The central thalamus has been demonstrated as a critical component in regulating arousal, sustained attention, working memory, and awareness¹. The central thalamic neurons maintain firing patterns involved in long-range cortico-cortical pathways and within cortico-straitopallidal-thalamocortical loop connections². Deep brain stimulation of central thalamus (CT-DBS) can enhance exploratory motor behaviors and cognitive performance in rats³. Moreover, the CT-DBS has been proposed as an experimental approach to produce consistent sustained regulation of forebrain arousal for several neurological diseases⁴. However, changes of functional connectivity evoked by the CT-DBS in related brain areas remain elusive. The combination of DBS and fMRI (BOLD response and resting-state) enables the study of regional responses to CT-DBS, with the potential to unambiguously study a specific neuroanatomical pathway/connectivity and monitor the treatment outcome. In this study, we aimed to characterize functional connectivity and BOLD response to CT-DBS. Our hypothesis was that the functional connectivity and BOLD responses could be enhanced by CT-DBS treatment in the reward and memory-related brain areas.MRI-compatible 16-channel neural probes (Fig. 1A) were stereotactically implanted into central lateral thalamus (AP 3.5, ML ±1.4 mm, and DV 5.0 mm) in male Sprague–Dawley rats (weighing 250-350 g, n=10). Before the behavioral task, five rats in CL-DBS group were stimulated with a bipolar square-wave current of 0.4 mA with 25 μs pulse-width at 100 Hz for 30 min and five rats in sham control group weren't stimulated. Rats were trained to obtain a water reward by pressing a lever during daily 5-h sessions, for 4 days at the most. For fMRI experiments, rats were anesthetized with 0.1 mg/kg Dexdomitor® subcutaneously. MRI was performed on a Bruker Biospec 7T system with a 30-cm diameter bore and a single-shot GE-EPI sequence (TR/TE=2000/20 ms, BW=200 kHz, 80×80 matrix, FOV=25×25 mm², thickness=1 mm) was used to acquire fMRI images. The resting-state data was acquired, totaling 260 scanning images for 10 dummy scanning and 250 images. Functional connectivity were calculated using average time course of all voxels within a 2 x 2 pixel region of interest (ROI) in hippocampus (HIP), primary motor cortex (M1), anterior cingulate cortex (ACC), caudate-putamen (CPu), parafascicular thalamic nucleus (Pf). Pearson’s correlation coefficient was then computed between all of the ROIs. BOLD functional images evoked by CT-DBS contained a total of 120 scanning for 10 dummy scanning and 110 images. The stimulation paradigm included 5 stimuli blocks and 6 rest blocks and additional 3 min minimum resting interval between trials. Correlation coefficient (CC) maps were performed by correlating BOLD pixel time courses to the stimulus paradigm with a significance level at p<0.05 (Bonferroni corrected).A T₂-weighted image showed that the position of probe with a minimum image distortion (Fig. 1B). The complete time of behavioral task in the CT-DBS group (16.75±1.67 h) was significantly shorter than those in the sham control group (8.56±2.63 h) (Fig. 2), suggesting that CT-DBS may increase the operant conditioning-learning effectiveness2. Before the CT-DBS, right CT-DBS produced positive BOLD responses in ipsilateral somatosensory cortex (SC) and negative BOLD responses in contralateral CPu and ACC (Fig. 3). Following behavioral task completed, BOLD responses evoked by right CT-DBS became negative in ipsilateral CPu and positive in contralateral SC (Fig. 3). BOLD fMRI showed that CT-DBS influenced bilateral SC, CPu and ACC and the BOLD responses changed between before and after CT-DBS, suggesting that continued daily 30-min CT-DBS may modulate neuronal striatal–thalamic connectivity. The rsfMRI showed that functional connectivity in the sham control group has no significant change in all ROIs between before CT-DBS and task complete (Fig. 4). In CT-DBS group, functional connectivity significantly increased in the CPu-Pf (+86%), CPu-Hip (+96%), and CPu-M1 (+53%) after task complete (Fig 4). The rsfMRI results indicate that CPu increases connectivity in Pf, Hip and M1 to increase cognitive learning. The activations may be produced by several neuropathological mechanisms, including cortico-cortical pathways², within cortico-straitopallidal-thalamocortical loop², brainstem to cortical and basal ganglia networks³, and CT to striatum and cortical layers⁵. However, as both BOLD fMRI and rsfMRI data showed CL-DBS indeed modulated functional connectivity in wide brain areas, these hypotheses merit further investigation. This study demonstrates significant changes in BOLD responses at bilateral SC, CPu, and ACC and rsfMRI at CPu regarding to Pf, Hip, and M1 as a result of CT-DBS. These changes showed the CT-DBS strengthened striatal–thalamic connectivity which suggests the enhancement of inter-regional connectivity may contribute to synaptic plasticity in striatum. CT-DBS fMRI reveals response not previously observed, and further investigation of this technique will permit further insight into the potential therapeutic mechanism of CT-DBS.