ࡱ > s u r q` R e bjbjqPqP 2p : : v8
| 2 1 F Z U o { 0 0 0 0 0 0 0 $ -5 h 7 H 1 F U F F 1 1 ) ) ) F R, ) F 0 ) ) ) N V]
' * ) R, 1 0 1 ) 7 ) 7 ) 7 ) ) 4 Q 1 1 )
1 F F F F r D r Supplemental information
Methods
Cell cultures
Astrocyte cultures of rat brain were prepared as described previously ADDIN EN.CITE Weinstein200120220217Weinstein, D. E.Albert Einstein College of Medicine, Bronx, New York, USA.Isolation and purification of primary rodent astrocytesCurr Protoc NeurosciCurr Protoc NeurosciUnit 3 5Chapter 32001May18428469http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18428469 1. Briefly, forebrains of new born rats (less than 12 h) were removed under sterile conditions, cut into small pieces and dissociated with 0.05% trypsin. After the dissociation procedure, the brain cells were cultures in 25-ml flasks in Eagles minimum essential medium, supplemented with 10% horse serum, 2% fetal calf serum, 0.6% glucose, 5 0 g / m l g e n t a m y c i n , a n d p e n i c i l l i n - s t r e p t o m y c i n ( 5 I U / m l a n d 5 g / m l , r e s p e c t i v e l y ) . C e l l c u l t u r e s w e r e k e p t a t 3 7 C i n a m o d i f i e d a t m o s p h e r e o f 5 % C O 2 i n a i r . T h e m e d i u m w a s c h a n g e d t w i c e a w e e k . I m m u n o c y t o c h e m i s t r y v e r i f i e d t h a t t h e c e l l s s t a i n e d p o s i t ively for the astrocytic marker glial fibrillary acid protein (GFAP).
For primary neuronal cultures ADDIN EN.CITE ADDIN EN.CITE.DATA 2, dissociated cells were plated on 35-mm dishes (106 cells) coated with l-polylysine and culture medium containing 10% fetal calf serum. After removing the last coating solution, cells were seeded in a serum-free medium consisting of 1:1 mixture of DME and F-12 nutrient (GIBCO), supplemented with glutamine (2mM), NaHCO3 (13mM), N-2-hydroxyethylpiperazine-N-2-ethanesulp h o n i c a c i d ( H E P E S , 5 m M , p H 7 . 4 ) , g l u c o s e ( 3 3 m M ) , p e n i c i l l i n - s t r e p t o m y c i n ( 5 I U / m l a n d 5 g / m l , r e s p e c t i v e l y ) , a n d a m i x t u r e o f s a l t a n d h o r m o n e s c o n t a i n i n g i n s u l i n ( 2 5 g / m l ) , t r a n s f e r r i n ( 1 0 0 g / m l ) , p r o g e s t e r o n e ( 2 0 n M ) , p u t r e s c i n e ( 6 0 M ) , a n d s o d i u m s e l e n i t e (Na2SeO3, 30 nM). Cells were cultured at 37C in a humidified atmosphere of 5% CO2 in air.
Preparation of Cytosolic Extracts and Crude Synaptic Membrane Fraction
For preparation of cytosolic extracts, hippocampal tissue was mechanically homogenized in lysis buffer on ice. Lysis was prepared in 50 mM Tris containing 50 mM HEPES (pH 7.4), EGTA (pH 8.0), 0.2% NP-40, 10 mM EDTA (pH 8.0), 15 mM sodium pyrophosphate, 100 mM - g l y c e r o p h o s p h a t e , 5 0 m M N a F , 1 5 0 m M N a C l , 2 m M s o d i u m o r t h v a n a d a t e , 1 m M P M S F a n d 1 m M D T T f o r 1 0 m i n . C e n t r i f u g a t i o n a t 1 6 0 0 0 g ( 4 C ) f o r 1 0 m i n i n a m i c r o c e n t r i f u g e r e m o v e d t h e n u c l e i a n d i n s o l u b l e f r a c t i o n s . T o e x t r a c t a c r u d e s y n a p t i c m e m b r a n e f r a ction from the hippocampus, we used the method of Danbolt et al. ADDIN EN.CITE Danbolt199020820817Danbolt, N. C.Pines, G.Kanner, B. I.Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brainBiochemistryBiochemistry6734-402928Amino Acid Transport System X-AGAnimals*Brain ChemistryCarrier Proteins/*isolation & purification/metabolismChromatography, AffinityChromatography, DEAE-CelluloseGABA Plasma Membrane Transport ProteinsGlycoproteins/*isolation & purification/metabolismIon Channels/*metabolismKineticsLiposomesMembrane Glycoproteins/*isolation & purification/metabolism*Membrane Proteins*Membrane Transport ProteinsMolecular WeightNerve Tissue Proteins/*isolation & purification/metabolism*Organic Anion TransportersPotassium/*metabolismRatsSodium/*metabolismWheat Germ Agglutinins1990Jul 171697765http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1697765 3. Briefly, hippocampal tissues were homogenized in ice-cold 0.32 M mannitol containing 1 mM EDTA (pH 7.4) and centrifuged at 1 000 g for 10 min at 4 C. The supernatant was then centrifuged at 27 000 g for 20 min at 4 C, and the pellet was suspended in 1 ml of hypotonic 1 mM TrisHCl containing 1 mM EDTA. This suspension was centrifuged at 27 000 g for 20 min at 4 C. The pellet was re-suspended in 1 ml of re-suspension buffer (100 mM P B S , p H 7 . 4 , 5 m M T r i s H C l , 1 m M M g S O 4 , 0 . 5 m M E D T A , 1 % g l y c e r o l ) a n d c e n t r i f u g e d a t 2 7 0 0 0 g f o r 2 0 m i n a t 4 C . T h e f i n a l p e l l e t w a s r e - s u s p e n d e d i n 2 0 0 l o f r e - s u s p e n s i o n b u f f e r .
B r a i n S e c t i o n P r e p a r a t i o n
A f t e r d e c a p i t a t i o n , t h e a n i m a l s b r a i n s w e r e removed quickly, and the bilateral hippocampus (both the ventral and dorsal sectors) were carefully isolated (within 3 min on ice), immediately frozen in liquid nitrogen and then stored at -80 (C until assay. For Immunocytochemical staining, rat brains were removed, postfixed overnight, and equilibrated in phosphatebuffered 30% sucrose. Free-floating 35- m s e c t i o n s w e r e c o l l e c t e d o n a M i c r o m c r y o s t a t a n d s t o r e d s u b m e r g e d a t - 2 0 C i n 2 4 - w e l l p l a t e s c o n t a i n i n g a c r y o p r o t e c t a n t c o m p o s e d o f g l y c e r o l , e t h y l e n e - g l y c o l , a n d p h o s p h a t e b u f f e r e d s a l i n e .
C o l o c a l i z a t i o n A n a l y s i s
T h e n u m b e r o f C x 4 3 + / N G 2 + c e l l s w e r e c o unted under a confocal microscope (Zeiss 510) ADDIN EN.CITE Hoehn2005252517Hoehn, B. D.Palmer, T. D.Steinberg, G. K.Department of Neurosurgery, Stanford University, Stanford, CA, USA.Neurogenesis in rats after focal cerebral ischemia is enhanced by indomethacinStrokeStroke2718-243612AnimalsAnti-Inflammatory Agents, Non-Steroidal/*pharmacologyBiological Markers/metabolismCell CountCell Division/drug effectsCell Lineage/drug effectsCerebral Cortex/pathologyCorpus Striatum/pathologyImmunologic Factors/pharmacologyIndomethacin/*pharmacologyInflammation/immunologyIschemic Attack, Transient/*pathology/physiopathologyLymphocyte Activation/drug effectsMaleMicroglia/drug effects/pathologyMonocytesNeurons/*drug effects/metabolismRatsRats, Sprague-Dawley2005Dec16282546http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16282546 4 by using split-panel and Z-axis analyses, and multichannel configuration with a 40 objective and electronic zoom of 1. Each cell was examined in its full z-dimension and only cells with a nucleus unambiguously associated with NG2 were scored as positive.
REFERENCES
ADDIN EN.REFLIST 1. Weinstein DE. Isolation and purification of primary rodent astrocytes. Curr Protoc Neurosci. 2001;Chapter 3:Unit 3 5
2. Rouach N, Glowinski J, Giaume C. Activity-dependent neuronal control of gap-junctional communication in astrocytes. J Cell Biol. 2000;149:1513-1526
3. Danbolt NC, Pines G, Kanner BI. Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain. Biochemistry. 1990;29:6734-6740
4. Hoehn BD, Palmer TD, Steinberg GK. Neurogenesis in rats after focal cerebral ischemia is enhanced by indomethacin. Stroke. 2005;36:2718-2724
5. Jiang KW, Gao F, Shui QX, Yu ZS, Xia ZZ. Effect of diazoxide on regulation of vesicular and plasma membrane gaba transporter genes and proteins in hippocampus of rats subjected to picrotoxin-induced kindling. Neurosci Res. 2004;50:319-329
6. Jiang K, Shui Q, Xia Z, Yu Z. Changes in the gene and protein expression of k(atp) channel subunits in the hippocampus of rats subjected to picrotoxin-induced kindling. Brain Res Mol Brain Res. 2004;128:83-89
Results
Manipulation of mitoKATP channels affected the PTX-induced convulsions
Animals, including those both for Western blot analysis and immunocytochemical staining (n=8-10 per group), were observed for recording the seizures. There was no spontaneous motor seizure recorded in any stage 4/5 seizure rat during the seizure-free interval, i.e. without a PTX re-kindling. All stage 4/5 seizure rats showed heterogeneous seizure frequencies, as the seizures were easily induced by subconvulsant PTX after an interval of 20 days. Activation of mitoKATP channels not only attenuated seizure severity, but also decreased the numbers of spike-wave discharges in a 4-hour EEG recording period (see Fig.S2A-C), which were similar to that described in our earlier work ADDIN EN.CITE ADDIN EN.CITE.DATA 5, 6.
Table S1. Pairs of primary and secondary antibodies used for double/triple staining
First couple Second couple Third couple
First Ab Second Ab First Ab Second Ab First Ab Second Ab
Cx43 FITC-AR Cx45 Cy3-AG GFAP Cy5-AP
Cx43 FITC-AR Cx45 Cy3-AG Map2 Cy5-AM
Cx43 FITC-AR Cx45 Cy3-AG NeuN Cy5-AM
DCX FITC-AG Cx43 Cy3-AR NeuN Cy5-AM
Cx43 FITC-AR Cx45 Cy3-AG
NG2 FITC-AM Cx43 Cy3-AR
GFAP FITC-AP Cx43 Cy3-AR
GFAP FITC-AP Cx45 Cy3-AG
Ab, Antibody; FITC-AR, FITC-conjugated anti-rabbit; FITC-AG, FITC-conjugated anti-goat; FITC-AM, FITC-conjugated anti-mouse; FITC-AP, FITC-conjugated anti-Guinea pig; Cy3-AG, Cy3-conjugated anti-mouse; Cy3-AR, Cy3-conjugated anti-rabbit; Cy5-AP, Cy5-conjugated anti-Guinea pig; Cy5-AM, Cy5-conjugated anti-mouse.
Figure legends
Fig. S1. A. A diagram showing the rostral-caudal region of interest. B. Representative pictures showing the method of semiquantification for Cx43/GFAP or Cx45/GFAP colabelling. Using ImageJ 1.41 with ColocalizeRGB and Measure RGB Plugin, colabelling was defined as in white color. Arrows indicate the colabelling. Scale bar=10(m. Cx, connexin; GFAP, glial fibrillary acid protein.
Fig.S2. Anticonvulsant action of Dia preconditioning on PTX-induced seizures. At 14 days after the last PTX administration of the 20-day protocol (2.25 mg/kg, one times a day for 20 days), rats were treated with Dia followed by a PTX re-injection. A. Animals were observed for 45 min after the PTX re-injection and seizures were recorded. Activation of mitoKATP channels with Dia attenuated the seizure severity. B. Sample electroencephalographic (EEG) recordings from PTX re-kindling, Dia+ re-kindling, and 5-HD+ re-kindling rats illustrate Dia-treated rats have less spike-wave discharges (SWDs) than PTX re-kindling and 5-HD+ re-kindling rats. C. Dia-treated rats had less SWD than PTX re-kindling and 5-HD+ re-kindling rats in a 4-hour recording period. Control, vehicle administration followed by a vehicle re-injection in normal rats; PTX, vehicle administration followed by a vehicle re-injection in PTX kindling rats; PTX re-kindling, vehicle administration followed by a PTX re-injection in PTX kindling rats; Dia+ re-kindling, DIZ administration followed by a PTX re-injection in PTX kindling rats; 5-HD+ re-kindling, 5-HD administration followed by a PTX re-injection in PTX kindling rats. Values are mean SEM; * P<0.05; n=8-10. Dia, Diazoxide; 5-HD, 5-hydroxydecanoate; PTX, picrotoxin.
Fig. S3. Representative immunocytochemical staining pictures of the Cx43 and Cx45 expressions in DG (A), CA1 ( ! . / u v 0 2 E c d s t Źvi\K\K\ !j h8 haf CJ KH UaJ h8 haf CJ KH aJ h8 hZ' CJ KH aJ h8 haf CJ H*aJ h8 haf CJ H*aJ j h8 haf CJ UaJ h8 haf CJ aJ !h8 h
5CJ KH \]aJ h8 h
5CJ aJ !h8 h 5CJ KH \]aJ h?y h'2j 5KH \]aJ h?y hc= 5KH aJ o( h?y hc= 5KH aJ ! / F ] v# # ' ' U0 `0 0 1 <2 2 3 4 4 4 0dh ^`0gd1 gdK2 0^`0gdK2
dh WD `gd1 dh 7$ 8$ H$ gd1 dh gd1
$dh a$gd1 e
+ / @ S T \ ] * ڿ̿zi\Mh8 h CJ KH PJ aJ h8 h CJ KH aJ !h8 h 5CJ KH \]aJ !h8 haf 5CJ KH \]aJ h8 haf CJ H*aJ h8 haf CJ KH PJ aJ h8 haf CJ H*KH aJ h8 haf CJ aJ h8 haf CJ KH aJ h8 haf CJ H*KH aJ !j h8 haf CJ KH UaJ 'j h8 haf CJ KH UaJ
A B A B C D ! ! ! ! ! ! N" P" " " " v# # # # $ pc h8 hR CJ KH aJ !h8 hR 5CJ KH \]aJ h8 hR 5CJ KH aJ h8 h CJ H*KH aJ h8 haf CJ H*KH aJ !j h8 h CJ KH UaJ h8 h CJ aJ h8 h CJ KH ]aJ h8 h CJ KH PJ aJ h8 h CJ KH PJ aJ h8 h CJ KH aJ $ $ $ $ $ $ &