PUMA-STEM Poster Session

Yesterday, two Slug lab members had their first chance to present their ongoing research projects, premiering their work at the 2022 PUMA-STEM summer research conference:

  • Jaqueline Gutierrez presented work on very long-lasting sensitization in Aplysia
  • Monica Lopez presented work on developing earthworms to study long-term sensitization

It was a great event, and both Jaqueline and Monica did themselves proud–they had really nicely designed posters, presented with confidence, and did a fantastic job fielding question.

Go slug lab!

Sluglab 2022 – Summer Photos

DU school photographer Ryan Pagelow stopped by this week to immortalize the intense work the Sluglab has been conducting this summer (thanks, Ryan!). Here are some cool images featuring Hannah Danha, Christian Gonzalez, Emma Gray, Jaquelin Gutierrez, Zayra Juarez, Monica Lopez, Steven Proutsos, Theresa Wilsterman, Jashui Zarate Tores, and Octavian Calin-Jageman.

Summer of ’22 — The SlugLab Roaring Back to Life

It’s July of 2022 and for the past 2 months the SlugLab has been lurching back into life.

For the first time since 2019, the sluglab welcomed a new cohort of summer research students: a record 10 students!

Start of the summer celebration with Tavi, Theresa, Emma, Jas, Zayra, Monica, and Jaquelin (all seated); Christian, Steven, and Hannah.

We’ve so far been confronting the many problems associated with getting the lab back up and running. All previous SlugLab students had graduated, so training had to start from scratch with everything: tank maintenance, siphon-withdrawal reflex measurement, sensitization training, dissections, RNA isolation, qPCR, and data analysis.

Not only has getting everyone up to speed been a challenge, there have also been many challenges to confront from raising up the lab from dormancy. We had challenges with our RNA isolation protocol, an unhealthy batch of animals, a tank that shut down mysteriously over the weekend (and a tank monitoring system that didn’t sound the alarm!), a new file sharing service imposed by the university (which has been terrible), a simulator set up wrong… it’s really been an uphill fight almost every step of the way.

While the list of challenges has been lengthy, it’s turned out to be a lot of fun overcoming them. Our new and large group of sluglab scientists has brought tremendous enthusiasm and camaraderie, a surprisingly deep level of artistic talent, donuts, and a whole lot of fun to the lab. We’ve been knocked down, but we’ve made funny memes about it, and got back up again.

Nothing but great concentrations in the SlugLab!

Maybe we’re also smiling because as we finally seem to have kick started the engine in the lab. We switched to hand homogenization and RNA yields have been amazing. We fixed the stimulator, got healthy animals, and doubled-down on training how to measure behavior, and viola–behavioral data has been pretty fantastic. With data starting to roll in we were finally able to have a lab meeting to work through how to analyze qPCR data, and students have been adding plate after plate of new data for us all to ponder.

At this point, it’s late July and things are really cooking! We have developed and pre-registered (https://osf.io/wvx6z/) an experiment to examine the transcriptional correlates of a very long lasting memory, and it looks like we might end the summer with all behavioral data and tissue collection complete (or at least close to it!). This is an exciting experiment. It’s very clear that forming new long-term memories changes gene expression. What is less clear, though, is if these transcriptional changes are needed to help create the memory, or if they are needed both to create and maintain the memory. Neuroscientists have generally assumed an important role in maintenance, and some models specifically imagine transcriptional feed-back loops that help perpetuate transcription to help maintain memory expression​1​. But this would be a costly way to store a memory. Maybe instead, memories can become transcriptionally independent–perhaps by re-allocating resources within a neuron rather than permanently increasing them.

Our lab has had some hints that transcription might not persist throughout maintenance, at least not for the form of long-term sensitization we study in Aplysia. First, we’ve found that transcriptional changes after sensitization fade within 5 days, 2 days earlier than the memory lasts​2​. This might mean that transcription isn’t needed for maintenance, but it could also mean that there is a slight lag between gene expression decaying and memory expression decaying (2 days isn’t that much of a gap). A second line of evidence is that we’ve found that re-activating a seemingly forgotten memory requires no new changes in gene expression (at least none we could detect), suggesting an uncoupling between memory expression and transcription​3​. This is all suggesting, but not at all definitive.

Now we are collecting data that might help illuminate what role (if any) transcription plays in maintaining a long-term sensitization memory. To do this, we’ve cranked up our training protocol to 11– we are training each animal for 4 consecutive days rather than 1. Work in the Byrne lab​4​ and other labs has suggested that this extended training protocol produces very long-lasting sensitization, and indeed we’re seeing robust behavioral expression 11 days after training (in our typical 1-day training protocol, behavior was almost always back to normal within 7 days). With this longer-lasting training protocol we can examine if transcription also lasts a long time (more than 5 days) or it it still fades quickly. Specifically, we’ll conduct microarray on samples harvested 1 and 5 days after the end of training, and compare the levels of gene regulation at those two time points. If we see that the widespread transcritptional changes at 1 day are still present at day 5, this would suggest a potential role in memory maintenance. However, if we see a decay in transcription at day 5, it would suggest something else is going on…. perhaps transcriptional changes are offset by compensatory mechanisms? Or perhaps memories can be maintained without an ongoing transcriptional change?

At this point we have no idea how the new study will work out… will transcription persist as long as behavior? Will it fade early? We don’t know, but we’re excited to find out. At this point, it looks like we might end the summer with all behavioral data collected and tissue harvested… so it won’t be too much longer now before we have an answer (hopefully).

It’s been a grueling but fantastic summer.

  1. 1.
    Zhang Y, Smolen P, Baxter DA, Byrne JH. The sensitivity of memory consolidation and reconsolidation to inhibitors of protein synthesis and kinases: Computational analysis. Learn Mem. Published online August 24, 2010:428-439. doi:10.1101/lm.1844010
  2. 2.
    Patel U, Perez L, Farrell S, et al. Transcriptional changes before and after forgetting of a long-term sensitization memory in Aplysia californica. Neurobiology of Learning and Memory. Published online November 2018:474-485. doi:10.1016/j.nlm.2018.09.007
  3. 3.
    Rosiles T, Nguyen M, Duron M, et al. Registered Report: Transcriptional Analysis of Savings Memory Suggests Forgetting is Due to Retrieval Failure. eNeuro. Published online September 14, 2020:ENEURO.0313-19.2020. doi:10.1523/eneuro.0313-19.2020
  4. 4.
    Wainwright ML, Byrne JH, Cleary LJ. Dissociation of Morphological and Physiological Changes Associated With Long-Term Memory in Aplysia. Journal of Neurophysiology. Published online October 2004:2628-2632. doi:10.1152/jn.00335.2004

The Sluglab is back!

Today we received a shipment of Aplysia–the first shipment we’ve had since February of 2020.

It’s been a long, frustrating, and anxiety-ridden time for the animal colony to be empty. It’s not that the lab has been inactive–in fact, we published what I think is our best paper ever just a few months ago​1​ . But it has been a long stretch without being able to provide the our typical level of involvement and excitement for our student researchers in the slug lab.

It feels really good to know that we are getting back on track. In fact, in addition to welcoming new slugs we’ve welcomed 5 new lab members: Lucas Eggers, Cynthia Espino, Daniel Mason, Delaney Mcriley, & Steven Proutsos. They join continuing member Melissa Nguyen to round out the Fall 2021 edition of the Sluglab. Let’s kick some a**! (scientifically)

First batch in a long time: Dr. Bob, Dr. C-J, and new lab member Cynthia Espino, October 2021

Our first project with this batch of animals will be to explore for epigenetic markers accompanying long-term sensitization.

Over the last summer, C-J has worked like crazy on protocols for measuring methylation. We’ve found that it is surprisingly easy to full yourself, to obtain signals due to non-specific binding. What we’ve settled on is a process to check specificity of primer sets exhaustively by using synthetic DNA that we can manually methylate. Using this approach we’re pretty sure a key CPG island in the CREB1 promoter is *not* methylated in either control or trained animals. And our summer results also identified a CPG island in the egr promoter that seems to be default methylated, but with no change after sensitization. Our goal with these new animals is to now survey other methylation sites in the promoters of highly learning-regulated transcripts. Having lab meetings back in person has been fantastic (masks, of course, and DU has a vaccine mandate which has been very well implemented); very excited to see where research involvement takes our latest batch of slug lab members.

  1. 1.
    Rosiles T, Nguyen M, Duron M, et al. Registered Report: Transcriptional Analysis of Savings Memory Suggests Forgetting is Due to Retrieval Failure. eNeuro. Published online September 14, 2020:ENEURO.0313-19.2020. doi:10.1523/eneuro.0313-19.2020

Sluglab 2019 – It will be an unforgettable summer

It’s summer and the slug lab is rocking. We have 8 students working in the lab (!), and a number of really exciting projects.

Here’s the lab photo to start the summer.

From left to right: Kiara Rana, Dr. C-J, Dr.Bob, Tania Rosiles, George Garcia, Annette Garcia, Hannah Gordon, Lorena Juarez, Monica Duron, and Melissa Nguyen

We knew this spring that we had recruited a special group of students in to the lab. So far the work this summer has confirmed our hunch–we’ve already completed two rounds of behavioral testing, students are making progress learning qPCR, and yesterday we had a great start to learning electrophysiology. I’m sure we’ll have our ups and downs, but it seems like we’re poised for a fun and productive summer.

Projects we’ll be working on include: 1) investigating if savings memories are re-formed or re-covered, 2) investigating the role of the peptide transmitter FMRF-amide in forgetting, 3) exploring the role of methylation in memory maintenance, and 4) some exciting pilot testing with a paradigm for sensitization in fruit fly larva, in collaboration with Scott Kreher in biology.

Our work this summer continues to be supported by the NIH (our current R15 expired at the end of May, but looks like it will be renewed starting July 1). Huzzah.

In addition, Dominican has received a generous donation from Joe Moskal to start the Moskal scholars program. Joe is a professor of biomedical engineering at Northwestern, a biotech entrepreneur, a Dominican trustee, and an all-around amazing guy. He generously helped Irina and me develop pilot data for our first grant and provided a sparkling letter of support… so it is no exaggeration to say he has already helped make the slug lab what it is today.

This year, Joe took the next step in his efforts to develop and broaden the biotech pipeline by funding the Moskal scholars program. Over the next five years this program will fund students interested in careers in the life and health sciences to spend a summer engaged in intensive research. The goal is for students to have the space, mentoring, and encouragement to develop their skills and passions in the science, and to launch them forward to great things.

Our first two Moskal scholars are Annette Garcia and Tania Rosiles. Tania will be spending her second summer in the slug lab–she’s already gained tremendous lab skills and helped co-author our recent paper on the long-term transcriptional response to sensitization ​(Patel et al., 2018)​. Annette is new to the lab, but was a star in Dr. C-J’s neurobiology class and has already been making big strides in the lab.

The inaugural Moskal Scholars: Annette Garcia and Tania Rosiles

Neither Irina nor I would be where we are today if we hadn’t been fortunate enough to have amazing summer experiences. For Irina it was a summer working at Loyola Medical School. For me, it was a summer at Carnegie Mellon. In both cases it was generous funding from sponsors that enabled us to forgo our usual summer jobs and spend 3 months in intense and life-altering contemplation and study. We are so excited and proud to pay that forward each summer with a new batch of slug lab recruits, and we’re extremely grateful to Joe Moskal for his generosity and support.

One of our annual summer traditions is having DU photographer Ryan Pagelow come to the lab for a group photo and some science B-roll. As always, he does an amazing job. Here’s this year’s album:

 
Google Photos Refresh Token invalid. Please authenticate from Photonic → Authentication.
Error encountered during authentication:
{
  "error": "invalid_grant",
  "error_description": "Bad Request"
}
See here for documentation.
  1. 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, 474–485. doi: 10.1016/j.nlm.2018.09.007

Updated word search and mirror-tracing tasks for Qualtrics

I finally had some spare time to document and post the mirror tracing and word-search tasks I developed for some replication work my students and I completed ​(Cusack, Vezenkova, Gottschalk, & Calin-Jageman, 2015)​.

Each task is (I think) pretty nifty, and I’ve had lots of emails about them over the past couple of years. I’ve finally posted both code bases to github along with working demos in Qualtrics and some rudimentary instructions. The code itself is not pretty–I was learning javascript and wrote most it during a conference I was attending in Amsterdam. Still, it works, and I’m sure it could come in handy.

The mirror-tracing task is just like it sounds–participants trace an image with their mouse or track pad but the mouse movements are mirrored, making it hard to stay in the line. You can vary task difficulty by changing line thickness. There is an expected weak negative correlation with age. The script can even posts the traced images back to your server, which is cool for making figures showing how groups differ with representative data.

The word-search task is also like it sounds. You can use pre-defined grids, or the script can generate a grid for you. I’ve used it to try priming for power (control vs. power-related words hidden in the grid) and to look at frustration (by having a grid that *doesn’t* have all the target letters…mean, I know).

  1. Cusack, M., Vezenkova, N., Gottschalk, C., & Calin-Jageman, R. J. (2015). Direct and Conceptual Replications of Burgmer & Englich (2012): Power May Have Little to No Effect on Motor Performance. PLOS ONE, e0140806. doi: 10.1371/journal.pone.0140806

Kids, Neurons, and Robots

At the end of February I (Dr. Bob) visited a local elementary school as part of the Oak Park Educational Foundation’s Science Alliance Program.

I was matched up with Sue Tressalt’s Third Grade Class at Irving Elementary. For an activity, I brought along the neuroscience program’s collection of Finch Robots, a set of laptops, and the Cartoon Network simulator I have been developing (Calin-Jageman, 2017, 2018). I introduced kids to the basic rules of neural communication, and they explored Cartoon Network, learning how to make brains to get the Finch Robots to do what they wanted (e.g. avoid light, sing when touched, etc.). It was a great class, and a ton of fun.

I’m proud of Cartoon Network, and the fact that it can make exploring brain circuitry fun. It’s simple enough that the kids were able to dive right in (with some help), yet complex enough that really interesting behaviors and dynamics can be modelled.

As a kid, my most formative experience in science was learning logo, the programming language developed by Seymour Papert and colleagues at MIT. Logo was fun to use, and it made me need/want key programming concepts. I clearly remember sitting in the classroom writing a program to draw my name and being frustrated at having to re-write the commands to make a B at the end of my name when I had already typed them out for the B at the beginning of my name. The teacher came by and introduced me to functions, and I remember being so happy about the idea of a “to b” function, and I immediately grasped that I could write functions for every letter once and then be able to have the turtle type anything I wanted in no time at all.

Years later I read Mindstorms and it remains, to my mind, one of the most important books on pedagogy, teaching, and technology. Papert applied Piaget’s model of children as scientists (he had trained with Piaget). He believed that if you can make a microworld that is fun to explore, children will naturally need, discover, and understand deep concepts embedded in that world. That’s what I was experiencing back in 2nd grade–I desperately needed functions, and so the idea of them stuck with me in a way that they never would in an artificial “hello world” type of programming exercise. Having been a “logo kid” it was amazing to read Mindstorms and recognize Papert’s intentionality behind the experiences I had learning Logo.

Anyways, bringing Cartoon Network to an elementary school for a day gave me a great feeling of carrying on a tiny piece of Papert’s legacy. The insights kids develop in just an hour of playing with neural networks are amazing–the idea of a recurrent loop made immediate sense to them, and that also sets up the idea that both excitation and inhibition are important. And, like in Logo, the kids were excited to explore–to know that their experience was not dependent on getting the ‘right’ answer but on trying, observing, and trying again.

The day was fun and even better I received a whole stack of thank-you cards this week. Reading through them has kept a smile on my face all week. Here’s a sample.

This kid has some great ideas for the future of AI

“I never knew neurons were a thing at all”–the joy of discovery
“Your job seems awesome and you are the best at it”—please put this kid on my next grant review panel.
  1. Calin-Jageman, R. (2017). Cartoon Network: A tool for open-ended exploration of neural circuits. Journal of Undergraduate Neuroscience Education : JUNE : A Publication of FUN, Faculty for Undergraduate Neuroscience, 16(1), A41–A45. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/29371840
  2. Calin-Jageman, R. (2018). Cartoon Network Update: New Features for Exploring of Neural Circuits. Journal of Undergraduate Neuroscience Education : JUNE : A Publication of FUN, Faculty for Undergraduate Neuroscience, 16(3), A195–A196. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/30254530

SlugLab Alum Derek Stek heading to medical school with a full ride scholarship

Graduates of the sluglab have been moving on to amazing careers. So we were excited to get the news that lab alumni and neuroscience major Derek Stek has just been offered a full scholarship to attend medical school at the School of Medicine and Public Health at the University of Wisconsin. Woo hoo!

The slug lab in summer of 2018. Derek is the tall guy on the left-side of the table; this was just before he shipped out to UCLA.

Derek spent the summer of 2017 working in the lab, and also did summer research programs at the University of Colorado (2016) and UCLA (2018). In the sluglab, Derek learned how to do extract RNA (which does *not* start by vortexing the DNA-ase) and conduct qPCR. He helped track the expression of several transcripts regulated after learning, and was a co-author on the lab’s most recent paper (Patel et al., 2018).

Derek was also a star outside of the classroom, playing a big part on the DU varsity basketball team. This year, as he finishes at DU, Derek has been working with children with autism and learning behavioral therapy.

Congrats, Derek!

  1. 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, 474–485. doi:10.1016/j.nlm.2018.09.007

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.