Is forgetting an active process? Some new evidence from Aplysia

The SlugLab has a new preprint from a really cool experiment we conducted this summer (2023). Check it out here ​(Calin-Jageman et al., 2023)​: https://osf.io/preprints/psyarxiv/xgfdk.

The results are a bit equivocal, but that’s the messiness of doing good science (“the data is the data”, as Bob’s PhD advisor was always fond of saying). In addition, we’re proud that this work has so many excellent student co-authors — well done Bryan, Elise, Zayra, Anna, Nelly, Leslie, Zayra, Jash, Elise, Dina and Theresa!

Now to the science. We’ve been studying the transcriptional changes that occur when Aplysia form long-term sensitization memories. One intriguing thing we’ve found, is that some of the transcriptional changes we observe would seem to work against the expression of sensitization ​(Conte et al., 2017)​. Specifically, one of the strongest transcriptional changes that occurs when sea slugs form a new sensitization memory is a strong and long-lasting up-regulation of a transcript encoding FMRFamide (FMRFa), a peptide neurotransmitter ​(Patel et al., 2018; Perez, Patel, Rivota, Calin-Jageman, & Calin-Jageman, 2017)​. This is strange begauce FMRFa is inhibitory and it generally works to depress synapses and specifically undoes the types of synaptic changes that help encode sensitization. Why would this be happening?

We’ve proposed the FMRFa is up-regulated because it is part of an active forgetting process — meaning a specific, biological pathway designed to erode/prune away memories. The idea would be that training produces transcriptional changes that encode sensitization but also produces a slower-developing increase in FMRFa, and that as FMRFa signalling increases it wears away the changes that maintain a sensitization memory, producing memory. Consistent with this hypothesis, we’ve found that FMRFa transcripts are up-regulated for a long time, even after sensitization memory seems completely forgotten.

To test the role of FMRFa signalling in forgetting, we gave animals sensitization training and then manipulated FMRFa signalling: boosting it with direct injections or blocking it with injections of a drug (4-BPB) that prevents arachidonic acide release, a key step in the G-protein-coupled-signaling that FMRFa triggers in Aplysia neurons. After these injections, we tracked forgetting of sensitization, measuring the strength of memory 4, 6, and 13 days after training.

What did we find? Well, inconsistent with our hypothesis we found that direct injection of FMRFa did alter forgetting at all. Bummer — sometimes you’re wrong! Or maybe we just didn’t use a strong enough dose, or the FMRFa didn’t get to the nervous system…. not sure. On the other hand, we found that 4-BPB slowed forgetting — animals in this condition had a stronger senstization memory 6-days after training than controls, and even had detectable levels of sensitization at day 13 (though no longer a clear difference from controls).

So, what does this mean? Well, it seems pretty clear that arachidonic acid plays some type of role in forgetting of sensitization. But FMRFa may not… or maybe it does but our FMRFa condition just wasn’t strong/direct enough. We’re going to repeat the study in reduced preps where we can control the drug application just a bit more strongly (though where we’ll have to rely on a physiological measure of memory strength). Excited to see where this goes.

  1. Calin-Jageman, R., Delgadillo, B. G., Gamino, E., Juarez, Z., Kurkowski, A., Musajeva, N., … Calin-Jageman, I. (2023). Evidence of Active-Forgetting Mechanisms:  Blocking Arachidonic Acid Release May Slow Forgetting of Sensitization in Aplysia. Center for Open Science. doi: 10.31234/osf.io/xgfdk
  2. Conte, C., Herdegen, S., Kamal, S., Patel, J., Patel, U., Perez, L., … Calin-Jageman, I. E. (2017). Transcriptional correlates of memory maintenance following long-term sensitization of Aplysia californica. Cold Spring Harbor Laboratory. doi: 10.1101/lm.045450.117
  3. Patel, U., Perez, L., Farrell, S., Steck, D., Jacob, A., Rosiles, T., … Calin-Jageman, I. E. (2018). Transcriptional changes before and after forgetting of a long-term sensitization memory in Aplysia californica. Elsevier BV. doi: 10.1016/j.nlm.2018.09.007
  4. Perez, L., Patel, U., Rivota, M., Calin-Jageman, I. E., & Calin-Jageman, R. J. (2017). Savings memory is accompanied by transcriptional changes that persist beyond the decay of recall. Cold Spring Harbor Laboratory. doi: 10.1101/lm.046250.117

New Review Article: Transcriptional Mechanisms of Long-Term Sensitization

Psychologists and neuroscientists have long been fascinated by memory: how do we learn and carry with us new skills and memories? One key insight is that lasting memories require both transcriptional change and neural plasticity.

Much of what we know abou tthe links between transcription and memory has been revealed through the study of long-term sensitization in Aplysia.

The sluglab has a a new review paper reviwing what we’ve learned from Aplysia, summarizing the state of the art of how sensitization memories are induced, encoded, and maintained . You can check it out here: https://osf.io/preprints/osf/urxk2

This review was a lot of work — but also a lot of fun to work on. This is a topic we know well — it’s the main thing the sluglab has studied over the past 15 years. But it was still incredible (and overwhelming) to get a chance to sit down and intensely re-read the many amazing papers that have explored this topic. Pulling it all together was tough, but rewarding; we especially appreciated being able to carefully explain the evidence behind the synchronization model of the induction of sensitization memory that has emerged from recent empirical an computational work.

Writing this review re-newed our appreciation for the incredible work of Gary Philips, the lab of Jack Byrne, Eric Kandel, and the many other folks who have studied sensitization in Aplysia. It was a real honor to be asked to write about all this work; we hope we’ve done it justice.

Writing this review was also fun because Theresa Wilsterman (DU class of 2023) worked up some really amazing figures — nice work, Theresa, and congrats on graduating!

Oh – it was also fun to make a preprint of this review using Quarto and RStudio — it was easy to produce a really beautiful document.

What is forgetting? Slug lab provides some new insights!

Today, the SlugLab can share an exciting new paper, with contributions from Tania Rosiles, Melissa Nguyen, Monica Duron, Annette Garcia, George Garcia, Hannah Gordon, and Lorena Juarez ​(Rosiles et al., 2020)​.

Where to even start?

  • Contributions from 7 student co-authors! It’s been such a long haul; we’re proud of each of you for sticking with it and for all your contributions to this paper.
  • This paper is a registered report: We first proposed the idea and the methods, even writing a complete analysis script. This was then sent to peer review (you know, when you can still do something if the reviewers turn up an issue or problem to consider!) and after some back and forth received an ‘in principle’ acceptance. Then we completed the work and the analysis and submitted it for one more round of review focused solely on the interpretation of the data. This approach to publication lets peer reviewers have a more meaningful impact on the project and it also helps combat publication bias. People tend to think of this model for replication research, but in our case we used a registered report because we wanted to establish a fair and valid test between two competing theories and to ensure that the approach and analysis were pre-specified.
  • This paper is exciting! We were able to test two very different theories of forgetting:
    • decay theory, which says that memories are forgotten because they physically degrade
    • retrieval failure, which says that memories don’t degrade at all, but simply become more difficult to retrieve due to interference

We found clear support for the retrieval failure theory of forgetting, something I (Bob) was completely not expecting.

So, what was the study actually about?

Even memories stored via wiring changes in the brain can be forgotten. In fact, the majority of long-term memories are probably forgotten. What does this really mean? Is the information gone, or just inaccessible?

One clue is from savings memory, the fact you can very quickly re-learn seemingly-forgotten information. Savings memory is sometimes taken to mean the original memory trace persists, but it could also be that it had decayed, and the remnants prime re-learning.

We noticed a testable prediction:

  • If forgetting is decay, savings re-encodes the memory and must involve the transcriptional and wiring changes used to store new information.
  • If forgetting is inaccessibility, savings shouldn’t involve transcriptional/wiring changes

To test this prediction, we tracked transcriptional changes associated with memory storage as a memory was first formed, then forgotten, then re-activated. We did this in the sea slug, Aplysia calinfornica as a registered report (with pre-registered design and analyses).

The memory was for a painful shock—this is expressed as an increase in reflexes (day 1, red line way above baseline). Sensitization is forgotten in about a week (day 7, reflexes back to normal), but then a weak shock produces savings (day 8, reflexes jump back up)

What’s happening in the nervous system? Our key figure shows expression of ~100 transcripts that are sharply up- or down-regulated when the memory is new. At forgetting, these are deactivated (all lines dive towards 0). At savings? No re-activation! (lines stay near 0)

Our results show that savings re-activates a forgotten memory without invoking *any* of the transcriptional changes associated with memory formation. This strongly suggests the memory is not rebuilt, but just re-activated—the information must have been there all along?!

Lots of caveats (see paper), but the results seem compelling (though surprising) to us. In particular, we used an archival data set to show we would have observed re-activation of transcription had it occurred. Transcriptional changes with savings are clearly negligible.

  1. Rosiles, T., Nguyen, M., Duron, M., Garcia, A., Garcia, G., Gordon, H., … Calin-Jageman, R. J. (2020). Registered Report: Transcriptional Analysis of Savings Memory Suggests Forgetting is Due to Retrieval Failure. Society for Neuroscience. doi: 10.1523/eneuro.0313-19.2020

The remarkably long-lasting fragments of memory

It was a whirlwind 2018. Irina and I are just now catching our breath and finding some time to update the lab website.

One awesome piece of news we forgot to publicize is that our latest paper came out in the August issue of Neurobiology of Learning and Memory (Patel et al., 2018).   This paper continues our work of tracking the molecular fragments of a memory as it is forgotten.  Specifically, we tracked 11 genes we suspected of being regulated *after* forgetting (Perez, Patel, Rivota, Calin-Jageman, & Calin-Jageman, 2017).  Things didn’t work out quite as well as we had expected: of our 11 candidate genes 4 didn’t show much regulation, meaning that our previous results with these genes were probably over-estimating their importance (curse you, sampling error!).  On the other hand, we replicated the results with the other genes and found that some of them are actually regulated for up to 2 weeks after the memory is induced, long after it seems forgotten.

Here are two key figures.  The first is the memory curve for sensitization in our Aplysia -it shows that after memory induction there is strong sensitization recall that decays within a week back to baseline.  Even though the memory seems gone, giving a reminder 2 weeks after learning rekindles a weak re-expression of the memory. That’s a classic “savings” effect.  

The next figure traces the time-course of memory-induced gene expression (levels of mRNA) for 6 specific genes, measured in the pleural ganglia that contains neurons known to be important for storing sensitization memory.  You can see that each of these transcripts is up- or down-regulated within 24 hours of learning, and that in each case this regulation lasts at least a week and sometimes out to 2 weeks.  So, just as the behavioral level of the memory fades but isn’t really completely gone, the some of the transcriptional events that accompany learning also seem to persist for quite some time. 

Why would this occur?  Perhaps these transcripts are part of savings…maybe they set the stage for re-expressing the memory?  Or maybe they are actually part of forgetting, working to remove the memory?  Or maybe both?  For example, one of the transcripts is encodes an inhibitory transmitter named FMRFamide.  It is really up-regulated by learning, which would normally work against the expression of sensitization memory.  So perhaps this helps suppress the memory (forgetting), but in a way that can be easily overcome with sufficient excitation (savings)… that’s an exciting maybe, and it’s the thing we’ll be working this summer to test.

As usual, we’re so proud that this paper was made possible through exceptional hard work from some outstanding DU student researchers: Ushma Patel, Leticia Perez, Steven Farrell, Derek Steck, Athira Jacob, Tania Rosiles, and Melissa Nguyen.  Go slug squad!

Patel, U., Perez, L., Farrell, S., Steck, D., Jacob, A., Rosiles, T., … Calin-Jageman, I. E. (2018). Transcriptional changes before and after forgetting of a long-term sensitization memory in Aplysia californica. Neurobiology of Learning and Memory, 155, 474–485. doi:10.1016/j.nlm.2018.09.007
Perez, L., Patel, U., Rivota, M., Calin-Jageman, I. E., & Calin-Jageman, R. J. (2017). Savings memory is accompanied by transcriptional changes that persist beyond the decay of recall. Learning & Memory, 25(1), 45–48. doi:10.1101/lm.046250.117

New preprint on the very long-lasting transcriptional response to learning

The sluglab has a new preprint out, currently under review at the Neurobiology of Learning and Memory.  We shows that both transcription and savings can persist for as long as 2 weeks after the induction of long-term sensitization, way beyond the decay of recall.  Interestingly, all the long-lasting transcriptional changes start within 1 day of training.  Lots of student co-authors on this one; it was a *lot* of work.  Looking forward to the reviews.

Memories fade..but something remains

Most long-term memories are ‘forgotten’–meaning that it becomes harder and harder to recall the memory.  Psychologists have long known, though, that forgetting is complex, and that fragments of a memory can remain.  For example, even after a memory seems forgotten it can be easier to re-learn the same material, something called ‘savings memory’.  That suggests that there is at least some fragment of a memory that persists in the brain even after it seems forgotten…but what?

Today our lab has published a paper shedding a bit of light on this long-standing mystery (Perez, Patel, Rivota, Calin-Jageman, & Calin-Jageman, 2017).  We tracked a sensitization memory in our beloved sea slugs.  As expected, memories faded–within a week animals had no recall of the prior sensitization.  Even more exciting, we found similar fragments of memory at the molecular level–there was a small set of genes very strongly regulated by the original training even though recall had fully decayed.

Why?  Do these persistent transcriptional changes help keep a remnant of the memory going?  Or are they actually doing the work of fully erasing the memory?  Or do they serve some other function entirely (or no function at all)?  These are some of the exciting questions we now get to investigate.  But for now, we have these fascinating foothold into exploring what, exactly, forgetting is all about in the brain.

As usual, we are enormously proud of the undergraduate students who helped make this research possible: Leticia Perez, Ushma Patel, and Marissa Rivota. Ushma, who wants to do science illustration, is making an incredible piece of artwork representing these findings.  A draft is shown above.  She submitted it for the cover of the journal, but sadly they journal selected a different image (boo!).  Still, a very exciting and proud day for the slug lab!

Perez, L., Patel, U., Rivota, M., Calin-Jageman, I. E., & Calin-Jageman, R. J. (2017). Savings memory is accompanied by transcriptional changes that persist beyond the decay of recall. Learning & Memory, 25(1), 45–48. doi: 10.1101/lm.046250117

An unforgettable experience talking about forgetting

Wow! Our lab just returned from the 2017 Society for Neuroscience meeting.  It was the typical maelstrom of neuroscience–with more than 20,000 neuroscientists bustling about trying to share the latest and greatest about their research.

This turned out to be an especially great year for the Slug Lab.  Leticia Perez, who has been working in our lab for the past two summers, submitted an abstract to present the work she and others in the lab have been doing on forgetting.  We’ve been really excited about the results of this project.  It turns out the SFN organizers were excited, too–they selected Leticia’s abstract for a 10 minute talk during a mini-symposium on the mechanisms of learning and memory.

Leticia absolutely crushed it–she gave a concise, clear, and exciting presentation on what happens in the Aplysia nervous system as a long-term memory is forgotten.  She handled the questions wonderfully, and was soundly congratulated by many researchers in the learning and memory community.  Of the 20,000+ in attendance, I’m willing to be she was the only undergraduate to give a talk at this year’s meeting.  It was *such* an accomplishment.

In case that wasn’t enough, Leticia also brought along a poster presenting the research.  She gave the poster at the pre-meeting on molecular and cellular neuroscience and at the undergraduate poster session.  Yes, that means she gave 3 presentations last weekend!  Wow!  And, again, all went wonderfully.

Part of the reason Leticia was able to attend the meeting to earn all this acclaim is that she was awarded an Excel scholarship through Dominican University–this paid her registration, hotel, and airfare to make it affordable to attend the meeting.  She still had to work like crazy to collect the data, refine the presentation, and clear her class schedule to attend.  Lab alumnnus Marissa Rivota also attended–so her and Leticia also got to see the capital and the White house.

We’re so proud of Leticia, and of the many other students who have worked so hard in the lab for the past summers to make this forgetting project such a success.  There will be a paper on it coming out very soon in Learning and Memory.  It’s tremendous work to do good science–we’re so happy to have wonderful students who want to get involved and excel.

Below are photos of Leticia giving her talk, giving her poster, and celebrating with me, Irina, and Marissa.  Congrats, Leticia!

Maintaining Memories, Changing Transcription

Under the right circumstances, a memory can last a lifetime.  Yet at the molecular level the brain is constantly in flux: the typical protein has a half-life of only a few hours to days; for mRNA a half-life of 2 days is considered extraordinarily long.   If the important biological molecules in the brain are constantly undergoing decay and renewal, how can memories persist?

The Slug Lab has a bit of new light to shed on this issue today.  We’ve just published the next in our series of studies elucidating the transcriptional changes that accompany long-term memory for sensitization in Aplysia.  In a previous paper, we looked at transcription 1 hour after a memory was induced, a point at which the nervous system is first encoding the memory.  We found that there is rapid up-regulation of about 80 transcripts, many of which function as transcription factors (Herdegen, Holmes, Cyriac, Calin-Jageman, & Calin-Jageman, 2014).

For the latest paper (Conte et al., 2017), we examined changes 1 day after training, a point when the memory is now being maintained (and will last for another 5 days or so).  What we found is pretty amazing.  We found that the transcriptional response during maintenance is very complex, involving up-regulation of >700 transcripts and down-regulation of <400 transcripts.  Given that there are currently 21,000 gene models in the draft of the Aplysia genome, this means more than 5% of all genes are affected (probably more due to the likelihood of some false negatives and the fact that our microarray doesn’t cover the entire Aplysia genome).   That’s a lot of upheaval… what exactly is changing?  It was daunting to make sense of such a long list of transcripts, but we noticed some very clear patterns.  First, there is regulation influencing growth: an overall up-regulation of transcripts related to producing, packaging, and transporting proteins and a down-regulation of transcripts related to catabolism.  Second, we observed lots of changes which could be related to meta-plasticity.  Specifically, we observed down regulation in isoforms of PKA, in some serotonin receptors, and in a phosphodiesterase.  All of these changes might be expected to limit the ability to induce sensitization, which would be consistent with the BCM rule (once synapses are facilitated, raise the threshold for further facilitation).  (Bienenstock, Cooper, & Munro, 1982).

One of the very intriguing findings to come out of this study is that the transcriptional changes occuring during encoding are very distinct from those occuring during maintenance.  We found only about 20 transcripts regulated during both time points.  We think those transcripts might be especially important, as they could play a key regulatory/organizing role that spans from induction through maintenance.  One of these transcripts encoded a peptide transmitter called FMRF-amide.  This is an inhibitory transmitter, which raises the possibility that as the memory is encoded, inhibitory processes are simultaneously working to limit or even erode the expression of the memory (a form of active forgetting).

There are lots of exciting pathways for us to explore from this intriguing data set.  We feel confident heading down these paths because a) we used a reasonable sample size for the microarray, and b) we found incredibly strong convergent validity in an independent set of samples using qPCR.

This is a big day for the Slug Lab, and a wonderful moment of celebration for the many students who helped bring this project to fruition: Catherine Conte (applying to PT schools), Samantha Herdegen (in pharmacy school), Saman Kamal (in medical school), Jency Patel (about to graduate), Ushma Patel (about to graduate), Leticia Perez (about to graduate), and Marissa Rivota (just graduated).  We’re so proud of these students and so fortunate to work with such a talented and fun group.

Bienenstock, E., Cooper, L., & Munro, P. (1982). Theory for the development of neuron selectivity: orientation specificity and binocular interaction in visual cortex. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 2(1), 32–48. [PubMed]
Conte, C., Herdegen, S., Kamal, S., Patel, J., Patel, U., Perez, L., … Calin-Jageman, I. E. (2017). Transcriptional correlates of memory maintenance following long-term sensitization of Aplysia californica. Learning and Memory, 24, 502–515. doi: 10.1101/lm.045450117 [Source]
Herdegen, S., Holmes, G., Cyriac, A., Calin-Jageman, I. E., & Calin-Jageman, R. J. (2014). Characterization of the rapid transcriptional response to long-term sensitization training in Aplysia californica. Neurobiology of Learning and Memory, 116, 27–35. doi: 10.1016/j.nlm.2014.07009

APS Presentations

APS was in Chicago this year, so the replicators I have been supervising were out in full force.

Clinton Sanchez presented his replications of a study claiming that analytic thinking promotes religious disbelief. [cite source=’doi’]10.1126/science.1215647[/cite]. His manuscript is having a rough time, but we’re hoping it will be out soon. Clinton is now in a MA program in Clinical Counseling at DePaul. Data from his project is here: https://osf.io/qc6rh/

Elle Lehmann presented a poster of her replications of a studies showing that red enhances perceived attractiveness of men rating women [cite source=’doi’]10.1037/0022-3514.95.5.1150[/cite] and women rating men [cite source=’doi’]10.1037/a0019689[/cite] . Elle’s paper is in submission–she found little to no effect for either gender. She’s now working on a meta-anlaysis which has become quite a project, but really interesting. She has graduated and will be applying for a Fullbright in the fall. Data from here project is here: https://osf.io/j3fyq/

Last but not least Eileen Moery presented a poster of her replications of a study which claimed that organic food makes you morally judgemental [cite source=’doi’]10.1177/1948550612447114[/cite]. Eileen’s studies were recently published [cite source=’doi’]10.1177/1948550616639649[/cite]. She found little to no effect of organic food exposure on moral judgements. She’s starting an MA program in clinical psych at IIT in the fall!. Data from here project is here: https://osf.io/atkn7/

Photos came out a bit blurry (new phone, but crappy camera!).

Elle and me at her poster.
Elle and me at her poster.

Clinton and Elle at his poster.
Clinton and Elle at his poster.

2014×3 – Transcriptional correlates of long-term habituation

Third paper of the year for the lab (gasp!) is now out in Learning and Memory [cite source=’doi’]10.1101/lm.036970.114[/cite].

The focus of the project is habituation, considered the simplest and most ancient form of memory. Long-term habituation requires changes in gene expression, but to date there is almost nothing known about what specific changes are required to encode and store a long-term habituation memory.

We’re not the first to try to tackle this issue, but it turns out to be a very difficult topic for study. Habituation is typically very site specific, occurring only at the site of training. This implies a relatively discrete set of neurons encode the memory, and that presents a real problem for qPCR and microarray analysis, because the signal from memory-encoding neurons could easily be washed out from signal from non-encoding neurons, glia, etc.

Our strategy was to develop a new, automated protocol for inducing long-term habituation over the entire body of an Aplysia. With the help of a tinker-toy set, a windshield-wiper motor, a relay box, an old computer with a parallel port, and some qBASIC programming (blast from the bast), we developed a slug car wash–an apparatus we could place over the tanks to repeatedly (though gently) brush Aplysia without any need for human intervention during training. We made a video to show off the system, which you can see here.

The slug car wash turns out to work great. We tracked the development of habituation over repeated rounds of training and saw a classic pattern of behavior–robust decreases in behavior at the end of each round of training, substantial overnight recovery (forgetting), but a progressive development of a persistently decreased response within 3 days of training. Importantly, we could observe habituated responding when stimulating the animal at the head, the siphon, or the tail. Moreover, the effect sizes were huge. So it was pretty clear that the slug car wash was producing the high impact we were looking for. In addition, we found that pattern of training really does matter–when training has breaks between sessions and is spaced out over 3 days it is extremely effective; massing all the same stimulation together into a single one-day session (at a slightly higher rate to squeeze it all in) produced neither long-term nor short-term habituation. This is a useful finding because it gave us an additional no-memory control, one which could ensure any molecular correlates identified are specific to memory formation, not just to the activity induced by brushing.

So what’s changing transcriptionally? We decided to focus on the pleural ganglia containing the VC nociceptors. These are relatively high-threshold neurons, and are probably not carrying the bulk of the activity induced by the brush. Unfortunately, though, no one yet knows *where* in the Aplysia nervous system to find the cell bodies of the low-threshold neurons that mediate light touch (probably in the periphery). Not to worry, though–we did record from the VCs in reduced preps and found that they do actually get some activation from the brush: about 1/4 fired APs, and most of the rest got lots of IPSPs from off-center stimulation.

To track transcriptional changes, we used the custom-designed microarray we recently developed in the lab [cite source=’pubmed’]25117657[/cite]. Some quick words about methods: We again used a large-ish sample size (n=8/group; can you believe that n=3/group is still common in microarray!?). We also used very high statistical standards by adopting the ‘treat’ function in limma which allows you to specify a reasonable null hypothesis (e.g. at least 10% regulation in either direction, rather than the standard practice of testing against a null of no regulation). Adopting a more reasonable null enables you to test for statistical and practical significance at the same time, and we’ve found that transcripts which pass such a rigorous test generalize very well to new samples. We’ve been finding R and limma surprisingly easy to use, which is pretty fantastic for free software.

Anyways, back to the data. The microarray results were a bit of a bummer. Out of over 20,000 transcripts tested, only *one* came up as strongly regulated. Bummer. Another 20 transcripts came up as regulated if you use a standard null hypothesis, but, as expected, none of these validated.

Although the microarray results were not what we hoped, we did further explore the one regulated transcript, and it turns out to be quite interesting. From sequence alignment, it seems to be an Aplysia homolog of cornichon, an auxiliary subunit for AMPA receptors. In invertebrates, cornichon seems to limit trafficking to AMPA receptors to the membrane and therefore reduces glugatmate-induced currents[cite source=’pubmed’]24094107[/cite]. Note that this is precisely the type of effect that could produce behavioral habituation. Moreover, one of the few known molecular correlates of long-term habituation is a decrease in surface expression of glutamate receptors [cite source=’pubmed’]14573539[/cite]. Fits perfectly!

To ensure that cornichon is truly regulated in our paradigm, we did some additional follow-ups. First, we used qPCR to check cornichon levels not only in the microarray samples but in an additional, independent set of samples. Sure enough, we confirmed up-regulation of cornichon in the pleural ganglia 1 day after training. In addition, we checked levels in massed animals, who display no memory after training. In this case, cornichon was actually slightly down, and was significantly different than in the regularly trained animals. So, cornichon is quite specifically and consistently up-regulated after long-term habituation training. As far as we know, this is the first specific transcriptional correlate of long-term habituation to be identified.

Needless to say, we’re quite proud of this work. It wouldn’t have been possible without two of the most talented undergrads we’ve had in the lab: Geraldine Holmes and Samantha (Sami) Herdegen. Geraldine was the most diligent slug trainer in the history of the lab. For this paper alone she ran over 48 animals, testing each 8 times a day for 3-5 days–that’s a whole lot of behavior to monitor! Sami, of course, has been the qPCR wizard in the lab, testing lots and lots and lots and lots of transcripts for regulation. It’s no surprise that both are on to bigger and better things, Geraldine is now in a PhD program in Canada and Sami is soon to start pharmacy school. We also had contributions from John Schuon (when he could fight his way in for some qPCR; now off to medical school), Ashly Cyriac (who helped start the project before heading off to pharmacy school), Jamie Lass and Catherine Conte. Congrats!

As has now become the norm for the lab, all the raw data from this study been posted online at the Open Science Framework: https://osf.io/6ew4i/.