Introduction
Materials and methods
Animals used for the morphological analysis
Generation of GluA3 and GluA4 KO mice
FRIL
Antibody characterization
Antigen | Description of Immunogen | Source, Host species, Cat.#, Clon or Lot | Concentration |
---|---|---|---|
GluA1–4 (pan-AMPAR) | Recombinant proteins corresponding to aa 724–781 of rat GluA flop | 3 μg/ml | |
GluA3 | Synthetic peptide of N terminus portion of mouse GluA3 corresponding to aa 394–408, (C)NEYERFVPFSDQQIS | Raised in Dr. Makoto Itakura’s laboratory. Rabbit, affinity purified polyclonal antibody | 6.4 μg/ml (FRIL) 0.5–1 mg/l (western blot) |
GluA4 | Synthetic peptide of N terminus portion of mouse GluA4 corresponding to aa 244–257, (C)FKDISLERFIHGGA | Raised in Dr. Makoto Itakura’s laboratory. Rabbit, affinity purified polyclonal antibody | 4.8 μg/ml (FRIL) 0.5–1 mg/l (western blot) |
Quantification of immunogold particles
Intrasynaptic distribution of gold particles within the IMP cluster
Identification of auditory nerve (AN) synapses on the replica
Identification of AN-BC synapses on replicas of the AVCN
Identification of AN-FC synapses on replicas of the DCN
AN-FC synapses
Measurement of the width of the postsynaptic membrane specialization from ultrathin sections
Data analysis
Results
Identification of AN synapses on bushy and fusiform cells
AVCN | DCN | |
---|---|---|
AN-BC | AN-FC | |
Mean | 0.031**** | 0.051 |
SEM | 0.001 | 0.003 |
Median | 0.028 | 0.042 |
Kurtosis | 0.60 | −0.01 |
Skewness | 0.89 | 0.87 |
Minimum | 0.005 | 0.008 |
Maximum | 0.078 | 0.123 |
CV | 0.50 | 0.56 |
Count | 94 | 83 |
Number and density of AMPAR subunits on AN-BC and AN-FC synapses
GluA1–4 (pan-AMPAR antibody labeling)
AVCN | DCN | |
---|---|---|
AN-BC | AN-FC | |
GluA1–4 | ||
Total gold | 1232 | 1004 |
IMP cluster N (positive/total) | 34/34 | 32/32 |
GluA3 | ||
Total gold | 734 | 231 |
IMP clusters N (positive/total) | 30/30 | 23/23 |
GluA4 | ||
Total gold | 305 | 635 |
IMP clusters N (positive/N) | 30/30 | 28/28 |
AN-BC synapses | AN-FC synapses | |
---|---|---|
GluA1–4 | 34 | 32 |
IMP-clusters (n) | ||
Number of gold particles/IMP-cluster | 36 ± 3 | 32 ± 3 |
Mean (±SEM) | 35 | 26 |
Median | 7–74 | 5–67 |
Range | ||
Density of gold particles/IMP-cluster (μm2) | 1100 ± 35 | 623 ± 47 |
Mean (±SEM) | 1034 | 623 |
Median | 732–1571 | 223–1257 |
Range | ||
GluA3 | ||
IMP-clusters (n) | 30 | 23 |
Number of gold particles/IMP-cluster | ||
Mean (±SEM) | 25 ± 2 | 10 ± 1 |
Median | 21 | 9 |
Range | 9–57 | 3–20 |
Density of gold particles/IMP-cluster (μm2) | ||
Mean (±SEM) | 946 ± 50 | 184 ± 19 |
Median | 954 | 179 |
Range | 500–1556 | 36–381 |
GluA4 | ||
IMP-clusters (n) | 28 | 28 |
Number of gold particles/IMP-cluster | ||
Mean (±SEM) | 10 ± 1.2 | 23 ± 3 |
Median | 9.5 | 19 |
Range | 1–22 | 5–44 |
Density of gold particles/IMP-cluster (μm2) | ||
Mean (±SEM) | 337 ± 34 | 561 ± 38 |
Median | 372 | 539 |
Range | 47–727 | 108–1091 |