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

    Harmonin enhances voltage-dependent facilitation of Cav1.3 channels and synchronous exocytosis in mouse inner hair cells

    Gregory FD, Pangrsic T, Calin-Jageman IE, Moser T, Lee A

    J. Physiol. (Lond.) 2013 Jul;591(Pt 13):3253-69

    PMID: 23613530

    Abstract

    Cav1.3 channels mediate Ca(2+) influx that triggers exocytosis of glutamate at cochlear inner hair cell (IHC) synapses. Harmonin is a PDZ-domain-containing protein that interacts with the C-terminus of the Cav1.3 α1 subunit (α11.3) and controls cell surface Cav1.3 levels by promoting ubiquitin-dependent proteosomal degradation. However, PDZ-domain-containing proteins have diverse functions and regulate other Cav1.3 properties, which could collectively influence presynaptic transmitter release. Here, we report that harmonin binding to the α11.3 distal C-terminus (dCT) enhances voltage-dependent facilitation (VDF) of Cav1.3 currents both in transfected HEK293T cells and in mouse inner hair cells. In HEK293T cells, this effect of harmonin was greater for Cav1.3 channels containing the auxiliary Cav β1 than with the β2 auxiliary subunit. Cav1.3 channels lacking the α11.3 dCT were insensitive to harmonin modulation. Moreover, the ‘deaf-circler’ dfcr mutant form of harmonin, which does not interact with the α11.3 dCT, did not promote VDF. In mature IHCs from mice expressing the dfcr harmonin mutant, Cav1.3 VDF was less than in control IHCs. This difference was not observed between control and dfcr IHCs prior to hearing onset. Membrane capacitance recordings from dfcr IHCs revealed a role for harmonin in synchronous exocytosis and in increasing the efficiency of Ca(2+) influx for triggering exocytosis. Collectively, our results indicate a multifaceted presynaptic role of harmonin in IHCs in regulating Cav1.3 Ca(2+) channels and exocytosis.

    Glutamate transporters EAAT4 and EAAT5 are expressed in vestibular hair cells and calyx endings

    Dalet A, Bonsacquet J, Gaboyard-Niay S, Calin-Jageman I, Chidavaenzi RL, Venteo S, Desmadryl G, Goldberg JM, Lysakowski A, Chabbert C

    PLoS ONE 2012;7(9):e46261

    PMID: 23049999

    Abstract

    Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.

    Harmonin inhibits presynaptic Cav1.3 Ca²⁺ channels in mouse inner hair cells

    Gregory FD, Bryan KE, Pangršič T, Calin-Jageman IE, Moser T, Lee A

    Nat. Neurosci. 2011 Sep;14(9):1109-11

    PMID: 21822269

    Abstract

    Harmonin is a scaffolding protein that is required for normal mechanosensory function in hair cells. We found a presynaptic association of harmonin and Ca(v)1.3 Ca(2+) channels at the mouse inner hair cell synapse, which limits channel availability through a ubiquitin-dependent pathway.

    Molecular microdomains in a sensory terminal, the vestibular calyx ending

    Lysakowski A, Gaboyard-Niay S, Calin-Jageman I, Chatlani S, Price SD, Eatock RA

    J. Neurosci. 2011 Jul;31(27):10101-14

    PMID: 21734302

    Abstract

    Many primary vestibular afferents form large cup-shaped postsynaptic terminals (calyces) that envelope the basolateral surfaces of type I hair cells. The calyceal terminals both respond to glutamate released from ribbon synapses in the type I cells and initiate spikes that propagate to the afferent’s central terminals in the brainstem. The combination of synaptic and spike initiation functions in these unique sensory endings distinguishes them from the axonal nodes of central neurons and peripheral nerves, such as the sciatic nerve, which have provided most of our information about nodal specializations. We show that rat vestibular calyces express an unusual mix of voltage-gated Na and K channels and scaffolding, cell adhesion, and extracellular matrix proteins, which may hold the ion channels in place. Protein expression patterns form several microdomains within the calyx membrane: a synaptic domain facing the hair cell, the heminode abutting the first myelinated internode, and one or two intermediate domains. Differences in the expression and localization of proteins between afferent types and zones may contribute to known variations in afferent physiology.

    Ca(v)1 L-type Ca2+ channel signaling complexes in neurons

    Calin-Jageman I, Lee A

    J. Neurochem. 2008 May;105(3):573-83

    PMID: 18266933

    Abstract

    Ca(v)1 L-type Ca2+ channels play crucial and diverse roles in the nervous system. The pre- and post-synaptic functions of Ca(v)1 channels not only depend on their intrinsic biophysical properties but also their dynamic regulation by a host of cellular influences. These include protein kinases and phosphatases, G-protein coupled receptors, scaffolding proteins, and Ca2+-binding proteins. The cytoplasmic domains of the main pore forming alpha(1) subunit of Ca(v)1 offer a number of binding sites for these modulators, permitting fast and localized regulation of Ca2+ entry. Through effects on Ca(v)1 gating, localization, and coupling to effectors, protein modulators are efficiently positioned to adjust Ca(v)1 Ca2+ signals that control neuronal excitability, synaptic plasticity, and gene expression.

    Ca2+-binding proteins tune Ca2+-feedback to Cav1.3 channels in mouse auditory hair cells

    Cui G, Meyer AC, Calin-Jageman I, Neef J, Haeseleer F, Moser T, Lee A

    J. Physiol. (Lond.) 2007 Dec;585(Pt 3):791-803

    PMID: 17947313

    Abstract

    Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Ca(v)1.3 voltage-gated Ca(2+) channels. Central to their role in this regard, Ca(v)1.3 channels in inner hair cells show little Ca(2+)-dependent inactivation, a fast negative feedback regulation by incoming Ca(2+) ions, which depends on calmodulin association with the Ca(2+) channel alpha(1) subunit. Ca(2+)-dependent inactivation characterizes nearly all voltage-gated Ca(2+) channels including Ca(v)1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Ca(v)1.3 in inner hair cells remains a mystery. Previously, we established calmodulin-like Ca(2+)-binding proteins in the brain and retina (CaBPs) as essential modulators of voltage-gated Ca(2+) channels. Here, we demonstrate that CaBPs differentially modify Ca(2+) feedback to Ca(v)1.3 channels in transfected cells and explore their significance for Ca(v)1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca(2+)-dependent inactivation of Ca(v)1.3. CaBP1 and CaBP4 both interact with calmodulin-binding sequences in Ca(v)1.3, but CaBP4 more weakly inhibits Ca(2+)-dependent inactivation than CaBP1. Ca(2+)-dependent inactivation is marginally greater in inner hair cells from CaBP4(-/-) than from wild-type mice, yet CaBP4(-/-) mice are not hearing-impaired. In contrast to CaBP4, CaBP1 is strongly localized at the presynaptic ribbon synapse of adult inner hair cells both in wild-type and CaBP4(-/-) mice and therefore is positioned to modulate native Ca(v)1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca(2+) channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Ca(v)1.3 Ca(2+) currents required for auditory transmission.

    Erbin enhances voltage-dependent facilitation of Ca(v)1.3 Ca2+ channels through relief of an autoinhibitory domain in the Ca(v)1.3 alpha1 subunit

    Calin-Jageman I, Yu K, Hall RA, Mei L, Lee A

    J. Neurosci. 2007 Feb;27(6):1374-85

    PMID: 17287512

    Abstract

    Ca(v)1.3 (L-type) voltage-gated Ca2+ channels have emerged as key players controlling Ca2+ signals at excitatory synapses. Compared with the more widely expressed Ca(v)1.2 L-type channel, relatively little is known about the mechanisms that regulate Ca(v)1.3 channels. Here, we describe a new role for the PSD-95 (postsynaptic density-95)/Discs large/ZO-1 (zona occludens-1) (PDZ) domain-containing protein, erbin, in directly potentiating Ca(v)1.3. Erbin specifically forms a complex with Ca(v)1.3, but not Ca(v)1.2, in transfected cells. The significance of erbin/Ca(v)1.3 interactions is supported by colocalization in somatodendritic domains of cortical neurons in culture and coimmunoprecipitation from rat brain lysates. In electrophysiological recordings, erbin augments facilitation of Ca(v)1.3 currents by a conditioning prepulse, a process known as voltage-dependent facilitation (VDF). This effect requires a direct interaction of the erbin PDZ domain with a PDZ recognition site in the C-terminal domain (CT) of the long variant of the Ca(v)1.3 alpha1 subunit (alpha1 1.3). Compared with Ca(v)1.3, the Ca(v)1.3b splice variant, which lacks a large fraction of the alpha1 1.3 CT, shows robust VDF that is not further affected by erbin. When coexpressed as an independent entity with Ca(v)1.3b or Ca(v)1.3 plus erbin, the alpha1 1.3 CT strongly suppresses VDF, signifying an autoinhibitory function of this part of the channel. These modulatory effects of erbin, but not alpha1 1.3 CT, depend on the identity of the auxiliary Ca2+ channel beta subunit. Our findings reveal a novel mechanism by which PDZ interactions and alternative splicing of alpha1 1.3 may influence activity-dependent regulation of Ca(v)1.3 channels at the synapse.