A METHOD FOR ENHANCING LONG-TERM MEMORY IN A SUBJECT AND USES THEREOF

Abstract: The present invention provides for a method to enhance longterm memory in a subject whose cAMP-responsive gene expression is repressed due to binding of a cAMP-response-element-binding-protein-2 to a protein or a DNA associated with cAMP-responsive gene expression, or both, which comprises administering to the subject a compound capable of interfering with such binding in an amount effective to interfere with binding of the protein or the DNA so as to thereby derepress cAMP-responsive gene expression in the subject and enhance the subject's long-term memory.

Description: A METHOD FOR ENHANCING LONG-TERM MEMORY IN A SUBJECT AND USES THEREOF This application claims the priority of U.S. Serial No. 08/656,811, filed June 3, 1996 the contents of which are hereby incorporated in its entirety by reference. The invention disclosed herein was made with Government support under Grants No. MH37134 and GM32099 from NIH. Accordingly, the U.S. Government has certain rights in this invention. Background of the Invention Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding Sequence Listing and the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. Introduction Memory acquisition has at least two components, a transient short-term memory lasting minutes to hours that can be followed by a more persistent and self-maintained long-term memory lasting days to years. Whereas short-term memory requires only covalent modifications of preexisting proteins, long-term memory requires the synthesis of new mRNA and proteins (Flexner et al . , 1963; Davis and Squire, 1984; Montarolo et al . , 1986;) and is accompanied by the growth of new synaptic connections (Bailey and Kandel, 1993 ) . The switch from short- to long-term memory can be studied on the molecular level in the gill-withdrawal reflex of the marine snail Aplysia . Following a single noxious stimulus to the tail, the animal acquires a short-term memory for the noxious stimulus lasting minutes, during which time both the amplitude and the duration of the gill-withdrawal reflex to tactile stimulation of the siphon is greatly enhanced (Pinsker et al . , 1970; Carew et al . , 1971) . Following five or more spaced sensitizing stimuli, the animal acquires a long-term memory lasting days or weeks (Carew et al., 1972; Pinsker et al. , 1973) . The short-term memory does not require new protein synthesis, whereas long-term memory is blocKed by inhibitors of protein and RNA synthesis (Montarolo et al. , 1986; Castellucci et al. , 1989; Bailey et al. , 1992) . A cellular representation of both types of memeory storage can be studied in cocultures of a single sensory neuron and a single motor neuron of the gill withdrawal reflex. Here one brief application of 5-HT, a modulatory transmitter released in vivo by mterneurons activated by sensitizing tail stimuli, produces short-term facilitation that results from a strengthening of preexisting synapt c connections between the sensory and motor cell by means of covalent modifications of preexisting proteins (Montarolo et al. , 1986; Rayport and Schacher, 1986) . By contrast, five applications of 5-HT, spaced by 20 mm, produce long-term facilitation that lasts for more than one day, is dependent on the synthesis of mRNA and protein, and is accompanied by an increase in the number of sensory neuron synaptic terminal varicosities in contact with the motor neuron (Montarolo et al . , 1986; Glanzman et al., 1990; Bailey et al. , 1992) . Summary of the Invention The present invention provides for a method to enhance long- term memory m a subject wnose cAMP-responsive gene Ξ expression is repressed due to binding of a cAMP-response- element-bind_.ng-proteir.-2 to a protein or a DNA associated with cAMP-responsive gene expression, or otr., wmcn comprises administering to the subject a compound capable of interfering with such binding m an amount effective to 0 interfere with binding of the protein or the DNA so as to thereby derepress cAMP-responsive gene expression in zhe subject and enhance the subject's long-term memory. Brief Description of the Figures Figures 1A and IB. Amino Acid Sequence of ApCREB-2. (A) The predicted ammo acid seσuence of ApCREB-2 deαuced from two independent clones isolated oy a yeast two-πycnd screen of an Aplysia CNS cDNA library The bZIP domain tnat interacts with ApC/EBP is boxed and laDeled I . W tnm tnis domain, hydrophobic residues of the leuc e zipper motif are shaded. The box labeled II delineates a second leucine heptad repeat. ApCREB-2 contains a consensus seσuence for PKC phosphorylation (aa 271-274, bold, underlined In addition, ApCREB-2 has putative consensus sequences for MAP Kinase in similar positions to those m human CREB-2 and mouse ATF-4 (aa 235-238 and aa 150-153, italics, underlined) . (SEQ ID NO 1) This sequence and ApCRΞB-2 cDNA sequence were deposited in GenBank. See deposit information nereinbelow. (Fig. IB) The C-terminal 79 ammo acids of ApCREB-2 containing the bZIP domain aligned with the bZIP domains of mouse ATF-4 (mATF-4) and human CREB-2 (hCREB-2) The numbers on the left margin refer to the adjacent ammo acids, and an asterisk marks the hydrophobic residues of the leucine zippers. Identical residues are boxed Witniπ the bZIP region the compared proteins are 50% identical A cyste e residue in the basic region conserved m most bZIP proteins is substituted for a tyrosine at position 323 (shaded) This substitution, as well as the tyrosine at position 361 (shaded) within the leucine zipper, is conserved among all three proteins: ApCREB-2, hCREB-2 and mATF-4. (SEQ ID NO 2) Figures 2A, 2B and 2C. Expression of ApCREB-2 in Aplysia Tissues. {Fig. 2A) Northern blot analysis of ApCREB-2 mRNA expression. The tissue used as tne source of RNA is indicated above each lane . Five μg of total RNA was loaded m each lane The arrowhead indicates the position of ApCREB-2 mRNA detected by hybridization with a full length ApCREB-2 cDNA probe (upper panel) and Aplysia actir. cDNA probe (lower panel) . ApCREB-2 mRNA is highly expressed in central nervous system (CNS) and g ll. (Fig 2B) Western blot analysis of bacterial-expressed recombinant ApCREB-2 (upper panel) and Aplysia CNS protein extract (lower panel) . 2 μg of recombinant ApCREB-2 or 15 μg of total protein isolated from Aplysia CNS were separated by SDS-PAGE, electroblotted, and probed with antiserum raised against recombinant ApCREB-2 (immune; , preimmune serum and immune ApCREB-2 antiserum premcubated with immobilized immunizing ApCREB-2 antigen (depleted immunej Multiple bands recognized by the antι-ApCREB-2 antibodies, indicated by arrowhead, are specifically blocked by depleting the immune serum with recombinant ApCREB-2. Positions of molecular mass markers kD are indicated. (Fig 2C) Analysis of ApCREB-2 protein expression Aplysia CNS using affmity-purifled antibodies. The specific ApCREB-2 signals are indicated by arrowheads. ( I V T ) 35 S-methιonme labeled ApCREB-2 protein m vi tro translated in rabbit reticulocyte lysate, separated by SDS-PAGE and visualized by fluorography. (W) Western blot of Aplysia CNS protein extract from animals exposed to 5-HT in vivo for 2 hr. 20 μg of total CNS extract were subjected to SDS-PAGE, electroblotted, and probed with affmity-purifled antι-ApCREB-2 antibodies. (IP) Immunoprecipitation, using affinity purified antι-ApCREB-2 antibodies, from total Aplysia CNS dissected from anesthetized animals and metabol cally labeled with 33 S-methionine. ApCREB-2 is expressed the Aplysia central nervous system and migrates on SDS-PAGE as multiple bands with an apparent molecular weignt of around 50 kD. Figures 3A and 3B. ApCREB-2 is Constitutively Expressed in the Sensory Neurons of Aplysia. (Fig 3A) A 450 bp of ApCREB-2 cDNA amplified oy T-PCF from Aplysia sensory neurons. RNA was isolated from cultures of approximately 200 Aplysia sensory neurons that had been exposed to none, one, or five pulses of 5-HT (10 μM' as indicated. As a control, one half of the RNA was processed omitting reverse transcπptase from the reaction '-RT) . ApCREB-2 RNA is constitutively expressed m Aplysia sensory neurons . (Fig 3B) Northern blot analysis of RNA isolated from CNS of Aplysia exposed to 5-HT m vivo . CNSs were dissected from anesthetized animals after exposing them in vivo to 5-HT (50 μM) for the time indicated above each lane. Eight μg of total RNA from each ti epomt was separated a 1% agarose gel, blotted and consecutively hybridized with probes specific for ApCREB-2, ApCREB-1, ApC/EBP, and ribosomal protein S4 transcripts. Unlike ApC/EBP mRNA, ApCREB-2 and ApCREB-1 mRNAs are constitutively expressed in the Aplysia central nervous system, and their steady-state level is not affected by exposure to 5-HT in vivo . Figures 4A and 4B. ApCREB-2 Is a Phosphoprotein In Vivo. (Fig 4A) Western blot of Aplysia CNS extracts from animals exposed to 5-HT in vivo for the time indicated above each lane. The blots were probed with antι-ApCREB-2, anti-rat CREB-1, and anti-ApC/EBP antibodies. The positions of these proteins are indicated by arrowheads. ApCREB-2 migrates as multiple bands. Note the appearance of a slower migrating ApCREB-2 specific signal in the course of 5-HT exposure. (Fig 4B) Western blot of Aplysia CNS extract from animals exposed to 5-HT m vivo for 90 m and mcuoated with phosphatase (CIP) or mock treated (M^ The positions of ApCREB-2 specific signals are indicated by arrows Note tne appearance of a faster migrating ApCREB-2 in SDS-PAGE after nospnatase treatment These data indicate that ApCREB-2 is a pnosphoprotem n vivo and tnat the pnospncrylat on _evel of ApCREB-2 changes following exposure to 5-HT Figures 5A and 5B. Effect of ApCREB-2 and ApCREB-1 Expression on CRE-Mediated Transcription in F9 Cells. (Fig 5A) Mouse F9 cells were transiently cotransfected with 1 μg of pGL3-CRE reporter plasmid along with 1 μg of each of the indicated expression plasmids- ApCREB-2 pRcRSV-ApCREB-2, ApCREB-l=pRcRSV-ApCREB-l, PKA = pRcRSV-PKA All transfections were adjusted to 4 μg total DNA with pRcRSV vector DNA. The luciferase activity of the reporter was normalized to (-galactosidase activity from 0.2 μg of the cotransfected RSV-lacZ expression plasmid. The relative luciferase activity was calculated by comparing the activities measured m cotransfection experiments to the activity of pGL3-CRE alone (arbitrarily set at 1.0) . Each bar represents the mean of at least nine independent transfections ± SEM ApCREB-2 αoes not activate transcription on its own, but represses the activation mediated by ApCREB-1 on a minimal CRE control region. (Fig 5B) Transient transfections of F9 cells using a 5xCRE-VIP-lacZ reporter plasmid The indicated expression constructs were cotransfected with 5xCRE-VIP-lacZ reporter as in (A) , except that relative (-galactosidase activity was calculated relative to 5xCRE-VIP-lacZ reporter and normalized to the luciferase activity of cotransfected pRSV-luc. ApCREB-2 and ApCREB-1 are activators of 5xCRE-VIP-lacZ reporter, but become mutual repressors when coexDressed. Figures 6A, 6B and 6C. ApCREB-2 Homodimers Bind to CRE (Seq ID No 3) . (Fig 6A) The sequence of DNA binding sites for ApCRΞE-2 (BSl) and ApC/EBP (BS2) . Optimal DNA binding sequences were selected from a pool of double-stranded random twentymers oy repeated binding and PCR amplification. In both BSl (Seq ID No 4) and BS2 (Seq ID No 5) , the homology to the CAAT box is underlined and the homology to the palindromic CRE site (above) is bold. (Fig 6B) Specificity of recombinant 6His-ApCREB-2 binding to symmetrical and asymmetrical CREs and CAAT binding sequences. Electrophoretic mobility shift assay with double-stranded oligonucleotides containing the CREs of the somatostatin gene (1) and its core palindrome (2) , PEPCK gene (3) , proenkephalin gene (4) and putative CRE of the ApC/EBP gene (5) . Also tested were the BSl selected ApCREB-2 DNA-binding sequence (6) , a composite CRE-CAAT binding site (7) , the C/EBP-binding APRE of the angiotensin gene (8) , the selected BS2 ApC/EBP DNA-binding site (9) , and the C/EBP-binding ERE of the fos promoter (10) . The arrowhead indicates the position of a specific ApCREB-2 shift and an asterisk marks the unbound oligonucleotide probes. The specificities of the shifts were confirmed by competition with 10 and 100 molar excess of nonradioactive oligonucleotides and mutants of the CRE and C/EBP sites (not shown) . Recombinant ApCREB-2 binds directly to the high-affinity somatostatin CRE as well as to BSl and the CRE from the proenkephalin gene, but does not bind to any of the CAAT sites tested. (Fig 6C) Interaction of in vi tro translated ApCREB-2 and ApC/EBP with bacterial-expressed glutathione S-transferase (GST) fusions of ApCRΞB-2. Glutathione-agarose oeadε saturated with equal amounts of bacterial-expressed GST-ApCREB-2 and its deletion mutants (indicated above lanes) were incubated with 35 S-labeled ApCREB-2 or ApC/EBP proteins, washed, and eluted bound proteins resolved by SDS-PAGE. ApCREB-2 forms weak homodimers, but that homodimεr formation can be greatly increased by deleting :ne N- erminal 260 ammo acids which contain the second leucine zipper. Figures 7A, 7B, 7C and 7D. Time Course of the Effect of Injection of ApCREB-2 antiserum on Short- and Long-Term Facilitation. (Fig 7A) Time course of EPSP amplitude changes recorded in motor neuron L7 in response to stimulation of the sensory neuron (expressed as percent change in the amplitude of the EPSP) after single and multiple applications of 5-HT to Aplysia sensory-motor neuron cocultures. Changes m EPSP amplitude after application of one 5 min pulse of 5-HT (1 x 5-HT, short-term facilitation) and one 5 mm pulse of 5-HT paired with injection of anti-ApCREB-2 antibodies (1 x 5-HT - CREB-2 Ab, both in bold lines) are compared to changes m EPSP amplitude induced by five pulses of 5-HT (5 x 5-HT) at 2 and 24 hr. While the EPSP facilitation decays rapidly after one pulse of 5-HT (with a return to base line after 10 mm) , pairing one pulse of 5-KT with injection of anti-ApCREB-2 antibodies induces a long-term facilitation paralleling that of 5 x 5-HT. This long-term facilitation is abolished by the application of the protein synthesis inhibitor anisomycin (1 x 5-HT + CREB-2 Ab + ANISO) or the RNA synthesis inhibitor actinomycin D (1 x 5-HT + CREB-2 Ab+ ACTINO) during the training. The difference in EPSP amplitude at 2 hr between 5 x 5-HT and 1 x 5-HT + CREB-2 Ab may reflect the transient protein synthesis-dependen , but RNA synthesis-independent component of long-term facilitation 2 hr after 5-HT stimulation (Ghirardi et al . , 1995) . The controls are either untreated (control , or injected with ApCREB-2 antiserum without 5-HT administration (CREB-2 Ab) . (Fig 7B) Comparison of the time course of the ΞPSP amplitude changes in the first 2 hr after application of a single 5 min pulse of 5-HT with or without injection of CREB-2 Ab. The control cells were not exposed to 5-HT. (Fig 7C) Example of EPSPs recorded in motoneuron L7 after stimulation of the sensory neuron before (0 hr) , 2 hr and 24 hr after 5-HT treatment. One pulse of 5-HT paired witn the injection of an ApCREB-2 antiserum induces a significant increase EPSP amplitude at 2 and 24 hr, but injection the preimmune serum (PRE-CREB-2 Ab) or depleted immune serum does not induce long term facilitation. (Fig 7D) Examples of EPSPs recorded at indicated times in cocultures injected with ApCREB-2 antiserum paired with one 5 mm pulse of 5-HT. Figures 8A, 8B f 8C and 8D. Summary of the Effects of Injection of ApCREB-2 antiserum on Short- and Long-Term Facilitation. (Fig 8A) Injection of ApCREB-2 antiserum paired with one pulse of 5-HT induces a facilitation at 2 hr that is blocked by anisomycin (ANISO) and actinomycin D (ACTINO) . The height of each bar corresponds to the mean percentage change ± SEM in EPSP amplitude tested 2 hr after one pulse of 5-HT or of seawater (control cultures, first bar) . CREB-2 and C/EBP indicate antisera raised against recombinant ApCREB-2 ana ApC/EBP proteins, respectively. CREB-2 depleted indicates the immune anti-ApCREB-2 serum depleted by incubation with oactεrial-expressed ApCREB-2 bound to agarose beads. PRE indicates matching preimmune serum. PAS is an antiserum raised against an Aplysia RNA binding Y box protein (Sκehel and Bartsch, 1994) . (Fig 8B) The facilitation induced by one pulse of 5-HT paired with ApCREB-2 antiserum injection lasts 24 r.r ana is dependent on RNA and protein synthesis. The neignt cf eacr. bar corresponds to the mean percentage change - SEM in EPSP amplitude tested 24 hr after one 5 mm pulse of 5-HT or of seawater (control cultures, first bar) . (Fig 8C) Long-term facilitation induced oy five pulses of 5-HT is not affected by ApCREB-2 antiserum injection The neight of each bar corresponds to the mean percentage cnange ± SEM m EPSP amplitude tested 24 hr after five 5 mm pulses of 5-HT or of seawater (control cultures, first 3 bars) The injection of ApCREB-2 antiserum (third bari as well as injection of normal rabbit serum (NRS, second oar; without exposure to 5-HT does not induce long-term facilitation. (Fig 8D) Short-term facilitation is not affected by injection of ApCREB-2 antiserum. The height of each bar corresponds to the mean percentage change SEM m EPSP amplitude tested 1 mm after one pulse of 5-HT or of sea water (control cultures, first bar) . Figure 9. Summary of Long-Term Functional and Structural Changes Evoked by One Pulse of 5-HT Paired With Injection of ApCREB-2 antiserum. For the functional changes, the height of each bar is the mean ± SEM of the percent change in the amplitude of the EPSP induced m motor neuron L7 following a single pulse of 5-HT and retested 24 hr later. For the structural changes, the height of each oar represents tne mean ± SEM of the percent change m the number of fluorescent varicosities per sensory neuron reexammed 24 hr after one pulse of 5-HT. Injection of the ApCREB-2 antiserum paired w th one pulse cf 5-HT results 24hr later in a significant enhancement of the EPSP amplitude and a concomitant significant increase ir. the number of varicosities . Figure 10. Examples of Structural Changes Evident 24 hr After One Pulse of 5-HT Paired With Injection of ApCREB-2 Antiserum. The fluorescent micrographs taken from the same regions of sensory neurites contacting the axon hillock of L7 before (1 and 3) and 24 hr after treatment (2 and 4) . Arrows (2) illustrate examples of some of the new varicosities present one day after one pulse of 5-HT paired with the injection of the ApCREB-2 antiserum. By contrast, cocultures exposed to one pulse of 5-HT in the absence of antiserum injection (4) showed no long-term increases in either the amplitude of the evoked EPSP or in the number of sensory neuron varicosities. All micrographs are composed of superimpositions of labeled sensory neurite images taken from all focal planes of the view area. As a result, the shape of individual varicosities may be obscured. Scale = 20 μm. The EPSPs, evoked before (0 hr) and after (24 hr) one pulse of 5-HT m the. pictured neurons are indicated m the inserts . Detailed Description of the Invention The present invention provides for a method tc ennance long- term memory m a subject whose cAMP-responsive gene expression is repressed due to binding of a cAMP-respcnse- element-bιndmg-protem-2 to a protein or a DNA associated with cAMP-responsive gene expression, or both, whicn includes administering to the subject a compound capable of interfering with such binding in an amount effective to interfere with binding of the protein or the DNA so as to thereby derepress cAMP-responsive gene expression m the subject and enhance the subject's long-term memory. Tne compound may be an anti-cAMP-response-eiement-pindmg- protem-2 antibody. The compound may be capable of altering phosphorylation of the cAMP-response-element-binding- protem-2. The compound may be an organic compound, a peptide, a peptide mimetic, a small molecule, or a nucleic acid. The protein associated with cAMP-responsive gene expression may include a cAMP-response-element-binding- protem-1, a C/EBP protein, an Aplysia ApC/EBP protein, a human C/EBP / S protein, an AF-1 protein, a c-jun protein, a fla protein, or a c-Fos protein. The administration may include mtralesional, intramuscular or intravenous injection; infusion; liposome mediated delivery; viral infection; gene bombardment; topical, nasal, oral, anal, ocular, cerebro-spinal, or otic delivery. Another embodiment of the subject invention may be a method for evaluating the ability of a compound to interfere with binding of a cAMP-response-element-bιndmg-proteιn-2 to a protein associated with cAMP-responsive gene expression in a cell which includes : (a) contacting the cell with the compound under suitanle cell culture conditions, (b) measuring the amount of unbound protein associated with cAMP-responsive gene expression n tne cell, (c) comparing the amount m step (b) witr. tne amount of unbound protein associated witn cAMP-responsive gene expression tne aosence of the compound, so as to thereby evaluate tne aoility of the compound to interfere with binding of the cAMP-response-element-Dindmg- protem-2 to the protein. Another embodiment of the present invention is a method for evaluating the ability of a compound to interfere with binding of a cAMP-response-element-bmdmg-proteιn-2 to a DNA associated with cAMP-responsive gene expression a cell which includes: (a) contacting the cell with the compound under suitable cell culture conditions; (b) measuring the amount of unbound DNA associated with cAMP-responsive gene expression in the cell; (c) comparing the amount m step (b) with the amount of unbound DNA associated with cAMP-responsive gene expression m the absence of the compound, so as to thereby evaluate the ability of the compound to interfere with binding of the cAMP-response- element-bmdmg-protein-2 to the DNA. Another embodiment of the present invention is a method for treating a subject with a long-term memory defect due to binding of a cAMP-response-element-bmdmg-proteιn-2 to a protein or a DNA associated with cAMP-responsive gene expression, or both, which includes administering to the subject a compound capable of interfering with such binding m an amount effective to interfere with the binding of the protein or the DNA so as to thereby treat the subject's long-term memory def ct. The long-term memory defect may include age-relateα memory loss, Alzheimer's Disease, amnesia, ischemia, snock, nead trauma, neuronal injury, neuronal toxicity, neuronal degradation, Parkinson's disease, or senility. The compound may comprises an anti-cAMP-response-element-oinding-prcte n- 2 antioody. The protein may include a cAMP-response- element-bιndmg-protem-1, a C/EBP protein, an Aplysia ApC/EBP protein, a human C/EBP3 protein, an AF-1 protein, a c-jun protein, a fla protein, or a c-Fos protein. The cAMP- response-element-bindmg-protem-2 may include human CREB2 transcription factor, murine ATF4 transcription factor, or Aplysia ApCREB2 transcription factor. The administration may include mtralesional, intramuscular or intravenous injection; infusion; liposome mediated delivery, viral infection; gene bombardment; topical, nasal, oral, anal, ocular, cerebro-sp al, or otic delivery. The present invention provides for a recombinant eukaryotic cell including a DNA encoding a cAMP-response-element- bmdmg-protem-2 not naturally present in the cell, operatively linked to a promoter capable of directed enhanced expression of the DNA, the DNA and the promoter being stably integrated into the genome of the eukaryotic cell. The present invention provides for a transgenic, non-numan mammal whose somatic and germ cells contain and express a DNA encoding a cAMP-response-element-bιndmg-proteιn-2 not naturally occurring in the non-human mammal, operatively linked to a promoter capable of directed enhanced expression of the DNA, the DNA and the promoter being stably integrated into the genome of the non-human mammal. The present invention further provides for a pharmaceutical composition which includes an effective amount of a compound capable of interfering with binding cf a cAMP- response-element-bmding-protem-2 to a protein associated with cAMP-responsive gene expression m a cell ana a pharmaceutically acceptable carrier. The carrier may oe a diluent, an aerosol, a topical carrier, an aqueous solution, a nonaqueous solution, or a solid carrier. The carrier may include an appropriate adjuvant, a herpes virus, a liposome, a microencapsule, a neuronal cell receptor l gand, a neuronal-specific virus, a polymer encapsulated cell, or a retroviral vector. As used herein cAMP-response-element-bmdιng-protem-2" encompasses CREB2, Aplysia CREB2 transcription factor protein, Aplysia CREB2 transcription factor nucleic acid, ApCREB2, human CREB2 transcription factor protein, human CREB2 transcription factor nucleic acid, murine ATF2 transcription factor protein, murine ATF2 transcription factor nucleic acid and natural variants thereof. Such variants encompass homologues of CREB2 m human, mouse, Drosophila, pig, dog, horse, monkey, C. Elegans and Aplysia. As used herein "subject" encompasses a mammal, a human, a primate, a dog, a swine, an aplysia or a mouse. Tranαenics This invention provides a transgenic nonhuman mammal whose somatic and germ cells contain and express a gene coding for a cAMP-response-element-binding-protem-2 not naturally occurring in the non-human mammal. The gene, having been introduced into the nonhuman mammal, or an ancestor cf the nonhuman mammal at the single cell stage or an embryonic stage, is operably linked to a promoter and integrated into the genome of the nonhuman mammal . One skilled m the art would be familiar with the experimental methods necessary to produce a transgenic mammal, e.g. Leder et al. , U. S. Patent No. 4,736,866 and Knmpenfort and Berns, U. S. Patent No. 5,175,384 and Wagner and Chen, U. S. Patent No. 5,175,385. Preferably, the nonnuman mammal may be a mouse. The gene may be a combination of human cAMP-response-eiement-Dindmg- protem-2 nucleic acid sequences and adjacent, homologous nonhuman mammal apolipoprotein- nucleic acid sequences. The promoter may be a nerve tissue specific promoter such as the mouse neurofilament-light gene promoter or the rat neuronal specific enolase promoter ;Forsε-Petter et al . , 1990) , which is effective for the expression of tne gene in neuronal cells of the brain. The human platelet-derived growth factor-/3 gene promoter, which is effective fcr the expression of the gene in cells of the brain may also be utilized. Other nerve tissue specific promoters which may be used are rat sodium channel gene promoter (Maue et al . , 1990) , the human APP gene promoter (Wirak et al . , 1991) and mouse mylein basic protein gene promoter (Readhead et al . , 1987) . A yeast artificial chromosome construct containing the human cAMP-response-element-bmdmg-protem-2 gene may also be utilized. This invention provides a nonhuman mammal wnose neuronal cells or glial cells or both, express a cAMP-response- element-binding-protein-2 gene. Preferably, the nonhuman mammal may be a mouse. The gene, having been introduced into the mouse by localized infection with retrovirus, is operably linked to a promoter. The retrovirus has an inducible retroviral vector consisting of a marker gene, a constitutive promoter and an inducible promoter. Retroviral-mediated gene transfer is a procedure known to individuals skilled m the art . Procedures for the mfection of neuronal progenitor cells have oeen established, see, for example, Levison and Goldman (1993) . CREB2-containing retroviral expression constructs may be introduced into fetal and neonatal animals by direct viral infection of subventπcular zone (primitive neuronal and glial precursor) cells (see Levison and Goldman, 1993) . In this protocol, the CREB2 constructs may be cloned downstream of a constitutive promoter (e.g. SV40) tandem with a beta-galactosidase gene under the control of the retroviral long terminal repeat (LTR) promoter. Thus, CREB-2-producing retrovirally infected cells will be specifically marked by 3-galactosidase enzymatic activity (i.e. blue stair, m tissue sections) . It would then be possible to searcn for effects of local CREB2 protein overexpression the intact animal brain on neuronal morphology, amyloid deposition, tau protein phosphorylation and determine whether these effects differ for each CREB2 isoform. If pathological changes are observed, then these animals would serve as a useful in vivo assay system for pharmacological agents. The transgenic nonhuman mammals may provide an experimental medium for elucidating aspects of long-term facilitation and memory and to serve as tools for screening drugs that may have potential application as therapeutic agents to prevent or limit memory defects. Transgenic nonhuman mammals provide both a prognostic and diagnostic means for the study of memory, in particular for determining the efficacy of pharmaceutical drugs in treating a subject. This invention is illustrated in the Experimental Details section which follows. These sections are set forth to aid in an understanding of the invention but are net intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter. EXPERIMENTAL DETAILS Example 1: Transcription factor CREB2/ATF4 as a repressor of memory and a potential target of drugs for improvement of memory formulation. A transcription factor CREB2 has been identified aε a repressor of long-term facilitation m Aplyεia neurons. Injection of anti-CREB2 antibodies into sensory neurons has been shown to interfere with CREB2 function and causes a single pulse of serotonin (5-HT) . This pulse of serotonin ususally induces only short term facilitation lasting minutes, however, under these conditions (witn ant -CRΞB2) , serotonin evokes facilitation lasting more than one day. This facilitation has the properties characteristic of long- term facilitation. Specifically, it requires transcription and translation, it induces growth of new synaptic connections and finally it occludes further facilitation by five pulses of 5-HT. It has been demonstrated that similar to its human ho ologue CREB2 , Aplysia CREB2 is a repressor of the transcriptional activator CREB1. Furthermore, both Aplysia CREB2 and its mammalian homologue ATF4 both heterodimerize, and are repressors of the transcriptional activators ApC/EBP and C/EBP3, respectively. Another transcriptional activator has also been identified, AF-1, which is necessary for the establishment of long-term facilitation. AF-1 has been demonstrated to interact with CREB2. Therefore, CREB2 blocks the transition from short term to long-term facilitation by interacting with transcriptional activators necessary for development of long-term facilitation. Long-term facilitation is a close cellular correlate to long-term memory Aplysia. It is possible, therefore, that CREB2 blocks the transition from snor -term memory to long-term memory in both Aplysia and mammals, including humans. Certain defects n memory formation, in particular the age-related memory loss, may represent in part the inability to remove such repression. Repression of memory mediated by CREB2 and the CREB2 protein itself can oe targets for developing drugs for use m tne treatment of memory defects. The use may extend to memory defects which are related to Alzheimers disease and other diseases or trauma. Example 2: CREB-2/ATF-4 as a Repressor of Long-Term Facilitation in AOlvsia : Relief of Repression Converts a Transient Facilitation into a Long-Term Functional and Structural Change Summary The switch from short- to long-term facilitation induced by behavioral sensitization in Aplysia involves CREB-like proteins, as well as the immediate-early gene ApC/EBP. Using the bZIP domain of ApC/EBP in a two-hybrid system, we have cloned ApCREB-2, transcription factor constitutively expressed in sensory neurons which resembles human CREB-2 and mouse ATF-4. ApCREB-2 represses ApCREB-1 mediated transcription in F9 cells. Injection of anti-ApCREB-2 antibodies into Aplysia sensory neurons causes a single pulse of serotonin (5-HT) , which induces only short-term facilitation lasting minutes, to evoke facilitation lasting more than one day. This facilitation has the properties of long-term facilitation: it requires transcription and translation, induces the growth of new synaptic connections, and occludes further facilitation by five pulses of 5-HT. In cell culture, as in the intact ganglion, botn snort- and long-term facilitation involve an enhancement of transmitter release induced by cAMP and mediated by the cAMP dependent protein Kinase (PKA) (Brunelli et al . , 1976, Scnacner et al., 1988; Scholz and Byrne, 1988; Ghirardi et al . , 1992) . With repeated pulses of 5-HT, whicn give rise to lon -terir facilitation, the intracellular cAMP concentration increases (Bernier et al . , 1982) and the catalytic subunit of PKA translocates to the nucleus of the sensory neurons (BacsKai et al., 1993) , where it appears to phosphorylate one or more cAMP response element-binding proteins (CREB-like transcription factors) , thereby activating cAMP-inducible gene expression (Kaang et al . , 1993) . Injection of an oligonucleotide containing the somatostatin cAMP response element (CRE) into the nucleus of a sensory neuron selectively blocks the long-term enhancement m synaptic strength induced by 5-HT without affecting the short-term process (Dash et al . , 1990) . An Aplysia homolog of CREB-1 has recently been cloned. ApCREB-1 has 42% homology with the mouse CREB-1 over the whole length of the protein, while the basic region/leucine zipper (bZIP) and the phosphorylation domain (P-DOX) , characteristic of CRΞB-1, are 96% and 90% identical, respectively. Similar to its mammalian homologues, ApCREB-1 binds to the CRE m vi tro and is a PKA dependent transactivator (Bartsch et al., in preparation) . In sensory neurons, 5-HT and cAMP induce the immediate-early gene ApC/EBP, a transcription factor necessary for the establishment and maintenance of the stable, self-maintained structural changes characteristic of the long-term memc y process (Bailey and Chen, 1983; 1988, 1989; Glanzman et a 1990) . There is apparent generality of CREB-1 as a component of the switch between short -term and long -term memory m Aplysia , Drosophila , mice and pernaps humans. (Ym et al. , 1994,1995; Bourtchuladze et al. , 1994., Petri et al. , 1995) It is a question as to whether ApCREB-1 and ApC/EBP can recruit additional transcription factors the sensory neurons following sensitizing stimuli. Results Cloning of ApCREB-2 by its interaction with ApC/EBP in the yeast two-hybrid system. The C-termma portion of ApC/EBP containing tne oasic region/leucme zipper (bZIP) domain was used to screen an Aplysia CNS specific cDNA library by tne yeast two-nyorid system (Fields and Song, 1989; Chien et al . , 1991) Two independent clones contained an identical open reading frame of 1134 bp encoding a putative 378 amino acids polypeptide (Figure 1A) . The 118 C-termmal ammo aciαs (ammo acids 260-378) of the predicted polypeptide contain a bZIP motif (Landschulz et al. , 1988, V son et al , 1989) that interacts with ApC/EBP (Fig 6C) In addition to interacting witn ApC/EBP, both the full-length 378 ammo acid protein and the C-termmal 118 ammo acid peptide interact with mammalian CREB-1 and c - fos proteins in the two-hyoπd system. The predicted polypeptide shows highest sequence homology to the ammo acid sequences of two transcription factors: human CREB-2 (hCREB-2) [ (Karpinski et al. , 1992) , also ATF-4 or TAXREB 67 (Hai et al . , 1989, Tsujimoto et al . , 1991)] and mouse ATF-4 (mATF-4) [ (Mielmcki and Pruitt , 1991) , also C/ATF (Vallejo et al . , 1993)] Therefore the Aplysia polypeptide has been termed ApCREB-2 Over the whole length of the protein ApCREB-2 shares 21% identical ammo acids with nCREB-2 and mATF-4. In the bZIP domain, ApCREB-2, mATF-4, and hCREB-2 are 50% identical (Figure IB) . Unlike hCREB-2 or mATF-4, ApCREB-2 contains a second heptad repeat of hydrophobic ammo acids near the N-terminus (aa 73-108) , which could potentially form a second leucine zipper (Figure 1A) . However, this domain does not mediate ApCREB-2 homodimerization or the interaction with the bZIP domain of ApC/EBP ( Fig 6 C ) . ApCREB-2 is expressed in the nervous system of Aplysia. ApCREB-2 is expressed at high levels in the CNS and the gill, but is detectable at low levels by Northern Plot m all Aplysia tissues tested (Figure 2A) . This relatively restricted expression of ApCREB-2 mRNA contrasts witr. tne more ubiquitous expression of its closest homologε, mATF-4 and hCREB-2 (Tsujimoto et al., 1991; Chevray and Nathans, 1992; Vallejo et al . , 1993; Jungling et al., 1994) . In Western blots of Aplysia CNS extracts, polycional ApCREB-2 antiserum raised against full length recombinant ApCREB-2 and the affinity-purified anti-ApCREB-2 antibody recognize a protein that migrates as multiple bands with an apparent molecular weight of around 50 kD (Figure 2B and 2C) . This molecular weight is higher than that 42 kD deduced from the ApCREB-2 cDNA, but it is consistent with the apparent molecular weight of in vi tro translated ApCREB-2 and ApCREB-2 i munoprecipitated from extracts of the Aplysia nervous system (Figure 2C) . Although both the preimmune antisera and the anti-ApCREB-2 antisera recognize additional proteins in Western blots of Aplysia CNS extracts, the major 50 kD signals are not detected by the preimmune serum (Figure 2B, lane 2) , or by the ApCREB-2 antiserum premcubated with recombinant ApCREB-2. (Figure 2B, lane 3) . The strong signal at 40 kD and several weaker signals are recognized by both immune, blocked immune and preimmune antisera. Therefore, preimmune sera has been used and blocked immune antisera has been used as matching controls in electrophysiological experiments described below. ApCREB-2 is constitutively expressed in sensory neurons. To determine whether ApCREB-2 is expressed in the neurons that exhibit long-term presynaptic facilitation, RT-PCR was used to examine the expression cf ApCREB-2 mRNA cultures of Aplysia sensory neurons. We detected ApCREB-2 mRNA ootn in nontreated cultures of sensory neurons and m cultures exposed to repeated pulses of 5-HT. (Figure 3A) ApCREB-1 mRNA was also detected untreated Aplysia sensory neurons by RT-PCR (Bartsch et al . , preparation) . In addition, tne steady state levels of ApCREB-2 and ApCREB-1 mRNAs were not affected either m vivo or m vi tro by exposure to 5-HT which induces the mRNA level of ApC/EBP (Figure 3B) . Furthermore the level of ApCREB-2 mRNA does not cnange following exposure to agents that increase cAMP (forsKolm, IBMX, 8-bromo cAMP and Sp-cA PS) , application of the PKA mnioitor Rp-cAMPS, calcium lontophore A23187, pnorbol esters, okadaic acid, the protein synthesis inhibitor aniso ycm or neuronal injury. These results indicate that ApCREB-2 and ApCREB-1 are coexpressed in sensory neurons and the steady state level of ApCREB-2 mRNA is not regulated by 5-HT, cAMP or cytoplasmic Ca 2* levels. ApCREB-2 is a substrate for protein kinases. The primary structure of ApCREB-2 has putative phosphorylation sites for both PKC and MAP kinases (Figure 1A) . Furthermore, ApCREB-2 is a substrate for PKC, MAP kinase, PKA, and CaM kinases m vi tro . The phosphorylation of ApCREB-2 m vi tro results m an increase of apparent molecular weight of the phosphoprotein m SDS gels. A similar shift m molecular weight is detected by Western blots of Aplysia CNS extracts isolated from animals exposed to 5-HT m vivo . Exposure to 5-HT does not affect the protein level of ApCREB-2 or ApCREB-1 in the Apiysia CNS. By contrast, ApC/EBP protein m the same CNS extracts becomes detectable only after 60 m of 5-HT exposure m vivo (Figure 4A) . The shift m apparent molecular weight of ApCREB-2 following 5-HT treatment in vivo presumably is the result of phosphorylation, since phosphatase treatment of the CNS extract results in the increased migration of ApCREB-2 in SDS-PAGE (Figure 4B) . ApCREB-2 is a repressor of ApCREB-l mediated activation in F9 cells. Human CREB-2 represses CREB-1 mediated transcriptional activation in CV-1 cells and neurons (Karpinski et al., 1992; Jungling et al . , 1994) . Whether ApCREB-2 could also function as a repressor of ApCREB-1 mediated transactivation in transfected undifferentiated mouse F9 cells was examined. The ability of ApCREB-2 and ApCREB-1 to regulate a minimal control region (a single CRE in front of a minimal SV40 promoter) of a pGL3-CRE luciferase reporter gene was first examined. ApCREB-1 activates this minimal CRE reporter in a PKA dependent manner (relative activation 2.13 ± 0.26 without and 10.50 + 1.42 with the PKA) and is repressed by ApCREB-2 upon cotransfection in the absence and presence of PKA catalytic subunit (relative activation 0.96 ± 0.15 and 1.64 ± 0.20, respectively) (Figure 5A) . The degree of ApCREB-1 repression by ApCREB-2 is dependent on the concentration of ApCREB-2. ApCREB-2 does not activate this minimal CRE reporter. In previous experiments, Kaang et al. (1993) demonstrated that a 5xCRE-VIP-lacZ reporter is activated by 5-HT and cAMP in Aplysia sensory neurons. We therefore also cotransfected ApCREB-2 and ApCREB-1 were cotransfected with this reporter. Upon cotransfection, ApCREB-2 again abolished the transcriptional activity of ApCREB-1 both in the absence and in the presence of PKA (relative activation 1.84 ± 0.25 and 4.12 ± 0.53, respectively, as compared to 2.13 ± 0.30 and 48.60 ± 5.23 for ApCREB-1 alone.) (Fig. 5B) . Similarly, ApCRE3-2 repressed transactivation mediated oy rat CREB-1 (relative activation 2.27 ± 0.3 and 5.15 z 0.62 as compared to 7.65 ± 0.65 and 68.82 T 7 3 for rat CRΞB-1 alone, without and with PKA, respectively) Thus this data suggest that ApCREB-2 can repress octn ApCREB-1 and rat CREB-1 mediated transactivation from a CRE and are consistent with the possibility tnat ApCREB-2 and ApCREB-1 may interact directly on the CRE. ApCREB-2 can be an activator in the absence of ApCREB-1. In addition to repressing transcription mediated oy ApCREB-1, it was found that ApCREB-2 can also function as an activator of tne 5xCRE-VIP-lacZ reporter gene in F9 cells ApCREB-2 transactivation F9 cells is stimulated by PKA to the level comparable with ApCREB-1 (relative activation 2.46 ± 0.2 without and 43.12 ± 5.1 with PKA ) (Figure 5B) . Both the full length ApCREB-2 protein and its N terminal 288 ammo acids are strong activators of the reporter UASg-lacZ gene when fused to the GAL4 DNA binding domain (relative activity 82% ± 27 and 59% ± 18, respectively, as compared to wild type GAL4) . Thus, ApCREB-2 has an internal transcription activation domain in its N-termmal 280 amino acids and can under certain circumstances be a transcriptional activator. Unlike ApCREB-1, ApCREB-2 cannot activate a minimal, single copy CRE driven construct (Figure 5A) Thus, tne activating and repressing potential of ApCREB-2 and ApCREB-1 on the CRE are not fully symmetrical. The ability of ApCREB-2 to activate from the CRE is more restricted than its ability to repress ApCREB-1 injury. ApCREB-2 is a CRE binding protein. The DNA binding specificity of ApCREB-2 was examined m a oinάing site selection assay using bacterial-expressed recompinant ApCREB-2 protein tc select optimal omdmg sequences from a pool of randomly generateα DNA targets . This assay identified a binding sequence (BSl for ApCRE3-2 whicn resembles tne CRE DNA-binding sequence of the CREB/CREM/ATF family of transcription factors, as well as the CAAT DNA omdmg motif of the C/EBP family cf transcription factors (Figure 6A) The DNA oin ng capaciiity of ApCREB-2 by electrophoretic mooility sr.iftε using both symmetrical and asymmetrical CREs and CAAT motifs of C/EBP binding sites was then reexammed Purified bacterial-expressed ApCREB-2 protein binds solution the symmetrical CRE of the somatostatin gene, the core CRE palindrome, the asymmetrical CRE of the enkephal gene and the BSl DNA binding site, . However, ApCREB-2 aid not oina to the asymmetrical CRE of the pnospnoenoipyruvate carboxyk ase gene or to the asymmetrical putative CRE site of the ApC/EBP gene In addition, ApCREB-2 a d not bind to any of the CAAT DNA-binding sites tested (Figure 6B) The binding affinity of ApCREB-2 to CRE was low, pernaps because of inefficient homodimer formation. Deleting the 260 N-termmal ammo aciαs of ApCREB-2, which removes the second leucine zipper motif, greatly increases the efficiency of homodimer formation. This αeletion does not affect heterodimer formation as both tne full-length ana the N-termmal αeletion mutant of ApCREB-2 heterodimerize with the same high affinity with ApC/EBP (Figure 6C) Thus, ApCREB-2 forms weak homodimers on CRE, perhaps due to the presence of an inhibitory structure m the N-terminal part of the protein. In contrast to its ability to form only weak homodimerizers we have found that ApCREB-2 can effectively heterodimerize with other b-ZIP transcription factors In some experiments, it was found that m addition to ApC/EBP, ApCREB-2 forms in vi tro heterodi ers with ApCREB-1, witn rat CREE-1 and with c-fos proteins. One pulse of 5-HT produces long-term facilitation when paired with injection of ApCREB-2 antiserum. In both the intact Aplysia and in neuronal cell culture, five pulses of 5-HT induce long-term facilitation the connections between the sensory and motor neurons lasting 24 hr or more. By contrast, a single pulse of 5-KT produces only a short-term facilitation lasting about 10 m (Figure 7A and B) . This single pulse increases the excitatory postsynaptic potential (EPSP) evoked in the motor ceil by stimulating a single sensory neuron by 76.7% at 1 mm after 5-HT exposure (± 5.4, n = 11 p < 0.01 compared to control cells) . This facilitation decays to 12.6% at 10 mm (±18.7, n = 5) , and to 2.83% at 30 mm (±8.8, n = 12) . Two hours after a single pulse of 5-HT the change in EPSP amplitude has returned to control level (-1% ± 5.7, n = 16) and it remains there at 24 hr (-4.12 % ± 6.09, n = 8) . To determine whether ApCREB-2 could also act as a functional repressor and parallel its action as a transcriptional repressor of ApCREB-1 in transfection assays, we injected ApCREB-2 antiserum into the sensory neurons 1 hr before exposure to single or multiple pulses of 5-HT. In the presence of the antiserum, rather than producing the short-term facilitation lasting 10 min, one pulse of serotonin produced facilitation lasting more than 24 hour. This facilitation was robust; it was seen in 42 out of 43 cells. Moreover, the facilitation seen at 24 hr was comparable in magnitude to that seen at 24 hr with five pulses of 5-HT. The long-term facilitation following five pulses of 5-HT i:- seen as early as 2 hr after the first pulse (Figure 7A and Ghirardi et al. , 1995) . This early component of long-term facilitation has a larger amplitude than the facilitation evident at 24 hr, but it differs mechanistically from the facilitation evident at 24 hr that it is only partially (about 70%) dependent en RNA synthesis, although it is completely dependent on protein synthesis. When a single pulse of 5-HT is paired with the injection cf ApCREB-2 antiserum, there is also a significant facilitation present at 2 hr [+100.95% (± 12.52, n = 43 p < 0.01). and this facilitation persisted 24 hr later, when the synaptic potential is still significantly facilitated [-r70.05% (__ 7.65, n = 42, p < 0.01) (Figures 7A, 8A and B) ] . By contrast cultures treated with one pulse of 5-HT and injected with serum depleted of anti-ApCREB-2 antibody or with preimmune serum shows no significant facilitation, e tner at 2 hr (+19.64% ± 10.56, n = 11 and -13.75% ± 4.0Ξ, n = 12, respectively) , or at 24 hr (-4.27% ± 9.70, n = 11 and + 1.5% ± 11.57, n = 12, respectively) compared to the nonin ected control cells not exposed to 5-HT (-6.21% ± 2.10, n = 14 at 2 hr, and -0.33% + 4.56, n = 9 at 24 hr) (Figures 8A and B) . As an additional control, a single pulse of 5-HT was paired with the injection of two unrelated antibodies (anti-ApC/EBP Ab, anti-PAS Ab ) . In both cases no significant facilitation was found either at 2 hr (+11.8% + 5.51, n = 5 and +1.87% + 6.29, n = 8, respectively) or at 24_hr (+1.8% + 9.77, n = 5, and +3.62% ± 11.97, n = 8, respectively) (Figures 8A and B) . These results were obtained using a single ApCREB-2 antiserum. However, injection of a second independently raised ApCREB-2 antiserum yielded comparable results. Facilitation produced by one pulse of 5-HT paired with injection of ApCREB-2 antiserum has the properties of transcriptionally-dependent long-term facilitation. Long-term facilitation induced by repeated pulses of 5-HT requires protein and RNA synthesis. (Montarolo et al. , 1986; Bailey et al . , 1992) . The effect of the protein synthesis hibitor anisomycm and the RNA synthesis mnioitor act omycm D was examined on the synaptic modifications produced at 2 and 24 hr after the injection of ApCRΞB-2 antiserum paired with the application of a single pulse cf 5-HT. Incubating sensory-motor neuron cocultures with anisomycm during a single pulse of 5-HT PIOCKS tne increase amplitude of synaptic potential after injection witn ApCREB-2 antiserum, both at 2 hr after exposure +19.17% z 6.85, n= 18) and at 24 hr (+3.67% + 5.54, n = 18) (Figures 7A, 8A and 8B) Similar results were ootamed using actmomycm D, where the facilitation at 2 hr was reduced to +10.12% (± 5.10, n = 24) , and at 24 hr to 7.87% »± 5.84, n = 24, Figure 7A, 8A and 8B) . If one pulse of 5-HT m the presence of ApCREB-2 antiserum phenocopies long-term facilitation, the injection of antibody should also occlude the effects of five pulses of 5-HT. In the cocultures injected with ApCREB-2 antiserum, the facilitation measured 24 hr after five pulses of 5-HT was not significantly greater (+74.5% ± 13.56, n= 20) than the facilitation obtained n cells exposed to five pulses of 5-H and not injected with antibody (+79.6% + 12.95, n = 15) , or cells treated with five pulses of 5-HT and injected with normal rabbit serum (+91.6% ± 21.31, n = 10) (Figure 8C) . Injection of ApCREB-2 antiserum alone, witnout 5-HT treatment, induced a slight decrease m the strength of the connection (-5.11% ± 5.03, n = 9) , similar to that found in the control cocultures. In conclusion, the facilitation produced by one pulse of serotonin the presence of the antibody has properties similar to that induced by five pulses of 5-HT and occludes the effect of five pulses. The facilitation produced at 2 hr by five pulses of 5-HT is completely blocked by inhibitors of protein synthesis, but IΞ only partially blocked by inhibitors of transcription (Ghirardi et al . , 1995) . This suggests that five pulses of 5-HT modulate both transcription and translation. Since ApCREB-2 presumably acts only on the transcriptional component of long-term facilitation, one might predict that the pairing cf one pulse of 5-HT with injection cf ApCREB-2 antiserum with would produce less facilitation at 1 hr than 5 pulses of 5-HT. The facilitation at 2 hr produced by one pulse of 5-HT in the presence of ApCREB-2 antibody is approximately 30% less than that produced by five pulses of 5-HT (Figure 7A) . Similarly, injection of CRE oligonucleotides, which would also likely to affect only the transcriptional component of 2 hr facilitation produced a comparable inhibition at 2 hrs [EPSP increase to - 66.29% ( 18.76, , thus supporting the suggestion tnat the role of ApCREB-2 is specific to the transcriptional response to 5-HT . Facilitation induced by one pulse of 5-HT paired with injection of ApCREB-2 antiserum has two distinct phases The facilitation induced by one pulse of 5-HT paired with injection of ApCREB-2 antiserum shows two temporal stages: the first phase is similar both in amplitude and time course to the short-term facilitation induced by one pulse of 5-HT in the absence of the antibody (Figure 7A and B) . Thus, m the presence of ApCREB-2 antiserum the facilitation also peaks at 1 min and decays at 30 min to 33.3% (± 10.44, n = 20) , but instead of decaying progressively and completely back to baseline at 30 min as in uninjected cells, a second phase of facilitation emerges. At 1 hr this facilitation reaches +72% (±19.74, n = 11) and at 2 hr it is about +100.95% (±12.52, n = 43) . This second phase presumably represents the gradual induction of the transcriptional components of long -term facilitation. Injection of ApCREB-2 antiserum does not affect short-term facilitation. The effect of ApCREB-2 antiserum injection on short-term facilitation induced by one pulse of 5-HT was next investigated. One minute after exposure of one pulse of 5-HT the nonmjected ceils showed a facilitation cf - 79.43% '._. 7.06, n = 7) , the ApCREB-2 antiserum injected ceils snowed a facilitation of +96.17% (±32.35, n = 6) and the cells injected with normal rabbit serum had a facilitation of +76.45% (±12.03, n = 11) ( Figure 8D) . All these values are significantly different from the decrease EPSP amplitude due to homosynaptic depression observed in non-5-HT- reated control cells (-25.5% ±5.59, n = 8, p < 0.01) . Th s, as with other agents that specifically affect the long-term process, ApCREB-2 antiserum has no effect on tne snort-term process. One pulse of 5-HT paired with injection of the ApCREB-2 antiserum induces the growth of new synaptic connections Long-term memory for sensitization of the gill-withdrawal reflex is associated with the growth of new synaptic connections between the sensory neurons and their follower motor neurons (Bailey and Chen, 1983, 1988) . The duration of this structural change parallels the behavioral retention of the memory (Bailey and Chen, 1989) . Similar changes can be observed in sensory-motor neuron cocultures where five pulses of 5-HT produce a long-lasting (24 hr) mcrease the number of sensory varicosities contacting the motor neuron (Glanzman et al. , 1990; Bailey et al, 1992) . To determine whether ApCREB-2 can also act as a repressor of the morphological changes that accompany long-term facilitation, the ApCREB-2 antiserum was injected into sensory neurons and examined the consequences of one pulse of 5-HT on long-term changes both the strength of the sensory-motor neuron connection and on the numoer of fluorescently-iaoeied sensory neuron varicosities contacting the motor neuron (Figures 9 and 10) . The pairing of a single pulse of 5-HT with the injection of ApCREB-2 antiserum 1 hr before training induced significant increases, 24 nr after the injection, in botn tne strength of the sensor\ -motor neuron connection (66% ± 12, n = 8, p < 0.001) , and m tne number of sensory neuron varicosities contacting the postsynaptic motor neuron (59% ± 8, n = 8, p < 0.001) By contrast, control cells receiving ust one pulse of 5-HT and no injection of antiserum showed no facilitation (-31.5% + 6, n = 6) and no mcrease m the number of sensory neuron varicosities (-10% ± 7, n = 6) 24 hr following training (Figure 9) . Figure 10 contains examples of raw data taken from individual cocultures and illustrates the tτιarκed long-term increases m both the amplitude of the evoked EPSP and the number of fluorescently-labeled sensory neuron varicosities elicited by one pulse of 5-HT in the presence of antibody to ApCREB-2. As is the case with the structural changes induced by conventional 5 x 5-HT long-term training in vi tro, the application of one pulse of 5-HT paired with injection of ApCREB-2 antiserum now results in the formation of new sensory neuron varicosities in contact with the motor neuron as well as new neuritic outgrowth. The magnitude of both the long-term functional and structural changes are comparable to those seen at 24 hr following five pulses of 5-HT (Glanzman et al. , 1990; Bailey et al . , 1992) Discussion A bZIP transcription factor, ApCREB-2, has been cloned which is homologous to human CREB-2 Hai et al. , 1989; Tsujimoto et al., 1991: Karp ski et al, 1992) and mouse ATF-4 (Mielnicki and Pruitt, 1991; Vallejo et al . , 1993) . ApCREB-2 represses the activation mediated by ApCREB-1 m mouse F-9 cells. Following injection into the presynaptic sensory neurons of two specific ApCREB-2 antisera, one pulse cf 5-HT, wnich nor ally induces short-term presynaptic facilitation that does not require RNA or protein synthesis, produces long-term facilitation that lasts more than a day, requires both transcription and translation, and is accompanied oy a growth of new synaptic connections. Although the parallel between the inhibition of ApCREB-1 mediated transactivation in the F9 cells, and the inhibitory action of ApCREB-2 in the sensory neurons, is suggestive, the functional repression by ApCREB-2 in the induction and maintenance oi long-term facilitation does not necessarily mean it occurs by means of transcriptional repression. Since ApCREB-2 can activate transcription on its own, it is conceivable that ApCREB-2 may be a repressor only indirectly and that it functions in sensory neurons by activating expression of genes that are themselves inhibitory for the induction of long-term facilitation. Furthermore, the possibility cannot be ruled out that the anti-ApCREB-2 antibodies activate ApCREB-2 rather than blocking it. Nevertheless, the idea that ApCREB-2 acts as a direct repressor of long-term facilitation is favored. The data so far are most consistent with the idea that the anti-ApCREB-2 antibodies prevent ApCREB-2 from interacting with transcriptional activators (such as ApCREB-1) . ApCREB-2 resembles human CREB-2 in both sequence and repression of ApCREB-1 ApCREB-2 resembles most closely human CREB-2 and mouse ATF-4 in its primary amino acids sequence and in its binding (albeit with low affinity) to the CRE (Hai et al . , 1989; Karpmski et al . , 1992) . Furthermore, interaction of ApCREB-2 witn Ap/CEBP resembles the interaction of ATF-4 and C/EBP(Valle o et al. , 1993) . In addition, ApCREB-2 represses ApCREB-1 mediated transactivation m F9 cells, thus resembling the repression of CREB-1 by numan CRΞΞ-2. (Karpinski et al . , 1992, Junglmg et al . , 1994 ,-j In fact, ApCREB-2 can substitute for human CREB-2 aε a represser of mammalian CREB-1 mouse F9 cells The mechanisms whereby ApCREB-2 mediates transcriptional repression of ApCREB-1 are not yet elucidated. However, its action seems to be distinct from the known mduciole and constitutive repressors of CRE-mediated transactivation For example, unlike the inducible repressors ICER or E4BP4 (Molina et al . , 1993, Cowell et al . , 1992) , ApCREB-2 is constitutively expressed and can act as a transcriptional activator. This ability to activate transcription also distinguished ApCREB-2 from the constitutive repressors of CRE-mediated transactivation exemplified by CREM and ApCREB-2 also lacks other features characteristic of the CREMs, such as the highly conserved KID domain (Foulkes and Sassone-Corsi, 1992;) In addition to being a repressor of ApCREB-1, ApCREB-2 can also be an activator The finding that ApCREB-2 can botn repress and activate transcription further demonstrates that the distinction between activators and repressors is not strict but is critically dependent on the particular promoter, on the recruitment of the specific second messenger pathways, and on the repertoire of the transcription factors available (Hai et al., 1989; Valle o et al . , 1993; Lemaigre et al . , 1993;. Ellis et al . 1995) . ApCREB-2 is an activator as a GAL4 fusion protein yeast and a PKA dependent transactivator when cotransfected with the 5xCRE-VIP-lacZ reporter gene However, ApCREB-2 does not activate transcription from the minimal CRE-SV40 regulatory region m pGL3-CRE reporter gene althougn it can repress tne transactivation by ApCREB-1 from this minimal reporter The reason for this difference is not clear Perhaps for effective transactivation, ApCREB-2 requires multiple CRE elements, alternatively, ApCREB-2 may interact w th additional regulatory elements m 5xCRE-VIP-lacZ reporter that are unrelated to the CRE. Induction of long-term memory requires the coordinated regulation of both CREB-1 and CREB-2 The data herein provide evidence that ApCREB-2 is a functional repressor of long-term facilitation These data provide the first molecular evidence for a possible role of f nctional repressors n memory storage Overexpression of an inhibitory form of Drosophila CREB-1, dCREB-2b, blocks the formation of long-term memory transgenic flies (Ym et al., 1994) . Recently, Ym et al. (1995) demonstrated that overexpressing the activating form of Drosophila CREB-1 (dCREB-2a) greatly reduces the number of training trials needed to establish long-term memory This ga of function, where a single massed training trial is sufficient to achieve long-term memory which normally requires spaced training trials, greatly strengthens the earlier evidence from Drosophila (Yin et al . , 1994, 1995) , Aplysia (Dash et al . , 1990; Kaang et al., 1993) , and mice (Bourtchuladze et al . , 1994) that CREB-1 is of central importance m initiating the long-term memory formation. The results Aplysia point to a parallel importance for ApCREB-2 n this process. Injection of anti-ApCREB-2 antibodies paired with a single training trial, which normally produces only short-term facilitation, results n induction of long -term facilitation. This ga of function resembles overexpression of the dCREB-2a activator in Drosophila . These findings suggest the interesting possibility that removal of the ApCREB-2-mediated repression may be limiting in regulating the long-term increase m synaptic strength. A possible mechanism for the physiological role of ApCREB-2 There are a number of ways by which a transcription factor such as ApCREB-2 could directly repress (for recent review, see Johnson, 1995) . First, ApCREB-2 could act directly to inhibit the basal transcriptional machinery. Since ApCREB-2 can be an activator on its own, this is unlikely. Second, ApCREB-2 could compete for the DNA binding sequence with ApCREB-1 (or another activator) . Since the affinity of the ApCREB-2 homodimers for CRE is much lower than that of ApCREB-1, this is also unlikely. Therefore, the possibility that ApCREB-2 might mediate repression by interacting directly with ApCREB-1 (or another activators) to form an inactive heterodimer is favored. Both ApCREB-1 and ApCREB-2 are coexpressed in the sensory neurons. Moreover, ApCREB-2 can form heterodimers on a CRE with rat CREB-1 in vi tro . However it remains to be determined whether ApCREB-2 also heterodimerizes with ApCREB-1 m vivo . How might the repression of ApCREB-1 by ApCREB-2 be relieved? Since a change in the amount of the ApCREB-2 protein after exposure to 5-HT is not detected, the relief of repression most likely does not involve targeted degradation of the ApCREB-2 protein. More likely the repressive action of ApCREB-2 is relieved by a covalent modification induced by the repeated pulses of 5-HT. According to this view, the physiological role of ApCREB-2 ay be : First it may prevent the long-term process frorr oeing turned on adventitiously without repeated exposures to 5-HT Second, it may regulate the amplitude of synaptic cnange by integrating the activation of ApCREB-1 by PKA with signals from additional second messenger pathways. The induction of long-term facilitation and the concomitant structural changes induced by a single pulse of 5-HT paired with injection of anti ApCREB-2 antibody is consistent with the idea that a single pulse of 5-HT is sufficient to fully induce the activating pathway. The finding that ApCREB-2 transcriptionally represses ApCREB-1 in the presence of cotransfected catalytic subunit of PKA in F9 ceils indicates that another pathway besides PKA (a pathway not active m undifferentiated F9 cells) must mediate its derepression. This suggests the interesting possibility that additional second messengers and kinases or phosphatases may be involved n relieving the repression. The pathways regulating stimulation of the activator and relief of the repressor may have distinctive kinetics. Such differences in kinetics could define the optimal time window separating training trials and account for the well established difference between massed and spaced training. In cell culture, as in the intact animal, spaced training (5 pulses of 5-HT separated by 20 mm) trial is more effective m triggering long-term facilitation than massed training (5 pulses of 5-HT not separated at all but given continuously over 25 mm) . The synaptic potential is facilitated by +80% (12.95, n=15) after 5 spaced pulses as compared with only + 39% (19, n=16) after 25 minutes of continuous exposure to 5-HT Perhaps the reason that spaced training is more effective than massed training is that only spaced training allows coordinated activation of ApCREB-1 and derepression Of ApCREB-2. That 5-HT triggers different signaling pathways in a coordinated way PKA to stimulate the activator and possibly others to relieve the repressor should not be taken tc indicate that each of these pathways cannot be engaged alone by other transmitter signals acting on surface receptors. Certain odulatory transmitters might act selectively to relieve repression. Such a priming action on memory might allow for one trial learning. One trial learning: Flashbulb memories Dual control of activators and repressors by different second messenger pathways could provide a beginning insight into to a range of features characteristic of memory, ranging from amnesia to photographic memory. For example, a characteristic feature of age-related memory loss (benign senescent forgetfulness) is the inability to consolidate long-term memories (Petersen et al . , 1992) . This aging defect, therefore, may represent not only a weakening stimulation by activators, but perhaps also an inability to relieve repression. Conversely, genetically endowed differences in the activity of the repressor m relation to the activator could prime the storage process and contribute to exceptional memory. Although long-term memory typically requires repeated spaced training, it occasionally occurs following a single exposure. One trial learning is particularly well-developed in certain rare individuals (memoπsts) with exceptional memory. For example, the famous me orist D. C. Shershevski, studied by A. R. Luna (1968) , seemed never to forget anything he had learned following a single exposure, even after more than a decade. More commonly, memorists have more restricted capabilities: they may be exceptionally good in remembering certain specific types of knowledge and not others (Brown and Deffenbacher, 1995) . There are people with astonishing memory that is selective for visual images, for musical scores, for chess games, for poetry, or for faces. But photographic memory is not limited to memorists. The most common type cf photographic memory, flashbulb memory, is a detailed and vivid memory most people store on one or another occasion and retain for a lifetime (Brown and Kulik, 1977; Conway, 1995; Neisser, 1982) . Flashbulb memories, such as the memory of where you were when President Kennedy was assassinated, preserve knowledge of an event in an almost indiscriminate way, much as a photograph preserves all the details of the scene. Initial studies on flashbulb memories focused on important historical events. But there is now good evidence that autobiographical details of surprising and important defining personal events are retained with the same vivid clarity for details (Conway, 1995) . How are the details of these dramatically personal and historical events stored? These surprising and emotionally-charged events are thought to recruit the amygdala and the major arousal systems of the brain the serotonergic, noradrenergic, dopaminergic, and cholinergic odulatory systems (McGaugh et al., 1993) . One potential consequence of the action of 'these modulatory systems might be to relieve repression and thereby prime the memory system. It is therefore of particular interest that these modulatory systems can play a significant role in the CREB-related learning in Aplysia , Drosophila , and mice. Experimental Procedures General Methods Standard manipulations of E. coli , S . cerevisiae , proteins and nucleic acids, were performed essentially as described m Maniatis et al. (1989) , Ausubel et al . , (1994) and Harlow and Lane (1988) . Plasmids and Cloning Cloning was generally done by PCR using Ultima DNA polymerase (Perkin Elmer) . The Aplysia CNS specific cDNA library was constructed in pGADIO and the ApC/EBP bZIP domain (amino acids 151-286) was cloned in pMA424 (Ma and Ptashne, 1987) . Subsequent subcloning was carried out m pASl and pACT2 plasmids (Durfee et al . , 1993) . The initiation codons of ApCREB-2, ApC/EBP and ApCRΞB-1 were replaced by an Ncol restriction site by PCR and cloned m the Ncol-Sacl site of pGEX-KG (Guan and Dixon, 1991) or pET-30 (Novagen, modified by replacing the Ndel-Ncol fragment by a synthetic oligonucleotide encoding the initiating methionine followed by six histidmes) . The mammalian expression constructs pRcRSV-ApCREB-2 and pRcRSV-ApCREB-1 were made by subcloning the corresponding cDNAs in pRcRSV (Invitrogen) . The reporter pGL3-CRE was made by cloning a single CRE palindrome into a pGL3 promoter luciferase reporter plasmid (Promega) . The plasmids pRcRSV-PKA C-αl expressing the PKA catalytic subunit and pRcRSV-CREB341 expressing the wild type rat CREB were utilized. Aplysia CNS cDNA Library Construction, Two-Hybrid Screening in Yeast and GAL4 DNA Binding Domain Activation Assay The Aplysia CNS cDNA library was synthesized in pGADIO using random hexamers and the BRL cDNA synthesis kit. Two libraries constructed from size fractionated cDNAs with average inserts >2 kb (5 x 10 6 independent clones each) were used in the two-hybrid screening as described previously (Fields and Song., 1989, Durfee et al . , 1993, Ausubei et al. , 1994) . The transcriptional activation properties of ApCREB-2 and its interaction with other proteins in the two-hybrid system was analyzed using the full length ApCREB-2 and its deletion mutants subcloned pASl and pACT2 vectors. The transcriptional activity of ApCREB-2/GAL4 DNA binding domain fusions was determined as decribed (Ma and Ptashne, 1987; Durfee et al. , 1993) . To analyze the protein interactions, ApCREB-2, ApC/EBP, rat CREB, c-fos and deletion mutants of these proteins in pASl and pACT2 were cotransformed into S. cerevisiae Y190 , and the expressed /3-galactosidase quantified as above. Purification of Recombinant Proteins The induction and purification of GST fusion proteins were done as described (Frangioni and Neel, 1993) . 6His-ApCREB-2 fusion protein was expressed and purified using the QIAexpress system (Qiagen, denaturing protocol) . The bound 6His-ApCREB-2 protein was renatured on the Ni-NTA resin and eluted with 250 mM imidazole. Antisera Production, Depletion and Affinity Purification Two rabbit antisera were raised (BABCO) against GST-ApCREB-2 and one against GST-ApC/EBP fusion proteins. The anti-ApCREB-2 antisera were depleted of ApCREB-2 specific antibodies by incubation with an equal volume of glutathione-agarose saturated (3 μg/μl) with GST-ApCREB-2 fusion protein. The matching controls for Western blots and electrophysiological experiments were prepared by parallel incubation of the immune antisera with glutathione-agarose saturated with GST. The antibodies were affinity purified on the GST-ApCREB-2, GST-ApC/EBP proteins coupled to Affi Gel (Bio Rad) . Prior to purification, the antisera were peadsorbed on the GST-Affigel. Western Blotting 20 μg of Aplysia CNS extract were separated o 10% SDS-PAGE, electroblotted to PVDF membranes (Immobilon-P, Millipore) The membranes were probed with affinity purified anti-ApCREB-2 , anti-ApC/EBP antibodies or ant rat CREB antiserum (UBI) followed by anti-rabbit-HRP and visualized by chemiluminescence (ECL, Amersham) . Immunoprecipita ion The CNS ganglia removed from anesthetized Aplysia were labeled with 35 S-methionin , overnight at 18°C, homogenized in 10 mM Tris pH 7.2, 350 mM NaCl, 0.5% Triton X-100, 50 mM β-glycerophosphate, 25 mM NaF, 1 mM NaVO < , 2 mM DTT, 1 mM PMSF, 5 mM benzamidine and 10 μg/ml each of chymostatin, leupeptin, antipain and pepstatin A. After diluting 1:1 with 2 x RIPA , the extract was precleared with Protein A-Sepharose for 1 hr at 4°C, incubated with affinity purified anti-ApCREB-2 antibody for 1 hr at 4°C followed by Protein A Sepharose (Pharmacia) . The immunoprecipitated proteins were resolved by 10% SDS-PAGE, and visualized by fluorography (Amplify, Amersham) . Phosphatase Treatment of Aplysia CNS Extracts Both phosphatase and mock buffer cocktails contained 20 mM MgCl 2 , 0.5 mM EGTA, 1 mM PMSF, 5 mM benzamidine and 10 (g/ml each of chymostatin, leupeptin, antipain and pepstatin A. The phosphatase mix contained 2 U of calf intestinal phosphatase, the mock mix 20 mM NaF and 20 mM β-glycerophosphate . These cocktails were added to 40 μg of the CNS extracts and incubated at 37°C for 30 min. The reactions was stopped by the addition of SDS sample buffer and the proteins were visualized by Western Blotting w th affinity purified anti-ApCREB-2 antibodies. RNA Extraction from Sensory Neuron Cultures and RT-PCR Cultures of approximately 200 Aplysia sensory neurons, established by the dissociation of the pleural sensory cluster in a single dish, were exposed to 10 uM 5-HT for 5 min once or five times separated by 20 min. After washing with ASW, cells were lysed by 100 μl of the guanidium thiocyanate solution and the RNA was isolated. For RT-PCR, the isolated RNA was treated with RNAse free DNAse (Boehringer) , reextracted as above and processed using RT-PCR kit (Boehringer) . The sequence of the primers used were TTCCGCTTTCCATAAGTCGA (Seq ID No 6) and ACCTGAAAATGATATTGTAC (Seq ID No 7) . DNA Binding Site Selection Optimal recognition sequences for DNA binding of ApCREB-2 and ApC/EBP were determined by a PCR assisted binding site selection method (Norby et al. (1992) . The oligonucleotides which bound in 150 mM KCl (standard isotonic condition) or 400 mM KCl (resembling Aplysia cell os olarity) were eluted by 1M KCl, diluted and PCR amplified. After 13 cycles of binding and PCR amplification the amplified products were cloned in Bluescript (Stratagene) and sequenced. In each of the 48 independent clones from both the low and high salt conditions the BSl binding sequence (for ApCREB-2) or BS2 (for ApC/EBP) was present. In Vitro Protein Binding Assay 35 S methionine-labeled ApCREB-2 and ApC/EBP were translated in the TNT rabbit reticulocyte lysate (Promega) . Ten ul of the lysates containing the m vi tro translated proteins were mixed with 25 μl of Glutathione-Sepharose beads saturated with Glutathione S-transferase (GST) or GST fusion proteins in 400μl of PBS and mixed for 1 hour at room temperature. The bound complexes were washed thoroughly witn 20 rl cf 0.1% Triton X-100 PBS on a minicolumn (Wizarα, Promeσa , eluted m SDS sample buffer and resolved by 10% SDS-PAGE Electrophoretic DNA Mobility Shift Assays (EMSA) The sequences listed show one strand of the double stranded oligonucleotides used in the gel mobility shift assays The sequences in capital letters correspond to: the somatostatin gene CRE (SOM CRE) gatccggcGCCTCCTTGGCTGACGTCAGAGAGAGAGA (Seq ID No 8) , the palindromic core CRE (CRE) gatccggcTGACGTCAtcaagcta (SEQ ID NO 9) , the phosphoenolpyruvate carboxykmase gene CRE-l (PEPCK CRE) gatccCCTTACGTCAGAGGCGA (SEQ ID NO 10) , the enκepnalm gene CRE (ENK CRE) gatccggcGCGGGGCTGGCGTAGGGCCTGCGTCAGCTGCA (SEQ ID NO 11) , the ApC/EBP gene putative CRE (Ap CRE) GAGTGGCATCTACGTCAAGGCTTC (SEQ ID NO 12) , the ApCREB-2 DNA binding sequence (BSl CRE) gatccggcAGTATTGCGTCATCtcaagcta (SEQ ID NO 13) , the composite CRE-CAAT site (CRE-C/EBP) gatccggcTGACGCAATtcaagcta (SEQ ID NO 14) , the angiotens gene acute phase response element (ANG-APRE) gatccACAGTTGTGATTTCACAACCTGACCAGA (SEQ ID NO 15) , the ApC/EBP DNA-binding sequence (BS2 C/EBP) gatccggcACTATTGCGCAATCtcaagcta (SEQ ID NO 16) and the C/EBP/3 binding sequence of the c -foε promoter (ERE) gatcCATATTAAGGACATGCCG (SEQ ID NO 17) . The EMSA assays were performed as described in Ausuoel et al. (1994) using the high ionic strengtn TGE buffer, 200 ng of recombinant 6Hιs-ApCREB-2, 200 ng of poly (dl-dC) (Pharmacia) , and 25 fmol of 32 P end-labeled double-stranded oligonucleotide probes. F9 Cell Culture, Transfections and Reporter Gene Assays Undifferentiated mouse F9 cells were transfected using Lipofectamine (BRL) . The β-galactosidase and luciferase activities were quantitated by cnemiiuminescence (Gaiacton Plus, Tropix, and a Luciferase assay kit, Promega 1 m a Turner 20e lummometer. Aplysia Cell Culture and Electrophysiology Aplysia sensory neurons from the pleural ganglia of adult animals (80-100 g) were cocultured with the motor neuron L7 from juvenile animals (0.5-4 g) . After 4-5 days in culture, the strength of the synaptic connections between the sensory and motor cell was measured electrophysiologically, as previously described (Montarolo et al . 1986, , Albermi et al . , 1994) . The motor neuron was impaled with a glass microelectrode filled with 2.5M KCl (10 MΩ (resistance) and its membrane potential was held at 30 mV below its resting value. The EPSP was evoked by extracellular stimulation of the sensory neuron and the data were stored on a 4 channel tape recorder. Induction of Facilitation and Antisera Injection Two protocols were used to induce synaptic facilitation in Aplysia cocultures. In the first, after testing the initial EPSP amplitude, 10 μM 5-HT was applied for 5 mm (single pulse) . The EPSP was retested 1 min (short-term facilitation) , 2 or 24 hr (long-term facilitation) after the washout of the 5-HT. The amount of facilitation was calculated as the percentage change in EPSP amplitude recorded before and at the different time points after the s gle 5-HT application. In the otner group of experiments, long-term facilitation was evoked by five exposures to 10 μM 5-HT for 5 mm each, at 20 mm intervals (5 pulses) The facilitation was calculated as the percentage cnange in EPSP amplitude 24 hr after the five pulses of 5-HT. The antisera, adjusted to the osmolaπty of Aplysia neurons et al. , 1994) , were pressure injected into the sensory neurons 1 hr before 5-HT treatment. Where indicated, anisomycm (10 μM) or actmomycm D (50 μg/ml) was added to the cocultures 1 hr before the 5-HT pulse and was present continuously during the 5-HT treatment. All data are presented as mean percentage change + SEM in the EPSP amplitude measured after treatment, as compared with its initial pretreatment amplitude. A one way analysis of variance and Newman Keuls multiple range test were used to determine the significance of the EPSP changes . Dye Injection, Cell Imaging and Quantification of Structural Changes Individual sensory neuron were cocultivated with a single motor cell . Glass micropipettes were filled with a 6% sterile-filtered solution of the fluorescent dye 5 (6) -carboxyfluorescem (chromatographically purified; Molecular Probes) m 0.44 M KOH (pH 7.0, resistance 50-90 M. The dye was injected into the sensory neurons immediately after measuring the initial EPSP amplitude by 0.4-0.6 nA hyperpolarizing current pulses (500 ms duration at 1 Hz) for 4-6 mm, phase contrast and fluorescence images of the same view areas were taken both before and 24 hr after treatment as previously described (Glanzman et al . , 1990; Bailey et al. , 1992) . Varicosities were identified according to criteria previously established for sensory neurons m vivo (Bailey et al., 1979; Bailey and Chen, 1983, 1988) , and in vi tro (Glanzman et al . , 1990;; Bailey et al . , 1992) and included all slightly elongated spheres of approximately 2-3 μm or more in diameter. Sequences: Accession Number 1123036 Definition: Aplysia californica bZIP transcription factor (ApCREB2) mRNA, complete eds. GenBank Name: ACU40851, Accession: U40851 NCB Seq ID: 1123036 Citation D Bartsch, M Ghirardi , P Skehel, K Karl, S Herder, M Chen, C Bailey & E Kandel (1995) . Aplysia CREB2 represses long-term facilitation: relief of repression converts transient facilitation into long-term functional and structural change. Cell 83, 979-992. MEDLINE identifier: 96107336 Coding region function: bZIP transcription 1123036: 190..1326 factor; repressor. Sequence 1336 nt, linear ma (SEQ ID NO 18) 1 acgctacaga acggtcaaga aatataatgt gtgcaaagat gtctgcttag 51 gaagacacgg cgacgtccgc ccccaagggc aatcggcaca atggcaacct 101 ttcatgatgt attcctagct acggctatct cgcttctcta ttggacggat 151 ttatttatca catagaagac tcgtatacca aactctacga tggagctgga 201 cctttggagc gaagattttc aactggccag ggaatggggg ctggaaatgc 251 cagtcgtcca gaccgatggc cagttcggtg acctcaaatc aaccagtcgt 301 catggtggcg acgaatctct aagtttgcag ccccagggcg ctacactgaa 351 gttggaaccc tttgaggaag atgtccttgg tgcagagtgg atggagtcgt 401 ccgatctcgg ctcttttctg gatgctttgg gtgacaacca tgagcggctg 451 catccgttcg agtcaaactt gctcgagttc acttctctga tcactcctga 501 tgattcgacg gtgtcaaagg acattctcag ctcaactctt cagtttccaa 551 ctcaaccagt gaacatccct ttatatgcaa gtcatggggc cgaagatttc 601 tctgcagaga ctgagtttga gaaccacctg tcgcctccag attctccgga 651 gcaggtagcc cctgtcataa atctagaacc agttgaactc actgcgagcc 701 atatgacggt gatctcacct gatggcttgt tgggtggcat ggaactσgct 751 tcagaaagct taacatttac cgaactagac tttgtgaact tcaatgacag 801 tgctgttggt tcaattggcg gtgctgaaga acttcttggc tccccactgt 851 cagttgatga tgtggaaagt acaatatcat tttcaggtcc atcgtcgcca 901 gaaaccagcc agagcagcat cattgaatca agtcctgaat tgtacaaagt 951 tatctctacc tcgtccattg atgcatctaa gcgtttctct ccatactctc 1001 gttcctccaa gtccaagcaa tctgtcaaga cttcagacgc taaggcacct 1051 cgtaaaacga ggacaccggc gcagcctgtg ccagaacatg tcatcatgga 1101 acatttggac aaaaaggaca gaaagaagct tcagaacaag aatgctgcca 1151 ttaggtatag gatgaagaag aagggggagg ctcagggcat caaaggggag 1201 gaacaggaat tagaagaact caacacaaag cttaagacta aggtcgatga 1251 cttgcaaaga gaaatcaagt acatgaaaaa tttaatggaa gatgtttgca 1301 aggcgaaagg tattcagctt aaatagtggg aagggt Accession Number 1123037 Definition ApCREB2 Protein Name: ApCREB2 1123037: [ Whole ] NCBI Seq ID: 1123037 Citation D Bartsch, M Ghirardi, P Skehel, K Karl, S Herder, M Chen, C Bailey & E Kandel (1995) . Aplysia CREB2 represses long-term Facilitation: relief of repression converts transient facilitation into long-term functional and structural change. Cell 83, 979-992. MEDLINE identifier: 96107336 Citation Data Submission: D. Bartsch (1995) . Coding region function: bZIP transcription 1123036: 190..1326 factor; repressor. Sequence 378 aa (SEQ ID NO 1) 1 meldlwsedf qlarewglem pwqtdgqfg dlkstsrhgg desislqpqg 51 atlklepfee dvlgaewmes sdlgsfldal gdnherlhpf es lleftsl 101 itpddstvsk dilsstlqfp tqpvniplya shgaedfsae tefenhlspp 151 dspeqvapvi nlepveltas hmtvispdgl lggmelases Itfteldfvn 201 fndsavgsig gaeellgspl svddvestis fsgpsspets qssiiesspe 251 lykvistssi daskrfspys rsskskqsvk tsdakaprkt rtpaqpvpeh 301 vimehldkkd rkklqnknaa iryrmkkkge aqgikgeeqe leelntklkt 351 kvddlqreik ymknlmedvc kakgiqlk Example 3 : CREB2 as a negative regulator of neuronal growth, neuronal differentiation and neuronal re- differentiation. The blockage of activity of ApCREB2 by antibody paired with a pulse of 5-HT induces long-term facilitation which is accompanied by growth of new synaptic connections. One view of this process is that 5-HT may be functioning as a growth factor and thus may be capable of the induction of synaptic growth. Since neuronal growth involves the migration of growth cones and the establishment of neuronal connections, the induced synaptic growth may be considered a form of neuronal differentiation as known to be present in the early stages of neuronal development. This process may be considered re-differentiation since it is possible that this growth factor-like activity may occur subsequent to the initial development following a trauma situation. This re- differentiation would rebuild the synaptic connections in order to facilitate _the formation of long-term memory. Therefore, CREB2 and its mammalian homologue, ATF4 , would be a negative regulator in this process. The above may be tested in a cellular model of neuronal differentiation, specifically the rat pheochromocytoma cells PC12. The cell line is widely used as a model for neuronal differentiation and also for apoptosis, which is programmed neuronal cell death. PC12 cells are undifferentiated in culture and can be differentiated in vitro by exposing them to nerve growth factor (NGF) and cAMP analogs. Withdrawal of these compounds from the cell culture conditions leads to programmed cell death. It has been investigated whether ATF4 plays a role in the neuronal differentiation of PC12 cells. It has been demonstrated that in the course of PC12 cellular differentiation, the amount of ATF4 is dramatically reduced (likely by reducing its transcription) in cells exposed to NGF or cAMP analogs. In addition, overexpression of ATF4 in PC12 cells by transfecting expression constructs with constitutive CMV promoter and ATF4 cDNA prevents the PC12 cells from undergoing neuronal differentiation. Taken together, these data support the proposal that the ATF4 gene is a regulator (negative regulator) of neuronal differentiation and that a long term memory acquisition may be a particular form of neuronal differentiation. It is important to also study the role of ATF4 in apoptosis. Since both differentiation and cell death in PC12 cell culture involve similar signal transduction pathways, it is reasonable that ATF4 may be involved in this process as well. Thus, compounds which are capable of interfering with the binding of a protein or a DNA with a cAMP-response-element- bιndιng-proteιn-2 may be useful memory improvement and also m neuronal regeneration after injury and potentially cell death in several human diseases including stroke and Aizneimer's disease. If ATF4/CREB2 is a negative regulator in neuronal differentiation, compounds or pnarmaceutical compositions which target the action of ATF4/CREE2 could improve neuronal regeneration after injury, either alone or in combination with growth factors like NGF. Furthermore, interfering with ATF4 function could slow down damage induced by stroke and maybe some progressive neuronal diseases with extensive cell death, like Parkinson's diseae or Alzheimer's disease. REFERENCES Alberini, C, Ghirardi, M., Metz, R. , and Kandel, E. R. (1994) . C/EBP is an immediate-early gene required fcr the consolidation of long-term facilitation in Aplysia . Cell 76, 1099-1114. Ausubel, F.M. , Brent, R. , Kingston, R.E., Moore, D.D. , Seidman, J.G. , Smith, J.A and Struhl, K. (1994) . Current Protocols m Molecular Biology (New York, John Wiley and Sons) . Bacskai, B. J., Hochner, B., Mahaut-Smith, M. , Adams, £. R., Kaang, B.-K., Kandel, E. R., and Tsien, R. Y. (1993) Spatially resolved dynamics of cAMP and protein Kinase A subunits in Aplysia sensory neurons. Science 260, 222-226. Bailey, C. H. , and Chen, M. (1983) . Morphological basis of long-term habituation and sensitization in Aplysia . Science 220, Sl-93. Bailey, CH. and Chen, M. (1988) . Long-term memory in Aplysia modulates the total number of varicosities of smge identified sensory neurons. Proc. Na tl . Acad . Sci . USA 85, 2373-2377. Bailey, C. H. and Chen, M. (1989) . Time course of structural changes at identified sensory neuron synapses during long-term sensitization m Aplysia . J. Neurosci . 9, 1774-1780. Bailey, C. H. , Montarolo, P., Chen, M., Kandel, E. R. , and Schacher, S. (1992) . Inhibitors of protein and RNA synthesis block structural changes that accompany long-term neterosynaptic plasticity m Apiysia . Neuron 9 , 749-758. Bailey, C. H. , and Kandel, E. R. (1993) . Structural changes accompanying memory storage. Annu. Rev. Physioi . 55, 397-426. Bailey, C. H. , Thompson, E. B., Castellucci, V. F. , and Kandel, E. R. (1979) . Ultrastructure of the synapses of sensory neurons that mediate the gill-withdrawal reflex m Aplysia . J. Neurocytol . 8, 415-444. Bernier, L., Castellucci, V. F., Kandel, E. R., and Schwartz, J. H. (1982) . Facilitatory transmitter causes a selective and prolonged increase adenosme 3 ' : 5' -monophosphate in sensory neurons mediating the g ll and siphon withdrawal reflex in Aplysia. J. Neurosci . 2 , 1682-1691. Bourtchuladze, R., Frenguelli, B., Blendy, J. , Cioffi, D. , Schutz, G., and Silva, A. J. (1994) . Deficient long-term memory in mice with a targeted mutation of the cAMP responsive element-binding protein. Cell 79, 59-68. Brown, E., and Deffenbacher, K. (1995) . Forgotten mnemonists. J. Hist . Behav. Sci . 11, 342-349. Brown, R. , and Kulik, J. (1977) . Flashbulb memories. Cognition 5, 73-99. Brunelli, M., Castellucci, V., and Kandel, E. R. (1976) . Synaptic facilitation and behavioral sensitization in Aplysia : Possible role of serotonin and cyclic AMP. Science 194, 1178-1181. Carew, T. J. , Castellucci, V. P. , and Kandel, E. R. (1971) . An analysis of dishabituation and sensitization of the gill-withdrawal reflex in Aplysia . Int . J. Neuroεci . 2, 79-98. Carew, T. J., Pinsker, H. M., and Kandel, Ξ. R. :i972) . Long-term habituation of a defensive withdrawal reflex Aplysia . Science 175, 451-454. Castellucci, V. F. , Blumenfeld, H., Goelet, P., and Kandel, E. R. (1989) . Inhibitor of protein synthesis blocks long-term behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia . J. Neurobiol . 20, 1-9. Chevray, P. M. , and Nathans, D. (1992) . Protein interaction cloning in yeast: Identification of mammalian proteins that react with the leucine zipper of Jun. Proc . Natl . Acad. Sci . USA 89, 5789-5793. Chien, C. T., Bartel, P. L. , Sternglanz, and Fields, S. (1991) . The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl . Acad . Sci . 88, 9578-9582. Conway, M. (1995) . Flashbulb Memories (Hillsdale, N.J., Erlbaum) . Cowell, I. G., Skinner, A., and Hurst, H. C. (1992) . Transcriptional repression by a novel member of the bZIP family of transcription factors. Λol . Cell Biol . 12, 3070-3077. Dash, P. K. , Hochner, B., and Kandel, E.R. (1990) . Injection of cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation. Na ture 345, 718-721. Davis, H. P., and Squire, L. R. (1984) . Protein synthesis and memory: A review. Psychol . Bull . 96. 518-559. Durfee, T., Becherer, K. , Chen, P.-L., Yeh, S.-H., Yang, Y., Kilburn, A. E., Lee, W.-H., and Elledge, S. J. (1993) . The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555-569. Ellis, M.J., Lindon, A.C., Flint, K.J., Jones, Ν.C. , and Goodbourn, S. (1995) Activating transcription factor-1 is a specific antagonist of the cyclic adenosine 3 * .5 * - onophosphate (cAMP) response element-binding protein-1-mediated response to cAMP. Mol Endocrinol 9:255-265. Fields, S., and Song, 0. K. (1989) . A novel genetic system to detect protein-protein interactions. Nature 340, 245-246. Flexner, J. B., Flexner, L. B., and Stellar, E. (1963) . Memory in mice as affected by intracerebral puromycin. Science 141 , 57-59. Foulkes, Ν., and Sassone-Corsi, P. (1992) . More is better; Activators and repressors from the same gene. Cell 68 , 411-414. Frangioni, J. V., and Νeel, B. G. (1993) . Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Analytical Biochemistry 210, 179-187. Ghirardi, M. , Braha, 0., Hochner, B. , Montarolo, P G. , Kandel, E. R. , and Dale, N. (1992) . Roles of PKA and PKC facilitation of evoked and spontaneous transmitter release at depressed and nondepressed synapses m Aplysia sensory neurons. Neuron 9, 479-489. Ghirardi, M. , Montarolo, P. G. , and Kandel, E. R. (1995) . A novel intermediate stage m the transition between short- and long-term facilitation m the sensory-to-motor neuron synapse of Aplysia . Neuron 14, 413-420. Glanz an, D. L. , Kandel, E. R. , and Schacher, S. (1990) . Target-dependent structural changes accompanying long-term synaptic facilitation m Aplysia neurons. Science 249, 799-802. Guan, K.-L., and Dixon, J. E. (1991) . Eukaryotic proteins expressed m Escherichia coli : An improved thrombm cleavage and purification procedure of fusion proteins with glutathione S-transferase . Analy . Biochem . 192, 262-267. Hai, T. W., Liu, F., Coukos, W. J., and Green, M. R. (1989) . Transcription factor ATF cDΝA clones: An extensive family of leucine zipper proteins able to selectively form DΝA binding heterodimers . Genes Dev. 3 , 2083-2090. Harlow, E., and Lane, D. (eds.) (1988) . Antibodies. (Cold Spring Harbor, Ν.Y. , Cold Spring Harbor Press) . Johnson, A. D. (1995) . The price of repression. Cell 81, 655-658. Junglmg, S., Cibelli, G., Czardybon, M., Gerdes, H. H., and Thiel, G. (1994) . Differential regulation of chromogranm B and synapsm I gene promoter activity by cAMP and cAMP-dependent protein kinase. Eur . J. Biocne . 226, 925-935. Kaang, B.-K., Kandel, E. R. , and Grant, S. G. N. (1993) . Activation of cAMP-responsive genes by stimuli that produce long-term facilitation in Aplysia sensory neurons. Neuron 10, 427-435. Karp ski, B. A., Morle, G. D., Huggenvik, J., Uhler, M. D., and Leiden, J. M. (1992) . Molecular cloning of human CREB-2: An ATF/CREB transcription factor that can negatively regulate transcription from the cAMP response element. Proc . Na tl . Acad . Sci . USA 89, 4820-4824. Landschulz, W. H., Johnson, P. F. , and McKnight, S. L. (1988) . The leucine zipper: A hypothetical structure common to a new class of DΝA binding proteins. Science 240, 1759-1764. Lemaigre, F. P., Ace. C. I., and Green, M. R. (1993) . The cAMP response element binding protein, CREB, is a potent inhibitor of diverse transcriptional activators. Nucleic Acids Res . 21, 2907-2911. Luria, A. R. (1968) . The Mind of a Mnemonist (London, Basic Books) . Ma, J., and Ptashne, M. (1987) . A new class of yeast transcriptional activators. Cell 51 , 113-119. Mamatis, T., Sambrook, Z., and Fritsch, E. F. (1989; . Molecular Cloning. (Cold Spring Harbor, N.Y., Cold Spring Harbor Press) . McGaugh, J. , Introini-Collison, II, Cahill, L., Castelianc, C , Dalmaz, C, Parent, M. , and Williams, C. '1993 1 . Neuromodulatory systems and memory storage: Role of tne amygdala. Behav. Brain Res . 58, 81-90. Mielnicki, L. M., and Pruitt, S. C. (1991) . Isolation and nucleotide sequence of a murine cDNA homologous to human activating transcription factor 4. Nuclei c Acids Res . 19, 6332. Molina, C. A., Foulkes, N. S., Lalli, E. , and Sassone-Corsi, P. (1993) . Inducibility and negative autoregulation of CREM: An alternative promoter directs the expression of ICER, an early response repressor. Cell 75, 875-886. Montarolo, P. G. , Goelet, P., Castellucci, V. F., Morgan, J., Kandel, E. R., and Schacher, Ξ. (1986) . A critical period for macromolecular synthesis m long-term heterosynaptic facilitation m Aplysia . Science 234, 1249-1254. Neisser, V. (1982) . Snapshots or benchmarks? In Memory Observed: Remembering in Natural Contexts, V. Neisser, ed. (San Francisco, Freeman) , pp. 43-48. Norby, P. L. , Pallisgaard, N., Pedersen, F. S., and Jorgensen, P. (1992) . Determination of recognition-sequences for DNA-binding proteins by a polymerase chain reaction assisted binding site selection method (BSS) using nitrocellulose immobilized DNA binding protein. Nucleic Acids Res . 20, 6317-6321. Petersen, R., Smith, G., Kokmen, E., IvniK, R. , and Tangalos, E. (1992) . Memory function normal ag g. Neurology 42, 396-401. Petπ , F., Giles, R. H., Dauwerse, H. G., Saris, J J., Hennekam, R. C. M., Masuno, M. , Tommerup, N. , Van Ommen, G. J. B., Goodman, R. H., Peters, D. J. M. , and Bruening, M. H. (1995) . Rubinstein-Taybi syndrome caused by mutations m the transcriptional co-activator CBP. Nature 376, 348-351 PmsKer, H., Kupfermann, I., Castellucci, V., and Kandel, E R. (1970) . Habituation and dishabituation of the gill-withdrawal reflex in Aplysia. Science 167, 1740-1742. Pinsker, H. M., Hening, W. A., Carew, T. J., and Kandel, E. R. (1973) . Long-term sensitization of a defensive withdrawal reflex in Aplysia . Science 182, 1039-1042. Rayport, S. G., and Schacher, S. (1986) . Synaptic plasticity m vi tro : Cell culture of identified neurons mediating short-term habituation and sensitization. J. Neurosci . 6 , 759-763. Schacher, S., Castellucci, V. F., and Kandel, E. R. (1988) . cAMP evokes long-term facilitation in Aplysia sensory neurons that requires new protein synthesis Science 240, 1667-1669. Scholz, K. P., and Byrne, J. H. (1988) . Intracellular in j ection of cyclic AMP induces a long-term reduction of neuronal potassium currents. Sci ence 240, 1664-1667. Skehel, P. A. , and Bartsch, D. (1994) . Characterization of a Y-box factor from Aplysia californica. Gene 145, 231-235. Tsu i oto, A., Nyunoya, H., Morita, T. , Sate, T., and Shimotohno, K. (1991) . Isolation of cDNAs for DNA-bmdmg proteins which specifically bind to a tax-responsive enhancer element m the long terminal repeat of human T-cell leukemia virus type I. J. Virol . 65, 1420-1426. Vallejo, M., Ron, D., Miller, C. P., and Habener, J. F. (1993) . C/ATF, a member of the activating transcription factor family of DNA-binding proteins, dimerizes with CAAT/enhancer-binding proteins and directs their binding to cAMP response elements. Proc . Na tl . Acad . Sci . USA 90, 4679-4683. Vinson, C. R. , Hai, T., and Boyd, S. M. (1993) . Dimeπzation specificity of the leucine zipper-contaming bZIP motif on DNA binding; Prediction and rational design. Genes Dev. 7, 1047-1058. Vinson, C. R. , Sigler, B., and McKnight, S. L. (1989) . Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science 246, 911-916. Yin, J. C. P. , Del Vecchio, M. , Zhou, H. , and Tully, T. (1995) . CREB as a memory modulator: Induced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila. Cell 81, 107-115. Yin, J. C. P., Wallach, J. S., Del Vecchio, M., Wilder, E. L. , Zhou, H., Quinn, W. G. , and Tully, T. (1994) . Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila. Cell 79, 49-58. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Bartsch, Dusan Kandel, Eric R. Ghirardi, Mirella (ii) TITLE OF INVENTION:A METHOD FOR ENHANCING LONG-TERM MEMORY IN A SUBJECT AND USES THEREOF (iii) NUMBER OF SEQUENCES: 20 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Cooper & Dunham LLP (B) STREET: 1185 Avenue of the Americas (C) CITY: New York (D) STATE: New York (E) COUNTRY: U.S.A. (F) ZIP: 10036 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: White, John P. (B) REGISTRATION NUMBER: 28,678 (C) REFERENCE/DOCKET NUMBER: 50865/JPW/JML (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 212-278-0400 (B) TELEFAX: 212-391-0525 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 379 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Met Glu Leu Asp Leu Trp Ser Glu Asp Phe Gin Leu Ala Arg Glu Trp 1 5 10 15 Gly Leu Glu Met Pro Val Val Gin Thr Asp Gly Gin Phe Gly Asp Leu 20 25 30 Lys Ser Thr Ser Arg His Gly Gly Asp Glu Ser Leu Ser Leu Gin Pro 35 40 45 Gin Gly Ala Thr Leu Lys Leu Glu Pro Phe Glu Glu Asp Val Leu Gly 50 55 60 Ala Glu Trp Met Glu Ser Ser Asp Leu Gly Ser Phe Leu Asp Ala Leu 65 70 75 80 Gly Asp Asn His Glu Arg Leu His Pro Phe Glu Ser Asn Leu Leu Glu 85 90 95 Phe Thr Ser Leu Ile Thr Pro Asp Asp Ser Thr Val Ser Lys Asp Ile 100 105 110 Leu Ser Ser Thr Leu Gin Phe Pro Thr Gin Pro Val Asn Ile Pro Leu 115 120 125 Tyr Ala Ser His Gly Ala Glu Asp Phe Ser Ala Glu Thr Glu Phe Glu 130 135 140 Asn His Leu Ser Pro Pro Asp Ser Pro Glu Gin Val Ala Pro Val Ile 145 150 155 160 Asn Leu Glu Pro Val Glu Leu Thr Ala Ser His Met Thr Val Ile Ser 165 170 175 Pro Asp Gly Leu Leu Gly Gly Met Glu Leu Ala Ser Glu Ser Leu Thr 180 185 190 Phe Thr Glu Leu Asp Phe Val Asn Phe Asn Asp Ser Ala Val Gly Ser 195 200 205 Ile Gly Gly Ala Glu Glu Leu Leu Gly Ser Pro Leu Ser Val Asp Asp 210 215 220 Val Glu Ser Thr Ile Ser Phe Ser Gly Pro Ser Ser Pro Glu Thr Ser 225 230 235 240 Gin Ser Ser Ile Ile Glu Ser Ser Pro Glu Leu Tyr Lys Val Ile Ser 245 250 255 Thr Ser Ser Ile Asp Ala Ser Lys Arg Phe Ser Pro Tyr Ser Arg Ser 260 265 270 Ser Lys Ser Lys Gin Ser Val Lys Thr Ser Asp Ala Lys Ala Pro Arg 275 280 285 Lys Thr Arg Thr Pro Ala Gin Pro Val Pro Glu His Val Ile Met Glu 290 295 300 His Leu Asp Lys Lys Asp Arg Lys Lys Leu Gin Asn Lys Asn Ala Ala 305 310 315 320 Ile Arg Tyr Arg Met Lys Lys Lys Gly Glu Ala Gin Gly Ile Lys Gly 325 330 335 Glu Glu Gin Glu Leu Glu Glu Leu Asn Thr Lys Leu Lys Thr Lys Val 340 345 350 Asp Asp Leu Gin Arg Glu Ile Lys Tyr Met Lys Asn Leu Met Glu Asp 355 360 365 Val Cys Lys Ala Lys Gly Ile Gin Leu Lys Met 370 375 (2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 73 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : Leu Asp Lys Lys Asp Arg Lys Lys Leu Gin Asn Lys Asn Ala Ala Ile 1 5 10 15 Arg Tyr Arg Met Lys Lys Lys Gly Glu Ala Gin Gly Ile Lys Gly Glu 20 25 30 Glu Gin Glu Leu Glu Glu Leu Asn Thr Lys Leu Lys Thr Lys Val Asp 35 40 45 Asp Leu Gin Arg Glu Ile Lys Tyr Met Lys Asn Leu Met Glu Asp Val 50 55 60 Cyε Lys Ala Lys Gly Ile Gin Leu Lys 65 70 (2) INFORMATION FOR SEQ ID NO:3 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH 8 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY- linear (ii) MOLECULE TYPE: DNA (genomic) (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:3 : TGACGTCA (2) INFORMATION FOR SEQ ID NO:4 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH. 14 base pairs (B) TYPE: nucleic ac d (C) STRANDEDNESS single (D) TOPOLOGY: linear (n) MOLECULE TYPE: DNA (genomic) (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:4. AGTATTGCGT CATC 14 (2) INFORMATION FOR SEQ ID NO: 5 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ll) MOLECULE TYPE: DNA (genomic) (Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ACTATTGCGC AATC 14 (2) INFORMATION FOR SEQ ID NO:6 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ill MOLECULE TYPE: DNA (genomic) (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:6: TTCCGCTTTC CATAAGTCGA 20 (2) INFORMATION FOR SEQ ID NO: 7 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) MOLECULE TYPE: DNA (genomic) (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:7. ACCTGAAAAT GATATTGTAC 20 (2) INFORMATION FOR SEQ ID NO: 8 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 8 : GATCCGGCGC CTCCTTGGCT GACGTCAGAG AGAGAGA 37 (2) INFORMATION FOR SEQ ID NO:9 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ll) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GATCCGGCTG ACGTCATCAA GCTA 2 4 (2) INFORMATION FOR SEQ ID NO:10: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ll) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GATCCCCTTA CGTCAGAGGC GA 22 (2) INFORMATION FOR SEQ ID NO:11: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GATCCGGCGC GGGGCTGGCG TAGGGCCTGC GTCAGCTGCA 40 (2) INFORMATION FOR SEQ ID NO:12: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION- SEQ ID NO:12 : GAGTGGCATC TACGTCAAGG CTTC 24 (2) INFORMATION FOR SEQ ID NO:13: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GATCCGGCAG TATTGCGTCA TCTCAAGCTA 30 (2) INFORMATION FOR SEQ ID NO:14: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ( l) SEQUENCE DESCRIPTION. SEQ ID NO:14: GATCCGGCTG ACGCAATTCA AGCTA 25 (2) INFORMATION FOR SEQ ID NO:15: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GATCCACAGT TGTGATTTCA CAACCTGACC AGA 33 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GATCCGGCAC TATTGCGCAA TCTCAAGCTA 30 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GATCCATATT AAGGACATGC CG 22 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1336 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: ACGCTACAGA ACGGTCAAGA AATATAATGT GTGCAAAGAT GTCTGCTTAG GAAGACACGG 60 CGACGTCCGC CCCCAAGGGC AATCGGCACA ATGGCAACCT TTCATGATGT ATTCCTAGCT 120 ACGGCTATCT CGCTTCTCTA TTGGACGGAT TTATTTATCA CATAGAAGAC TCGTATACCA 180 AACTCTACGA TGGAGCTGGA CCTTTGGAGC GAAGATTTTC AACTGGCCAG GGAATGGGGG 240 CTGGAAATGC CAGTCGTCCA GACCGATGGC CAGTTCGGTG ACCTCAAATC AACCAGTCGT 300 CATGGTGGCG ACGAATCTCT AAGTTTGCAG CCCCAGGGCG CTACACTGAA GTTGGAACCC 360 TTTGAGGAAG ATGTCCTTGG TGCAGAGTGG ATGGAGTCGT CCGATCTCGG CTCTTTTCTG 420 GATGCTTTGG GTGACAACCA TGAGCGGCTG CATCCGTTCG AGTCAAACTT GCTCGAGTTC 480 ACTTCTCTGA TCACTCCTGA TGATTCGACG GTGTCAAAGG ACATTCTCAG CTCAACTCTT 540 CAGTTTCCAA CTCAACCAGT GAACATCCCT TTATATGCAA GTCATGGGGC CGAAGATTTC 600 TCTGCAGAGA CTGAGTTTGA GAACCACCTG TCGCCTCCAG ATTCTCCGGA GCAGGTAGCC 660 CCTGTCATAA ATCTAGAACC AGTTGAACTC ACTGCGAGCC ATATGACGGT GATCTCACCT 720 GATGGCTTGT TGGGTGGCAT GGAACTGGCT TCAGAAAGCT TAACATTTAC CGAACTAGAC 780 TTTGTGAACT TCAATGACAG TGCTGTTGGT TCAATTGGCG GTGCTGAAGA ACTTCTTGGC 840 TCCCCACTGT CAGTTGATGA TGTGGAAAGT ACAATATCAT TTTCAGGTCC ATCGTCGCCA 900 GAAACCAGCC AGAGCAGCAT CATTGAATCA AGTCCTGAAT TGTACAAAGT TATCTCTACC 960 TCGTCCATTG ATGCATCTAA GCGTTTCTCT CCATACTCTC GTTCCTCCAA GTCCAAGCAA 1020 TCTGTCAAGA CTTCAGACGC TAAGGCACCT CGTAAAACGA GGACACCGGC GCAGCCTGTG 1080 CCAGAACATG TCATCATGGA ACATTTGGAC AAAAAGGACA GAAAGAAGCT TCAGAACAAG 1140 AATGCTGCCA TTAGGTATAG GATGAAGAAG AAGGGGGAGG CTCAGGGCAT CAAAGGGGAG 1200 GAACAGGAAT TAGAAGAACT CAACACAAAG CTTAAGACTA AGGTCGATGA CTTGCAAAGA 1260 GAAATCAAGT ACATGAAAAA TTTAATGGAA GATGTTTGCA AGGCGAAAGG TATTCAGCTT 1320 AAATAGTGGG AAGGGT 1336 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 74 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Leu Asp Lys Lys Leu Lys Lys Met Glu Gin Asn Lys Thr Ala Ala Thr 1 5 10 15 Arg Tyr Arg Gin Lys Lys Arg Ala Glu Gin Glu Ala Leu Thr Gly Glu 20 25 30 Cys Lys Glu Leu Glu Lys Lys Asn Glu Ala Leu Lys Glu Lys Ala Asp 35 40 45 Ser Leu Ala Lys Glu Ile Gin Tyr Leu Lys Asp Leu Ile Glu Glu Val 50 55 60 Arg Lys Ala Arg Gly Lys Lys Arg Val Pro 65 70 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 74 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 0: Leu Asp Lys Lys Leu Lys Lys Met Glu Gin Asn Lys Thr Ala Ala Thr 1 5 10 15 Arg Tyr Arg Gin Lys Lys Arg Ala Glu Gin Glu Ala Leu Thr Gly Glu 20 25 30 Cys Lys Glu Leu Glu Lys Lys Asn Glu Ala Leu Lys Glu Arg Ala Asp 35 40 45 Ser Leu Ala Lys Glu Ile Gin Tyr Leu Lys Asp Leu Ile Glu Glu Val 50 55 60 Arg Lys Ala Arg Gly Lys Lys Arg Val Pro 65 70

Claims: What is claimed is: 1. A method to enhance long-term memory a subject whose cAMP-responsive gene expression is repressed due to binding of a cAMP-response-element-binding-protem-2 to a protein or a DNA associated with cAMP-responsive gene expression, or both, which comprises administering to the subject a compound capable of interfering with such binding in an amount effective to interfere with binding of the protein or the DNA so as to thereby derepress cAMP-responsive gene expression m the subject and enhance the subject's long-term memory. 2. The method of claim 1, wherein the compound is an anti- cAMP-response-element-binding-protein-2 antibody. 3. The method of claim 1, wherein the compound is capable of altering phosphorylation of the cAMP-response- element-binding-protein-2. 4. The method of claim 1, wherein the compound is an organic compound, a peptide, a peptide mimetic, a small molecule, or a nucleic acid. 5. The method of claim 1, wherein the protein associated with cAMP-responsive gene expression comprises a cAMP- response-element-binding-protein-1, a C/EBP protein, an Aplysia ApC/EBP protein, a human C/EBP3 protein, an AF- 1 protein, a c-jun protein, a fla protein, or a c-Fos protein. 6. The method of claim 1, wherein the administration comprises intralesional , intramuscular or intravenous injection; infusion; liposome mediated delivery; viral infection; gene bombardment; topical, nasal, oral, anal, ocular, cerebro-spinal , or otic delivery. 7. A method for evaluating the ability of a compound to interfere with binding of a cAMP-response-ele en - binding-protein-2 to a protein associated with cAMP- responsive gene expression in a cell which comprises: (a) contacting the cell with the compound under suitable cell culture conditions; (b) measuring the amount of unbound protein associated with cAMP-responsive gene expression in the cell; (c) comparing the amount in step (b) with the amount of unbound protein associated with cAMP-responsive gene expression in the absence of the compound, so as to thereby evaluate the ability of the compound to interfere with binding of the cAMP-response-element- binding-protein-2 to the protein. 8. The method of claim 7, wherein the compound is an anti- cAMP-response-element-binding-protein-2 antibody. 9. The method of claim 7, wherein the compound is capable of altering phosphorylation of the cAMP-response- element-binding-protein-2. 10. The method of claim 7, wherein the compound is an organic compound, a peptide, a peptide mimetic, a small molecule, or a nucleic acid. 11. A method for evaluating the ability of a compound to interfere with binding of a cAMP-response-element- binding-protein-2 to a DNA associated with cAMP- responsive gene expression in a cell which comprises: (a) contacting the cell with the compound under suitable cell culture conditions; (b) measuring the amount of unbound DNA associated with cAMP-responsive gene expression in the cell; (c) comparing the amount in step (b) with the amount of unbound DNA associated with cAMP-responsive gene expression in the absence of the compound, so as to thereby evaluate the ability of the compound to interfere with binding of the cAMP-response-element- binding-protein-2 to the DNA. 12. The method of claim 11, wherein the compound is an anti-cAMP-response-element-binding-protein-2 antibody. 13. The method of claim 11, wherein the compound is capable of altering phosphorylation of the cAMP-response- element-binding-protein-2. 14. The method of claim 11, wherein the compound is an organic compound, a peptide, a peptide mimetic, a small molecule, or a nucleic acid. 15. A method for treating a subject with a long-term memory defect due to binding of a cAMP-response-element- binding-protein-2 to a protein or a DNA associated with cAMP-responsive gene expression, or both, which comprises administering to the subject a compound capable of interfering with such binding in an amount effective to interfere with the binding of the protein or the DNA so as to thereby treat the subject's long- term memory defect. 16. The method of claim 15, wherein the long-term memory defect comprises age-related memory loss, Alzheimer's Disease, amnesia, ischemia, shock, head trauma, neuronal injury, neuronal toxicity, neuronal degradation, Parkinson's disease, or senility. 17. The method of claim 15, wherein the compound is an anti-cAMP-response-element-binding-protein-2 antibody. 18. The method of claim 15, wherein the compound is capable of altering phosphorylation of the cAMP-response- element-binding-protein-2. 19. The method of claim 15, wherein the compound is an organic compound, a peptide, a peptide mimetic, a small molecule, or a nucleic acid. 20. The method of claim 15, wherein the protein comprises a cAMP-response-element-binding-protein-1, a C/EBP protein, an Aplysia ApC/EBP protein, a human C/EBPS protein, an AF-1 protein, a c-jun protein, a fla protein, or a c-Fos protein. 21. The method of claim 15, wherein the cAMP-response- element-binding-protein-2 is human CREB2 transcription factor, murine ATF4 transcription factor, or Aplysia ApCREB2 transcription factor. 22. The method of claim 15, wherein the administration comprises intralesional, intramuscular or intravenous injection; infusion; liposome mediated delivery; viral infection; gene bombardment; topical, nasal, oral, anal, ocular, cerebro-spinal, or otic delivery. 23. A recombinant eukaryotic cell comprising a DNA encoding a cAMP-response-element-binding-protein-2 not naturally present in the cell, operatively linked to a promoter capable of directed enhanced expression of the DNA, the DNA and the promoter being stably integrated into the genome of the eukaryotic cell. 24. A transgenic, non-human mammal whose somatic and germ cells contain and express a DNA encoding a cAMP- response-element-binding-protein-2 not naturally occurring in the non-human mammal, operatively linked to a promoter capable of directed enhanced expression of the DNA, the DNA and the promoter being stably integrated into the genome of the non-human mammal. 25. A pharmaceutical composition which comprises an effective amount of a compound capable of interfering with binding of a cAMP-response-element-binding- protein-2 to a protein associated with cAMP-responsive gene expression in a cell and a pharmaceutically acceptable carrier. 26. The pharmaceutical composition of claim 25, wherein the carrier is a diluent, an aerosol, a topical carrier, an aqueous solution, a nonaqueous solution, or a solid carrier . 27. The pharmaceutical composition of claim 25, wherein the carrier comprises an appropriate adjuvant, a herpes virus, a liposome, a microencapsule, a neuronal cell receptor ligand, a neuronal -specific virus, a polymer encapsulated cell, or a retroviral vector.

Inventors: BARTSCH DUSAN, GHIRARDI MIRELLA, KANDEL ERIC R, BARTSCH, DUSAN, GHIRARDI, MIRELLA, KANDEL, ERIC, R.
Applicants: UNIV COLUMBIA, THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF
Application year: 1997
Main classification: A61K39
IPC codes: A61K31, A61K38, A61K39, A61K45, A61K48, A61P25, A61P43, C07K14, C07K16