The next day, the brains were dissected out, removed, and cryoprotected with 30% sucrose at 4C. Frozen transverse (horizontal) sections were made of 50 μm thickness on a sliding microtome and collected in 0.1 M PBS. Sections were mounted on glass slides and mounted with Vectashield
mounting medium with DAPI (Burlingame, CA, 5-HT Receptor USA) for visualization of nuclei. Sections were imaged in the NIS-Elements software (Nikon Instruments, Inc., Melville, NY, USA) using a Nikon DS-Fil color digital camera on a Nikon E400 microscope equipped with TRITC, FITC, and DAPI fluorescence cubes. RESULTS HISTOLOGIC VALIDATION OF CHANNEL EXPRESSION AND ELECTRODE PLACEMENT Channelrhodopsin-2 expression in the MS (Figure Figure2A2A, green) and hippocampus (Figure Figure2B2B, red) was robust upon histologic evaluation. From the MS, axonal projections to the hippocampus (Figures 2A, C) were readily apparent, coinciding with the passage of the electrodes (Figure Figure2C2C, red and white arrows) and the hippocampal pyramidal cell
layer (yellow arrow). The NeuroNexus array also passed alongside the expressing pyramidal cell layer of the hippocampus (Figure Figure2B2B). Consequently, we would expect our recordings to appropriately reflect the influence of optogenetic stimulation on these respective neuron populations. FIGURE 2 Robust expression of ChR2 on transverse section histology and verification of electrode placement. (A) AAV5-hSyn-ChR2-EYFP injection into the medial septum (MS) produced robust ChR2-EYFP expression (green). Axons from the MS express along the septohippocampal … VALIDATION OF HIPPOCAMPAL RESPONSE TO PULSATILE STIMULATION PATTERNS IN THE MEDIAL SEPTUM To
validate the effectiveness of the platform, we first explored the LFP response in the dorsal hippocampus to square-wave pulsatile stimulation of the MS (Figure Figure33). The MS has been stimulated electrically previously, producing a stimulus-frequency specific response (McNaughton et al., 2006) that we hypothesized we would recapitulate. At 50 mW/mm2, stimulation of the MS produced readily visible delayed pulsatile responses in the hippocampal Dacomitinib LFP in both the CA1 and CA3 layers during the stimulation epoch (Figures Figures1B1B and and3A3A). These responses did not persist into the post-stimulation epoch, but instead were highly time-locked to the stimulus onset and offset. In order to examine the waveform of the LFP response, a peristimulus average was constructed by determining the mean LFP signal between 5 ms preceding and 40 ms following onset of each stimulus pulse. These were calculated across every stimulation parameter to produce the mean (solid line) and SD (shaded area; Figure Figure3B3B). As expected, the stimulation parameter specifications had a large impact on response waveform amplitude, shape, and timing. Increasing the amplitude of the stimulus pulse tended to generate a quicker time to peak response.
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