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. Author manuscript; available in PMC: 2026 Feb 20.
Published in final edited form as: Science. 2024 Jul 25;385(6707):409–416. doi: 10.1126/science.adk7411

Neurons for infant social behaviors in the mouse zona incerta

Yuexuan Li 1,2, Zhong-Wu Liu 2, Gustavo M Santana 1,3, Ana Marta Capaz 4, Etienne Doumazane 4, Xiao-Bing Gao 2, Nicolas Renier 4, Marcelo O Dietrich 1,2,3,*
PMCID: PMC12917754  NIHMSID: NIHMS2130646  PMID: 39052814

Abstract

Understanding the neural basis of infant social behaviors is crucial for elucidating the mechanisms of early social and emotional development. Here, we report a specific population of somatostatin-expressing neurons in the zona incerta (ZISST) of preweaning mice that responds dynamically to social interactions, particularly with their mother. Bidirectional neural activity manipulations in pups revealed that widespread connectivity of preweaning ZISST neurons to sensory, emotional, and cognitive brain centers mediates two key adaptive functions associated with maternal presence: reduction of behavior distress and facilitation of learning. Overall, these findings reveal a population of neurons in the infant mouse brain that coordinate the positive effects of the relationship with the mother on infant’s behavior and physiology.

In humans, as in other mammals, infants have an inborn tendency to form an attachment bond with their mother or surrogate (1, 2). This bond plays a critical role in the infant’s development and serves as a safety net from which the infant can explore the surrounding environment (3). The mother serves as a secure base that helps alleviate distress responses and enables the infants to learn and form associations that are key to their development (4–6). Identification of the neurons in the infant brain that respond to the social relationship with the mother is paramount for understanding the mechanisms underlying brain and behavior development.

The infant’s response to their mother requires the integration of diverse sensory inputs. Of known brain regions involved in this process, the zona incerta (ZI) serves as an integrative node for both external stimuli like somatosensory, visual, and auditory cues, as well as interoceptive signals (7, 8). The ZI displays distinct developmental traits: It establishes dense projections to other brain regions during the early postnatal period, which retract post-weaning (9, 10). We therefore reasoned that neurons in the ZI may integrate the early social experiences of the infant, particularly with the mother, to modulate social behavior.

Somatostatin neurons in the zona incerta respond to social interaction in preweaning pups

We first tested whether ZI neurons in preweaning mice respond to social interactions with their mother. To record the activity of ZI neurons, we performed fiber photometry in sixteen-to-eighteen-day old pups (P16-P18) (11). To gain access to specific populations of ZI neurons (7), an adeno-associated virus (AAV) expressing the calcium sensor jGCaMP7s in a Cre-recombinase-specific manner was selectively injected into the ZI of newborn mice expressing Cre in excitatory (Vglut2) and inhibitory (parvalbumin-PV and somatostatin-SST) ZI neurons (Fig. 1A).

Fig. 1. ZISST neurons respond to social interactions in preweaning mice.

Fig. 1.

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(A) Newborn mice are injected with an AAV to express jGCaMP7s in Vglut2-, PV-, or SST-neurons in the ZI; when sixteen-to-eighteen days old, fiber photometry is used to measure neuronal activity. (B) Representative image of the infection of ZI neurons and position of the fiber optic used for fiber photometry. (C) Average z-score of ZI neuron activity in response to interaction with the mother (Vglut2, n = 5; PV, n = 5; SST, n = 10). (D) Mean z-score calculated from ‘C’ during the 20 seconds of social interaction with the mother (Vglut2: t4 = 0.09, P = 0.92; PV: t4 = 0.1.74, P = 0.15; SST: t9 = 7.89, P = 0.00002; one sample t test). (E) Similar to ‘D’: mean z-score of ZISST neuron activity in response to social interactions with unfamiliar social stimuli (mother, n = 7; adult female, n = 10; adult male, n = 10), in addition to the mother (n = 15) and an inanimate object (n = 10) (F4, 47 = 15.84, P < 10−7; one-way ANOVA). We did not observe active sucking behavior during recordings with the mother and non-lactating female. (F) Similar to ‘E’: response to social interactions with siblings (n = 10) and peers (n = 7) compared with the mother and object (F3, 38 = 19.60, P < 10−7; one-way ANOVA). In E-F, P values calculated using the Holm-Sidak’s multiple comparisons test. Line graphs for neuron recordings represent mean ± SEM. Box plot denotes minimum, first quartile, median, third quartile, and maximum values. Round symbols in box plots represent individual data. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

We recorded the activity of ZI neurons in pups during interaction with their mother (Fig. 1B and fig. S1). Introduction of the mother selectively increases the activity of somatostatin-expressing neurons, but not other neuron types (Fig. 1C–D), and the activity remains elevated during social interaction (Fig. 1C). Given the specificity of the response of the somatostatin-expressing neurons (ZISST), we focused our subsequent experiments in characterizing their dynamics.

First, we tested the effect of social interactions with unfamiliar conspecifics, including lactating female, non-lactating female, and male, and compared these responses to an inanimate object. All the three social stimuli induced activation of ZISST neurons compared to an object, but to a lesser extent than the mother (Fig. 1E). In addition, we tested the response of ZISST neurons to siblings and same age peers. Similar to social interaction with unfamiliar adults, interactions with siblings and peers increased the activity of ZISST neurons, an effect that was not as robust as the mother (Fig. 1F). Thus, ZISST neurons in pups respond to social interactions with conspecifics, with the largest responses directed to the mother.

Social contact activates ZISST neurons

We next tested whether the magnitude of the response to social interaction with the mother changed after increasing periods of social isolation. Social interactions with the mother after 10 minutes, or 3, 6, or 12 hours, of isolation equally increased the activity of ZISST neurons (fig. S2), suggesting that these neurons track the presence of the mother rather than the need state of the pup for the mother.

We then tested whether different forms of social interaction and contact can differentially modulate the activity of ZISST neurons (Fig. 2A) (12, 13). Regardless of the type of social interaction, the activity of ZISST neurons increased at the time of social contact, but not in response to contact with a toy (Fig. 2B–D). These results suggest that direct social contact between pup and mother activates ZISST neurons.

Fig. 2. Social contact activates ZISST neurons.

Fig. 2.

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(A) Classification of social interaction types. (B) Example trace of fiber photometry recording of ZISST neurons in pups interacting with their mother. Shaded area is when the mother is introduced to the chamber. Each social contact is labeled in the panel and three contact events are depicted in the panel. (C) ZISST neuron activity (dF/F0 aligned to starting time of contact and subtracted from baseline) in response to bilateral contact (n = 14 trials) or to pup-snout-to-dam-anogenital (n = 15 trials), pup-snout-to-dam-other-body-part (n = 25 trials), dam-snout-to-pup-body (n = 13 trials) contacts compared to contact with an inanimate toy (n = 25 trials) (trials from n = 6 mice). (D) Peak dF/F (compared to mean −1s prior to contact) for all types of social and non-social contacts (H(5) = 46.68, P < 10−8; Kruskal-Wallis test with Dunn’s multiple comparisons test). (E) Recording of ZISST neuron activity in pups on a linear track. In trial 1, the linear track is empty. In trial 2, a stuff mouse toy is placed at the opposite end of the track. In trial 3, the anesthetized mother is placed at the end of the track. (F) Representative tracking and neuronal activity data for one pup. (G) Neuronal activity data aligned to the point of door opening (left panels) and contact (right panels) (n = 5 mice). (H) Mean Z score from (G) before and after door opening [effect of door opening (F1, 4 = 11.89, P = 0.02); trial (F2, 8 = 0.3, P = 0.74); event × trial (F2, 8 = 0.02, P = 0.97)) and contact [effect of door opening (F1, 4 = 49.18, P = 0.002); trial (F2, 8 = 4.47, P = 0.05); event × trial (F2, 8 = 26.76, P = 0.003); two-way ANOVA]. (I) Effect of anosmia, whisker trimming, or both on the response of ZISST neurons to social interactions with the mother compared to intact pups (intact, n = 15; anosmia, n = 8; whisker trimmed, n = 7; or both, n = 8) (F3, 34 = 6.27, P = 0.001; one-way ANOVA). In H-I, ANOVA was followed by Holm-Sidak’s multiple comparison test. Line graphs for neuron recordings represent mean ± SEM. Box plot denotes minimum, first quartile, median, third quartile, and maximum values. Round symbols in box plots represent individual trials in D and individual mice in I. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

These experiments were performed during free interactions between the pup and mother. To better isolate the contribution of pup’s behavior to the changes in the activity of ZISST neurons, we designed a linear track test (Fig. 2D–E) (14). Opening the gate caused a small increase in the activity of ZISST neurons across all three trials (Fig. 2F–G). Social contact with the mother, but not contact with a furry toy, robustly increased the activity of ZISST neurons (Fig. 2F–G). Together, these experiments demonstrate that ZISST neurons in pups respond to social interactions with the mother, especially to direct social contact.

ZISST neurons integrate olfactory and whisker sensory signals

Olfaction and tactile sensing by the whiskers are two primary sources of sensory information for developing rodents (15–19). We therefore examined the contribution of olfactory and whisker sensing for the response of ZISST neurons to interactions with the mother. We analyzed the activity of ZISST neurons after removal of olfactory inputs (anosmia by methimazole (20–22)), whisker inputs (whisker trimming), or both. Anosmia or deprivation of whisker sensing alone did not change the magnitude of the activation of ZISST neurons (Fig. 2I). However, concurrent removal of olfactory and whisker inputs reduced neuronal activation induced by social interaction (Fig. 2I). These results suggest that pups integrate multisensorial information that is conveyed to ZISST neurons during social interactions with mother.

ZISST neurons modulate distress responses in socially isolated pups

Following a period of social isolation, the reunion of an infant with their mother alleviates distress responses. Two of these distress responses are the production of ultrasonic vocalizations (USVs), which elicits maternal care and attention in mice (23–25), and corticosterone release. Because maternal reunion and contact activate ZISST neurons (Figs. 1–2), we predicted that increasing the activity of ZISST neurons in socially isolated pups would alleviate distress responses.

To activate ZISST neurons, we injected the ZI of neonatal SST-IRES-Cre mice with AAVs to express hM3Dq (26) or tdTomato in ZISST neurons (Fig. 3A–B and fig. S3). Slice electrophysiology experiments confirmed that chemogenetic activation of hM3Dq-expressing ZISST neurons increased their firing rates (fig. S4). On P11, an age in which mouse pups vocalize at high rates (27–30), we separated the pups from the home cage and measured the emission of USVs. Ten minutes before separation, all pups were injected with the hM3Dq ligand, clozapine-N-oxide (CNO). As expected, maternal separation triggered the emission of USVs in control pups with an average rate of ~56 USV/min (Fig. 3C). Activation of ZISST-hM3Dq neurons reduced the rate of vocalizations to less than 11 USV/min, a pronounced 5-fold reduction (Fig. 3C). We then reunited both ZISST-tdTomato and ZISST-hM3Dq pups with their anesthetized mother to minimize the influence of maternal behaviors on pup behavior (12). Reunion with the mother decreased USV production in control mice to ~20 USV/min (Fig. 3D). In ZISST-hM3Dq pups, reunion failed to further reduce the rate of vocalizations compared to the separation period (Fig. 3D).

Fig. 3. ZISST neurons modulate infant’s response to social isolation and reunion.

Fig. 3.

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(A) Schematic depicting protocol for measuring pup vocalizations. CNO (1 mg/kg, i.p.) is injected 10 min prior to the isolation. (B) Representative image of the viral expression in ZISST neurons. (C) Number of USVs emitted during isolation in control mice (n = 13) and ZISST-hM3Dq mice (n = 19) quantified in 5-min bins [effect of time (F3.82, 114.61 = 14.98, P < 10−8); group (F1, 30 = 92.29, P < 10−9); time × group (F11, 330 = 9.18, P < 10−13); two-way ANOVA followed Sidak’s multiple comparison test]. (D) Number of USVs emitted during the last 5 min of isolation and during reunion with the anesthetized mother, in control (n = 10) and ZISST-hM3Dq mice (n = 14) [effect of time (F1,22 = 31.26, P < 10−4); group (F1, 22 = 35.78, P < 10−5); time × group (F1, 22 = 31.76, P < 10−4); two-way ANOVA followed Sidak’s multiple comparison test]. (E-H) UMAPs of individual USVs during isolation (control, n = 25473; ZISST-hM3Dq, n = 12390) and reunion (control, n = 727; ZISST-hM3Dq, n = 859). (I) Locomotor activity and distance travelled in P11 mice during isolation (control, n = 8; ZISST-hM3Dq, n = 8). (J) Ethograms of manually classified behaviors aligned to the emission of USVs by control and ZISST-hM3Dq mice. (K) Quantification of USVs emitted while pups were engaged in different behaviors (n = 4/group). (L) Plasma corticosterone concentration in fifteen-day-old control (n = 15) and ZISST-hM3Dq (n = 12) mice injected with CNO 10 min prior to isolation and then socially isolated for 60 min. Home-cage control mice remained undisturbed in the nest (n = 8) (F2, 32 = 11.39, P = 0.0002; one-way ANOVA followed Holm-Sidak’s multiple comparison test). (M) Similar to (A), newborn mice are injected with an AAV to express Kir2.1 or tdTomato in SST neurons in the ZI; on postnatal day 11, ultrasonic vocalizations are recorded when pups are socially isolated for 60 min and then reunited with the anesthetized mother for 5 min. (N) Number of USVs emitted during the last 5 min of isolation and during reunion with the anesthetized mother, in control (n = 12) and ZISST-Kir2.1 mice (n = 17) [effect of time (F1,27 = 9.48, P = 0.004); group (F1, 27 = 2.83, P = 0.10); time × group (F1, 27 = 13.60, P = 0.001); two-way ANOVA followed by Holm-Sidak’s multiple comparison test]. (O) Newborn mice are injected with an AAV to express the inhibitory light-sensitive opsin stGtACR2 or tdTomato in SST neurons in the ZI; on postnatal day 13, ultrasonic vocalizations are recorded when pups are socially isolated for 10 min and then reunited with the anesthetized mother; during reunion, the LED is turned ON for 1 minute and then turned OFF. (P) Raster plot showing the USVs before, during, and after photo-inhibition of ZISST neurons (control, n = 7; stGtACR2, n = 6). (Q) Quantification of USVs before, during, and after photoinhibition (per minute) [effect of time (F2, 22 = 44.86, P < 10−7); group (F1, 11 = 59.63, P < 10−5); time × group (F2, 22 = 33.79, P < 10−6); two-way ANOVA followed Holm-Sidak’s multiple comparison test]. Line graphs represent mean ± SEM. Box plot denotes minimum, first quartile, median, third quartile, and maximum values. In D, I, K, L, and N, symbols represent individual data. In Q, symbols represent mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

To further investigate the effect of ZISST neuron activation on vocalizations, we employed an unsupervised approach, whereby we segmented the spectrograms of each USVs and projected them into a UMAP (fig. S5) (12, 31). While the distribution of USVs did not show clear clusters (fig. S5) (32), the distribution of USVs of control pups clearly displayed regions of high density (Fig. 3E). The high-density regions shifted when control pups were reunited with their anesthetized mother (Fig. 3F). Activation of ZISST-hM3Dq neurons elicited a shift in the profile of USVs during the isolation period that was similar to the reunion period of control pups (Fig. 3G). The vocal repertoire of ZISST-hM3Dq pups after reunion resembled their own vocal repertoire during isolation and that of control pups during reunion (Fig. 3H). A supervised method to classify USVs based on their spatiotemporal features (33) found similar results (fig. S6). Finally, activation of ZISST neurons during, rather than prior to, maternal separation also reduced vocalizations (fig. S7).

We also investigated whether the activation of ZISST neurons affected other behaviors in pups (34) (fig. S8) and found that it did not influence the total distance traveled (Fig. 3I) or the number of USVs emitted during each behavior (Fig. 3J–K). Taken together, these behavioral results suggest that the activation of ZISST neurons changes pup vocalizations towards a state resembling when they are with their mother.

In addition to vocalizations, maternal separation activates the endocrine stress response, increasing corticosterone release. To investigate the effects of ZISST neurons on the endocrine stress response of pups, we chemogenetically activated these neurons and measured blood corticosterone in fifteen-day-old pups, an age at which maternal separation strongly activates this axis (35).

ZISST-tdTomato and ZISST-hM3Dq pups received an injection of CNO ten minutes before maternal separation. In control mice, maternal separation almost doubled blood concentration of corticosterone compared to pups that remained in the home cage (control: 98.8 ± 19.3 ng/ml, n = 8; isolation: 176.7 ± 13.05, n = 15; Fig. 3L and fig. S9). The activation of ZISST neurons blunted this increase in corticosterone (85.8 ± 15.7, n = 12; Fig. 3L). Together, the effects of ZISST neuron activation on blood corticosterone and vocalizations suggest a broader function of these neurons in alleviating the distress response of the infant.

We next tested whether the activity of ZISST neurons is required for the effects of maternal reunion on infant’s distress responses. We first reduced the excitability of ZISST neurons by expressing a mutated Kir2.1 channel (36, 37) (figs. S10–S11). At P11, littermate pups expressing either ZISST-tdTomato or ZISST-Kir2.1 were socially isolated, followed by reunion with their anesthetized mother (Fig. 3M). During the separation period, both groups emitted similar amounts and profiles of USVs (fig. S12). In response to reunion with the mother, the reduction in USV production, but not corticosterone concentration, was blunted in ZISST-Kir2.1 pups (Fig. 3N and fig. S13). Thus, the normal excitability of ZISST neurons is required for the rapid effects of maternal reunion on pup USV production but not for its effects on corticosterone.

To test whether reducing the activity of ZISST neurons during reunion could trigger the emission of USVs by the pup, we used an optogenetic approach to briefly silence ZISST neurons. We injected newborn SST-IRES-Cre mice with AAVs encoding the anion-conducting channelrhodopsin, stGtACR2 (38) or tdTomato (Fig. 3O and figs. S14–S15). On P12, we implanted bilateral fiber optic cannulas above the ZI. One day later, we separated both control and ZISST-stGtACR2 pups from the home cage for ten minutes and then photo-inhibited ZISST neurons for one minute during reunion with the anesthetized mother. This brief silencing of ZISST neurons increased the rate of vocalizations (Fig. 3P–Q), suggesting that the ongoing activity of these neurons during social interactions with the mother is required to alleviate the behavioral distress response of the infant.

The activity of ZISST neurons facilitates learning of positive associations

The interaction of the pup with the mother is conducive to learning: Pups learn to associate cues with the presence of the mother and form positive associations with these cues (5, 6). We therefore asked if ZISST neurons can facilitate the formation of positive associations in infants.

We designed an associative learning paradigm for mouse pups in which they learn to associate a neutral odor with the presence of their mother (fig. S16). Using similar paradigm, we first aimed to test if the inhibition of ZISST neurons during conditioning with the mother can affect the strength of the learned association. To inhibit ZISST neurons, we expressed the inhibitory receptor, hM4Di (or control tdTomato) in ZISST neurons (figs. S17–S18). At P17, we began the conditioning protocol (Fig. 4A). Control and ZISST-hM4Di pups were first separated from their mothers and, subsequently, injected with the agonist of hM4Di. Pups were then individually reunited with an anesthetized mother and paired with a toy coated with a colorless odorant (hexanoic acid) (39). Next day, pups were tested for their preference for the toy used during conditioning or a novel toy. As expected, control pups conditioned with anesthetized mother showed a strong preference for the conditioned toy (preference index = 0.82, 95% CI [0.78, 0.86]; Fig. 4B). Remarkably, ZISST-hM4Di pups did not display a preference for either toy (preference index = 0.57, 95% CI [0.45, 0.69]; Fig. 4B). Therefore, during social contact of the pup with the mother, the activity of ZISST neurons is required for the pup to fully learn new positive associations.

Fig. 4. ZISST neurons facilitate learning.

Fig. 4.

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(A) Schematic depicting behavioral assay for associative learning upon inhibition of ZISST neurons. During conditioning, pups (ZISST-tdTomato and ZISST-hM4Di) are injected with C21 (3 mg/kg, i.p.). (B) Preference index calculated based on the amount of time that pups spend exploring the toy scented with odorant versus a novel toy (0.5 denotes iso-preference; > 0.5 denotes preference for the scented toy). Statistical difference from iso-preference (0.5) was tested using one-sample t test: ZISST-tdTomato (n = 11, t10 = 17.78, P < 10−8) and ZISST-hM4Di (n = 10, t9 = 1.40, P = 0.19). Statistical difference between groups was tested using unpaired t test: t19 = 4.48, P = 0.0003. (C) Schematic depicting behavioral assay for associative learning upon activation of ZISST neurons. During conditioning, pups (ZISST-tdTomato and ZISST-hM3Dq) are injected with CNO (1 mg/kg, i.p.). (E) Statistical difference from iso-preference using one-sample t test: ZISST-tdTomato (n = 13, t12 = 0.52, P = 0.60) and ZISST-hM3Dq (n = 14, t13 = 6.78, P = 0.00001). Statistical difference between groups using unpaired t test: t20 = 3.36, P = 0.002. Box plot denotes minimum, first quartile, median, third quartile, and maximum values. Round symbols in box plots represent individual mice. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Next, we aimed to test if the activation of ZISST neurons during conditioning without the mother could facilitate learning. We expressed hM3Dq (or tdTomato) in ZISST neurons as before (Fig. 3). ZISST-tdTomato and ZISST-hM3Dq pups were first separated from their mothers and then received an injection of the agonist of hM3Dq. Ten minutes after the injection, the toy coated with odorant was presented to each pup individually, but without the mother (Fig. 4C). Control ZISST-tdTomato pups did not show a preference for either toy (preference index = 0.47, CI 95% [0.38, 0.57]; Fig. 4D). However, ZISST-hM3Dq pups showed a preference for the conditioned toy (preference index = 0.63, CI 95% [0.58, 0.67]; Fig. 4D), an effect that was however weaker than pups conditioned with the mother (fig. S16). Thus, similar to how pups learn in the presence of their mother, the activation of ZISST neurons in socially isolated pups can facilitate the formation of positive associations in infant mice.

The ‘fan-in’ and ‘fan-out’ connectivity of ZISST neurons

The ability of ZISST neurons to respond to social interactions and influence an array of responses suggest an integration with various neural networks. To map the connectivity of ZISST neurons in pups, we first assessed their long-range connections by expressing channelrhodopsin-2 fused with mCherry in these neurons (Fig. 5A). At P14, we characterized their arborization using immunohistochemistry for mCherry. ZISST neurons extended their projections to several brain areas (fig. S19), with the strongest density of fibers observed in the caudoputamen, anterior cingulate area, primary motor area, ventromedial thalamus, and the periaqueductal gray (Fig. 5B–C). Several of these regions match previously reported areas in adult mice (40), underscoring the early establishment of this fan-out circuit architecture and the capacity for ZISST neurons to influence multiple neural circuits from infancy.

Fig. 5. Downstream and upstream connectivity of ZISST neurons in preweaning pups.

Fig. 5.

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(A) Schematic for tracing downstream targets from ZISST neurons: Cre-dependent AAV that expresses ChR2-tdTomato is injected at P0 and brains are collected at P14; representative image showing the endogenous expression of mCherry in the ZI of an injected mouse. (B) Representative images of the main projections of ZISST neurons. (C) Quantification of projection density using an intensity score (from 0 to 5, from weakest to strongest). Top regions are shown from three mice. (D) Schematic for monosynaptic retrograde labeling of ZISST neurons; Cre-dependent AAV that expresses TVA-mCherry and G-protein is injected at P0 (helper virus) and EnvA pseudotyped G-deleted rabies virus expressing GFP is injected at P14; brains are collected at P20. Detail of the injection site, 100 μm horizontal projection at the level of the zona incerta, showing the mCherry+ starter cells (red) and GFP+ input cells (green). (E) GFP+ cells seen in a whole brain projection (side ipsilateral to the injection shown), revealing a sparse distribution of cells in the anterior cortex, thalamus and hypothalamus. (F) Atlas-aligned location of the segmented GFP+ cells in a horizontal whole brain projection and on a coronal slice at the level of the hypothalamic pre-optic area. The cells are shown as large circles. (G) Regional counts of the GFP+ cells from two brains (only the top counts shown). (H) Diagram representing the circuit architecture of ZISST neurons in preweaning pups with a ‘fan-in’ and ‘fan-out’ organization. CPu, caudoputamen; VL, ventral medial nucleus of the thalamus; VM, ventral anterior-lateral complex of the thalamus; Re, nucleus of reuniens; Cg, cingulate cortex; M, motor cortex; HDB, horizontal limb of the diagonal band nucleus; IC, inferior colliculus; PAG, periaqueductal gray; DR, dorsal raphe nucleus; ZI, zona incerta; LH, lateral hypothalamic area; MeA, medial amygdalar nucleus; BMA, basomedial amygdalar nucleus. Scale bars are 500 μm (A, B, and D) and 1 mm (E and F). In C and G, color coding refers to large brain regions such as hypothalamus and thalamus (see fig. S21 for details).

Next, to establish the main inputs to ZISST neurons, we performed monosynaptic retrograde tracing (41). We first injected an AAV virus expressing Cre-dependent TVA receptor fused with mCherry and the optimized G protein in the ZI of newborn SST-Cre mice (Fig. 5D). Two weeks later, G-deleted rabies virus expressing GFP and pseudotyped with the envelope protein from avian sarcoma/leukosis virus subtype A (EnvA), which binds to TVA receptor for cell entry, was injected into the ZI (Fig. 5D). Starter cells (TVA-mCherry+ and GFP+) were found in the ZI only at the level of the injection site (Fig. 5D). Whole-brain imaging (42) revealed a ‘fan-in’ circuit architecture of ZISST neurons (Fig. 5E). GFP+ cells were located in many brain regions (Fig. 5F–G and fig. S20–21), with the strongest labeling observed in integrative areas of the thalamus (such as the ventromedial, paracentral, mediodorsal, and reticular nuclei of the thalamus) and hypothalamus (particularly in the paraventricular nucleus and lateral hypothalamus). In the midbrain, the reticular nucleus and periaqueductal grey were also strongly labeled. We further corroborate these findings through immunohistochemistry in brain slices (fig. S22). Collectively, our findings unveil a ‘fan-in’ and ‘fan-out’ circuit architecture of ZISST neurons, with many regions of reciprocal connectivity, rendering these neurons as an integrative node for the modulation of infant’s responses (Fig. 5H).

Discussion

Our findings reveal that ZISST neurons are selectively tuned to social interactions between mouse pups and their mothers. Mothers exert a powerful influence over infant’s responses, dampening both behavioral distress (crying) and physiological stress (cortisol or corticosterone release) (3, 43–48). We found that ZISST neuron activation reduces vocalizations and corticosterone concentration during isolation, thereby mimicking the mother’s soothing effects. Disrupting ZISST neuron activity selectively impaired the rapid behavioral effects of maternal reunion without altering corticosterone responses. This dissociation between behavioral and endocrine regulation aligns with previous work showing dissociable freezing behavior and endocrine responses during fear extinction (49), and suggests distinct neural mechanisms governing these aspects of the infant’s response to maternal contact. As ZISST neurons do not directly innervate the PVH, which control corticosterone release (50), their effects on corticosterone likely occur via indirect pathways.

The presence of the mother not only reduces distress, but also facilitates the learning of positive associations (5, 6, 51, 52). Even under threatening conditions, such as electric shock, the presence of the mother prevents corticosterone release, and pups learn positive associations (5, 52, 53). This suppression of corticosterone release is thought to be critical for learning as it prevents the activation of the amygdala. Because we identified ZISST neuron projections to parts of the amygdala, future research should clarify the role of this and other projections in infant learning during threat.

Our results contrast with the effects of ZISST neuron activation in adult mice, which increases anxiety-like and fear-like responses (40, 54). The differential effects of ZISST neurons in infants and adults reinforce the notion that neural circuits adapted to support the distinct needs of individuals across development (55). These findings position the ZI as an important brain region for understanding the developmentally-regulated functions of neurons.

The ZI exhibits distinct developmental characteristics. During early development, it establishes dense projections to various brain regions, some of which retract post-weaning (9, 10). Additionally, the activity of ZISST neurons influences cortical development (56), which is intriguing because the protracted development of the cortex is a major feature of mammalian brain evolution. Another hallmark of mammals is the unique bond between the infant and its mother. In light of our findings, it is tempting to consider the ZI as a nexus that intertwines these two defining characteristics of mammalian biology.

In summary, this work establishes that somatostatin-expressing neurons in the mouse ZI are key modulators of the infant’s social responses and learning. The richness of the infant’s social experiences provides the foundation for cognitive and emotional development. Disruptions in social development can occur as a result of child maltreatment as well as in neurodevelopmental conditions such as autism spectrum disorder. The findings provide an entry point to study infant-specific responses during neurotypical and neurodivergent development.

Supplementary Material

Supplementary material
MDAR Reproducibility Checklist

Materials and Methods

Figs. S1 to S22

References (58–65)

MDAR Reproducibility Checklist

Acknowledgments:

We thank members of the Dietrich lab, Alicia Che, Daniel Mucida, Ivan de Araujo, Luis Portela, and Amber Alhadeff for critical feedback on the project and on the manuscript. We thank the Janelia GENIE project for the jGCaMP7s plasmid. We thank Qingchun Tong for the Kir2.1 plasmid. We thank David Bruin for copyediting the manuscript.

Funding:

National Institute of Mental Health of the National Institutes of Health (R01MH125008 and R01MH130825) and Smith Family Foundation to MOD. The new directions in research were, however, only possible by discretionary funds from the Yale School of Medicine and the support from the Department of Comparative Medicine to MOD. Coordination for the Improvement of Higher Education Personnel (CAPES) to GMS. China Scholars Program to YL. Marie Skłodowska-Curie Action under the European Union’s Horizon 2020 research and innovation program (Grant Agreement 953327) to AMC. Wellcome Leap Foundation to ED. ERC-consolidator grant “VIRGINs” (grant agreement 101125555) to NR.

Footnotes

Competing interests: Authors declare that they have no competing interests.

Data and materials availability:

All code related to this study is available at: https://github.com/yxl95/zona_incerta_infant_social_behavior. The data that support the findings of this study is available on Zenodo (57).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material
NIHMS2130646-supplement-Supplementary_material.pdf (9.9MB, pdf)
MDAR Reproducibility Checklist
NIHMS2130646-supplement-MDAR_Reproducibility_Checklist.pdf (178.4KB, pdf)

Data Availability Statement

All code related to this study is available at: https://github.com/yxl95/zona_incerta_infant_social_behavior. The data that support the findings of this study is available on Zenodo (57).

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