Introduction
Materials and methods
Animals
Neonatal hypoxia
Clozapine treatment
Test on prepulse inhibition and tissue preparation
Molecular investigations
RNA isolation and reverse transcription
In vitro transcription and labeling
Preparation of DNA (“glutamate”) Chips
Hybridization and scanning of slides
Data processing
Quantitative real-time PCR (qRT-PCR)
Sequences reading from 5′ -end | ||
---|---|---|
Gene | Forward primer | Reverse primer |
CPLX1 | GCAGGGCATACGAGATAAGT | TGGTGGGGTCAGTGATGGCAGTA |
NPY | CGCTCTGCGACACTACAT | CTCAGGGCTGGATCTCTTG |
NRXN1 | AGGGCGTCAGCTCACAATCTTCAA | TCTGCCGAGCCTGGGTATGGT |
SNAP25 | AGTGGCGTTTGCTGAATGAC | TGCTGGTATGACTTAATCTTGACA |
STX1A | TGTCCCGAAAGTTTGTG | GCGTTCTCGGTAGTCT |
Results
Microarray experiments
CPU | FR | PAR | TE | ||||
---|---|---|---|---|---|---|---|
Genelist t-test fdr correction | Genelist t-test fdr correction | Genelist t-test fdr correction | Genelist t-test fdr correction | ||||
The factor “hypoxia” regulated the following genes in comparison with “normoxia” | The factor “hypoxia” regulated the following genes in comparison with “normoxia” | The factor “hypoxia” regulated the following genes in comparison with “normoxia” | The factor “hypoxia” regulated the following genes in comparison with “normoxia” | ||||
UP | DOWN | UP | DOWN | UP | DOWN | UP | DOWN |
ATF2 | ATF2 | CAPN2 | ADCY3 | ||||
ATF4 | AURKB | CAPON | ADCY6 | ||||
ATP2A2 | CALM2 | CASK | ATP2A2 | ||||
CPX1 | CAP2 | EAAT3 | BDNF | ||||
CPX2 | CAPN8 | GABBR1 | BNPI | ||||
GAP43 | CPX1 | GAPDH | CAPN2 | ||||
GAPDH | DEAD BOX | GNA12 | CAPN6 | ||||
GATA4 | DLGAP2 | LOC294679 | CASQ2 | ||||
GRM2 | DUSP1 | LOC362704 | CPX1 | ||||
ITPR1 | FYN | MAPK3 | DAT1 | ||||
MARK1 | GAPDH | PIPS | EAAT3 | ||||
MPPS7 | GNAO | PLD2 | ERK7 | ||||
MYD116 | MAPK14 | PLEKHC1 | GABRG3 | ||||
NM_017261 | MAPK3 | POU3F4 | GAD67 | ||||
NOS2 | MRPS7 | PPP1R14A | GADD45A | ||||
NRXN1 | NELL2 | PPP2R2C | GAP43 | ||||
PKN1 | NPY | PPP5C | GATA4 | ||||
PLD1 | NOS2-1149 | PREFOLDIN5 | GNAO | ||||
PLEKHC1 | NRXN1 | PRKCZ | GPSM1 | ||||
PPM1B | PACSIN2 | PTPRA | GRM8 | ||||
PPP1CC | PIM3 | PTPRR | ID3 | ||||
PPKCC | PLAT | RAB14 | ITPR3 | ||||
PTP4A1 | PPP1R14B | RAB3A | LOC294679 | ||||
PTPRA | PPP1R14C | RAB3B | LOC362704 | ||||
PTPRN | PTP4A2 | RN.25045 | MGC105762 | ||||
PTPRN2 | PTPRD | RN.28284 | NPY | ||||
RAB10 | PTPRN | SHANK1 | NR2F2 | ||||
RN.28284 | PTPRN2 | SLC2A1 | NRXN3 | ||||
SACM1L | RAB3A | SLC6A12 | NUP54 | ||||
SLC1A3 | SLC1A3 | SLK | PIPS | ||||
SLC1A3 | SLC6A12 | SRC | PLD2 | ||||
SLC1A6 | SNAP25 | STAT3 | PNOC | ||||
SLC32A1 | SOD1 | SYT1-1511 | PPM1G | ||||
SNAP25 | SQSTM1 | TRANSC.LOCUS | PPP1R10 | ||||
STAT3 | SRC | PPP2R2C | |||||
SYN1 | STK39 | PPP2R5B | |||||
VAMP2 | STX1A | PPP4R1 | |||||
TRANSC.LOCUS | PPP5C | ||||||
PRKCH | |||||||
PRKCZ | |||||||
RAB10 | |||||||
RGS3 | |||||||
RN.137813 | |||||||
RN.15926 | |||||||
RN.7389 | |||||||
SLC1A6 | |||||||
SLC32A1 | |||||||
SLC6A12 | |||||||
SMAGP | |||||||
SNAP25 | |||||||
SP1 | |||||||
SQSTM1 | |||||||
STAT3 | |||||||
STK6 | |||||||
STX1A | |||||||
SYN1 | |||||||
SYN2 | |||||||
TCF12 | |||||||
VAMP1 |
Clozapine treatment and qRT-PCR results
Normoxic | Hypoxic | ||||||
---|---|---|---|---|---|---|---|
Region | Gen | Reg.-Dir. | P-value | Region | Gen | Reg.-Dir. | P-value |
CPU | CPX1 | CPU | CPX1 | up | 0.026 | ||
FR | CPX1 | down | 0.043 | FR | CPX1 | ||
PAR | CPX1 | PAR | CPX1 | up | 0.012 | ||
TE | CPX1 | TE | CPX1 | ||||
CPU | NPY | CPU | NPY | ||||
FR | NPY | FR | NPY | ||||
TE | NPY | TE | NPY | ||||
CPU | NRXN1 | CPU | NPY | ||||
FR | NRXN1 | FR | NRXN1 | ||||
PAR | NRXN1 | PAR | NRXN1 | ||||
TE | NRXN1 | TE | NRXN1 | ||||
CPU | SNAP25 | CPU | NRXN1 | ||||
FR | SNAP25 | FR | SNAP25 | ||||
PAR | SNAP25 | PAR | SNAP25 | ||||
TE | SNAP25 | TE | SNAP25 | ||||
FR | STX1A | down | 0.015 | FR | SNAP25 | ||
PAR | STX1A | up | 0.048 | PAR | STX1A | up | 0.002 |
TE | STX1A | TE | STX1A |
Discussion
Complexin1
Gene | Region | Entity | Normoxia | Normoxia | Normoxia | Hypoxia | Hypoxia | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Entity | Without Clozapine | With Clozapine | Without Clozapine | With Clozapine | |||||||||||||||
N
| rho |
P
|
N
| rho |
P
|
N
| rho |
P
|
N
| rho |
P
|
N
| rho |
P
|
N
| rho |
P
| ||
CPX1 | CPU | 23 | 0.35 | 0.11 | 12 | −0.38 | 0.23 | 6 | −0.89 |
0.02
| 6 | 0.03 | 0.96 | 5 | 1 | 0 | 12 | −0.38 | 0.23 |
NPY | TE | 23 | 0.1 | 0.65 | 12 | 0.000 | 1.000 | 6 | 0.89 |
0.02
| 6 | −0.31 | 0.54 | 5 | 0.3 | 0.62 | 12 | 0.000 | 1.000 |
NRXN1 | CPU | 23 | 0.07 | 0.77 | 12 | 0.7 | 0.01 | 6 | 0.600 | 0.21 | 6 | 0.66 | 0.16 | 5 | 0.2 | 0.75 | 12 | 0.7 |
0.01
|
SNAP25 | FR | 20 | 0.02 | 0.93 | 9 | 0.02 | 0.97 | 4 | −1.000 |
0.000
| 5 | 0.1 | 0.87 | 5 | −0.3 | 0.62 | 9 | 0.02 | 0.97 |