Dual Regulation of Adenylate Cyclase by Ca*+/Calmodulin and Transmitter

The Journal of Neuroscience, September 1991, 7 I(9): 2655-2665

Biochemical Studies of Stimulus Convergence during Classical Conditioning in Aplysia: Dual Regulation of Adenylate Cyclase by Ca*+/Calmodulin and Transmitter

Thomas W. Abrams,‘,’ Kevin A. Karl,’ and Eric R. Kandell ‘Howard Hughes Medical Institute, Center for Neurobiology and Behavior, Columbia University, College of Physicians and Surgeons, New York, New York 10032 and 2Department of Biology and The Institute of Neurological Sciences, The University of Pennsylvania, Philadelphia, Pennsylvania 19104-8018

Activity-dependent facilitation is a mechanism of associa- These several results suggested that the dually regulated tive synaptic plasticity that contributes to classical condi- adenylate cyclase might underlie the temporal requirements tioning in Aplysia. Previous studies of activity-dependent for effective classical conditioning in this system. facilitation in the mechanosensory neurons of Aplysia suggested that the Ca*+ influx during paired spike activity en- During associative learning, memories are formed and future hances the transmitter-stimulated, CAMP-dependent, presyn- behavior is altered as a result of an association made between aptic facilitation in these cells. Moreover, paired activity was two or more events. Perhaps the simplest form of associative found to potentiate the activation of the adenylate cyclase learning is classicalconditioning, where the temporal pairing of by transmitter. It was therefore proposed that the Ca*+/cal- one event, the conditioned stimulus, with a second event, the modulin-sensitive cyclase may serve as a site of interaction unconditioned stimulus, alters the responseto the first event. between the inputs from the conditioned and unconditioned Thus, the animal learns that the conditioned stimulus predicts stimuli. These studies were carried out to test whether a the unconditioned stimulus (Rescorla, 1988). Ca*+/calmc-dulin-sensitive adenylate cyclase in the Ap/ysia Where in the nervous system do the associative interactions CNS has the properties necessary to mediate such an as- between the conditioned and unconditioned stimuli occur dur- sociative interaction. ing learning? Are these associative interactions an emergent Three lines of evidence indicate that the same cyclase property of complex neural circuits specifically organized for molecules that are sensitive to Ca*+/calmodulin are also learning, or do they take place among small groups of neurons stimulated by receptor to facilitatory transmitter via the stim- or perhaps even within individual cells? Recent analysis of ulatory G-protein, G,: First, calmodulin inhibitors reduced mechanismsof learning in Aplysia and Drosophila have sug- stimulation of the cyclase by facilitatory transmitter. When gestedthat associative changescan result from allosteric inter- membranes had been preexposed to one of these inhibitors, actions at particular molecular loci within singleneurons (Dudai trifluoperazine, the addition of exogenous calmodulin par- and Zvi, 1984; Livingstone et al., 1984; Abrams and Kandel tially reversed the inhibition. Second, when G, had been 1985, 1988; Ocorr et al., 1985; Yovell and Dudai, 1987). In activated by GTP+, so that it persistently activated the cat- classicalconditioning of the defensivewithdrawal reflex ofrlply- alytic unit of the cyclase, stimulation of the cyclase by Ca2+ sia, the convergent regulation of adenylate cyclaseby Ca2+/cal- was greatly amplified, suggesting that the two inputs interact modulin and modulatory transmitter has been proposed to un- in activating a common population of the enzyme. Third, derlie associative changesin the monosynaptic component of solubilized cyclase activity that bound to calmodulin-Se- the reflex (Abrams, 1985; Abrams and Kandel, 1985, 1988). In pharose in a Ca*+-dependent manner was stimulated by G,, this training paradigm, the conditioned stimulus, a weak siphon which had been partially purified from Ap/ysia CNS, as well touch which produces only a small reflex withdrawal, is pre- as by Ca*+/calmodulin. Having demonstrated dual activation sentedimmediately before the unconditioned stimulus, a mod- of the cyclase, we have explored the dependence of cyclase erate tail shock which elicits a strong defensive withdrawal re- activation on the temporal pattern of Ca*+ and transmitter sponse.After a seriesof thesepairings, the responseto that same addition. Optimal activation required that a pulse of Ca*+ weak conditioned stimulus is substantially enhanced,as if the temporally overlap the addition of facilitator-y transmitter. animal has learned that the siphon touch predicts the noxious tail stimulus (Carew et al., 1981; Hawkins et al., 1986). At the cellular level, the unconditioned stimulus activates Received Dec. 14, 1990; revised Mar. 5, 1991; accepted Mar. 22, 1991. modulatory input to the siphon sensory neurons, the neurons We are grateful to Yoram Yovell and Lise Eliot for their helpful comments on that comprise the afferent pathway for the conditioned stimulus. the manuscript and to Yadin Dudai for his thoughtful suggestions throughout this study. We thank John Bilezikian, who generously provided membranes of the S49 This modulatory input triggers presynaptic facilitation of syn- cycm lymphoma, and Murray Smigel, whose suggestions aided us in developing aptic transmissionfrom the siphon sensoryneurons to postsyn- the protocol for reconstituting G, with the catalytic unit of the cyclase. Correspondence should be addressed to Thomas W. Abrams, Department of aptic interneurons and motoneurons, thus strengtheningthe af- Biology, The University of Pennsylvania, Philadelphia, PA 19 104-60 18. ferent input to the reflex. The responseof thesesensory neurons Copyright 0 1991 Society for Neuroscience 0270-6474/91/l 12655-11$03.00/O to the facilitatory input is enhanced if the sensoryneurons are 2666 Abrams et al. * Dual Regulation of Cyclase in Aplysia caused to fire action potentials immediately prior to their re- have given inconsistent results. While a number of studiesof ceiving the facilitatory input (Hawkins et al., 1983; Walters and cyclasein mammalian brain membranessuggested that the stim- Byrne, 1983a). Because activity-dependent facilitation in the ulatory G-protein activates the samecyclase molecules that are siphon sensory neurons is likely to be an important contributor stimulated by Ca2+/calmodulin(Brostrom et al., 1977, 1978a,b, to the associative changes produced by conditioning (Hawkins Seamonand Daly, 1982; Malnoe et al., 1983; Gnegy et al., 1984; et al., 1983), we probed the underlying associative interactions Harrison et al., 1988, 1989), other studies suggesteda lack of on both a cellular and a biochemical level. convergence (MacDonald, 1975; Lynch et al., 1977; Salter et Conventional presynaptic facilitation of nondepressed syn- al., 198 1). Sanoand Drummond (198 1) reported that solubilized aptic connections from siphon sensory neurons, which occurs cyclase from cerebral cortex could be separatedinto two frac- when facilitatory input arrives in the absence of paired spike tions, one that was Ca2+/calmodulinsensitive and unaffected by activity, is mediated by an increase in CAMP levels (Brunelli et activators of G,, and a second fraction that was stimulated by al., 1976; Bemier et al., 1982; Castellucci et al., 1982). During G, but was not stimulated by Ca2+. More recently, however, activity-dependent enhancement of presynaptic facilitation, severalstudies of solubilized,partially purified mammalian brain paired spike activity in the sensory neurons enhances the CAMP- cyclase have indicated that the calmodulin-sensitive catalytic dependent changes, including the modulation of presynaptic unit is also activated by G, (Heideman et al., 1982; Yeager et membrane currents that contributes to synaptic facilitation al., 1985; Rosenberget al., 1987). In mammals, whether the (Hawkins et al., 1983; Walters and Byrne, 1983b; Hawkins and cyclaseis dually activated may depend upon the specifictissue; Abrams 1984). Ocorr et al. (1983, 1985) and Abrams et al. for example, mammalian sperm cyclase is stimulated by cal- (1984) found that, in response to brief applications of the fa- modulin but is not known to be activated by either hormones cilitatory transmitter 5-HT, the rise in CAMP in sensory neurons or G, (Gross et al., 1987). In invertebrates, the issueof dual is enhanced if the sensory neurons were caused to fire action activation remains unresolved; the only relevant analysiswas potentials or were depolarized just before they were exposed to on Drosophila by Livingstone (1985), who concluded that the transmitter. Electrophysiological studies revealed that Ca2+ in- calmodulin-sensitive population of cyclasewas transmitter influx during a train of action potentials was critical for enhance- sensitive. Convergent activation is an essentialrequirement for ment of the facilitation response by the paired spike activity the hypothesis,described above, that Ca2+influx and facilitatory (Abrams, 1985). These several findings suggested that activity- transmitter interact at the level of the cyclase. We therefore dependent enhancement might occur because the brief elevation carried out experiments designedto determinewhether the same of intracellular Ca*+ during a paired train of action potentials adenylate cyclase in Aplysia CNS that is activated by Ca2+/ increases the synthesis of CAMP stimulated by facilitatory trans- calmodulin is also stimulated by facilitatory transmitter. We mitter. According to this view, the conditioned stimulus is rep- show here that convergent, dual activation of the cyclasedoes resented within the sensory neuron by Ca2+ influx, and the un- occur. We also describe initial experiments designedto explore conditioned stimulus is represented by the action of facilitatory whether activation of the cyclaseis affected by temporal overlap transmitters, such as 5-HT; the interaction of Ca2+ influx and between a transient elevation in CaZ+and arrival of transmitter. facilitatory transmitter might occur within the adenylate cyclase system. The possibility that Ca2+ was modulating the activation of Materials and Methods adenylate cyclase by transmitter seemed plausible in light of the Preparation of tissue. Aplysia californica, weighing100-200 gm, were anesthetizedby iniection of isotonicM&l,. and their centralaanelia finding of Brostrom et al. (1975) and Cheung et al. (1975) that, wereremoved.-Most experiments were d&e%n desheathedpleural gin- in mammalian brain, some of the adenylate cyclase is stimulated glia, the gangliawith the largestpopulation of mechanosensoryneurons by Ca2+, via the Ca2+-dependent regulatory protein calmodulin. (Walterset al., 1983).A smallnumber of experimentswere done on Eliot et al. (1989) demonstrated that the majority of Aplysia isolatedpleural ganglion sensory neuron clusters. In both cases,the neural cyclasecan be stimulated by Ca2+/calmodulin.Yovell et neurons(without sheath)were homogenizedon ice in a glass-Teflon homogenizer.In contrast,in experimentson the entireCNS, the ab- al. (1986) examined the sensitivity of the cyclase to Ca2+/cal- dominal,cerebral, pleural, and pedalganglia were trimmed to reduce modulin and found that this enzyme would be stimulated by the amountof non-neuraltissue (sheath) and homogenizedon ice in a the normal rise in intracellular Ca2+ that occurs during and glass-glasshomogenizer. The homogenate,without the largepieces of immediately after a brief train of action potentials. To explore sheath,was rehomogenizedin a glass-Teflon homogenizer as above. With all three types of material, the homogenate was then centrifuged whether the Ca2+/calmodulin-sensitive adenylate cyclase in at 1000 x g for 2 min to remove large particles. The supernatant was Aplysia neurons provides a molecular site of convergencebe- centrifuged at 30,000 x g for 20 min. In experiments using the entire tween the conditioned and the unconditioned stimuli, we have CNS, the pellet was rehomogenized in homogenization buffer (see be- asked whether the same adenylate cyclase molecules that are low) and centrifuged again. The final pellet was rehomogenized in a stimulated by Ca2+/calmodulinare also activated by transmitter. small volume, 20 ~1 per desheathed pleural ganglion and 500 ~1 per CNS. In most experiments, homogenization was done in the absence When the calmodulin-sensitive cyclasein mammalian brain of EGTA so that membranes retained substantial endogenous calmodu- was first described, there was disagreementabout whether the lin (Lynch et al., 1977). samecyclase that is stimulated by Ca2+/calmodulinis also ac- Assay of calmodulin inhibitors. Potential calmodulin inhibitors were tivated by a receptor for transmitter or hormone. Activation of screened by testing for inhibition of Ca2+/calmodulin activation of phos- adenylate cyclaseby a receptor has been shown to be mediated phodiesterase in Aplysia neural tissue. Desheathed pleural ganglia were homogenized in a glass-glass homogenizer and centrifuged at 13,000 x by a coupling protein, the stimulatory G-protein, G,, which g. Phosphodiesterase activity in the supematant was assayed (Kauvar, binds GTP when a receptor becomesoccupied with agonist; G,, 1982) with free Ca2+ at 10 nM or 1 mM to assess Ca*+ stimulation. Ca2+ in turn, activates the catalytic unit of the cyclase(Schramm and (in the presence of endogenous calmodulin) stimulated phosphodies- Selinger, 1984). Studies that have tested for dual activation of terase activity by approximately 60%. Compounds tested for their inhibition of &+ stimulation included melitini trifluoperazine (TFP, Sig- the cyclaseby attempting to determine whether the stimulatory ma), R2457 1 (calmidazolium; Boehringer Mannheim), and W7 G-protein interacted with calmodulin in activating the enzyme (Seikagaku America, St. Petersburg, FL). R2457 1, trifluoperazine, and The Journal of Neuroscience, September 1991, 77(9) 2657

W7 were maximally effective at concentrations of 5 phi, SO-100 PM, matography buffer (with 8 mM CHAPS) to remove free GTPyS, then and 1 mM, respectively; these efficacies are consistent with those for aliquoted and frozen. inhibition of Aplysia calmodulin-dependent protein kinase reported by Calmodulin-binding adenylate cyclase was isolated by calmodulin- DeRiemer et al. (1984). Melitin (Sigma) was ineffective in the range of Sepharose affinity chromatography (Westcott et al., 1979) using the concentrations that we tested [up to 10 PM, which is 10 times the max- procedure described by Eliot et al. (1989) with minor modifications. imally effective concentration in vertebrate studies (Barnette et al., 1983)]. Prior to each reconstitution experiment, pleural ganglia from 50 animals The efficacies of R24571 and trifluoperazine in inhibiting Ca2+/cal- were desheathed and homogenized in homogenization buffer with 5 mM mod&n stimulation of adenylate cyclase were then verified. (A small EGTA. The material was centrifuged and rehomogenized twice in the number of experiments were also conducted using W7; W7 produced EGTA buffer to deplete endogenous calmodulin. The final pellet was similar effects as the other two compounds, though it required substan- resuspended in cyclase solubilization buffer and centrifuged once again. tially higher concentrations.) The pellet was suspended in 2 ml solubilization buffer with 0.1 mg/ml S&&z&s. All buffers contained 50 mM K-HEPES, 1 mM dithiothreitol HSA and 1.5% Lubrol and shaken at 0°C for 70 min, with occasional (DTT). and motease inhibitors (10 &ml leuuentin. 10 &ml anrotonin. vortexing. After solubilization, 10 mM MgCl, and 1 mM ATP were and 50 &ml PMSF). Except whenindicated, homog&izatibn buffer added, and the material was centrifuged 1 hr at 100,000 x g. The was pH 8.0 and contained no additional components. Other buffers supernatant was diluted with 1 ml binding buffer, brought to 2 mM contained additional components, listed below. Cyclase solubilization CaCl, and 30 mM KCl, and incubated, with rotation, with 1 ml of buffer consisted of 1 mM EDTA and 250 mM sucrose, pH 7.5. G-protein calmodulin-Senharose (Pharmacia) for 3 hr at 4°C. The resin was then solubilization buffer consisted of 1 mM EDTA, 250 mM sucrose, 10 mM transferred to a column and washed with 80 ml of binding buffer. The MgCl,, and 800 mM NaCl, pH 8.0. CHAPS chromatography buffer calmodulin-binding cyclase was then eluted with 4 ml of elution buffer. consisted of 10 mM CHAPS, 10 mM MgCl,, 1 mM EDTA, 0.1 mg/ml The eluted material from calmodulin-Sepharose was thoroughly de- human serum albumin (HSA, Schwarz-Mann), and 10 PM ATP, pH salted and concentrated by ultrafiltration on Centricon 30 filters in 8.0. Calmodulin-Sepharose binding buffer consisted of 8 mM CHAPS, CHAPS chromatography buffer, without ATP, that contained 10 /lo 30 mM KCl, 2 mM CaCl,, 5 mM MgCl,, and 0.1 mg/ml HSA, pH 7.5. forskolin (Behring Diagnostics) to stabilize the enzyme (Smigel, 1986). Calmodulin-Sepharose elution buffer consisted of 25 mM KCl, 8 mM Reconstitution of the fraction containing G, with the fraction con- CHAPS, 5 mM MgCl,, 5 mM EGTA, 1 mM EDTA, and 0.1 mg/ml HSA, taining calmodulin-binding cyclase was achieved with a protocol adapt- pH 7.5. cyc- reconstitution buffer consisted of 10 mM MgCl,, 1 mM ed from Smigel (1986). Aliquots of 50 ~1 of this material were added EDTA, 180 mM NaCl, 0.1 mg/ml HSA, and 10 pM ATP, pH 8.0. to equal volumes (50 ~1) of 1.5 m&ml dioleolylphosphatidylcholine (in Reconstitution buffer consisted of 10 mM MgCl, and 1 mM EDTA, pH reconstitution buffer). This mixture was shaken with 20 mg of SM-2 8.0. In the temporal pairing experiments, pleural ganglia were homog- beads (Bio-Rad) for 15 min at 4°C and then diluted with 300 ~1 of the enized in 50 mM K-HEPES, pH 7.5 (with 1 mM DTT and protease reconstitution buffer with 1 PM forskolin and again shaken 20 min at inhibitors), plus 200 PM EGTA, 60 PM CaCl,, 6 mM MgCl,, and 10 PM 4°C. The material was centrifuged at 13,000 x g for 1 min, and the GTP and resuspended after centrifugation in 2.5 mM EGTA, 160 PM supematant (without SM-2 beads) was added in 125-~1 aliquots to tubes CaCl,, and 6.8 rnr+r MgCl,, pH 7.5 (free Ca2+, approximately 5 nM). In with 5.5 mg of SM-2 beads each. To these tubes, either 13 ~1 of the these pairing experiments, elevations in Cal+ were achieved by adding activated G, or an equal volume of 8 mM CHAPS chromatography buffer with CaCl, and MgCl, to bring the total concentrations of Ca2+ buffer was added; the tubes were shaken for 20 min at 4°C. Caz+/cal- to 800 PM, of MgZ+ to 6.8 mM, and of EGTA to 1.8 mM (free Ca*+, modulin was then added to selected tubes. The cyclase in each tube was approximately 0.1-0.2 PM, after an initial transient of > 100 PM free then assayed in triplicate. Ca2+); free MgZ+ remained approximately 5 mM throughout the exper- Adenylatecyclase assays.Synthesis of cyclic AMP was assayed by a iment. To terminate the Ca2+ pulse, buffer with EGTA and MgCl, was modification of the method of Salomon (1979). The reaction was carried added to bring the total concentrations of Ca*+ to 640 PM, of Mg*+ to out with membranes or solubilized cyclase in a 50-~1 volume containing 8.3 mM, and of EGTA to 10 mM (free Ca2+, approximately 5 nM). For the following: 50 mM K-HEPES (pH 7.6), 50 U/ml creatine phosphoki- the trifluoperazine preexposure experiments, Ca2+/EGTA buffers that nase, 5 mMcreatine phosphate, 0.1 mg/ml HSA, 1 mM 3-isobutyl-l- yielded a free-MgZ+ concentration of 2 mM and a range of free Ca*+ methvlxanthine (IBMX). 2 x lo4 dnm ‘H-cvclic AMP (New England, concentrations were prepared according to the method developed by Nuclear, #TRK304), and 2-30 x - lo6 dpm @P-ATP (Ameriham; Yovell et al. (1986). #PB 17 1). Concentrations of nucleotides, divalent cations, and trans- Reconstitution.A fraction from Aplysiu CNS containing stimulatory mitters varied among experiments and are noted elsewhere. Because G-protein was prepared using a modification of the protocol described Aplysia neural tissues yielded limited amounts of membrane protein by Hanski et al. (1981). Nervous systems from 50 animals were ho- and low total cyclase activity, 32P-cAMP synthesized per assay tube was mogenized in homogenization buffer with 1 mM EDTA, pH 8.0. The increased by using low concentrations of cold ATP, typically 20 PM. membranes were centrifuged (30,000 x g for 20 min) and rehomogen- Assay mixtures were incubated 2-6 min at 25°C. Assays of solubilized ized and recentrifuged two more times. The membranes were then re- cyclase were carried out for 10 min. In preliminary experiments, syn- suspended in G-protein solubilization buffer and centrifuged once again. thesis of cyclic AMP was found to be linear with time over the durations The pellet was suspended in 1 ml of the same buffer with 1.25% cholate used. Cyclic AMP synthesized was isolated by successive cation ex- and 0.1 mg/ml HSA and shaken at 0°C for 70 mitt, with occasional change (Dowex 5OW-X4, Bio-Rad) and neutral alumina (Sigma) chro- vortexing. After solubilization, 10 mM MgCl, and 1 mM ATP were matography. 3H-cyclic AMP included in the assay solution permitted added, and the material was centrifuged at 100,000 x g for 1 hr. The normalization for recovery in calculating cyclic AMP synthesized. supematant was diluted in CHAPS chromatography buffer and concentrated to approximately 1 ml by centrifugation in a Centricon 30 tube. The concentrated material was fractionated by size-exclusion FPLC at 4°C in CHAPS chromatography buffer using a Superose 6 column and Results a Superose 12 column, in series (each column was 30 cm in length, Exogenous calmodulin can overcomeeffects of calmodulin Pharmacia). Fractions were assayed for cyclase activity, and those con- inhibitors in reducing stimulation of the cyclaseby transmitter taining cyclase were discarded. The fractions were also assayed for the presence of G,cu, by its fluoride-dependent stimulation of the cyclase As an initial approach to determining whether, in the Aplysia activity in membranes from the cycm strain of the S49 lymphoma cell CNS, calmodulin influences activation of adenylate cyclase by line, a mutant lacking G,ol (Ross et al., 1978). Reconstitution of G, with transmitter, we tested the effects of calmodulin inhibitors on cyc- membranes was done as follows: 3 ~1 of each fraction were added transmitter stimulation. Two inhibitors, R2457 1 and TFP, were to 12 ~1 of cyc- membranes (0.35 mg/ml protein), then diluted with 10 ~1 of cyc- reconstitution buffer and incubated for 15 min at 0°C. A found in preliminary experiments to be effective in blocking second incubation was done with 25 UM ATP. with and without fluoride Ca2+ stimulation of calmodulin-dependent phosphodiesterase (50 mM NaF with 60 PM AlF, as an activator) for 10 min at 30°C. and cvclase in ADlvsia (seeMaterials and Methods). In further Cyclase activity was then assayed by addition of 3ZP-ATP and other prcl&nary experiments, the stimulation of the cyclase by the assay components. The peak fractions containing G$a (see Fig. 4) were facilitatory transmitter 5-HT in the presence of 100 PM free Ca2+ then preactivated by incubating them with 100 PM GTP-# in the presence of 60 mM MgZ+ for 100 min at 30°C. The preactivated material was found to be decreased by either 4 PM R2457 1 or 100 PM was desalted by ultrafiltration on Centricon 30 filters in CHAPS chro- TFP. 2666 Abrams et al. - Dual Regulation of Cyclase in Aplysia

One explanation of these results is that the transmitter-activated cyclase in Aplysia CNS could also be regulated by Ca2+/ calmodulin. Alternatively, these inhibitors might have inter- fered with transmitter stimulation in some nonspecific manner. To explore further whether calmodulin influences the stimulation of cyclase by transmitter, we examined whether the effect of TFP could be reversed by the addition of exogenous calmodulin. To determine whether exogenouscalmodulin can re- store transmitter stimulation, membraneswere preexposed to 200 PM TFP prior to the assay, then centrifuged and resuspended in buffer without TFP. BecauseTFP is lipophilic, this procedure leaves substantial amounts of the inhibitor in the membrane (seebelow); this TFP in the membraneseffectively reduced Caz+ stimulation of the cyclase even after they were resuspendedin buffer lacking TFP. If the TFP in the membraneswere exerting its effects nonspecifically by inhibiting somecomponent of the adenylate cy- 50 , clase complex other than calmodulin, we would expect that -8 -7 -6 -5 -4 -3 exogenouscalmodulin would not improve stimulation by transmitter. On the other hand, if the TFP were influencing trans- log [Cal mitter stimulation by binding to and inhibiting the endogenous calmodulin, we would expect that exogenouscalmodulin, added 100 B to the TFP-free buffer, might interact with the cyclaseand over- 1 come the effects of the inhibitor in the membranes (Gross et n=5 al., 1987). PI 75 1 Two Ca*+/EGTA buffers were selectedfor theseexperiments, one in which free Caz+ was 3 x lOmEM, which gave no CaZ+ stimulation, and one in which free Ca2+was 3 x 1O-6M, which gave optimal Caz+stimulation (Fig. 1A). In contrast to the stimulation observed in untreated membraneswith Ca*+concentrations of l-100 PM, cyclase activity in TFP-pretreated mem- branesshowed modest inhibition by 3 I.IM Ca2+(data not shown). After addition of 3 PM exogenousbovine brain calmodulin, cyclase activity in pretreated membranes showed stimulation by 3 PM Caz+,though lessthan in untreated membranes(30% h/CM 5-HT 5-HT & 5-HT + vs 67% ‘stimulation in one experiment in which membranes with and without TFP pretreatment were compared, also cf. Fig. CdCM CaKM lA,B). In the absenceof exogenouscalmodulin, stimulation by Figure I. Exogenous calmodulin partially reverses effects of TPP. A, 10 PM 5-HT in pretreated membranes was invariably weak. Ca2+ sensitivity of cyclase. To determine optimal levels of Ca2+ to use Addition of Ca*+/calmodulin improved this 5-HT stimulation. in these experiments, membranes from Aplysia CNS were assayed in a In five experiments on separatepreparations, the simultaneous series of buffers prepared according to the methods of Yovell et al. stimulation of the cyclase by the two ligands was consistently (1988). Free Mg*+ in assays was 2 mM; added calmodulin was 3 PM. Data are from a single experiment. Cyclase activity is expressed as greater than the sum of the separatestimulations by Ca2+/cal- percentage of basal with 10 nM free Ca*+ in the presence of calmodulin. modulin and by transmitter (Fig. 1B). Although this difference B, Ca*+/calmodulin and 5-HT stimulation of membranes preexposed was small (19.6 f 4.3%, mean f SEM), it was significant (p < to TFP. After exposure to 200 NM TFP, membranes from Aplysia CNS 0.02, two-tailed t test), suggestingthat Ca2+/calmodulinand fa- were centrifuged and resuspended in buffer without the calmodulin inhibitor (it was necessary to remove the TFP from the buffer before cilitatory transmitter interact in activating some of the same adding exogenous calmodulin so that the inhibitor would not saturate cyclase molecules. the exogenous calmodulin). Assays were conducted in either 30 nM free An alternative explanation for this reversal of inhibition by Ca*+ (basal conditions) or 3 PM free Ca2+ (high Caz+ conditions). Stim- exogenouscalmodulin could be that the added Ca*+/calmodulin ulation of cyclase by Ca2+ with exogenous calmodulin (Ca/CM), by 10 binds and removes TFP located within the membrane. Rather PM S-HT, or by 5-HT simultaneously with Ca2+/calmodulin (5-HT & &/CM) are expressed as percentage above basal activity. The sum of than substituting for the endogenouscalmodulin in interacting the separate stimulations by 5-HT and Ca2+/calmodulin is also shown with the cyclase, the calmodulin added might merely decrease (5-HT + Ca/CM). Average basal cyclase activity was approximately the effective concentration of the inhibitor. To test for this pos- 15 pmol cAMP/mg.min. Data are means k SEM for five separate sibility, CNS membraneswere preexposed to 200 PM 3H-TFP, experiments, each assayed in quadruplicate. The simultaneous stimulation by the two ligands was significantly greater than the sum of the centrifuged, and resuspendedin buffer without TIP, with the independent stimulations by the two ligands separately (p < 0.02, two- identical procedure usedwith the experimental cyclaseprepa- tailed t test). (In the absence of added calmodulin, the &crease in free ration. By centrifuging thesemembranes once more and count- Ca*+ from 30 nM to 3 MM resulted in a 14.3 k 3.9% inhibition of cvclase ing the pelletsand supematants,we calculatedthe relative amount activity; data not shown.) Assays were conducted with 20 PM ATP in of inhibitor remaining in the membranesafter incubation with the presence of 10 PM GTP. and without Ca2+and calmodulin. The membranesconcentrated The Journal of Neuroscience, September 1991, f I(9) 2659

Table 1. Interactions between Ca2+/calmodulin and G, in stimulating 1000 - adenylate cyclase = a Control + GTPyS GTPyS stimulation 2 800 - Low Ca*+ 3.6 137 133 e9 High Ca*+ 6.7 188 181 Ca2+stimulation 3.1 51 - c 600 - 5 Membrane pellets from whole CNS were resuspended in homogenization buffer with 10 rnM MgC1, and divided into two equal portions. One portion (+ GTPyS) 5 400 - was preactivated by incubating it for 3 min at room temperature with 100 PM GTPyS plus 100 MM 5-HT. The other portion (control) was similarly incubated without transmitter or guanyl nucleotide. Both portions of membrane were then z centrifuged twice more before resuspension for assay.Values are pmol CAMP/ 5 200 - mg.min.

0’ I I I I I I I the inhibitor so that, after resuspensionin TFP-free buffer, they 0 -7 -6 -5 -4 -3 -2 contained a minimum of 5 mM TFP (basedon an estimate of log [GTP] the volume of the pellets obtained by centrifuging the resus- Figure 2. GTP dependenceof cyclasestimulation by transmitter.Cy- pended membranes).Exposure to calmodulin in the presence clase activity in membranes from the entire CNS was assayed with and of 3 PM CaZ+resulted in a 14-20% decreasein the amount of without 20 PM S-HT at four different concentrations of GTP. Note that the inhibitor remaining within the membranes. Such a small 5-HT stimulation is negligible with 100 nM GTP and improves with decreasein the TFP concentration within the membrane frac- increasing GTP. Cyclase activity is expressed as percent of basal activity tion is unlikely to reduce effectively any nonspecificeffects the (without added auanvl nucleotides or 5-HT). which was 5.1 Dmol CAMP/ mg.min. Data are the mean -t SD for three replicates from a single inhibitor may have, unlessthe TFP is selectively removed from experiment. A similar dependence of transmitter stimulation on GTP a site at which it acts nonspecifically to inhibit the cyclase.While was observed in two other experiments. Assays were conducted with not conclusive, the reversal of the TFP inhibition of 5-HT stim- 20 PM ATP. ulation by exogenouscalmodulin suggeststhat the TFP is exerting its action specifically, by inhibiting endogenouscalmodulin. stimulations by G, and Caz+/calmodulin, the dual stimulation was lessthan multiplicative. Nevertheless,these results suggest Interaction betweenG, and Ca2+lcalmodulin in activating dual activation of a single speciesof cyclase. If G, and Ca*+/ cyclase calmodulin were activating separatecyclases, then the absolute These TFP preexposure experiments suggestedthat the same stimulation of CAMP synthesis by Ca*+ would be identical adenylate cyclase can be influenced by two signals, Ca2+/cal- whether or not an independent, calmodulin-insensitive species modulin and transmitter. Another way to test whether suchdual of cyclasehad been activated by G,. Clearly the two stimulations activation occurs is to determine whether the Ca*+/calmodulin- interact and are not independent, consistent with convergent sensitivecyclase can alsobe activated by the stimulatory G-pro- activation of the Ca2+/calmodulin-sensitiveadenylate cyclase. tein, G,. In those systemsthat have been studied, receptor has been found to be coupled to the catalytic unit of cyclase by Reconstitution of stimulatory G-protein with the calmodulin- means of G, (Schramm and Selinger, 1984). Although G, has binding cyclase not been directly shown to mediate receptor activation of the The possibility of dual activation can be tested more directly cyclasecatalytic unit in Aplysia, Castellucci et al. (1983) found by isolating the cyclasethat binds to calmodulin and determin- that, in sensoryneurons, cyclase-mediated effects of transmitter ing whether this same enzyme can be activated by the stimu- were blocked by GDPPS, and Volterra et al. (1987) found that latory G-protein. The calmodulin-sensitive cyclasewas isolated GTPrS mimicked these effects; moreover, Vogel et al. (1989) from other speciesof adenylate cyclasethat may exist in Aplysia have identified immunoreactive G, in Aplysia CNS. Indeed, we CNS by calmodulin-Sepharoseaffinity chromatography (West- found transmitter stimulation of cyclase from Aplysia CNS re- cott et al., 1979; Yeager et al., 1985; Eliot et al., 1989). In an quired the presenceof the guanyl nucleotide GTP, which is attempt to enrich the experimental preparation for the mechano- necessaryfor G,-mediated activation (Fig. 2). These several re- sensory neurons that show activity-dependent facilitation, we sults suggestthat, as in other systems, receptor activation of carried out theseexperiments on desheathedpleural ganglia,the cyclaseoccurs via an intermediary G-protein that binds GTP. ganglia with the greatest abundance of these neurons. Solubi- To examine whether the sameCa2+/calmodulin sensitive cy- lized material from pleural ganglion membraneswas incubated clase is also activated by G,, we determined whether G, alters with calmodulin-Sepharosein the presenceof Ca2+,the resin the Ca2+stimulation of the enzyme. For this purpose,we preac- waswashed to remove unbound material, and calmodulin-bind- tivated G, by incubating membraneswith the nonhydrolyzable ing cyclaseactivity was then eluted with EGTA-containing buff- GTP analog GTP$S. As shown in Table 1, after activation by er (Fig. 3; see also Materials and Methods). As expected, the GTP-& the Ca2+stimulation increaseddramatically, by more calmodulin-binding cyclaseactivity was stimulated by addition than 17-fold. The stimulation by GTPyS was also greater, by of 200 PM Ca2+plus bovine brain calmodulin (seeFig. 5). To about 36%, in the presenceof Ca2+than in its absence.It should determine whether this calmodulin-binding adenylate cyclase be noted that, though the dual activation by G, together with interacted with the stimulatory G-unit, we prepareda crude G,- Ca*+/calmodulin was greater than the sum of the two separate containing fraction from solubilized Aplysia CNS membranes. 2660 Abrams et al. - Dual Regulation of Cyclase in Aplysia

NEURAL TISSU;HOMOGENIZED 2000 1 CENTRIFUTD 30,000 g I - Gs activity MEMBRANE PELLET RESUSPENDED Absorbance I + + 1500 SOLUBILIZED IN SOLUBILIZED IN 1% LUBROL ‘O mM YOLATE 1 GEL PERMEATION INCUBATED WITH FPLC IN CHAPS CALMODULl$S!~PHAROSE 1000 + FRACTION:ASSAYED HSA 1 “YH cyc - 4 CYCLASE G FRACTIONS ACTIVATED ELUTED WITH EGTA WITH GTP Y S 500 0 (CALMODULIN-BINDING (Gs aSUBUNIT) CATALYTIC UNIT) 3 1 DILUTED IN DIOLEOLYL~HOSPHATIDYLCHOLINE 0 DETERGENT REMOVED WITH SM-2 BEADS 0 10 20 30 40 50 Figure 3. Protocol for reconstitution of G, with calmodulin-binding FRACTION NUMBER adenylate cyclase in phospholipid. Starting material for partial purification of calmodulin-sensitive adenylate cyclase was desheathed Aplysiu Figure 4. Partial purification of G, from Aplysia CNS membranes. pleural ganglia. Starting material for partial purification of G, was entire CNS membranes were solubilized in cholate. diluted into CHAPS buffer. Aplysia CNS. Additional details are described in Materials and Methods. and fractionated by gel-permeation FPLC. ‘Fractions (400 bl vol) were assayed for the presence of G,cu by reconstituting aliquots of each with membranes from S49 lymphoma mutant cyc-, which lacks G,(Y. Re- constituted membranes were then assayed with or without fluoride, an activator of G%oI.cyc- stimulation is the cyclase stimulation by fluoride, The solubilized material was fractionated by size-exclusion as percent above activity in the absence of fluoride; no fluoride stim- chromatography. Fractions containing G,cuwere identified using ulation occurs in the absence of G,. The continuous curve indicates the cyc- lymphoma mutant, which lacks GSa (Fig. 4; see also absorbance at 280 nm. Arrows indicate the elution times of peaks rep- Materials and Methods). The fractions containing the peak Gsal resenting HSA and myoglobin, which were included with injection of activity were preactivated using the nonhydrolyzable GTP an- solubilized material as molecular-weight markers. Some G, activity eluted, together with cyclase activity, in the first 8 ml; these early fractions alog GTPyS (Fig. 3). Without such preactivation (or without an were not assayed in the experiment shown in this figure. Cyclase activity alternative activator, fluoride), these fractions were unable to was negligible in those fractions that were assayed, fractions 25-50. The activate cyclase (data not shown). The preactivated, partially two peaks of G, activity may represent the intact c~& heterotrimer, purified G, was reconstituted with the calmodulin-Sepharose which would be expected to elute before HSA, and the dissociated G, eluate in phospholipid (Smigel, 1986). By itself, the G, fraction unit, which would be expected to elute between HSA and myoglobulin. had no detectable cyclase activity. However, addition of the preactivated G, fraction stimulated the calmodulin-binding cyclase 3.6 +- l.l-fold (mean rt SEM) in five experiments on can be activated by a stimulatory G-protein. The cyclaseactivity separatepreparations (Fig. 5). In all of these reconstitution ex- that we have examined was the activity that binds specifically periments, while cyclasestimulation was always observed, the to calmodulin in a Ca*+-dependentmanner (Eliot et al., 1989). quantitative stimulation by the G,-containing fraction and by Moreover, while the G,-containing fraction was not fully pu- Ca2+/calmodulin was quite variable; this was possibly a con- rified, stimulation of the calmodulin-binding cyclase by this sequenceof either inconsistentreconstitution into lipid or vary- fraction required its preactivation with GTP$; this arguesthat ing amountsof degradation ofthe solubilized enzyme. Whatever the observed stimulation of the calmodulin-binding cyclase is the cause of this variability, it prevented us from reliably as- mediated by a guanyl nucleotide binding protein, probably some sessingwhether dual stimulation by the G, fraction combined speciesof the stimulatory G-protein, G,. with Ca2+/calmodulin was greater or less than the sum of the separatestimulations. While we do not yet know which factors Temporal requirements for effective Ca2+-transmitter pairing affected whether or not synergistic stimulation was observed, During classical conditioning of the defensive withdrawal reflex, we did note that the synergismtended to be stronger when both the conditioned stimulus must be temporally paired with the individual stimulations were weaker. unconditioned stimulus for associativelearning to result (Carew In these reconstitution experiments, becauseof the limited et al., 1981, 1983; Hawkins et al., 1986). Similarly, in cellular amount of membrane proteins available from the ApZysia CNS, experiments, action potentials in the sensoryneurons must oc- neither component was a purified protein; for example, the pleu- cur immediately before the cells receive facilitatory input for ral ganglion membranesfrom which we purified the calmodulin- activity-dependent enhancement of the facilitation responseto binding cyclase provided only approximately 20 pg of solubi- occur (Hawkins et al., 1983; Clark, 1984). We therefore wanted lized membrane protein. Although neither G,a nor the catalyst to examine whether convergent activation of the dually acti- was fully purified, theseexperiments do provide evidence that vated cyclaseby CaZ+and transmitter could underlie this tem- the Ca2+/calmodulin-sensitivecyclase from Aplysia neural tissue poral specificity. Does the elevation in Ca*+ need to overlap The Journal of Neuroscience, September 1991, 7 l(9) 2661 temporally with the addition of 5-HT for it to enhance stimu- 16 - lation of cyclase? In order to explore these issues, initial experiments on temporal interactions were conducted with traditional methods in test tubes, in which it was not possible to terminate the exposure to stimulatory transmitter once the transmitter had been added. We tested the effects of brief Ca2+ pulses that were paired or unpaired with 5-HT addition. Experiments were done on membranes from desheathed pleural ganglia that were resuspended in a Ca2+/EGTA buffer with a low level of free Ca2+, approximately 10 nM. To pulse Ca2+ in an assay tube, we pipetted in additional Ca2+ to bring the free Ca2+ in the buffer to approximately 0.1-0.2 FM and, 10 set later, pipetted in additional EGTA to return the free Ca*+ to the initial concentration of approximately 10 nM. (Free Mg2+ was maintained at 5 mM throughout.) For paired stimulation, we added 5-HT in the middle of the Ca2+ pulse; for unpaired stimulation, we added 5-HT 5 set after the end of the pulse. Tubes were vortexed within 0.5 set after each addition. Assays were initiated at the same time as the o- CYCLASE G CYCLASE CYCLASE addition of the stimulating ligands and terminated 2.5 min later. FRACTION FRACTION +G + CaPa Although the differences were modest, we consistently observed differences in cyclase stimulation between paired and Figure5. Stimulatory G-protein activates the calmodulin-binding cy- unpaired Ca2+ and 5-HT addition (Fig. 6). Membranes that clase catalytic unit. EGTA eluate from calmodulin-Sepharose was de- received Ca2+ paired with addition of 5-HT showed significantly salted and reconstituted into phospholipid vesicles, and cyclase activity was assayed alone or with addition of activated G,-containing fraction greater stimulation of cyclase activity than did membranes that or 200 NM Ca2+ plus 0.3 PM calmodulin. Calmodulin-binding cyclase received unpaired Ca2+ and 5-HT (55.6 + 10.0% stimulation was stimulated both by activated G, and by Ca2+/calmodulin (CuICM). above basal for paired vs. 32.6 f 5.8% stimulation above basal The G fraction, containing activated G,, was reconstituted into dioleo- for unpaired; the mean difference, with stimulation compared lylphosphatidylcholine vesicles to which the calmodulin-Sepharose eluate had not been added. Note that the G, fraction by itself had negligible within experiments, was 23.0 + 5.9%; p < 0.02, two-tailed t cyclase activity. Data for CAMP synthesized are means f SD of four test; data are means + SEM). In other experiments, cyclase replicates from a single, representative reconstitution experiment. Qual- stimulation when a Ca2+ pulse was paired with 5-HT addition itatively similar results were obtained in a total of five other experiments. was compared with stimulation with 5-HT alone. Again, the Assays were without free guanyl nucleotides. [Because total cyclase ac- paired Ca2+ pulse significantly enhanced activation by 5-HT tivity from these solubilized pleural ganglion membranes was quite low, assays were conducted with relatively large amounts of 32P-ATP (up to (e.g., in one series of experiments, stimulation was 62.9 f 5.2% 3 x 10’ cpm per tube) and reduced cold ATP concentrations (8 PM for paired CaZ+ and 5-HT vs. 37.4 + 4.1% for 5-HT alone; p ATP). Under these assay conditions, basal cyclase activity yielded more < 0.05). than 1500 cpm 32P-cAMP per tube; recovery was approximately 80%, One possible explanation for the apparent enhancement of and blanks were less than 100 cpm.] stimulation when Ca*+and 5-HT overlapped temporally is that there was simply summation of the two separatestimulations by 5-HT and Ca2+.However, such summation cannot account for the differential cyclase stimulation observed because,in the that Aplysia CNS contains a speciesof adenylate cyclasethat is presenceof GTP, Ca*+stimulation is extremely modest (Novell stimulated by Ca2+ via calmodulin; the existence of a Ca*+/ et al., 1986); these brief, IO-set pulses of CaZ+produced no calmodulin-sensitive cyclasewas confirmed by the experiments detectable stimulation when delivered alone. In fact, in similar describedabove (see,e.g., Figs. lB, 5). experiments without 5-HT in which free Ca*+was increased(as A critical requirement for the hypothesis that the Ca2+/cal- in the pulse experiments), but then left elevated throughout the modulin-sensitive cyclase serves an associative role during remainder of the 2.5-min assay, stimulation was only 6.4 + learning is that the enzyme can be dually regulated. It wasthere- 1.5% of basalactivity. Such minimal stimulation by Ca*+could fore essentialto determine whether the sameadenylate cyclase not explain the enhanced cyclase stimulation observed with moleculesthat can be modulated by Ca2+influx into the sensory pairing. neuronsduring activity elicited by the conditioned stimulus can also be activated by facilitatory transmitter releasedby the un- Discussion conditioned stimulus. To addressthis issue, we have reconsti- The cyclaseas a molecular site of stimulus convergence tuted in phospholipid the Ca*+/calmodulin-sensitive cyclase, Activity-dependent presynapticfacilitation is an associativeform isolated using calmodulin-Sepharose,together with a fraction of synaptic plasticity that has been implicated in classicalcon- containing stimulatory G-protein, G,, from Aplysia CNS. These ditioning in Aplysia. Studies of activity-dependent facilitation reconstitution experiments demonstratedthat the catalytic unit have suggesteda specific molecular locus for the associative of cyclasethat binds to calmodulin in a Ca2+-dependentmanner changes in the sensory neurons. Investigations on intact is effectively activated by G,. Addition of Ca2+/calmodulinalso mechanosensory neurons indicated that the associative en- stimulated this same cyclase. This stimulation by calmodulin hancement of synaptic transmission might result from inter- is most likely direct, given that the enzyme binds to calmodulin- actions betweeninputs from the conditioned and unconditioned Sepharose;alternatively, it could be mediated by a Ca*+/cal- stimuli converging at the Ca*+/calmodulin-sensitive adenylate modulin-dependent kinase present both in the membrane prep- cyclase.Eliot et al. (1989) and Yovell et al. (1986) demonstrated aration and in the eluate from calmodulin-Sepharose. 2662 Abrams et al. * Dual Regulation of Cyclase in Aplysia

presenceof GTP (Fig. 2). Thus, the observation that the Ca2+/ calmodulin-sensitive catalyst can be activated by a G,-containing fraction suggeststhat it can be also activated by transmitter via the stimulatory receptorslinked to G,. The Caz+/calmodulin- sensitiveadenylate cyclasesimilarly isolated from bovine brain 5-HT 1 is also activated by the stimulatory G-protein; Heideman et al. (1982) and Yeager et al. (1985) found that, after separation with calmodulin-Sepharose,the bovine catalytic unit remains coupled to G, and is stimulated by ligands that activate G,. In contrast, the solubilized Aplysia cyclase appears to separate readily from G,, becauseit usually showslittle or no sensitivity 5-HT - to ligands that activate G, after calmodulin-Sepharosechro- matography (L. S. Eliot, unpublished observations). Thus, it was necessaryin this study to reconstitute the catalyst with a G, fraction to demonstrate activation by the guanyl nucleotide- binding protein. Additional evidence that the Ca2+/calmodulin-sensitivead- n=5 enylate cyclase is also activated by transmitter and stimulatory G-protein comes from the observation that, under some conditions, transmitter and G, interact with Ca*+/calmodulin in stimulating the cyclase in homogenized neural membranes.If the two stimuli were activating separateenzymes, the absolute stimulation by one would be unaffected by activation by the other. As discussedbelow, the magnitude of this interaction, or synergism, varied substantially, depending upon experimental conditions. Nevertheless,the fact that under somecircumstanc- es these interactions occurred must mean that Ca2+and transmitter converge in stimulating a single speciesof cyclase. Becausethe Aplysia enzyme is dually regulatedby calmodulin and by the stimulatory G-protein, it provides a potential site of convergence between the conditioned stimulus, which causes activity and Ca*+ influx in the sensory neurons, and the unconditioned stimulus, which causesrelease of facilitatory trans- PAIRED - UNPAIRED PAIRED mitter. This dual regulation of the cyclase contrasts with the UNPAIRED conclusion by Livingstone (1985), studying Drosophila cyclase, Figure 6. Temporal requirements for effective Ca*+-transmitter pair- that a singlespecies of cyclasecannot by itself mediate stimulus ing. A, Protocol for CaZ+ pulses paired and unpaired with facilitatory convergence during conditioning and that more complex mo- transmitter. Homogenized desheathed pleural ganglia were resuspended lecular cascadesmust be postulated to account for the critical in a Ca*+-EGTA buffer with 5 nM free Ca*+. During the assay, each tube roles of Ca2+influx and the Ca2+/calmodulin-sensitivecyclase contained the membranes from approximately one pleural ganglion. Ca2+ transients were achieved by pipetting Cai+ into a tube, followed in associative changesduring learning. 10 set later bv addition of EGTA to reduce the free CaZ+ to the initial low level (see Materials and Methods). Addition of 20 PM 5-HT was Can Ca’+ and facilitatory transmitter act synergistically in done either in the middle of the Ca*+ pulse (PAIRED) or 5 set after the activating the calmodulin-sensitive cyclase? EGTA addition (UNPAIRED). Assav of cvclase activitv was terminated 2.5 min after 5-HT addition: Tubes were vortexed immediately after If Ca2+influx and facilitatory transmitter act associatively to each addition. Assays included 20 PM ATP and 10 FM GTP, and no produce an enhancedfacilitation responseby converging on the added calmodulin (membranes were homogenized and centrifuged in a cyclase,they may be expected to stimulate the adenylate cyclase bufferdesigned to retainendogenous membrane-bound calmodulin). B, synergistically. We consider an activation to be synergisticwhen Cyclase stimulation by paired and unpaired CaZ+ pulses: results of ex- the absolute stimulation by two ligands acting simultaneously periments carried out according to protocol in A. Mean of the difference, within eachexperiment, between stimulation with pairedCa*+ pulse is greater than the sum of the two separatestimulations pro- and stimulation with unpaired Ca2+ pulse (right bar) was significantly duced by each ligand acting alone; thus, synergistic stimulation greater than 0 (*, p < 0.02, two-tailed t test). Data are means + SEM is greater than additive stimulation. Synergistic activation by for five experiments, consisting of four or five replicates each. Average two stimulating inputs that overlap in time could underlie the basal cyclase activity was approximately 16 pmol cAMP/mg.min. requirement for the pairing of the conditioned and unconditioned stimuli. Alternatively, in the absenceof synergism, an associativerequirement for dual activation of the cyclasecould We have not yet carried out the complete reconstitution with result from a nonlinear step in the facilitation responsedown- receptor, G-protein, and catalyst. However, in all systemsthat stream from the synthesis of CAMP (e.g., a process with a dis- have been sufficiently studied, the stimulatory G-protein serves crete threshold) that necessitatedthe coincidence of the two as a coupling protein, binding GTP and activating the catalyst stimuli to achieve a minimum required level of CAMP (see when agonist binds to the appropriate receptor (Schramm and discussionby Yovell and Dudai, 1987). Studiesthat have sought Selinger, 1984). As in other systems, stimulation of Aplysia to identify synergism between the stimulatory G-protein and neural cyclaseby the facilitatory transmitter 5-HT requiresthe Ca2+/calmodulin in activating cyclase in mammalian brain The Journal of Neuroscience, September 1991, 1 I(9) 2663 membranes have yielded inconsistent results (MacDonald, 1975; ed. Ideally, exposure to facilitatory transmitter should be tran- Brostrom et al., 1977, 1978b; Treisman et al., 1983). Similarly, sient; in electrophysiological experiments, no activity-dependent in the CNS of Aplysia, we have found that Ca2+/calmodulin and enhancement of the facilitation response is observed when 5-HT either transmitter or guanyl nucleotides may produce stimula- exposures last more than a few set (Abrams, 1985; T. W. Abrams, tion that is either greater than or less than additive stimulation, unpublished observations). Recent studies have used a modi- depending upon assay conditions. For example, when solubi- fication of the perfused membrane cyclase assay methodology lized cyclase and a G,-containing fraction were reconstituted in ofYovel1 et al. (1987) to examine the activation ofApZysia neural phospholipid, dual stimulation by the preactivated G, fraction cyclase by temporally paired transient stimuli (Yovell and and Caz+/calmodulin was greater or less than additive, depend- Abrams, 1988). These results are consistent with the results of ing upon the individual experiment; our preliminary observa- the Ca*+ pulse experiments described here in suggesting that, tions suggested that the synergism tended to be stronger when whereas there is no synergism when Ca2+ and transmitter are the individual effects of Ca*+/calmodulin and G, were weak. In present for the duration of an assay, Ca2+/calmodulin may in- contrast, in experiments on intact membranes, GTPyS and Ca*+/ teract to enhance activation by 5-HT when the stimuli are suf- calmodulin consistently produced more than additive stimu- ficiently brief (Yovell and Abrams, unpublished observations). lation of the cyclase (Table 1). Eliot et al. (1989) also observed Despite the substantial temporal limitations in the present synergistic activation of solubilized bovine brain cyclase by non- protocol, one interesting observation has emerged: a Ca2+ pulse hydrolyzable guanyl nucleotides and Ca2+/calmodulin. Reliable, that ended 5 set before 5-HT was added did not noticeably though weak, synergism was also observed when TFP preex- enhance the stimulation of the cyclase, suggesting that Ca2+/ posure was used to inhibit endogenous calmodulin (Fig. 1B). calmodulin effects on cyclase activation decay in less than 5 set The parameters that affect the extent to which synergism is (Fig. 6, unpaired pulse). Such a rapid decay of the effects of Ca2+ observed under different assay conditions must still be eluci- is consistent with the narrow interstimulus interval (< 2 set) for dated. Nevertheless, these experiments, and those in which puls- effective stimulus pairing that has been observed behaviorally es of CaZ+ were paired with 5-HT, suggest that at least under (Hawkins et al., 1986). some conditions Ca2+/calmodulin may regulate the activation In addition to requiring temporal proximity for effective pair- of cyclase by transmitter. As discussed below, under normal ing of conditioned and unconditioned stimuli, a molecular conditions (where G, is activated by the natural guanyl nucle- mechanism for associative plasticity during conditioning would otide GTP, and in the absence of calmodulin inhibitors), syn- be expected to display the sequence requirement observed in ergistic activation may be most dramatic when the activating behavioral and physiological studies of classical conditioning; stimuli are transient. that is, the conditioned stimulus, or activity, and Ca*+ influx must precede the unconditioned stimulus, or facilitatory trans- Temporal requirements for eflective stimulus pairing mitter. Results indicating a possible Ca*+-5-HT sequence re- Perhaps the most interesting prediction from behavioral and quirement for optimal cyclase activation have recently been electrophysiological studies about associative activation of ad- obtained using the perfused membrane cyclase methodology enylate cyclase is that there should be precise temporal require- mentioned above (Yovell and Abrams, 1988). ments for the pairing of Ca2+ and transmitter to achieve optimal cyclase activation: (1) Ca2+ and transmitter must overlap tem- Possible additional sites of stimulus convergence porally, and (2) activation by Ca*+ must begin before activation Although we have identified one molecular site at which activity by transmitter. and Ca*+ influx can enhance the CAMP-mediated facilitation Ideally, the interaction between stimuli would be explored response, undoubtedly there are other sites of interaction. For with transient increases in Ca2+, comparable to the rise in in- example, in several cell types, phosphorylation by the CaZ+/ tracellular Ca*+ during a brief (~0.5 set) train of action poten- phospholipid-dependent kinase (kinase C) increases transmitter tials, and with transient exposures to facilitatory transmitter, activation of the cyclase by inactivating the inhibitory G-pro- such as would be produced by a short (l-l .5 set) unconditioned tein, Gi (Jakobs et al., 1985; Katada et al., 1985). Thus, this is stimulus. The techniques used in the present study were inad- a second possible mechanism by which Ca2+ could enhance equate for a thorough temporal analysis. Nevertheless, our pre- CAMP synthesis. Downstream from adenylate cyclase and CAMP liminary attempts to test the effects of temporal coincidence synthesis, there are additional possible sites at which Ca2+ could indicated that Ca2+ pulses that overlap in time with 5-HT ad- modulate the facilitation response. Such interactions could di- dition were more effective in activating cyclase than Ca2+ pulses minish, as well as potentiate, CAMP-mediated effects, as Kramer that ended before the addition of transmitter (Fig. 6). Thus, this and Levitan (1988) and Kramer et al. (1988) have described in initial study suggests that Ca*+/calmodulin may serve to prime neuron R15 in Aplysia. In this cell, Ca2+ influx antagonizes the cyclase for optimal stimulation by facilitatory transmitter. modulation by CAMP by activating the Ca2+/calmodulin-sen- There are, however, two important limitations to the procedure sitive phosphodiesterase. Similarly, Aplysia sensory neurons used in these initial experiments: First, durations of stimuli and contain a calmodulin-sensitive phosphodiesterase (T. W. Abrams intervals between stimuli were long compared with physiolog- and K. A. Karl, unpublished observations) as well as a cal- ical stimuli. For instance, the lo-set Ca*+ rise in the test tube modulin-sensitive cyclase. The predominant effect of Ca2+ el- is probably much longer than the CaZ+ transient produced by a evations on CAMP-mediated processes may depend on the rel- 0.5-set train of action potentials in the sensory neuron (M. Spira, ative abundance of the two enzymes and on the distribution of H. Blumenfeld, and S. Siegelbaum, personal communication). CaZ+ within the cell; a small influx Ca2+ may be localized near Second, in our experiments, 5-HT, once added, remained in the the membrane and may primarily activate the cyclase. The role membranes for the duration of the assay. In contrast, in the played by other sites of interaction between activity and the ganglion, the concentration of free facilitatory transmitter prob- facilitation response in activity-dependent facilitation must be ably begins to decline once the unconditioned stimulus has end- explored in future cellular and biochemical studies. 2664 Abrams et al. - Dual Regulation of Cyclase in Aplysia

Bemier L, Castellucci VF, Kandel ER, Schwartz JH (1982) Facilitatory Allosteric interactions and associative learning transmitter causes a selective and prolonged increase in adenosine 3’: 5’-monophosphate in sensory neurons mediating the gill and siphon Following the initial characterization of proteins that had central withdrawal reflex in Aplysiu. J Neurosci 2: 1682-l 69 1. roles in neuronal function, such as the ACh receptor, the sug- Brostrom CO, Huang YC, Breckemidge BM, Wolff DJ (1975) Iden- gestion was made that allosteric interactions at a single molecule tification of a calcium-binding protein as a calcium-dependent reg- could underlie associative changes during learning (Stent, 1973; ulator of brain adenylate cyclase. Proc Nat1 Acad Sci USA 72:64-68. Heidmann and Changeux, 1982; Changeux and Heidmann, Brostrom CO, Brostrom MA, Wolff DJ (1977) Calcium-dependent adenylate cyclase from rat cerebral cortex. Reversible activation by 1987). Recently, two particular examples of such allosteric in- sodium fluoride. J Biol Chem 252:5677-5685. teractions have been described in studies of learning and syn- Brostrom MA, Brostrom CO, Breckenridge B, Wolff DJ (1978a) Cal- aptic plasticity in simple neural systems. First, the analysis of cium-dependent regulation of brain adenylate cyclase. Adv Cyclic activity-dependent facilitation gave rise to the hypothesis that Nucleotide Res 9:85-99. convergence of the conditioned stimulus and unconditioned Brostrom MA, Brostrom CO, Wolff DJ (1978b) Calcium-dependent adenylate cyclase from rat cerebral cortex: activation by guanine nu- stimulus at the Ca*+/calmodulin-sensitive adenylate cyclase could cleotides. Arch Biochem Biophys 191:341-350. be responsible for the associative synaptic changes. The present Brunelli M, Castellucci V, Kandel ER (1976) Synaptic facilitation and study indicates that, in Aplysia, the Ca2+-sensitive cyclase has behavioral sensitization in Aplysia: possible role of serotonin and some of the properties required for it to play such an associative cyclic AMP. Science 194: 1178-l 18 1.. Carew TJ. Walters ET. Kandel ER (198 1) Classical conditionina in a role. Additional support for an associative role for the cyclase simple- withdrawal reflex in Aply& ca&mica. J Neurosci 1: l-426- comes from the finding that the Drosophila mutant rutabaga, 1437. which shows poor learning and memory, has a defect in the Carew TJ, Hawkins RD, Kandel ER (1983) Differential classical con- function of its Ca2+/calmodulin-sensitive adenylate cyclase (Du- ditioning of a defensive withdrawal reflex in Aulvsia_ . californica. Sci- dai and Zvi, 1984; Livingstone et al., 1984; Yovell et al., 1986). ence 219:397400. Castellucci VF, Naim A, Greengard P, Schwartz JH, Kandel ER (1982) Second, recent studies of the mechanism of associative long- Inhibitor of adenosine 3’:5’-monoohosphatedenendent nrotein ki- term synaptic potentiation in hippocampus suggest that nase blocks presynaptic facilitation in kplysia. J Neurosci 2: 1673- associative activation of the glutamate-gated channel may be 1681. responsible for strengthening the synaptic connections (Collin- Castellucci VF, Bemier L, Schwartz JH, Kandel ER (1983) Persistent activation of adenylate cyclase underlies the time course of short- gridge et al., 1983; Harris et al., 1984; Kelso et al., 1986; Sastry term sensitization in Aplysia. Sot Neurosci Abstr 9: 169. et al., 1986; Wigstrom et al., 1986). Temporal coincidence of Changeux JP, Heidmann T (1987) Allosteric receptors and molecular glutamate binding and postsynaptic depolarization is required models of learning. In: Synaptic function (Edelman GM, Gall WE, for effective activation of the NMDA-gated Ca2+ conductance Cowan WM, eds),-pp 549-661. New York:‘ Wiley. Cheuna WY. Bradham LS. Lvnch TJ. Lin YM. Tallant EA (1975) (Nowak et al., 1984; MacDermott et al., 1986); the resulting Protein activator of cyclic 3<5’-nucleotide phosphodiesterase of bo: postsynaptic Caz+ influx may serve to trigger synaptic plasticity vine or rat brain also activates its adenylate cyclase. Biochem Biophys (Lynch et al., 1983; Malenka et al., 1988). Thus, in both Aplysia Res Commun 66:1055-1062. sensory neurons and hippocampus, associative changes in syn- Clark GA (1984) A cellular mechanism for the temporal specificity of aptic connections may not require a complex neuronal circuit, classical conditioning of the siphon-withdrawal response in Aplysia. 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