LETTERS TO NATURE released by high levels of emotional arousal can produce memory impairment, Findings of animal experiments indicate that memory storage is influenced by drugs affecting many neuro- modulatory systems, including a-adrenergic systems!" Studies to date have not investigated the selective involvement of other systems in regulating emotionally influenced memory ins human subject Although clinical reports suggest that fblockers may induc, memory loss in some patients", controlled laboratory investi- ‘gations have so far generally failed to find consistent effects of Beblockers on memory". However, previous studies generally investigated memory for relatively unemotional events and typ- ically studied only short-term memory. Future research using B-blockers with different affinities for §, and Bs receptors and differential effectiveness in passing the blood-brain barrier should clarify the role of the adrenergic system in regulating long-term memory for emotional experienc 53. Mcoagy J. Lis Renato moon aa hey: Research aes ee ara ne anne sia Changes in reliability of synaptic function as a mechanism for plasticity Charles F. Stevens & Yanyan Wang Laboratory for Molecular Neurobiology, Howard Hughes Medical Institute, Salk Institute, 10010 North Torrey Pines Road, L3 Jolla, Califoria 92210, USA SyNarric transmission in the hippocampus is rather unreliable, ations in synaptic strength seen in long-term potentiation (L,TP)* and long-term depression” (L'TDY? We find that L-TP increases i y, and LTD decreases it, both without a change in the size of those postsynaptic currents that do occur. Thus LTD is a functional inverse of LTP. We have used “minimal stimulation’, a method for activating only one or a few synapses: the stimulus intensity is reduced to a level that produces a postsynaptic response of uniform latency fand shape in a target neuron with a probability of less than about 0.5. This method permits us to separate {40 components ‘of synaptic strength: reliability, the fraction of stimuli that pro- 704 duce & postsynaptic current, and potency. the average peak size of the postsynaptic current when one does occur Whole-cell recording” provides a signal-to-noise ratio that is adequate for clearly distinguishing between a release failure and 4 success in almost all instances. ‘This is illustrated in Fig. | ‘hich reveals clear separation betwen transmitter release and no release Data for typical LTP experiments are illustrated in Figs ta and 2a e. Separate averages were made (before and after the induction of LTP) for all responses. all stimulation trials for ‘hich postsynaptic current was detected, and all records that represented Failures in synaptic transmission (Fig. 2a). Further, non-overlapping groups of about 25 sugcessve traces were wver ‘aged throughout the experiment, and failure probabilities were estimated forthe same groups of traces: these group averages reveal stable LTP following the tetanie stimulus (Fig. 2). and 2 sustained post-etanic decrease in the failure rate (Fig. 20) ‘The group average potency is roushly constant throughout the experiment (Fig. 2), and the histograms of excitatory postsyn aptic potential (eps..) peak amplitude for the control and LTP periods (Fig. 2c) are not significantly different (Kolmogoroy Smirnov test, P>0.1) Tn this instance, stable LTFP is represented as an increase in the average response. a decrease in the synaptic failure rate and no appreciable change in the potency. As with the LTP experiment, LTD is represented by'a decrease in the average response size (Fig. a, am inerease in the failure rate (Fig. 30), and no appreciable change in synaptic potency (Fig. 3a. d.e) Although we find. as reported previously that LTP is difi- cult to induce after about 20 min of recording in the whole-cell mode. LTD seems not to "wash out’. That is, we could produce LTDas late as 80 min after the statt of whole-cell recording, the longest time tested. In six experiments, LTD reversed previously established LTP (data not shown), To compare data fram different experiments, we note thatthe average peak eps. 1. is (by definition) the product of the probability, thata release will cur and the average size «of a response (when release does occur): 1/= i, the subscript j is 0 before the induction of LTP or LTD and 1 after, The probe ability ofa failure j,= (1 ~,) is determined directly from experi- ment ass the average poteney 4). Synaptic plasticity Sis defined asthe ratio Sr, ry Inplasticity were only a change in synaptic reliability Qwith no change in poteney), then a de Would equal I and S would be just the ratio of success probabilities wii. Fay dy. A plot of S worsus the ratio inp would then fall on a straight line with slope 1, and a plot ofthe ratio ays would be constant (=I). Our experiments provide the data reajuired for testing the extent to which plasticity is determined by changes in reliability and changes in poteney as we estimate di fy and yy diet The unfilled circles in Fig, 4a and b represent data from 26 experiments hike the ones illustrated in Figs 1, 2 and 3, Cleary. the ratio w/in Falls along the diagonal in Fig. 4, and the ratio 4 y's roughly constant in Fig. 4b. For these experiments, then wwe conclude that the mechanism of LTP and LTD is a change in synaptic reliability with no appreciable change in poteney The experimental situation here isa little different from previ ous investigations of LTP that activated a population of sye fipses so that all or most failures are obscured. Because we have activated fewer synapses than was usual for earlier studies, we felt that we must demonstrate thatthe plasticity examined here exhibits the same properties as standard LTP. LTP is sometimes elicited by the “pairing” stimulation model instead of by tetanic stimulation. In these “pairing” experiments low-frequency stimulation is paired witha postsynaptic depokar zation imposed on the cell". Data from fixe "palring’ exper tents, plotted in Fig. 4e and d as unfilled triangles, conform to the behaviour of synapses potentiated with tetanie stimulation LTP is blocked by the presence of the N-methyl-p-aspartate (NMDA) receptor antagonist AP-S", and by hyperpolartzation NATURE - VOL 371 - 20 OCTOBER 1994FIG. 1 a, Amplitude of synaptic currents as a function of time during the experiment to estimate the uncertainty in classifying traces as release’ or ‘no release’. Each point was calculated by averaging the curtent over a fixed window 2:ms long centred atthe time that core: Sponded to the peak curtent. Diamonds are from traces on which release wes apparent. and dots from traces without detectable release (currents everaged over the same 2s window). Tetanic stimulation was gen at the time indicated by the arrow, and produced LTP of 41708. Three typical traces of eurent asa function of time, ane without release and two with release, are superimposed In the inset. The cali bration bars in b inset apply to these traces. b, Amplitude histogram for the data presented in 3. The filed histogram was derived from all ofthe races, represented by diamonds in which the experimenter could Setect a synaptic current, and the open bars from all of the rolease failures (Gots in a). The smooth histogram was derived from traces measured in the same way (and from the same experiment) except no stimulus was present to evoke synaptic current. The ‘ailures histogram (uniled bars) and the ‘no stimulus’ histogram (smooth tine) are not Signiicantly diferent (Kolmogoroy-Smimov test, P>0.1), The inset presents average current as function of time from (1) all data in the files histogram, (2) all data in the untied bar histogram, and (3) all ata fom the line histogram. We estimate from the overlaps of the releases’ and “alures'nistograms, and from uncertainties in making eosiors with visual classification and inthis and other experiments, that less than about 5% of the races would have been misclassified In-most experiments, the classification of traces as “elease’ or 'no release’ was done visually by the experimenter so that every trace was Sorutnized. Most classfation was done bling in the sense that the experimenter didnot know if the records came from an LTP or an LTD experiment or ifthe traces being classified were obtained betore or after the condoning event (tetanus, pairing or low-frequency stimulation. In about 20% of the instances a second experimenter classified the traces Independently, clasifcations by two independent experiments agree to within a few per cent. The calibration bars apply to the three inset ‘waces in a and b, Vertical bar, 3 pA: Rorizontal bar, 5 ms. METHODS. Slices were prepared as in refs 16 and 27. Transverse hip- pocampal slices (~400 ym thick) were obtained from rats (P1d-P26, most P15-P24, Harlan Sprague-Dawley). The tempersture in the recording chamber was 31.5 0.5 °C. The extracellular solution is com. posed of (in mM): NaCl, 120; KCl, 38; NaH,PO., 1.25; NaHCO, 26; MeCls, 1.3: CaCl,, 2-2.5, Pieratoxin (50 4M) was added to the bath LETTERS TO NATURE & 2300 Relative frequency e.p.s.c amplitude (pA) % Solution in ~60%6 ofthe experiments. Electrodes for whole-cell recorcing were filed with in mM) caesium gluconate, £30; CsCl, 5; EGTA, 0:5; MgCl, 2: Na-ATP, 2; GTP, 0.2: NaCl, 5; HEPES’ 10; pH 7.25. When using the perforated patch technique, the pipette solution also contains 4180 pg mi * nystatin. E1ps.cs were evoked bypassing current through fa bipolar tungsten electrode at 0.25 Hz while holding the neuron's membrane potential at ~70 mv. Tetanic stimulation consisted of three bursts, each at 100 He witha 200 ms duration and an iterburst interval (F105, that were applied while the target neuron’s membrane potential ‘was maintained at 20 mV. LTP was alsa induced by @ paring protocol Uwith 25 stimu applied at 0.2 He while maintaining the neuron's mem brane potential at 0 mv. @ FG. 2 9, average records of caret 8 2 futon of Movs 5 time with current and time given by the calibration bars uw forai races te lot toes were taken rom 4a txperment sing heme pocecing te tetanus anc Foie the righthand traces from @ final 10-min period 30 min after the tetanus, The top traces are averages of synap- tic currents (all events, N= 110 left, N= 450 right), the middle traces aro averages of those records for which no release was detected (failure, N=72 left, N=67 Fight), and the bottom traces are’ averages of records {or which transmitter release was detected (release, N= ‘36 left, N~ 83 right. b, Synaptic strength as @ function Of time (min) during the experiment demonstrating LTP for a sample neuron. Average peak synaptic current, including falures, was found by averaging across non- ‘overlapping grouss of successive Incvidual peak syn ‘apie currents and Is potted as a rato to the average fof the five baseline points. The same time base is also Used for c and d, Tetanic stimulation was presented, Using the some stimulus svength, atthe time indicated by the arrow (also in ¢ and a). The horizontal line ater the tetanic. stimulation represents LTP of 2.18. c, Release failure rate, estimated over the same groups of successive individual aces as used forthe average ‘synaptic stength in b, 282 function of time. The failure fate decreased from 0.6 to 0.2. 0, Average amplitude (of the peak synaptic current for these tnals on which ‘lease was detected, as @ function of time: averages ‘ver the same groups of successive traces as used in and c.e, Histograms presenting the reatve frequency of peak elpssc. (when release occurred) before (bar, N=18%) and after stable LTP has been established (smooth line, N=339). The two histograms are not Si nificantly different (Koimogorov-Smirnov test, P> 0.1). NATURE - VOL 371 20 OCTOBER 1904 ol... | ° os Fe Ty $08 ere bos Boal tee yewd 20 imeimn) | pee ay E | Eo trices Poe coped g : ° 10 20 30 40 50 ° 5 0 Time (min) eps. amplitude (pA) 705LETTERS TO NATURE FIG, 3 a, Averages fr a typical experiment as for Fig a 2a All vents, N=150 lek, N= 180 rit; failure, N= 658 lof, N~ 108 right; release, N~82 lef, N72 right. », Characteristics of responses for an LTO experiment pitied in the same way as the LTP data in Fig. 2. Low foqueney stimulation le indieated by bars in be, and {TD of 0.51 incleated by the ine afer te low-frequency stimulation. ¢, Failure rate inereases from 0.6 t0 0.77. €, Histogram of peak synaptic currents before low-tre- ‘quency stimulation (open bars, N'=80) and after (sold line, N=100). The amplitude histograms are not Sig: nificantly different (Kolmogorov-Smirnov test, P> 0.1). METHODS. LTO was inducee by apalyng stimuli at 1 He for 10-12 min while maintaining the membrane poten- Lal at ~TOmW. The magnitude of LTP or LTD was typ ‘cally determines between 15 to 35min afer the conditioning event, Access resistance (25 to 30 M2) and input resistance (100-300 MO) were monitored ‘rroughout experiments and only recordings with stable ‘access and input resstance were used for date analysis 10 os 18 FB poo Failure rate e2oo, 38 ‘release success or fallure was usually determined by eye, rather than by setting an arbitrary threshoid, to ensure that any changes in the smallest response size were detected. A failure was determined by te following o 10 10 20 30 40 50 Time (min) 20.30 40 80 Criteria: (2) the recorded Wace ts not eiscernbly diferent from background noise level, (2) the time course oF shape of recorded ‘ace Is not consistent with. the establisned time course or shape for the epp.s.cs from ‘that neuron In some cells, a smal fraction of recorded ‘races (5%) could not be fairly judged as ether a fai: Ute ora success and were rected. The distribution and average of ep... amplitudes in our study are compat- e.p.s.c. amplitude (ratio of bs! g Relative frequency lle wth those of spontaneous miniature €.p... O° ‘single fore e:ps.c. in CAL reporteo previously". :AP- 5 (GRE), picrotonin (Aldrich) and nystain (Sigma). ° 10 20 30 alll sen e.p.s.c. amplitude (pA) 40 50 ° Time (min) FIG. 4 a, Ratio of success Minimal stimalus probablites (afte/before ‘conditioning » stimulation consisting of tetanus, oF low-frequency stimula tion} as @ function of syn apie strength rato fatter? before) calculated fom averages such a8. illus ‘rated in the top traces (of Figs 2a and 3a. Un- files cncies are trom 12 LIP synaptic strengin ratio>1) and 14 UID {synaptic Strength ratio <1) experiments, Filled ccioles. are from ATP ‘experiments, of the sort ihustated in e ang f that Release probability ratio Response size ratio Interleaved strong. and minimal stimuli at te ‘some stimulation site in ‘the slice. b, Ratio fatter before conditioning stimulation) of average peak e.p:.c. for tals on whieh release vecured, obtained from averaged traces like those ius trated at the bottom of Figs 2a and 3a. asa function of the synaptic ‘strength ratio (rom average traces like thase atthe top af Figs 29 and 3a). Symbols nave the same meanings as those in a.c, LTP experiments, ‘as 3. Diamonds are from 5 LTP experiments done in the presence of AP-5 (93) or with the membrane potential maintained at ~80 mv ‘uring the tetanic stimulation (1~ 2). Unfilled tangles are from 5 ‘experiments in which ‘pairing’ ater than tetanic stimulation was the Conditioning event. Two ofthese experiments used weaky trong condi tioning stmt fs in @and f), Uniled squares are from six experiments that used perforated patch recording. d. Same type of plat as b with ‘symbo's having the same meaning as c.e, Peak synantic current as a Tunetion of time during an LTP experiment with minimal stimulation. ‘Standard tetanic stimulation with nigh intensity just before the 20 min time point Insets are averages (over 10 min) of traces from before and 30 min after the tetanic stimulation, witn curent and time calibrations, ranges trom tals on which release was detected and dots for ° o 1 2 3 0 706 1 Relative synapte strengths (S) eps campitude (pA) 2 3 Time (min) nowelease tra0es. f, AS for ¢, but €.9.5.cs evoked by strong stimulus ‘applied to the same stimulating electrode. No release failures were Seen with the stiong stimulus. Note thatthe ordinate in ds 10 times greater than tne ordinate inc. In these experiments, minimal ana strong Stimuli were applied alternatively to the stimulation site; the stimulus ‘uration for minimal stimulation was reduced to 50 us and the stimulus ‘uration for strong stimulation was 100 ys with the same current a5 ‘minimal stimulation. NATURE - VOL 372 - 20 OCTOBER 1994of the postsynaptic cell during the conditioningevent"™"*", Data from five experiments that used hyperpolarization or AP-5 10 block LTP, plotted as untilled diamonds in Fig. 4e and d. all fall near S= 1. wy /wo= and ay /do= |. Thus, LTP is produced tunder the conditions of our experiments by “pairing” as well as tetanic stimulation, and LTP is blocked by hyperpolarization and AP-S) Although we are aware of no firm evidence that genuine LTP requires the activation of synuptic population, one can imagine some intersynaptic cooperative effect for which plasticity ‘mechanisms would be different from those that are invoked when only one or two synapses are involved, as is the case for our preceding experiments. To test this, we have done the Following experiment on six cells, Ata single stimulation site, microscopic ‘and macroscopic stimuli are interleaved with a pattern of five ‘minimal stimuli (failure rate >0.5) followed by one strong stimu: lus. After obtaining a baseline response size with this repeating 5/1 pattern, LTP was produced with the strong stimulus and then the 5 weak/I strong stimulation pattern was resumed. Typ ical LTP was produced in the population of synapses activated by the strong stimulus, and LTP as we have measured it above \was produced in the subset of synapses activated by the minimal stimulus (Fig. 4e,.) Data from these experiments (filled circles for tetanic stimul- tion in Fig. 4a, , and two unfilled triangles for “pairing” in Fig 4e and d) show that when LTP is elicited in a population of synapses, the modification of synaptic strength in the subset of synapses activated by the weak stimulus occurs by a change in synaptic reliability not potency All of the experiments presented above used standard whoie- call recording which can gradually change the concentration of small intracellular molecules: for example, LTP is known to ‘wash out’ afier about 20 min, Could we have washed out some- LETTERS TO NATURE hat is required for increasing potency? To test this, we did six LTP experiments using the perforated patch technique! and found that, as for the other experiments. LTP involves @ change in reliability without modification of potency (squares in Fig. de and a), Although many different mechanisms are used to modify syn: aaptic strength, we conclude that, under the conditions of our experiments, LTP and LTD operate by’ modifying synaptic rei ability. not poteney. Whatever the precise mechanism by which it is achieved, the fact that symapses ai le and that synaptic plasticity reflects modifications in reliability rather than potency has implications for information processing. The brain rust use a design that is fault tolerant and produces reliable results when constructed with unreliable communications links that modily the strength of connections by chan, and not po! eR ig ie seas ata Cloning of an avermectin- sensitive glutamate-gated chloride channel from Caenorhabditis elegans Doris F. Cully, Demetrios K. Vassilatis**, Ken K. Liu, Philip S. Paress, Lex H. T. Van der Ploeg’, James M. Schaeffe & Joseph P. Arena Department of Cellular Biochemistry and Physiology and * Department of Genetics and Molecular Biology, Merck Research Laboratories, Rahway, New Jersey 07065-0300, USA Th avermectins are a family of macrocyclic lactones used inthe conteol of nematode and arthropod parasites. Ivermectin (2223- er blindness in humans", Abamectin (avermectin By.) isa miticide and insec- ticide used in erop protection’. Avermectins interact with verte- bbrate and invertebrate GABA receptors™’ and invertebrate sglutamate-gated chloride channels". The soil nematode Cacnor- habiitis elegans has served as a useful model to study the mechan- ism of action of avermectins''"*. AC. elegans messenger RNA expressed in Xenopus oocytes encodes an avermectin-sensitive glutamate-gated chloride channel", To elucidate the structure and properties of this channel, we used Xenopus ooeytes for expres- NATURE - VOL 371 - 20 OCTOBER 1994 sion cloning of two functional complementary DNAs encoding an avermectin-sensitive glutamate-gated chloride channel. We find that the electrophy siological and structural properties ofthese pro- Jns indicate that they are new members of the ligand-gated ion ‘channel superfari Xenopus oocytes injected with C. elegans poly(A)” RNA exhibit a rapidly activating reversible ghutamate- and irreversible ivermectin 4"-O-phosphate (IVMPO,)-sensitive — current (Fig, 1a)", A dizectional cDNA library was constructed from size-fractionated RNA and sereened by expression in Xenopus oocytes. Glutamate: and TVMPO,-sensitive currents were observed after injection of i vitye RNA from a pool of 5.000 eDNAs (Fig. 1a). Subjractionation yielded two cDNA clones (PGluCha and pGluCl-f) that expressed glutamate- and IVMPO,-sensitive currents (Fig. 1a) ‘Oocytes simultaneously expressing GluCl a and proteins. exhibited rapidly activating reversible glutamatc- and irrevers- ible IVMPO,-sensitive currents comparable with those found in oocytes injected with poly(A)” RNA (Fig. 1a). The time for maximal activation of IVMPO,sensitive current was 42425 for GluCl wand f compared to 36+35 for poly(A) RNA. The desensitization of the glitamate-sensitive current observed in oocytes injected with poly(A)” RNA was also observed in oocytes injected with GluCl @ and Bat glutamate concentrations greater than TmM., The individual: subunits, GluCla or GluCl-p, expressed functions homomerie channels which were selectively responsive to IVMPO, or glutamate, respectively (Fig. 1a). The time course for IVMPO, activation of homomeric GluClar channels was 181s, which is faster than that observed for GluCl @ and and poly(A)” RNA (P0001), ‘The dose-response curves indicate that coexpression of GluCl ‘@ and i result in changes in the effector concentration for half maximum response (EC) for glutamate and in the Hill 107