| Part1 Temporal subtraction method for removing hemodynamic signal during widefield fluorescent imaging of epileptiform activityObjective: Wide-field fluorescent imaging is increasingly being utilized to examine distributed population activity in neuronal networks.This research aims to establish a method to eliminate the functional hemodynamic contamination from the fluorescence data in in vivo experiments.Approach: We describe a temporal subtraction method to separate hemodynamic changes using simultaneously recorded fluorescent and hemodynamic signals,based on the temporal delay in the decay of the hemodynamic signal.Results: Using the temporal window after the fluorescent signal has terminated but the hemodynamic change still exists,we calculate and subtract the difference between the signals.Using the hemodynamic subtraction technique,artifacts can be successfully removed from the raw fluorescent images.Conclusions: This temporal subtraction technique is demonstrated during both interictal and ictal epileptiform events and shown to be superior to the more commonly used division method.Part2 Mesoscopic mapping of the interictal hemodynamic response in awake and anesthetized miceObjective: Neurovascular coupling-based technologies(e.g.,fMRI and SPECT)are often used to estimate the spatial distribution of epileptic activity in clinical practice.However,the mechanisms of neurovascular coupling during pathological epilepsy are largely unknown.Previous data were mainly obtained on anesthetized animals little data exist in awake models.We investigated the epileptic neurovascular coupling using optical imaging to generate spatial maps of neuronal activity,neurotransmitter activity,and intrinsic optical spectroscopy to measure oxy-hemoglobin(HbO),deoxy-hemoglobin(Hbr),and total hemoglobin(HbT),in vivo during interictal spikes(IIS)in anesthetized and awake mouse neocortex to examine their correlations.Approach: Transgenic mice expressing GCaMP6f(n=13)and iGluSnFR(n=10)in the subset of excitatory neurons were employed in the study.A 5x7 mm craniotomy window was created over both hemispheres and the window was sealed with clear silicone-based polydimethylsiloxane(PDMS)film for long-term imaging.Three weeks after the surgery,the mice will be fixed on a clamp for imaging in awake and anesthetized animals.5m M bicuculline(0.5ul)was injected into the neocortex to induce interictal spikes(IIS).We used simultaneous calcium(illumination at 470 nm)and intrinsic signal at 530 nm and 610 nm imaging to record the neuronal,neurotransmitter,and hemodynamic changes.To explore the role of the thalamus during the IISs process,our study chose to use TTX to inhibit the contralateral ventral posteromedial nucleus(VPM)and ventral posterolateral nucleus(VPL).Results:(1)In both awake and anesthetized mice,IISs were recorded in the LFP a few minutes after BMI injection.IIS induced a monophasic calcium increase in the injection side,with higher amplitude and faster dynamics in awake mice than those of anesthetized mice.(2)In the BMI injection hemisphere,the HbT signal showed a monophasic increase triggered by IIS with a lower amplitude and slower dynamic in anesthetized mice than awake mice.The HbO and Hbr signals showed biphasic waveform and anesthesia dramatically dampened the amplitude and delayed the time course.(3)In the contralateral cortex,anesthesia has minimal impact on the calcium signal but a profound impact on the neurovascular coupling mechanisms,causing a significant decrease in amplitude and delay in the hemodynamic responses.(4)In the awake animals,the amplitude of the HbT and HbO signals were linearly correlated to the pre-spike interval.(5)Anesthesia greatly reduces the spatial extent of hemodynamic changes in the contralateral side,determined with a modified Chen-Bee method.In both awake and anesthetized mice,HbT best present the spatial extend of neuronal activity.(6)Silencing the contralateral thalamus with TTX injection in awake mice also greatly reduced the hemodynamic response in the contralateral cortex,which is similar to the anesthesia.Anesthetic effects on the thalamus may play a role in blunting the contralateral hemodynamic response and impact neurovascular coupling.(7)The spatial propagation of glutamate signal in both hemispheres was larger than that of calcium signal,which was more significant under anesthesia.Conclusions: IIS induces more robust hemodynamic changes in awake mice than anesthetized mice.The maximal hemodynamic changes overestimate the neuronal activity during IIS in awake mice,which is different from anesthetized mice.Our data suggest that anesthesia profoundly infects neurovascular coupling and that results obtained under anesthesia cannot necessarily be extrapolated to the awake state. |
