Activation of neural connection from the SCN to the striatum mediated by SCN lesions
Anterograde tracers are widely used in neural circuit analysis. To explore whether the SCN projects to the striatum (Str), AAV1-hSyn-eGFP, which expresses GFP with a nuclear localization signal, was employed. AAV1 has been demonstrated to mediate anterograde transsynaptic tagging, allowing for neuronal circuit probing. Connection from the SCN to the Str would therefore lead to the expression of nuclear GFP in Str neurons.
In this study, we injected the anterograde tracer virus AAV1-hSyn-eGFP into the SCN (Fig. 1a). Anterograde tracing results showed that eGFP exhibited green fluorescence in the SCN, confirming effective viral delivery (Fig. 1b). Between 3 to 4 weeks following injection, neuron in the striatum also expressed green fluorescence, indicating neural connection from the SCN to the striatum (Fig. 1c).
Then, we performed bilateral SCN lesions on C57BL/6J mice using stereotactic techniques and direct electrical current (1.0 mA for 60 seconds). Following SCN lesions, we observed an increase in c-Fos expression and fluorescence intensity in the striatum compared to the sham group (Fig. 1d–f). This indicates that lesions to the SCN can activate neural connection from the SCN to the striatum.
SCN lesions and conditional knockout of Bmal1 induced circadian rhythm disorder
To evaluate the effects of suprachiasmatic nucleus dysfunction on circadian rhythm disorders, we performed bilateral SCN lesions and administered the AAV2/5-hSyn-Cre-GFP virus for a conditional Bmal1 knockout (cKO). An iButton device was implanted in each mouse to continuously monitor body temperature (Fig. 2a). Nissl staining demonstrated substantial structural damage to SCN neurons in the lesion group compared to the sham group (Fig. 2b). Immunofluorescence confirmed successful Bmal1 knockout, indicated by significantly diminished Bmal1 fluorescence in the SCN (Fig. 2e).
Temperature monitoring under constant environmental conditions revealed that the sham group displayed a pronounced peak in body temperature during the middle of the subjective night. Conversely, the SCN lesion group exhibited erratic temperature fluctuations with dual peaks during the subjective day and night, suggesting altered temperature rhythms (Fig. 2c, d). Similarly, the conditional knockout (cKO) group showed a disrupted circadian temperature pattern with two less pronounced peaks and significantly reduced temperature oscillation amplitude, unlike the control group which maintained a normal circadian rhythm with a typical nocturnal temperature peak (Fig. 2f, g). These findings collectively suggest that both SCN lesions and the conditional knockout of Bmal1 in SCN neurons contribute to SCN dysfunction, leading to significant disruptions in circadian rhythms.
SCN lesions and conditional knockout of Bmal1 induced anxiety- and depression- like behaviors
To evaluate the impact of SCN lesions on anxiety and depression, behavioral assessments were conducted 14 days post-lesion. In the open field test (OFT), compared with sham mice, SCN lesion mice spent less time and made fewer entries in the center, although their overall activity levels did not differ (Fig. 3a, b). In the elevated plus maze (EPM), compared to sham mice, SCN lesion mice spent less time and made fewer entries in the open arms, while spending more time in the closed arms (Fig. 3c, d). Also, compared with sham mice, SCN lesion mice demonstrated a less sucrose preference rate in the SPT (Fig. 3e), a longer immobility time in TST (Fig. 3f) and FST (Fig. 3g).
Behavioral tests were initiated 3 weeks after the virus injection to evaluate the effect of the Bmal1 knockout on anxiety- and depression-like behaviors. In OFT, compared with control mice, cKO mice spent less time and made fewer entries in the center, although their overall activity levels did not differ (Fig. 3h, i). In the elevated plus maze (EPM), compared to control mice, cKO mice spent less time and made fewer entries in the open arms, while spending more time in the closed arms (Fig. 3j, k). These findings suggested anxiety-like behaviors in cKO mice. Also, compared with control mice, cKO mice demonstrated a less sucrose preference rate in the SPT (Fig. 3l), a longer immobility time in TST (Fig. 3m) and FST (Fig. 3n).
Overall, the above results indicate that dysfunction in the suprachiasmatic nucleus, whether induced by physical lesions or genetic manipulation via Bmal1 knockout, leads to significant anxiety- and depression-like behaviors.
SCN lesions and conditional knockout of Bmal1 altered the oscillation amplitude of circadian genes in the striatum
The expression levels of circadian genes in the striatum were assessed at different times of the day (ZT0, ZT6, ZT12, ZT18, ZT24) using RT-qPCR to evaluate the effect of bilateral SCN lesions and conditional knockout of Bmal1. Compared with sham group, the oscillation amplitude of Bmal1, Clock, Cry1 and Cry2 was significantly decreased in SCN lesion group (Fig. 4a–d), while that of Per1 and Per2 significantly increased (Fig. 4e, f). Furthermore, the total mRNA levels of Bmal1, Clock, Per1, and Per2 genes in the striatum were significantly increased in SCN lesion group (Fig. 4a, b, e, f), while the total mRNA expression levels of Cry1 and Cry2 significantly reduced in SCN lesion group (Fig. 4c, d).
Compared with control group, cKO group demonstrated a decrease oscillation amplitude of Bmal1 and Clock in the striatum (Fig. 4g, h), while this of Per1 and Per2 significantly increased (Fig. 4l, l). Furthermore, compared with control group, cKO group demonstrated an increase total mRNA level of Bmal1, Clock, Per1, and Per2 in the striatum (Fig. 4g, h, k, l). However, the expression level of Cry1, oscillation amplitude of Cry1 and Cry2 did not significantly change in the cKO group (Fig. 4i, j).
Taken together, these findings demonstrate that SCN lesions and Bmal1 knockout significantly disrupt the oscillation amplitude and total mRNA level of circadian genes in the striatum, underlining the profound influence of SCN integrity on circadian regulation within this brain region.
SCN lesions and conditional knockout of Bmal1 upregulated the BDNF-TrkB pathway in the striatum
Brain-derived neurotrophic factor (BDNF) plays an important role in the pathogenesis of anxiety and depression [29]. In this study, we used qRT-PCR and Western blot to assess circadian changes of BDNF in the striatum. In the sham group, BDNF mRNA levels increased at night and decreased during the day. In contrast, in the SCN lesion group, the mRNA levels of BDNF, especially during the day, significantly increased. Furthermore, the oscillation amplitude of BDNF also showed a significant increase in this group (Fig. 5a–c). Given the pivotal role of the BDNF-TrkB pathway in regulating the mental illness [30], we next assessed the expression levels of key proteins in this pathway, including BDNF, TrkB, CREB, ERK, p-CREB, and p-ERK1/2. Our results indicate that SCN lesions lead to increased expression levels of BDNF, TrkB, p-CREB, and p-ERK1/2 (Fig. 5d–h).
Additionally, compared with control group, cKO group demonstrated increase total mRNA level and oscillation amplitude of BDNF in the striatum (Fig. 5i–k). Compared with control group, cKO group demonstrated an increase protein expression level of BDNF, TrkB, p-CREB, p-ERK1/2 in the striatum (Fig. 5l–p), while the total protein level of CREB and ERK1/2 were unchanged.
Taken together, these results collectively indicate that disturbances in the SCN, whether through SCN lesions or Bmal1 knockout, lead to upregulation of the BDNF-TrkB pathway in the striatum. This suggests that the Bmal1 gene plays a crucial role in maintaining normal SCN function and regulating the BDNF-TrkB pathway in the striatum, potentially influencing the pathophysiology of anxiety and depression.
TrkB receptor antagonist ANA-12 reversed anxiety- and depression-like behaviors in SCN Lesion and cKO mice
The TrkB receptor is the primary receptor for BDNF, which is required for neuronal survival, development, and plasticity. ANA-12 is a potent, selective TrkB antagonist, which can direct and selective binding to TrkB and inhibited processes downstream of TrkB [31].
In OFT, compared to SCN lesion mice, SCN lesion + ANA-12 mice exhibited anti-anxiety and antidepressant effects, such as an increased time spent in the center and a higher number of center entries (Fig. 6a, b). In EPM, SCN lesion + ANA-12 mice demonstrated an increased time spent in open arms, a higher number of open arms entries and a decreased time spent in close arms (Fig. 6c, d). Also, compared with SCN lesion mice, SCN lesion + ANA-12 mice demonstrated a higher sucrose preference rate in the SPT (Fig. 6e), a shorter immobility time in TST (Fig. 6f) and FST (Fig. 6g).
Similarly, compared with cKO mice, cKO + ANA-12 mice showed anti-anxiety and depression effects, such as an increased time spent in the center and a higher number of center entries in OFT (Fig. 6h, i). In EPM, cKO + ANA-12 mice demonstrated an increased time spent in open arms, a higher number of open arms entries and a decreased time spent in close arms (Fig. 6j, k). Also, compared with cKO mice, cKO + ANA-12 mice demonstrated a higher sucrose preference rate in the SPT (Fig. 6l), a shorter immobility time in TST (Fig. 6m) and FST (Fig. 6n).
These results indicate that dysfunction of the suprachiasmatic nucleus may promote anxiety and depression-like behaviors through activation of the BDNF-TrkB signaling pathway. The observed therapeutic effects of ANA-12 underscore its potential for mitigating these behaviors by inhibiting TrkB.