NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion

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Contents lists available at ScienceDirect

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NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion

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Tali Rosenberg a, Alina Elkobi a, Daniela C. Dieterich b, Kobi Rosenblum a,c,⇑

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Sagol Dept. of Neurobiology, University of Haifa, Haifa 3498838, Israel Institute for Pharmacology and Toxicology, Medical Faculty, Otto-von-Guericke University Magdeburg, Leipziger Strasse 44, Magdeburg 39120, Germany c Center for Gene Manipulation in the Brain, University of Haifa, Haifa 3498838, Israel b

a r t i c l e

i n f o

Article history: Received 27 August 2015 Revised 6 January 2016 Accepted 9 January 2016 Available online xxxx Keywords: CTA Taste learning Consolidation NMDAR Proteasome

a b s t r a c t Taste information is processed in different brain structures in the mammalian brain, including the gustatory cortex (GC), which resides within the insular cortex. N-methyl-D-aspartate receptor (NMDAR) activity in the GC is necessary for the acquisition of conditioned taste aversion (CTA) but not positive novel taste learning. Previous studies have shown that taste memory consolidation requires intact protein synthesis in the GC. In addition, the direct involvement of translation initiation and elongation factors was documented in the GC during taste learning. However, protein expression is defined by protein synthesis, degradation, and localization. Protein degradation is critical for the consolidation and reconsolidation of other forms of learning, such as fear learning and addiction behavior, but its role in corticaldependent learning is not clear. Here, we show for the first time that proteasome activity is specifically increased in the GC 4 h following experiencing of a novel taste. This increase in proteasome activity was abolished by local administration to the GC of the NMDA antagonist, APV, as well as a CaMKII inhibitor, at the time of acquisition. In addition, local application of lactacystin, a proteasome inhibitor, resulted in impaired CTA, but not novel taste learning. These results suggest that NMDAR-dependent proteasome activity in the GC participates in the association process between novel taste experience and negative visceral sensation. Ó 2016 Published by Elsevier Inc.

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1. Introduction

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Creating long term memories involves the stabilization of short term labile information in a nonlinear process, termed memory consolidation, that requires protein synthesis (Kandel, Dudai, & Mayford, 2014). Protein synthesis-dependent memory consolidation occurs in a certain time window, shortly after acquisition, during which long term memory (LTM) is susceptible to inhibition (Dudai, 2012). Stable LTM can be modified via a process termed reconsolidation, by recalling the memory. Similarly to consolidation, the reconsolidation process is also sensitive to protein synthesis inhibitors (Bonin & De Koninck, 2015). However, during the past decade, it has been shown that in addition to protein synthesis, protein degradation also plays a central role in the formation of LTM for different types of learning paradigms subserved by different brain regions (Jarome & Helmstetter, 2013). Protein degradation, which is essential for

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⇑ Corresponding author at: Sagol Dept. of Neurobiology, University of Haifa, Haifa 3498838, Israel. Fax: +972 4 8249654. E-mail address: [email protected] (K. Rosenblum).

normal function both on the cellular and organism levels, is performed by lysosomes, proteasomes, and specific proteases. Most cellular proteins are degraded by the ubiquitin proteasome system (UPS) (Rock et al., 1994). The UPS involves ubiquitin tagging of the protein targeted for degradation in an ATPdependent series of enzymatic reactions. Proteins tagged with a poly-ubiquitin chain linked to the ubiquitin lysine-48 (K48 ubiquitination) are recognized by the proteasome and degraded into short peptides and amino acids. The proteasome found in mammals, the 26S proteasome, consists of a 20S core catalytic subunit harboring trypsin, chymotrypsin, and post-glutamine hydrolase-like activities; and a 19S regulatory subunit that recognizes and recruits K48 ubiquitinated proteins, and facilitates their entrance to the catalytic core in an ATP-dependent manner. The regulatory subunit also has iso-peptidase activity, removing the poly-ubiquitin chain and allowing the recycling of ubiquitin (Ciechanover, 2013). Recently, ubiquitin-independent 20S protein degradation was found to degrade unfolded, native proteins with large (over 30 amino acids) intrinsically disordered regions (IDRs), and entirely disordered

http://dx.doi.org/10.1016/j.nlm.2016.01.002 1074-7427/Ó 2016 Published by Elsevier Inc.

Please cite this article in press as: Rosenberg, T., et al. NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.01.002

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proteins (IDPs) (Ben-Nissan & Sharon, 2014). The present study focuses on ubiquitin-dependent proteasomal degradation. In the past decade it has been shown that protein degradation by the UPS is mediated by N-Methyl D-Aspartate receptor (NMDAR) and CaMKII signaling. On the cellular level, stimulation of hippocampal culture has been shown to induce recruitment of active proteasome particles to synaptic spines via the NMDAR and CaMKII signaling (Bingol & Schuman, 2006; Djakovic, Schwarz, Barylko, DeMartino, & Patrick, 2009; Djakovic et al., 2012). However, in a different study, NMDAR activation reduced ubiquitin-dependent protein degradation (Tai, Besche, Goldberg, & Schuman, 2010). Finally, behavioral experiments showed that ubiquitin-dependent proteasome activity in the amygdala following fear learning was induced via activation of the NMDAR and CaMKII phosphorylation of proteasome subunit Rpt6 (Jarome, Kwapis, Ruenzel, & Helmstetter, 2013). NMDAR signaling is involved in synaptoneurogenesis, different forms of translation regulation, as well as different forms of learning, including taste learning (Gal-Ben-Ari et al., 2012). Blockade of NMDAR in the gustatory cortex (GC, one of the brain regions subserving taste learning) with (2R)-amino-5-phosphonopentanoate (APV) impairs conditioned taste aversion (CTA), a robust association of novel taste with visceral malaise (Barki-Harrington et al., 2009; Gildish et al., 2012; Inberg et al., 2013; Rappaport et al., 2015; Rosenblum, Berman, Hazvi, Lamprecht, & Dudai, 1997). This negative association serves as a defense mechanism against poisoning, and leads to reduced consumption of the associated taste in later encounters. However, attenuation of neophobia, a paradigm that consists of several encounters with a novel taste during which the consumption of the novel taste increases as it becomes more familiar (Bureš, Bermudez-Rattoni, & Yamamoto, 1998), is not sensitive to APV (Parkes, De la Cruz, Bermudez-Rattoni, Coutureau, & Ferreira, 2014). A recent study has shown that while injection of proteasome inhibitors to the GC does not affect CTA, this form of learning is impaired by simultaneous inhibition of proteasome activity in the amygdala and the GC (Rodriguez-Ortiz, Balderas, SaucedoAlquicira, Cruz-Castaneda, & Bermudez-Rattoni, 2011). Other studies have also shown that administration of proteasome inhibitors to rodents affect consolidation and reconsolidation (Sol Fustinana, de la Fuente, Federman, Freudenthal, & Romano, 2014). However, the link between proteasome inhibition and NMDAR activation and their effect on taste learning has not been clear. Here, we tested the hypothesis that ubiquitin-dependent protein degradation in the GC is correlated with taste learning and necessary for it. First, we identified a time point in which ubiquitin-dependent proteasome activity in the GC changes following taste learning. next, we assessed the involvement of the NMDAR in the process. Finally, we determined the behavioral implications of inhibiting proteasome activity in the GC on novel taste learning, and the negative associative taste learning paradigm, CTA.

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2. Materials and methods

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2.1. Subjects

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Adult male Wistar rats weighing 200–250 g (Harlan, Jerusalem, Israel) were maintained on a 12-h light/dark cycle. All procedures were performed in strict accordance with the University of Haifa regulations and the US National Institutes of Health guidelines (NIH publication number 8023).

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2.2. Micro-surgery and micro-infusion

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Micro-infusion into the insular cortex was performed via chronically implanted cannulae, as previously described (Ounallah-Saad,

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Sharma, Edry, & Rosenblum, 2014). Briefly, rats were anesthetized with Equithesin (0.3 ml/100 g) (2.12% w/v MgSO4, 10% v/v ethanol, 39.1% v/v 1,2,-propranolol, 0.98% w/v sodium pentobarbital, and 4.2% w/v chloral hydrate), restrained in a stereotactic apparatus (Stoelting, USA) and implanted bilaterally with a 10 mm guide stainless steel cannula (23 gauge) aimed at the rat gustatory cortex (anteroposterior, +1.2 mm relative to bregma; lateral, ±5.5 mm; ventral, 5.5 mm (Paxinos & Watson, 2005)). The cannulae were positioned in place with acrylic dental cement and secured by two skull screws. A stylus was placed in the guide cannula to prevent clogging. Following the microsurgery, animals were injected i.m. with an antibiotic and Dypiron to ease their pain, and were allowed to recuperate for one week. For micro-infusion, the stylus was removed from the guide cannula, and a 28 gauge injection cannula, extending 1.0 mm from the tip of the guide cannula, was inserted. The injection cannula was connected via PE20 tubing to a Hamilton micro-syringe driven by a micro-infusion pump (Harvard PHD 2000). Micro-infusion was performed bilaterally in a 1.0 lL volume per hemisphere, delivered over 1 min. The injection cannula was left in position before withdrawal for an additional 1 min to minimize dragging of the injected liquid along the injection tract.

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2.3. Pharmacology

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For the behavioral set of experiments, the rats were bilaterally injected either with lactacystin (10 lM, Enzo life science, Farmingdale, NY, USA) or with vehicle (aCSF containing 124 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 26 mM NaHCO3, 10 mM D-glucose, and 2.4 mM CaCl2, and brought to a concentration of 10 lM) 20 min before CTA or attenuation of neophobia. For the biochemical experiments, the rats were bilaterally injected with NMDAR blocker (2R)-amino-5-phosphonopentanoate (APV, Sigma–Aldrich, Israel) (50 lM) 20 minutes before incidental taste learning or CTA (as described in Elkobi, Ehrlich, Belelovsky, Barki-Harrington, & Rosenblum, 2007). CaMKII inhibitory peptide TatCN21 or Tat control peptide (0.3 nM/lL, a kind gift from Dr. Ulrich Bayer) was bilaterally injected 20 min before incidental taste learning. The rats were decapitated 2, 4, 6, or 8 h after CTA or incidental taste learning, and insular cortices were dissected and subjected to proteasome activity assay and western blotting.

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2.4. Behavior

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2.4.1. Conditioned taste aversion (CTA) CTA was performed as described previously (Merhav and Rosenblum, 2008; Rosenblum, Meiri, & Dudai, 1993; Stern, Chinnakkaruppan, David, Sonenberg, & Rosenblum, 2013). Saccharin (0.1% w/v, sodium salt, Sigma, Israel) was used as the unfamiliar taste in training [i.e., the conditioned stimulus (CS)], and injection of LiCl (0.15 M, 2% body weight, i.p.) as the malaise-inducing agent [unconditioned stimulus (UCS)]. At the beginning of the behavioral experiment, the rats were trained for 3 days to get their daily water ration once a day for 20 min from two pipettes, each containing 10 mL of water. On the conditioning day, they were allowed to drink the saccharin solution instead of water from similar pipettes for 20 min, and 40 min later were injected with LiCl. Under these conditions, 2 days after training the conditioned rats preferred water to saccharin in a multiple choice test situation (three pipettes with 5 mL of saccharin each and three with 5 mL of water each), whereas non-conditioned rats preferred saccharin to water. The behavioral data are presented in terms of aversion index, defined as [mL water/(mL water + mL saccharin)] consumed in the test; 0.5 was considered chance level, and the higher the aversion index, the more the rats prefer water to the conditioned taste.

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Please cite this article in press as: Rosenberg, T., et al. NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.01.002

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2.4.2. Attenuation of neophobia Rats were separated into individual housing cages and subjected to a 3-day water-restriction training period, in which once a day, for 20 min, they were offered 20 mL of water from two pipettes, each containing 10 mL. On the fourth day, the control group received water, whereas the experimental group was exposed to a novel taste (0.1% (w/v) sodium saccharin) (Rosenblum et al., 1993; Yefet et al., 2006). After two successive days of water restriction training, the rats were tested in a multiple-choice test as described above for the CTA procedure. The behavioral data are presented in terms of preference index, expressed as a percentage, [mL saccharin/(mL water + mL saccharin)]  100, in which the quantities are those consumed during each test.

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2.5. Biochemical procedures

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2.5.1. Measuring ubiquitin-dependent proteasome activity In order to assess changes in proteasome activity, we measured the chymotrypsin-like activity of the proteasome. Brain samples snap-frozen in liquid nitrogen were homogenized in a lysis buffer containing 20 mM Tris, 0.32 M sucrose, 5 mM MgCl2, 2 mM ATP, 2 mM DTT, and 0.2% triton. Total lysate was sampled for SDS PAGE and western blotting and the rest of the sample was centrifuged at 15,000g  15 min. Protein amount was quantified using the bicinchoninic acid assay (Pierce BCA Protein Assay Kit, Life Technologies, Thermo Scientific, NY) and equal amounts of protein were added to a buffer containing 50 mM Tris, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 0.1 mM suc-LLVY-AMC (a synthetic peptide degraded via the chymotrypsin-like activity of the 20S proteasome), incubated in the dark at 37 °C for 30 min, and fluorescence intensity was measured at kex = 380 nm, kem = 440 nm, using a microplate reader (Infinite M200 Pro, Tecan, Switzerland). Initial experiments included samples incubated with lactacystin, serving as blanks. Since there was no difference between the results of experiments using lactacystin blanks or non-sample blanks (consisting of the homogenization buffer added to the reaction buffer; paired t test t29 = 0.009, p = 0.993), we omitted the lactacystin blanks from the results, and used non sample blanks.

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2.5.2. SDS PAGE and western blotting Aliquots of the homogenate in SDS Laemmli sample buffer were subjected to SDS-PAGE and Western blot analysis. After electrophoresis and electroblotting onto a nitrocellulose membrane, the blots were blocked in freshly prepared Tris-buffered saline solution containing 0.1% Tween 20 (TBST) with 3–5% BSA for 1 h at room temperature, with agitation. The blots were incubated with primary antibody (ubiquitin K48, 1:2000, Merck, Darmstadt, Germany; b actin, 1:6000, Santa-Cruz Biotechnology, Dallas, TX; PSMB4 (a proteasome catalytic subunit) 1:1000, abcam, San-Francisco, CA, USA) overnight at 4 °C. After three 5 min washing steps with TBST, the blots were incubated for 1 h at room temperature with the corresponding secondary antibody (goat-anti-Rabbit (IgG) HRP conjugated; and rabbit anti-goat (IgG) HRP conjugated, 1:10,000, Jackson ImmunoResearch West Grove, PA, USA)). The blots were then washed three times in TBST and treated with an enhanced chemiluminescence (EZ ECL) kit (Bet Haemek, Israel). 2.5.3. Statistical analysis All grouped data are presented as mean ± SEM. Comparisons between data of two independent groups were analyzed by unpaired Student’s t test and the differences between the variances of groups were corrected following Levene’s test for equality of variances. Multiple group comparisons were assessed using one way analysis of variance (ANOVA), repeated measures ANOVA, and two-way ANOVA, as indicated. Follow-up analyses were conducted using Tukey’s, Sidak’s, and Dunnett’s multiple comparisons.

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All comparisons were conducted using two-tailed tests of significance. The null hypothesis was rejected at the P < 0.05 level. Data analysis was performed using GraphPad Prism 6.

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3. Results

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3.1. Proteasome activity in the gustatory cortex (GC) is increased 4 h following novel taste consumption

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Novel taste learning, also referred to as incidental taste learning or attenuation of neophobia, is considered as a positive taste learning paradigm, in which there is no association of the taste with additional stimuli, and over time the subjects consume more of the presented taste (Bureš et al., 1998). To examine the effect of novel taste learning on ubiquitin-dependent proteasome activity, rats were exposed to either a novel taste (saccharin 0.1%) or a familiar taste (water), and were sacrificed at different time points following taste consumption (2, 4, 6, and 8 h). Proteasome activity was assessed in the gustatory cortex (GC) and occipital cortex (OC), and results of each time point were normalized to the control, water consuming group, and analyzed using multiple t-tests (Fig. 1a–c). The results indicate a significant increase in proteasome activity, specifically 4 h following novel taste consumption (novel saccharin 139.1% ± 4.7%; water 100% ± 1.6%; t28 = 4.2, p = 0.0003), but not after 2 h (novel saccharin 97.2% ± 10.8%; water 100% ± 9.7%; t16 = 0.19, p = 0.85), 6 h (novel saccharin 113.1% ± 10.7%; water 100% ± 10.0%; t20 = 0.89, p = 0.38), or 8 h (novel saccharin 99.0% ± 9.8%; water 100% ± 1.7%; t15 = 0.08, p = 0.94; Fig. 1a). The increase in proteasome activity 4 h after novel saccharin consumption was localized to the GC and was not evident in the OC in any of the time points examined (2 h: novel saccharin 104.5% ± 4.8%; water 100% ± 2.7%; t16 = 0.81, p = 0.46; 4 h: novel saccharin 98.0% ± 15.6%; water 100% ± 5.1%; t28 = 0.12, p = 0.91 6 h: novel saccharin 97.9% ± 8.6%; water 100% ± 31.3%; t20 = 0.89, p = 0.38; 8 h: novel saccharin 106.5% ± 10.7%; water 100% ± 15.8%; t15 = 0.31, p = 0.76; Fig. 1c). To assess the effect of taste familiarization on proteasome activity 4 h following taste consumption, rats were exposed to a novel taste (saccharin 0.1%, single exposure), a familiar taste (water), or a familiarized taste (saccharin 0.1%, exposure for 10 days). Results were normalized to the water group (Fig. 1d–g) and analyzed using one-way ANOVA with Tukey’s multiple comparisons. The results show that chymotrypsin-like proteasome activity was significantly higher in the novel saccharin group compared to both familiar saccharin (novel saccharin 140% ± 7.2%; familiar saccharin 114% ± 3.3%; p = 0.02) and water (water 100% ± 4.1%; p = 0.0005; F2,11 = 15.3, p = 0.0007). Moreover, proteasome activity did not differ between the water and the familiar saccharin groups (water 100% ± 4.1%; familiar saccharin 114% ± 3.3%; p = 0.22). The samples used to measure proteasome activity were also analyzed for K48 ubiquitination (Fig. 1f and g). One-way ANOVA and Tukey’s post hoc comparisons revealed that similarly to proteasome activity, K48 ubiquitination did not differ between the water and the familiar saccharin groups (water 1.0 ± 0.04; familiar saccharin 0.99 ± 0.10; p = 1.0), and was significantly higher in the group exposed to novel saccharin compared to familiar saccharin (novel saccharin 1.4 ± 0.12; p = 0.02) and water group (p = 0.02; F2,11 = 6.9, p = 0.01; Fig. 1f). These results suggest that the increase in GC proteasome activity 4 h after novel taste consumption is specifically correlated with novel but not familiar taste consumption.

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3.2. The increase in proteasome activity following novel taste consumption is NMDAR-dependent

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To explore the hypothesis that the increase observed in proteasome activity in the GC 4 h after taste learning is NMDAR-

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Fig. 1. Novel taste consumption induces an increase in GC proteasome activity. (a) Time dependency of proteasome activity following novel taste consumption – schematic representation of the experimental protocol. (b) Increased proteasome activity in the GC 4 h after saccharin (0.1%) consumption (n = 15) compared with water (n = 15; ⁄⁄⁄ p = 0.0003), but not 2 h (saccharin = 10, water n = 8; p = 0.85), 6 h (saccharin n = 11, water n = 11; p = 0.38), or 8 h (saccharin n = 9, water n = 8; p = 0.93). (c) There are no changes in OC proteasome activity 2 h (saccharin n = 10, water n = 8; p = 0.46), 4 h (saccharin n = 15, water n = 15; p = 0.91), 6 h (saccharin n = 11, water n = 11; p = 0.95), or 8 h (saccharin n = 9, water n = 8; p = 0.76) following novel taste consumption. (d) Proteasome activity 4 h following consumption of novel or familiar saccharin, schematic representation of the experimental protocol. (e) Novel saccharin (n = 5) induces an increase in GC proteasome activity 4h after consumption compared to familiar saccharin (n = 4; ⁄ p = 0.02) or water (n = 5; ⁄⁄⁄ p = 0.0005). Familiar saccharin (n = 4) compared to water (n = 5) does not induce changes in proteasome activity (p = 0.22). (f) Novel saccharin (n = 5) induces an increase in K48 ubiquitination 4 h after consumption compared to familiar saccharin (n = 4; ⁄ p = 0.02) or water (n = 5; ⁄ p = 0.02). Familiar saccharin (n = 5) compared to water (n = 5) does not induce changes in K48 ubiquitination (n = 4, 5; p = 1). (g) K48 ubiquitin and actin representative blots. Results are presented as mean ± SEM (⁄ p < 0.05; ⁄⁄⁄ p < 0.001).

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dependent, rats were bilaterally stereotaxically microinjected into the GC with NMDAR antagonist (2R)-amino-5-phosphonopenta noate (APV, 50 lM) or artificial cerebrospinal fluid (aCSF as vehicle solution). Twenty minutes later, the rats consumed water or saccharin for 20 min, and were sacrificed 4 h following consumption. Chymotrypsin-like proteasome activity and K48 ubiquitination were measured and normalized to the vehicle-microinjected water consuming group, and analyzed using two-way ANOVA with Tukey’s post-hoc comparisons to determine treatment (vehicle vs. APV) and novel taste (water vs. saccharin) effects (Fig. 2a–e). The results show that the correlative increase observed in proteasome activity following novel taste consumption was completely abolished when NMDARs in the GC were blocked using local injection of APV before learning: the APV-microinjected, saccharin consuming group did not differ from the

APV-microinjected, water consuming group (APV-saccharin 102.3% ± 6.8%; APV-water 102.4% ± 9.4%; p = 1.0), or from the vehicle-microinjected, water consuming group (vehicle–water 100% ± 7.4%; p = 1.0). As in the previous section, the vehicle–saccharin group showed an increase in proteasome activity compared to the vehicle–water group (vehicle–saccharin 139.8% ± 10.1%; p = 0.01), as well as the APV-saccharin group (p = 0.02). There was a significant APV effect (F1,33 = 4.42, p = 0.04) as well as novel taste effect (F1,33 = 5.02, p = 0.03), and a clear interaction between the APV and novel taste (F1,33 = 5.69, p = 0.02; Fig. 2b). Protein ubiquitination assessment showed similar results: The APV-saccharin group did not differ from the APV-water group (APV-saccharin 0.93 ± 0.14; APV-water 1.15 ± 0.23; p = 0.97), or from the vehicle–water group (vehicle–water 1.0 ± 0.11; p = 0.98). As in Section 3.1, the vehicle-novel saccharin group

Please cite this article in press as: Rosenberg, T., et al. NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.01.002

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Fig. 2. APV and TatCN21 prevent the increase in GC proteasome activity 4 h after novel saccharin consumption. (a) Schematic representation of the experimental protocol. (b) GC proteasome activity does not change 4 h after novel saccharin consumption in rats injected with APV (50 lM) (n = 9) compared to the vehicle-injected, water consuming group (n = 9; p = 1). APV injected to water consuming rats (n = 9) had no effect on proteasome activity compared to the vehicle-injected, water consuming group (n = 9; p = 1). Novel saccharin consumption induced an increase in proteasome activity of saline-injected rats (n = 10) compared to the saline-injected, water consuming group (n = 9; ⁄ p = 0.01) as well as the APV-injected, novel saccharin group (n = 9; ⁄ p = 0.02). (c) There were no differences in fluid intake between different tastes (F1,33 = 0.06; p = 0.8), or different treatments (F1,33 = 0.8; p = 0.4). (d) GC K48 ubiquitination does not change 4 h after novel saccharin consumption in rats injected with APV (50 lM) (n = 9) compared to the vehicle-injected, water consuming group (n = 9; p = 0.98). APV injected to water consuming rats (n = 9) had no effect on K48 ubiquitination compared to the vehicleinjected, water consuming group (n = 9; p = 0.84). Novel saccharin induced an increase in K48 ubiquitination of saline-injected rats (n = 10) compared to the saline-injected water consuming group (n = 9; ⁄⁄ p = 0.004) as well as the APV injected novel saccharin group (n = 9; ⁄ p = 0.01). (e) Proteasome catalytic subunit PSMB4 levels does not change between the different tastes (F1,33 = 0.27; p = 0.61), or different treatments (F1,33 = 0.01; p = 0.92). (f) K48 ubiquitin, PSMB4 and actin representative blots. (g) Schematic representation of the experimental protocol. (h) GC proteasome activity does not change 4 h after novel saccharin consumption in rats injected with TatCN21(Tat) 0.3 nM (n = 5) compared to the Tat control-injected, water consuming group (n = 5; p = 1). Tat injected to water consuming rats (n = 5) had no effect on proteasome activity compared to the vehicle-injected, water consuming group (n = 5; p = 0.99). Novel saccharin induced an increase in proteasome activity of the Tat control group (n = 5) compared to the Tat control water group (n = 5; ⁄ p = 0.01) as well as the Tat novel saccharin group (n = 5; ⁄⁄ p = 0.0097). Results are presented as mean ± SEM. ⁄ p < 0.05; ⁄⁄ p < 0.01; # represents treatment effect p < 0.05.

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showed an increase in K48 ubiquitination compared to the vehicle–water group (vehicle–saccharin 1.3 ± 0.14; p = 0.004), as well as the APV-saccharin group (p = 0.01). There was a significant treatment effect (F1,33 = 4.2, p = 0.049) and novel taste effect (F1,33 = 4.3, p = 0.047), as well as interaction between the APV and novel taste (F1,33 = 10.74, p = 0.002; Fig. 2d and f). Of note, insufficient saccharin intake in the treatment group may produce similar results (Merhav and Rosenblum, 2008). However, two-way ANOVA analysis of taste solution consumption revealed that there was no significant treatment effect (F1,33 = 0.81, p = 0.37) or a novel taste effect (F1,33 = 0.06, p = 0.8), i.e., APV did not affect volume intake (Fig. 2c). Taken together, these results suggest that that the increase observed in proteasome

activity as well as in K48 ubiquitination 4 h following novel taste consumption is NMDAR-dependent. NMDAR-dependent increase in proteasome activity may be mediated via CaMKII phosphorylation of Serine 120 in the proteasome Rpt6 subunit. This increase in proteasome activity was documented previously following fear learning in the amygdala (Jarome et al., 2013). In order to assess whether this increase in proteasome activity is also CaMKII-dependent, we injected CaMKII inhibitory peptide TatCN21 (Tat; 1 lL/hemisphere; 0.3 nM/lL) or Tat control (Buard et al., 2010) 20 min before novel taste learning, and measured proteasome activity 4 h later (Fig. 2g and h). Results were analyzed using two way ANOVA and Tukey’s multiple comparisons. There was a clear novel taste effect (F1,15 = 7.67,

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p = 0.014) and a clear treatment effect (F1,15 = 7.43, p = 0.016). Posthoc comparisons show a significant difference between the water and saccharin groups for the Tat control group (p = 0.01), however, there was no difference between the water and saccharin groups treated with Tat (p = 1). In addition, proteasome activity in the animals treated with Tat control saccharin was significantly higher than in animals treated with Tat and saccharin (p = 0.0097), and there is no difference between the water groups (p = 0.99). There was a significant interaction between the treatment and the novel taste (F1,15 = 7.07, p = 0.02; Fig. 2h). Therefore, the increase in GC proteasome activity 4 h following novel taste learning is not only NMDAR-dependent but also CaMKII-dependent. This NMDAR–CaMKII effect could also be the outcome of increased translation of the proteasome itself. To test this possibility, we blotted the samples from the APV experiment against PSMB4, one of the catalytic subunits of the proteasome (Fig. 2e and f). There was no difference in the amount of PSMB4, and two way ANOVA showed that there was no significant novel taste effect (F1,33 = 0.27, p = 0.61) or an APV effect (F1,33 = 0.01, p = 0.92; Fig. 2e).

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In order to examine whether a negative learning paradigm affects GC proteasomal activity and protein ubiquitination similarly to a positive one, the conditioned taste aversion (CTA) paradigm was used. CTA is a negative form of taste learning in which the subjects associate novel taste (conditioned stimulus, CS) with a negative value, i.e., malaise (unconditioned stimulus, US), creating an aversive conditioned response to the associated taste (Bureš et al., 1998; Rosenblum et al., 1993). To study the effect of CTA on GC proteasome activity and protein ubiquitination, samples were taken from rats which consumed either water or 0.1% saccharin (novel taste) and were i.p. injected with either the malaiseinducing agent LiCl (0.15 M) or saline (0.9% NaCl). Results were normalized to the water consuming group injected with saline, and two-way ANOVA with Tukey’s post-hoc comparisons was used to determine treatment effect (LiCl vs. saline) and novelty effect (water vs. saccharin). The LiCl-injected, saccharin consuming (CTA) group showed a significant increase in GC proteasome activity compared to the LiCl-injected, water consuming group (CTA 118.9% ± 4.1%; LiCl–water 92.4% ± 2.4%; p = 0.004), indicating that on its own, LiCl does not increase proteasome activity. Moreover, there was a non-significant decrease in proteasome activity of the CTA group compared with the saline-injected, saccharin consuming group (saline–saccharin 134% ± 8.1%; 0.15). The LiCl water group also showed a non-significant reduction in activity compared to the saline-injected water group (saline-water 100% ± 2.9%; p = 0.70). Similarly to the results in Figs. 1 and 2b, GC proteasome activity was increased in the saline-injected saccharin group compared to the saline-injected water group (p = 0.0005). There was a significant treatment effect (F1,21 = 5.3, p = 0.03), indicating that the LiCl reduces proteasome activity, and a significant novelty effect (F1,21 = 38.3, p < 0.0001). There was no interaction between the LiCl and the novelty (F1,21 = 0.56, p = 0.46) (Fig. 3b). Blotting against K48 ubiquitin and actin produced similar results (Fig. 3c). The CTA group showed a significant increase in K48 ubiquitination compared to the LiCl–water group (CTA 1.34 ± 0.02; LiCl–water 0.9 ± 0.05; p = 0.005). GC K48 ubiquitination was increased in the saline–saccharin group compared to the saline-water group (saline–saccharin 1.44 ± 0.09; salinewater 1.0 ± 0.08; p = 0.007). There was a significant treatment effect (F1,21 = 4.7, p = 0.048) and a significant novelty effect (F1,21 = 37.2, p < 0.0001). There was no interaction between LiCl and novelty (F1,21 = 0.45, p = 0.51; Fig. 3c).

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Total fluid consumption was measured in order to rule out the possibility that the effect of CTA on GC proteasome activity and protein ubiquitination is due to changes in consumption. Twoway ANOVA showed no treatment effect (F1,21 = 0.98, p = 0.33) or novelty effect (F1,21 = 2.397, p = 0.14) (Fig. 3e). These results indicate that the increase in proteasome activity and protein K48 ubiquitination is correlated with CTA in a similar way to novel taste consumption, which serves as the CS in CTA learning, i.e., both positive and negative learning forms result in increased proteasomal activity and K48 ubiquitination. Next we assessed whether the increase in proteasome activity following CTA is NMDAR-dependent. Rats were microinjected with APV (50 lM/vehicle (aCSF)) 20 min before CTA or water consumption followed by i.p. injection of saline. The animals were sacrificed 3 h after the i.p. injection (overall, 4 h after fluid consumption), and GC chymotrypsin-like proteasome activity and protein K48 ubiquitination were measured (Fig. 3f–i). Results were normalized to the vehicle-microinjected, water drinking, saline i.p. injected (aka water–vehicle) group, and analyzed using one-way ANOVA with Tukey’s post-hoc comparisons. Blockade of the NMDAR using APV completely abolished the increase observed both in GC proteasome activity and protein K48 ubiquitination following CTA (Fig. 3g and h). The CTA-APV group showed significantly lower proteasome activity compared to the CTA-vehicle group (CTA-APV 89.0% ± 9.9%; CTA-vehicle 130.2% ± 8.0%; p = 0.006), and did not differ from the water–vehicle control group (water–vehicle 100% ± 4.6%; p = 0.62). The CTAvehicle group presented significantly higher proteasome activity than the water–vehicle group (p = 0.04) (F2,13 = 7.7, p = 0.006). Blotting against K48 ubiquitin and actin produced similar results (Fig. 3h). K48 ubiquitination was significantly lower in the CTA-APV group compared to the CTA-vehicle group (CTA-APV 0.92 ± 0.07; CTA-vehicle 1.31 ± 0.08; p = 0.004), and showed similarity to the water–vehicle group (water vehicle 1.0 ± 0.06; p = 0.73). Protein K48 ubiquitination was significantly increased in the CTA-vehicle group compared to the water–vehicle group (p = 0.02) (F2,13 = 8.8, p = 0.004). To rule out the possibility that the changes in proteasome activity and protein K48 ubiquitination resulted from differences in fluid consumption, total fluid intake was measured and analyzed using one-way ANOVA. No difference was found in fluid intake between the groups (F2,13 = 1.1, p = 0.3; Fig. 3j). Altogether, the results above demonstrate that the increase in GC proteasome activity following novel taste consumption, as well as CTA, which includes the CS (novel taste), is NMDAR-dependent.

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After establishing that NMDAR-dependent proteasome activity occurs following novel taste consumption (Figs. 1 and 2) and CTA (Fig. 3), we set to find out if this increase is also necessary for these positive and negative forms of taste learning. Since we found that the increase in proteasome activity in the GC occurs following CTA and novel taste consumption, which serves as the CS in CTA, but not following LiCl injection, which serves as UCS, we hypothesized that proteasome inhibition in the GC would disrupt CTA (Fig. 4). Since inhibition of proteasome activity via administration of high concentrations (over 20 mM) of lactacystin promotes cell death (McNaught, Olanow, Halliwell, Isacson, & Jenner, 2001), we used a low concentration of the proteasome inhibitor, lactacystin, to induce weak proteasomal inhibition and avoid cell death. To find a lower concentration of lactacystin which would inhibit proteasome activity efficiently, we incubated rat GC lysate samples with lower concentrations of lactacystin (0.5, 1, 2.5, 5, 7.5, 10 lM) for 40 min. Results were analyzed via one-way ANOVA with Dun-

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Fig. 3. CTA induces an increase in GC proteasome activity 4 h after consumption, which is abolished by APV. (a) Measuring proteasome activity following CTA, schematic representation of the experimental protocol. (b) CTA group (n = 7) shows an increase in proteasome activity 4 h after novel saccharin consumption compared with the LiCl– water group (n = 6; ⁄⁄ p = 0.004), as well as the saline-water group (n = 6; ⁄ p = 0.049). There was no significant difference between the saline–saccharin group (n = 6) and the CTA group (n = 7; p = 0.15). (c) K48 ubiquitination is increased 4 h after CTA (n = 7) compared to the LiCl–water group (n = 6; ⁄⁄ p = 0.005) as well as the saline-water group (n = 6; ⁄ p = 0.029). There was no significant difference between the saline–saccharin group (n = 6) and the CTA group (n = 7; p = 0.74). (d) K48 ubiquitin and actin representative blots. (e) There were no differences in fluid consumption between different tastes (F1,21 = 2.39; p = 0.14) or between different treatments (F1,21 = 0.98; p = 0.33). (f) Measuring proteasome activity 4 h after CTA following APV injection; schematic representation of experimental protocol. (g) APV (50 lM) injected 20 min before CTA abolishes the increase in proteasome activity 4h after CTA. Proteasome activity in the CTA-APV group (n = 5) is significantly lower than in the CTA-vehicle group (n = 6; ⁄⁄ p = 0.006), and does not differ from the vehicle–water group (n = 5; p = 0.62). Proteasome activity in the CTA-vehicle group (n = 6) is increased compared to the vehicle– water group (n = 5; ⁄ p = 0.042). (h) K48 ubiquitination in the CTA-APV group (n = 5) is significantly lower than in the CTA-vehicle group (n = 6; ⁄⁄ p = 0.005), and does not differ from the vehicle–water group (n = 5; p = 0.73). K48 ubiquitination in the CTA vehicle group (n = 6) is increased compared to the vehicle–water group (n = 5; ⁄ p = 0.021). (i) Representative blots of K48 and actin. (J) There were no differences in fluid consumption between groups (F2,13 = 1.13; p = 0.35). Results are presented as mean ± SEM.⁄ p < 0.05; ⁄⁄ p < 0.01; ⁄⁄⁄ p < 0.001. # represents treatment effect p < 0.05.

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nett’s post-hoc comparing each concentration to the non-inhibited control samples. A significant decrease in chymotrypsin-like proteasome activity was observed using 5–10 lM lactacystin (n = 3, p < 0.001; Fig. 4a). Therefore, a dosage of 10 lM lactacystin was selected for stereotaxic administration into the GC. Local microinjection of lactacystin (10 lM) to the GC significantly reduced proteasome activity 4 h following injection (lactacystin 71% ± 4.2%; vehicle 100% ± 6.9%; t7 = 2.5, p = 0.04) and

5 h (lactacystin 78% ± 3.6%; vehicle 100% ± 5.7%; t7 = 3.1, p = 0.02) but not 1 h (lactacystin 105.6% ± 14.1; vehicle 100% ± 9.7; t8 = 0.32, p = 0.75), 2 h (lactacystin 87.7% ± 10.5; vehicle 100% ± 18.7; t7 = 0.66, p = 0.53), or 6 h following injection (lactacystin 93.8% ± 11.9%; vehicle 100% ± 20.3%; t3 = 0.26, p = 0.80; Fig. 4b and c). To test the hypothesis that weak inhibition of proteasome activity would disrupt CTA, lactacystin (10 lM)/ vehicle (aCSF) was

Please cite this article in press as: Rosenberg, T., et al. NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.01.002

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Fig. 4. Proteasomal inhibition impairs CTA but does not affect taste preference. (a) Proteasome inhibitor lactacystin ex vivo calibration. Different doses (0–10 lM) of lactacystin added directly to tissue lysate in a microplate show significant reduction in activity compared to control for 5 lM (n = 3; ⁄⁄⁄ p = 0.0007), 7.5 lM (n = 3; ⁄⁄⁄ p < 0.0001), and 10 lM (n = 3; ⁄⁄⁄ p < 0.0001). (b) Lactacystin 10 lM in vivo calibration; schematic representation of experimental protocol. (c) Following lactacystin (10 lM) injection, there is a significant reduction in proteasome activity 4 h and 5 h compared to the vehicle-group (4 h: lactacystin n = 4, vehicle n = 5, ⁄ p = 0.04; 5 h: lactacystin n = 4, vehicle n = 5; ⁄ p = 0.02), but not 1 h (lactacystin n = 5, vehicle n = 5; p = 0.75), 2 h (lactacystin n = 4, vehicle p = 5; p = 0.53), or 6 h (lactacystin n = 4, vehicle n = 4; p = 0.80) following injection. (d) Lactacystin injected immediately before CTA; schematic representation of experimental protocol. (e) Lactacystin (10 lM) injected 20 min before CTA (n = 8) significantly reduced the aversion index compared to the control group (n = 7; ⁄⁄ p = 0.0062). (f) Lactacystin (10 lM) injected 20 min before CTA (n = 8) had no effect on saccharin consumption compared to the control group (n = 7; p = 0.55). (g) Lactacystin injected 20 min before AN; schematic representation of experimental protocol. (h) Lactacystin (10 lM) injected 20 min before taste consumption had no effect on AN (F1,19 = 0.002; p = 0.96). There was a significant time effect (F1,19 = 28.5; p < 0.0001), i.e. AN was intact in both groups. Results are presented as mean ± SEM. ⁄ Indicates p < 0.05; ⁄⁄ p < 0.01; ⁄⁄⁄ p < 0.001.

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bilaterally stereotaxically microinjected shortly (20 min) before CTA. Aversion index was measured 72 h following CTA, and results were analyzed using t-test (Fig. 4d and e). Lactacystin local injection to the GC significantly reduced the aversion index (Lactacystin 66.1% ± 8.6%; vehicle 96.2% ± 0.7%; t13 = 3.26, p = 0.006; Fig. 4e). To confirm that the reduction in aversion was not due to insufficient saccharin consumption in the lactacystin group, saccharin raw intake data was analyzed using t-test. The results show that there was no difference in saccharin consumption between groups (Lactacystin 11.7 ± 0.6; vehicle 12.2 ± 0.7; t13 = 0.62, p = 0.55) (Fig. 4f). These results indicate that the increase in GC proteasome activity is not only correlative, but necessary for CTA. This increase in proteasome activity following CTA occurs even in the absence of the UCS, i.e. LiCl injection, following novel taste consumption, and thus may be necessary also for incidental taste

learning. We hypothesized that similar inhibition of proteasomal activity would affect novel taste memory per se. Therefore, lacracystin (10 lM)/vehicle (aCSF) was bilaterally stereotaxically microinjected to the GC 20 min before subjection of the animals to the attenuation of neophobia paradigm (Fig. 4g and h). Attenuation of neophobia is a positive form of taste learning. Since subjects are generally more suspicious toward novel tastes (neophobic), they tend to increase consumption of a novel taste after familiarization with it over time. Attenuation of neophobia refers to the increase in consumption of the taste after the first (novel) encounter, and serves as a measurement of positive taste learning (Bureš et al., 1998). During the first day (novel day), animals microinjected with lactacystin/vehicle were given a choice between water and novel saccharin. 72 h later, the animals were given a choice between

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water and saccharin again (Familiar day). Fluid consumption was measured during the novel and familiar day, preference index was calculated, and the results were analyzed by two-way repeated measures ANOVA with Sidak’s post-hoc comparisons (Fig. 4g and h). Lactacystin (10 lM) had no effect by itself (F1,19 = 0.002, p = 0.96), nor was there an interaction between lactacystin and the novel taste (F1,19 = 0.87, p = 0.36). The increased saccharin consumption due to attenuation of neophobia was significant (F1,19 = 28.5, p < 0.0001) both in the lactacystin group (novel 44.8% ± 2.1%, familiar 58.3% ± 0.3%, p = 0.0004) and the vehicle group (vehicle novel 46.7% ± 2.4%, familiar 56.2% ± 2.2%, p = 0.01; Fig. 4h). Thus, these results refuted the hypothesis that increased proteasome activity in the GC is also necessary for novel taste learning.

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A major finding of this study is that proteasome activity is increased 4 h following taste learning in the cortex, a brain structure assumed to store long term memories (Frankland & Bontempi, 2005). The temporal phase is in line with a previous study, which demonstrated an increase in the chymotrypsin-like proteasome activity in the hippocampus 4 h following avoidance learning (Lopez-Salon et al., 2001). Interestingly, the increase in proteasome activity we observed in the cortex was necessary for the associative taste learning paradigm, CTA, but not for positive taste learning. It has been previously shown that consolidation of other forms of negative associative learning, such as fear learning, is interrupted by proteasomal inhibition in the prefrontal cortex (Reis, Jarome, & Helmstetter, 2013) and the amygdala (Jarome, Werner, Kwapis, & Helmstetter, 2011; Jarome et al., 2013). Studies which examined the effect of hippocampal proteasome inhibition on hippocampal-dependent forms of learning have shown that different dosages are required for consolidation and reconsolidation in the hippocampus (Lee et al., 2008; Sol Fustinana et al., 2014). Here, we showed attenuation of CTA via effective proteasome inhibition using lactacystin. These results are in contradiction to a former study reported no effect of proteasome inhibition in the GC with a different dosage injected shortly before a modified version of CTA (Rodriguez-Ortiz et al., 2011). This conflict can be explained by the properties of different proteasome inhibitors. While lactacystin proteasome-specific and irreversible, its effect is mild compared to its active derivative, clasto b lactone. In addition, since the lactacystin has to be converted to its active form, its inhibitory effect is slower than clasto b lactone (Dick et al., 1996). Since different inhibitors promote different effects, we measured the effect of low dosage lactacystin (10 lM) in vivo (Fig. 4b and c), and found it to be effective only 4 h and 5 h after injection. This delayed reduction in proteasome activity is likely the outcome of using lactacystin, which has to be converted to clasto b lactone prior to its activation. Interestingly, there is a distinction between negative associative taste learning, CTA, which is sensitive to NMDAR-dependent proteasome activity in the GC and attenuation of neophobia, which is not (Parkes et al., 2014). Our results suggest that increased proteasome activity 4 h following novel taste consumption is necessary for the association process but not for incidental taste memory. Although an increase in proteasome activity occurred following either consumption of novel taste alone or combined consumption of novel taste and visceral malaise (via LiCl injection), there was a small, but significant LiCl effect, but no interaction between the novelty and LiCl (Fig. 3a–c). This implies that LiCl does not influence GC proteasome activity via the NMDAR. However, the association of novel taste and malaise requires this NMDARdependent increase in proteasome activity.

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Proteasome inhibitors have also been shown to influence associative but not positive gastric learning in the context of addiction. For example, proteasome inhibition in the nucleus accumbens core has been shown to impair animal performance in several associative behavioral paradigms of opiate addiction: morphine induced locomotor sensitization, morphine conditioned place preference, intra-ventral tegmental area morphine self-administration and intra-venous heroin self-administration. In contrast, proteasome inhibition does not affect animal performance in the nonassociative learning paradigm of discrimination learning rewarded by palatable foods (Massaly, Frances, & Mouledous, 2014). The strength of the negative association formed during CTA is time-dependent. Increasing the inter-time interval (ITI) between novel taste (CS) and the injection of the LiCl (US) results in decreased aversion. Moreover, the activation of synaptosomal CaMKII in the GC occurs shortly after taste learning, and persists for 3 h (Adaikkan & Rosenblum, 2015). These findings suggest that there is a critical period following novel taste consumption during which visceral negative stimulation can be associated with the taste to form a strong CTA trace. This time window following novel taste consumption is accompanied by an increase in CaMKII activity, which is also upstream to proteasome activation. Several recent studies have explored the molecular mechanism by which activation of the NMDAR induces activation of the proteasome and its downstream consequences. For example, chemical NMDAR activation-induced LTD requires intact ubiquitination rather than proteasome activity (Citri, Soler-Llavina, Bhattacharyya, & Malenka, 2009). A different study showed that NMDAR stimulation attenuated proteasome activity (Tai et al., 2010). Other studies showed that NMDAR stimulation in hippocampal culture induces CaMKII-dependent phosphorylation of proteasome subunit Rpt6, which in turn activates the proteasome, leading to its recruitment into synapses (Djakovic et al., 2009, 2012). In vivo, NMDAR dependent-CaMKII activation in the amygdala results in increased phosphorylation of proteasome subunit Rpt6 and facilitation of proteasome activity. This increase is also necessary for the memory of fear (Jarome et al., 2011, 2013). In a similar way to protein degradation, NMDAR can activate different cellular responses including the mTOR/S6K pathway, BDNF translation, eEF2 phosphorylation, and translation downregulation (Gal-Ben-Ari et al., 2012; Gildish et al., 2012). The involvement of NMDAR signaling in several protein translation/ degradation pathways may point to a possible role that this receptor plays in proteostasis, i.e., the combined action of several cellular pathways which generates a certain translation/degradation homeostatic status. This status cannot be determined via a single protein. Rather, it is a certain proteome in a certain subcellular compartment, which can determine the fate of a given phenotype or a memory in our case (Rosenberg et al., 2014). It is possible that different types of learning, such as CTA or attenuation of neophobia, subserved by the same brain region, require different proteostatic modulations. Here, we identified the specific role which NMDAR-dependent proteasome degradation plays in maintaining taste memory trace for future association with visceral information.

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Funding and disclosure

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This work was supported by German-Israeli Foundation DIP (RO3971/1-1) and ISF (1003/12) for KR.

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5. Uncited references

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Berman, Hazvi, Rosenblum, Seger, and Dudai (1998), Lin, Arthurs, and Reilly (2015) and Schachtman et al. (2003).

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We thank Dr. Hwan-Ching Tai for the protocols of the proteasome activity assay buffers, and Dr. Nurit Livnat Levanon and Dr. Dasha Krutauz from the laboratory of Prof. Michael Glickman for their kind help in establishing the proteasome activity assay protocol. We thank laboratory members of KR, specifically Dr. Shunit Gal Ben-Ari and Sara Murru.

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References

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Adaikkan, C., & Rosenblum, K. (2015). A molecular mechanism underlying gustatory memory trace for an association in the insular cortex. eLife, 4. http://dx.doi.org/ 10.7554/eLife.07582. Ben-Nissan, G., & Sharon, M. (2014). Regulating the 20S proteasome ubiquitinindependent degradation pathway. Biomolecules, 4(3), 862–884. Berman, D. E., Hazvi, S., Rosenblum, K., Seger, R., & Dudai, Y. (1998). Specific and differential activation of mitogen-activated protein kinase cascades by unfamiliar taste in the insular cortex of the behaving rat. The Journal of Neuroscience, 18(23), 10037–10044. Bingol, B., & Schuman, E. M. (2006). Activity-dependent dynamics and sequestration of proteasomes in dendritic spines. Nature, 441(7097), 1144–1148. Bonin, R. P., & De Koninck, Y. (2015). Reconsolidation and the regulation of plasticity: moving beyond memory. Trends in Neurosciences, 38(6), 336–344. Buard, I., Coultrap, S. J., Freund, R. K., Lee, Y. S., Dell’Acqua, M. L., Silva, A. J., et al. (2010). CaMKII ‘‘autonomy” is required for initiating but not for maintaining neuronal long-term information storage. The Journal of Neuroscience, 30(24), 8214–8220. Bureš, J., Bermudez-Rattoni, F., & Yamamoto, T. (1998). Conditioned taste aversion: Memory of a special kind. Oxford; New York: Oxford University Press. Ciechanover, A. (2013). Intracellular protein degradation: From a vague idea through the lysosome and the ubiquitin–proteasome system and onto human diseases and drug targeting. Bioorganic & Medicinal Chemistry, 21(12), 3400–3410. Citri, A., Soler-Llavina, G., Bhattacharyya, S., & Malenka, R. C. (2009). N-methyl-Daspartate receptor- and metabotropic glutamate receptor-dependent long-term depression are differentially regulated by the ubiquitin–proteasome system. European Journal of Neuroscience, 30(8), 1443–1450. Dick, L. R., Cruikshank, A. A., Grenier, L., Melandri, F. D., Nunes, S. L., & Stein, R. L. (1996). Mechanistic studies on the inactivation of the proteasome by lactacystin: A central role for clasto-lactacystin b-lactone. Journal of Biological Chemistry, 271(13), 7273–7276. Djakovic, S. N., Marquez-Lona, E. M., Jakawich, S. K., Wright, R., Chu, C., Sutton, M. A., et al. (2012). Phosphorylation of Rpt6 regulates synaptic strength in hippocampal neurons. The Journal of Neuroscience, 32(15), 5126–5131. Djakovic, S. N., Schwarz, L. A., Barylko, B., DeMartino, G. N., & Patrick, G. N. (2009). Regulation of the proteasome by neuronal activity and calcium/calmodulindependent protein kinase II. Journal of Biological Chemistry, 284(39), 26655–26665. Dudai, Y. (2012). The restless engram: Consolidations never end. Annual Review of Neuroscience, 35, 227–247. Elkobi, A., Ehrlich, I., Belelovsky, K., Barki-Harrington, L., & Rosenblum, K. (2007). ERK-dependent PSD-95 induction in the gustatory cortex is necessary for taste learning, but not retrieval. Nature Neuroscience, 11(10), 1149–1151. Frankland, P. W., & Bontempi, B. (2005). The organization of recent and remote memories. Nature Reviews Neuroscience, 6(2), 119–130. Gal-Ben-Ari, S., Kenney, J. W., Ounalla-Saad, H., Taha, E., David, O., Levitan, D., ... Rosenblum, K. (2012). Consolidation and translation regulation. Learning & Memory, 19(9), 410–422. Gildish, I., Manor, D., David, O., Sharma, V., Williams, D., Agarwala, U., ... Rosenblum, K. (2012). Impaired associative taste learning and abnormal brain activation in kinase-defective eEF2K mice. Learning & Memory, 19(3), 116–125. 24. Jarome, T. J., & Helmstetter, F. J. (2013). The ubiquitin–proteasome system as a critical regulator of synaptic plasticity and long-term memory formation. Neurobiology of Learning and Memory, 105, 107–116.

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Jarome, T. J., Kwapis, J. L., Ruenzel, W. L., & Helmstetter, F. J. (2013). CaMKII, but not protein kinase A, regulates Rpt6 phosphorylation and proteasome activity during the formation of long-term memories. Frontiers in Behavioral Neuroscience, 7, 115. Jarome, T. J., Werner, C. T., Kwapis, J. L., & Helmstetter, F. J. (2011). Activity dependent protein degradation is critical for the formation and stability of fear memory in the amygdala. PLoS One, 6(9), e24349. Kandel, E. R., Dudai, Y., & Mayford, M. R. (2014). The molecular and systems biology of memory. Cell, 157(1), 163–186. Lee, S. H., Choi, J. H., Lee, N., Lee, H. R., Kim, J. I., Yu, N. K., ... Kaang, B. K. (2008). Synaptic protein degradation underlies destabilization of retrieved fear memory. Science, 319(5867), 1253–1256. Lin, J. Y., Arthurs, J., & Reilly, S. (2015). Gustatory insular cortex, aversive taste memory and taste neophobia. Neurobiology of Learning and Memory, 119, 77–84. Lopez-Salon, M., Alonso, M., Vianna, M. R., Viola, H., Mello e Souza, T., Izquierdo, I., ... Medina, J. H. (2001). The ubiquitin–proteasome cascade is required for mammalian long-term memory formation. European Journal of Neuroscience, 14(11), 1820–1826. Massaly, N., Frances, B., & Mouledous, L. (2014). Roles of the ubiquitin proteasome system in the effects of drugs of abuse. Frontiers in Molecular Neuroscience, 7, 99. McNaught, K. S., Olanow, C. W., Halliwell, B., Isacson, O., & Jenner, P. (2001). Failure of the ubiquitin–proteasome system in Parkinson’s disease. Nature Reviews Neuroscience, 2(8), 589–594. Ounallah-Saad, H., Sharma, V., Edry, E., & Rosenblum, K. (2014). Genetic or pharmacological reduction of PERK enhances cortical-dependent taste learning. The Journal of Neuroscience, 34(44), 14624–14632. 29. Parkes, S. L., De la Cruz, V., Bermudez-Rattoni, F., Coutureau, E., & Ferreira, G. (2014). Differential role of insular cortex muscarinic and NMDA receptors in one-trial appetitive taste learning. Neurobiology of Learning and Memory. Paxinos, G., & Watson, C. (2005). The rat brain in stereotaxic coordinates. Elsevier Academic Press. Rappaport, A. N., Jacob, E., Sharma, V., Inberg, S., Elkobi, A., Ounallah-Saad, H., ... Rosenblum, K. (2015). Expression of quinone reductase-2 in the cortex is a muscarinic acetylcholine receptor-dependent memory consolidation constraint. The Journal of Neuroscience, 35(47), 15568–15581. 25. Reis, D. S., Jarome, T. J., & Helmstetter, F. J. (2013). Memory formation for trace fear conditioning requires ubiquitin–proteasome mediated protein degradation in the prefrontal cortex. Frontiers in Behavioral Neuroscience, 7, 150. Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., ... Goldberg, A. L. (1994). Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell, 78(5), 761–771. Rodriguez-Ortiz, C. J., Balderas, I., Saucedo-Alquicira, F., Cruz-Castaneda, P., & Bermudez-Rattoni, F. (2011). Long-term aversive taste memory requires insular and amygdala protein degradation. Neurobiology of Learning and Memory, 95(3), 311–315. Rosenberg, T., Gal-Ben-Ari, S., Dieterich, D. C., Kreutz, M. R., Ziv, N. E., Gundelfinger, E. D., et al. (2014). The roles of protein expression in synaptic plasticity and memory consolidation. Frontiers in Molecular Neuroscience, 7, 86. Rosenblum, K., Berman, D. E., Hazvi, S., Lamprecht, R., & Dudai, Y. (1997). NMDA receptor and the tyrosine phosphorylation of its 2B subunit in taste learning in the rat insular cortex. The Journal of Neuroscience, 17(13), 5129–5135. Rosenblum, K., Meiri, N., & Dudai, Y. (1993). Taste memory: The role of protein synthesis in gustatory cortex. Behavioral and Neural Biology, 59(1), 49–56. Schachtman, T. R., Bills, C., Ghinescu, R., Murch, K., Serfozo, P., & Simonyi, A. (2003). MPEP, a selective metabotropic glutamate receptor 5 antagonist, attenuates conditioned taste aversion in rats. Behavioural Brain Research, 141(2), 177–182. Sol Fustinana, M., de la Fuente, V., Federman, N., Freudenthal, R., & Romano, A. (2014). Protein degradation by ubiquitin–proteasome system in formation and labilization of contextual conditioning memory. Learning & Memory, 21(9), 478–487. Stern, E., Chinnakkaruppan, A., David, O., Sonenberg, N., & Rosenblum, K. (2013). Blocking the eIF2a kinase (PKR) enhances positive and negative forms of cortexdependent taste memory. The Journal of Neuroscience, 33(6), 2517–2525. Tai, H. C., Besche, H., Goldberg, A. L., & Schuman, E. M. (2010). Characterization of the brain 26S proteasome and its interacting proteins. Frontiers in Molecular Neuroscience, 3.

Please cite this article in press as: Rosenberg, T., et al. NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion. Neurobiology of Learning and Memory (2016), http://dx.doi.org/10.1016/j.nlm.2016.01.002

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