Olig2-specific loss-of-function Slc35a2 results in hypomyelination and spontaneous seizures methods
Aim. Evidence-backed execution summary for Olig2-specific loss-of-function Slc35a2 results in hypomyelination and spontaneous seizures methods from Olig2-specific loss-of-function Slc35a2 results in hypomyelination and spontaneous seizures.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Objective
reagent used in the protocol.
- Use
- Malformations of cortical development represent major causes of drug-resistant epilepsy, with mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy recognized as a distinct pathological entity. Pathogenic X-linked SLC35A2, encoding the uridine diphosphate-galacto...
Significance
reagent used in the protocol.
- Use
- Our results demonstrate that loss of SLC35A2 function in oligodendrocytes is sufficient to reproduce pathological and clinical features observed in human mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy. Spontaneous seizures in the absence of direct neuronal genetic abnormalit...
Slc35a2 cKO mice have reduced number of oligodendrocytes, an intact neuronal architecture, and gliosis in the corpus...
reagent used in the protocol.
- Use
- Slc35a2 conditional knockout (cKO) mice do not have neuronal architecture abnormalities and have gliosis. Neuronal nuclei (NeuN) and glial fibrillary actin protein (GFAP) immunohistochemistry was conducted on paraformaldehyde-fixed tissue at the genu of corpus callosum (gCC) level. (A) Representative low magni...
Slc35a2 cKO mice demonstrate hypomyelination throughout cortical layers of cortex and corpus callosum
reagent used in the protocol.
- Use
- To visualize the myelination, we stained for 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase), a membrane-associated enzyme in myelin sheaths. We examined three timepoints (P15, P21, and P60) spanning the myelination development in mice (Figure ). CNPase protein e...
Slc35a2 cKO mice demonstrate hypomyelination throughout cortical layers of cortex and corpus callosum
reagent used in the protocol.
- Use
- Slc35a2 conditional knockout (cKO) mice express hypomyelination patterns throughout several timepoints in myelination. 2′,3′-Cyclic nucleotide-3′-phosphodiesterase (CNPase) immunohistochemistry was performed using 30-µm-thick, paraformaldehyde-fixed brain ti...
Methods
A conditional knockout (cKO) mouse model was generated using an Olig2-specific promoter. Behavioral assessments included neurological scoring, open field, locomotor habituation, and pentylenetetrazole-induced seizure testing in 95 mice (31 female controls, 22 female cKO, 26 male controls, 16 male cKO), a...
- Use
- A conditional knockout (cKO) mouse model was generated using an Olig2-specific promoter. Behavioral assessments included neurological scoring, open field, locomotor habituation, and pentylenetetrazole-induced seizure testing in 95 mice (31 female controls, 22 female cKO, 26 male controls, 16 male cKO), a...
Video- EEG monitoring
Mice were recorded for up to 24 h in custom-made, electrically shielded boxes with simultaneous EEG and video using Nihon Koden recording systems (Nihon Kohden America). More details are included in the.
- Use
- Mice were recorded for up to 24 h in custom-made, electrically shielded boxes with simultaneous EEG and video using Nihon Koden recording systems (Nihon Kohden America). More details are included in the.
EEG analysis
The digitized EEG signals were reformatted to a signal channel (left-right motor cortex epidural electrodes). EEG recordings were analyzed by visual inspection for the presence of interictal epileptiform discharges and EEG seizure patterns, then correlated with synchronized video of mouse behavior during the i...
- Use
- The digitized EEG signals were reformatted to a signal channel (left-right motor cortex epidural electrodes). EEG recordings were analyzed by visual inspection for the presence of interictal epileptiform discharges and EEG seizure patterns, then correlated with synchronized video of mouse behavior during the i...
Statistical analyses
Prism 10 (GraphPad Software) was used to conduct statistical analyses and data visualization for all data. In brief, Welch t -tests and two-way and three-way analyses of variance (ANOVAs) were used for parametric data, and Friedman test was used for nonparametric data. More details regarding the da...
- Use
- Prism 10 (GraphPad Software) was used to conduct statistical analyses and data visualization for all data. In brief, Welch t -tests and two-way and three-way analyses of variance (ANOVAs) were used for parametric data, and Friedman test was used for nonparametric data. More details regarding the da...
Motor deficits in Slc35a2 cKO mice worsen over time
Slc35a2 conditional knockout (cKO) mice suffer from motor deficits indicated by open field and locomotor habituation tests. (A) Postnatal day 30-60 randomly selected mice were subjected to a weekly 28-point NeuroScore test to measure overall motor function. Male cKO ( n = 5-6) mice exh...
- Use
- Slc35a2 conditional knockout (cKO) mice suffer from motor deficits indicated by open field and locomotor habituation tests. (A) Postnatal day 30-60 randomly selected mice were subjected to a weekly 28-point NeuroScore test to measure overall motor function. Male cKO ( n = 5-6) mice exh...
Motor deficits in Slc35a2 cKO mice worsen over time
To further assess motor activity, we conducted open field and locomotor habituation tests (Figure ). In the open field test, we observed a significant difference between male and female cKO in the distance traveled (mean = 1537.7 and 3088.5 cm, respectively) and velocity (mean = 2...
- Use
- To further assess motor activity, we conducted open field and locomotor habituation tests (Figure ). In the open field test, we observed a significant difference between male and female cKO in the distance traveled (mean = 1537.7 and 3088.5 cm, respectively) and velocity (mean = 2...
Slc35a2 cKO mice have more severe induced seizures and exhibit spontaneous seizures
To assess spontaneous epileptic activity without PTZ, we implanted epidural electrodes in control ( n = 8; four females and four males) and cKO ( n = 8; six females and two males) mice and conducted continuous video-EEG recordings sessions lasting 20-24 h for each animal....
- Use
- To assess spontaneous epileptic activity without PTZ, we implanted epidural electrodes in control ( n = 8; four females and four males) and cKO ( n = 8; six females and two males) mice and conducted continuous video-EEG recordings sessions lasting 20-24 h for each animal....
Slc35a2 cKO mice have reduced number of oligodendrocytes, an intact neuronal architecture, and gliosis in the corpus...
Olig2 expression via fluorescent reporter in P21 mice confirmed cell-specificity of Slc35a2 deletion in oligodendroglial lineage cells (Figure ). In the genu of the corpus callosum (gCC), we observed positive Olig2-reporter colocalized with CC1 and Olig2, markers oligodendrocytes. The Olig2-r...
- Use
- Olig2 expression via fluorescent reporter in P21 mice confirmed cell-specificity of Slc35a2 deletion in oligodendroglial lineage cells (Figure ). In the genu of the corpus callosum (gCC), we observed positive Olig2-reporter colocalized with CC1 and Olig2, markers oligodendrocytes. The Olig2-r...
Statistical analyses
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Prism 10 (GraphPad Software) was used to conduct statistical analyses and data visualization for all data. In brief, Welch t -tests and two-way and three-way analyses of variance (ANOVAs) were used for parametric data, and Friedman test was used for nonparametric data. More details regarding the da...
Slc35a2 cKO mice demonstrate hypomyelination throughout cortical layers of cortex and corpus callosum
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Slc35a2 conditional knockout (cKO) mice express hypomyelination patterns throughout several timepoints in myelination. 2′,3′-Cyclic nucleotide-3′-phosphodiesterase (CNPase) immunohistochemistry was performed using 30-µm-thick, paraformaldehyde-fixed brain ti...
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Motor deficits in Slc35a2 cKO mice worsen over time
Slc35a2 conditional knockout (cKO) mice suffer from motor deficits indicated by open field and locomotor habituation tests. (A) Postnatal day 30-60 randomly selected mice were subjected to a weekly 28-point NeuroScore test to measure overall motor function. Male cKO ( n = 5-6) mice exhibited severe ataxic gait and failed to climb a 45-degree slope when compared to their littermates ( n = 15-17; Friedman test, p =.037). There was no detectable difference between female cKO ( n = 5-6) and control mice ( n = 12-16). All other comparisons are not significant (ns). (B) Open field test supported motor deficits in both male and female cKO. In distance traveled (cm), there are significant differences in male cKO versus all other genotypes (two-way analysis of variance [ANOVA], F 1...
Slc35a2 cKO mice have more severe induced seizures and exhibit spontaneous seizures
To evaluate seizure susceptibility in Slc35a2 cKO mice, both female and male mice were administered pentylenetetrazole (PTZ), a γ-aminobutyric acid antagonist, via intraperitoneal injection at a dose of 45 mg/kg body weight (Figure ). Female control ( n = 5) demonstrated tonic-clonic seizures (Racine = 4-6) but did not progress to tonic extension with respiratory arrest (Racine = 7). This was significantly different from littermate female cKO mice, whereas four of five cKO mice developed severe tonic-clonic seizures with respiratory arrest (Racine = 7; two-way ANOVA, F 1, 17 = 13, p =.0022; Figure ). Control male mice responded to the PTZ injection with neck jerking (Racine = 3) and short clonic seizures (Racine = 4) that did not evol...
Slc35a2 cKO mice demonstrate hypomyelination throughout cortical layers of cortex and corpus callosum
Slc35a2 conditional knockout (cKO) mice express hypomyelination patterns throughout several timepoints in myelination. 2′,3′-Cyclic nucleotide-3′-phosphodiesterase (CNPase) immunohistochemistry was performed using 30-µm-thick, paraformaldehyde-fixed brain tissue. Two to three sections per animal were used as technical replicates, and one section was used as a secondary antibody control by omitting the primary antibody. (A-C) CNPase, an enzyme found in the myelin sheath, is abundant and widespread at postnatal day (P) 15, P21, and P60 in littermate control mice ( n = 3 per timepoint per sex). cKO mice ( n = 3 per timepoint per sex) had patches of hypomyelination throughout the motor cortex as well as corpus callosum (CC), anterior commissure (AC), lateral septal (LS), and caudate/putamen (CPu) re...
Measurement outputs
What raw and processed outputs should exist?
To characterize electroencephalographic (EEG) phenotypes, a total of eight control (four female and four male) and eight cKO mice (six female and two male) were implanted with e...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
The digitized EEG signals were reformatted to a signal channel (left-right motor cortex epidural electrodes). EEG recordings were analyzed by visual inspection for the pre...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Slc35a2 conditional knockout (cKO) mice suffer from motor deficits indicated by open field and locomotor habituation tests. (A) Postnatal day 30-60 randomly selected mice...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Slc35a2 conditional knockout (cKO) mice have lower seizure induction and develop severe spontaneous seizures. (A) Adult (postnatal day [P] 90-150) female and male mice wer...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Prism 10 (GraphPad Software) was used to conduct statistical analyses and data visualization for all data.
from paperScoring or quantification
Quantify the primary readouts for this experiment: To characterize electroencephalographic (EEG) phenotypes, a total of eight control (four female and four male) and eight cKO mice (six female and two male) were implanted with e...; The digitized EEG signals were reformatted to a signal channel (left-right motor cortex epidural electrodes). EEG recordings were analyzed by visual inspection for the pre...; Slc35a2 conditional knockout (cKO) mice suffer from motor deficits indicated by open field and locomotor habituation tests. (A) Postnatal day 30-60 randomly selected mice...; Slc35a2 conditional knockout (cKO) mice have lower seizure induction and develop severe spontaneous seizures. (A) Adult (postnatal day [P] 90-150) female and male mice wer....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Prism 10 (GraphPad Software) was used to conduct statistical analyses and data visualization for all data. In brief, Welch t -tests and two-way and three-way a...; Next, we aimed to characterize the behavioral phenotypes of Slc35a2 cKO mice. We utilized a 28-point NeuroScore encompassing several assessments in gait, climbing, and ove...; Slc35a2 conditional knockout (cKO) mice suffer from motor deficits indicated by open field and locomotor habituation tests. (A) Postnatal day 30-60 randomly selected mice...; To further assess motor activity, we conducted open field and locomotor habituation tests (Figure ). In the open field test, we observed a significant difference between m...
from paperReporting output
Report representative outputs alongside summary comparisons for To characterize electroencephalographic (EEG) phenotypes, a total of eight control (four female and four male) and eight cKO mice (six female and two male) were implanted with e..., The digitized EEG signals were reformatted to a signal channel (left-right motor cortex epidural electrodes). EEG recordings were analyzed by visual inspection for the pre..., Slc35a2 conditional knockout (cKO) mice suffer from motor deficits indicated by open field and locomotor habituation tests. (A) Postnatal day 30-60 randomly selected mice..., Slc35a2 conditional knockout (cKO) mice have lower seizure induction and develop severe spontaneous seizures. (A) Adult (postnatal day [P] 90-150) female and male mice wer....
inferred from protocolStructured statistical methods
Prism 10 (GraphPad Software) was used to conduct statistical analyses and data visualization for all data. In brief, Welch t -tests and two-way and three-way a...; Next, we aimed to characterize the behavioral phenotypes of Slc35a2 cKO mice. We utilized a 28-point NeuroScore encompassing several assessments in gait, climbing, and ove...; Slc35a2 conditional knockout (cKO) mice suffer from motor deficits indicated by open field and locomotor habituation tests. (A) Postnatal day 30-60 randomly selected mice...; To further assess motor activity, we conducted open field and locomotor habituation tests (Figure ). In the open field test, we observed a significant difference between m...
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Evidence quotes (3)
Slc35a2 conditional knockout (cKO) mice suffer from motor deficits indicated by open field and locomotor habituation tests. (A) Postnatal day 30-60 randomly selected mice were subjected to a weekly 28-point NeuroScore test to measure overall motor function. Male cKO ( n = 5-6) mice exhibited severe ataxic gait and failed to climb a 45-degree slope when compared to their littermates ( n = 15-17; Friedman test, p =.037). There was no detectable difference between female cKO ( n = 5-6) and control mice ( n = 12-16). All other comparisons are not significant (ns). (B) Open field test supported motor deficits in both male and female cKO. In distance traveled (cm), there are significant differences in male cKO versus all other genotypes (two-way analysis of variance [ANOVA], F 1, 36 = 11.36, p =.0018). Sex differences were significantly different (two-way ANOVA, F 1, 36 = 9.905, p =.0033). In velocity (cm/s), a similar trend was found as the distance traveled where male cKO mice were overall much slower than the o...
To evaluate seizure susceptibility in Slc35a2 cKO mice, both female and male mice were administered pentylenetetrazole (PTZ), a γ-aminobutyric acid antagonist, via intraperitoneal injection at a dose of 45 mg/kg body weight (Figure ). Female control ( n = 5) demonstrated tonic-clonic seizures (Racine = 4-6) but did not progress to tonic extension with respiratory arrest (Racine = 7). This was significantly different from littermate female cKO mice, whereas four of five cKO mice developed severe tonic-clonic seizures with respiratory arrest (Racine = 7; two-way ANOVA, F 1, 17 = 13, p =.0022; Figure ). Control male mice responded to the PTZ injection with neck jerking (Racine = 3) and short clonic seizures (Racine = 4) that did not evolve into generalized tonic-clonic seizures (Figure ). Conversely, male cKO mice developed significant severe tonic-clonic seizures (Racine = 5-7) with tonic extension and respiratory arrest (two-way ANOVA, F 1, 17 = 13, p <.0001; Fi...
Slc35a2 conditional knockout (cKO) mice express hypomyelination patterns throughout several timepoints in myelination. 2′,3′-Cyclic nucleotide-3′-phosphodiesterase (CNPase) immunohistochemistry was performed using 30-µm-thick, paraformaldehyde-fixed brain tissue. Two to three sections per animal were used as technical replicates, and one section was used as a secondary antibody control by omitting the primary antibody. (A-C) CNPase, an enzyme found in the myelin sheath, is abundant and widespread at postnatal day (P) 15, P21, and P60 in littermate control mice ( n = 3 per timepoint per sex). cKO mice ( n = 3 per timepoint per sex) had patches of hypomyelination throughout the motor cortex as well as corpus callosum (CC), anterior commissure (AC), lateral septal (LS), and caudate/putamen (CPu) regions of the brain, indicated by black squares and high magnification images of each region of interest (ROI). This hypomyelination pattern progressed throughout P21 and P60. (D) Semiquantitative analysis using automated thresholding to determine area covered by stain was performed in ImageJ and ave...
Machine-readable layer
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