were subjected to higher-intensity CFC (1.0 mA, 2 s) one day before the OF and consolation assays (fig. S1B). Under this condition, although freezing with gazing was higher compared with naive OF observers (fig. S1E, G), the increase was smaller compared with the moderate-CFC condition (Fig. 1E, G and fig. S2C, D). These results suggest that excessive fear memory may disrupt the perception of others’ affective states and diminish motivation for prosocial engagement. This lack of modulation by higher-intensity of CFC may reflect the nonlinear scalability of empathy, as empathic responses are not necessarily proportional to the demonstrator’s distress. Indeed, recent work indicates that affective state transmission can follow a nonlinear trajectory, where extreme distress in others does not necessarily elicit stronger empathic or prosocial responses (44). Because the high-CFC (1.0 mA) abolished prosocial allogrooming (fig. S1J, K), we used moderate-CFC (0.5 mA) for subsequent Exp OF assays.

We analyzed jumping and freezing behaviors of demonstrators during naive and Exp OF with different experimental settings. We found that demonstrators had no distinction in jumping and freezing among different experiment groups, confirming that observers’ responses were not attributable to variations in demonstrator performance (fig. S2E, F). Additionally, we quantified demonstrator behaviors during the consolation assay (fig. S2G). During reunion, the amount of demonstrator self-grooming was consistently higher than baseline, but not different across observer floor type or CFC shock intensity.

ACC theta modulates empathy and prosociality

Previous studies have shown that 5–7 Hz theta oscillations in the ACC are essential for vicarious freezing during naive OF (16, 20). However, it remains unknown whether these theta oscillations can be enhanced by shared fear experience, and whether these theta oscillations are involved in prosocial behavior. To address these questions, we conducted local field potential (LFP) recordings in the ACC during OF and consolation assays (Fig. 2 and figs. S4, S7).

Fig. 2. ACC 5–7 Hz theta modulates affective empathy and prosocial behavior in experienced observers.

Fig. 2.

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(A) Representative image showing electrode implantation (yellow arrow) into the ACC. Scale bar, 200 μm. (B) Schematic of LFP recordings during Exp OF and consolation assays. OB, observer; DM, demonstrator. (C–I) OB behaviors during habituation and conditioning periods in Exp OF. (C) Four different behavioral events selected for analysis. (D) Percentage of freezing with gazing or without gazing quantified in 1-minute time bins. (E) Mean freezing percentage with gazing or without gazing. (F, G) Z-scored relative power spectrograms aligned to the onset of freezing without gazing (F) or with gazing (G). Black dashed lines indicate behavioral onset; white dashed lines indicate the 5–7 Hz range. (H) Temporal changes in 5–7 Hz relative power during each event. (I) Mean z-scored 5–7 Hz power from t = 0 to t = 2 s (gray shaded region in panel h). (J–O) OB behaviors during baseline and reunion periods in consolation assay. (J, K) Duration of self-grooming (J) and allogrooming (K). (L, M) Z-scored relative power spectrograms aligned to the onset of self-grooming (L) and allogrooming (M). (N, O) Mean z-scored 5–7 Hz power from t = 0 to t = 2 s during self-grooming (N) and allogrooming (O). (P–R) DM results during open-field test. (P) Representative heatmaps from three experimental groups. (Q) Time spent in each zone. (R) Total distance traveled. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. Statistical details are provided in table S1. Panels A–C, F, and G were created with BioRender.com.

In naive OF observers, ACC theta power increased during freezing with gazing in the conditioning period, compared to freezing without gazing, immobility with gazing, and immobility without gazing (fig. S4F to I). This suggests that ACC theta activity increase is selectively enhanced in the presence of demonstrator-directed visual cues during freezing.

We next examined whether ACC theta power also increases during prosocial allogrooming in the consolation assay (fig. S4J to O). Although naive OF observers displayed increased self-grooming and allogrooming during reunion compared with baseline (fig. S4J, K), ACC theta power remained unchanged (fig. S4L to O). These results indicate that in naive animals, ACC theta oscillations are selectively engaged during observational fear, but not during prosocial behavior.

We next tested whether theta modulation is also involved in observers with prior fear experience. In the Exp OF group, 5–7 Hz theta oscillations in the ACC increased during freezing with gazing compared to freezing without gazing, immobility with gazing, and immobility without gazing, consistent with the pattern observed in the naive OF group (Fig. 2F to I). Exp OF observers also exhibited a robust increase in theta power during allogrooming (Fig. 2M, O). In contrast, although self-grooming behavior was elevated during the reunion (Fig. 2J, K), it was not accompanied by increased theta power (Fig. 2L, N), indicating that ACC 5–7 Hz oscillations are specifically engaged during prosocial comforting rather than self-directed relief.

To determine whether allogrooming exerts a consoling effect on demonstrators, we conducted an open-field test (OFT) immediately after the reunion period (Fig. 2 and fig. S4). Using ezTrack, an open-source tracking software (45), we found that demonstrators receiving extensive allogrooming from Exp OF observers spent more time in the center zone during the OFT than demonstrators in other groups, suggesting an anxiolytic effect of allogrooming (Fig. 2P to R). To further validate this finding with a more specific measure of anxiety, we performed the elevated plus maze (EPM) test immediately after the reunion session (fig. S5) (12, 46). Demonstrators that received more allogrooming again showed reduced anxiety-like behavior, spending more time in the open arms than demonstrators left alone (fig. S5H, I). Moreover, plasma corticosterone amounts (47) were reduced in male but not in female animals (fig. S5J, K). We examined potential sex differences in observational fear and consolation assays and found no differences between male and female mice (fig. S6). Together, these findings indicate that shared experience enhances empathy-driven prosocial allogrooming with a genuine consoling effect on distressed partners, closely associated with 5–7 Hz theta oscillations in the ACC.

To assess whether other frequency bands contribute to these behaviors, we conducted additional spectral analyses across a wider frequency range (fig. S7). We did not identify any modulation outside the 5–7 Hz band during freezing with gazing or prosocial allogrooming in either naive or Exp OF groups. These results indicate that ACC 5–7 Hz theta oscillations represent a selective and generalizable neural signature underlying both observational fear and prosocial allogrooming, particularly in observers with prior fear experience.

Orexin encodes empathy and prosociality

Given that 5–7 Hz theta oscillations in the ACC are critically involved in both observational fear and prosocial allogrooming—particularly in observers with prior fear experience—we next investigated whether orexin neurons also contribute to these behaviors.

To address this, we conducted fiber photometry recordings using OxLight1 (48)—a genetically encoded orexin sensor—during the OF and consolation assays (Fig. 3A, B and fig. S8A, B). In the Exp OF group, orexinergic activity in the ACC increased during freezing with gazing, but not during freezing without gazing or during the habituation period (Fig. 3F to I). This robust increase in ACC-projecting orexinergic signals was likewise observed during reunion allogrooming, but not during self-grooming, in the consolation assay (Fig. 3L to O). These selective increases paralleled the previously observed enhancement of 5–7 Hz ACC theta oscillations during freezing with gazing and reunion allogrooming in the Exp OF group (Fig. 2). In contrast, naive OF observers showed only transient increase in orexin activity during habituation (fig. S8F), likely reflecting arousal-related rather than affective state-related signaling (49-52). Furthermore, ACC-projecting orexinergic activity remained unchanged in naive OF observers during both freezing with gazing (fig. S8H, I) and allogrooming (fig. S8M, O). Together, these results suggest that ACC-projecting LHAOrx neurons are selectively recruited during observational fear and prosocial allogrooming, specifically in observers with prior fear experience, in line with increases in ACC theta oscillations.

Fig. 3. Orexin neurons are selectively recruited during affective empathy and prosocial behavior in experienced observers.

Fig. 3.

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(A) Representative image showing OxLight1 (a genetically encoded orexin sensor) expression and optic cannula implantation (yellow dashed lines) into the ACC. Scale bar, 200 μm. (B) Schematic of OxLight1 signal recordings during Exp OF and consolation assays. OB, observer; DM, demonstrator. (C–I) OB behaviors during habituation and conditioning periods in Exp OF. (C) Four different behavioral events selected for analysis. (D) Percentage of freezing with gazing or without gazing quantified in 1-minute time bins. (E) Mean freezing percentage without gazing or with gazing. (F–I) Z-scored OxLight1 signals during Exp OF (black dashed lines, behavior onset). (F, H) Temporal changes in z-scored OxLight1 signals during freezing without gazing (gray line) or with gazing (blue line) in habituation (F) and conditioning (H). (G, I) Mean z-scored OxLight1 signals from t = 0 to t = 2 s in each period (gray shaded regions in panel F and H). (J–O) OB behaviors during baseline and reunion periods in consolation assay. (J, K) Duration of self-grooming (J) and allogrooming (K). (L, M) Temporal changes in z-scored OxLight1 signals during self-grooming (L) and allogrooming (M). (N, O) Mean z-scored OxLight1 signals from t = 0 to t = 2 s (gray shaded regions in panel L and M). In D–F, H, and J–M, data are presented as mean ± SEM. Box plots in G, I, N, and O, indicate the median (center line), upper and lower quartiles (box limits), and minimum to maximum values excluding outliers (whiskers); outliers are displayed as individual points beyond the whiskers. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. Statistical details are provided in table S1. Panels A–C, F, and H were created with BioRender.com.

Orexin-driven theta controls empathy and prosociality

To determine whether ACC-projecting LHAOrx neurons causally modulate observational fear and allogrooming behavior via 5–7 Hz theta oscillations, a Flp-dependent retrograde adeno-associated virus expressing hM4Di (AAVrg-hSyn-fDIO-hM4Di-mCherry) was unilaterally injected into the ACC of Orexin-Flp mice (53), followed by electrode implantation in the same region. The LHAOrx–ACC circuit was chemogenetically inhibited by systemic administration of clozapine-N-oxide (CNO) prior to the OF session, and LFP recordings were performed during the Exp OF and consolation assays (fig. S9A, B).

Inhibition of the ACC-projecting orexinergic pathway reduced changes in 5–7 Hz theta power during freezing with gazing in the Exp OF assay (fig. S9H to O), indicating that LHAOrx inputs modulate ACC theta oscillations during observational fear. Behaviorally, allogrooming duration during the consolation assay was decreased in the hM4Di group compared with the control group, whereas self-grooming increased (fig. S9P, Q), suggesting a behavioral shift from prosocial motivation to self-directed coping. Consistently, theta power during allogrooming—but not during self-grooming—was reduced in the hM4Di group (fig. S9R to U), further supporting a selective role for ACC theta activity in prosocial behavior.

Given that the Exp OF observers displayed selective enhancement of ACC theta power during freezing with gazing, we next sought to manipulate the ACC-projecting orexinergic circuit specifically during moments when the observers gazed at the demonstrators, to test whether this selective inhibition could influence observational fear and prosocial allogrooming. To this end, we unilaterally injected a Flp-dependent anterograde adeno-associated virus expressing NpHR3.3 (AAV8-nEF-Coff/Fon-NpHR3.3-EYFP) into the LHA of Orexin-Flp mice and optogenetically inhibited axon terminals in the ACC by implanting a combined fiber-optic and electrode (optrode). Real-time, gaze-dependent optogenetic inhibition of the ACC-projecting orexinergic circuit was applied whenever the observers gazed at the demonstrators—regardless of whether they were freezing—during the conditioning period in Exp OF. LFP was also simultaneously recorded during the behavioral assays (Fig. 4A, B). To enable real-time, gaze-dependent optogenetic inhibition, we implemented DeepLabCut (54), a machine-learning-based tracking software, to annotate observer (left and right ears) and demonstrator (center body) positions for real-time gaze detection. Gazing was defined as a head direction within ±60° of the demonstrator, which automatically triggered laser-mediated inhibition of the ACC-projecting orexinergic inputs.

Fig. 4. LHAOrx–ACC inputs drive affective empathy and prosocial behavior through theta oscillations in experienced observers.

Fig. 4.

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(A) Representative image of viral expression in the LHA (bottom right) and optrode implantation into the ACC (top right) of Orexin-Flp mice. Scale bars, 200 μm. (B) Schematic of optogenetic inhibition of orexin neurons during conditioning period, with concurrent LFP recordings in Exp OF and consolation assays. OB, observer; DM, demonstrator. (C–N) OB behaviors during habituation and conditioning periods in Exp OF. (C, D) Percentage of freezing without gazing (C) or with gazing (D) quantified in 1-minute time bins. (E, F) Mean freezing percentage without gazing (E) or with gazing (F). (G–J) Z-scored relative power spectrograms aligned to the onset of freezing in each group (black dashed lines indicate behavioral onset; white dashed lines indicate the 5–7 Hz range). (K, M) Temporal changes in 5–7 Hz relative power during each behavior in control (K) and NpHR (M) groups. (L, N) Mean z-scored 5–7 Hz power from t = 0 to t = 2 s (gray shaded regions from panel k or m). (O–V) OB behaviors during baseline and reunion periods in consolation assay for control and NpHR groups. (O, S) Duration of self-grooming (O) and allogrooming (S). (P, Q, T, U) Z-scored relative power spectrograms aligned to the onset of self-grooming (P, Q) and allogrooming (T, U) in each group. (R, V) Mean Z-scored 5–7 Hz power from t = 0 to t =2 s during self-grooming (R) and allogrooming (V). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. Statistical details are provided in table S1. Panels A, B, and G–J were created with BioRender.com.

This temporally precise and behavior-specific inhibition reduced freezing with gazing in the NpHR group (Fig. 4C to F) and suppressed 5–7 Hz ACC theta power (Fig. 4G to N) during the Exp OF assay. Although the ACC-projecting orexinergic inhibition was restricted to observer gazing during the conditioning period, the NpHR group exhibited reduced allogrooming during the reunion period, whereas self-grooming remained unaffected (Fig. 4O, S). Consistently, ACC theta power during allogrooming was reduced in the NpHR group (Fig. 4T to V), whereas theta power during self-grooming remained unchanged (Fig. 4P to R).

To examine whether these effects depend on prior fear experience, we next performed the same real-time, gaze-dependent optogenetic inhibition of ACC-projecting orexinergic neurons during OF and consolation assays, with simultaneous LFP recordings from the ACC in naive observers (fig. S10). In contrast to experienced observers, this manipulation did not alter freezing with gazing during naive OF, allogrooming behavior during the consolation assays, or 5–7 Hz ACC theta power during either assay compared with the control group.

Taken together, these results demonstrate that ACC-projecting orexinergic neurons are necessary for experience-dependent observational fear (Exp OF) and prosocial allogrooming, acting through orexin-dependent 5–7 Hz theta oscillations in the ACC.

Discussion

Our findings reveal a previously unrecognized orexin-dependent neuromodulatory mechanism linking affective empathy with prosocial comforting behavior through coordinated 5–7 Hz theta oscillations. Specifically, we identify a shared neural circuit in which orexinergic neurons in the lateral hypothalamus (LHAOrx) project to the anterior cingulate cortex (ACC), modulating 5–7 Hz theta oscillations that underlie both freezing with gazing during experience-dependent observational fear (Exp OF) and prosocial allogrooming during consolation assays. Disruption of this pathway—via chemogenetic or optogenetic inhibition—suppressed ACC theta power and reduced both behaviors, thereby establishing a causal role for orexin-driven theta oscillations in affective empathy and prosocial behavior. Observers with prior fear experience (Exp OF) selectively recruited this LHAOrx–ACC circuit during both Exp OF and consolation assays (Figs. 3, 4, and fig. S9), whereas naive OF observers showed no substantial recruitment of this circuit during either behavior (figs. S4, S8, S10). These findings support the importance of shared experience in recruiting this empathy-related pathway.

Although both naive and experienced observers exhibit increases in ACC 5–7 Hz theta oscillations during OF, the underlying mechanisms of theta power enhancement likely differ between the two groups. For example, spectral analyses revealed distinct patterns: naive mice lacked the prominent 4 Hz oscillations observed in the experienced observers (fig. S4G and Fig. 2G). Given that 4 Hz oscillations in the BLA have been associated with self-fear expression (55) and fear memory recall, and that ACC–BLA synchrony is engaged during OF (20), the emergence of 4 Hz oscillations in the ACC of experienced observers may reflect the engagement of self-fear-related neural activity associated with the BLA. Moreover, optogenetic inhibition of ACC-projecting orexinergic neurons in naive observers did not alter freezing with gazing, allogrooming and ACC theta power (fig. S10), suggesting that this pathway is selectively engaged when observers have prior fear experience. Together, these results suggest that the observed differences between naive and experienced observers, is due to the naive observers engaging orexin-independent mechanisms rather than there being a difference in the tone of orexinergic modulation.

The presence of ACC 4 Hz activity in experienced observers likely reflect an experience-dependent reinforcement of self-fear-related ACC–BLA synchronization, whereby prior fear experience may strengthen ACC–BLA coupling and promotes the emergence of a 4 Hz oscillatory component during observational fear. Inhibition of ACC-projecting orexinergic neurons did not affect lower-frequency activities (~4 Hz), whereas 5–7 Hz theta oscillations were selectively reduced in the Exp OF group (Fig. 4 and fig. S9). In both the hM4Di group, in which LHAOrx–ACC inputs were chemogenetically silenced prior to the OF session, and the NpHR group, in which optogenetic inhibition was applied specifically during observer gazing at the demonstrator during the conditioning session, 3–5 Hz activity was preserved throughout the Exp OF assay (Fig. 4J and fig. S9L, M). This frequency dissociation suggests that whereas 5–7 Hz oscillations are selectively associated with empathy-related behavior, 3–5 Hz may reflect self-experienced fear or general arousal. No sustained increase in 3–5 Hz power was observed during allogrooming in orexin-inhibited observers.

Although our LFP recordings were confined to the ACC, these findings align with previous studies implicating ~4 Hz prefrontal-amygdala synchrony in self-experienced fear (55). In line with these frequency-specific effects, intracerebroventricular administration of orexin A increases theta (4–10 Hz) and beta (16–48 Hz) power while decreasing delta (1–4 Hz) and alpha (10–16 Hz) (31), indicating a consistent experimental outcome, where orexin differentially impacts delta and theta band activities. Together, our results highlight the LHAOrx–ACC pathway as a key circuit orchestrating affective empathy and prosocial behavior via 5–7 Hz theta oscillations.

ACC theta may share mechanistic features with the cholinergic-dependent type 2 theta described in the hippocampus (56, 57), which depends on septal cholinergic and GABAergic projections modulated by orexinergic signaling (30, 32). A previous study showed that septal GABAergic modulation can facilitate type 2 theta in both the hippocampus and ACC during observational fear (20). Further investigation of the precise relationship between the septo-hippocampal circuit and the direct orexinergic inputs projecting to the ACC would be an important next step in understanding the role of hippocampal type 2 and ACC 5–7 Hz theta oscillations. This investigation could also shed light on how orexin-driven modulation of 5–7 Hz ACC theta oscillations can selectively affect observers with prior fear experience.

A recent study by Luo et al. (28) performed whole-cell recordings and circuit manipulations in the ACC—at the same coordinates used in our experiments—and showed that activation of orexin terminals selectively increases the excitability of CaMKIIα+ pyramidal neurons through an orexin-2-receptor–dependent mechanism, without affecting either somatostatin- or parvalbumin-expressing interneurons. These findings suggest that orexin primarily targets excitatory neurons within the ACC. Previous work has shown that bidirectional ACC–BLA excitatory connections are essential for observational fear by synchronizing 5–7 Hz theta oscillations (20), suggesting that orexin may act as an upstream modulator of this ACC–BLA excitatory neuron driven 5–7 Hz theta oscillations to coordinate affective empathy-related network dynamics.

Supplementary Material

SupplementaryMatrerial
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Materials and Methods

Figs. S1 to S10

Table S1

Movies S1 to S2

Acknowledgments:

The authors thank Dr. Jinhee Baek for his technical assistance. Schematics of behavioral paradigms and events were created with BioRender.com. The authors also acknowledge the Stanford University Cell Sciences Imaging Facility – Neuroscience Microscopy Service (Stanford CSIF NMS) for microscopy equipment access.

Funding:

National Institutes of Health grant RF1MH114227 (to JHL)

National Institutes of Health grant DP1NS116783 (to JHL)

National Institutes of Health grant R01 AG064051 (to JHL)

National Institutes of Health grant R01 EB030884 (to JHL)

Footnotes

Competing interests: JHL is a founder, shareholder, and consultant for LVIS. JGK and JHL are inventors on a pending patent application titled “Methods and Systems for Modifying Empathy by Modulating an Orexinergic Pathway to Modulate Theta Oscillations” (Application # 63/798,442).

Data and materials availability: All data are available in the main text or the supplementary materials. Orexin-Flp mice (RBRC11555) are available from Nagoya University (Akihiro Yamanaka) under a material transfer agreement with RIKEN BioResource Research Center (BRC).

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