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/.

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 [cite source=’pubmed’]25486125[/cite] 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 [cite source=’pubmed’]25117657[/cite]. 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/.

    New Publication – Microarray analysis of sensitization

    We’ve got a new paper out [cite source=’pubmed’]25117657[/cite] 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/.

    Registered Replication In Social Psychology – Our contribution and amazing special issue

    Tracy Caldwell and I have a new paper out which we’re very proud of [cite source=’doi’]10.1027/1864-9335/a000190[/cite]–a registered replication of a previous finding that superstition can cause a substantial improvement in skilled performance [cite source=’doi’]10.1177/0956797610372631[/cite].

    We conducted 2 high-power and extremely precise replication attempts of this finding, pre-registered our design, sample targets, and analysis in advance. What we found was disappointing–performance did not strongly improve in the superstition conditions despite successful manipulation of feelings of luck. But the overall approach of conducting registered replications–well, that was a real treat.

    We’re especially proud that the paper is in a special issue dedicated just to registered replications of social psychology. We will also be presenting the paper at a Social Psych conference in Amsterdam this summer!

    NeuronBank: A Tool for Cataloging Neuronal Circuitry

    Katz PS, Calin-Jageman R, Dhawan A, Frederick C, Guo S, Dissanayaka R, Hiremath N, Ma W, Shen X, Wang HC, Yang H, Prasad S, Sunderraman R, Zhu Y

    Front Syst Neurosci 2010;4:9

    PMID: 20428500

    Abstract

    The basic unit of any nervous system is the neuron. Therefore, understanding the operation of nervous systems ultimately requires an inventory of their constituent neurons and synaptic connectivity, which form neural circuits. The presence of uniquely identifiable neurons or classes of neurons in many invertebrates has facilitated the construction of cellular-level connectivity diagrams that can be generalized across individuals within a species. Homologous neurons can also be recognized across species. Here we describe NeuronBank.org, a web-based tool that we are developing for cataloging, searching, and analyzing neuronal circuitry within and across species. Information from a single species is represented in an individual branch of NeuronBank. Users can search within a branch or perform queries across branches to look for similarities in neuronal circuits across species. The branches allow for an extensible ontology so that additional characteristics can be added as knowledge grows. Each entry in NeuronBank generates a unique accession ID, allowing it to be easily cited. There is also an automatic link to a Wiki page allowing an encyclopedic explanation of the entry. All of the 44 previously published neurons plus one previously unpublished neuron from the mollusc, Tritonia diomedea, have been entered into a branch of NeuronBank as have 4 previously published neurons from the mollusc, Melibe leonina. The ability to organize information about neuronal circuits will make this information more accessible, ultimately aiding research on these important models.

    Altering cAMP levels within a central pattern generator modifies or disrupts rhythmic motor output

    Clemens S, Calin-Jageman R, Sakurai A, Katz PS

    J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 2007 Dec;193(12):1265-71

    PMID: 17972082

    Abstract

    Cyclic AMP is a second messenger that has been implicated in the neuromodulation of rhythmically active motor patterns. Here, we tested whether manipulating cAMP affects swim motor pattern generation in the mollusc, Tritonia diomedea. Inhibiting adenylyl cyclase (AC) with 9-cyclopentyladenine (9-CPA) slowed or stopped the swim motor pattern. Inhibiting phosphodiesterase with 3-isobutyl-1-methylxanthine (IBMX) or applying dibutyryl-cAMP (dB-cAMP) disrupted the swim motor pattern, as did iontophoresing cAMP into the central pattern generator neuron C2. Additionally, during wash-in, IBMX sometimes temporarily produced extended or spontaneous swim motor patterns. Photolysis of caged cAMP in C2 after initiation of the swim motor pattern inhibited subsequent bursting. These results suggest that cAMP levels can dynamically modulate swim motor pattern generation, possibly shaping the output of the central pattern generator on a cycle-by-cycle basis.

    Potentiation phase of spike timing-dependent neuromodulation by a serotonergic interneuron involves an increase in the fraction of transmitter release

    Sakurai A, Calin-Jageman RJ, Katz PS

    J. Neurophysiol. 2007 Oct;98(4):1975-87

    PMID: 17686912

    Abstract

    In the mollusk, Tritonia diomedea, the serotonergic dorsal swim interneuron (DSI) produces spike timing-dependent neuromodulation (STDN) of the synaptic output of ventral swim interneuron B (VSI) resulting in a biphasic, bidirectional change of synaptic strength characterized by a rapid heterosynaptic potentiation followed by a more prolonged heterosynaptic depression. This study examined the mechanism underlying the potentiation phase of STDN. In the presence of 4-aminopyridine, which blocks the depression phase and enhances transmitter release from VSI, rapidly stimulating VSI led to a steady-state level of transmitter depletion during which potentiation by DSI or serotonin (5-HT) was eliminated. Cumulative plots of excitatory postsynaptic currents were used to estimate changes in the size and replenishment rate of the readily releasable pool (RRP) and the fraction of release. 5-HT application increased transmitter release without altering replenishment rate. The magnitude of 5-HT-evoked potentiation correlated with the increase in the fraction of release. A phenomenological model of the synapse further supported the hypothesis that 5-HT-induced potentiation was caused by an increase in the fraction of release and correctly predicted no change in frequency facilitation. A dynamic version of the model correctly predicted the effect of DSI stimulation under a variety of conditions. Finally, depletion of internal Ca(2+) stores with cyclopiazonic acid showed that Ca(2+) from internal stores is necessary for the 5-HT-induced potentiation. The data indicate that 5-HT released from DSI increases the fraction of the RRP discharged during VSI action potentials using a mechanism that involves Ca(2+) extrusion from internal stores, resulting in time- and state-dependent neuromodulation.

    Parameter space analysis suggests multi-site plasticity contributes to motor pattern initiation in Tritonia

    Calin-Jageman RJ, Tunstall MJ, Mensh BD, Katz PS, Frost WN

    J. Neurophysiol. 2007 Oct;98(4):2382-98

    PMID: 17652417

    Abstract

    This research examines the mechanisms that initiate rhythmic activity in the episodic central pattern generator (CPG) underlying escape swimming in the gastropod mollusk Tritonia diomedea. Activation of the network is triggered by extrinsic excitatory input but also accompanied by intrinsic neuromodulation and the recruitment of additional excitation into the circuit. To examine how these factors influence circuit activation, a detailed simulation of the unmodulated CPG network was constructed from an extensive set of physiological measurements. In this model, extrinsic input alone is insufficient to initiate rhythmic activity, confirming that additional processes are involved in circuit activation. However, incorporating known neuromodulatory and polysynaptic effects into the model still failed to enable rhythmic activity, suggesting that additional circuit features are also required. To delineate the additional activation requirements, a large-scale parameter-space analysis was conducted (~2 x 10(6) configurations). The results suggest that initiation of the swim motor pattern requires substantial reconfiguration at multiple sites within the network, especially to recruit ventral swim interneuron-B (VSI) activity and increase coupling between the dorsal swim interneurons (DSIs) and cerebral neuron 2 (C2) coupling. Within the parameter space examined, we observed a tendency for rhythmic activity to be spontaneous and self-sustaining. This suggests that initiation of episodic rhythmic activity may involve temporarily restructuring a nonrhythmic network into a persistent oscillator. In particular, the time course of neuromodulatory effects may control both activation and termination of rhythmic bursting.

    Behavioral adaptation of the Aplysia siphon-withdrawal response is accompanied by sensory adaptation

    Calin-Jageman RJ, Fischer TM

    Behav. Neurosci. 2007 Feb;121(1):200-11

    PMID: 17324064

    Abstract

    Behavioral adaptation is a decrease in behavioral responsiveness due to a sustained stimulus and is a key component to an organism’s overall adjustment to its ambient environment. The authors examined the relationship between behavioral adaptation and sensory adaptation in the siphon-withdrawal reflex (SWR) of Aplysia californica. Sensory input to the SWR circuit was measured via en passant extracellular recordings from the siphon nerve in semi-intact preparations. The authors characterized sensory activity in response to water turbulence, an ethologically relevant stimulus that produces behavioral adaptation of the SWR. The authors found that water turbulence elicits a continuous sensory response that adapts to a low-level steady state. Consistent with behavioral measures, this sensory adaptation attenuates the evoked response to siphon taps delivered during water turbulence. Comparing trends in both behavioral and sensory adaptation revealed similar dynamics during turbulence but divergent trends during recovery. Specifically, behavioral recovery was inversely related to the duration of environmental change; recovery of sensory adaptation was not. This supports the authors’ hypothesis that environment-driven behavioral adaptation reflects an interaction between sensory adaptation and additional dynamic processes.

    A distributed computing tool for generating neural simulation databases

    Calin-Jageman RJ, Katz PS

    Neural Comput 2006 Dec;18(12):2923-7

    PMID: 17052151

    Abstract

    After developing a model neuron or network, it is important to systematically explore its behavior across a wide range of parameter values or experimental conditions, or both. However, compiling a very large set of simulation runs is challenging because it typically requires both access to and expertise with high-performance computing facilities. To lower the barrier for large-scale model analysis, we have developed NeuronPM, a client/server application that creates a “screen-saver” cluster for running simulations in NEURON (Hines & Carnevale, 1997). NeuronPM provides a user-friendly way to use existing computing resources to catalog the performance of a neural simulation across a wide range of parameter values and experimental conditions. The NeuronPM client is a Windows-based screen saver, and the NeuronPM server can be hosted on any Apache/PHP/MySQL server. During idle time, the client retrieves model files and work assignments from the server, invokes NEURON to run the simulation, and returns results to the server. Administrative panels make it simple to upload model files, define the parameters and conditions to vary, and then monitor client status and work progress. NeuronPM is open-source freeware and is available for download at http://neuronpm.homeip.net . It is a useful entry-level tool for systematically analyzing complex neuron and network simulations.