:\nbra Univ Veı Fak Derg. 49,177-182.2002
Electrotonic synapses İn young rat İnferİor colliculus
Ramazan BAl}, Gary G. R. GREEN
2,Adrian REES
2,David
.I.
SANDERS
2i Departmenı of Physiology. !'acıılly of Veıerinary Medicine. \1ııstafa Kemal University. Antakya/Haıay: : Dep~lrtıııcnl of
Physiology. Medical SchooL. Newcastle University. "Jeweastle
Summary: Biocytın was injecıed into neurons in frontal sliees of inferİor eollicııliıs (IC) from raıs. Eıghtecn oııt of 76
intraeellular injection of bioeytin inlü single neurons resulted in staining of more than one cel i (23%). Longer bioeytı n ınjectıon
times resulted in a higher incidenee and increased numher of coupled eclis. One minute of hıoeytin inJection resulted ın 16v;() ıncıdence raıe of eoupling. whereas 5-i(J minute of injeetion resulled ın 28%. There was no diff'ercnee ın ıhe incidcnee of dye coııpling when the sliee was ineubated in the bath for long or short period .., after the bioeytın injection. I-Iowcver. ıhe dye coııpling was eorrelated with the age of aniıııal; ıhe ineidenee rate of the eouplıng among stained cells was 2X"!r: ııı
ıe
slıce preparaııon, froııı 13 day-old rats. whereas the raıe wası
5% in theı
6 day-old ones. These results strongly suggesı the preselKT of eleeırotonıe [oupling beı\\/een neurons of 13-16 day-old rat inferior eolliculus.Kcl' words: Biocytin. elcetrotonıe synapse, inferior eollieulus. inıraeellular sıaining
Genç sıçanların kollikulus inferiör'ünde bulunan elektriksel kavşaklar
Özet: Sıçanlardan elde edilen kollikulus inferiör'iin ön kesitlerindeki sinir hücrelerine hiyosaytın hoyası enjekte edıldı. Yetıııı~ allı enjeksıyonıın on sekizinde hirden fazla hiiere hoyandı (%23). Enjeksiyon süresi lIzadıkça. hirden fazLı hüere hoyanması olayında ve hoyanan hiiere sayısında da artl~ gözlendi. Bir dakikalık enjeksiyon °..1..16. buna kar~ııı 5-10 dakikalık enjeksıyon ıse '/r2X'lık oranda hirden fazla hücre hoyanınasıyla sonuçlandı. Biyosaytin injeksıyonundan sonra keSillerin kayıt odasında \.,ısa \-eya L1lun'tırell İnklihe edılmesi birden fazla sinir hiieresı hoyanması oranıııda herhangi bir fark oluşıurmadı. Bununla hırlikte. hırden Lız'" hllClT
boyaıııııası olayı hayvanın yaşı ile ilişkıli bulundu. On üç günllik sıçanlardan elde edilen kollikulus ınfcriör kesitlerınde hıı or~ın 'j,,2X iken 16 günliik sıçanlarda Vk i5 olarak saptandı. Bu sonuçlar. sıçan kollieulus inferiiir sinİr hücreleri arasıııda elektrıksel kav~~ıkların varlığım hiiyiik oranda ortaya koyar.
Anahıar kelimeler: Biyosaytın. elektriksel kavşak. hücre içı boyama. kollikulus inferiiir
Introduetion
The inferior collieulus (ıe) is an auditory nucleus, in which all ascending and descending auditory pathways l11ake an obligaıory synapse. it is ıhought to be involvcd in inıegration of binaural sound information. Interaetions beı ween neurons in mammalian nervous systems were helievcd to be mediated cxclusively by chemical synapses, buı in recent ycars it has heen shown that elccırotonic coupling mayaıso exist in a numher of adult mal11nıalian brain structures, such as, hippocampus (34). supraopıic neurons (15), striatal neurons (6.16,24,28).
There are several ways to demonstrate existence of gap-juncıions, including elcctrophysiological techniques, e.g. simultaneous recordings from pairs of cells to test directly for electrical continuity (20). visualization of gap junclions, morphologieal correlates of electrotonic
coupling. using either transmission electron microscopy (TEM), freeze-fracture techniques (5). or immunoreactivity lO gap junctions (::l) or demonstration of dye transfer heıween couplcd cells through gap junctions, namely, dye coupling. The dye coupling method using dyes with low molecular weight «1000 Dalton), e.g., Lucifer yel10w
(33) and biocytin (12) has provided a parlicularly successful method and has been useel commonly as an index for studying electrotonie coupling (i 1.21.22.25. 36) Although gap junctions have not heen rcporıed 111 the
ıe,
a gene encoding connexin (Cx::l6). a gap juncııon protein, whieh is synthesi/ed hy neurons only, has heen found to he expressed in neurons of adult rat infcrim colliculus (9,28)In this study, it was aimed lo study ıhe possihlc presenee of electrotonie coupling in the inl'erim c()lliculus of young rats using dye coupling as an index
rm
gap junction.Materials and Methods
Dissection of anirnalFor intracel1ular staining. sliees from ral ınferior colliculus were used. Seventy-six Wisıar ralS heıween ı:1 to 16 day s of age were used for the expcrırm:nts. lmmediately after arat was killed by ccrvical dıslocaıion, the he ad was immcrsed in cold (4-8~C) oxygenaıed Na-free artificial eerebrospinal l1uid (S-aCSF) (soelııım
Ramazan Bal - Gary G. R. Grccn - Adrian Rccs - David 1. Sanders
iııns were replaeed hy sucrose (ın an equimolar basis) (2). Having reIlloved the scalp anel other soft tissues. the dnrsal naniuIll was eul in order to expose the hrain. The \\ho\c. hrain \vas rapidly remO\cd and placed in freshl)' oxygenaıed eold S-aCSF. The hrain stem containing the İnferinr eoııieulııs and superim eollieulus was freed from ıhe resi of ıhe hrain by ClH1İng ıransversally using a paır of scissors. Anoıher transverse euı was ımıde in the remaining bloek heıween ıhe inferior coııiculus and the cerebellum and ıhe hlock caudal to the IC was discarded. Af ter carduııy removing ıhe rine arteries o\'er the surface of the inferior colliculus, using a pair of fine forceps. the speciınen \vas Illounıed with a cyanoacrylate glue. with ıhe superİor eoııiculus end down, onlo a mounting block. This bloek was fixecl onto a Yibraıome tray, which was filled with cold conıinuously oxygenated S ..aCSF (3-W'C). Slices (300 ~ın in thick) were ıhen cul using a series 1000 YİbralOllll: (Technical Produets International Inc.). The slices \vere lransferred into fresh continuously oxygenated S-~ICSF The time heıween ıhe cervical dislocation and ıhe compleıion of slİcing WdS typicdllY !css than 7 min.
Elcct fophysioIogy
The slices were kepı in S-aCSF for at least 1 hour LO
~ıllo\\. recovery from lraumd. A slice was then transferred ıo the recording chamher. The slice was perfuscd at a rale of (j 7 ınllmin wiıh arıificial cerdırospinal fluid (aCSF) Clınıaining (in mM): NaCL. 124; KCl. 5; KH2P04, 1.2;
CaCl:. 2.4: MgSO~, l.3: NaHCO,. 2(j; glucose. 10 Aıı chemieals were obtained from BDH and were of AnalaR gr~ıde. The pH of aCSI-' \Vas 7.4 (bet\Veen 7.35 and 7.45) subsequenı ıo saıuration with 957, O:, sril. CO2 at :15~C.
The osmolarİly of the aCSF was in the range between .,O~ ..' 15 IIıO,ın. Sliccs were viewed ıhrough an Olympus microscope wiıh a ınaximum ınagnification of 50X and illuıııinaled from above using a fiber optic lighı source. For slable recordings. ıip resistance of the most suitable ınicroelectnıdes ranged hetween 140 and IRA MOhm. Buı only those ınİeroeIectrodes ıhaı had the ability lo pass currenıs of ?2 nA were uscd. The rccording were performcd using an Axoc\amp-2A amplifier (Axon Instnıments Inc .. Burlinghaıııe).
Biocytin injcction and histology
Intraceııular staııııng was made with micro. clecıroctes J"illed wiıh 2% solution oj" hioeytin in 2 M K-;lCel<lle, hulTercd ıo pH of 7.4 wiıh acctic acid. Ai the cm! of each sııccessful recording. biııeytin was iniecıecl into ıhe inıpalcd cell hy ionıophoresis using depolarizİng currenl pulses oj"
ı
nA al1lplitude wiıh d duty cycle of 2()O nıs fnr varying durdtiOlı betweenı
30 min. Intraceııularstaining was carried out if the cell had reSIİng menılıranc potentials more negative than -48 mY. Thc posilH'n of the injecteel neuron was noteel on a skcıch ol the slice. More than ten minuıes after the final inıeclion of bi(ıcylin. the slice was removed from ıhe rec(1rding chaıııbn and ıhen fixeel for at !casl 24 hours in 2'1('.gluıaralelehyde 2';; paraformalelehyde in O. i M sodıul1l phosphaıc huller (pH 7.4). The sliccs \Vere then ıerı in .,0';; SULTose ın ..;odıuııı phosphate huller (0.1 M, pH 7A) Im ah,ıut =.4 hnu!", lur cryoprntecıion and \Vere then cut inlo 50-(ıO pın secıions on a freezing microtome. The secıions were incuhaıed for 2-3 hours at rnom ıemperaturc or overııighı al
re
wiıh avidin D-I-/RP (Yecto!" Laboraıories) 20 ~l inı
o
ını soclium phosphaıe buller (O. i !'vi, pH 7.4) wiıh i "f' ıriton X-I 00. Sections wc re rinsed in soctium phosphate hulTn (O.] M. pH 7.4) and the HRP was then n;;ıcted using ıhe DAB-nickel/coball inlensification meıhod i i). Seelıons were rinsed \vith sodium phosphaıe bulleı (i) i ;vl. pH 7.4) and were ıhen ınounted onlo gelaıine-nı;ııed slıdes. After elrying ıhem in air, ıhey were dehydrated. coıınıcr stained with neutral red and coverslipped.Results
Twenty-seven out of 7(ı hioeyıin inıeeliı)ns ınıo neurons in both the cortex regions anct ıhe central nucleus of the IC resulled in staining of more ıhan one ccl\. dye eoupling (Figure I). In so me cases, hoıh ıhe dyecl1llplcd ceııs were equally weıı stained so that fine slnıctures .;uch as deneıritic spines and fine dendritic arb(ıı-jz.ations wne obvious in the coupled cells. in oıhers ıhe neurnn int,ı which biocyıin was iniecıect could he Ix:ııcr disıinguished by its darker appearance rcsulling froııı hellLT filling.
Three dimensional analysis of the coupIed cclls showed that the point of Clmtacl betwcen ıhe dyc-couplcd neurons appeared to vary and included dendro-dendrjııc. soma-somatie and dendro-somatic conlaeh ~ıııcl their dendritic arborisations ovcrlapped exıensıvely ıhgure \ ancl 2). The mımhcr oj" coupled cells re'u!ıing fmııı each injeeıion and lhe incidcnce of Cl,uplın:~ \\as corrdıteli \vith ıhe duraıion of the biocyLİn injeclions: ıhe longer injeetions gave rise lo more couplcd cclls anel a higher inciclence of coupling (rablc i). A bioeyıin inıecliOlı o!" one minute eluraıion. ıhe ıypical duraıion f(ır rilIing a cell. resulled in only two coupIed ceııs at a 16
ı';1,. incidencc.
whereas an inieclion tiıne of 5 lo LO minııle gmc rİsc lo up to four coupled ceııs at a 2X'i;, incidenceFigure :1 shows ıhe physiologie<ı1 heh~ı\'ıor ol ıhe eoupled neurons. There \Vas no cnrrc!aıion ı'et\\.ceıı coupling anel different firing p;ıtlerııs: ıhe dye c(lllplilıgs were seen in neurons with onset. regular (hgure .,ii.) ;ıml
Ankara C'niv Yel Fak Derg. -i9. 2002 17()
81
A
i
~~ui~
I
LV
\1
5()ms
-55 mV -
'\----i
Figure 2. Three c1lınensional analy'!.s or IhL' coııplcd cclls. .'\ Tlıere arc two cel!.s stained with hiocyl1n ınjcclion. Thı'cc YICW' or the eoupled eclis t<ıken rroııı dillcrent roı~ıııon' anglcs wnc shown. B: Another coupled eells were show il al ıwo dıllerenı roıations. The somas or the eells \Yere indicııcd hı, the ~ııTOWS. Tlıere are numerous poinı of possi hle eoıılacls hcl WCL:1li\i'"
cclls. which are shov.ıı hy [he arrowhe<ıels.
FigıırL' I. A phoıoıııicrograph or coııpleo neun.lIls slaineo with Illıracellıllar hiocytın III ılıe inkrior collicıılus. Intraccllular ıoıııophorıııc ınieeıion or hıocyl1n into a singlc ncuron resulted ın sı~ıııııng or nlOIT ılı~ul oııe eell. 1'wo LIye coupled neurons are ındıcııcd wııh arrows. Cıiihration: 33 ı.ılll.
Figure 3. Elcctrophysiologieal rcsponsc' recordeel rmııı iwo
couplcd eells. A: In response LO 02 n/\ cılıTeııl. ıhe cL'11lıleLi ~ıelion poıeııtial regıılarly. B: .J\noıhcr uıupk" ccll ıespoııdcd wiıh an acbrıing finng paııern ın rc.spoıısc ıo+().2 n.'\ nlITCIlI
whether neighhoring eells picked up ıhe dye from ıhe extraeellular space. the injection \Vas earricd ouı Im the standard duration and then it was noted that no oıher eell \Vas stained. In another six control experimenıs hiocyıin was injected into the extraeellular space through microclccırodcs of similar ıip resistdnee kır i() ınin in t\VO sliccs with the same ionıophoreıie CIIITCn!s. Ncı intracellular uptJke of the dye \\'as ohsel'\u\ ;ıs ~Lrcsult <ll'
adapting (Figure 3B) temporal firing patterns. In order to ıesI the possihility that this corI'ciation might be the rcsult of keeping ıhe slices Iımger in the bath after in.iection of bıocytin herore fixation and was not the result of the Iııııger hiocyıin injection. the injected sIices were kept m the baıh fm 1\\'0 dillerenı time periods. As show n in Tablc 2. no eorrelation \Vas found bet\veen long and short periods of incuhation and wiıh the pattem and incidenee of ıhe eoupling.
The dye coııpling was found lo he corrclated with ıhe age of aninıa!. The incidenee rate of the eoupIing anıı.'ng sıained celIs was 2X% in
ıe
sliee preparations ıaken fml1l J3 dayolel rats. whereas ıhe rate \Vas iy/, in thc 16 day-old group (correlation codfieient=
-o.t)ı).In ımler ıo prevenı artdacıua] coupling. the following precauıil1ns \Vere taken: onlyone nemon was injected with bioeytin in cach collieulus or the stajned cells were ~L!!cas! i mm ap art in a colliculus and penetrations were temıinated if the acıion potential amplitude fell belowsm,
of the aelion potentia!'s initial amplitude or if the ccll stopped firing. Ho\Vever. in !'İve eclis. it \Vas clear from ıhe recording ıhaı ıhe nıicroe1eetrode had come out of the eell during the hiocytin injeelion. In order to testB
-61
i
i i
i
i
i ~
180
Green - Adrian Rees - David J.Sanders Ramazan Bal - Gary G. R. J
.' f dye eoup\ıng. The cileel of chıratıon of hioeytin ınjeetion on number and p,ıttcrn o .
'Lıble
ı.
.
Total Numher wıthDuralion of lota\ . f 2 eoupled number numbeı o
bıoeytin injcetıon eouplings eells of staining 7 4 25 'i,IO mın 6 6 22 3 ınin 5 5 i ıııı n 19 ;\Iuınher with 3-4 eauplcd cclls 3 iIlerdcnee ıaıe of ~ouplıng 'lXS;, :2(/~(~ 16S; Perıocl of incubation in bath aftcr starting injection prıor to fixation 20-30 min 50.1 ıO min Total ımmber of staınings 51 25 12 6 LO 5 2 Incıdence ralC of coupling 13.5% 24%
this proeeclure and conversely no extraeellular dye was ohserved after single eell inieetions when the preeautions given ahove \\iere followed.
Discussion and Conclusion
The results reported here of dye-coupling among infcrior eollieulus eells supparıs the existeııee of clecırieal eoupling among these eclis though direct denıonstratioıı of elcetrotonie eoupling is absent. On the basis of Ihe control experiments and the preeautions taken. it is unlikcIy thaı bioeytin Ieaked out of the eclis and was subsequently ıaken up hy neighhoring neurons or ıhat there was inadvertent staining due to possible ıCakage during previous hrief ecll penetrations while searching for a stahlc eell. Furıhermore, the ohservation thaı somc of the dye-eoupled eclis were more than 0.1 ının apart rules out this possihility, beeause there were a numher of unstained eells hetween these stained Ileurons.
On the other hanel. it eould he argued iıı the dye-eoupled eases that dye erossed ehemiea! synapses. This arguınenı has. in faet. been suggested to explain the dye eoupling oecurring as a result of injeetion of Proeion yellow into singlc neuroııs but not Lueifer yellow or bioeytin revealed eoupling (19). it is reported that hıoeytin does not eross ehemieal synapses (l8). The observation that the pattem and incidence of dye eoupling was nol found \o he eorrelaled wiıh Ihe ineuhation duraIİon of ıhe sliees hetween hioeyıin inieelion and fixalion eould he regarded as evidenee againsı this possihility. Therefore, on Ihe basis of these data presenıed here and with the light of ıhe evidenee ıhat the gap juneIİon gene (Cx36) has heen expressed in adult rat inferior eollieulus (9,28). it is suggested ıhaı gap .iunetions prohahly exisı in the rat inferior collieulus, in
the same way thal exisıcnce has been suggesıed on an ultrastructural basis in coehkar nucleus (26.32.33) and primary auditory eortex (30,31). Gap Junelions mal' he an importanı physiological mechanism İn ıhe audiıory paıhway sinee conducıion beıween eells through gap .iuncıions is fasıer ıhan ıhal ıhrough ehemicı] synapsis. The hmetioning of vertehraıe hearing is enıeia!!y dependent on the ahiliıy of neurons tu eııende timing Human can distinguish 2e differences in the location of sound source in the horizonta! plane, \",'hich İndieates ıhaı ıhey can deıecı differenees of iimicroseconds (23). This ability may be dependent on gap junctional conductance
it has heen shown ıhat gap junctional eonductanee is not a static mechanism but a dynamie one eontrolled by several factors. including neurotransmittcrs. pH. voltage_ adenosine 3. 5-eyclie monophosphaıc etc. (ı6,28). For example. dopamine and histamine. neurotransnıiıters. whieh are considered lO have modulatory acıions on chemical transmission in many slnıelures of ıhe mammal-ian brain, have been shown lo cnhanee e!eeırolonic coupling via gap .iunetions (15,24.271. Although iı is nol known whether dopamine functions as a nellrotransmiııer in the
ıe
of rat, ıhere are repons thaı growing numbers of neurotransmitters have similar modulaıory erfeeıs. as re-viewed hy Jefferys (16) and Perez Velazqucı and Cari en(28).
However, it is pure speculaıion to allrihule any funeıion to the gap junctions at this slage. Therc are growing evidenee which suggcsıs 1hat gap .iunclions in malure mammalian nerveous system muy be a residul' iert from their role in embryonie development: they may be present in a dormanı stale waiting to be aetivaıed İn abnormaI hrain rhyıhms (8.13.14.17,35) They nıİghl have also been reaclivated from a dormant developmenı
Ankara Üniv Vet Fak Derg. 49. 2002 i X i
meehanisnı hy the physieal danıage to the neumns during s!ıeing. wiıh dendmıomy (7,14). Electrotonic coupling is widcspread throughout ıhe developing nervous system huı theyare ıhoughı to he transicnı, disappearing at the end of first few postmlta! weeks in most types of neurons (4.R.
ı
7,29). Thcir function in the early development may be ıhe transport of messenger molceules between neurons. patlcm formation, neurogenesis, neuronal diffcrenıiaıion and eireuit fornıation (R.I O.2R,35,.17).ıı is eoncluded that the gap juncıions revealed by dye eoupling in ıe preparation of
ı
3-16 day-old rats are nıore likely lo he a residue len from their role 111 embryonic developmenl. There are two pieees of cvidence for this statemenl. first, the incidenee rate of the coupling among stained cclls was higher in ı 3 day-old rats ıhan thal in the 16 day-old ones; this age may ((lincide wiıh the transition to normal ehemieal conınııınieaıion. Second, expressiOlı of the gene for Cx36 in adulı rat ıc is 100v compared lo sonıe other hrain areas in whieh gap .iunctions are proved to exist.Acknowledgements
WC are gratcful to Dr. Steve MeHanwell for unliıniteduse of his freezing microtomc.
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17. Kandlcr K. Kalz LC (1995): Neıımnal c(llll'lllıg lIııci wl-COUp/lılgiıııhe del'f'loplııg Ilavous .1'.1"111'117.Cmr Opın I\c-urohioi. 5. 98- 105.
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Exp Braın Res. 35. 37
ı
-385.20. Llinas R, Yarom Y (1981): F:!nımphrswlogı' of mll!J1-!J1alialı ııı/i>rior oliı'arr Ileuroıı£'s iııı'ıım. DII/ereııl ırpes oj vol/(lge-depeııdeııı ümic uıııduet(llıces. J Physiol. 315. 549-567 .
2 I. MaeVicar BA. Dudek FE (1981): Lif'cimıoıııc colıplUlg helwe£'ıı prramidal cells: adirtTI df'!J1o!1.llmtlolıIII ml
/l/p-pocmnpal slices. Scieııcc. 2.13. 7X2. 7X5,
22. Mann-Melzer 1', Yarlım Y (i ')99): EinlmıolılC uIIl/JIllıg ilılemCls ıviıh iilırilisic prolJerlin lo geııemle sllIflıl"mllZnl auivily IIIc£'l'£,I)f'llar IIf'llVork.\ of illllIhiıol'\' 1))lel'lleUmIıS .i
Neurosci. 19.3298-3306
23. Oerlcl D i
ı
(97): Ellcodilıg of limiııg /ilılıe !lmiıı .\Iem ull-diıorr Ilııclei o/l'f'I'/£'hmıes. i"cıımn. 19. 959-')(,2.24. Onn SP, (;racc AA (i 994): /Jre coUpllılg heıwı>eıımı slri-a/(llııeıırmıs recorded iıı I'iı'o: coml}(lrımeıııul orgwıızalıoıı aııd in odıı la ıi(l{ı !Jrdoıııııniıı!:'. J i"cıırophySlul. 71. ~L)17.
1934.
25. Onn SP, Graee AA (1999): Allnalloıı' m elnlllll,/ıI.IIIIIII-gicul uctil'itl' uııd dı'e col/pliııg o/slrtlllul sımıı' ııııdU.lfllIIl
Il£'l/rons iııdOpıılııilıe-deııel'l'(/{ed rul sınııııııı! recol'ded III
l'il'(I. Syııapse. 33. 1- 15.
26. Pcinado A, Yuste R. Katz LC (ı993) Gaıı jUlICIIIIIWI COlnlıııınicuıiolı und ıh£' del'£'lopl11l:'ll/ "Oout! conııls iıı ııe-"COl'In. Cerch Cortex. 3. 48~-49X.
27. Pereda A, Triller A, Korn H, Faher DS (1')92)
D,,-p(//niııf' £'Ilh(//ıees hoıh elecırolıınic colı{'lmg ııııd dıeıııiert! £'xcilalorr posısrnupııc 1}()lelılw{' lll ııUwd \I'lıl1/lses
182 R"ımvan 13,,1- Gary G. R. Grccn - Adrian Recs - David 1.Sandcrs
28. Perez-Vclazııuez .IL, Carlen PL (2()()O): GupjUllC/lOll ... .\\ lıehl'Oll1 uııd Sel711l'es. Trcıııls Ncul'Osci. 23. 68-74. 29. Rozental R. Srinivas M. Gokhan S, Urhan M,
Der-miclzcl R, Kcsslcr .lA. Spray DC, Mehler MF (2000) Fell71JIi1'11i eıpl'es.lloıı or 1If'III'OIlUI coııııexiııs dıınııg
hip-Pocull7/,lıloıııogell\' Braııı Res 13l"<lınRes Rcv. 32. 57-71.
]11. Smith
nE.
Moskowitz:'i
(1970): Ulımsll'lıcrlıl'f' or 100:el'LV oi ıhe Pl'ill1lllT ıııililion' (ol'ln or ıhe slllıil'l'f'! 117olıkey.
!\clIn.ısı:ıcnce. 4. ]40-]59.
] i Sotclo C, (;enlschev T, Zamora A.i (1976): Gııp JIIIIC'
/1011' iıı l'elıll'ul (:I1(:hlf'1I1' IıI1e/elı.1 ııl'ıhe ml. A pııssihle ne\\' e \1/117/'11' ııl' elecrmıoııic 1{IıIC/ilılı.l iii ıhe eNS. ;-":c. 1Il"oscıcncc. i.5.7.
}2. Sotdu (~, Korn H (i LJ7X): ,\1orphn/ogicu/ ("orrelates oL
(~{eLınelıl ııııd oıhel' ılıleI'U(/lOII.I ıhnmgh Imv-l'esisıuIıCf' P"I/lI'.'''.I'.1 1)<'1"'.-1'1'11III'UI'IIIISol' ıhl' l'el'lehmıe ceıııral
111'1'-FIIliS SI'.III'II7 ını Rcı CylOl. 55. (,7. i 07.
:n.
Slewart \V\V (1978): Fwıc:ıi{}/wl C{}/llıl'clilıııs helwl'eıı ulis LLSI'eı'euled hıdl'f'-COııplıııg wilh (i highll' jluol't'.\cell/ 1WI,hlllIlhl/7idf' lracel'. Ccll. 14. 741-759.3-1. Taylor CI', Dudek FE i
ı
0S2): A ph"ıolıı,~/(III ıesI {ii/' I'leUl'Ololıic couphııg 1)('1,,'l'elı CAi l'I"'UI/7«III/ c(~"s ii! ml hipPoC(//l1pul shces. Brain Rcs. 235, ]51 357.35. Warner A (1992): Gup jUlıcliolıS iıı df'l't'IIIIJ1'1I'III-'U
llel'l-pelIiı'I'. Scın Ccll Biol. 3. S 1-91.
3(ı Yang QZ. Hatton Gl (1999): Nıınc ııwlı' I'{ti c(;'\1I).
dl'pl'ııdeııı IIl1'chOlllsl/7.1 IIICJnıses d"e eııııpl,l[.~ illit! 1'\.
cililhilin' o/rul .ıupn/IJIJ/lc lıuc!t'Iı.1l1eUI'OIlI J ~Clıl"o,CI. 1~.
4270-4279.
37. Yuste R, Pcinado A. Katz LC 11(02) NewııliııI dli/llillll' iııdeFl'lopolg IIf'Ocol'le.l. Scicncc. 257.ôô5-(ı(,l)
ReCl'il'ed 13 Sı'pıemha 20()1 / Acupınl 28,VOlt'IlI"t'!
:znoı
Correspondence address:
Yl'd. f)oç.'. DI'. Nll/ncc.un /3ul
Velt'I'inel' Fllklillesl, Fltmlo{; AnulJl/llIı nt/It MııSIII/U Kell71l1 Üııil'ersileSl, 3/()40 A1!Iokm//-Iul(iI bııııil: rctll71171iiı.hill {£ıle.\'ciıf'. Ci,117/I'hul@m/,u.edu.lI'
