morphological and functional remodelling of the neuromuscular junction by skeletal muscle pgc-1α - wacom pen tablet

Nerve and Muscle joint (NMJ)
It has a high degree of morphological and functional plasticity.
In mature muscles, the relative level of physical activity is the main determinant of NMJ function.
Classical motor neurons
The mediated activation pattern of muscles is considered to be NMJ plasticity and subsequent fiber-
Type determination in muscle.
We use muscles.
Specific genetically modified animals with peroxide object proliferation
Co-activated receptor γ
Activation factor 1 Alpha (PGC-1α)
As a genetic model for training mice to clarify the contribution of muscles to activity
Induction adaptation of NMJ.
We found muscles.
Specific expression of PGC-
1α promotes re-modeling of NMJ, even without increased physical activity.
It is important that these plastic changes are not limited to the rear
The synaptic structure, but extends to the regulation of the morphology and function of the presynaptic cells.
Therefore, our data suggest that skeletal muscle meat significantly contributes to the adaptation of NMJ after physical activity.
All the experiments were conducted in men, 6-to 8-week-old wild-type (WT)
And genetically modified mice (
All mice obtained from in-house breeding)
Where PGC-
The expression of 1 Alpha is composed of muscle creative kinase (MCK)promoter (
Designated MCK mice, not WT buddies).
The animals are kept in a room with a 12/12 light/dark cycle, are fed standard laboratory food and water is available.
All animal experiments are approved by the agency and the Swiss state veterinary authorities in accordance with the guidelines of the European Council Directive on the care of experimental animals.
Directly freeze the separated muscles in liquid nitrogen.
According to the manufacturer's protocol, total RNA was extracted using TriReagent and RNase-free DNase (Invitrogen).
Adjust concentration to reverse 1 μg RNA
Use the superscript RT (Invitrogen)
Primer with random hexamer (Roche). Real-
Time PCR using Power Sybr Green Master Mix (
Applied Biological system
Use step one Plus light receiver (
Applied Biological system.
According to the Delta method, with WT samples as a reference, the relative expression level of each gene of interest is estimated according to the expression of TATA-
As a binding protein for calibration. Real-
Time PCR primers are shown in.
To extract protein, digest frozen muscles or tissues with lysate buffer (
Tris HCl 20 mM, 138 mM sodium chloride, potassium chloride month.
5%, 1% v/v of glycerin, NP40 v/v)
Cocktail served with a protein inhibitor (Roche)
Quantitative protein concentration Determination Kit with BCA protein (
Pierce, Thermofisher).
By SDS-
PAGE, transfer to the polyammonium diammonium film and with-
Synaptic antibody (Abcam, Ref.
ab53166, 45u2009kD), anti-Antibody SCN4A (
Novus bio, Ref NBP1-19008, 208kda kd)
And SV2A antibodies (
Novus bio, Ref NBP1-46367, 88u2009kD).
Expression (
Northern cell signal, Ref 05-829, 55kda kd)
Used as a loading control.
The relative strength of the band of interest was analyzed using image J, and expressed with respect to the band strength of tubulin.
Set the WT/tubulin level to 100%.
Able to measure the expression of pre-
Synaptic markers from muscle samples, part of which were stained by Alexa Fluor 488-
Coupling alpha-
Snake toxin and Micro
Anatomy under the eyes microscope
The isolated synaptic part is then treated as other samples for protein or RNA extraction.
For immune fluorescence, soleus, EDL, diaphragm and SCM were isolated.
The collected muscles were fixed in methanol for 10 minutes at 20 °c and washed with phosphate
Buffer saline, incubated at room temperature for 2 h in a closed solution (phosphate-
Supplement 1% bovine serum protein, 5% horse serum, buffer saline of 1% Triton-X100, 0. 1% NaN3).
The whole muscle was then incubated at 4 °c for the night with a mixture of primary and Alexa Fluor 488coupled α-bungarotoxin (
Molecular Probes, reference B13422)
Dilute 1/10 in a closed solution.
Antibody against neurofilament protein (Chemicon-
Reference AB1987)
Dilution at 1/1 and multi-price resistance
Synapses (
References m73 15)at 1/200.
After 4 hours of incubation with sufficient secondary antibodies, muscle integrity
Install the medium on the slide with fluorescence (Dako).
Observe samples under a co-Focusing microscope (DMI6000, Leica)
The NMJ structure was studied by using the maximum intensity projection of the chimney.
The same settings are reserved for all samples (
Thickness, number of layers, gain and offset of scan area).
The features of the NMJ architecture are based on the definitions given by Sanes and Lichtman.
More than 100 NMJs ()
There are at least three animals. )were counted. Pre-
The synaptic variables of NMJ include the number of branches identified at the end of the nerve, the total length of these branches, the average length of each branch and the complexity of the branch, by multiplying the number of branches by the total length of these branches, and divide the number by the 100 described earlier.
The LAL and TVA muscles were completely anatomical for the electrophysiological records, and the nerve branches were fixed at 2-
Ml room, in curing silicone rubber bed (
Sylgard of Dow Corning
Preparation under continuous flow of 5% CO/95% O gas mixture, with mm NaCl, 5 kcal mm h2o, 2 cacmm CaCl, 1 mM MgCl, 25mm
Using Image J software and TEM images to determine the synaptic fold length of a single NMJs at X 8,500 magnification.
Using Wacom CTH-a rough measure of the length from the edge of the synaptic to the end of each fold470 pen tablet.
The length of synaptic fold and the number of synaptic fold per NMJ (= 9-16 per mouse)
Average each mouse and then between mice of the same genotype (
WT = 4 in SCM muscle, MCK = 4).
The volume and surface density of the mitochondrial in NMJ are calculated according to the method previously described by Weibo and digitally adapted to Adobe Photoshop.
Using Wacom CTH-manual identification and overview of mitochondrial and cristae structures in NMJ470 pen tablet.
Determine the volume density in the test point system using the D64 grid (
= 16, = 64, '= 1024)
TEM images using X 8,500 magnification.
Volume density ()
The ratio defined in equation 1 as a test point located within the mitochondrial ()
And the total number of test points in NMJ ().
Using the D576 grid to measure the Cristae density of the mitochondrial in an equal magnification in the test point system (
= 16, = 576, '= 9216).
Cristae density of a single mitochondrial ()
Defined in equation 2 as the total number of intersections between the inner mitochondrial membrane and the Test line ()
Divided by the product of the number of points in the mitochondrial ()
And the actual length of the fine test line in μ m (d).
Cristae density was determined in one mitochondrial randomly selected in each NMJ, with a total of 9-16 NMJ per mouse.
NMJ data for Cristae and bulk density averaged each mouse and subsequently between mice of the same genotype (
WT = 4 in SCM muscle, MCK = 3).
The activity of ache was determined by Amplex red ache/ache test kit (
Molecular Probe).
Each reaction contains 200 μm Amplex red reagent containing 1 μ l ml mpo, 0.
1 μm and 100 μm ach.
Ach produced from AChE activity is oxidized by the enzyme to react with the red reagent of the amplifier to generate a fluorescent product detected at 590 nm (
= 3 in each experimental group).
Anesthesia of mice with fluoride.
Use the key point EMG machine to record the electrical performance of the calf (
Meridian, Neurolite, Switzerland).
In short, direct repeated stimulation of the dorsal nerve through a series of 15 stimuli (0.
The duration is 04 u2009 ms, the amplitude is 10 ma)
Under the ultra-extreme condition of 50 hz, two single pole needle electrodes were used, and the action potential in the calf muscle response was recorded using the needle electrodes placed directly in the muscle abdomen.
For WT and MCK mice, the decreasing percentage of amplitude and area is average (
= 4 in each experimental group).
Electrical stimulation and in-cell recording were performed as mentioned earlier.
To put it simply, the nerve is stimulated by suction the electrode.
The stimulus is square.
Wave pulses with variable frequency.
A glass micro-electrode filled with 3 µm potassium is attached to an in-cell recording amplifier (
Cygnus technical neural data IR283)
Used to exercise individual muscle fibers near the ends of the nerve.
As mentioned earlier, muscle and mEPPs are recorded from different NMJs in the muscles.
Prevent muscle contraction by adding in bath 3-4 μm μ-
Conotoxin GIIIB (
Alomone Lab)
Specific blocking of Muscle Voltage
Sodium channel.
As mentioned earlier, the data is analyzed.
The EPP amplitude is normalized to-70 mv and the nonlinear summation is corrected.
Samples were prepared and processed by the microscope center of the University of Basel (
ZMB Basel, Switzerland).
Fix the muscles to more than 3% formaldehyde, 0.
Propolis of 5%-
Buffer solution for 1 u2009 h, then incubated in 1% silotin oxide
Buffer solution.
Slides are dehydrated in the graded EtOH series (50–100%)
, Infiltrated in 100% acetone, embedded in Ethernet, and thin continuously-
Pre-sections were stained with uranium acetate and lead acetate.
The samples were analyzed on the transmission electron microscope Moragni 268D (Philips)
At 80 kV.
The result is represented by an average. e. m.
Unless otherwise stated.
Comparison of MCK and WT samples using student tests (two-tailed)
And-value