Journal: Brain
Article Title: Medial septal GABAergic neurons reduce seizure duration upon optogenetic closed-loop stimulation
doi: 10.1093/brain/awab042
Figure Lengend Snippet: MSGNs and their connections across the hippocampus remain despite hippocampal sclerosis. ( A ) Top : Schematic of viral expression and cannula placement. Cre-dependent GFP and mRuby conjugated to synaptophsyin were expressed in MSGNs by AAV injection into medial septum of VGAT::cre mice. A cannula was implanted for kainate or vehicle (saline) unilateral injection over rostral-dorsal hippocampus. Numbers correspond to approximate hippocampal rostral-caudal levels imaged for puncta analysis. Bottom : Experimental timeline. ( B ) Representative GFP-mRuby AAV expression in MSGNs and staining for Neurotrace and GABA in saline and kainate treated mice. Scale bar = 50 µm. Examples of neurons expressing GFP, mRuby or GABA (arrows) and neurons not expressing GFP, mRuby or GABA (arrowheads). We found 96.16 ± 6.46% of mRuby-GFP expressing cells co-expressed GABA. ( C ) Neuronal populations in saline and kainate treated mice. Horizontal lines indicate mean values [mean ± standard error of the mean (SEM)]. Points correspond to values from individual mice. Populations were not significantly decreased in kainate-treated mice when compared to saline-treated controls (two-way ANOVA, P = 0.58, F = 0.31, df = 1, n of saline and kainate treated mice per cell type, respectively: mRuby-GFP labelled cells in VGAT::Cre mice n = 6, 7; GABA n = 3, 5; PV, CB, ChAT n = 4, 4). ( D ) Representative hippocampal sections of rostral-caudal levels stained with fluorescent Neurotrace are shown in a saline-injected mouse, and the contralateral and ipsilateral hippocampi of a kainate-injected mouse. Scale bars = 200 µm. Expression of GFP (green) in MSGN axons across the hippocampus and sclerosis in rostral slices ipsilateral to kainate injection. ( E ) Putative synaptic terminals expressing mRuby in the hilus at second rostral-caudal level contralateral and ipsilateral to kainate injection (dashed white boxes in D ). Scale bar = 10 µm. ( F ) Density of synaptic terminals across rostral-caudal levels in the hilus and the entire hippocampus in saline-treated mice and contralateral and ipsilateral hippocampi of kainate-treated mice. Bars indicate mean (mean ± SEM). Points correspond to values from individual mice. Synaptic density did not decrease in kainate-treated mice (two-way ANOVA, P = 0.57, F = 0.87, df = 11, n = 5 mice per treatment). Puncta counts were reported normalized to the number of virus-labelled cells in medial septum.
Article Snippet: Adeno-associated virus (AAV) expressing either mRuby conjugated to synaptophysin and membrane-bound green fluorescent protein (GFP) under the control of the synapsin promotor (AAV-hSyn-Flex-mGFP-2A-Synaptophysin-mRuby, Addgene plasmid 71760, serotype 1/2, packaged into AAV), channelrhodopsin-2 (ChR2) conjugated to mCherry [AAV-EF1a-DIO-hChR2(H134R)-mCherry-WPRE-pA, serotype 5, Addgene plasmid 20297 purchased from University of North Carolina Vector Core, USA] or mCherry (AAV-EF1a-fDIO-mCherry, serotype 5, Addgene plasmid 114471, purchased from University of North Carolina Vector Core, USA) was injected through a craniotomy (0.6 mm rostral, 0.0 mm caudal to bregma).
Techniques: Expressing, Injection, Saline, Staining, Virus
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![MSGNs and their connections across the hippocampus remain despite hippocampal sclerosis. ( A ) Top : Schematic of viral expression and cannula placement. Cre-dependent <t>GFP</t> and mRuby conjugated to synaptophsyin were expressed in MSGNs by AAV injection into medial septum of VGAT::cre mice. A cannula was implanted for kainate or vehicle (saline) unilateral injection over rostral-dorsal hippocampus. Numbers correspond to approximate hippocampal rostral-caudal levels imaged for puncta analysis. Bottom : Experimental timeline. ( B ) Representative GFP-mRuby AAV expression in MSGNs and staining for Neurotrace and GABA in saline and kainate treated mice. Scale bar = 50 µm. Examples of neurons expressing GFP, mRuby or GABA (arrows) and neurons not expressing GFP, mRuby or GABA (arrowheads). We found 96.16 ± 6.46% of mRuby-GFP expressing cells co-expressed GABA. ( C ) Neuronal populations in saline and kainate treated mice. Horizontal lines indicate mean values [mean ± standard error of the mean (SEM)]. Points correspond to values from individual mice. Populations were not significantly decreased in kainate-treated mice when compared to saline-treated controls (two-way ANOVA, P = 0.58, F = 0.31, df = 1, n of saline and kainate treated mice per cell type, respectively: mRuby-GFP labelled cells in VGAT::Cre mice n = 6, 7; GABA n = 3, 5; PV, CB, ChAT n = 4, 4). ( D ) Representative hippocampal sections of rostral-caudal levels stained with <t>fluorescent</t> Neurotrace are shown in a saline-injected mouse, and the contralateral and ipsilateral hippocampi of a kainate-injected mouse. Scale bars = 200 µm. Expression of GFP (green) in MSGN axons across the hippocampus and sclerosis in rostral slices ipsilateral to kainate injection. ( E ) Putative synaptic terminals expressing mRuby in the hilus at second rostral-caudal level contralateral and ipsilateral to kainate injection (dashed white boxes in D ). Scale bar = 10 µm. ( F ) Density of synaptic terminals across rostral-caudal levels in the hilus and the entire hippocampus in saline-treated mice and contralateral and ipsilateral hippocampi of kainate-treated mice. Bars indicate mean (mean ± SEM). Points correspond to values from individual mice. Synaptic density did not decrease in kainate-treated mice (two-way ANOVA, P = 0.57, F = 0.87, df = 11, n = 5 mice per treatment). Puncta counts were reported normalized to the number of virus-labelled cells in medial septum.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9369/pmc08219369/pmc08219369__awab042f1.jpg)