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. (10.1126/science.1215647). 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 (10.1037/0022-3514.95.5.1150) and women rating men (10.1037/a0019689) . 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 (10.1177/1948550612447114). Eileen’s studies were recently published (10.1177/1948550616639649). 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.

Bibliography

2014×3 – Transcriptional correlates of long-term habituation

Third paper of the year for the lab (gasp!) is now out in Learning and Memory (10.1101/lm.036970.114).

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 (25117657). 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(24094107). 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 (14573539). 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/.

Bibliography

Sluglab Strikes Again – New paper tracing dynamics of learning-induced changes in transcription

A nice way to wrap up 2014–we have a new paper out (25486125) where we trace learning-induced changes in transcription over time and over different location in the CNS. We think it’s a nice follow-up to the microarray paper, because:

  • We show that some transcriptional changes are likely occuring in interneurons and motor neurons, not just in the VC nociceptive sensory neurons.
  • We found some transcripts which, like Egr, are rapidly *and* persistently up-regulated by sensitization training (GlyT2, VPS36, and an uncharacterized protein known for now as LOC101862095). We’re interested in such transcripts because they could be related to memory maintenance
  • We were able to better test the notion that CREB supports memory maintenance. So far, our evidence continues to go against this hypothesis, with no long-lasting changes detected in the VC sensory neurons nor in the pedal ganglia.
  • As a methodological point, we found that microdissecting out the VC cluster really really improves signal:noise for identifying transcriptional changes induced by learning. This is exciting–most work on the molecular mechanisms of memory uses tissue samples representing homogenous cell types. Zooming in on a single cell type of known relevance for storing the memory really enhances the power of the analysis.
  • We re-rested the four novel transcripts identified in our microarray paper from earlier this year (25117657). All four validated again! Moreover, all 4 were specifically up-regulated in the VC nociceptors (and some elsewhere as well). Another good indication that we’re on the right track with our microarray approach.
  • Another 3 student co-authors on this paper! We’re especially proud of Sami, Catherine, and Saman.
  • The paper is free on PLOSE ONE: http://dx.plos.org/10.1371/journal.pone.0114481. Also, you can download our raw data to examine for yourself at the Open Science Framework: https://osf.io/ts9ea/.

    Bibliography

    New Publication – Microarray analysis of sensitization

    We’ve got a new paper out (25117657) with the first of what we hope will be a series of studies using microarray to track the transcriptional changes following long-term sensitization training. This paper looks at the changes that occur immediately (1 hour) after training. It provides lots of details and data to validate the microarray design we developed, but also identifies a set of 81 transcripts that are strongly regulated after learning. Best of all, for a microarray paper, we use a large sample size (n = 8) and show using a subset of transcripts that most generalize to a completely independent sample. Among the changes we fully validated are up-regulation of a c/ebp-gamma (what the what!?), a glycine transporter, and a subunit of ESCRTII. The rest of the gene list that we’re working on has some exciting possibilities, too.

    Another thing to be proud of, is our three student co-authors on the paper.

    The paper is free for the next 50 days via this link, then it goes behind a paywall for 305 days, then it will be in PubMedCentral for free again (strange, right?). All the raw data is available on the Open Science Framework: https://osf.io/8pgfh/.

    Bibliography

    An Aplysia Egr homolog is rapidly and persistently regulated by long-term sensitization training

    Cyriac A, Holmes G, Lass J, Belchenko D, Calin-Jageman RJ, Calin-Jageman IE

    Neurobiol Learn Mem 2013 May;102:43-51

    PMID: 23567107

    Abstract

    The Egr family of transcription factors plays a key role in long-term plasticity and memory in a number of vertebrate species. Here we identify and characterize ApEgr (GenBank: KC608221), an Egr homolog in the marine mollusk Aplysia californica. ApEgr codes for a predicted 593-amino acid protein with the highly conserved trio of zinc-fingered domains in the C-terminus that characterizes the Egr family of transcription factors. Promoter analysis shows that the ApEgr protein selectively recognizes the GSG motif recognized by vertebrate Egrs. Like mammalian Egrs, ApEgr is constitutively expressed in a range of tissues, including the CNS. Moreover, expression of ApEgr is bi-directionally regulated by changes in neural activity. Of most interest, the association between ApEgr function and memory may be conserved in Aplysia, as we observe rapid and long-lasting up-regulation of expression after long-term sensitization training. Taken together, our results suggest that Egrs may have memory functions that are conserved from mammals to mollusks.

    Transcriptional changes following long-term sensitization training and in vivo serotonin exposure in Aplysia californica

    Bonnick K, Bayas K, Belchenko D, Cyriac A, Dove M, Lass J, McBride B, Calin-Jageman IE, Calin-Jageman RJ

    PLoS ONE 2012;7(10):e47378

    PMID: 23056638

    Abstract

    We used Aplysia californica to compare the transcriptional changes evoked by long-term sensitization training and by a treatment meant to mimic this training, in vivo exposure to serotonin. We focused on 5 candidate plasticity genes which are rapidly up-regulated in the Aplysia genus by in vivo serotonin treatment, but which have not yet been tested for regulation during sensitization: CREB1, matrilin, antistasin, eIF3e, and BAT1 homolog. CREB1 was rapidly up-regulated by both treatments, but the regulation following training was transient, falling back to control levels 24 hours after training. This suggests some caution in interpreting the proposed role of CREB1 in consolidating long-term sensitization memory. Both matrilin and eIF3e were up-regulated by in vivo serotonin but not by long-term sensitization training. This suggests that in vivo serotonin may produce generalized transcriptional effects that are not specific to long-term sensitization learning. Finally, neither treatment produced regulation of antistasin or BAT1 homolog, transcripts regulated by in vivo serotonin in the closely related Aplysia kurodai. This suggests either that these transcripts are not regulated by experience, or that transcriptional mechanisms of memory may vary within the Aplysia genus.