Female 16p11.2 +/− mice exhibited social novelty deficit
We performed several behavioral tests to determine whether 16p11.2+/− female mice (16p) have any motor and cognitive dysfunctions compared with the wildtype (WT) female littermates. In the open field test, the distance traveled and the time spent in the center were not altered, demonstrating similar locomotion (Unpaired t test, t(12) = 1.883, P = 0.084) and anxiety levels (Unpaired t test, t(12) = 0.193, P = 0.851) in the female 16p11.2+/− and WT mice (Supplementary Fig. 1a-c). In the novel object recognition (NOR) test, the recognition index was also similar, suggesting that the 16p11.2+/− female mice had a comparable level of cognitive memory to the WT female mice (Unpaired t test, t(12) = 0.626, P = 0.543, Supplementary Fig. 1d-f). However, in the three-chamber test, the 16p11.2+/− female mice showed normal social ability (Unpaired t test, t(12) = 1.533, P = 0.151) but impaired social novelty ability (Unpaired t test, t(12) = 5.712, P < 0.001, Supplementary Fig. 1g-i). In summary, the 16p11.2+/− female mice had normal locomotion and cognitive memory, but impaired social novelty ability.
Amelioration of social novelty deficit of 16p11.2 female mice by 40 Hz light flicker
The 40 Hz light flicker visual stimulation entrains with oscillations of the visual and higher-order brain cortices, such as the hippocampus, to alleviate cognitive dysfunctions in Alzheimer's and stroke. However, it remains unknown as to the effect of 40 Hz light flicker on neurological deficits in ASD. We subjected groups of female 16p11.2+/− and WT mice to 40 Hz light flicker treatment at 1 hour daily for 14 days. On the 8th and 15th days, we performed the open field and three-chamber tests. In the open field test, the distance traveled and time spent in the open-field center were not altered by the 40 Hz light flicker treatment, suggesting that the light flicker did not affect the locomotion (One-way ANOVA, F(3, 28) = 1.645, P = 0.201) and anxiety levels (One-way ANOVA, F(3, 28) = 1.698, P = 0.19) of both female 16p11.2+/− and WT mice (Fig. 1a-c). Moreover, the three-chamber test result also showed that the 40 Hz light flicker was ineffective in altering the social ability of both groups of mice (One-way ANOVA, F(3, 37) = 1.58, P = 0.211, Fig. 1e). In contrast, the 40 Hz light flicker significantly alleviated the social novelty deficit (One-way ANOVA, F(3, 37) = 6.611, P = 0.0011, Tukey's post hoc test, PWT vs. 16p = 0.001, P16p vs. 16p+LED7 = 0.048, P16p vs. 16p+LED14 = 0.02, Fig. 1d, f). Taken together, these data showed that the long-term 40 Hz light flicker treatment is an effective method to ameliorate the social novelty disability of the 16p11.2+/− female mice.
Suppression of elevated local field potential (LFP) by 40 Hz light flicker in 16p11.2 +/− female mice
To further understand the potential mechanisms of 40 Hz light flicker on ameliorating the social novelty disability of the 16p11.2+/- female mice, we determined changes in the LFP power in the PFC region. Following the recognized terminology, the LFP power wave frequency was divided into several bands, including the delta (1–4 Hz), theta (4–8 Hz), alpha (8–13 Hz), beta (13–30 Hz), low gamma (30–50 Hz), and the high gamma (50–80 Hz). The LFP power of the untreated 16p11.2+/− female mice was increased in all frequency bands compared with WT female mice. Interestingly, after 14 days of 40 Hz light flicker treatment, the PFC's LFP power of the 16p11.2+/− female mice decreased to a level similar to that of the untreated WT mice (Fig. 2a, b). Detailed analyses of LFP power bands showed that all of them were reversed to the untreated levels of the WT female mice following the 40 Hz light flicker treatment (Kruskal-Wallis test, p < 0.001, Dunn's post hoc test, PWT vs. 16p < 0.001, P16p vs. 16p+LED14 < 0.001, Fig. 2c-h). These data indicated that 40 Hz light flicker modulated the PFC neural network excitation state in the 16p11.2+/− female mice, which may contribute to the amelioration of the social novelty disability of the 16p11.2+/− female mice.
Reduction of excessive excitatory neurotransmission and excitatory synapses by 40 Hz light flicker treatment in 16p11.2 +/− female mice
We measured the spontaneous excitatory postsynaptic current (sEPSC) and spontaneous inhibitory postsynaptic current (sIPSC), which represented the excitatory and inhibitory neurotransmission, respectively. We found that the sEPSC frequency, but not the amplitude, was significantly increased in 16p11.2+/− female mice. Interestingly, the 40 Hz light flicker treatment for 7 and 14 days significantly reduced the sEPSC frequency (One-way ANOVA, F(3, 52) = 6.013, P = 0.0014, Tukey's post hoc test, PWT vs. 16p = 0.0037, P16p vs. 16p+LED7 = 0.01, P16p vs. 16p+LED14 = 0.004, Fig. 3a-b) without affecting sEPSC amplitude (One-way ANOVA, F(3, 52) = 1.181, P = 0.326, Fig. 3c). In contrast, neither the frequency, nor the amplitude of sIPSC was altered in 16p11.2+/− female mice compared with the WT mice. Treatment for 7 and 14 days with 40 Hz light flicker only showed induction of sIPSC amplitude on the 14th day (One-way ANOVA, F(3, 53) = 4.759, P = 0.0052, Tukey's post hoc test, P16p vs. 16p+LED14 = 0.011, Supplementary Fig. 2). These data showed that the 40 Hz light flicker reduced the excitatory neurotransmission in 16p11.2+/− female mice.
To exclude the possibilities that reduced excitatory neurotransmission was due to an effect on the altered numbers of neurons, or the intrinsic differences in excitability of PFC neurons of the 16p11.2+/− female mice, first, we determined the numbers of NeuN positive neurons and PV positive inhibitory interneurons. Results showed that the 40 Hz light flicker did not alter the numbers of these neurons in 16p11.2+/− female mice (Supplementary Fig. 3). Second, we measured the firing rate of pyramidal neurons in PFC of the 16p11.2+/− female mice, which showed a reduced level compared with the WT mice, confirming a previous report 8. Interestingly, 40 Hz light flicker did not reverse the reduced firing rate of the 16p11.2+/− female mice PFC neurons (Two-way RM ANOVA, F(3, 43) = 9.359, P < 0.001, Tukey's post hoc test, PWT vs. 16p < 0.001, P16p vs. 16p+LED7 = 0.288, P16p vs. 16p+LED14 = 0.213, Supplementary Fig. 4a-c). Third, the 40 Hz light flicker had no impact on the rheobase current and the resting membrane potential of the PFC pyramidal neurons of the 16p11.2+/− female mice (One-way ANOVA, F(3, 43) = 3.579, P = 0.021, Tukey's post hoc test, PWT vs. 16p = 0.03, P16p vs. 16p+LED7 = 0.07, P16p vs. 16p+LED14 = 0.783, Supplementary Fig. 4d, e).
To further understand the altered neurotransmission, we also measured the paired-pulse ratio (PPR), which represented the release probability of neurotransmitters. Interestingly, no difference in PPR between the WT and 16p11.2+/− female mice occurred. Furthermore, the 40 Hz light flicker for 14 days did not affect the PPR (Two-way RM ANOVA, F(2, 35) = 0.972, P = 0.388, Fig. 3d-f).
Could the altered synapses in the 16p11.2+/− female mice be accounted for the changed excitatory transmission by the rhythmic 40 Hz light flicker? To answer this, we stained the excitatory synapse using a VGLUT1 antibody. We found the number of VGLUT1 positive boutons significantly increased in the 16p11.2+/− female mice, while 40 Hz light flicker for 14 days reduced the VGLUT1 positive boutons (One-way ANOVA, F(2, 153) = 6.769, P = 0.0015, Tukey's post hoc test, PWT vs. 16p = 0.0015, P16p vs. 16p+LED14 = 0.023, Fig. 3g, h). Furthermore, using Golgi staining, we also found that the dendritic spines were increased in 16p11.2+/− female mice, which was reduced by 40 Hz light flicker (One-way ANOVA, F(2, 95) = 5.985, P = 0.0036, Tukey's post hoc test, PWT vs. 16p = 0.0043, P16p vs. 16p+LED14 = 0.021, Fig. 3i, j).
Together, these data showed that the 40 Hz light flicker reduced excitatory transmission by reducing the excessive excitatory synapses in the 16p11.2+/− female mice without affecting the neuronal excitability and density.
Rhythmic 40 Hz flicker did not enhance the microglia-dependent synaptic pruning
To determine how light flicker treatment altered the PFC synapses, we hypothesized that 40 Hz light flicker might activate microglia to prune excessive excitatory synapses in the 16p11.2+/− female mice. Indeed, previous studies using AD mouse model showed that the 40 Hz light flicker could activate microglia to reduce amyloid plaques 14. To test this hypothesis, we first stained the PFC with Iba1, a microglia marker. We found that the number of microglia did not change in 16p11.2+/− female mice compared with the WT mice. The 40 Hz light flicker treatment for 14 days also did not affect the microglia number (Supplementary Fig. 5a, b). Interestingly, the branch length, branch number, and brunch junction numbers increased in the 16p11.2+/− female PFC, indicating a more complex microglia morphology than the WT mice. However, 40 Hz light flicker did not affect microglia morphology, indicating that 40 Hz light could not affect synaptic pruning through microglia activation (Supplementary Fig. 5c-f).
Second, the level of CD68 expression was determined, which is a marker for activated microglia. Using co-localization studies, we found that both Iba1 intensity and CD68 volume in microglia were reduced in 16p11.2+/− female mice, while 40 Hz light flicker did not affect Iba1 intensity and CD68 volume in microglia (Supplementary Fig. 5g, h), serving as another piece of evidence. Third, we measured microglia phagocytosis and found that the VGLUT1 volume in microglia was reduced, indicating reduced microglia phagocytosis in 16p11.2+/− female mice. Rhthymic 40 Hz light flicker also did not affect microglia phagocytosis (Supplementary Fig. 5i, j). Fourth, the level of expression of GFAP, MAG, and MBP was also determined. The degree of astrogliosis and myelination did not change in 16p11.2+/− female mice compared with the WT mice and which was neither affected by 40 Hz light flicker (Supplementary Fig. 6). Collectively, these data showed that the 40 Hz light flicker did not activate microglia to engulf and prune excessive excitatory synapses.
Induction of adenosine release by 40 Hz light flicker to suppress A receptor-dependent excitatory transmission
Adenosine is a potent neuromodulator that strongly suppresses excitatory transmission but has only a minor effect on inhibitory transmission 42, 43. We hypothesized that 40 Hz light flicker might induce adenosine production to regulate excitatory transmission. We used an adenosine sensor to show the level of adenosine in the PFC of WT female mice and found the peak of fluorescent adenosine signal was very low without light flicker, while 40 Hz light flicker could increase its intensity (t(4) = 3.121, P = 0.036) and frequency (t(4) = 2.383, P = 0.076, Fig. 4a-d). Using the microdialysis technique as we previously described 44, we also found that 40 Hz light flicker for one hour significantly increased the level of adenosine in the PFC of WT female mice (Fig. 4e).
Importantly, the in-vitro application of adenosine could significantly reduce sEPSC frequency in 16p11.2+/− female mice, which was blocked by the A1 receptor antagonist, DPCPX (One-way ANOVA, F(2, 32) = 5.853, P = 0.0068, Tukey's post hoc test, PACSF vs. Ade = 0.043, PAde vs. Ade+DPCPX = 0.0057, Fig. 4f-h). In brain slices of 16p11.2+/− female mice, we also tested the effect of blocking the A2A receptor or A1 receptor on excitatory transmission. The A2A receptor antagonist ZM241385 did not affect the sEPSC frequency, but the A1 receptor antagonist DPCPX significantly elevated the sEPSC frequency (One-way ANOVA, F(2, 38) = 21.1, P < 0.001, Tukey's post hoc test, PACSF vs. ZM241385 = 0.761, PACSF vs. DPCPX < 0.001, Supplementary Fig. 7). Both ZM241385 and DPCPX did not affect sEPSC amplitude (Supplementary Fig. 7).
Adenosine A receptor-dependent reduction of excessive excitatory transmission and alleviation of social novelty deficits
The following experiments were designed to determine whether the rhythmic 40 Hz light flicker evoked adenosine release could contribute to the alleviation of neurological deficits through its cognate receptors. The adenosine receptor consists of excitatory receptors, including A2A and A2B, and inhibitory receptors, including A1 and A3. Co-localization of VGLUT1 with either A1 or A2A receptors showed that A1 did not merge with VGLUT1, while A2A was strongly co-localized with VGLUT1, suggesting that A2A receptors were mostly located in presynaptic sites, while A1 receptors were located in the soma or postsynaptic sites (Supplementary Fig. 8).
In the open field test, compared to the control group, the A2A receptor antagonist SCH58261 reduced the locomotion and increased the anxiety level, while in contrast, the A1 receptor antagonist DPCPX did not affect locomotion and anxiety (One-way ANOVA, locomotion, F(2, 26) = 4.893, P = 0.016, Tukey's post hoc test, P16p + Veh14+LED14 vs. 16p+SCH14+LED14 = 0.038, P16p + Veh14+LED14 vs. 16p+DPCPX14+LED14 = 0.973; anxiety, F(2, 26) = 4.824, P = 0.017, Tukey's post hoc test, P16p + Veh14+LED14 vs. 16p+SCH14+LED14 = 0.013, P16p + Veh14+LED14 vs. 16p+DPCPX14+LED14 = 0.208, Fig. 5a-c). In the three-chamber test, both SCH58261 and DPCPX had no effects on social ability (One-way ANOVA, F(2, 20) = 0.172, P = 0.844) (Fig. 5e). Interestingly, while SCH58261 had no effect on social novelty preference, DPCPX blocked the 40 Hz light flicker's prosocial effect on social novelty preference (One-way ANOVA, F(2, 20) = 6.776, P = 0.006, Tukey's post hoc test, P16p + Veh14+LED14 vs. 16p+SCH14+LED14 = 0.614, P16p + Veh14+LED14 vs. 16p+DPCPX14+LED14 = 0.039, Fig. 5d, f), demonstrating A1 receptor's role in modulating social novelty deficits.
We also measured the sEPSC and found that SCH58261 did not affect the sEPSC frequency and amplitude compared to the control group. In contrast, DPCPX blocked the 40 Hz light flicker's effect on sEPSC, with significantly increased both the sEPSC frequency and amplitude (One-way ANOVA, frequency, F(2, 67) = 6.236, P = 0.003, Tukey's post hoc test, P16p + Veh14+LED14 vs. 16p+SCH14+LED14 = 0.836, P16p + Veh14+LED14 vs. 16p+DPCPX14+LED14 = 0.0047; amplitude, F(2, 67) = 7.436, P = 0.0012, Tukey's post hoc test, P16p + Veh14+LED14 vs. 16p+SCH14+LED14 = 0.971, P16p + Veh14+LED14 vs. 16p+DPCPX14+LED14 = 0.003, Fig. 5g-i). We also measured the PPR. Both SCH58261 and DPCPX had no effects on PPR (Two-way RM ANOVA, F(2, 26) = 0.054, P = 0.9480, Fig. 5j, k). Furthermore, we stained the excitatory synapse using VGLUT1 and found that only DPCPX blocked the 40 Hz light flicker's effect on excitatory synapses with increasing VGLUT1+ boutons surrounding neurons compared to the control group (One-way ANOVA, F(2, 159) = 25.77, P < 0.001, Tukey's post hoc test, P16p + Veh14+LED14 vs. 16p+SCH14+LED14 = 0.893, P16p + Veh14+LED14 vs. 16p+DPCPX14+LED14 < 0.001, Fig. 5l, m).
To further demonstrate the role of the A1 receptor, an adeno-associated virus (AAV) expressing the shRNA to A1 receptor (see Methods) was injected into the PFC of 16p11.2+/− female mice. After 3 weeks of expression, the positive neurons were identified as expressing the reporter eGFP (Fig. 6a). The level of A1 receptor expression was significantly reduced in the PFC of 16p11.2+/− female mice compared to the control group (Unpaired t test, t(268) = 14.74, P < 0.001, Fig. 6b). We then used these mice to perform several behavioral tests.
In the open field test, compared to the control group, knockdown of A1 receptor had no effects on both locomotion and anxiety (Fig. 6c-e). In the three-chamber test, knockdown of the A1 receptor had no effects on social ability (Unpaired t test, t(11) = 1.161, P = 0.27), but blocked the 40 Hz light flicker's prosocial effect on social novelty preference (Unpaired t test, t(11) = 2.649, P = 0.023, Fig. 6f-h). We also measured the sEPSC, and found that compared to the control group, knockdown of A1 receptor could block the 40 Hz light flicker's effect on sEPSC, with significantly increasing the sEPSC frequency (Unpaired t test, t(47) = 2.458, P = 0.018) without changing sEPSC amplitude (Unpaired t test, t(47) = 0.803, P = 0.426, Fig. 6i-k). We also measured the PPR and found that knockdown of A1 receptor had no effects on PPR (Two-way RM ANOVA, F(1, 28) = 0.026, P = 0.873, Fig. 6l, m). Furthermore, we stained the excitatory synapse using VGLUT1 antibody and blocked the 40 Hz light flicker's effect on excitatory synapses with increasing VGLUT1+ boutons surrounding neurons compared to the control group (Unpaired t test, t(87) = 2.469, P = 0.016, Fig. 6n, o).
Taken together, these data demonstrated that rhythmic 40 Hz light flicker reduced excessive excitatory neurotransmission of 16p11.2+/− female mice through increasing adenosine release in the PFC. Elevated adenosine, through its cognate A1 receptor, suppresses excitatory transmission, alleviating the social novelty deficits.