Dock4 KO mice exhibit social deficits

A Dock4 KO mouse line was established, and the loss of Dock4 expression in the mice was verified using quantitative real-time PCR (qRT-PCR) and western blotting (Fig. 1a, Supplementary Figs. 2 and 3). The Dock4 transcript was completely absent in the KO brain and reduced by approximately half in the HET brain (Fig. 1b, Supplementary Fig. 3d). Transcripts of 10 other Dock-family members, Dock 1–3 and 5–11, did not vary extensively (Fig. 1b). In accord with previous observations [27], Dock4 was found to be expressed in various brain regions and highly enriched in the hippocampus (Fig. 1c). No Dock4 was detected in any brain region or other organs in KO mice, whereas reduced amounts of Dock4 were found in the corresponding HET tissues (Fig. 1c, Supplementary Fig. 3f).

Fig. 1 Dock4 KO mice exhibit altered social behavior. a Strategy used for generating Dock4 KO mice. b Brain mRNA levels of Dock4 and other Dock-family members were assessed using quantitative RT-PCR; mRNA levels of KO mice were normalized to those of WT mice. n = 6 WT or KO mice; *P < 0.05, ***P < 0.001, unpaired t test. c Dock4 protein expression in different brain regions of WT, HET, and KO mice; α-tubulin was used as a loading control. d Nissl staining of sagittal brain sections from 5-month-old WT and KO mice. Brain lateral ventricles were slightly enlarged in KO mice (arrows). Scale bar, 2 mm. e Brain weight measurements from 5-month-old WT, HET, and KO mice. *P < 0.05, one-way ANOVA with Bonferroni’s Multiple Comparison Test. f Distance traveled by WT, HET, and KO mice in open-field test. Hyperactivity was displayed by a small population of HET (~1.7%; 3/172) and KO (~9.1%; 4/44) female mice that exhibited stereotyped circling behavior. ***P < 0.001, unpaired t test. g Ultrasonic vocalizations of pups at P3, P6, P9, and P12 during maternal separation for 5 min. n = 28, 28, 29, and 26, respectively for WT; n = 39, 41, 38, and 38, respectively for HET; n = 13, 15, 15, and 13, respectively for KO. *P < 0.05, one-way ANOVA with Bonferroni’s Multiple Comparison Test. h Pup-retrieval assay was performed to evaluate maternal behavior of virgin female mice. Latency of retrieving the first pup and all 3 pups was measured in a 10-min trial. *P < 0.05, **P < 0.01, one-way ANOVA with Bonferroni’s Multiple Comparison Test. i Representative heatmap of movement of male and female WT, HET, and KO mice during social approach and social novelty phases of the Three-chamber test. j, k Duration spent in sniffing different cups, measured for male (j) and female (k) WT, HET, and KO mice during social approach. l, m Duration spent in sniffing different cups, measured for male (l) and female (m) WT, HET, and KO mice during social novelty preference. E, empty cup; S1, cup containing stranger mouse #1; S2, cup containing stranger mouse #2. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant, unpaired t test. n values of each group are displayed on the corresponding bars of the bar charts. Error bars: SEM Full size image

Despite showing slightly lower natality (Supplementary Fig. 4a), Dock4 HET and KO mice exhibited similar growth rate (weight gain; Supplementary Fig. 4b, c), ingestion, and mating as WT mice did. Brain architecture in Dock4 KO mice was grossly normal, with slight enlargement of the lateral ventricles (Fig. 1d, Supplementary Fig. 4d–g), and the brain was slightly heavier in female KO mice than in female WT mice (Fig. 1e). Notably, a small population of female HET (1.7%) and KO (9.1%) mice showed autism-like hyperactivity and stereotyped behavior, such as continual circling in the home cage and open field (Fig. 1f, Supplementary Fig. 5a, Supplementary Videos 1–3). The male HET and KO mice and the majority of the female HET and KO mice displayed mostly normal locomotion in the open field (Fig. 1f) and did not show stereotyped behaviors (Supplementary Fig. 5b, c). This suggests that Dock4 deficiency might contribute to a risk factor of stereotyped behavior particularly in females. Because the number of stereotyped mice was small, these mice were excluded in the subsequent studies.

To investigate language communication in Dock4 KO mice, we evaluated ultrasonic vocalizations in pups when isolated from their dam within the first two weeks after birth. Interestingly, Dock4 KO pups emitted more calls at postnatal days 3, 6, 9, and 12 than their WT littermates (Fig. 1g). The total call duration was also longer in KO pups than in WT pups, but the average peak frequency of all calls was similar in both genotypes (Supplementary Fig. 6a, b). This altered vocal behavior was reminiscent of some ASD mice models [33,34,35], suggesting abnormal communication in Dock4 KO mice. We next performed tests to assess several features of the social behavior. First, the ability of nesting, a nature in mice that tightly correlates with social behaviors such as parenting and reproduction, was evaluated. As a result, mice of all genotypes performed equally well in nest building (Supplementary Fig. 6c–e). Second, maternal behavior was measured in a pup-retrieval assay by using virgin female mice. Relative to WT littermates, KO females spent significantly longer time to retrieve the pups (Fig. 1h), suggesting poorer maternal behavior of KO females. Finally, social approach and social preference behaviors were assessed using the Three-chamber test [36]. In the social approach phase, WT, HET, and KO mice all performed normally, spending more time exploring the stranger conspecific than the object (Fig. 1i–k; Supplementary Fig. 6f, g). However, in the social novelty phase, both male and female KO mice and HET female mice failed to distinguish between familiar and newly introduced unfamiliar conspecifics (Fig. 1i, l, m; Supplementary Fig. 6h, i). Together, characterizations of the ASD core symptoms revealed that Dock4 KO mice exhibited both language and social deficits, and displayed increased tendency of stereotyped behavior.

Elevated anxiety and defective learning and memory in Dock4 KO mice

Besides the core symptoms, ASD subjects commonly present co-occurring symptoms that include anxiety and intellectual disability [37, 38]. We measured anxiety behavior in elevated zero-maze. Relative to WT mice, Dock4 KO mice traveled a markedly shortened total distance (Fig. 2a). Moreover, the anxiety levels were higher in KO males than KO females: the KO males not only spent less time in the open sections of the maze (Fig. 2b), but also entered the open area less frequently (Fig. 2c). Furthermore, the KO males exhibited reduced mobility in both the open and closed sections, as indicated by the decreased travel distance and velocity (Supplementary Fig. 7a–d). To measure cognitive ability, mice were subjected to several learning and memory tasks, including novel object recognition test, Y-maze test, and Morris water-maze test. In the novel object recognition task, female KO mice failed to distinguish the novel object from the familiar one (Fig. 2d). In the Y-maze task, working memory was tested by measuring the alternate exploring behavior among the three arms of the maze: KO males displayed decreased alternation, and their arm-entry numbers were significantly higher than those of WT males (Fig. 2e, f), which suggests defective working memory in KO males. In a distinct Y-maze paradigm, spatial recognition memory between familiar and novel arms was tested. KO males failed to distinguish between the two arms, suggesting that KO males show aberrant spatial recognition (Fig. 2g, h). In the Morris water-maze task, WT, HET, and KO mice displayed similar latency in locating the submerged platform over a 7-day training period (Supplementary Fig. 7e–h). Collectively, these results indicate that Dock4 KO mice manifest all three core domains of ASD symptoms, and display elevated anxiety and impaired object and spatial learning (Supplementary Table 4).

Fig. 2 Dock4 KO mice display elevated anxiety and impaired learning and memory. a–c WT, HET, and KO mice were tested in an elevated zero-maze, and the following variables were measured and analyzed: (a) Total distance traveled; (b) duration (% of total time) spent in open sections; and (c) number of entries into open sections. *P < 0.05, **P < 0.01, one-way ANOVA with Bonferroni’s Multiple Comparison Test. (d) Novel object recognition memory of WT, HET, and KO mice was examined at 24 h after training. Female KO mice showed aberrant recognition memory. F, familiar object; N, novel object. ***P < 0.001, ns, no significant, unpaired t test. e, f Alternation (e) and number (f) of arm entries of WT, HET, and KO mice during 8 min of free exploration in Y-maze test. ***P < 0.001, unpaired t test. g, h Duration (g) and number of entries (h) in novel or familiar arm, measured for WT, HET, and KO mice in 5-min trials in Y-maze. F, familiar arm; N, novel arm. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant, unpaired t test. n values of each group are displayed on the corresponding bars of the bar charts. Error bars: SEM Full size image

Dock4 deficiency in hippocampus leads to social deficit and impaired excitatory synaptic transmission

The hippocampus, in which Dock4 is highly expressed, plays a central role in integrating brain networks for social and recognition memories [39]. To investigate the effect of Dock4 in hippocampus, we specifically deleted Dock4 in hippocampus by adeno-associated virus (AAV)-mediated delivery of Cre into hippocampal CA1 region of Dock4fl/fl mice (Fig. 3a, Supplementary Fig. 8a, b). Intriguingly, these hippocampal conditional KO mice failed to distinguish between familiar and newly introduced unfamiliar conspecifics in Three-chamber test, recapitulating the social deficit in whole-body KO mice (Fig. 3b–d, Supplementary Fig. 8c–e). This result indicates that social novelty preference requires Dock4-dependent regulation of hippocampal function. We then explored the synaptic basis in the hippocampus that underlies the aberrant social behavior in Dock4 KO mice. The amplitude of miniature excitatory synaptic currents (mEPSCs) recorded in hippocampal CA1 pyramidal neurons was smaller in KO mice than in WT mice (Fig. 3e–g), whereas miniature inhibitory synaptic currents (mIPSCs) were unaltered in KO mice (Fig. 3h–j). To determine whether excitatory/inhibition (E/I) balance is affected by Dock4 deficiency, we recorded both evoked EPSC and IPSC from same pyramidal cells. Notably, whereas IPSC showed no change, EPSC amplitude reduced remarkably in KO cells (Fig. 3k–m). As a result, the E/I ratio of KO cells was substantially smaller than that of WT cells (Fig. 3n), suggesting a shift of E/I balance toward inhibition in KO hippocampal CA1 pyramidal neurons. To examine whether the reduced EPSC in KO neurons is resulted from compromised presynaptic function, paired-pulse ratios (PPRs) of EPSCs were measured. Normal paired-pulse facilitation was observed in both WT and KO neurons (Fig. 3o, p), suggesting that the diminished excitatory synaptic transmission in Dock4 KO hippocampus is probably attributed to postsynaptic dysfunctions.

Fig. 3 Dock4 deficiency in hippocampus leads to social deficit and impaired excitatory synaptic transmission. a Adeno-associated virus (AAV)-Cre was bilaterally injected into the hippocampal CA1 region of Dock4fl/fl mice. b Representative heatmap of movement of Dock4fl/fl + AAV-vector or Dock4fl/fl + AAV-Cre mice in the Three-chamber test at 4 weeks after virus injection. c, d Duration spent by two groups of mice in sniffing different cups during social approach (c) and social novelty (d) phases. E, empty cup; S1, cup containing stranger mouse #1; S2, cup containing stranger mouse #2. n = 11 Dock4fl/fl + AAV-vector mice (6 males and 5 females), and n = 8 Dock4fl/fl + AAV-Cre mice (4 males and 4 females). *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant, unpaired t test. e Representative (left) and average (right) traces of mEPSCs recorded from WT (black) and KO (red) hippocampal CA1 pyramidal cells. Magnified traces from the boxed regions are shown below. f, g Cumulative probability of mEPSC amplitude (f) and inter-event interval (g) in WT (15 cells, 4 mice) and KO (15 cells, 3 mice) groups. Insets: average mEPSC amplitude (f) and frequency (g) from WT and KO groups *P < 0.05, two sample Kolmogorov-Smirnov test. #P < 0.05, unpaired t test. h Representative (left) and average (right) traces of mIPSCs recorded from WT (black) and KO (red) hippocampal CA1 pyramidal cells. i, j Cumulative probability of mIPSC amplitude (i) and inter-event interval (j) in WT (18 cells, 3 mice) and KO (15 cells, 3 mice) groups. Insets: average mIPSC amplitude (i) and frequency (j) from WT and KO groups. k Representative traces of evoked EPSC and IPSC in same CA1 pyramidal cells from WT (black) and KO (red) mice. l, m The maximal EPSC (l) and IPSC (m) recorded from WT (20 cells, 6 mice) and KO (21 cells, 5 mice) groups. ***P < 0.001, ns, not significant, unpaired t test. (n) The calculated E/I ratio from WT and KO groups. ***P < 0.001, unpaired t test. o Representative traces of paired-pulse ratio (PPR) of EPSCs at different inter-stimulus intervals recorded from WT (black) and KO (red) mice. p The plot of PPR vs. different inter-stimulus intervals (WT: 10 cells, 4 mice; KO: 10 cells, 4 mice). Error bars: SEM Full size image

Disrupted AMPA and NMDA receptor functions in Dock4 KO hippocampus

To examine whether the excitatory postsynaptic function is impaired in KO hippocampus, the input-output properties of AMPAR- and NMDAR-mediated EPSCs were measured separately. Notably, the EPSCs mediated by both receptors were significantly decreased in KO cells after stimulation at a series of intensities, with the reduction of NMDAR-EPSCs being more prominent (Fig. 4a, b). Accordingly, the ratio of NMDAR-EPSCs to AMPAR-EPSCs was lowered by Dock4 deficiency (Fig. 4c). We also measured the functional synaptic composition of NMDAR subunits by isolating NMDAR-EPSCs blocked by the GluN2B antagonist ifenprodil (3 μM); the blockage of NMDAR-EPSCs by ifenprodil was significantly weaker in KO neurons than in WT neurons, which suggests that KO neurons contain less synaptic proportion of GluN2B-containing receptors (Fig. 4d). Because NMDAR function is important for the induction of both long-term potentiation (LTP) and long-term depression (LTD), we examined whether these two forms of synaptic plasticity are altered by Dock4 deficiency. Whole-cell recordings were used to examine LTP at Schaffer collateral-CA1 synapses induced by a pairing protocol, which revealed that LTP was significantly reduced in KO neurons as compared with that in WT neurons (Fig. 4e). Application of APV, an NMDAR antagonist, completely blocked the induction of LTP in either WT or KO neurons, suggesting that only NMDA-dependent LTP was induced using this protocol (Fig. 4e). On the other hand, LTD was induced either by low frequency stimulation or by application of NMDA. Whereas both forms of LTD were hardly induced at adult as reported [40], they are similarly induced in WT and KO hippocampus at young postnatal age, suggesting that LTD is intact in KO mice (Supplementary Fig. 9).

Fig. 4 AMPAR and NMDAR-dependent synaptic transmission is impaired in Dock4 KO hippocampus. a, b KO neurons displayed a downward input-output curve of both AMPAR-EPSC (a) (25–30% decrease at strongest stimulation relative to WT; n = 10 cells from 3 WT mice; n = 12 cells from 4 KO mice, P < 0.001, two-way AVOVA) and NMDAR-EPSC (b) (30–35% decrease at strongest stimulation relative to WT; n = 26 cells from 6 WT mice; n = 24 cells from 5 KO mice, P < 0.001, two-way AVOVA). Representative traces are shown in the right panel of each graph. c NMDAR-EPSC to AMPAR-EPSC ratios from WT (13 cells, 5 mice) and KO (15 cells, 5 mice) groups. Representative traces are shown on the right. *P < 0.05, unpaired t test. d KO mice showed a reduction in the proportion of synaptic GluN2B. Values are presented as change of NMDAR-EPSC (% of baseline) after application of ifenprodil, an GluN2B inhibitor. Representative traces before and at 20–30 min after application of ifenprodil are shown on the right. n = 10 cells from 4 WT mice; n = 14 cells from 8 KO mice. P < 0.05, unpaired t test. e Time-course changes of EPSC amplitude (% of baseline) after a pairing protocol (0 mV, 2 Hz, 360 pulses; arrow), with or without application of APV, measured from WT (Ctr, 12 cells, 5 mice; APV: 9 cells, 4 mice) and KO (Ctr: 14 cells, 6 mice, APV: 13 cells, 6 mice) groups. P = 0.0241, WT vs. KO; P = 0.00001, WT + APV vs. WT; P = 0.0001, KO + APV vs. KO, unpaired t test. Representative traces before and at 25–35 min after pairing are shown on the right. f Representative Golgi staining images of dendritic spines of hippocampal CA1 and dentate gyrus (DG) neurons in WT and KO mice. Scale bar, 5 μm. g Spine density was decreased in both the hippocampal CA1 and DG regions of KO mice. n = 3 WT and KO pairs. ***P < 0.001, unpaired t test. h Expression levels of AMPAR and NMDAR subunits in the synaptosomal fraction and total lysate of the hippocampus of WT and KO mice. GluA2, GluN1, GluN2A, and GluN2B subunits were decreased at the synapse as well as in the whole hippocampus. α-tubulin served as a loading control. i Alteration of synaptosomal and total expression of indicated proteins in the KO hippocampus, expressed as fold-change of that in WT. n = 4 WT and KO pairs, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test. Error bars: SEM Full size image

We next investigated whether the impairment of excitatory synaptic transmission was associated with altered synaptic number and expression of AMPARs and NMDARs. Dendrite arborization was normal in KO hippocampus (Supplementary Fig. 10), but KO neurons had significantly decreased dendritic spines in both CA1 and dentate gyrus (DG) when compared to WT neurons (Fig. 4f, g). Notably, the synaptic expression of several AMPAR and NMDAR subunits (including GluA2, GluN1, GluN2A, and GluN2B) was markedly reduced in the KO hippocampus (Fig. 4h, i). Therefore, the diminished AMPAR- and NMDAR-mediated EPSCs can potentially be attributed to the decreased synaptic content of the corresponding receptors. To further examine whether the reduction of these receptor subunits was limited to the synapse, we measured their global expression in the hippocampus. Intriguingly, GluA2, GluN1, GluN2A, and GluN2B still showed 10–30% reduction in expression (Fig. 4h, i). Reduction of some AMPAR and NMDAR subunits was also observed in prefrontal cortex, but not striatum and cerebellum of KO mice (Supplementary Fig. 11). In contrast to the protein level changes, the mRNA levels of these receptor subunits were unaltered (Supplementary Fig. 12). These results suggest that the receptor decrease at the protein level might be due to protein homeostasis regulation.

Dock4 maintains normal protein synthesis of AMPAR and NMDAR subunits in a Rac1-dependent manner

The diminished expression of glutamate receptors could be resulted from excessive protein degradation or attenuated mRNA translation. To test these possibilities, we used primary cultured hippocampal neurons. Indeed, shRNA-mediated Dock4 knockdown recapitulated the findings in the KO hippocampus: we again observed reduced protein expression of GluA2, GluN1, GluN2A, and GluN2B (Fig. 5a, b). Notably, treatment with MG132, an inhibitor of proteasome-mediated protein degradation, failed to restore the receptor expression (Fig. 5a, b), which indicates that the reduction in protein levels was potentially caused by abrogated protein synthesis/mRNA translation.

Fig. 5 Dock4 maintains normal expression of glutamate receptor subunits in a Rac1-dependent manner. a Dock4 was knocked down in hippocampal neurons by using Dock4 shRNA (Dock4-sh) or a scrambled shRNA (Dock4-scr) as a control at 5 DIV, and the proteasome inhibitor MG132 (2 μM) was added 24 h before protein samples were collected at 9 DIV. b Expression level of each receptor subunit was quantified and normalized to Dock4-scr group. *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, one-way ANOVA with Bonferroni’s Multiple Comparison Test from 3 independent experiments. c Hippocampal neurons from WT or KO littermates were treated with puromycin at 9 DIV, and the puromycin-labeled, newly synthesized polypeptides were analyzed through Western blotting. d Puromycin-labeled protein levels were quantified and normalized to those of WT hippocampal neurons. *P < 0.05, paired t test from 3 WT and KO pairs. e, f Dock4 cDNA was transfected into Neuro-2a cells, which were then treated with the Rac1 inhibitor NSC23766 (50 μM). The levels of puromycin-labeled proteins were examined (e) and were quantified and normalized to the levels in the vector group (f). **P < 0.01, ns, no significant, one-way ANOVA with Bonferroni’s Multiple Comparison Test from 3 independent experiments. g, h cDNAs encoding full-length (FL) Dock4 and its truncation mutants were transfected into Neuro-2a cells. Puromycin-labeled proteins were examined (g) and were quantified and normalized to the levels in the vector group (h). *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant, one-way ANOVA with Bonferroni’s Multiple Comparison Test from 3 independent experiments. i, j Rac1-WT and Rac1-CA (G12V) cDNAs were transfected into Neuro-2a cells. Puromycin-labeled proteins were examined (i) and were quantified and normalized to the vector group (j). *P < 0.05, **P < 0.01, one-way ANOVA with Bonferroni’s Multiple Comparison Test from 3 independent experiments. k, l Rac1-GTP (activated form of Rac1) levels in Dock4 WT and KO hippocampus were examined (k) and quantified (l). n = 3 independent experiments. *P < 0.05, unpaired t test. m, n Dock4 was knocked down in rat hippocampal neurons and WT-Rac1 was delivered into the Dock4-knockdown neurons by using a lentiviral vector. Puromycin-labeled protein levels were analyzed (m) and were quantified and normalized to the level in the Dock4-scr group (n). *P < 0.05, #P < 0.05, ns, no significant, one-way ANOVA with Bonferroni’s Multiple Comparison Test from 3 independent experiments. o, p Expression levels of AMPAR and NMDAR subunits were examined (o) after Dock4 knockdown plus Rac1 overexpression in hippocampal neurons. AMPAR and NMDAR subunit levels were quantified and normalized to the levels in the Dock4-scr group (p). *P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01, ###P < 0.001, ns, no significant, one-way ANOVA with Bonferroni’s Multiple Comparison Test from 3 independent experiments. α-tubulin (a, c, o) and GAPDH (e, g, i, k, m) served as loading controls. Error bars: SEM Full size image

To measure protein synthesis, we used puromycin, an aminonucleoside antibiotic structurally similar to aminoacyl-tRNAs, to label newly synthesized polypeptides [41, 42]. Notably, global protein synthesis was significantly lower in hippocampal neurons cultured from Dock4 KO mice than WT littermates, as indicated by diminished labeling of puromycin (Fig. 5c, d). Because Dock4 functions as a Rac1 GEF, we tested whether Dock4 affects protein synthesis through Rac1 activation; Dock4 overexpression induced protein synthesis, but this effect was negated by the Rac1 inhibitor NSC23766 (Fig. 5e, f). Accordingly, a Dock4 truncated mutant lacking the entire Rac1-activating domain (945VS) failed to promote protein synthesis (Fig. 5g, h), whereas overexpression of WT-Rac1 or its constitutively active (CA) mutant induced protein synthesis (Fig. 5i, j). Notably, the active (GTP-bound) form of Rac1 was drastically decreased in the KO hippocampus (Fig. 5k, l), which suggests that Rac1 activation is diminished when Dock4 is deficient. To verify whether Dock4-Rac1 signaling is a crucial mechanism underlying the protein synthesis of AMPAR and NMDAR subunits, we replenished activated Rac1 in Dock4-deficient neurons by lentivirus-mediated delivery of WT-Rac1. Intriguingly, Rac1 overexpression restored global protein synthesis to normal levels in Dock4-deficient neurons (Fig. 5m, n), and more importantly, the overexpression reversed Dock4 knockdown-mediated reduction of GluA2, GluN1, GluN2A, and GluN2B (Fig. 5o, p). Collectively, our results revealed that Dock4 is critical for the normal expression of AMPARs and NMDARs, which probably occurs through Rac1-dependent protein synthesis.

Social behavior is restored in Dock4 KO mice by increasing Rac1 activity or NMDAR function

To test whether Rac1 overexpression rescues the behavioral deficits in Dock4 KO mice, lentiviral particles expressing WT-Rac1 or control vector were bilaterally injected into the hippocampal CA1 region of Dock4 KO mice (Fig. 6a). A large population of CA1 pyramidal neurons were successfully infected 4 weeks after injection, and the surgery did not affect the locomotion of the animals (Fig. 6a–c, Supplementary Fig. 13a). Notably, synaptic transmission properties, including mEPSC and LTP, were largely rescued in Rac1-expressing KO neurons (Fig. 6d–g). We next measured whether social deficits in Dock4 KO mice were rescued by Rac1. Intriguingly, Rac1-injected mice showed normal social novelty preference, whereas vector-injected KO mice failed to distinguish unfamiliar from familiar conspecifics (Fig. 6h–j, Supplementary Fig. 13b, c). Thus, replenishing Rac1 in the hippocampal CA1 region was sufficient for correcting defective social preference caused by Dock4 deficiency.

Fig. 6 Impaired social behavior of Dock4 KO mice is rescued by enhancing Rac1 activity or NMDAR function. a Rac1-lentivirus was bilaterally injected into the hippocampal CA1 region of Dock4 KO mice. b GFP signals observed in CA1 pyramidal neurons at 4 weeks post-injection confirmed successful Rac1 or control vector expression. Scale bar, 1000 μm. c Higher-magnification view of boxed CA1 area in b. Scale bar, 40 μm. d Representative (left) and average (right) traces of mEPSCs recorded from KO pyramidal cells expressing either vector (KO + vector) or Rac1 (KO + Rac1). e, f Cumulative probability of mEPSC amplitude (e) and inter-event interval (f). Insets: average mEPSC amplitude (e) and frequency (f). n = 15 cells from 4 KO + vector mice; n = 18 cells from 4 KO + Rac1 mice. *P < 0.05, two-sample Kolmogorov–Smirnov test. #P < 0.05, unpaired t test. f mEPSC frequency from KO + vector or KO + Rac1 cells. g Time-course changes of EPSC amplitude (% of baseline) after a pairing protocol (0 mV, 2 Hz, 360 pulses; arrow), measured from KO + vector (12 cells, 6 mice) or KO + Rac1 (9 cells, 7 mice) groups. P = 0.021, unpaired t test. Representative traces before and at 25–35 min after pairing are shown on the right. h Representative heatmap of movement of KO + vector or KO + Rac1 mice in the Three-chamber test at 4 weeks after virus injection. i, j Duration spent by KO + vector or KO + Rac1 mice in sniffing different cups during social approach (i) and social novelty (j) phases. E, empty cup; S1, cup containing stranger mouse #1; S2, cup containing stranger mouse #2. n = 8 KO + vector mice (4 males and 4 females), and n = 7 KO + Rac1 mice (4 males and 3 females). **P < 0.01, ***P < 0.001, ns, no significant, unpaired t test. k Outline of experimental design for D-cycloserine (DCS) treatment. l Representative heatmap of movement of KO mice in social novelty phase at 90 min after administration of DCS (20 mg/kg; KO + DCS) or vehicle (KO + Veh). m Duration spent by KO + Veh or KO + DCS mice in sniffing different cups during social novelty phase. n = 10 KO + Veh mice (5 males and 5 females), and n = 10 KO + DCS mice (5 males and 5 females). *P < 0.05, unpaired t test. n values of each group are displayed on the corresponding bars of the bar charts. Error bars: SEM Full size image

As described earlier, in Dock4 KO mice, the NMDAR/AMPAR ratio and NMDAR-dependent LTP were decreased, and concomitantly, NMDAR subunit expression was diminished (Fig. 4c, e, h, i). Therefore, NMDAR hypofunction might represent a major functional cause for the behavioral deficits in the KO mice. To test this hypothesis, we used D-cycloserine (DCS), a partial agonist of NMDAR that holds potential for use in treating autism-like behaviors [43, 44]. Notably, intraperitoneal injection (i.p.) of DCS rapidly restored normal social novelty preference of KO mice (Fig. 6k–m, Supplementary Fig. 13e), but the effect was diminished by 7 days after treatment (Supplementary Fig. 13d, f, g). We also tested the effect of potentiating AMPAR function by PF-4778574, a positive allosteric modulator of AMPAR previously shown to restore social behaviors in ASD mouse models [45]. However, i.p. of PF-4778574 had no effect on correcting social deficit in Dock4 KO mice (Supplementary Fig. 13h–j). These results suggest that Dock4-Rac1 signaling-mediated NMDAR function, at least in the hippocampal CA1 region, is essential for social novelty preference.