Serotonin Stimulation of Camp-Dependent Plasticity in Aplysia Sensory Neurons Is Mediated by Calmodulin- Sensitive Adenylyl Cyclase

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Serotonin stimulation of cAMP-dependent plasticity in Aplysia sensory neurons is mediated by calmodulin- sensitive adenylyl cyclase

Allison H. Lina, Jonathan E. Cohena, Qin Wana, Katelyn Niub, Pragya Shresthaa, Steven L. Bernsteinc, and Thomas W. Abramsa,b,1

aDepartment of Pharmacology, bProgram in Neuroscience, and cDepartment of Ophthalmology, University of Maryland School of Medicine, Baltimore, MD 21201-1559

Edited* by Eric Richard Kandel, Columbia University, New York, NY, and approved July 13, 2010 (received for review April 2, 2010) Calmodulin (CaM)-sensitive adenylyl cyclase (AC) in sensory neu- SNs. In these studies, we focused on the biochemical properties and rons (SNs) in Aplysia has been proposed as a molecular coincidence regulation of the two AC isoforms that are expressed in Aplysia detector during conditioning. We identiﬁed four putative ACs in SNs. One of these isoforms, AC-AplA, is stimulated by Ca2+/CaM, Aplysia CNS. CaM binds to a sequence in the C1b region of AC- whereas the second, AC-AplC, is directly inhibited by Ca2+. AplA that resembles the CaM-binding sequence in the C1b region Knockdown experiments revealed that the Ca2+/CaM-sensitive of AC1 in mammals. Recombinant AC-AplA was stimulated by Ca2+/ isoform is responsible for the great majority of 5-HT-induced CaM. AC-AplC is most similar to the Ca2+-inhibited AC5 and AC6 cAMP-mediated plasticity in SN somata. This demonstrates that the in mammals. Recombinant AC-AplC was directly inhibited by Ca2+, CaM-sensitive AC is indeed dually regulated in those neurons where independent of CaM. AC-AplA and AC-AplC are expressed in SNs, it has been hypothesized to function as an associative integrator. whereas AC-AplB and AC-AplD are not. Knockdown of AC-AplA demonstrated that serotonin stimulation of cAMP-dependent plas- Results ticity in SNs is predominantly mediated by this CaM-sensitive AC. In an initial effort to identify AC isoforms expressed in Aplysia We propose that the coexpression of a Ca2+-inhibited AC in SNs,

CNS, we used degenerate primers corresponding to sequences NEUROSCIENCE together with a Ca2+/CaM-stimulated AC, would enhance the as- within the two cytoplasmic regions that are highly conserved 2+ sociative requirement for coincident Ca influx and serotonin for among transmembrane ACs in metazoans, the C1a and C2a re- effective stimulation of cAMP levels and initiation of plasticity me- gions (19, 20). We isolated cDNA clones from Aplysia CNS that diated by AC-AplA. corresponded to three distinct sequences based on restriction map analysis and sequencing. Full-length sequences of these coincidence detector | associative learning | synaptic plasticity | classical putative AC transcripts, AC-AplA, AC-AplB, and AC-AplC, were conditioning | calcium generated by 5′ and 3′ rapid amplification of cDNA ends (RACE) (GenBank accession nos. AY843025, AY843026, and hree decades ago, early experimental evidence from two sim- AY843027). The membrane-associated ACs of multicellular Tple invertebrates, Aplysia and Drosophila (1, 2), demonstrated animals have two transmembrane domains, each with approxi- that cAMP plays an important role in learning. Based on studies mately six membrane-spanning α-helices, separated by a cyto- of classical conditioning in Aplysia, calmodulin (CaM)-sensitive plasmic C1 domain. A second cytoplasmic domain, C2, is located adenylyl cyclase (AC) was proposed to play an associative role in at the C terminus and a third, relatively short cytoplasmic se- learning (3–5). CaM-sensitive AC was similarly implicated in quence is at the N terminus (21). Analysis of these putative conditioning in Drosophila (6, 7). Subsequently, CaM-sensitive AC Aplysia AC isoforms indicated a similar structure, with two hy- was also found to be important in learning in mammals; mouse drophobic domains containing five to seven membrane-spanning mutants that lack CaM-sensitive ACs 1 and 8 displayed deficits in α-helices per domain, similar to predictions for mammalian ACs memory (8, 9), and overexpression of AC1 in mice improved (Fig. S1). Because in transmembrane ACs the C1a and C2a cy- memory (10). toplasmic regions interact to create the nucleotide binding site The CaM-sensitive AC in Aplysia was hypothesized to serve required for catalytic activity, we predict there is an even number as an associative coincidence detector that integrates two signals of membrane crossings, namely six. A fourth putative AC, AC- 2+ during classical conditioning: (i)Ca influx during sensory AplD, was identified through a search of the Aplysia EST data- neuron (SN) activity triggered by the conditioned stimulus and (ii) base (22) and a full-length sequence was obtained by RACE serotonin (5-hydroxytryptamine; 5-HT), released by modulatory (GenBank accession no. HM030824). Analysis of this transcript interneurons during the unconditioned stimulus. This coincidence indicated a similar topology (Fig. S1D). The predicted C2 do- detector concept was based on cellular electrophysiology and main of AC-AplA is nearly three times the length of the other C2 – biochemical assays (5, 11 14). Evidence suggesting that CaM- domains (Table S1). All metazoan transmembrane ACs contain sensitive AC may function as a coincidence detector has also been highly conserved sequences in the C1a and C2a regions that obtained in Drosophila (15) and in studies of the mammalian AC1 (16). Nevertheless, the proposed role of CaM-sensitive AC as

a coincidence detector during learning has not been directly Author contributions: A.H.L., J.E.C., S.L.B., and T.W.A. designed research; A.H.L., J.E.C., tested in Aplysia or any other system. Q.W., K.N., P.S., and T.W.A. performed research; A.H.L., J.E.C., Q.W., P.S., and T.W.A. Although CaM-sensitive AC was implicated in synaptic plasticity analyzed data; and A.H.L., J.E.C., K.N., S.L.B., and T.W.A. wrote the paper. during learning in Aplysia more than 25 years ago, this enzyme from The authors declare no conﬂict of interest. Aplysia had not been characterized at the molecular level. Recent *This Direct Submission article had a prearranged editor. studies have raised questions about the role of the cAMP cascade Data deposition: The sequences reported in this paper have been deposited in the Gen- in associative facilitation at Aplysia SN-to-motor neuron (MN) Bank database (accession nos. AY843025, AY843026, AY843027, and HM030824). synapses (17, 18). We have investigated which AC isoforms are 1To whom correspondence should be addressed. E-mail: [email protected]. expressed in Aplysia CNS and in SNs in particular, and determined This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. whether a CaM-sensitive AC is coupled to 5-HT receptors in the 1073/pnas.1004451107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1004451107 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 together form the ATP-binding pocket in the catalytic core the other cytoplasmic domains (Fig. S5). The C1b region of AC- (Fig. S2) (23–25). The four putative Aplysia ACs share this AplA contains a sequence with similarity to the CaM-binding conserved pattern of residues (Fig. S2). All four C1a regions region of mammalian AC1 (28) (Fig. 1). CaM-binding motifs are contain the characteristic GDCYYC sequence (19, 20), which is relatively diverse, and include the 1-8-14 pattern of hydrophobic conserved in all transmembrane ACs in metazoans that we have residues (35, 36). AC1 contains a 1-8 motif, where the 1 and 8 examined, including Trichoplax (Fig. S2C). Moreover, in the residues are both required for CaM binding (28). Both AC-AplA purine-binding pocket of the C2a region, all four predicted and rutabaga AC contain a 1-8-15 pattern of hydrophobic resi- sequences contain the lysine and aspartate residues that typify dues within the C1b region (37). A 25-aa peptide from AC-AplA ACs, in contrast to guanylyl cyclases (24, 26). containing this 1-8-15 sequence bound CaM. To determine which AC-AplA and AC-AplC Resemble AC Isoforms That Are Regulated by hydrophobic amino acids mediated CaM binding, we mutated the Ca2+. To identify the CaM-sensitive AC in Aplysia, which had been motif in the AC-AplA C1 domain at positions 1 and 8 (F498S/ proposed to play an associative role in classical conditioning, V505D), positions 8 and 15 (V505D/V512D), or positions 15 we compared the four Aplysia sequences with the C1a and C2a alone (V512D). CaM binding was eliminated in both the F498S/ regions of all nine mammalian and four of the Drosophila transmembrane ACs. In mammals, AC1 and AC8 are stimulated by Ca2+/CaM (27–29). ACs 5 and 6 are highly similar isoforms that are directly inhibited by Ca2+ (30). ACs 2, 4, and 7 are closely related, Ca2+-independent ACs. AC3 is inhibited by CaM kinase II (31). AC9 is regulated by CaM kinase II, PKC, and calcineurin (32, 33). The C1a region of AC-AplA is particularly similar (72%) to rutabaga, the Ca2+/CaM-sensitive AC in Drosophila (34) (Fig. S3), which suggested that AC-AplA may be the Ca2+/CaM-sensitive AC in Aplysia CNS (Fig. S4). AC-AplA is also similar to ACs 1, 5, and 6, as is rutabaga AC. However, AC-AplC is the Aplysia isoform most similar to AC5 and AC6 (Fig. S4). Based on this analysis, we predicted that AC-AplA might be stimulated by Ca2+/CaM and that AC-AplCmightbeCa2+-inhibited. The C1a region of AC-AplBis not highly similar to any single group of ACs, whereas the C2a domain of AC-AplB is most similar to members of the AC2/4/7 group (Figs. S3 and S4). AC-AplD has a relatively low similarity to all of the mammalian ACs; however, it is most similar to AC9 and to Dro- sophila AC 35C, the Drosophila homolog of AC9 (19) (Fig. S4). None of the identiﬁed Aplysia AC isoforms resembled AC3.

AC-AplA Contains a Ca2+/CaM-Binding Domain. To determine which cytoplasmic domains interact with CaM, we expressed the N- terminal and C1 and C2 domains of each of the four putative ACs. In CaM overlay assays, we observed that CaM bound to the C1 domain of AC-AplA, in a Ca2+-dependent manner, but not to

Fig. 2. Ca2+ regulation of AC-AplA and AC-AplC. (A) AC-AplA is stimulated by Ca2+/CaM. (A1) AC activity in membranes from High Five insect cells infected with baculovirus encoding either AC-AplAorβ-gal, assayed in EGTA/Ca2+/Mg2+ buffers with free Ca2+ concentrations ranging from 5 nM to 350 μM, in the presence of 1 μM CaM. Absolute AC activity from a representative experiment (mean ± SD of five replicate assays). Note both AC- AplA and native insect AC (β-gal) display stimulation by Ca2+, although the absolute maximal activity of the cells expressing recombinant Aplysia AC is substantially greater. (A2) Calculated activity of AC-AplA as a function of [Ca2+]. To calculate the Ca2+ stimulation of AC-AplA, for each Ca2+ concentration, the absolute activity in membranes from cells infected with β-gal virus was subtracted from the activity in membranes from cells infected with AC-AplA virus. Data represent means ± SEM of AC activity from four separate experiments using membranes from independent populations of High Five cells; each membrane preparation was assayed in five replicates. AC Fig. 1. CaM-binding sequence within C1b region of AC-AplA. (A)(Upper) activity at each Ca2+ concentration was normalized to the basal activity at Alignment of the CaM-binding domain of human AC1 and the corre- 5nMCa2+.(A3) The stimulation of AC-AplAbyCa2+ is CaM-dependent. Data sponding sequences in AC-AplA and rutabaga AC; the 1-8 or 1-8-15 pattern are means ± SEM of data from three experiments on separate populations of 2+ of hydrophobic residues is indicated by shading. (Lower) Mutations of these cells (F3,6 = 23.7, P = 0.001). (B) AC-AplC is inhibited by Ca , independently of three hydrophobic residues in AC-AplA are shown with substitutions at CaM. (B1) AC activity in membranes from High Five cells infected with positions 1 and 8 (mutant 1), 8 and 15 (mutant 2), or 15 alone (mutant 3). (B) baculovirus encoding either AC-AplCorβ-gal, assayed at a range of free Ca2+ CaM overlay assays to determine which hydrophobic residues in the 1-8-15 concentrations from 5 nM to 44 μM, in the presence of 1 μM CaM. Absolute motif in the C1b region of AC-AplA (amino acids 498–512) are critical for AC activity from a representative experiment (mean ± SD of five replicate CaM binding. Coomassie-stained gel (Left). CaM overlay assays in the pres- assays). (B2) Activity of AC-AplC, calculated as in A2. Note the activity of AC- ence of 1 mM Ca2+ (Center) or 10 mM EGTA (Right). Note the double AplC decreased as the concentration of free Ca2+ increased. (B3) The in- mutants F498S/V505D and V505D/V512D both eliminated CaM binding, hibition of AC-AplCbyCa2+ is independent of CaM. Data are means ± SEM of

whereas the single mutation V512D did not affect CaM binding, suggesting data from three experiments on separate populations of cells (F3,6 = 42.8, P < that residues 1 and 8 are necessary for CaM binding. Note also that CaM 0.001). In A1, A2, B1, and B2, some error bars are smaller than symbols. *P < binding was Ca2+-dependent. 0.05 for pairwise, posthoc comparisons with 50 nM Ca2+ + CaM.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1004451107 Lin et al. Downloaded by guest on September 28, 2021 V505D and V505D/V512D mutants (Fig. 1B). In contrast, CaM binding was not substantially affected in the V512D mutant, in- dicating that residue 15 does not contribute to the afﬁnity for CaM. Thus, the CaM-binding motif of AC-AplAhasa1-8pattern of hydrophobic residues, as does mammalian AC1 (28).

AC-AplAIsaCa2+-/CaM-Regulated AC, Whereas AC-AplC Is Inhibited by Ca2+. To explore Ca2+ regulation of the hypothesized CaM- sensitive and Ca2+-inhibited ACs AC-AplA and AC-AplC, we expressed these two AC isoforms in High Five insect cells. Ex- pression was conﬁrmed with isoform-speciﬁc antibodies (Fig. S6). Membranes from High Five cells expressing AC-AplAor AC-AplC displayed a 6.0 ± 1.5-fold (n = 4) and 7.3 ± 2.5-fold (n = 3) increase, respectively, in basal AC activity as compared with membranes from control cells infected with baculovirus encoding β-gal (Fig. 2 A1 and B1). AC-AplA and AC-AplC were assayed with Ca2+/Mg2+/EGTA buffers with free Ca2+ ranging from 50 nM to ≥40 μM. In the presence of CaM, we observed 3.1 ± 0.4- fold stimulation as Ca2+ increased to 1.7 μM(n = 4) (Fig. 2A). No stimulation was seen in the absence of CaM (Fig. 2A3). AC- AplC was inhibited when Ca2+ was increased above 100 nM; inhibition reached a plateau by 10 μMCa2+ (39.2 ± 4.5% of basal activity, n = 3) (Fig. 2B). This inhibition of AC-AplC was CaM-independent (Fig. 2B3).

Ca2+-Regulated AC Isoforms Are Enriched in Aplysia Sensory Neurons. To assess which of the AC isoforms are expressed in Aplysia

mechanosensory neurons, we carried out quantitative real-time NEUROSCIENCE PCR (qRT-PCR) assays for all four AC isoforms. Assays were linear down to <10 copies (Fig. S7). Assays on cDNA from SN clusters gave a wide range of values for AC-AplA and AC-AplC, depending on the population of animals. AC-AplA message was present at >1000 copies per SN. AC-AplCmRNAvariedfrom10to>1000 copies per SN. In contrast, AC-AplB and AC-AplD were present at <1 copy per SN, consistent with contamination from other cells, such as glia. Thus, it appears that both AC-AplA and AC-AplCare expressed in SNs, whereas the other two AC isoforms are not. The expression of AC-AplA and AC-AplC was confirmed by Fig. 4. cAMP-dependent modulation of SN action potential by 5-HT is medi- immunoblot on desheathed pleural ganglia, which contain the ated by AC-AplA. SNs in the pleural ganglion ventro-caudal cluster were injected largest population of these mechanoreceptor SNs (Fig. 3). In either with antisense morpholino or dsRNA for AC-AplAorAC-AplBorantisense morpholino for AC-AplC. Alexa 594 dextran was included with morpholinos or both pleural ganglion and CNS membranes, the antibody against ≈ dsRNA to identify injected neurons. Injection of control morpholino or Alexa 594 the C2b domain of AC-AplA stained a major band at 115 kDa; dextran served as a control. Because AC-AplB is not expressed in SNs, morpholino in many immunoblots, an additional weaker band was visible at or dsRNA for AC-AplB was used as a third control. After injection, clusters were 200 kDa (Fig. 3A), similar to the predicted size of 208 kDa (Fig. maintained in culture for 4 d before electrophysiology. Spike-broadening responses to 5-HT were measured in the presence of 100 mM TEA and 20 μM nifedipine; under these conditions, 5-HT-induced spike broadening is mediated entirely by cAMP-dependent phosphorylation (40, 41) (SI Methods). SNs injected with either morpholino or dsRNA for AC-AplA showed significantly less spike broadening than control neurons. (A) Traces recorded before and after 3–4min of exposure to 10 μM 5-HT; each pair of traces is recorded from a different SN. (B) Group data for 5-HT-induced spike broadening for SNs treated with either dsRNA or morpholino. SNs injected with morpholino for AC-AplAshowedsig- nificantly less broadening of the action potential than control SNs injected with either Alexa dextran (P < 0.001), control morpholino (P = 0.008), or morpholino for AC-AplB(P =0.002;allP values for individual pairwise, posthoc comparisons

were made with the Sidak adjustment; SI Methods)(**indicatesP < 0.01) (F4,28 = 5.31 P = 0.003). SNs injected with dsRNA for AC-AplA showed signiﬁcantly less broadening of the action potential than control SNs injected with Alexa dextran

Fig. 3. Immunoblot of AC-AplA and AC-AplC in pleural ganglion and CNS (P = 0.003) or dsRNA for AC-AplB(P =0.006)(F4,28 =9.71,P =0.001).Therewasno membranes. (A) Antibodies against sequences in the C2b domains of AC- signiﬁcant (NS) reduction in the spike-broadening response of SNs injected with AplA and AC-AplC recognize bands in membranes from pleural ganglia, morpholino for AC-AplC(P = 0.416, 1.0, and 0.982 for Alexa control, control which are enriched for SNs. Preincubation with antigen peptide blocked morpholino, and AC-AplB morpholino, respectively, for pairwise, posthoc staining. The high molecular weight bands recognized by the anti-AC-AplA comparisons). (C) Effect of double knockdown of AC-AplCandAC-AplA. To antibody are indicated by arrowheads. (B)Toconﬁrm the identity of the further assess whether some of the action potential broadening induced by 5-HT predominant protein recognized by anti-AC-AplA antibody, which is sub- might be mediated by AC-AplC, we tested whether double knockdown using stantially smaller than the predicted molecular weight, we probed CNS morpholinos for both AC-AplCandAC-AplA produced greater reduction in the preparations with antibodies against two distinct domains of AC-AplA. Note spike-broadening response, as compared with AC-AplA morpholino alone. SNs that the C1b and C2b antibodies recognized identical bands. (This same injected with both morpholinos showed no greater reduction in spike broad- lower molecular weight band has been observed in immunoblots on pleural ening compared with SNs injected with AC-AplA morpholino alone. The data for ganglion sensory neuron clusters.) AC-AplA morpholino are from B (** indicates P < 0.01).

Lin et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 S6). To distinguish whether the lower molecular weight band in 100 mM tetraethylammonium (TEA) and 20 μM nifedipine represents a truncated form of AC-AplA or an unrelated protein, (at these concentrations, TEA blocks K+ channels and nifedi- we compared immunoblots of CNS with antibodies against two pine blocks Ca2+ channels that are modulated by PKC; SI different domains of AC-AplA (Fig. 3B). Both antibodies stained Methods) (40, 41). Control SNs were injected either with Alexa a band at 115 kDa, suggesting that this smaller 115-kDa protein, 594 dextran alone, which was used to identify all injected neu- which includes the C2b region, represents the primary form of rons, or with control morpholino. Because AC-AplB is not AC-AplA that is expressed in CNS. This band is comparable in expressed in SNs, morpholino for AC-AplB served as an addi- size to the other Aplysia and mammalian AC isoforms (Table S1 tional control. In SNs injected with AC-AplA morpholino, spike and Fig. S6). The smaller AC-AplA band was not due to pro- broadening was reduced significantly by 72–80%, depending on teolysis during preparation (SI Methods). We speculate that the the control (F4,28 = 5.3, P = 0.003; Fig. 4). Injection of mor- 115-kDa band represents a functionally active AC with a short pholino for AC-AplC resulted in a nonsignificant reduction in C2 domain produced by proteolytic processing. In contrast, ru- spike broadening. In SNs in which expression of AC-AplA was tabaga AC, which also has a long C2 domain (Table S1), is >200 knocked down, there was some remaining spike broadening kDa and does not appear to be expressed in a truncated form (114%). It seemed possible that this residual 5-HT response (34). The antibody against the C2b domain of AC-AplC stained might be mediated by AC-AplC. To test this hypothesis, we a band at ≈125 kDa, similar to the predicted size (Fig. 3A). injected SNs with a combination of morpholinos for AC-AplA and AC-AplC. This combination produced no greater reduction AC-AplA Is Expressed in SN Somata, Processes, and Growth Cones, in spike broadening as compared with AC-AplA morpholino Whereas AC-AplC Is Primarily Expressed in SN Somata. Using im- alone. To confirm the contribution of AC-AplA to 5-HT-initiated munocytochemistry, we determined the expression pattern of modulation in SNs, we used dsRNA as an alternate means to AC-AplA and AC-AplC in SNs (Fig. S8). SNs were cultured ei- knock down expression. Injection of dsRNA for AC-AplA sig- ther alone or together with the L7 MN. AC-AplA immunore- nificantly reduced spike broadening by 75–77% relative to the activity was observed in SN somata and processes. Both the Alexa control or dsRNA for AC-AplB(F2,18 = 9.714, P = 0.001) postsynaptic MNs and regions where pre- and postsynaptic pro- (Fig. 4). We did not attempt to further explore the possible cesses overlapped showed similarly intense staining. Interestingly, modest contribution of AC-AplC to the spike-broadening re- intense AC-AplA staining was observed in growth cones. AC-AplC sponse; given the large contribution of AC-AplA, which accounted immunoreactivity was also observed in SN and MN somata and for ≈75% of the broadening response, and the variability in spike processes; however, staining in processes and growth cones was broadening across experiments, it would be difficult to detect noticeably weaker than in somata (Fig. S8). a possible small contribution of AC-AplC. In summary, AC-AplA accounts for the great majority of 5-HT-initiated spike broadening AC-AplA Is Coupled to 5-HT Receptors in Aplysia SNs. We asked in the SNs. which of the AC isoforms are coupled to 5-HT receptors in the SNs. Pairing activity and Ca2+ influx with 5-HT enhance two Discussion forms of 5-HT-initiated, cAMP-dependent modulation in SNs: Four Putative ACs Are Expressed in Aplysia CNS. We identified four spike broadening and increased excitability (3, 14, 38). We distinct putative AC isoforms in CNS. The secondary protein therefore predicted that in SNs, 5-HT receptors are coupled to structures predicted from all four transcripts resemble the struc- the Ca2+/CaM-sensitive AC, AC-AplA. To test the role of spe- ture of transmembrane ACs in metazoans (Fig. S1). Moreover, all cific AC isoforms in 5-HT-initiated modulation in SNs, we four predicted proteins share highly conserved sequences typically knocked down expression of AC-AplA or AC-AplC by injecting found in metazoan ACs (Fig. S2). The presence of these key morpholino antisense (39). To assess the consequence of AC features of metazoan ACs suggests that all four genes code for knockdown, we measured an effect of 5-HT known to be me- functionally active AC enzymes. We focused on the functional diated entirely by cAMP: broadening of the SN action potential properties of the two isoforms that are expressed in Aplysia SNs:

Fig. 5. Coexpression of Ca2+-inhibited AC, together with dually regulated Ca2+/CaM-sensitive AC, enhances associative specificity of stimulation of cAMP pathway. (A) In the absence of Ca2+ influx or 5-HT, AC-AplC shows a low level of basal activity (curved arrow), producing modest levels of cAMP (red triangles). (B)Ca2+ influx during unpaired activity activates CaM, stimulating AC-AplA. At the same time, Ca2+ (green circles) binds to AC-AplC (green arrow), inhibiting the basal activity of this AC isoform (broken curved arrows represent loss of basal cAMP synthesis). The direct Ca2+ inhibition of AC-AplC reduces the net rise in cAMP levels when AC-AplA is stimulated by Ca2+ influx alone. (C) When activity and Ca2+ influx are paired with the arrival of 5-HT (hexagon), activation of AC- AplA is enhanced (thick curved arrow). With pairing there is a greater net increase in cAMP levels, resulting in effective activation of PKA.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1004451107 Lin et al. Downloaded by guest on September 28, 2021 AC-AplA and AC-AplC. Heterologous expression of either of AC, AC-AplA. Because AC-AplC immunoreactivity was weaker these transcripts increased cAMP-synthesizing activity (Fig. 2), in SN processes than in somata, we suggest that at presynaptic demonstrating that each codes for an enzymatically active AC. sites, AC-coupled 5-HT receptors also activate primarily AC- AplA. As a consequence, most or all of the stimulation of cAMP 2+ 2+ AC-AplAIsStimulatedbyCa /CaM, Whereas AC-AplC Is Inhibited by Ca . synthesis by 5-HT is subject to modulation by Ca2+/CaM. We Our primary goal was to identify and characterize the Ca2+/CaM- 2+ conclude that in the SNs, the CaM-sensitive AC is able to pro- sensitive AC in Aplysia SNs. The dually regulated Ca /CaM- vide a locus for associative integration of Ca2+ influx and 5-HT, sensitive AC has been hypothesized to function during classical as had been predicted earlier based on cellular studies (13, 14). conditioning as a molecular site of associative convergence for Ca2+ fl in ux accompanying SN activity and the modulatory neurotrans- A Possible Role for AC-AplC in Associative Integration. Whereas the mitter 5-HT. Of the 12 cytoplasmic domains of the cloned Aplysia role of a CaM-regulated AC may be important in associative in- AC isoforms, only the C1 domain of AC-AplA bound CaM. 2+ tegration of SN activity and modulatory inputs, the functional role Apl 2+ Recombinant AC- A was stimulated by Ca /CaM more than of a Ca -inhibited AC in the SNs is less obvious. The coex- 3-fold. Recombinant AC-AplC was directly inhibited by submi- pression of the Ca2+-inhibited AC-AplC and the CaM-stimulated cromolar Ca2+, much like AC5 and AC6 (42). Because inhibition 2+ AC-AplA in the presynaptic sensory neurons suggests a possible was detected as Ca increased just above 100 nM, AC-AplC 2+ should be modulated by small increases in Ca2+ with very modest role for Ca inhibition in enhancing the function of AC-AplAas a coincidence detector. Earlier biochemical and cellular studies spike activity. Although the C1a region of AC-AplB does not 2+ closely resemble any single group of ACs, its C2a domain is on Aplysia neurons observed that activity and Ca alone are relatively similar to ACs 2, 4, and 7 (Figs. S3 and S4). AC2 is unable to effectively activate the cAMP cascade (38, 51). Rather stimulated by PKC (21, 43). Lorenzetti et al. (44) have found in remarkably, cAMP-dependent kinase makes no contribution to B51 neurons that PKC increases cAMP levels, and that these posttetanic potentiation produced by trains of 40 action potentials cells express AC-AplB. They suggest that during operant condi- at these SN-MN synapses (52), suggesting that even intense ac- tioning, a PKC-modulated AC in B51 serves as a coincidence tivity, which increases presynaptic Ca2+ levels substantially, does detector for dopamine and for Ca2+ influx triggered by bursts of not effectively increase cAMP levels. Coexpression of a Ca2+- activity. Thus, AC-AplB, much like AC-AplA, may serve as an inhibited AC with the CaM-stimulated AC could make Ca2+ associative integrator during conditioning. AC-AplD is more a relatively ineffective stimulator of cAMP levels because in-

similar to AC9 than other mammalian isoforms (Fig. S3). All hibition of AC-AplC should at least partially offset the stimulation NEUROSCIENCE four Aplysia AC isoforms have clear orthologs in Lottia gigantean, of AC-AplA. In contrast, when a Ca2+ increase is paired with a prosobranch gastropod mollusk (24). Lottia, like most inver- arrival of 5-HT, CaM should potentiate the stimulation of AC- tebrates, has an ortholog of mammalian AC3. As yet, no AC3- AplAbyGs (Fig. 5). Thus, the coexpression of AC-AplC and AC- type AC has been found in Aplysia. AplA should work to minimize the nonassociative effect of activity and Ca2+ influx on cAMP levels, while permitting the associative Apl Apl Apl Both AC- A and AC- C Are Expressed in SNs, and AC- AIs activation of AC-AplAbyCa2+ and transmitter. The mammalian Enriched in Presynaptic Processes and in Growth Cones. Based on homolog to AC-AplC, the Ca2+-inhibited AC5, is highly expressed qRT-PCR, both AC-AplA and AC-AplC are expressed in SNs, but in regions of mammalian CNS, such as striatum (42); however the not AC-AplB or AC-AplD. The expression of AC-AplA and AC- 2+ AplC in SNs was confirmed by immunoblot and immunocyto- possible functional role of Ca inhibition of AC5 is not un- derstood. We propose that in mammalian brain, as in Aplysia CNS, chemistry. Immunocytochemistry on cultured neurons revealed 2+ that AC-AplA and AC-AplC are expressed both presynaptically in Ca -inhibited AC may function to enhance the associative re- 2+ fl SNs and postsynaptically in L7 motor neurons. In SNs, immuno- quirement for coincident Ca in ux and transmitter for effective reactive AC-AplA was equally observed in presynaptic processes elevation of cAMP levels mediated by CaM-sensitive AC. and in somata, whereas AC-AplC was preferentially localized to Methods somata. This suggests that AC-AplA may play important roles in short-term synaptic modulation, as well as in regulation of tran- AC Cloning. Total RNA was extracted from Aplysia CNS using TRizol (Invi- fi scription cascades that are important in long-term synaptic plas- trogen), puri ed on an RNAqueous column (Ambion), treated with DNase H, ticity. Interestingly, activation of PKA at presynaptic loci has been and reverse-transcribed using random hexamers. A degenerate cloning strategy using highly conserved sequences within the C1a and C2a regions implicated in synaptic tagging in the establishment of long-term was used to identify Aplysia AC isoforms (see SI Methods for details). Am- synaptic facilitation (45). AC-AplA is also expressed in growth plified PCR products were cloned into pCR2.1, and distinct clones were cones. This CaM-sensitive AC may serve to mediate interactions fi ′ ′ 2+ identi ed based on restriction mapping and DNA sequencing. 5 and 3 RACE between Ca signals and the cAMP pathways, both of which have was performed using a SmartRace Kit (Clontech). To verify each AC RACE been implicated in growth cone guidance (46, 47). sequence, PCR was performed on three independent cDNA preparations, using gene-specific primers located within the 5′ and 3′ UTRs. Sequences for In SNs, 5-HT Stimulation of cAMP Levels Is Predominantly Mediated by AC- AplA, -B, and -C were deposited in GenBank (accession nos. AY843027, AC-AplA. CaM-sensitive AC has been hypothesized to function as AY843025, and AY843026). a molecular convergence site for Ca2+ influx and 5-HT in SNs during associative synaptic plasticity (11). These two stimuli co- Antigen Peptide Selection, Antibody Production, and Purification. Antibodies incide temporally during classical conditioning when SN activity were generated in rabbits (Covance) against thyroglobulin-conjugated precedes a tail shock, which releases 5-HT. We explored whether peptides (Bio-Synthesis) corresponding to nonconserved sequences within the CaM-sensitive AC, AC-AplA, mediates the rise in cAMP the hypervariable C2b regions of each AC or the calmodulin-binding region of levels stimulated by 5-HT (48–50). SNs treated with morpholino AC-AplA. Antibodies were affinity-purified on antigen peptide columns or dsRNA for AC-AplA showed a 72–80% reduction in the before use for immunoblotting and immunocytochemistry (see SI Methods cAMP-dependent spike-broadening response to 5-HT. In con- for details). trast, morpholino for AC-AplC did not significantly affect the ACKNOWLEDGMENTS. Drs. Steven Munger and Frank Margolis provided spike-broadening response (Fig. 4). assistance with the design of molecular biology experiments and Drs. Tony Thus, in the SN somata, 5-HT stimulation of the cAMP Gover and Brian Hagen provided help with confocal imaging. This research pathway is largely or exclusively mediated via the CaM-sensitive was supported by National Institutes of Health Grant MH-55880.

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