Downloaded from learnmem.cshlp.org on October 10, 2021 - Published by Cold Spring Harbor Laboratory Press Identification of Specific mRNAs Affected by Treatments Producing Long-Term Facilitation in Aplysia Raymond E. Zwartjes, 1 Henry West, 1 Samer Hattar, 1 Xiaoyun Ren, 1 Florence Noel, 2 Marta Nufiez-Regueiro, 1 Kathleen MacPhee, 1 Ramin Homayouni, 1 Michael T. Crow, 3 John H. Byrne, 2 and Arnold Eskin 1'4 1Department of Biochemical and Biophysical Sciences University of Houston Houston, Texas 77204-5934 2Department of Neur0bi010gy and Anatomy University of Texas-H0ust0n Medical School Houston, Texas 77030 3Ger0nt010gy Research Center National Institute on Aging National Institutes of Health Baltimore, Maryland 21224 Abstract by sensitization training. Furthermore, stimulation of peripheral nerves of Neural correlates of long-term pleural-pedal ganglia, an in vitro analog of sensitization of defensive withdrawal sensitization training, increased the reflexes in Aplysia occur in sensory incorporation of labeled amino acids into neurons in the pleural ganglia and can be CaM, PGK, and protein 3. These results mimicked by exposure of these neurons to indicate that increases in CaM, PGK, and serotonin (5-HT). Studies using inhibitors protein 3 are part of the early response of indicate that transcription is necessary for sensory neurons to stimuli that produce production of long-term facilitation by 5-HT. long-term facilitation, and that CaM and Several mRNAs that change in response to protein 3 could have a role in the 5-HT have been identified, but the molecular generation of long-term sensitization. events responsible for long-term facilitation have not yet been fully described. To detect additional changes in mRNAs, we Introduction investigated the effects of 5-HT (1.5 hr) on Evidence has been accumulating for some time levels of mRNA in pleural-pedal ganglia indicating that protein synthesis is required for the using in vitro translation. Four mRNAs were formation of long-term memory (Davis and Squire affected by 5-HT, three of which were 1984). More recently, the use of models for learn- identified as calmodulin (CaM), ing that can be studied at both the behavioral and phosphoglycerate kinase (PGK), and a novel molecular levels indicates changes in gene tran- gene product (protein 3). Using RNase scription are required for the induction of long- protection assays, we found that 5-HT term memory (Nelson and Alkon 1990; Anokhin increased all three mRNAs in the pleural and Rose 1991; Bourtchuladze et al. 1994; Yin et al. sensory neurons. CaM and protein 3 mRNAs 1994). Additional evidence for the involvement of were also increased in the sensory neurons transcription has come from studying sensitization, a form of nonassociative learning, in Aplysia. Com- ponents of the defensive withdrawal reflexes, 4Corresponding author. which are modified by sensitization, can be studied LEARNING & MEMORY 4:478-495 91998 by Cold Spring Harbor Laboratory Press ISSN1072-0502/98 $5.00 L E A R N / N G & M E M O R Y 478 Downloaded from learnmem.cshlp.org on October 10, 2021 - Published by Cold Spring Harbor Laboratory Press MRNA REGULATION AND LTF in vitro using isolated ganglia or cultured sensory modulin (CAM), phosphoglycerate kinase (PGK), and motor neurons (for review, see Castellucci and and a novel gene (protein 3). CaM, PGK, and pro- Schacher 1990; Byrne et al. 1993). The induction tein 3 mRNAs also increased in pleural sensory neu- by serotonin [5-hydroxytryptamine hydrochloride rons following treatment with 5-HT, although only (5-HT)] of long-term facilitation (LTF) at the con- CaM and protein 3 were increased by sensitization nections between sensory and motor neurons in training. In addition, an in vitro analog of training culture was blocked by inhibitors of transcription increased incorporation of labeled amino acids or translation applied during the inducing treat- into all three proteins in sensory neurons. These ments (Montarolo et al. 1986). results indicate that CaM and protein 3 could have The results from subsequent studies further a role in the generation of long-term sensitization. support a role for transcription in the generation of LTF. The transcription inhibitor actinomycin D Materials and Methods blocked 5-HT-produced increases in the incorpora- tion of labeled amino acids into proteins (Barzilai et 5-HT TREATMENT OF GANGLIA AND BEHAVIORAL al. 1989) and blocked the persistent phosphoryla- SENSITIZATION tion of proteins found 24 hr after 5-HT treatments Twelve animals were used for each IVT experi- (Sweatt and Kandel 1989). In addition, the injec- ment, and four to six animals were used for ribo- tion of an oligodeoxynucleotide containing a cAMP nuclease protection assays (RPAs). Isolated ganglia response element (CRE) into sensory neurons in culture blocked the induction of LTF by 5-HT were treated for 1.5 hr with 5 ~M 5-HT (Sigma, St. (Dash et al. 1990). Finally, CCAAT/enhancer-bind- Louis, MO) and then frozen, essentially as de- scribed in Noel et al. (1991) but without [35S]me- ing protein (C/EBP) is induced by 5-HT, and inhib- thionine. Sensitization training consisted of four iting its function blocks LTF (Mberini et al. 1994). The results of the above studies with inhibitors blocks of electrical shocks over 1.5 hr delivered to indicate that transcription is, in some way, in- the posterior body wall (Scholz and Byrne 1987; volved in the production of LTF. To fully under- W.L. Lee, M. Aguirre, L.J. Cleary, and J.H. Byrne, unpubl.). Animals were anesthetized immediately stand LTF and the role of transcription, it is neces- after treatment; ganglia were then removed and sary to know the pathway through which 5-HT affects gene expression and the mechanism by frozen. which genes regulated by 5-HT mediate the pro- duction of LTF. In addition to C/EBP, several other RNA EXTRACTION FROM GANGLIA AND IVT mRNAs affected by 5-HT have been identified. RNA was extracted from ganglia by homogeni- Most of the proteins encoded by these mRNAs zation in phenol (Ambion, Austin, TX) and SDS have not been clearly linked to LTF. Ubiquitin hy- (Sigma) at 65~ followed by two extractions with droxylase was shown to be important for the es- phenol/chloroform (50:50) and precipitation with tablishment of LTF (Hegde et al. 1997). Aplysia ethanol. The ethanol precipitate was reconstituted tolloid may also contribute to LTF (Liu et al. 1997; in water and reprecipitated with 2 M sodium ac- Zhang et al. 1997). However, many aspects of the etate (pH 6). Poly(A) + RNA was isolated on an oli- mechanisms for the generation of LTF remain un- go(dT) column as described by Sambrook et al. clear. (1989). IVT was done using rabbit reticulocyte ly- To identify additional proteins that might con- sate (nuclease treated, minus methionine; Promega tribute to the production of LTF, we investigated Corp., Madison, WI) in the presence of [35S]me- changes in levels of mRNA following treatment thionine essentially following the supplier's proto- with 5-HT. We used in vitro translation (IVT) of col, except that 5 mM cAMP (Sigma) was added to cellular mRNAs followed by two-dimensional poly- prevent inactivation of eukaryotic initiation factor acrylamide gel electrophoresis (2-D PAGE) of the 2 (Gross et al. 1988). Reactions were carried out at protein products. This technique allows the deter- 30~ for 2 hr. Canine pancreatic microsomal mem- mination of the relative amount of translatable branes were obtained from Promega and used as mRNA for individual proteins within a complex instructed by the supplier. mixture of mRNAs (Colbert and Young 1986, 1987; Coleclough et al. 1990; Miles et al. 1992). We 2-D PAGE found that 5-HT altered four mRNAs in pleural- pedal ganglia, three of which we identified: cal- Miquots of IVT lysate were added to 6 volumes L E A R N I N G & M E M O R Y 479 Downloaded from learnmem.cshlp.org on October 10, 2021 - Published by Cold Spring Harbor Laboratory Press Zwartjes et aL of solubilization buffer and analyzed by 2-D PAGE, for 1 hr. The hybridization buffer contained 100 as described in Zwartjes and Eskin (1990) except mM KCI, 20 mM HEPES (pH 7.0), 1 mM EDTA (Min- that the ampholytes (Pharmacia, Piscataway, NJ) shull and Hunt 1986). The synthesized oligodeoxy- used were pH 2.5-5, 4-6.5, and 3-10 in a ratio of nucleotide was complementary to nucleotides 1:2:2. In addition, gels were treated with Autofluor 238-261 of the protein-coding region of the Aply- (National Diagnostics, Atlanta, GA) before drying. sia CaM mRNA (Swanson et al. 1990). One volume The fluorographs were first analyzed visually, and of 2x RNase H reaction buffer [16 U/ml of RNase spots were designated as changing or unchanging, H, 100 mM KC1, 40 mM Tris-C1 (pH 7.6), 3 mM followed by analysis using a computerized densi- MgC12, 100 mg/ml glycogen, 2 mM dithiothrietol tometer (DNA Proscan, Nashville, TN). The ratios (all from Sigma)] was added to each sample. of optical densities of the visually identified un- Samples were incubated at 37~ for 1 hr and pre- changing spots from experimental and control gels cipitated with ethanol. The RNA was translated in were determined to confirm that they were not vitro and the proteins analyzed by 2-D PAGE as affected by the treatment. The optical densities of described above. the visually identified changing spots were then determined and normalized for any discrepancy in gel loading by expressing them as a percentage of WESTERN BLOTS the sum of the optical densities of the verified un- changing spots. The difference in values between For CaM, two pleural-pedal ganglia were incu- experimental and control spots was then deter- bated with [35S]methionine (0.15 laCi/ml) for 2 hr.