Two-photon Ca2+ imaging in layer 2/3 of somatosensory cortex in vivo in living awake mice
We first injected an ultra-sensitive genetically encoded calcium indicator, GCaMP6f , into the layer 2/3 of somatosensory cortex (Fig. 1a, b). A chronic cranial window on cortex was established to observe neuronal activity in the awake mouse in vivo at least one month (Fig. 1d-f). Healthily control wild-type C57BL/6 mice exhibited spontaneous neuronal activity at different cortical depths (Fig. 1g). We induced EAE with myelin oligodendrocyte glycoprotein (MOG residues 35–55) injected into the mice via subcutaneous injection . The values of the disease clinical score were recorded every day after EAE induction. According to the clinical scores, we classified the process of the disease to three stages: ‘preclinic’, ‘relapse’, and ‘remission’ stage (Fig. 1a, c). In the preclinic stage, there is no clinical symptom in EAE mice, while in the relapse and remission stage, the obvious symptom came up and lasted several weeks, and the healthy wild-type mice did not exhibit any clear clinical symptoms.
Arctigenin relieves EAE symptoms
Arctigenin was injected intraperitoneally every day (10 mg/kg) from the beginning after EAE induction to determine its effects on EAE. The Arctigenin-treated mice had a delayed onset of clinical symptoms, approximately five days (Fig. 2a) compared with the vehicle-treated control mice. Besides, the Arctigenin-treated group had evidently reduced clinical scores throughout the disease. The severity of disease was assessed by cumulative and maximum clinical scores (Fig. 2b, c). EAE mice without Arctigenin treatment had significantly higher cumulative clinical score (40.55 ± 6.42) than Arctigenin-treated mice (22.00 ± 4.07) (Fig. 2b). These results suggest that Arctigenin extenuates the inflammatory impairment and relieves EAE mice’s clinical symptoms.
Emergence of more hyperactive neurons in early EAE
To chronically monitor neurons activity in layer 2/3 of somatosensory cortex, we performed two-photon calcium imaging at 8:00 a.m. daily for about 40 days. Representative calcium images of the same region and activity traces of neurons at four time points in different periods of the experiment are showed (Fig. 3a, b). In healthily control mice, around 12% of neural population exhibited hyperactivity (>6 transients / min) throughout entire chronic recordings. Moreover, in EAE mice, the fraction of hyperactive cells also stayed at the same level with control group both in the relapse (13%) and remission stage (14%). However, it is noteworthy that, on the 7th day post EAE induction, we observed a significant increase (up to 40%) in the mean fraction of hyperactive neurons (Fig. 3c). This extraordinary proportion of hyperactive neurons indicates the change of cortical activity pattern in early EAE.
Arctigenin restricts the increase in fraction of hyperactive at preclinical stage and reduced silent neurons at remission stage of EAE in vivo
Based on the calcium transients, cells were further classified to three categories, inactive cell ( no Ca2+ transient in recording time), normal cell ( 1- 6 Ca2+ transients/min), and hyperactive cell (> 6 Ca2+ transients/min) to observe the details of cortical activity in EAE (Fig. 4a, b). The distribution of hyperactive cell in activity map shows more hyperactive neurons in vehicle treated EAE mice on the 7th day after induction, while the increase was not obvious in Arctigenin treated mice (Fig. 4a). In addition, from the chronic recording of three kinds of cells, we found that the fraction of hyperactive cells abruptly increased as early as three days after EAE induction and remained high level for about a week. However, Arctigenin treatment made this increase slow and mild (Fig. 4e). Furthermore, the more inactive cells and less normally active cells appeared during late period in vehicle treated EAE mice while Arctigenin reversed these change (Fig. 4c, d). Apart from that, we summarized the fraction of three category cells in the preclinical stage (day 1 to day 10 after induction) and remission stage (day 19 to day 30 after induction) (Figure 4f, g). Notably, the percentage of hyperactive cells as well as normal cells in vehicle-treated EAE group was significantly increased compared to control mice at preclinical stage and Arctigenin reversed the increase to a considerable degree (Fig 4f). Meanwhile, at the remission stage of EAE, the fraction of inactive cells was obviously increased in vehicle treated mice and the normally active cells were reduced significantly, which means cortical silence during late period of disease. However, Arctigenin compromised these effects of EAE induction (Fig 4g). Taken together, these results demonstrate that Arctigenin limited the growing in numbers of cortical hyperactive neurons at preclinic stage of EAE. Besides, Arctigenin treatment reduced the number of silent cells and facilitated normal activity of cells at remission stage.
Arctigenin limits a surge in calcium transient amplitude of cortical neurons at preclinical EAE and reversed calcium influx at remission stage
To characterize the change of intracellular calcium concentration during EAE and Arctigenin treated process, we analyzed the mean amplitude of calcium transient in regions of view. Consistent with previous results, on the 7th day post EAE induction, the calcium transient amplitude increased significantly in vehicle treated EAE mice compared with Arctigenin treated group. Moreover, it’s quite unexpected that there were enhancements of calcium transient amplitude in control mice and Arctigenin treated group, while an obvious decrease in vehicle treated mice (Fig. 5a). From the day 6 after induction, the amplitude in vehicle treated EAE group suddenly increased and gradually dropped to the original level, then further decreased to a very low level at late EAE stage. But the increase of calcium transient amplitude in Arctigenin treated mice was slow and moderate in early EAE, like the increase of hyperactive cells described above. What’s more, the amplitude of control and Arctigenin treated mice experienced a slight enhancement in late period. This enhancement may be due to chronic and repeated touching mice to operate two-photon imaging (Fig. 5b). In addition, we summarized the amplitude of three groups in preclinical, relapse and remission stage respectively. And we found that the amplitude in vehicle treated mice was significantly increased at preclinical stage, while Arctigenin administration inhibited this preclinical increase (Fig. 5c). However, at the relapse stage, the amplitude began to growing in Arctigenin treated mice and exceeded vehicle treated EAE mice (Fig. 5d). Besides, the amplitude of calcium transient in vehicle treated group significantly decreased at remission stage but Arctigenin reversed this change to some extent (Fig. 5e). Collectively, these results indicate that more calcium influx accompanied with neuronal activity at preclinic EAE stage resulted in functional deficits of cortical cells at remission stage. However, Arctigenin exerted neuronal protection effects through delaying and attenuating the abnormal calcium influx at preclinical stage, indeed decreased silent cells caused by EAE induction.
Restoration of normal functional connectivity and phase synchronization of cortical network by Arctigenin administration at preclinical and remission stage
Functional connectivity is defined as a key indicator of temporal correspondence of calcium transients between two individual neurons. Synchrony is the temporal consistence of distributed neuronal activity, which serves as ability of transferring information among neurons. We used these two parameters to estimate temporal network activity patterns through calculating correlation and synchrony matrices quantifying network functional connectivity (Fig. 6a, left) and synchronization (Fig. 6a, right) among neurons in imaged regions respectively. One week after EAE induction, both of functional connectivity and synchronization of vehicle treated EAE mice were extremely higher in comparison to Arctigenin treated group (Fig. 6a-c). Indeed, at entire preclinical stage, the cortical functional connectivity and synchronization in vehicle treated mice increased starkly and Arctigenin inhibited the abnormal increase (Fig. 6d, f). However, after a long duration of steady enhancement, the functional connectivity continued to decrease and reached to the lowest level on the day 15 post induction in vehicle treated EAE mice (Fig. 6b). It’s worthy to note that from the end of relapse stage, the network connectivity was precipitously elevated and maintained a high level throughout entire remission stage in vehicle treated group (Fig. 6e). Intriguingly, the network synchronization was remained a low level at remission stage in vehicle treated mice, indicating deficits in information transfer (Fig. 6g). But Arctigenin rescued these abnormal effects of EAE induction. In conjunction with the data above, these results suggest the cortical hyperactive microcircuit activity at preclinical stage of EAE, while Arctigenin significantly prevented network hyperactivity and reversed maladaptive functional connectivity and decreased synchrony upon recovery at remission stage.
Arctigenin blunts increased frequency of AMPA receptor-mediated sEPSCs in preclinic EAE
Activation of glutamate AMPA type receptors could trigger calcium influx. Excessively activation of AMPA receptors may result in glutamate excitotoxicity, might contributing to the neuropsychological disorders in MS [28, 29]. We hypothesis that Arctigenin attenuates cortical hyperactivity may act through AMPA receptor mediated synaptic transmission. We recorded AMPA sEPSCs of single neurons in acute cortical brain slices of healthy controls and EAE mice in preclinical stage (day7 to day9 after induction). The frequency of sEPSCs was significantly increased in cells of vehicle-treated EAE mice (0.26 ± 0.03 Hz) in comparison to control mice (0.12 ± 0.01 Hz) and the frequency of sEPSCs was significantly decreased to control level in cells of actigenin-treated EAE mice (0.12 ± 0.01 Hz) (Fig. 7a, c). However, there is no difference in the mean amplitude of sEPSCs between the three groups (Fig. 7b, d). The increased frequency of AMPA receptors mediated sEPSCs, but not amplitude, can be probably explained by a presynaptic enhancement of glutamate transmission, which is compatible with increased neuron activity indicated by Ca2+ transients in preclinic stage in vivo.