D E N D R I T I C S P I N E S I N T H E H I P P O C A M P U S O F
G E N E T I C A B S E N C E E P I L E P S Y R A T S ( G A E R S ) :
A N U L T R A S T R U C T U R A L O U A N T I T A T I V E A N A L Y S I S
Serap Şirvancı, M .D .* / Şükrü M id illio ğ lıı, M .D .* / Charles K. Meshul, Ph.D.**
Fi!ix O nat, M .D .,P h .D .,M / Tangül Şan, Ph.D.*
* D e p a rtm e n t o f H is to lo g y a n d E m b ryo lo g y, S c h o o l o f M e d icin e , M arm ara U niversity, Is ta n b u l, Turkey.
* * E le c tro n M ic ro s c o p y L a b o ra to ry a n d D e p a rtm e n t o f B e h a v io ra l n e u ro s c ie n c e a n d P atholog y, VA M e d ic a l C enter, O regon H ealth S cie n ce U n ive rsity, P o rtla n d , OR, USA.
* * * D e p a rtm e n t o f P h a rm a c o lo g y a n d C lin ic a l P h a rm a co lo g y, S c h o o l o f M e d icin e , M a rm a ra U n iv e rs ity , Is ta n b u l, Turkey.
ABSTRACT
O b je c tiv e : Th e aim of the present study w a s to a n a lyze the a re a of glutam ate im m unoreactive (-ir) dendritic sp in e s in C A 3 and dentate gyrus hilar regions in G e n e tic A b se n ce E p ile p sy R ats from S trasb o u rg ( G A E R S ) h ippocam pus by electron m icro sco p y in order to investigate p o ssib le g lutam ate-in d u ced m orphological ch a n g e s in dendritic sp in e s.
M e th o d s: G A E R S and non-epileptic control (N E C ) rats w ere used. Th e brains w ere removed after perfusion fixation and C A 3 and dentate hilar regions of the hippocam pus w ere studied using immunogold electron m icroscopical m ethods. The area of dendritic sp in es w as m easured with the program NIH Image A n alysis.
R e s u lt s : Th ere w as no significant difference in the mean number of glutamate-ir sp ines making asym m etric sy n a p se s per m ossy terminal in CA 3 and dentate hilar regions com pared to the control group. No significant difference in the area of
glutamate-ir dendritic spines making asym m etric syn a p se s with m ossy fiber term inals between N E C and G A E R S groups w as found.
C o n c lu s io n : Dendritic sp in e s in the hippocampus seem not to be affected in absence epilepsy, a s opposed to those in temporal lobe epilepsy (T L E ). Th is may be due to differences in pathogenetic m echanism s between T L E and absence epilepsy.
Key
W ords:
H ippocam pus, G A E R S , Ultrastructure, Dendritic spineINTRODUCTION
Typical absence epilepsy is characterized by a sudden interruption of behavioral activity and re sp o n siv e n e ss a sso cia te d with bilaterally synchronous spike and w ave discharges in the E E G . G e n e tic a b se n c e ep ilep sy rats from Strasbourg (G A E R S ), a well-established model
M a r m a r a M e d ic a l J o u r n a l 2 0 0 3 ; 1 6 ( 3 ) : 1 9 5 - 2 0 0 Correspondence to: Serap Şirvancı, M.D., - Department o f Histology and Embryology,
School o l Medicine, Marmara University, Haydarpaşa, 34668 İstanbul, Turkey e.mail address: ssirvanci@yahoo.com
of human absence epilepsy on the b asis of neurophysiological, pharm acological and genetic studies (1,2), is a strain of W istar rats with genetically determined se izu re s. Although spike and w ave discharges have been demonstrated to originate from the thalam us and cortex, other brain regions such a s the hippocam pus may also be involved in the c e llu la r and m o lecular m echanism s underlying a b sen ce epilepsy. In a previous study, b asal levels of extracellu lar glutamate were shown to be three tim es higher in G A E R S compared to non-epileptic controls. In addition, no difference in G A B A levels within the hippocampus between G A E R S and the non epileptic control group w as reported in the sam e study (3). Metabolic activity w as increased in many brain regions of adult G A E R S and in limbic structures of immature G A E R S including the hippocampus (4).
Spines were proposed to have roles both in storing memory and protecting neurons from toxic insults associated with the raised C a +2 that follows synaptic activity (5). Th ey also mediate synaptic plasticity and more than 9 0 % of dendritic spines in the m am m alian C N S are contacted by excitatory sy n a p se s (6). It w as reported that e x c e ssiv e activation of postsynaptic glutamate receptors cau sed formation of cell body swelling, chromatin clumping, development of dendritic varicosities and swelling or loss of dendritic spines (7). Alterations in postsynaptic intracellular C a +2 concentration regulates the morphology of dendritic sp ines and se izu res cause spine shrinkage and an increase in spine density (8 ). S p in e d en sity in the proxim al dendrites of the granule cells in the hippocampus of T L E patients w as shown to be increased (9). Dendritic spine degeneration w a s also reported in different epilepsy models such a s the kainate (10,11), pilocarpine (12,13) and tetanus toxin induced m odels (1 4 ,1 5 ). Although dendritic shrinkage in CA1 and C A 3 c pyramidal neurons in tetanus toxin-induced se izu res w a s reported in previous stu d ies, other reports have shown dendritic hypertrophy of b asal dendrites of granule cells in dentate gyrus in both animal models and hum ans (9,16-18).
It has recently been reported that glutamate immunoreactivity in m ossy term inals in the CA 3 region in the hippocam pus w a s significantly increased in G A E R S com pared to the non
epileptic control group (19). Th e present study w as performed to analyze the area of glutamate imm unoreactive (-ir) dendritic sp in es in C A 3 and dentate g yrus hilar reg io ns in G A E R S hippocampus by electron m icroscopy in order to investig ate p o ssib le glutam ate-induced morphological ch ang e s in dendritic spines.
MATERIALS AND METHODS
Adult, non-epileptic control W istar rats (N E C ) and G A E R S (250-300 g, 6-12 months old) having a b se n ce se izu re s on E E G w ere used. T h e anim als were housed in a temperature-controlled room (20± 3°C) with a 12 h light / dark cycle and fed a stan d ard diet. Full app roval for the experim ental study w a s obtained from the Animal C are and U se Committee of M arm ara University (4 0 .2 0 0 0 .mar).
For ultrastructural ob servatio n and im m unocytochem istry, N E C (n=4) and G A E R S (n=4) rats w ere sa c rific e d by tran sao rtic perfusion with a fixative solution (2 .5 % glutaraldehyde, 0 .5 % p araform aldehyde, and 0 .1 % picric acid in 0.1 M H E P E S buffer, pH 7.3). Th e anim als w ere decapitated and the entire brain left overnight in the sam e fixative at 4 °C . Th e brains w ere w ashed se veral tim es in 0.1 M H E P E S , pH 7 .3, and cut into 300 pm slic e s using a vibratome in the following day. Th e C A 3 and dentate gyrus regions of the hippocam pus w ere dissected and incubated in 1% osmium tetroxide / 1 .5 % potassium ferricyanide for 30 min at room tem perature. Th e sam p le s w ere then w ashed several tim es in deionized w ater and stained en block with aqueous 0 .5 % uranyl acetate for 30 min at room tem perature in the dark. T h e tissue w a s then dehydrated in a graded se rie s of ethanol, cle are d in pro p ylene oxide and embedded in Epon for 24 h at 6 0 °C . Semi-thin sections (1 pm) w ere cut on a Leica Ultracut R ultramicrotome and stained with toluidine blue and viewed with the light m icroscope for proper orientation. Th e tissue w a s then thin sectioned (60 nm), collected on 200 m esh nickel grids coated with a C o at-Q u ick ‘G ’ pen (K iyo ta International, Elk G ro ve, IL, U SA ) and air-dried for 3-4 h.
The grids containing thin sections were w ashed in Tris-buffered salin e, pH 7 .6, containing 0.1 %
Dentritic spines in the hippocampus of GAERS
Triton X-100 (T B S T 7.6 ) and incubated in the prim ary antibody overnight in a moist cham ber at room te m p e ratu re . T h e anti-glutam ate antibody (B io g e n e sis) w a s diluted 1 :1 .6 0 0 .0 0 0 in T B S T 7 .6 . A spartate (1 mM) w a s added to the antibody solution to prevent cross-reactivity with asp artate within the tissu e . The sam p le s were w ash e d se ve ra l tim es in T B S T 7.6 and T B S T 8.2 and incubated for 90 min in goat anti-rabbit IgG
F i g . l : D endritic spines synapsing with m ossy term inals in control
(a ,b ,c ) and G A E R S (d,e) groups in CA3 (a ,b ,d ) and dentate hilar (c,e) regions. (MT: m ossy term inal: D: dendritic shaft:
d : dendritic spine: MOT: m ossy fiber collateral term inal;
B la c k /w h ite a r ro w : a s y m m e tric s y n a p se s b etw een d e n d ritic sp in e s and m ossy te rm in a ls; B la c k /w h ite a r ro w h e a d : dense core vesicle). Bar: 500 nm.
(Sigm a) conjugated to 10 nm gold, which w as diluted 1:50 in T B S T 8 .2. Th e sam p le s w ere then washed in T B S T 7.6 and deionized water. The sections were counterstained with uranyl acetate and lead citrate before being view ed and photographed on the tran sm issio n electron microscope. All of the sam p les w ere processed simultaneously with the sam e antibody solutions to avoid day-to-day differences. Sections were viewed and photographed using a J E O L 1200 E X transm ission electron m icroscope.
The photographs w ere taken randomly within the sam e region of the stratum lucidum layer of the C A 3 subfield w h ere the s y n a p s e s betw een m ossy fiber term inals and dendritic sp ines of CA 3 pyramidal neurons occur and within the hilar region of the dentate gyrus of the hippocam pus. Asym m etric sy n a p se s w ere identified by an accum ulation of synap tic v e s ic le s within the presynaptic nerve term inal and a prominent postsynaptic density which is wider than the width of the synaptic cleft.
For the quantitative a n a lysis, the area of dendritic spines w as determined using the program NIH Image A n alysis. Th e number of sp ines per m ossy fiber term inal w a s a lso ca lcu la te d and a comparison w as made between G A E R S and the control group. The data w ere analyzed using a Mann-Whitney U-test and are presented as mean ± standard deviation.
a-V) o E E 3 Z
F ig .2 : N u m b e r of g lu ta m a te -ir d e n d ritic sp in e s m a kin g asym m e tric synapse per m ossy term inal in C A3 and d e n ta te hilar regions in G A E R S and NEC. (NEC: N on epile p tic control rat).
F ig .3 : T he area of g lu ta m a te -ir d e n d ritic spines in C A 3 and d e n ta te hilar regions in G A E R S and NEC. (N EC: N o n epileptic co n tro l rat).
RESULTS
In the present study, the area of glutamate -ir dendritic sp in e s and the num ber of sp in e s m aking a sym m e tric s y n a p s e s per m o ssy terminal in C A 3 and hilar regions in G A E R S hippocampus w ere m easured. Th ere w a s no significant difference in the mean number of glutam ate-ir sp in e s m aking asym m e tric syn a p se s per m ossy terminal in C A 3 and dentate hilar regions com pared to the control group (m ean number of sp ines per m ossy terminal ± S D : C A 3 ;
control
[n=30], 2,33± 1,30;GAERS
[n=37], 2 ,1 6± 1,0 9 ; D G ;control
[n=45], 1 ,5 3 ± 0 ,6 6 ;GAERS
[n=23], 1 ,8 3 ± 0 ,8 3 ). No significant difference in the area of glutamate-ir dendritic sp ines making asym m etric syn a p se s with m ossy fiber term inals between N E C and G A E R S groups w as found (m ean area [pm2] ± S D : C A 3 ;control
[n=70], 0,24± 0,16;GAERS
[n=80], 0 ,2 7 ± 0 ,2 4 ; D G ;control
[n=69], 0,21 ±0,12;GAERS
[n=42], 0,25± 0,14).DISCUSSION
In the present study, a possible dendrotoxic effect of the increase in glutamate level in the C A 3 region on the number and area of dendritic sp in e s of C A 3 pyram idal n eu ro n s w a s investig ated . W e also e xam in e d the sa m e p aram eters in the dentate hilar region. No difference w as found either in the number or the area of dendritic sp ines in both regions between G A E R S and N E C groups. T h is is the first study
Dentritic spines in the hippocampus of GAERS
which reported a histomorphologic analysis of dendritic sp ines in G A E R S hippocam pus.
An in c re a se in the glutam ate d ensity in m ossy fiber term in als in the C A 3 region of G A E R S hippocam pus co m pared to N E C an im als has recen tly been reported (1 9 ). A previo us study e x a m in e d the e a rly c o n s e q u e n c e s of g lu tam ate re c e p to r a ctiv a tio n on d en d ritic sp in e s y n a p s e s and sh o w ed that cultured h ip p o cam p a l n e u ro n s e x p o se d to NM DA exhibited lo ss of dendritic sp in e s. A functional relation betw een glutam ate receptor activation and the a ctin -m ed iated sh a p e of dendritic sp in e s w a s su g g e ste d (2 0 ). A nother study using te tan u s toxin model of T L E , dendritic sh rin kag e in the ap ical and b asal dendrites of CA1 pyram idal n eu ro n s w a s o b served 8 w e e k s after the injection of the toxin (1 4 ). P re vio u s stu d ie s sho w ed that CA1 and C A 3 c pyram idal neurons and hilar n eu ro n s w ere lost in T L E a sso c ia te d with A m m o n’s horn s c le ro s is and dendritic coiling, nodulation and sp ine lo ss w ere also o b se rve d (1 4 ,1 5 ,2 1 ,2 2 ). In co ntrast, the p re se n t study did not sho w a n y e vid e n ce of dendritic sw e llin g (7) or sh rin kag e (14) a s p re vio u sly reported and the num ber of sp in e s m aking a sym m e tric sy n a p se per m o ssy fiber term inal w a s not sig n ifican tly different from the control group in both dentate and C A 3 regions in G A E R S hippocam pus.
Th e data obtained in the present study suggests that although an in c re a se in the glutam ate content of m o ssy fiber term in als h a s been previously dem onstrated in G A E R S strain (19), dendritic sp ines in the hippocam pus seem not to be affected in a b se n ce epilepsy. Th is may be due to differences in pathogenetic m echanism s between T L E and a b sen ce epilepsy.
A c k n o w le d g e m e n ts
The authors thank Cynthia Moore for assistan ce during routine tissu e preparation and immunolabeling for electron m icroscopy. The authors also thank the Brain R e se a rc h Society (Beyin Arastirm alari Dernegi - B A D , Istanbul, T u rke y) for providing a b u rsary. C K M w a s supported by the Department of Veterans Affairs Merit R eview Program .
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