Journal: Protein Science : A Publication of the Protein Society

Article Title: Rapid directed molecular evolution of fluorescent proteins in mammalian cells

doi: 10.1002/pro.4261

Figure Lengend Snippet: Intracellular brightness and photostability of mCardinal, TagRFP658, and emiRFP2 in live cultured hippocampal mouse neurons and in vivo in zebrafish larvae. (a,b) Representative fluorescence images of primary cultured mouse hippocampal neurons expressing TagRFP658 at (a) 14 and (b) 23 days in vitro (DIV; n = 53 and 33 neurons, respectively, from two independent cultures). Imaging condition: excitation 631/28 nm from an LED, emission 664LP. (c) Representative light sheet image of head of zebrafish larvae at 4 days postfertilization expressing TagRFP658 in neurons ( n = 10 fish from two independent injections). Imaging conditions: excitation 638 nm from a laser, emission 665LP. (i, ii) High‐magnification images of the respective regions shown in white boxes in e. Scale bars, 50 μm. (d) Relative fluorescence of cultured mouse hippocampal neurons expressing mCardinal and TagRFP658 ( n = 78 and 85 neurons, respectively, from two independent cultures for each protein; one‐way analysis of variance [ANOVA]). Imaging conditions as in (a). Box plots with notches are used in this figure (see Figure for the full description). (e) Raw photobleaching curves for mCardinal (dashed line) and TagRFP658 (solid line) in primary cultured mouse hippocampal neurons ( n = 9 and 7 neurons, respectively, from one culture each; one‐way ANOVA). Imaging condition: excitation 631/28 nm from an LED at 70 mW/mm 2 , emission 664LP. (f) Representative fluorescence images of cells transfected with pAAV‐CAG‐mCardinal‐P2A‐GFP (top), pAAV‐TagRFP658‐P2A‐GFP (middle), and pAAV‐emiRFP2‐P2A‐GFP ( n = 39, 33, and 41 neurons from three, two, and three independent transfections from one culture each for mCardinal, TagRFP658, and emiRFP2, respectively, for Cy5 channel and n = 15 and neurons from one independent transfection from one culture each for mCardinal and emiRFP2, respectively, for Cy5.5 channel). Imaging conditions: Cy5 channel: excitation 635/22 nm from 637 nm laser, emission 730/140 nm; Cy5.5 channel: excitation 680/13 nm from 680 nm laser, emission 710 LP; GFP channel: excitation 478/24 nm for an LED; emission 535/46 nm. Images in Cy5 and Cy5.5 were taken under matching excitation intensity (66 mW/mm 2 ) and the same exposure time (100 ms). The dynamic range of fluorescence intensity in Cy5 and Cy5.5 channels are identical across all images. Scale bar, 20 μm. (g) Near‐infrared (NIR)‐to‐green fluorescence ratio for mCardinal, TagRFP658, and emiRFP2 for the experiment shown in (f). (h) Intracellular photostability of mCardinal, TagRFP658, and emiRFP2 in Cy5 and Cy5.5 channels ( n = 8, 7, and 9 neurons from three, two, and three independent transfections from one culture each for mCardinal, TagRFP658, and emiRFP2, respectively, under Cy5 excitation and n = 5 neurons from one transfection from one culture for emiRFP2 under Cy5.5 excitation). Imaging conditions the same as in (a)

Article Snippet: Mouse primary hippocampal neurons were prepared from postnatal day 0 or 1 Swiss Webster (Taconic) mice (both male and female mice were used) and cultured as previously described.

Techniques: Cell Culture, In Vivo, Fluorescence, Expressing, In Vitro, Imaging, Transfection

Journal: bioRxiv

Article Title: Co-activation of selective nicotinic acetylcholine receptor subtypes is required to reverse hippocampal network dysfunction and prevent fear memory loss in Alzheimer’s disease

doi: 10.1101/2024.07.08.602576

Figure Lengend Snippet: a) Average traces of GCaMP6f signals in excitatory (EX) and inhibitory (IN) cells and summary data of normalized peak amplitude in each condition (n = number of cells excitatory cells = 57 and inhibitory cells =33 from 4 pups in 2 independent cultures). * p < 0.05, two-tailed student’s t-test. Average traces of GCaMP6f signals and summary graphs of normalized peak amplitude in each condition in b) excitatory (n = number of cells, CTRL = 27, αBTx = 10, DhβE = 15, and αCTx = 15 from 4 pups in 2 independent cultures) and c) inhibitory cells (n = number of cells, CTRL = 14, αBTx = 9, DhβE = 16, and αCTx = 16 from 4 pups in 2 independent cultures). Average traces of GCaMP7s signals and summary data of normalized peak amplitude in each condition in d) PV+ (n = number of cells, CTRL = 30, αBTx = 38, DhβE = 32, and αBTx+DhβE =43 from 6 pups in 3 independent cultures) and e) SST+ cells (n = number of cells, CTRL = 27, αBTx = 21, DhβE = 24, and αBTx+DhβE = 30 from 6 pups in 3 independent cultures). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. one-way ANOVA, Tukey test. An arrow indicates photostimulation. n.s.: not significant.

Article Snippet: As the majority of cells in hippocampal cultures are excitatory neurons , we expressed GCaMP6f (Addgene #40755) under the control of the CMV promoter by transfection for imaging excitatory cells, and GCaMP6f under the control of the GABAergic neuron-specific enhancer of the mouse Dlx (mDlx) gene (Addgene #83899) was transfected for imaging inhibitory interneurons as shown previously .

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Co-activation of selective nicotinic acetylcholine receptor subtypes is required to reverse hippocampal network dysfunction and prevent fear memory loss in Alzheimer’s disease

doi: 10.1101/2024.07.08.602576

Figure Lengend Snippet: a) A simplified local inhibitory circuit in the hippocampus. PV+ interneurons mainly provide somatic inhibition to pyramidal excitatory neurons (Pyr), while SST+ interneurons provide dendritic inhibition. This contributes to hippocampal oscillations and cognitive function. α7-, α4β2-, and α3β4-nAChRs predominantly control nicotinic cholinergic signaling in PV-, SST-, and pyramidal cells, respectively. b) Aβ selectively inhibits α7- and α4β2-nAChRs in inhibitory cells, but not α3β4-nAChRs, in turn reduces inhibitory inputs to excitatory cells, which contributes to disruptions in hippocampal oscillations and consequent learning and memory in AD. c-d) Single stimulation of each nAChR subtype by PNU-282987 (PNU), an α7 agonist, or RJR-2403 Oxalate (RJR), an α4β2 agonist, is unable to reverse the Aβ-induced adverse effects on network activity and memory. e) Co-stimulation of α7- and α4β2-nAChRs sufficiently restores normal hippocampal oscillations and memory.

Article Snippet: As the majority of cells in hippocampal cultures are excitatory neurons , we expressed GCaMP6f (Addgene #40755) under the control of the CMV promoter by transfection for imaging excitatory cells, and GCaMP6f under the control of the GABAergic neuron-specific enhancer of the mouse Dlx (mDlx) gene (Addgene #83899) was transfected for imaging inhibitory interneurons as shown previously .

Techniques: Inhibition, Control, Activity Assay

Journal: Cell

Article Title: Multimodal charting of molecular and functional cell states via in situ electro-sequencing

doi: 10.1016/j.cell.2023.03.023

Figure Lengend Snippet: (A) Schematics illustrating in situ electro-seq of neural patches. (B) Representative voltage traces showing spike-bursting dynamics of mouse hippocampal neurons (i) with the bursting activity (ii) and single spike train (iii) highlighted. (C) Detected spike trains from continuous recording (left panel) and single spikes (right panel) from the dashed box highlighted region. (D) Overlapped 3D cell-type and electrode maps. Grey color labels each individual electrode. (E) Identified electrically recorded neurons. Colors label spikes identified from each neuron highlighted by white arrows. Zoomed-in image shows one neuron that was simultaneously recorded by four electrodes. (F) UMAP visualizations of all the sequenced cells. (G) Heatmap showing the electrophysiological features and marker gene expression profiles. (H) Box and dot plots showing the peak-trough ratio between excitatory and inhibitory neurons. n = 20 for excitatory neurons, n = 15 for inhibitory neurons, ** p < 0.01, two-tailed, unpaired t test.

Article Snippet: Mouse primary hippocampal neurons , Broad Institute of MIT and Harvard , C57BL/6 mice.

Techniques: In Situ, Activity Assay, Marker, Gene Expression, Two Tailed Test

Journal: Cell

Article Title: Multimodal charting of molecular and functional cell states via in situ electro-sequencing

doi: 10.1016/j.cell.2023.03.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse primary hippocampal neurons , Broad Institute of MIT and Harvard , C57BL/6 mice.

Techniques: Recombinant, Microscopy, In Situ, Sequencing, Software