To combine optical Ca²⁺ recordings, as cellular readout of neuronal activity, with BOLD fMRI in a rat absence epilepsy model, in order to obtain seizure maps of the entire rat brain with enhanced sensitivity and specificity.Absence seizures are non-convulsive, frequently occurring, brief periods of impaired consciousness which are accompanied by large-amplitude synchronized spike-and-wave discharges (SWD). Electrophysiology studies in patients and animal models indicate that the thalamocortical network is involved in seizure generation and generalization. Previously, fMRI has been combined with electrical methods (e.g. LFP or EEG) to create seizure maps of epilepsy models^1,2. However, electrical recordings are disturbed during MRI acquisition and are based on the activity of extended populations of neurons. We follow another route and directly combine fiber-based optical Ca²⁺ recordings that are not affected by magnetic or RF fields with fMRI³. Ca²⁺ recordings are correlated with neuronal spiking and allow for the local assessment of suprathreshold activity of defined neuronal populations, when genetically encoded calcium indicators (GECI) are used.Adult female GAERS (Genetic Absence Epilepsy Rats from Strasbourg) rats were virally transduced with the GECI GCaMP6 (AAV1.Syn.GCaMP6f.WPRE.SV40, Penn Vector Core, USA) in the upper lip field of S1 (S1Ulp). Imaging experiments were conducted at least 4 weeks after virus injection. Using a custom-built optical setup, excitation light from a blue laser at 488 nm was delivered through an optical fiber implanted into S1Ulp. Fluorescent light was guided to an avalanche photodetector using the same fiber. Experiments were performed on a 9.4 T small animal MRI (Bruker Biospec). Animals were relaxed and ventilated, and anesthesia was switched from isoflurane to Narcolept (i.p. injection of 15 µg/kg fentanyl and 1.5 mg/kg droperidol every 20 min). The Ca²⁺ signal was monitored. When SWDs had established, typically after 90 to 120 min, synchronized BOLD fMRI and Ca²⁺ recordings were performed using a GE-EPI with TR 1 s, TE 18 ms, resolution 0.32 x 0.35 mm², slice thickness 1.2 mm for 30 min. Additional simultaneous Ca²⁺ (S1Ulp) and LFP (M1 ipsilateral and S1HL) recordings were obtained outside the magnet. After the experiments, animals were transcardially perfused and brains were removed for histological validation of GCaMP expression. A custom-written algorithm was implemented under MATLAB to detect SWDs in Ca²⁺-traces. SWD onset times and durations were used for an event-related analysis of the fMRI data in SPM8 with different hemodynamic response functions (HRFs). A HRF was modelled with data from stimulation experiments in rats and found an earlier onset time and duration compared to the canonical hemodynamic response function implemented in SPM (onset at 1 s, peak at 2 s, and decline to baseline at 5 s). This early onset HRF was shifted by 2 s to test for delayed responses (late onset HRF).Viral transduction of GCamP6 resulted in robust labelling of neurons in S1Ulp in an area of 1 mm³ (Fig. 1 left). Simultaneous LFP and Ca²⁺ recordings confirmed that SWDs were reflected as trains of short spikes in Ca²⁺ signal with a frequency of 5 -7 Hz (Fig. 1 right). SWD durations varied between 2 and 90 s with gaps lasting 2 to 70 s. SWDs were present during 40-80 % of the 30 min examinations. Analysis of fMRI data revealed strong activation in cortex, striatum and thalamus depending on the hemodynamic response function used (Fig. 2). With the early onset model function, mostly activation in cortex was observed. Using the delayed response model function, a negative BOLD response in striatum was detected.Simultaneous Ca²⁺ recordings during BOLD fMRI allow for undisturbed detection of neuronal population responses during epileptic seizures in GAERS. BOLD maps of SWDs showed large scale activations with delayed responses in subcortical areas in agreement with earlier studies combining electrophysiological recordings with fMRI^1,2. Ca²⁺ recordings with genetically encoded calcium indicators reflect the supra-threshold activity of defined neuronal populations, and thus in contrast to electrophysiological recordings allow for a cell-specific correlation with the hemodynamic response and provide a tool to obtain seizure maps with higher specificity.