The Journal of Physiology Water Transport Controversies Special Issue terminals spontaneous GABA release from rat medial preoptic nerve otg-ae Ca voltage-gated of activation the by triggered is release neurotransmitter potential-evoked action that established well is view the is a process that has been extensively studied. Accordingly, The release of neurotransmitter from central nerve terminals Cell Membranes, Institute of Cytology, Russian Academy of Sciences, St Petersburg 194064, Russia Department of Integrative * Medical Biology, Section for Physiology, Umeå University, S-901 87 Umeå, Laboratory Sweden of and I † * *† and *, Staffan Evgenya *†, Johansson Malinina David Haage Michael Druzin Dual and opposing roles of presynaptic Ca © The Physiological Society 2002 Journal of Physiology epilepsy (Hirsch Ca to the evoked neurotransmitter release, the requirement of and neurotoxins (for review see drugs Bouron, 2001). neuron, In used contrast the clinically neuropeptides, of neurotransmitters, activity electrical the including various means by modulated be can spontaneous release release, neurotransmitter potential-dependent action Like morpi ltrl ceoi (Andjus sclerosis lateral amyotrophic of pathophysiology the in involved be may transmission (Rhee Miles, 2000) and the modulation of nociceptive transmission 2000), the regulation of neuronal impulse firing (Cohen & (Jensen plasticity synaptic maintenance of synaptic structures (McKinney al. et (Hirsch information synaptic carry may release of type this that suggesting presented, been recently has evidence neuro- However, understood. well yet spontaneous not is release transmitter the of significance physiological The 1955). released Katz, & Castillo del 1952; 1950, Katz, & also (Fatt are transmitter of spontaneously in the absence of presynaptic impulse activity quanta that known Miledi, 1968; Miledi, 1973). However, it has also long been and local rise in intraterminal Ca 2+ o tigrn te cin potential-independent action the triggering for 1999; Staley, 1999) and play important roles in the in roles important play and 1999) Staley, 1999; t al. et washout of Cd of washout GABA- inhibitory (miniature) mediated postsynaptic currents spontaneous (mIPSCs). Similar effects of on mIPSC frequency frequency were recorded upon the increased paradoxically blockers rnmte rlae y xctss B suyn dsoitd rotc ern wt functional with neurons preoptic dissociated for signal studying adhering nerve terminals, we here trigger show that presynaptic Ca By a exocytosis. as established by well release is transmitter terminal nerve presynaptic the into influx Calcium S-901 87 Ume author Corresponding coupled to Ca parallel Ca parallel n h cnrl f pnaeu tasitr ees. hs apiain f aiu Ca various of application Thus, release. transmitter spontaneous of control the in (2002), (Received 18 December 2001; accepted after revision 23 April 2002) 00. n diin sotnos synaptic spontaneous addition, In 2000). et al. 2+ 542.1 hnes n sbeun Ca subsequent and channels 1999; Cossart , pp. 2+ å , Sweden. 131–146 influx through channels coupled to the exocytotic machinery and through channels through and machinery exocytotic the to coupled channels through influx 2+ 2+ -activated K t al. et or EGTA from the external solution. The results are explained by a model with model a by explained are results The solution. external the from EGTA or Email: [email protected] S. Johansson: Department of Integrative Medical Biology, Section for Physiology, Ume Physiology, for Section Biology, Medical Integrative of Department Johansson: S. 2+ 99 Kombian 1999; concentration (Katz & et al. + 2001). t al. et channels at a distance from the release site. et al. 97 and 1997) 2+ 1999), influx tal. et ertasitr ees i ls cer Tu, n several in Ca of independent was release Thus, spontaneous studies, clear. less is release neurotransmitter gated Ca terminals, the role of Ca nerve central from release neurotransmitter spontaneous of regulation of mechanisms the clarify to efforts intense release. Our results lead us to the conclusion that Ca by one predominant type of high-threshold Ca high-threshold of type predominant one by mediated mainly is release GABA depolarization-evoked that shown have we preparation, this of studies previous dissociated to In adhering (MPN). nucleus preoptic medial rat the from neurons terminals nerve from release GABA spontaneous investigate we study, present the In different types of presynaptic Ca presynaptic of types different release was influenced by Ca 1997; Brussaard example see also Capogna Ca voltage-gated low-threshold blocking by changed be also see Rhee see nlx a b idrcl, eaiey otold i Ca via controlled negatively indirectly, be may influx this frequency, release spontaneous high a for crucial is effects of Ca potentiating paradoxical seemingly the describe we Here, L-type whereas Q-type, or P- channels are not N-, involved (Haage either be may that opposing partly roles. Although and influx through dual some Ca play may terminal presynaptic the into ny eky eedn o Ca on dependent weakly only 2+ channels (Bao 2+ 2+ et al. et influx plays dual and opposing roles channels (Koyama 2+ channel blockers on the spontaneous GABA 2000). The rate of spontaneous release can release spontaneous of rate The 2000). et al. et al. 2+ 1999), but in other studies spontaneous 2+ influx for 1998) or high-threshold voltage- is still not clear. et al. 2+ et al. influx through a number of 2+ 1993; Vaughan & Christie, 2+ et al. 1999). However, despite Ft & az 15; for 1952; Katz, & (Fatt 2+ DOI: 10.1113/jphysiol.2001.015610 channels (for example (for channels å University, channel 1998). onic Channels of www.jphysiol.org 2+ 2+ channels channel 2+ influx 2+ , or , 2+ The Journal of Physiology Water Transport Controversies Special Issue n eprtr 2–3°C). temperature (21–23 room at performed were good experiments All ms. in was, 10 than less time cases, exchange solution the by and controlled valves, was solenoid solutions between Switching solutions. test of or solution extracellular standard of application continuous eight-barrelled or four- a pipette positioned of 100–200 outlet common the with system, glass pipette (see Edwards extracellular solution (for composition see below) applied from a of surfaces standard the differential of stream a by cleaned case, were neurons identified visually latter and the In optics. objectives contrast interference water-immersion microscope with FS Axioskop equipped Zeiss upright an using studied were slices in neurons whereas microscope, 25 Axiovert Zeiss inverted % were accepted. about Dissociated 20 cells were studied using an monitored repeatedly during was the experiments. step Slow changes voltage in series mV resistance less than _5 a to response in changes in series resistance, the time course of capacitative current 6 Johansson 16; = several neurites of length up to about 100 10–15 measuring isolated were 1991). No enzymes were used. cells The dissociated cells had cell the bodies case, of the slice at the site of the latter medial preoptic nucleus (cf. Vorobjev, the In mechanically by application of a vibrating glass rod to the surface used. were 250–300 slices terminals, synaptic adhering with neurons ~150 slices, intact in neurons from recordings For prepared. were area the brain was removed and coronal slices containing the preoptic g) male Sprague-Dawley rats were killed (50–120 by decapitation, – M voltage-clamp 2–5 under (Rae measured were technique perforated-patch B amphotericin the using conditions currents Whole-cell Electrophysiology described been have preparation cell elsewhere (Karlsson for used methods The Cell preparation ethics committee for animal research. Ethical approval of the procedures described was given by the local METHODS Ca other through influx 132 eodn ehiusyeddvle f1 M 3 recording techniques ± yielded values of 18 and preparation same the with estimation previous cells, studied the of all for determined not was resistance series the Although amplitude of the recorded signals under steady voltage conditions. was notused,duetoitsintroductionofextranoiseandthesmall personal dB). processor-based Series resistance compensation kHz (_3 pass filtered Pentium at 2–10 or 486 low- were which signals, electrical record to used were computer a via controlled the and Instruments) Axon interface, from all 6.03-8; 1200 (version software pCLAMP Digidata a amplifier, 200B Axopatch has and been (1992) subtracted in Neher all potential by values given. described An Axopatch as 200A or measured was potential extracellular solution (see below), were used. The liquid-junction xre b efcs n h mmrn ptnil i Ca via potential membrane the on effects by exerted dependent K et al. et m V m thick slices were used. For further dissociation of single , when filled with intracellular solution and immersed in 1991). Borosilicate glass pipettes with a resistance of resistance a with pipettes glass Borosilicate 1991). + channels. t al. et m a ter ogs ae, n otn n or one often and axes, longest their at m et al. 01. ute, o vi sde large sudden avoid to Further, 2001). et al. m 1997; Haage m from the studied cell, was used for 2+ 1989). A gravity-fed fast perfusion channels. The latter control is control latter The channels. et al. m m. 1998). In short, young V ma ± (mean M. Druzinand M. others Druzinand m S thick m . E . 2+ M .; - oxygenated (95% O oxygenated (95% from neurons in slice preparations, the extracellular solution was recordings For NaOH. with 7.4 to adjusted was pH and added, routinely were Israel) Jerusalem, Labs, Alomone from or Sigma eue h bcgon nie We Cs When noise. background the reduce 10 glucose. Glycine (3 Glycine glucose. 10 lcnt, . NC, . MgCl 1.2 NaCl, 3.0 gluconate, etd txn aai, acspie caydtxn and charybdotoxin calciseptine, apamin, toxins peptide rm sok ouin 1 m (10 solution stock a from dihydro- The prepared were 8644 K Bay and Sigma. nimodipine nifedipine, pyridines from were (TEA) and tetraethylammonium paxilline nimodipine, nifedipine, methiodide, bicuculline Alomone from Labs or alternatively purchased from Latoxan (Valence, were France), Bay K 8644; MVIIC and GVIA conotoxins iehl upoie, a add o fnl ocnrto of concentration final a 120 to added was sulphoxide), dimethyl from a stock solution (6 mg amphotericin B dissolved in 100 in dissolved B amphotericin mg (6 solution stock a from prepared (Sigma), B Amphotericin CsOH. with 7.2 to adjusted hrbooi, oie eu abmn 00 wv ws also was w/v) % added to (0.01 both experimental and with albumin control solutions. experiments serum In bovine charybdotoxin, solution. extracellular in dissolved toxins were the peptide in The as solutions. concentration test same dihydropyridine-containing the the achieve to to added solution control routinely was dimethylsulphoxide experiments adjusted to 7.2 with KOH. As an alternative to K used for filling the patch pipettes, contained (m frequency of spontaneous synaptic currents h sadr etaellr ouin sd s oto contained control (m as used solution extracellular standard The Solutions and chemicals tews) ih Cs with otherwise) stated (unless experiments following the made we mIPSCs, pipette-filling solution. To better quantify the frequency of the membrane of composition ion the on depends also and potential with somewhat varies which noise, current The adhering. are frequency of detected mIPSCs terminals depends on the background nerve presynaptic which to neurons, MPN dissociated the from recorded be can ‘miniature’ GABA-mediated postsynaptic currents (mIPSCs) (Haage reported earlier As Organic Ca RESULTS as presented are data The standard experiment. error of mean ± ( kept means each and level in noise the constant above set was currents postsynaptic inspection. The amplitude threshold for detection of spontaneous using Clampfit and Mini Analysis software with consequent visual spontaneous currents were detected were and counted semi-automatically presentations Analysis (Synaptosoft, Inc.) graphic and Origin (Microcal Mini Software) software. The Instruments), of (Axon Clampfit production using performed and analysis All Analysis eaiey ag diig oc fr Cl for a force give driving to chosen large was relatively voltage The mV. _4 at clamped voltage cell postsynaptic the with and noise, the background of reduction significant in resulted which solution, m M g amphotericin B per millilitre intracellular solution. The solution. intracellular millilitre per B amphotericin g : 3 al 50Kl 10 CaCl 1.0 KCl, 5.0 NaCl, 137 ): 2+ channel blockers increase the 2 , 5% CO , 5% m + M elcn K replacing ) and tetrodotoxin (TTX; 2 (TTX; tetrodotoxin and ) 2 ). The standard intracellular solution, M 2 10 GA 1 Hps p was pH Hepes; 10 EGTA, 1.0 , n iehlupoie. n all In dimethylsulphoxide). in S t al. et . E . 2 M 12 MgCl 1.2 , .), unless stated otherwise. + n h ppte filling pipette the in 98, spontaneous 1998), + _ a ue, H was pH used, was ihu risking without M + 2 ): 140 potassium , Cs 1 Hps and Hepes 10 , J. Physiol. + was used to M m , from , 542.1 v m - l The Journal of Physiology Water Transport Controversies Special Issue ioiie ee u t GABA to due were nimodipine by triggered events current all the that suggesting nearly mIPSCs, abolished reversibly methiodide bicuculline m 140 with perfusion transient by terminals presynaptic the of depolarization by evoked be can currents synaptic of burst a described, earlier As limit for mIPSCs was a pA. peak amplitude of ~5 at positive voltages. Under these conditions, the detection directed outwardly also thus and cations by non-selectively post- carried commonly are latter excitatory the since currents, miniature synaptic for mIPSCs the mistaking hratr h efc dcie t a oe tay level, steady more a min. The subsequent addition of 100 to reached after ~1 declined effect the ( from Hz thereafter 0.8 ± 3.8 2 many-fold of value peak Fig. a to Hz increased 0.13 ± 0.43 transiently frequency 1 (Fig. markedly increased iln slto; e Mtos t a ouin containing solution 100 a to Methods) see solution; filling with control extracellular solution (using standard pipette perfusion from switching Upon here. described mIPSCs 1998), the situation is markedly different for the spontaneous J. Physiol. the L-type Ca L-type the depolarization-induced synaptic current is not affected by recorded. was frequency reduced not a % did 8 in and significantly, frequency change the % 19 in whereas nimodipine, to neurons clearly was it cells, all in seen not was frequency mIPSC the on 2 (Fig. m Figure 1. Ca MVIIC (1.0 Nimodipine (100 dissociated neurons increase the frequency of mIPSCs in Currents presented in membrane mV. potential clamped at _4 Current recorded with the postsynaptic resulting increase in mIPSC frequency. at the time indicated by arrows. Note the were added to the external solution, starting recorded from the same cell, (1.0 two different cells. M A A nimodipine, the frequency of mIPSCs unexpectedly obtained in the majority of cells. In 73% of the 350 the of 73% In cells. of majority the in obtained . h pa ws ece wti 1–0s and s 10–20 within reached was peak The ). m ). Although the potentiating effect of nimodipine of effect potentiating the Although ). 542.1 tested the mIPSC frequency increased in response M , C ) and calciseptine (1.0 2+ m 2+ channel blocker nifedipine (Haage nifedipine blocker channel M , channel blockers B ), m M v , -conotoxin GVIA A A ), and v A -conotoxin B C . n vrg, h mIPSC the average, On ). were and m M M D A , D from C. lhuh the Although KCl. eetr activation receptor ) Dual roles of presynaptic Ca n 16; = et al. et m M o u peaain f iscae nuos w applied we 100 neurons, dissociated of preparation our to To rule out the possibility that the observed effect was due pyridine Bay K 8644 (10 8644 K Bay pyridine oetae mPC rqec ws eae t a lc of Ca block a to related was frequency mIPSC potentiated .1Hz, ± 0.31 (0.73 frequency baseline s 60 previous the twice > was Hz) 0.65 ± (1.76 frequency peak The clear. was nimodipine to reaction the reversible, poorly and prominent less was (see Methods). Although the increase in mIPSC frequency ic Ca Since steep concentration gradient reaching the recorded neurons. less and slice the into penetration slow a with solutions, related to the more restricted diffusion of the experimental probably are preparations slice the in washout of course time slow the and frequency The mIPSC in conditions. increase smaller physiological under seen be also may frequency mIPSC on nimodipine of effect potentiating large-conductance Ca Fagni currents: type above effects of Ca of effects above block K block may also 8644, K Bay agonist the as well as nimodipine, yiie Ca pyridine number of earlier studies have demonstrated that dihydro- on K on effects dihydropyridine-mediated by explained be could findings our that possible seem may it Although 1997). h mPC rqec i al he cnetain tested concentrations three all in frequency mIPSC the increased nimodipine antagonist the whereas mIPSCs, of 2+ m + channels. First, application of the agonist dihydro- agonist the of application First, channels. M 2+ channels, several lines of evidence suggest that the that suggest evidence of lines several channels, nimodipine to MPN neurons in a slice preparation + influx n 2+ currents in some, but not all, preparations (A- preparations all, not but some, in currents = 8; Fig. 2 Fig. 8; = s nw t tigr rnmte rlae the release, transmitter trigger to known is 2+ hne atgnss eg nfdpn and nifedipine e.g. antagonists, channel 2+ B et al. et channel blockers were surprising. A surprising. were blockers channel 2+ ). Thus, the results suggest that the that suggest results the Thus, ). -activated currents:Avdonin m 1994; Mlinar & Enyeart, 1994; Enyeart, & Mlinar 1994; M ) did not affect the frequency the affect not did ) et al. 133 The Journal of Physiology Water Transport Controversies Special Issue rqec. hs apiain f 1.0 of application mIPSC the Thus, potentiate frequency. did all which blockers channel K on effect blocking the pharmacology, unusual of currents K rectifier-like delayed on report one in since channels 100 and 10 (1.0, 134 1991). Stronger evidence for an effect on Ca uig u a iet fet f iyrprdns n K on dihydropyridines of effect direct a out ruling Ca general for all dihydropyridines, but only for those that are not is frequency mIPSC on effect potentiating the Thus, (in 4 of 7 cells tested; not further quantitatively analysed). nifedipine (100 nifedipine provided by the use of other, non-dihydropyridine, Ca non-dihydropyridine, other, of use the by provided VI, bokr f - P ad -ye Ca Q-type and P- N-, of blocker a MVIIC, lc b bcclie ehoie Fg 1 (Fig. methiodide bicuculline by block baseline, previous of % 54 resulted ± in an increased frequency of mIPSCs (296 iia,atog mle,efc 14±2 %, 21 ± (144 effect smaller, although similar, seen at the application of the N-type Ca + 2+ channels was not shared by Bay K 8644 (Valmier 8644 K Bay by shared not was channels channel antagonists. This is, however, not completely dissociated neurons ( (1.0, 10 and 100 and of BMI (100 caused by nimodipine and the block caused by addition of bicuculline methiodide (BMI; 100 (BMI; methiodide bicuculline of addition by caused block the and nimodipine by caused A (100 number of mIPSCs was measured during 10 s intervals in 17 cells. 17 in intervals s 10 during measured was mIPSCs of number Figure 2. Effect of Ca membrane potential mV. was Error clamped bars at indicate _4 cells ( , change in mIPSC frequency in dissociated neurons caused by nimodipine (100 nimodipine by caused neurons dissociated in frequency mIPSC in change , m M m n ), also potentiated the mIPSC frequency mIPSC the potentiated also ), m M 9). Note the lack of = effect of Bay K 8644. ) also affects the mIPSC frequency. Note the similar, although smaller, reaction to nimodipine as in M n (i. 2 (Fig. ) = 10), which were also sensitive to sensitive also were which 10), = m m M M ) or Bay K 8644 (10 ) on mIPSC frequency in 10 cells, measured as in A C ). The number of mIPSCs was measured as in . nte antagonist, Another ). 2+ channel blockers on the frequency of mIPSCs m 2+ M channel blocker B 2+ n 2 and channels was v 2+ -conotoxin n m channels, M = 7) was 7) = M. Druzinand M. others Druzinand ) on min) mIPSC in frequency the (measured same for group 1 of D et al. et . A ). 2+ D, + + effects of v o s a oe o-eetv bokr f high-threshold Ca of blocker non-selective more a use To Effects of Cd (1.0 calciseptine peptide channel-blocking Cd mle a euaoy oe lo o peyatc L-type presynaptic for also channels in controlling role spontaneous transmitter release. regulatory a implies fet f h Ltp Ca L-type the of effect channels (Haage channels release, whereas no evidence was found for a role of L-type onto MPN neurons, mainly N-, P- and Q-type Ca release GABA depolarization-evoked of studies previous n nrae n IS feuny 15±1 %, 11 ± (175 frequency mIPSC in increase an bokn efc o hg-hehl Ca high-threshold on effect blocking a which all were found substances, to potentiate above the mIPSC the frequency, is of property known common The i.1 Fig. ee on t mdae h Ca the mediate to found were cntxn VA (1.0 GVIA -conotoxin 2+ 2+ , whichblocksL-,N-,P-andQ-typechannelsto v channels, we proceeded to investigate the effects of effects the investigate to proceeded we channels, D -conotoxin MVIIC ( S . E ). . M A . B, , in eight cells. A . For all recordings the postsynaptic 2+ nasiepeaain nimodipine preparation, slice a in , Ni et al. et 2+ and EGTA on mIPSC frequency 1998). However, the potentiating the However, 1998). C, v m 2+ m -CTx MVIIC; 1.0 M effects of nimodipine M ). Note the increase the Note ). hne bokr fud here found blockers channel Fg 1 Fig. ; 2+ m nlx rgeig the triggering influx C M . h Ltp Ca L-type The ). ). The ). m m 2+ M M ) J. Physiol. hnes In channels. ) also caused also ) 2+ channels n 542.1 =2; 2+ The Journal of Physiology Water Transport Controversies Special Issue presence of a response, although significantly reduced, to reduced, significantly although response, a of presence remaining, % 23 ± (96 and did not significantly affect the response to nimodipine %, 11 ± 81 (to frequency mIPSC the of reduction small a only caused cells of series same the to channels, 200 of addition upon However, channels. R-type also extent some rqec dd o ices, u rte dcesd to although apotentiatingeffectofnimodipine(100 decreased ( rather % 11 but ± 63 increase, not did frequency J. Physiol. 200 3 control (Fig. to solution added was nimodipine when obtained ( % that 11 ± 38 to reduced was frequency mIPSC was added to the Cd nimodipine when present still was frequency mIPSC the m M m M Ni 542.1 Cd 2+ n wih any lcs o-hehl Ca low-threshold blocks mainly which , and of nimodipine (100 nimodipine of and A when added to control solution. Note that nimodipine evoked similar responses in Ni Figure 3. Effects of Cd 23m (2.3 conditions anddatapresentation similartothoseinFig.2 presentation similar to those in those to similar presentation 23m (2.3 the reduced mIPSC frequency, as well as the increase in mIPSC frequency after washout of Cd solution. Note the qualitatively similar effects compared with those seen with the application of Cd of application the with seen those with compared effects similar qualitatively the Note solution. effects of nimodipine (100 nimodipine of effects in those to similar presentation data and conditions Recording and in control solution. = 11) of that in control (Fig. 3 (Fig. control in that of 11) = 2+ , effects of Cd to the extracellular solution, the mIPSC the solution, extracellular the to A M M and 2+ ), Cd (100 nimodipine with and solution, control the to added ) containing solution, this increase in n D 2+ 1 Fg 3 Fig. 11; = 2+ . n otat apiain of application contrast, In ). (200 (200 m m M M ) orNi m 2+ ), and of nimodipine (100 M , Ni m ) in the presence of Ni of presence the in ) M B 2+ Dual roles of presynaptic Ca ) on the mIPSC frequency (measured for 1 min) in the presence of EGTA of presence the in min) 1 for (measured frequency mIPSC the on ) 2+ A and EGTA on mIPSC frequency and on response to nimodipine and (200 . Data from 11 cells. 11 from Data . A ). Further, ). m n D M = 11) of 11) = . (The ). ). Theeffectsare indicatedrelativetotheresponse tonimodipine n m 11) = M ) on 2+ 2+ m , on mIPSC frequency. Recording conditions and data and conditions Recording frequency. mIPSC on , M nimodipine in 200 in nimodipine C ) in the presence of Cd hehl Ca high- of threshold block non-complete a of findings earlier with influx through L-type channels remains. This is consistent Ca high-threshold most blocks by Cd by The above results showed that a rapid non-selective block, mIPSC frequency.) , number of mIPSCs in control solution, in EGTA in solution, control in mIPSCs of number , more selective block of either of several Ca not cause potentiation of the mIPSC frequency although a with that in Cd in that with below, showing a smaller response to nimodipine compared This was further supported by the experiments with EGTA Foehring, 1995; Sundgren-Andersson & Johansson, 1998). os Tu, lkl itrrtto i ta atog the although that is interpretation likely a Thus, does. A . Datafrom11cells. 2+ 2+ , of most high-threshold Ca high-threshold most of , A influx . Data from 15 cells. 15 from Data . m 2+ M ) added to the EGTA-containing the to added ) 2+ urns y 100 by currents as well as a larger reduction of baseline of reduction larger a as well as m 2+ , on mIPSC frequency. Note B M effectsofNi , Cd 2+ -containing solution 2+ D , summary of the of summary , suggests that although Cd although that suggests 2+ 2+ m 2+ . Recording M (200 hnes sm Ca some channels, 2+ 2+ Cd channel types does types channel in m 2+ M 2+ A. ), Lrno & (Lorenzon channel types 135 2+ 2+ The Journal of Physiology Water Transport Controversies Special Issue Ca reduced a by triggered is frequency mIPSC potentiated 136 observations on the effects of Cd of effects the on observations in mIPSC frequency after washout of Cd We further made observations suggesting that the increase free Ca release when Ca dramatic increase in probability for spontaneous transmitter frequency before addition of Cd Hz, 0.9 ± (1.6 levels to increased transiently frequency mIPSC the expected, other Ca other Effects similar to those seen with Cd Fig. 3 a rdcd n h peec o ET (o 2±1 of % 13 ± control, 52 (to EGTA of presence the in reduced was idea of a ‘partial’ block of the Ca the of block ‘partial’ a of idea uig ahu o Cd of washout During plcto f23m application of 2.3 reduced influx of Ca Fig. 3 Hz, 0.13 Hz ± and in control of 0.03 0.53 ± of 0.16 EGTA in frequency baseline the with compared washout; after s 10 first the during Hz 0.5 after ± immediately (2.0 EGTA of washout observed was frequency of increase nimodipine-evoked effect in control, % of 1 markedly reduced in the ± presence of EGTA (to 12 response to nimodipine (in the presence of Cd the in cells between variability the on based is conclusion This mechanism. same the on depended nimodipine by one-way ANOVA procedure the cells were classified into classified were cells the procedure ANOVA one-way 2+ influx through some Ca some through influx A C 2+ ). and 2+ ocnrto f~0 n concentration of ~100 n = 15), the response to 100 to response the 15), = channels is required for the effect. Additional effect. the for required is channels D similar to those in Fig. 2 responsive cells. Correlation marked by arrows in arrows by marked Correlation cells. responsive Figure 4. Effects of Cd solution. The cells were grouped on basis of a significant response to nimodipine (100 nimodipine to response significant a of basis on grouped were cells The orltd ih n nrae mPC rqec atr ahu o Cd of washout after frequency mIPSC increased an with correlated sensitive cells also showed some response in Cd in response some showed also cells sensitive ). Together, these observations suggest that a 2+ becomes available again. M 2+ n A, EGTA, which was expected to give a triggers some process leading to the = 11) well above the 60 s baseline s 60 the above well 11) = 2+ nimodipine-responsive cells ( cells nimodipine-responsive we a ata bok a be may block partial a when , 2+ 2+ channels, influx through influx channels, M A 06 .4Hz, 0.14 ± (0.61 2+ 2+ . The mIPSC frequency . 2+ 2+ n seem to support this support to seem and of nimodipine in the presence of Cd channel population. channel 15), but a dramatic = m were recorded upon M 2+ nimodipine was nimodipine and that caused 2+ ). Using a M. Druzinand M. others Druzinand n n n 11; = 6). = 15; = 2+ B, A (200 non-responsive cells ( cells non-responsive and ( two groups depending on whether they showed a significant the response to nimodipine and the effect of Cd solution. After this classification, a clear correlation between Due to the apparent role of Ca Relation between [Ca n pasbe oe ta cud xli a increased an explain Ca by caused frequency mIPSC could that model plausible One controlling mIPSC frequency A hypothetical model of presynaptic mechanisms without blocker was only weakly dependent on [Ca eain ewe [Ca between relation reasonably effect of the depolarization. to similar was nimodipine of effect the respect, this terminals on MPN presynaptic neurons (Fig. 5 of depolarization KCl-mediated by evoked xenl Ca external the between relation the determined experimentally we nimodipine as well as the effect of washing out Cd to response the underlying factor causative common a of 0.75, was test Spearman a by obtained coefficient correlation clear response to nimodipine did not (Fig. 4 P response to 100 to response in as well as agents blocking of absence the in frequency frequency after washout of Cd of washout after frequency mIPSC in increase significant a showed also nimodipine to response significant a showed that Cells evident. was oi rlto t [Ca to relation bolic hyper- roughly sublinear, a showed nimodipine of effect m < 0.05) response to nimodipine when added to control B M . Recording conditions and data presentation data and conditions Recording . ), and that the sensitivity to nimodipine was nimodipine to sensitivity the that and ), P < 0.05, good agreement with the previously established 2+ 2+ wih a nt en n h non- the in seen not was which , n 2+ ocnrto, [Ca concentration, 11.) This correlation suggests = the presence on mIPSC frequency m n M = 5). Note that nimodipine- that Note 5). = m nimodipine. Whereas the frequency the Whereas nimodipine. M 2+ 2+ ) when added to control to added when ) ] 2+ o ] o ] Fg 5 (Fig. o n te otyatc current postsynaptic the and and mIPSC frequency 2+ 2+ 2+ influx in the above effects, A , whereas cells without a without cells whereas , channel blockers involves blockers channel . hs eain a in was relation This ). B 2+ ; Haage ] o ad h mIPSC the and , A et al. J. Physiol. and 2+ 1998). In 2+ B washout 2+ . ). (The ] o 542.1 , the The Journal of Physiology Water Transport Controversies Special Issue Ca (Robitaille & Charlton, 1992; Robitaille 1992; neuro- Charlton, & (Robitaille of regulation in release evoked for mainly reported been has transmission involvement their although terminals (seereviewsbyJackson,1995;Meir between the Ca the between separation functional/spatial the on depends hypothesis the depolarization, and the resulting Ca resulting the and depolarization, the by activated be would they blocked), (not available were Ca J. Physiol. ml-odcac Ca small-conductance membrane would lead to a reduced K reduced a to lead would membrane odcac Ca conductance of Ca block a that imagine thus We 1998). Tavalin, & (Marrion sufficient Ca If terminal. presynaptic the of depolarization consequent channels) whereas N-type Ca N-type whereas channels) small neurons, Johansson & Århem, 1994). (For clarity, (For 1994). Århem, & Johansson neurons, very small for situation the (cf. currents small to response in small membrane the area, to is likely due to fluctuate resistance quite considerably membrane high a of of consequence probability a as terminal, presynaptic the of potential membrane the the increase would transmitter release (Fig. 6 channels those K neurons, L-type Ca L-type neurons, that even in individual membrane patches of hippocampal require a long distance. It has recently been demonstrated (Fig. 6 + 2+ 2+ hnes a be dmntae i sm nerve some in demonstrated been has channels -activated K 2+ channels that couple to Ca to couple that channels channels that couple to K A ). Such separation is clearly possible, and does not 542.1 2+ 60 s after nimodipine application). Note the weak dependence of mIPSC frequency on external Ca external on frequency mIPSC of dependence weak the Note application). nimodipine after s 60 control solution. Each point represents data from 15 m cells, except for 1.0 (squares) andmIPSCfrequency attheapplicationofnimodipine(100 A amplitude of the GABA Ca Figure 5. Relation between external Ca saturating Ca circles; data from Haage from data circles; 4mV. Error bars represent _4 of nimodipine (100 nimodipine of channels coupled to the exocytotic machinery , relation between external Ca external between relation , 2+ 2+ + 2+ , as described by Haage channels. The presence of Ca channels that trigger exocytosis and the and exocytosis trigger that channels atvtd K -activated 2+ channels may selectively couple to couple selectively may channels 2+ 2+ B atvtd K -activated ocnrto f01 m concentration of 0.15 and 2+ + m + C channels in the presynaptic channels couple to large- to couple channels M hnes B channels) (BK channels 2+ ). It should be noted that , triangles) as in as triangles) , A -activated K -activated receptor-mediated synaptic m current evoked by the application of 140 et al. et et al. S + . E + Dual roles of presynaptic Ca et al. et . conductance and conductance 1998). The smooth line is described by a hyperbolic equation with a half- a with equation hyperbolic a by described is line smooth The 1998). 2+ M 2+ (1998). . hnes (SK channels influx through influx B, concentration and mIPSC frequency in standard extracellular solution extracellular standard in frequency mIPSC and concentration et al. relation between external Ca 1993). Our 1993). 2+ + M -activated A channels , with superimposed relation between external Ca external between relation superimposed with , n aiu urn/IS rqec 1 fta t10m % and of maximum that current/mIPSC at frequency 1.0 117 1999), 2+ , mIPSC frequency and response to nimodipine Ca Fig. 6.) Therefore, it should be expected that high-threshold simplified the in illustrated not is situation dynamic this presynaptic Ca presynaptic of presence the requires above model hypothetical The Effects of K o ailn 41±25%, 245 ± for paxilline(491 7 (Fig. frequency mIPSC in increase significant a induced separately, tested when (Schneider addition in channels the less selective K %, 35 ± mIPSC frequency (217 %, 89 ± 265 e vnm pmn (1.0 apamin venom bee xrclua Ca extracellular on depends frequency mIPSC basal the that findings the that most of the time remain well polarized. This explains TA 1 m 10 (TEA, 0 n 200 %, 321 ± (694 frequency n mIPSC in increase dramatic several SK channel types (Hugues lhuh hrbooi my fet te tps f K of types other affect may charybdotoxin although K hnes (Miller channels BK K mIPSC increased and frequency. We tested both these requirements by depolarization applying presynaptic to hypothesis iscorrect,blockingthesechannelsshouldlead (Further arguments for the model are given in Discussion.) + = 17; Fig. 7 Fig. 17; = 2+ channel blockers to our preparation. Application of the channels may be activated occasionally even in terminals M 2+ ) and paxilline (1.0 paxilline and ) 2+ influx and mIPSC frequency at the application M + n m channel blockers on mIPSC frequency Fg 7 Fig. ; A 5 fr ailn) A iia ices in increase similar A paxilline). for 15, = M 2+ M ). We also applied charybdotoxin (ChTx; charybdotoxin applied also We ). , triangles;measuredduringfirst 2+ -dependent K -dependent Ca + n o hg-hehl Ca high-threshold on and 2+ channel blocker tetraethylammonium : 54 cells. Holding potential A . hs rsls hs ugs that suggest thus results These ). t al. et A ), although the effect was smaller was effect the although ), m m n M M 10,forcharybdotoxinand = , wl-nw bokr of blocker well-known a ), 95 Knaus 1985; ), which both block mainly block both which ), n + 2+ et al. et 13) was observed with = channels. Further, if the if Further, channels. and the peak the and et al. M K + 1989). Both drugs, Both 1989). 1982), resulted in a (filled 2+ in M t al. et 2+ channels. 1994), 137 + The Journal of Physiology Water Transport Controversies Special Issue hr ae ned Ca indeed are there 138 hs canl rsls n n nrae feuny of frequency hypothesis. increased above an the of support in release, transmitter in spontaneous results channels these synaptic terminals on the MPN neurons, and that blocking 2+ atvtd K -activated + hnes n h pre- the in channels M. Druzinand M. others Druzinand that applicationofCa is hypothesis above-presented the by prediction Another external K Effects of nimodipine on mIPSC frequency in high eoaie t na 0m, hr Ca where mV, 0 near to depolarized are terminals presynaptic the when frequency mIPSC the + concentration type Ca that outside the release site, N- and P/Q- type, at the transmitter release site. Note gated K release site. Ca and P/Q-types, located outside the which differs from that in fluctuate around a mean value in potential of the terminal is expected to simplification and that the membrane release site. Note that the figure is a channels leads to activation of Ca release site, and the Ca molecules to the Ca is limiting rapid access of large blocking space and the synaptic cleft. This barrier extrasynaptic part of the extracellular a diffusion barrier between the transmitter release. Note the presence of contains Ca control conditions. The terminal A nerve terminal on MPN neuron Figure 6. Hypothetical model of Ca ( nimodipine ( channels outside the release site by (right). channels are coupled to SK channels causes activation of Ca depolarization. The depolarization controls Ca The membrane potential, in turn, influence on the membrane potential. channels (left), whereas L-type Ca C , the presynaptic nerve terminal in ) is followed by closure of nearby 2+ 2+ -gated K channel blockersshouldnotraise 2+ + B channels that exert major channels are coupled to BK and 2+ 2+ + B channels, of N-, P- or Q- channels, of L- and N-, channels and C, 2+ ) or influx through these blocking of the Ca v 2+ -conotoxin MVIIC channels at the 2+ 2+ influx triggers channels at the 2+ B and nlx through influx J. Physiol. C 2+ A . 2+ - 2+ 542.1 The Journal of Physiology Water Transport Controversies Special Issue release-triggering Ca ISs a vr hg (6±5Hz, 5 100 of ± addition after (16 s 10–20 high very was mIPSCs et h vldt o te rdcin Fr hs K this, For prediction. the of validity the test J. Physiol. 0 mV. In the K the In mV. 0 near to terminals presynaptic the depolarize to expected high external K external high a by terminals presynaptic the depolarized therefore We substituted for Na for substituted ih ouin te IS feuny a rdcd to reduced ( was % 4 frequency ± 27 mIPSC the solution, rich 542.1 n Ca o ioiie fe 2 i o penuain ih GAA ( EGTA-AM with preincubation of min 21 after nimodipine to 1min in BAPTA-AM ( 21 (squares) and after addition of 100 of addition after and (squares) S Figure 7. Effects of K presence of nimodipine. Data from 7 cells. 1min of preincubation with either 100 21 A, paxilline (1.0 of K The number of s mIPSCs period was immediately measured before during and a just 60 after the application (100 each). Data presented relative to preceding baseline measured during a 60 s period immediately before immediately period s 60 addition of100 a during measured baseline preceding to relative presented Data each). in cells (6 neurons of groups two to application nimodipine of period s 60 a during measured was mIPSCs ) f ht eoe ioiie application nimodipine before that of 7) = . E . + 2+ + fet o te K the of effects M -rich external solution, the frequency of frequency the solution, external -rich + concentration (cf. Haage (cf. concentration . m buffer BAPTA-AM ( BAPTA-AM buffer channel blocker. + M in the external solution, a procedure a solution, external the in ). Frequency of mIPSCs in m an external solution containing 140 2+ channels isalreadyatamaximum. m M m ; M n + m 5 n E 1 m 15) and TEA (10 = nimodipine tothecontrolsolution andtoCa hne bokr aai (1.0 apamin blockers channel M B, + D nimodipine to the K the to nimodipine effect of a high external K channel manipulation on mIPSC frequency ). For all data, recording conditions similar to those in Fig. 2 D ) on mIPSC frequency and on the response to nimodipine. The number of number The nimodipine. to response the on and frequency mIPSC on ) n Dual roles of presynaptic Ca ) However, 7). = et al. et m M nimodipine (triangles). Note the reduced mIPSC frequency in the in frequency mIPSC reduced the Note (triangles). nimodipine 1998) to 1998) m M C M , n + and EGTA-AM or 100 13) on mIPSC frequency. = Data presented relative to control. was D + - + , effects of the slow Ca ocnrto 10m concentration (140 m M ; findings were thus consistent with the the hypothesis Again, above. solution. control to added when nimodipine 7 (Fig. nimodipine on mIPSC frequency o et hte te ioiieidcd nrae of increase nimodipine-induced Ca increased to due is frequency mIPSC the whether test To Intracellular Ca For that we applied the cell-permeable Ca These forms of BAPTA and EGTA enter the terminals by terminals the enter EGTA and BAPTA of forms These synaptic intracellular Ca intracellular synaptic not alter extracellular Ca extracellular alter not n 7, hrbooi (hx 20n 200 (ChTx, charybdotoxin 17), = m 2+ B M 2+ , n otat ih h ptnitn efc of effect potentiating the with contrast in ), C influx BAPTA-AM. Note the block of response ad h rdcd epne asd by caused response reduced the and ) buffer-containing solutionafter 10and 2+ M M buffer EGTA-AM ( K ) on the response to nimodipine 2+ + without Ca buffers reduce the effect of 2+ A 2+ concentration was manipulated. was concentration . Error bars represent , BAPTA-AM and EGTA-AM. and BAPTA-AM , 2+ channel blocker C ) and the fast M ; n 10), = 2+ 2+ influx, the pre- the influx, buffers that do 139 The Journal of Physiology Water Transport Controversies Special Issue f1 m 10 of 8 Fig. Hz; 1.1 ± 5.6 (peak charybdotoxin K the using by channels If the Ca channel blockers on the mIPSC frequency? channels explain the potentiating effects of the Ca Does closure of the presynaptic Ca 100 with perfusion of min 21 (Cummings esterases unspecific by cleavage following intracellularly accumulate and uptake 140 effect of the Ca the of effect 19±25H ws band n E, hra the whereas TEA, Hz ( 1.8 ± in was 8.7 solution control obtained in frequency peak was corresponding Hz 2.5 ± of 11.9 frequency peak A solution. control to applied when eas o coue f K of closure of because could then also be used to identify the specific K n mediators of effects due to reduced Ca nrtria Ca intraterminal suggests that the effect of nimodipine depends on a rise in AM on the nimodipine-evoked increase of mIPSC frequency ( % common reducingeffectofbothBAPTA-AMandEGTA- 8 ± 27 to nimodipine, of effect were in agreement with our general hypothesis. o28±12Hz, 1.2 ± 2.8 to icesdfo . . o24±09Hz, 0.9 ± 0.2 to 2.4 ± (increased from 1.2 TEA of presence the in masked or reduced was MVIIC Ca target their an therefore and caused frequency, mIPSC alone above, in blockers increase these noted of As each of paxilline. application and TEA charybdotoxin, as indicated below, we expect that other K other that expect we below, indicated as K depolarization, as suggested above, then blockage of these EGTA-AM, 100 ( control in of observed % that 14 ± 29 only was frequency mIPSC on nimodipine iia eut eeotie ntepeec f20n 8 200 Fig. of Hz; presence the 1.4 in obtained were ± results Similar 6.4 of peak a (to frequency mIPSC the increasing in effective was nimodipine application), lo n h peec o 1.0 of presence the in Also The situation for situation The sensitive to these toxins, are also present in the nerve terminals. f ioiie Atog apiain of application Although nimodipine. of rmtcly nrae te IS feuny n the in frequency mIPSC presence of 1.0 the increased dramatically MVIIC (down-pointing triangles in Fig. 8 . . Hz, 0.9 ± 4.5 n Hz, 1.7 ± 8.0 at (peak larger was nimodipine of effect the that the effects of nimodipine were mediated by K novd W teeoe tepe t ietf te K the identify to attempted therefore We involved. sensitive to any of these four K + 17;Fig.8 = 2+ 5 ta we apid o oto slto (ek at (peak solution control to applied when than 15) = channels by other agents may be expected to mask the mask to expected be may agents other by channels -dependent K M 2+ TEA, nimodipine was even more effective than effective more even was nimodipine TEA, channel blockers increase the mIPSC frequency n A n r20n ) or200 15; Fig. 8 = 2+ m 15; Fig. 8 = M m n 2+ M channel blockers. Such masking effects masking Such blockers. channel + v pmn(rm06±02t . . Hz, 1.4 ± 0.2 to 3.4 ± apamin (from 0.6 = 10; Fig. 8 Fig. 10; = channels seemed possible as candidate -conotoxin MVIIC differed from that from differed MVIIC -conotoxin BAPTA-AM significantly reduced the ocnrtos Tu, hs results these Thus, concentrations. M C n D hrbooi fo . 0.3 ± charybdotoxin (from0.8 ). Similarly, in 1.0 + = 6; Fig. 7 Fig. 6; = ). Therefore, it seems unlikely + + m hne bokr apamin, blockers channel B channel blockers. However, m M ), the effect the ), hnes n resulting and channels M EGTA-AM the effect of effect the EGTA-AM pmn 2mn pre- min (2 apamin n 2+ t al. et 2+ ; i. 7 Fig. 6; = C B -activated K influx. ). In the presence the In ). ). In similarity to similarity In ). A–D n + 13; Fig. 8 = 96. After 1996). channels, not channels, m v v ihn1 s ) within 10 -conotoxin -conotoxin M + + paxilline, channels D channels M. Druzinand M. others Druzinand . The ). + A 2+ C ). M ) + and nearly abolished in the presence of 1.0 of presence the in abolished nearly and Our findings above show that block of Ca of block that show above findings Our Effects of long-duration application of channels. K the of presence the in even concentration, nimodipine MVIIC on mIPSC frequency and on response to then, are the release-triggering Ca release-triggering the are then, hnes ciae K activates channels ag-odcac Ca large-conductance of that matches apamin, or charybdotoxin not but TEA paxilline. Thispharmacologicalprofile,withsensitivityto Fg ) s orc, h fnig ipy ht h L-type the K that unidentified yet imply as to findings coupled are channels the correct, is hypothesis 6) general (Fig. our If blocked. are depolarization, lces sd Aog hs canl my e Ca be may channels these Among used. blockers zones only affected by the small blocking ion Cd L-type channels are not coupled to the same K nimodipine-sensitive the that suggests TEA and paxilline of presence the in nimodipine of effect potentiated The et al. of to the type presence of a another of (Wang neuron hypothalamic terminals neurohypophysial the ees, lhuh u hpteia mdl ugss that Ca suggests model hypothetical our although transmitter release, spontaneous in increase paradoxical a give xetd hn K when expected is depolarization larger A depolarizing. is nimodipine of action the that support additional provides however, effect, v ht hr ae eann K remaining are there that individual where mIPSCs could not be resolved.) frequency These results thus suggest high extremely the to due and min apamin (Fig. for 8 5 that were pretreated cells with a in mixture frequency of TEA, mIPSC charybdotoxin in increase dramatic a caused rqec a a osqec o riig h etra K mIPSC external the increased raising of consequence an a as and frequency terminals presynaptic the paxilline. In this case, we should expect a depolarization of or TEA charybdotoxin, apamin, by blocked not are that h Ca the that hypothesis the with consistent is paxilline or TEA of The reduced effect of this of reversibility poor substance, compared the between different groups of to cells.) due were, conditions of effects (The 0. 4 Hz, ± 0.2 to 1. 2 ± (increased from 1.0 blockers. Indeed, application of K of application Indeed, blockers. conotoxin MVIIC, we do expect that N-, P- or Q-type Ca no L-type channels at the release site, but in the case of and expect we nimodipine nimodipine, of case the In MVIIC? blockers conotoxin large-molecular the by ciae K activated -conotoxin MVIIC-sensitive channels. The potentiated The channels. MVIIC-sensitive -conotoxin 2+ hnes r as rqie t tigr ees. Why release. trigger to required also are channels 2000). 2+ nlx through influx + hnes ht neat ih -ye Ca L-type with interact that channels v b cntxn VI udr different under MVIIC -conotoxin 4 subunit (Behrens + + v 2+ -conotoxin MVIIC in the presence atvtd K -activated hnes estv t TA and TEA to sensitive channels hnes wih counteract which channels, v E cntxn MVIIC-sensitive -conotoxin + ). (The effect was not quantified hnes nestv t the to insensitive channels et al. et + 2+ rc 10m (140 -rich 1992) and may be due be may and 1992) channels at the active the at channels + hnes on in found channels et al. n 2+ v m 15; Fig. 8 = J. Physiol. -conotoxin channels can channels + 2000; Meera M channels as M + + 2+ ) solution ) channels paxilline channel and not 542.1 D v v 2+ 2+ 2+ ). - - + - The Journal of Physiology Water Transport Controversies Special Issue involves different time courses of toxin effects on the Ca Dunlap see review (for (Haage channels are contributing to the release in this preparation J. Physiol. on the control of the Ca the of control the on and machinery release-triggering the of control mediated et al. et 542.1 1998) as well as in many other preparations other many in as well as 1998) conotoxin MVIIC, in the presence of the K in the presenceofamixtureapamin (1.0 Error bars represent Figure 8. Effects of K frequency upon application of K A– C were pre-treated with the mixture of K of mixture the with pre-treated were conotoxin MVIIC ( in Fig. 2 ) andpaxilline(1.0 A D, and effects of the K A . B E. differs from that in that from differs et al. et Effect of raising the external K 2+ -activated K -activated 1995). A likely explanation likely A 1995). v + S -CTx MVIIC; 1.0 . m channel blockers apamin (1.0 E M . M + ; channel blockers on response to nimodipine . D ) onmIPSCfrequencyandthe responsestonimodipine(100 + Dual roles of presynaptic Ca channels. A slow A channels. + C -rich solution. For all data, recording conditions similar to those in Fig. 2 and m + D -channel blockers for 5 min. Note the dramatic increase of mIPSC of increase dramatic the Note min. 5 for blockers -channel M . Note also the large effect of nimodipine, but small effect of effect small but nimodipine, of effect large the also Note . m ). Data from 17 ( + + M -channel blockers in ocnrto t 4 m concentration (to 140 ,caydtxn(hx 0 n ), charybdotoxin(ChTx;200 2+ - m M ; ie ore tm cntn > . i) of min) 1.5 > constant (time course time MVIIC-mediated block of K of block MVIIC-mediated A tal. et Wheeler see example (for slow is transmission synaptic on effect blocking the preparations other in Also 1998). (Haage preparation this for noted been previously ,caydtxn(hx 0 n ), charybdotoxin (ChTx, 200 A ), 10 ( 1994), in contrast to the fast potentiating effect on effect potentiating fast the to contrast in 1994), 2+ C influx and B ), 13 ( M D at the time indicated by an arrow) in . Data presentation similar to those C ) and 15 ( M n E 1 m ) andTEA(10 D ) cells. Note the scale M ; + B -evoked GABA release has release GABA -evoked ,TA(0m ), TEA (10 m M M ) andto ). Thecells v v M A v - - . ; -conotoxin et al. et 141 The Journal of Physiology Water Transport Controversies Special Issue i. 2 Fig. s; 10 within (maximum here reported frequency mIPSC 142 example, to separate locations of the Ca the of locations separate to example, e etd h pooe rqieet f functional Ca MVIIC-sensitive conotoxin of requirement proposed the tested We ie fr h nmdpn-vkd nrae n mIPSC in increase nimodipine-evoked frequency. We thus applied the for site) VI, ml in o hg mblt, uh s Cd as such mobility, high of ions small MVIIC, of the toxin at the two locations. In contrast to concentration different consequently and access different with 6, Fig. in suggested as systems, different two the to ucl rah h rlae ie n h snpi cet and cleft synaptic the thereby block the in Ca site release the reach quickly ioiie maue rltv t te aa mIPSC basal the in to frequency relative (measured nimodipine 9 (Fig. min 27–28 after ( % 5 ± 53 to frequency mIPSC basal of mIPSC frequency, the toxin induced a slow gradual decline nimodipine-evoked on effect potentiation of mIPSC the frequency. After an initially as increased well mIPSC basal as on frequency effect the investigated and min 28 for estv Ca sensitive h fnig tu soe that showed thus findings The % of that 12 in control solution ± ( was 44 n h peec of presence the each in preceding immediately time with gradually declined nimodipine) interval of application s 60 the during 0min in 20 D . ifrne i tm cus cud e u, for due, be could course time in Differences ). v 2+ -conotoxin MVIIC, the response to nimodipine slow decline (after the initial increase) in mIPSC frequency in the presence of presence the in frequency mIPSC in increase) initial the (after decline slow iue . fet o ln-uain plcto of application long-duration of Effects 9. Figure was measured during a 60-s period immediately before addition of nimodipine (in the presence of presence the (in nimodipine of addition before immediately period 60-s a during measured was during a 60-s period of nimodipine application. Data presented relative to preceding baseline. The baseline application of rsne rltv t control. to relative presented A, change in mIPSC frequency caused by long duration application of frequency and on response to nimodipine. bars represent prolonged after nimodipine to response preincubation with of decrease considerable the Note bar). 4th to 2nd for MVIIC, 1.0 hnes otiue t te aa mIPSC basal the to contributed channels v m cntxn VI-otiig solution MVIIC-containing -conotoxin M ) in 5 cells. The number of mIPSCs was measured during 60-s periods with 4-min intervals. Note the 2+ v cntxn VI (i. 9 (Fig. MVIIC -conotoxin influx necessary for release. A v S ). Further, also the response to response the also Further, ). . -conotoxin MVIIC ( E v . M -conotoxin MVIIC (1.0 . v 2+ -conotoxin MVIIC. For all data, recording conditions similar to those in Fig. 2 channels (at the release the (at channels v cntxn MVIIC- -conotoxin 2+ channels coupled channels n B, n = 5) of control of 5) = 5). = eue rsos t nmdpn (100 nimodipine to response reduced v v -CTx MVIIC; 1.0 -conotoxin B M. Druzinand M. others Druzinand 2+ . After ). may , m v M - ) fet f ioiie lo eedd n functional Ca on MVIIC-sensitive conotoxin depended also nimodipine of effect the that showed further the findings The on frequency. mIPSC effect potentiating the than course time slower frequency, and that the blocking effect showed a considerably m Ca several of existence the for evidence work, present the In DISCUSSION roles of Ca was put in efforts to explain the apparent dual and opposing transmitter release was demonstrated. The main emphasis spontaneous controlling in types channel these of several of rat MPN neurons was presented. Further, a contribution of ao prs f h ao hv be rmvd Thus, removed. been have axon although MPN neurons the can generate several types of Ca of parts major the presynaptic neuron, including cell body, dendrites and This preparation has the advantage that the major parts of functional adhering nerve terminals (cf. Haage with neurons dissociated using performed was study The with our proposed hypothesis. mediated by direct actions on the nerve terminal. be to likely are frequency mIPSC on effects observed the Therefore, excluded. be the can terminals nerve from the to soma impulses such of propagation study, present blocked (Sundgren-Andersson & Johansson, 1998), in the M eitd muss hn at Na fast when impulses mediated ) in 5 cells. The number of mIPSCs was measured 2+ and K v cntxn VI o bsl mIPSC basal on MVIIC -conotoxin 2+ + channel types in the presynaptic nerve terminals v influx in controlling the spontaneous release. -conotoxin MVIIC ( m M cue b long-duration by caused ) v -conotoxin MVIIC. Data MVIIC. -conotoxin v 2+ -CTx MVIIC; channels, in agreement in channels, + dpnet pks are spikes -dependent A v . Error -CTx J. Physiol. et al. 1998). 542.1 v - 2+ - The Journal of Physiology Water Transport Controversies Special Issue type Ca type v (calciseptine, blockers channel Ca of application that was study present the in finding unexpected major The blocked Ca Mechanisms for increased mIPSC frequency at Na without conditions ‘resting’ under open are types these of channels some that show frequency mIPSC on effects ion channel types are present in MPN nerve terminals. The The pharmacological evidence suggested that a number of Ca transmitter release the external K pnaeu tasitr ees. h peec o the of Ca P/Q-type and presence N- demonstrated previously The release. transmitter spontaneous of Parfitt & Madison (1993) and of Bao of and (1993) Madison & Parfitt of found no evidence for a contribution of Ni J. Physiol. lc o K of block nimodipine and nifedipine could possibly be due to direct (Haage MPN the in terminals presynaptic of depolarization by evoked release nxetd sne t s el salse ta Ca that was established well This is it currents. since unexpected, synaptic ‘miniature’ of frequency transmitter release, in our system. K the membrane potential, which is ‘overruled’ in via effect postulated the by explained easily is difference (Haage terminals nerve these from increase in mIPSC frequency caused by the Ca the by caused frequency mIPSC in increase ‘paradoxical’ the for account could that model simple A hehl Ttp Ca T-type threshold oe rprtos, h efcs f ahn ot Cd out washing of effects the preparations), some conotoxin GVIA and the even larger effect of (Haage mus-vkd rnmsin Frhr as dihydro- Ca affected L-type also calciseptine-sensitive and pyridine- channels Further, transmission. these impulse-evoked where conditions drug-free by Hu in hippocampal presynaptic terminals was recently presented sensitive BK channels. Interestingly, evidence for BK channels paxilline- and channels SK apamin-sensitive for evidence was there addition, In frequency. mIPSC the on MVIIC not contribute to transient K transient to contribute not study, present the in release transmitter neuro- spontaneous the in role prominent a play to appear interpreting our resultsintermsoftheproposed modelis when question main A 6. Fig. in presented was blockers eits rnmte rlae fr eiw e Bennett, see review Ca since and 1999), (for release transmitter mediates effects were caused by a reduced Ca the that suggest all nimodipine, of effect the with out EGTA washing of effect the of correlation the and EGTA, -conotoxin GVIA), the lack of effect of Bay K 8644 (L- 8644 K Bay of effect of lack the GVIA), -conotoxin + -evoked release where the membrane potential is is potential membrane the where release -evoked + 2+ dpnet pks n cnrl h feuny of frequency the control and spikes -dependent and K et al. 2+ et al. et 542.1 channel agonist that may block K block may that agonist channel + + 2+ (2001), although they were unable to find any channel types controlling spontaneous hnes te iia efc o ohr Ca other of effect similar the channels, 1998) was here supported by the effect of effect the by supported here was 1998) + influx concentration.) In contrast to the findings 2+ 2+ channel blockers reduce the GABA the reduce blockers channel 2+ t al. et channel blockers potentiated the potentiated blockers channel hnes o h spontaneous the to channels 98. hl te fet of effects the While 1998). + -evoked transmitter release transmitter -evoked v -conotoxin MVIIC and MVIIC -conotoxin 2+ influx. t al. et Dual roles of presynaptic Ca although they do they although et al. et 2+ -sensitive low- + v 98. (The 1998). channels in channels (1998), we (1998), -conotoxin 2+ 2+ 2+ the case of channels 2+ channels channel set by set influx 2+ v or 2+ - how a reduced Ca reduced a how xlnto. h ices o mPC rqec upon frequency Ca the mIPSC of application of increase The explanation. influx. The time course of the observed effects suggests an Ca the where cleft, instantaneously synaptic the of nearly membrane the to to spread expected is closure channel K by induced depolarization the blocked, are channels h Ca the of localization the that idea the support findings Recent (Fig. 6 accordance in release trigger and hra Ca whereas Ca effect potentiating of the that showed study present the barrier, space (Peters extracellular surrounding the to cleft synaptic the the from in structures the substances of diffusion that the with interfere may cleft synaptic suggested been earlier has It structures). these to contributing molecules adhesion cell different the of review a for 2001, Südhof, (see cleft synaptic the into diffusion for barrier the to contribute (Uchida synapses many at seen junctions adherens the and/or cleft synaptic the in 1988) Hashimoto, & (Ichimura fibrils and filaments the 1991), (Gray, 1959),thedenseorintermediateplaque 1971; Cotman & Taylor, 1972), the electron- pre- of Pfenninger, (see cells postsynaptic the to terminals synaptic attachment strong chemically and mechanically these drugs on the GABA release evoked by K by evoked release GABA the on drugs these of some of effects blocking the than quicker is considerably This s. 10–20 within obtained usually responses peak than the inhibiting effect (due to Ca the release machinery). When Ca small ions such as Cd as such ions small for reduced marginally only be may access the (although as such molecules, blocking large for slower be should cleft synaptic the in site release the exposed to the toxin-containing perfusion, whereas the outside membrane synaptic cleft.Thelatterchannelsshouldbemore terminal the in also expressed depolarization, where the effects of effects the where depolarization, and eeoe oe svrl iue (Haage minutes several over developed transmission contrast with the findings that findings the with contrast transmission Wheeler case of N-type channels) may block their target Ca GVIA, neat ih Ca with interact to has toxin the release, the block to that is preparation present the in difference the for explanation likely most 1994; Randall & Tsien, 1995; McDonough (Bargas seconds few a within cases some in rapidly, 2+ 2+ channels (not yet blocked), in turn, are quickly activated channels coupled to K to coupled channels v B 2+ 2+ cntxn VA a wl a of as well as GVIA, -conotoxin and v v influx hnes ihn h peyatc emnl may terminal presynaptic the within channels -agatoxin IVA and IVA -agatoxin et al. -conotoxin MVIIC on mIPSC frequency (due to 2+ C et al. ). hnes ope t K to coupled channels 1994). The slow blocking effects of synaptic 2+ 1991). Supporting the idea of a diffusion 2+ hnes t h snpi rlae site, release synaptic the at channels influx could cause an increased Ca increased an cause could influx 2+ 2+ ). The structures that structures The ). channel blockers was rapid, with rapid, was blockers channel + channels) was much quicker much was channels) v -conotoxin MVIIC (in the (in MVIIC -conotoxin with the proposed model proposed the with 2+ et al. et channels coupled to K 2+ v v channels coupled to cntxn MVIIC -conotoxin + -conotoxin MVIIC -conotoxin 1996) are likely to likely are 1996) hnes a be may channels v et al. aaoi IVA, -agatoxin dense material v (Peters t al. et provide the provide + -conotoxin 1996). The 2+ -mediated access to channels directly 1998; et al. et al. et 143 2+ + + The Journal of Physiology Water Transport Controversies Special Issue Further, findingsbyHirsch (Kombian transmission impulse-evoked of potentiation of absence the in potentiation short-term to subject be selectively may currents synaptic spontaneous see Bao channel blockers on these 1999), two types of release (for example Murphy, & separate controlissuggestedbydifferentialeffectsofCa (Prange release impulse-triggered transmitter spontaneous of probability the with parallel in regulated be can release of probability the Although neurotransmission Control of impulse-triggered of effect or maskedwhenTEApaxillinewerepresent.Thus,the In the case of channels involvedhavenotbeenunambiguouslyidentified. Ca presynaptic of presence the for evidence Although Types of Ca Different Ca of release. localization transmitter spatial to coupling their affect 144 of such spontaneous release. present findings Our suggest a 1999). novel mechanism Staley, for also the control (see release spontaneous of role physiological a for evidence providing thus intact, is release be may impulse-evoked release although activity seizure with associated spontaneous of dysfunction selective Ca by K the Ca the likely candidate. 2001). Chuhma also (see release transmitter to coupling al. Wu et by demonstrated clearly was terminal presynaptic akd y pmn caydtxn TA r paxilline. or Although TEA we were unable to block the proposed charybdotoxin, K apamin, by masked neurons observed in the present study was not blocked or MPN in frequency mIPSC on nimodipine of effect The synaptic BK channels in the MPN contain al. et 2000) although the sensitivity to paxilline is preserved (Hu as well as to iberiotoxin (Behrens iberiotoxin to as well as charybdotoxin to insensitivity introduces channels BK in Recently, it has been shown that the presence of a ev trias (Wang terminals nerve al. et II BK channel that is insensitive to charybdotoxin (Reinhart (SK1) insensitive to all K mediate the effect of nimodipine. The SK- showed that there are remaining K remaining are there that showed interacting with nimodipine-sensitive Ca nimodipine-sensitive with interacting dependent K + channels. A likely candidate K 2+ (1999) and also correlated with the effectiveness of effectiveness the with correlated also and (1999) 2001). Thus, it seems likely that some of the pre- the of some that likely seems it Thus, 2001). 99 ad s rsn i ohr (neurohypophysial) other in present is and 1989) 2+ et al. channels coupled to TEA- and paxilline-sensitive and TEA- to coupled channels v channel blockers were presented above, the K the above, presented were blockers channel -conotoxin MVIIC was at least partly mediated partly least at was MVIIC -conotoxin 2+ 1998). Recent findings also demonstrate that + v channels, as well as a depolarizing action of -activated K -conotoxin MVIIC, the effect was reduced + t al. et channel blockers 2+ et al. + channels hne sbye wti a within subtypes channel 92 Dopico 1992; versus (1999) demonstratethat + et al. et channel type is the type + channels, which can which channels, spontaneous 2000; Meera 2000; b used is the most channel subtype 2+ 4 subunits. t al. et channels, we channels, t al. et b + 4 subunit channels M. 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