(c,f) Backbone generation (c) and elimination (f) rates over 2-day intervals in wild-type or KO mice with a glass window reared in NC (wild-type: 12 intervals, five cells, five mice, 1526 spines; KO: 14 intervals, six cells, five mice, 2393 spines), KO mice raised in EE conditions (nine intervals, five cells, five mice, 1983 spines), KO mice that received intraperitoneal MK801 (10 intervals, five cells, four mice, 1233 spines), GM6001 (10 intervals, five cells, three mice, 1141 spines), or minocycline through drinking water (eight intervals, five cells, five mice, 1090 spines), in addition to mice that received the interface device with infusion of APV+ iVDCC (eight intervals, five cells, three mice, 996 spines). at a slower rate than processes governing enlargement and shrinkage4,5,6. Spine turnover has been traditionally observed following activity-dependent IL1 JC-1 plasticity induced by cytosolic increases in Ca2+ concentration2,6,7, but has also been identified in the absence of specific learning tasks3,8. Such intrinsic dynamics are reported to occur in a Ca2+-independent manner7,9,10. However, to the best of the authors knowledge, no study has directly investigated whether the baseline rate of spine turnover reflects non-specific learning under normal rearing conditions, or activity-independent intrinsic dynamics models of autistic spectrum disorder (ASD)11,12,13. Fragile X syndrome, the most prevalent monogenic form of ASD, is caused by the expansion of CGG repeats upstream of the coding region in the gene, leading to reduction of the fragile X mental retardation protein (FMRP). knockout (KO) mice present with many of the neural abnormalities observed in patients with fragile X syndrome, including abnormalities in dendritic spine morphology, synaptic plasticity, and learning and memory14,15,16,17,18. Moreover, spine turnover is similarly increased in KO mice, as observed in other models of ASD13,19,20. However, no studies have examined whether the increased rate of baseline turnover observed in ASD models reflects activity-dependent plasticity or activity-independent intrinsic dynamics, and therefore the mechanism responsible for increased spine turnover in ASD models remains largely elusive. With regard to previous neuroimaging techniques, studying the activity-dependent nature of basal spine turnover in the neocortex was difficult using methods such as cranial glass windows or thinned skulls. Because animals are unable to survive when cortical activity is abolished, neuronal Ca2+ signaling must be locally silenced in small regions, wherein the time-lapse imaging of dendritic spines can be performed. To resolve this issue, inhibitors of Ca2+ signaling were infused locally into the visual cortex via a microfluidic brain interface, and two-photon time-lapse imaging was performed in this region. Ca2+ signaling and learning-induced spine turnover were evaluated in wild-type and KO mice after treatment with Ca2+ signal inhibitors. Reports indicate that matrix metalloproteinase 9 (MMP9) KO rescues various abnormalities observed in KO mice, including structural spine abnormalities21. As MMP9 inhibitors have also been linked to changes in spine structure22,23,24, the effect of MMP9 inhibitor administration was also investigated with regard to increased spine turnover in KO and wild-type mice. Results Chronic infusion of the adult brain using a microfluidic JC-1 device The influence of activity on basal spine turnover was investigated using a brain interface device25 that enabled the infusion of Ca2+ inhibitors into the visual JC-1 cortex in adult mice (2C6 months old) (Fig. 1a,b). With regard to the surgical method, 20% mannitol was administered to allow the detachment and removal of the dura without directly touching the brain. To maintain a clear cranial window after open-dura surgery, the JC-1 dural blood vessels were coagulated to prevent bleeding prior to the removal of the dura mater (Supplementary Fig. 1aCc). Two-photon imaging was performed 1 day post-surgery, and chronic infusion was initiated immediately after the first imaging session, to avoid clogging of the inlet of device, using an osmotic pump implanted on the backs of mice (Fig. 1a). Open in a separate window Figure 1 Chronic and local blockade of Ca2+ signaling using a brain interface device in the mouse visual cortex.(a) A mouse with the interface device connected to an osmotic pump implanted on its back. (b) A magnified image of the device and schematic illustration. No drain was used, as the perfusion rate was slow (1.0?L/h). (c) A dendritic branch stained with GCaMP6s and superfused with artificial cerebrospinal fluid (ACSF), where the regions of interests (ROIs) for spine (red) and dendritic shaft (green) are indicated. (d,e,gCl) Typical Ca2+ transients obtained from mice infused with either ACSF (d,e; traces from ROIs in (c)), APV (g,h), or APV+ iVDCC (k,l) and mice who received MK801 intraperitoneally (i,j). Red traces represent those from spines, and black traces from the dendritic shafts. Dashed areas in (d,g,i,k) are magnified in (e,h,j,l), respectively. Blue bars in (e,h,j) indicate dendritic Ca2+ transients and the red bar in e indicates spine Ca2+ transients. (f) The percentage of the three patterns of Ca2+ transients that contain both spine and dendritic transients (open), only dendritic transients (blue), and no transients (black). The patterns were distinguished based on 4-min images of all spines JC-1 along an imaged dendrite. Data were.