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Accumulation of Polyribosomes in Dendritic Spine Heads, but Not Bases and Necks, During Memory Consolidation Depends on Cap-Dependent Translation Initiation

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Accumulation of Polyribosomes in Dendritic Spine Heads, but Not Bases and Necks, During Memory Consolidation Depends on Cap-Dependent Translation Initiation 1862 • The Journal of Neuroscience, February 15, 2017 • 37(7):1862–1872 Cellular/Molecular Accumulation of Polyribosomes in Dendritic Spine Heads, But Not Bases and Necks, during Memory Consolidation Depends on Cap-Dependent Translation Initiation Linnaea E. Ostroff,1,2 Benjamin Botsford,1* Sofya Gindina,1* Kiriana K. Cowansage,1 Joseph E. LeDoux,1,3 Eric Klann,1 and Charles Hoeffer4,5 1Center for Neural Science, New York University, New York, New York 10003, 2Allen Institute for Brain Science, Seattle, Washington 90109, 3Nathan Kline Institute for Psychiatric Research, Orangeburg, New York 10962, and 4Institute for Behavioral Genetics and 5Department of Integrative Physiology, University of Colorado, Boulder, Colorado 80303 Translation in dendrites is believed to support synaptic changes during memory consolidation. Although translational control mecha- nisms are fundamental mediators of memory, little is known about their role in local translation. We previously found that polyribo- somes accumulate in dendritic spines of the adult rat lateral amygdala (LA) during consolidation of aversive pavlovian conditioning and that this memory requires cap-dependent initiation, a primary point of translational control in eukaryotic cells. Here we used serial electron microscopy reconstructions to quantify polyribosomes in LA dendrites when consolidation was blocked by the cap-dependent initiationinhibitor4EGI-1.Wefoundthat4EGI-1depletedpolyribosomesindendriticshaftsandselectivelypreventedtheirupregulation in spine heads, but not bases and necks, during consolidation. Cap-independent upregulation was specific to spines with small, astrocyte- associated synapses. Our results reveal that cap-dependent initiation is involved in local translation during learning and that local translational control varies with synapse type. Key words: cap-dependent translation; local translation; pavlovian conditioning; polyribosomes; serial electron microscopy; structural plasticity Significance Statement Translation initiation is a central regulator of long-term memory formation. Local translation in dendrites supports memory by providing necessary proteins at synaptic sites, but it is unknown whether this requires initiation or bypasses it. We used serial electronmicroscopyreconstructionstoexaminepolyribosomesindendriteswhenmemoryformationwasblockedbyaninhibitor of translation initiation. This revealed two major pools of polyribosomes that were upregulated during memory formation: one pool in dendritic spine heads that was initiation dependent and another pool in the bases and necks of small spines that was initiation independent. Thus, translation regulation differs between spine types and locations, and translation that occurs closest to individual synapses during memory formation is initiation dependent. Introduction chemical and structural remodeling at synapses (Rosenberg et al., Long-term memory consolidation requires new gene expression, 2014; Bailey et al., 2015; Segal, 2016). It is thought that a signifi- culminating in protein synthesis, to support substantial bio- cant amount of this protein synthesis occurs locally in dendrites (Sutton and Schuman, 2006; Bramham and Wells, 2007; Wang et al., 2010; Liu-Yesucevitz et al., 2011). Local translation can pro- Received Oct. 25, 2016; revised Dec. 9, 2016; accepted Jan. 9, 2017. Author contributions: L.E.O., K.K.C., J.E.L., E.K., and C.H. designed research; L.E.O., K.K.C., and C.H. performed vide synapses with rapid access to new proteins, bypassing gene research; L.E.O., B.B., and S.G. analyzed data; L.E.O. wrote the paper. expression cascades and subsequent transport from the soma, This work was supported by National Institutes of Health Grants MH083583 and MH094965 (L.E.O.), NS034007 and can be a means of spatiotemporal protein targeting (Martin and NS047384 (E.K.), and NS086933 and DA036673 (C.H.); Simons Foundation Grant SFARI 27444 (C.H.); Brain and and Ephrussi, 2009; Holt and Schuman, 2013). However, how Behavior Research Foundation Grant 21069 (C.H.); and Alzheimer’s Association Grant MNIRGDP-12-258900 (C.H.). local translation fits into the larger scheme of regulated gene We are grateful to Elizabeth Perry and Robert Smith for expert technical assistance. *B.B. and S.G. contributed equally to this work. The authors declare no competing financial interests. Correspondence should be addressed to Linnaea E. Ostroff, Center for Neural Science, 4 Washington Place, Room DOI:10.1523/JNEUROSCI.3301-16.2017 809, New York, NY 10003. E-mail: [email protected]. Copyright © 2017 the authors 0270-6474/17/371862-11$15.00/0 Ostroff et al. • Cap-Dependent Local Translation during Learning J. Neurosci., February 15, 2017 • 37(7):1862–1872 • 1863 expression during memory consolidation is not well understood. cannula implantation, animals were anesthetized with a mixture of ket- Protein synthesis is a universal requirement for long-term mem- amine and xylazine (100 mg/kg and 6 mg/kg, i.p.) and secured in a ory across species, and several translational control mechanisms stereotaxic frame (David Kopf Instruments). Bilateral craniotomies were are known to mediate learning and their dysregulation to under- made, and 22 g stainless steel guide cannulae (Plastics One) were placed Ϫ Ϯ Ϫ lie intellectual disabilities in humans (Davis and Squire, 1984; relative to bregma ( 0.8 AP, 1.5 ML, and 1.6 mm DV) and secured with dental cement. Buprenorphine (2 mg/kg, i.p.) was used as a postop- McGaugh, 2000; Costa-Mattioli et al., 2009; Groppo and Richter, erative anesthetic. Animals were given at least 1 week to recover from 2009; Darnell, 2011; Gal-Ben-Ari et al., 2012; Buffington et al., surgery before experiments began and were handled briefly each day after 2014; Santini et al., 2014). RNA trafficking mechanisms, mean- surgery to acclimate them to having their cannulae manipulated by an while, have been the main focus of research on translation at experimenter. synapses (Hirokawa, 2006; Sossin and DesGroseillers, 2006; Behavior and drug infusion. Behavioral apparatus and paired training Doyle and Kiebler, 2011; Buxbaum et al., 2015). Whether protein and box control protocols were identical to those used by Ostroff et al. synthesis in dendrites is subject to the translational control mech- (2010). Briefly, animals were habituated to the training chambers (Coul- anisms that regulate learning is unknown. bourn Instruments) for 30 min on each of 2 d before training. The trained Translation is a complex, highly orchestrated process, with the rats were presented with five auditory tones (30 s, 5 kHz, 80 dB) cotermi- majority of regulatory mechanisms targeting the initiation step nating in footshocks (1 s, 0.7 mA) given at a variable interval over 32.5 (Sonenberg and Hinnebusch, 2009). Initiation mechanisms are min. The behavioral control rats were placed in the box but not pre- sented with tones or shocks. Immediately after training, rats were involved in synaptic plasticity and memory, and improper initi- infused with 10 ␮l per side of either 5 ␮g/␮l 4EGi-1 in 50% DMSO/10% ation is particularly implicated in a number of intellectual disor- ␤-propylcyclodextrin or vehicle. Infusions were given with a syringe ders (Costa-Mattioli et al., 2009; Darnell, 2011; Gal-Ben-Ari et pump (Harvard Apparatus) at a rate of 1 ␮l/min through 28 g stainless al., 2012; Buffington et al., 2014; Santini et al., 2014). The major steel injection cannulae projecting 1 mm past the tip of the guide cannu- rate-limiting step in initiation is binding of the eukaryotic initia- lae. The cannulae were left in place for 15 min to allow the drug to diffuse, tion factor eIF4E to eIF4G, which leads to recruitment of ribo- and the animals were returned to their home cages. Freezing during the somes to the 5Ј cap of the target mRNA (Groppo and Richter, tones was quantified to ensure that all animals displayed the expected 2009). Using the eIF4E–eIF4G interaction inhibitor 4EGI-1, we behavioral response during training. previously found that initiation of cap-dependent translation in Tissue preparation. All reagents were obtained from Electron Micros- the lateral amygdala (LA) is required for consolidation of aversive copy Sciences unless otherwise stated. Exactly 1 h after the first tone/ shock pairing or the equivalent time point for the control group, animals pavlovian conditioning memory (Hoeffer et al., 2011). Binding of were anesthetized with a lethal dose of chloral hydrate (1.25 g/kg, i.p.; eIF4E and eIF4G was increased during consolidation; in a sepa- Sigma) and perfused transcardially with saline containing 0.1 U of hep- rate study, we found that polyribosomes were upregulated in LA arin, 0.01 M sodium cacodylate, 154 mM NaCl (Sigma), 2 mM CaCl2, and dendrites as well (Ostroff et al., 2010). It is possible that initiation 4mM MgCl2 at pH 7.4 to clear the bloodstream, followed by 500 ml of triggered by learning is necessary for local translation at synapses fixative containing 2.5% glutaraldehyde, 2% paraformaldehyde, 0.1 M and that this, at least in part, explains why consolidation is initi- sodium cacodylate, 2 mM CaCl2,and4mM MgCl2 at pH 7.4 at a constant ation dependent. On the other hand, if dendritic translation after rate of 75 ml/min. The brains were removed and placed in vials of the learning serves to provide proteins quickly ahead of transport same fixative for 1 h and rinsed in the saline solution without heparin. ␮ from the soma, it could be regulated downstream of eIF4E– The left hemisphere of each brain was sectioned coronally at 70 mona
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