Journal:

Article Title: Improved Hepatic Gene Transfer by Using an Adeno-Associated Virus Serotype 5 Vector

doi: 10.1128/JVI.76.20.10497-10502.2002

Figure Lengend Snippet: Estimation of vector gene copy number in AAV-transduced livers by quantitative PCR (3 months after vector administration). A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems. Template for PCR was as follows: 100 ng of genomic DNA extracted from several random pieces of liver and subsequently combined for each individual animal. Each sample column represents an individual animal. standards, linearized plasmid pAAV-EF1α-hF.IX (0.01, 0.1, or 1 pg) mixed with 100 ng of genomic mouse DNA (extracted from untransduced animal); NC, negative control (template, genomic DNA from untransduced animal). Bands were analyzed by densitometric scanning, and intensities were quantitated with NIH Image 6.16 software. Shown is one representative blot. Ratios of band intensities (hF.IX band/mAAT band) are for the blot shown. Gene copy number estimates are average for two experiments.

Article Snippet: A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems.

Techniques: Plasmid Preparation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control, Software

Journal: bioRxiv

Article Title: Impaired developmental microglial pruning of excitatory synapses on CRH-expressing hypothalamic neurons exacerbates stress responses throughout life

doi: 10.1101/2021.07.21.453252

Figure Lengend Snippet: a. A representative confocal image of excitatory synaptic puncta and CRH-tdTomato+ neurons in the mediodorsal parvocellular (mpd) paraventricular hypothalamic nucleus (PVN). Inset shows puncta colocalizing vGlut2-647 and PSD95-488 on a CRH-tdTomato+ neuron. These puncta satisfied criteria for synapses (Imaris software, Bitplane, Zurich, Switzerland). Scale bar=10 µm. b. ELA increases the number of excitatory synapses onto CRH+ neurons in the PVN at postnatal day (P)10 (t 12.44 =2.95, p=0.01; Welch’s t-test). c. The ELA-induced increase in excitatory synapse number endures on neurons from mice aged 24-25 days (t 8.96 =3.69, p=0.005; Welch’s t-test). d. ELA leads to functional changes in excitatory synapses of presumed CRH-expressing neurons: Representative traces are epochs (10 s) of whole-cell voltage-clamp recordings of spontaneous inhibitory synaptic currents (sIPSCs; top) and spontaneous excitatory synaptic currents (sEPSCs; bottom) recorded from mpd PVN neurons derived from CTL (left) and ELA (right) mice. A subsection (blue shaded area; 1 s) of the recorded epoch is displayed on an expanded time scale below. Scale bars: sIPSC: y = 50pA, sEPSC: y = 10pA, x = 2 seconds and 200ms for top and bottom traces, respectively. e. ELA increases the frequency of sEPSCs in mpd PVN neurons (e; t 14 =2.28, p=0.04; unpaired t-test). f. ELA does not alter the frequency of sIPSCs; ( p >0.8). Data are mean ± SEM; * p <0.05.

Article Snippet: Sections were then incubated overnight at 4°C with rabbit anti-PSD95 antiserum (1:1,000, Invitrogen/ThermoFisher) and guinea pig anti-vGlut2 antiserum (1:12,000, Millipore) in PBS-T containing 2% normal donkey serum.

Techniques: Software, Functional Assay, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Impaired developmental microglial pruning of excitatory synapses on CRH-expressing hypothalamic neurons exacerbates stress responses throughout life

doi: 10.1101/2021.07.21.453252

Figure Lengend Snippet: a. Breeding strategy for the chemogenetic studies: CX3CR1-Cre+::Gq-DREADD+ mice were crossed with Gq-DREADD+ mice to generate ∼50% pups expressing Gq-DREADDs exclusively in microglia. b. Schematic of in vivo chemogenetic activation experiment: Litters of CX3CR1-Cre+::Gq-DREADD+ pups, born (postnatal day [P]0) and randomly assigned to CTL or ELA rearing conditions (P3) and received small, sustained-release CNO- or placebo-containing pellets under the skin (s.c.). One cohort of these mice was perfused on P10 for quantification of excitatory synapses (colocalized vGlut2+PSD95) onto mediodorsal parvocellular (mpd) paraventricular hypothalamic nucleus (PVN) cells. A separate cohort of mice provided adrenal gland weights, a measure of lifetime stress responses as adults. A third cohort provided baseline and stress-induced ACTH and corticosterone (CORT) levels. As adults, these mice experienced an acute multimodal stress (MAS60) and blood was collected at 30 and 60 min. following stress onset. c. Chronic chemogenetic microglial activation during postnatal days 3-10 in microglia-specific Gq-DREADDs in ELA mice decreased the number of excitatory synapses on mpd PVN neurons to control levels; this was not observed in ELA mice lacking microglial expression of Gq-DREADDs or in placebo-receiving mice (F 2,23.8 =3.76, p=0.04; Welch’s one-way ANOVA; p <0.05, Dunnett’s T3 multiple comparisons test). Because CNO treatment alone did not alter the number of excitatory synapses in microglial Gq-DREADD-expressing CTL mice ( p >0.6), CTL groups were combined for analysis. d. Adrenal weights of ELA-experiencing adult mice were higher than those of control mice, indicative of lifelong exposure to augmented stress-hormone release. Remarkably, chemogenetic microglial activation during the ELA epoch prevented the adrenal weight increase (F 3,24 =11.11, p<0.0001; one-way ANOVA; p <0.05, Holm-Sidak’s multiple comparisons test). e. At 30 min. from onset, MAS elicited a robust elevation of the stress hormone ACTH in both CTL and ELA mice. Chemogenetic activation of microglia in CTL mice blunted this response (F 3,20.6 =3.19, p=0.04; Welch’s one-way ANOVA; p<0.05, Dunnett’s T3 multiple comparisons test). At 60 min. after stress onset, ACTH levels dropped to 50% of peak values in CTL mice, but not in ELA mice (F 2,53 =3.45, p=0.04; one-way ANOVA; p <0.05, Holm-Sidak’s multiple comparisons test). Chemogenetic microglial activation in ELA mice ameliorated this prolonged elevation of ACTH levels, which returned to control levels by 60 min. ( p >0.8, Holm-Sidak’s multiple comparisons test). f. The ACTH decay index (30 min./60 min. ACTH levels) differed in ELA mice (F 3,21.5 =3.19, p =0.04; Welch’s one-way ANOVA; p =0.06, Dunnett’s T3 multiple comparisons test), and was partially restored in ELA mice whose microglia were chemogenetically activated ( p >0.2 vs. CTL, Dunnett’s T3 multiple comparisons test). These data indicate an aberrant prolongation of the neuroendocrine stress response in ELA mice, and the restoration of normal ‘shut-off’ mechanisms by early-life microglial activation. (CTL Gq+CNO mice were not included in the decay index analysis because their stress response was already blunted.) Data are mean ± SEM; * p <0.05.

Article Snippet: Sections were then incubated overnight at 4°C with rabbit anti-PSD95 antiserum (1:1,000, Invitrogen/ThermoFisher) and guinea pig anti-vGlut2 antiserum (1:12,000, Millipore) in PBS-T containing 2% normal donkey serum.

Techniques: Expressing, In Vivo, Activation Assay

Journal: bioRxiv

Article Title: Longitudinal multi-omics reveals pathogenic TSC2 variants disrupt developmental trajectories of human cortical organoids derived from Tuberous Sclerosis Complex

doi: 10.1101/2024.10.07.617121

Figure Lengend Snippet: TSC2 variants accelerate synapse formation. Shown are sample images (a) and quantification of SYN1 + (b) and PSD95 + (c) puncta density in both control and TSC organoids at Day 98, scale bars, 50 μm. Data are presented as mean ± s.e.m. (n = 13-14 for each line from three independent experiments, ** P < 0.01, **** P < 0.0001, one-way ANOVA). (d-j) TSC organoids exhibit enhanced neuronal network activity. (d) Raster plot of network spiking activity of control and TSC organoids, and parameters of weighted mean firing rate (e) , number of spikes per burst - Std (f) , burst frequency - Avg (g) , burst duration – Avg (h) , inter-burst interval - Avg (i) , and median ISI within burst – Avg (j) . Data are presented as mean ± s.e.m. (n = 8 for three control, n = 7 for three TSC, ** P < 0.01, *** P < 0.001, unpaired t-test). ISI, inter-spike interval.

Article Snippet: The following primary antibodies were used: anti-CTIP2 (rat, 1:500; Abcam, ab18465), anti-MAP2 (chicken, 1:500; Novus, nb300-213), anti-SYN1 (mouse, 1:500; Synaptic Systems, 106011), anti-PSD95 (rabbit, 1:500; Thermo Fisher, 51-6900), anti-NNAT (rabbit, 1:500; Abcam, ab27266), anti-S100B (rabbit, 1:500; Thermo Fisher, 710363) and anti-NEFL (rabbit, 1:500; Abcam, ab223343).

Techniques: Control, Activity Assay