Biography of an Experiment: Injection of the cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation
Dr. Eric R. Kandel, with Pramod K. Dash and Binyamin Hochner
Originally published in Nature, Volume 345, 21 June 1990, pgs. 718-721
Biography of an Experiment by Sara Berman '10 and Jill Geratowski '11
See the Study
Injection of the cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation
Eric R. Kandel, Pramod K. Dash, and Binyamin Hochner
Reproduced from Nature, Volume 345, 21 June 1990, pgs. 718-721
In both vertebrates and invertebrates, long-term differs from short-term in requiring protein synthesis during training (1,2). Studies of the gill and siphon withdrawal reflex in Aplysia indicate that similar requirements can be demonstrated at the level of sensory and motor neurons which may participate in memory storage.[Click here for a video of Dr. Kandel discussing how he came to study Aplysia]. A single application of serotonin (3), a transmitter that mediates sensitization, to individual sensory and motor cells in dissociated cell cultures lead to enhanced transmitter release from the sensory neurons that is independent of new macromolecular synthesis. Five applications of serotonin caused a long-term enhancement, lasting one or more days, which requires translation and transcription (2,3). Prolonged application or intracellular injection into the sensory neuron of cyclic AMP, a second messenger for the action of serotonin, also produced long-term increases in synaptic strength (4,5), suggesting that some of the gene products important for long-term facilitation are cAMP-inducible. In eukaryotic cells, most cAMP-inducible genes so far studied are activated by cAMP-dependent protein kinase (A kinase), which phosphorylates transcription factors that bind the cAMP-responsive element TGACGTCA. The cAMP-responsive element (CRE) binds a protein dimer of relative molecular mass 43,000 (43K), the CRE-binding protein (CREBP), which has been purified and shown to increase transcription when phosphorylated by the A kinase (6-11). Here we show that extracts of the Aplysia central nervous system and extracts of sensory neurons contain a set of proteins, including one with properties similar to mammalian CREBPs, that specifically bind the mammalian BRE sequence. Microinjection of the CRE sequence into the nucleus of a sensory neuron selectively blocks the serotonin-induced long-term increase in synaptic strength, without affecting short-term facilitation. Taken together, these observations suggest that one or more CREB-like transcription activators are required for long-term facilitation. [Click here for a video of Dr. Kandel describing a general overview of the paper].
We first determined whether the Aplysia central nervous system (CNS) contains a CREBP-like protein. Gel mobility shift assays on extracts of Aplysia CNS using a rat somatostatin CRE probe, gave three specifically retarded bands (Figure 1A). [Mouse-over here for an explanation of Figure 1]. The same number of retarded bands was seen when the CRE sequence from the vasoactive intestinal protein (VIP) gene was used as the probe (data not shown). A similar mobility shift pattern was also observed with extracts from Aplysia sensory neurons. Moreover, using antibody to the mouse CREBP, we have found cross-reactivity with a protein of relative molecular mass 43,000 from Aplysia CNS extract which is highly enriched after partial purification on a CRE affinity column (data, including the Western Blot, not shown).
Binding of Aplysia proteins to somatostatin CRE is sequence-specific. It is effectively inhibited to varying degrees by non-radioactive oligonucleotides containing the CRE sequences from somatostatin, VIP, fos, or enkephalin (Figure 1A). The differential inhibition seen may reflect either differences in the affinity of the protein for slightly different CRE sequences, or the contribution to binding affinity of the distinctive CRE flanking sequences (13, 14). By contrast, nonspecific linear plasmid DNA (PKSM13+) did not compete with binding, and only at a very high concentration (Figure 1A) was there some nonspecific decrease in binding. Two mutant oligonucleotides (TGAAGCCA and CTTAAGTG) with the same flanking sequences as the somatostatin probe also failed to compete with the binding. Moreover, the two AP-1 sequences (TGAGTCA) differing from the CRE sequence in only one nucleotide compete weakly at the concentrations we used (Figure 1B). Similar weak competition has been previously reported (8, 14, 15, 16).
To characterize the DNA-protein interaction at the CRE site, we carried out DNase I footprinting in parallel nuclear extracts derived from Aplysia CNS and HeLa cells. Addition of HeLa nuclear extract gave two specific retarded bands and one non-specific band in the gel shift assay (data not shown). Both the specific retarded bands are protected from DNaseI digestion at and around the CRE sequence as are the three retarded bands of Aplysia CNS extract (Figure 2). [Mouse-over here for an explanation of Figure 2]. Comparison of the two hypersensitive sites shows that DNAse protection by HeLa extract covers more nucleotides than the Aplysia extract. [Click here for a video of Dr. Kandel discussing the ethics of HeLa cell use].
Next we determined whether a protein like the CREBP is involved in long-term facilitation, which is transcription dependent and initiated by cAMP. As the site of plasticity for this long-term increase in transmitter release resides within the sensory neuron (17), we isolated sensory and motor neuron pairs in culture (18), and injected double-stranded somatostatin CRE sequence into the nucleus of the sensory neuron (Figure 3). [Mouse-over here for an explanation of Figure 3]. After injection, we treated the culture dish with five exposures of serotonin and monitored long-term facilitation by examining the connection 24 hours later. If the injected CRE sequence competed for binding of a CREPB-like protein, this protein might be prevented from activating the cAMP-inducible genes, despite the increase in cAMP produced by serotonin. A blockade of long-term facilitation might result (Figure 3B).
We found that cells injected with control (mutant) oligonucleotides showed an increase in synaptic strength one day after serotonin treatment (31% + or - 14% s.e.m., n = 7) which was comparable to that occurring in uninjected cells (38% + or - 13% s.e.m., n = 43) (Figure 4A,B). [Mouse-over here for an explanation of Figure 4]. By contrast, cells injected with somatostatin CRE did not show an increase in the excitatory postsynaptic potential (e.p.s.p.) 24 hours after serotonin treatment and were comparable to cells not exposed to serotonin and significantly different from control cells exposed to serotonin (7% + or - 8.5% s.e.m., n = 19, P < 0.05). Whereas the CRE oligonucleotides blocked long-term facilitation, these oligonucleotides had no effect on short-term facilitation, as tested either immediately after injection (data not shown) or 24 hours later (Figure 4C). Moreover, the effect on long-term facilitation was titratable. Injection of 20-fold less CRE blocked long-term facilitation only partially (n = 4).
To rule out a nonspecific reduction in the long-term facilitation attributable to a general effect on transcription, due to either titration of a general transcription factor(s) or some structural features specific to enhancer sequences, we also injected two other known enhancer sequences: (1) the consensus heat-shock element (HSE); and (2) the NF-kB enhancers (19,20). Neither injection of HSE, whose sequence is conserved from human to yeast, nor injection of NF-kB enhancer, affected the increase in synaptic strength seen 24 hours after serotonin treatment (Figure 4B). Gel shift assay shows that a heat-shock transcription factor is present in sensory neuron extracts and its binding can be induced by heat treatment.
To ensure that CRE injection did not simply produce its blockade by a general inhibition of transcription, we injected the CRE oligonucleotide into the nucleus of the R2 neuron, which is sufficiently large for us to examine the heat-shock response in the same cell. As the volume of R2 is about 100 times greater than that of sensory neurons, we injected a correspondingly greater amount of oligonucleotide. Induction of the family of heat-shock proteins was not affected, suggesting that the CRE oligonucleotide injection does not cause a general inhibition of transcription. (Similarly, injection of digested PKSM13+ plasmid DNA (average size 128 nucleotides), or of the vehicle, failed to block the increase in synaptic strength of sensory neurons.)
[Click here for a video of Dr. Kandel discussing the roles of CRE and CREB in long-term memory]. Our results, based on mobility shift and western blot assays, indicate that the nervous system of Aplysia contains a CREBP-like protein of relative molecular mass 43K. Moreover, the selective blockade of long- but not short-term facilitation by the binding of CREBP, in a sequence-specific manner, implicates CREBP as one component of the molecular machinery for setting up long-term facilitation. Although the sensory neuron-motor neuron system still precludes a direct examination of the mechanism by which binding of CREBP blocks long-term facilitation, titration of CREBP by excess CRE in 3T3 cells blocks induction of cAMP-inducible genes (21). We therefore think it likely that the CRE oligonucleotide works by inhibiting the action of CREBP, either on cAMP-inducible genes or on constitutively expressed genes that encode proteins that turn over rapidly (22). It is also possible that the oligonucleotide works by titrating general transcription factor that binds to CREBP, however we think this is less likely as injection of the HSE oligonucleotide does not block long-term facilitation.
These experiments also provide additional support (4,5) for the importance of the cAMP pathway in initiating long-term facilitation. The data do not exclude, however, the possibility that other second messenger systems also participate in the long-term process. Indeed, we and others have found that CREBP can be phosphorylated by the C kinase, by calcium-calmudulin-dependent protein kinase II, and by casein kinase (unpublished results) as well as by the A kinase. Moreover, CREBP is probably not the only protein factor important for the induction of the long-term process.
Nonetheless, as binding of CREBP seems to be an early step in long-term facilitation, and is a target for both cAMP-dependent and other kinases, purification of CREBP from Aplysia and characterization of the upstream region to which CREBP might bind in genes induced by serotonin should allow further study of the causal steps involved in et induction of the long-term process. Moreover, the approach used here to block gene function by intranuclear oligonucleotide injection might be generally useful in studying gene action in nerve cells. [Click here for a video of Dr. Kandel describing the importance of this paper with regard to CREB].
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Our Interview with Dr. Kandel
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The purpose of this project is to expand upon the contents of a seminal research article such that its scientific methods and results become more accessible to the public. We wished to go beyond the article, gaining insight on the rationale behind the experimentation and on the implications for the research. Because of our shared interest in the the brain and behavior, we chose to explore an article in the field of Neuroscience. From the 1950s through the present day, possibly the most influential Neurobiologist has been Dr. Eric Kandel. His contribution to the study of the molecular mechanisms behind memory storage has elucidated an entire pathway and set the stage for a stream of research investigating the molecular underpinnings of behavior.
After consulting with our advisors, Professors Andrea Morris and Rob Fairman, and subsequently discovering that Dr. Kandel both is an HHMI investigator and has a son who graduated from Haverford, we decided that Dr. Kandel would be an ideal researcher to interview for our Biography of an Experiment. We emailed him and he not only graciously accepted our proposal, but also sent us a copy of his Nobel Lecture and a thorough review detailing his contributions to the field of Neuroscience. The paper that we ultimately chose, with Dr. Kandel's advice and guidance, is a seminal one because it provides some of the first evidence that the cAMP-dependent protein kinase A can phosphorylate a CREB-like protein and in doing so contribute to the process of long-term facilitation. After many months of reading and communicating with Dr. Kandel, we traveled to the New York State Psychiatric Institute for an in-person interview. The outcome of our work is presented here.
We would like to extend our gratitude to a number of people whose time, energy, and support made this project possible:
Jenni Punt, whose inspiring idea to learn more about a seminal research article, digging deeper into the rationale behind and the content of the science, led to the start of the "Biography of an Experiment" series of projects.
David Moore for designing the webpage template and having patience in helping us with all computer-related issues.
Rob Fairman for his support of and investment in the project throughout.
Andrea Morris for her guidance in initiating our process.
Dr. Ruth Guyer for helping us to refine our interview questions and skills.
HHMI Interdisciplinary Science Scholars Progam for funding our research and travels for the interview.
Special thanks to Dr. Eric Kandel for his time, patience, dedication, and interest in us as young scientists.
Sara Berman, class of 2010, is a Biology major with a concentration in Neural and Behavioral Sciences. In the summer of 2008 and 2009, through the HHMI Interdisciplinary Science Scholars Program, Sara worked in the Morris Lab investigating the role of nitric oxide in retinal axon guidance in Xenopus, the African clawed frog. Research from these summers is the basis for her senior thesis research entitled "Nitric Oxide as a Putative Retinal Axon Pathfinding and Target Recognition Cue in Xenopus laevis." Next year, Sara will be traveling to Martinsried, Germany to research transmembrane proteins implicated in axon guidance in the fly, funded by a Fulbright scholarship.
Jill Geratowski, class of 2011, is also a Biology major with a concentration in Neural and Behavioral Sciences, and a Psychology minor. Funded in the summer of 2009 by the HHMI Interdisciplinary Science Scholars Program, she worked in the Fairman Lab studying the folding potential of a peptide synthesized in an effort to gain insight on aggregative mechanisms involving glutamine repeats. Her thesis work will be in the Hoang Lab, studying insect gastrulation in comparison to vertebrate neurulation. After she graduates from Haverford, Jill plans to attend medical school.
On campus, Sara and Jill are both Biology TAs, leading weekly discussion groups for the introductory Biology students, and peer tutors. Sara serves on the Student Study Abroad Advisor Board. Jill served on Haverford's Honor Council from 2008 to 2009, and has been working for the Audio/Visual department since her freshman year.