Looks like we’ll be funded!

Doing science isn’t always cheap. The sluglab has been very fortunate to have had multiple rounds of funding from the National Institute of Health to support our research. Specifically, we’ve been supported by R15 grants, a grant program designed specifically for undergraduate-focused labs. These grants are much smaller than the R01s obtained at larger universities (an R01 can be for up to $1 million/year for 5 years; an R15 is a total of $300,000 over 3 years), but they provide an opportunity to fund research with undergraduate researchers (one big complaint about this program, though, is the short timeline — R15s are on a much shorter cycle even though it typically takes *more* time to finish projects in an undergrad-focused lab).

The sluglab has had the good fortune to have strong R15 funding from the NIH. We got started in 2009 with an R15 to study long-term habituation. That project went well ​(Holmes et al., 2014)​, but also showed us that we could get more accomplished studying long-term sensitization. So in 2014 we applied for and received funding to study the formation and forgetting of sensitization memory. This project went so well that we renewed it in 2018.

This year (2024), in February, we applied to renew the project again. Putting together a grant application is *tough* — you have to review your progress from the previous grant and then make a good case that you have additional important, clear research questions along with the ability to answer those questions. Fortunately, our last round of funding was really productive — we mapped the transcriptional bases of savings memory ​(Rosiles et al., 2020)​, found experimental validation for our dual-process model of forgetting ​(Calin-Jageman, Gonzalez Delgadillo, et al., 2024; Perez, Patel, Rivota, Calin-Jageman, & Calin-Jageman, 2017)​, and wrote a new synthesis on the transcriptional mechanisms of sensitization ​(Calin-Jageman, Wilsterman, & Calin-Jageman, 2024)​. We also worked hard to develop some really strong new research projects, proposing to compare forgettable and unforgettable forms of sensitization memory with single-cell resolution and to test our theory of forgetting at the cellular level. With a ton of coffee and work, we developed our renewal application and sent it in.

And then we waited. Submission in February gets your grant reviewed in mid-June. You get your scores within a few days, then written comments within a few weeks, then there is a council meeting several months later at which actual funding decisions are made. It is quite a process.

It’s now July, so we still have a ways to go before council and the official decision… but we are excited to share that our grant was very highly scored — in the top 4% of grants reviewed this cycle. That’s not a guarantee, but we can still feel pretty good about ultimately being funded. So: huzzah — the sluglab will have the funding needed to continue training both sea slugs and undergrads, at least for the next several years to come. Can’t wait to see all we can accomplish on this next grant.

  1. Calin-Jageman, R. J., Gonzalez Delgadillo, B., Gamino, E., Juarez, Z., Kurkowski, A., Musajeva, N., … Calin-Jageman, I. E. (2024). Evidence of Active-Forgetting Mechanisms? Blocking Arachidonic Acid Release May Slow Forgetting of Sensitization inAplysia. Society for Neuroscience. doi: 10.1523/eneuro.0516-23.2024
  2. Calin-Jageman, R. J., Wilsterman, T., & Calin-Jageman, I. E. (2024). Transcriptional Regulation Underlying Long-Term Sensitization in Aplysia. Oxford University Press. doi: 10.1093/acrefore/9780190264086.013.499
  3. Holmes, G., Herdegen, S., Schuon, J., Cyriac, A., Lass, J., Conte, C., … Calin-Jageman, R. J. (2014). Transcriptional analysis of a whole-body form of long-term habituation inAplysia californica. Cold Spring Harbor Laboratory. doi: 10.1101/lm.036970.114
  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
  5. 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

Latest SlugLab paper now out at eNeuro!

The Sluglab’s latest paper is now live at eNeuro! You can find it here: ​(Calin-Jageman et al., 2024)​.

We’ve already blogged about the paper: it tested our hypothesis that we could manipulate forgetting by changing signalling of an inhibitory peptide neurotransmitter called FMRF-amide. Our hypothesis was informed by the fact that we observe a huge and long-lasting increase in FMRF-amide transcription when animals acquire a long-term sensitization memory ​(Conte et al., 2017; Patel et al., 2018)​. Given that FMRF-amide serves to inhibit withdrawal reflexes, we reasoned that it represents an active-forgetting process that could be manipulated.

Our results were equivocal. On the one hand, we found that blocking FMRF-amide did, indeed slow down forgetting. On the other hand, we obtained a very wide confidence interval: we can’t be sure it is a large/replicable effect. Moreover, boosting FMRF-amide did not seem to speed up foregetting, as we predicted. So: a very intriguing finding we’ll need to follow-up on, but not the most clear-cut evidence. It was great that we pre-registered our study and published at a journal that is open to all well-conducted results, so we didn’t have to feel any pressure to “pretty up” these somewhat ambiguous results or to make strong claims from what ended up being somewhat noisy data.

The best thing about this project was the great DU students who made the whole project happen in just one summer. Amazing! Here’s the crew celebrating at SFN this fall. Congrats!

Oh, and one of these great students, Theresa Wilsterman, made this fantastic illustration for the paper (and just got a job at Rush Medical!)

  1. Calin-Jageman, R. J., Gonzalez Delgadillo, B., Gamino, E., Juarez, Z., Kurkowski, A., Musajeva, N., … Calin-Jageman, I. E. (2024). Evidence of Active-Forgetting Mechanisms? Blocking Arachidonic Acid Release May Slow Forgetting of Sensitization inAplysia. Society for Neuroscience. doi: 10.1523/eneuro.0516-23.2024
  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

A new series on “Improving Neuroscience”

I (Dr. Bob) am excited to be organizing a new series of papers in eNeuro on Improving Neuroscience.

eNeuro has been leading the way for rigorous and reproducible research for some time. This new series will provide accessible, authoritative, hands-on tutorials for steps you can take to improve your research. We’ll cover sample-size planning, how to prove a negligible effect, per-registration, Bayesian methods, and much more.

If you have a topic you’d love to see covered or a tutorial you’d like to contribute, let me know!

Here is the editorial I wrote announcing this project ​(Calin-Jageman, 2024)​: https://www.eneuro.org/content/11/3/ENEURO.0048-24.2024. This all grew out of the editorial I wrote in 2019 about improving statistical practices in neuroscience ​(Calin-Jageman & Cumming, 2019)​.

Can’t say much yet, but already have a couple of great authors lined up to contribute tutorials. More soon!

  1. Calin-Jageman, R. J. (2024). NeweNeuroSeries: Improving Your Neuroscience. Society for Neuroscience. doi: 10.1523/eneuro.0048-24.2024
  2. Calin-Jageman, R. J., & Cumming, G. (2019). Estimation for Better Inference in Neuroscience. Society for Neuroscience. doi: 10.1523/eneuro.0205-19.2019

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.

Sluglab at SFN 2023

The Annual Meeting of the Society for Neuroscience is a dizzying conference — it is a multi-day science extravaganza attended by over 30,000 people. The main meeting has a talks on the latest and greatest in neuroscience, an endless series of poster sessions, and a huge exhibitors section where you can check out the newest in techniques. In addition, there are many sattelite meetings and socials. The Faculty for Undergraudate Neuroscience puts on a lot of great events: an undergraduate poster session, a social, and usually a teaching of neuroscience workshop as well.

Although the neuroscience meeting is overhwhelming (it’s a bit like the Total Perspective Machine that Douglas Adams imagined for The Hitchhiker’s Guide), we really missed being able to attend the conference during the covid years.

This year (2023), the sluglab got to return to the Society for Neuroscience conference in style. We presented 2 posters at the Molecular and Celluar Cognition Sattelite Meeting, 2 posters at the undergraduate poster session, and 1 poster on the main foor! All in all, that involved 7 students from the slug lab, all of whom got to travel to D.C. and drink from the science firehose that is SFN.

Here’s Theresa and Elise getting a bright idea while presenting their posters at the Mollecular and Cellular Cognition meeting.

Here is Jash presenting at the undergrad session:

And Anna:

Andd Zayra:

And Leslie:

And Bryan:

So proud of the sluglab; it was a fantastic conference and they kicked a**.

Here is the whole gallery:

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.

Slug Lab – Distinguished Service Awards from the Faculty for Undergraduate Neuroscience

At this year’s Society for Neuroscience meeting, Irina and I were honored for our contributions to the Faculty for Undergraduate Neuroscience (FUN).  Specifically, we were both given the annual Distinguished Service Award.  The honors were bestowed for our work organizing the FUN conference this past summer and for other work supporting the mission of undergraduate neuroscience education.

We’re so fortunate to be a part of FUN–it’s our favorite people all working towards a mission that is so very important.  Thanks for the great honor, and we’re looking forward to staying very involved with FUN.

Here’s a photo of Irina’s award.