Sucrose Ingestion Induces Rapid AMPA Receptor Trafficking (2013)

The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPA receptors containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca²⁺-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPA receptors. Electrophysiological, biochemical and quantitative electron microscopy studies revealed that sucrose training (7 days) induced a stable (>24 hr) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 hr) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7-day protocol of daily ingestion of a 3% solution of saccharin, a non-caloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multi-step GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.

Subjects and surgical procedures

The subjects were male Sprague-Dawley rats (Taconic; behavioral experiments) weighing 150–300 grams on arrival and female E18 pregnant Sprague-Dawley rats (Taconic; cell culture experiments). The rats were housed 2 per cage for behavioral experiments on a 12h/12h light-dark cycle (lights out at 18:00) and had access to food and water ad libitum at all times. All experimental procedures were approved by the New York University School of Medicine Institutional Animal Care and Use Committee and were performed in accordance with the “Principles of Laboratory Animal Care” (NIH publication number 85–23).

Sucrose training and locomotor measurements

Rats were transported to the test room 3 consecutive days for 2 h/day in their home cages. On the fourth day bottles containing water or 25% sucrose were introduced through the cage lid for 5 min. Bottles were then weighed. For all experiments, rats were required to drink at least 1 g of sucrose during the 5 min access within 3 days of training commencement to be included in the study; in fact, all rats met this criterion. After bottle removal, rats remained in the test room for 30 min before transport back to the animal facility. On the day of sacrifice, rats were rendered unconscious by CO₂, decapitated by guillotine, and tissue samples were collected on ice. For locomotor experiments, rats were placed in locomotor measurement chambers (Accuscan, Columbus, OH) for a total of 35-min. After 15-min in the chamber, a bottle with a bead stopper was introduced through the top of the chamber and stabilized. The bottle was removed from the top of the chamber after 5-min, and the rats remained in the chamber for an additional 15-min after bottle removal. This procedure was repeated identically for 7 consecutive days. Distance traveled was measured using the VersaMax System (Accuscan, Columbus, OH), which monitored animal activity via a grid of 16 × 16 infrared light beams that traverse the animal cage (42 × 42 × 30 cm) front to back and left to right. Information about beam status, scanned at a rate of 100 times per second, was stored to disk. Activity was expressed as ambulatory distance measured in cm during 12 different 3-min bins in a 35 min session (the final bin was 2-min).

Saccharin training

To compare sucrose-induced trafficking of GluA1 with the effects of saccharin ingestion, 12 adult male rats (250 g) were housed in the animal facility on a 12 hours light/dark cycle. All rats were then habituated to the test room by being transported to the test room, left for 2 hours, and transported back to the animal facility. One the 4th day (after 3 days of habituation), rats were given access bottles containing, water, sucrose, or saccharin. 4 rats were given access to a bottle containing water rested on the top of the cage with the spout protruding into the cage through the lid. Access time was 5 minutes, then the bottle was removed, and after 15 min additional minutes rats were transported back to the animal facility. 4 rats were given access to 25% sucrose solution and 4 rats were given access to 3% saccharin solution (Sweet’n Low). The volume of fluid consumed was measured. This procedure was repeated for 7 days. On the 7th day of drinking, immediately following bottle removal, rats were sacrificed and accumbens core was harvested and GluA1 levels were probed by Western blot.

Electrophysiology

Rats were sucrose trained as described above in clear plastic cages and, after bottle removal on day 7, were anesthetized with ketamine (100 mg/kg i.p.) and xylazine (10 mg/kg i.p.) and transcardially perfused with cold saline (mEPSC experiments) or decapitated immediately (rectification experiments). Brains were quickly removed into artificial cerebrospinal fluid (ACSF) consisted of the following (in mM): for mEPSC experiments: NaCl (118), KCl (2.5), CaCl₂ (3), MgCl₂ (1), NaHCO₃ (26), NaH₂PO₄ (1), D-glucose (10), osmolarity adjusted to 325 mOsm and aerated by 95% O₂/ 5% CO₂ (pH 7.4); for rectification experiments: 75 sucrose, 87 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 0.5 CaCl₂, 7 MgCl2 6 H₂O, 25 NaHCO₃, 10 dextrose, bubbled with 95% O₂ / 5% CO₂ (pH 7.4). Coronal slices (300μm thick) containing the nucleus accumbens were cut in ice-cold ACSF using a vibrotome (Leica, VT1200S) and kept submerged in ACSF(ACSF, in mM: 124 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 2.5 CaCl₂, 1.5 MgSO₄ 7H2O, 26 NaHCO₃, and 10 dextrose) for <30 min; then kept in a slice pre-incubator at room temperature for at least 1 hour to allow for recovery. For mEPSC experiments: a single slice was then transferred to a recording chamber in which it was held submerged by a nylon net at 32°C with a TC324B in-line solution heater and controller (Warner Instruments, CT). The chamber was continuously perfused by ACSF at a constant rate of 2 ml/min. Medium spiny neurons from the nucleus accumbens core region were identified under visual guidance using infrared-differential interference contrast video microscopy (Hamamatsu C5405) with an Olympus BX50WI upright microscope fitted with 40x long working distance water-immersion objective. Patch electrodes (4–6 MΩ) filled with an intracellular pipette solution consisting of (in mM): CsCl (145), HEPES (10), EGTA (0.5), and MgATP (5). Osmolarity was adjusted to 290 mOsm with sucrose, and pH was adjusted to 7.4 with CsOH. Miniature excitatory post-synaptic currents (mEPSCs) were recorded in the presence of bicuculline (10μM) and tetrodotoxin (1μM) using Axopatch 200B amplifier (Molecular Devices, CA) and digitized by Digidata 1322A (Molecular Devices, CA). For rectification experiments: slices were transferred to the recording chamber and perfused (2.0–2.5 ml min⁻¹) with oxygenated ACSF at 33–35°C containing 50 μm picrotoxin to isolate EPSCs. Somatic whole-cell recordings were made from core region medium spiny neurons in voltage-clamp with a Multiclamp 700B amplifier (Molecular Devices) using IR-DIC video microscopy. Patch pipettes (4–6 MΩ) were filled with intracellular solution (in mM: 125 Cs-gluconate, 2 CsCl, 5 TEA-Cl, 4 Mg-ATP, 0.3 GTP, 10 phosphocreatine, 10 HEPES, 0.5 EGTA, and 3.5 QX-314). Data were filtered at 2 kHz, digitized at 10 kHz, and analyzed with Clampfit 10 (Molecular Devices). Extracellular stimulation (0.01–1 ms, 5–150 μA, 0.2 Hz) was applied with a small glass bipolar electrode 0.05–0.5 mm from the recording electrode. After ~10 min of baseline recording, a solution containing Naspm (200 μM) was perfused into the bath for 10 min. Changes in EPSC amplitude were measured before and after drug application at holding potentials of −70, −50, −30, 0, +20, +40 and +60 mV. The rectification index (i_r) was calculated by correcting any potential shifts in reversal potential and computed from the following equation: i_r = (I₋₇₀ / 70) / (I₊₄₀ / 40), where I₋₇₀ and I₊₄₀ are the EPSC amplitudes recorded at −70 mV and +40 mV, respectively.

Subcellular fractionation and Western blotting

Accumbens were collected on ice as described above. When core and shell were separately dissected, separation was confirmed by probing synaptosome fractions for dopamine β-hydroxylase, an enzyme found in the terminals of axons to the shell but not the core (Sesack and Grace, 2010). Whole cell, synaptosome, and PSD fractions were prepared as described previously (Jordan et al., 2004). Synaptosomal pellets were resuspended in 200 μl 25 mM Tris with 1% Triton X-100, rocked at 4°C for 30-min, and centrifuged at 13,800 × g for 15-min in a microcentrifuge to pellet PSDs. The pellet containing crude PSDs was resuspended in 25 mM Tris with 2% SDS. Fractions were analyzed by Western blot on SDS-PAGE gels as described previously (Jordan et al., 2004). The following antibodies were used: dopamine β-hydroxylase (1:1,000, Abcam), GluA1 (1:1,000, Millipore), phosphor-Ser 845 GluA1 (1:1,000, Millipore), GluA2 (1:1,000, Millipore) and tubulin (1:10,000, Sigma).

Electron microscopy

On the day of tissue harvesting (day 7 of sucrose training), rats from 3 test groups (Water, Sucrose/Water, Sucrose; 3 rats/test group) were placed in locomotor measurement chambers for 15-min; at 15-min a bottle was introduced through the chamber top. Rats in the Water group received water, rats in the ucrose group received 25% sucrose, rats in the Sucrose/Water group, which had consumed 25% sucrose for 6 days, received water. Rats were deeply anesthetized with Nembutal (50 mg/kg i.p.) and transcardially perfused with 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde and 0.1% glutaraldehyde at a rate of 50 ml/min during the first 3-min, then at a rate of 20 ml/min for the succeeding 7-min. Tissue was prepared for postembed immunogold (PEG) labeling and images were captured as described previously (Nedelescu et al., 2010). Immunolabels were categorized according to their position relative to the PSD at asymmetric synaptic junctions as “cleft,” “near PSD” (within 1 PSD width from the PSD), “at PSD,” “intraspinous,” or “extrasynaptic membrane.” From each animal, 93 synapses were samples from the core of the accumbens. Random sampling was ensured by analyzing all of the first 93 randomly-encountered synapses as we sweeped systematically across the grid, then pooling the same number of synapses from each of the three animals that received identical ante mortem treatments. Two types of quantification were performed. One was to evaluate GluR1-immunoreactivity level, by tallying the number of PEG particles that occurred at discrete functional domains of the spine. The other was to assess the proportion of synapses labeled at the PSD by any number of PEG particles. Even synapses labeled by just 1 PEG particle were considered labeled, based on earlier work demonstrating specificity of the GluR1-PEG procedure (Nedelescu et al., 2010). Treatment effects on the proportion and level of GluR1-immunolabeling were analyzed by one-way ANOVA with planned post hoc comparisons (Fisher’s LSD). To eliminate experimental bias, the data were triple-blinded: one experimenter performed sucrose training and kept records of the animals in the three test groups, a second experimenter created the electron micrographs and assigned a new alphanumeric code to each micrograph and kept the code sealed, and three additional experimenters scanned the micrographs and quantified PEG particles. After PEG quantification was complete, the experimenters convened to reveal the identities of each micrograph.

Cannula implantation and intracranial injections

Intracranial injection was employed to deliver Naspm and APV to the accumbens core. For cannula implantation, as described previously (Carr et al., 2010), rats were deeply anesthetized with ketamine (100 mg/kg i.p.) and xylazine (10 mg/kg i.p.) and injected post-operatively with the analgesic benamine (1 mg/kg subcutaneous). Rats were stereotaxically implanted with two 26-gauge guide cannulae (PlasticsOne, Roanoke, VA) bilaterally in the accumbens core with coordinates: 1.6 mm anterior to bregma; 2.9 mm lateral to the sagittal suture, tips angled 8° toward the midline, 5.6 mm ventral to skull surface. Cannulae were held in place by dental acrylic and patency was maintained with an occlusion stylet. For intracranial injections, Naspm and APV solutions were loaded into two 30 cm lengths of PE-50 tubing attached at one end to 25-μl Hamilton syringes filled with distilled water and at the other end to 31-gauge injector cannulae, which extended 2.0 mm beyond the implanted guides. The syringes were mounted on the twin holders of a Harvard 2272 microliter syringe pump that delivered the 0.5 μl injection volumes over a period of 100 sec. One minute following completion of injections, injector cannulae were removed from guides, stylets were replaced, and animals were placed into locomotor testing chambers for sucrose training. Following animal sacrifice, cryogenic brain sections were analyzed for cannula localization; 2 out of 15 animals were excluded from the study because of improper cannula placement.

Statistical analysis

One-way ANOVA followed by Fisher post hoc tests was used for electron microscopy, immunocytochemistry and biotinylation experiments. Two-tailed Student’s t-tests were used for electrophysiology. For sucrose hyperactivity experiments, two-way ANOVA was used, followed by Fisher post hoc tests.

Characterization of a sucrose ingestion paradigm

We employed a sucrose ingestion paradigm to investigate the effects of a natural, orosensory reward on synaptic transmission (Figure 1A). Adult male rats were transported to a test room on three consecutive days. On the fourth day (first day of training), the rats were placed in a locomotor measurement chamber. After 15 minutes of locomotor activity measurement in the chamber, bottles containing either water (for Water animals) or 25% sucrose solution (for Sucrose animals) were introduced into the measurement chambers through holes in the lid of the chamber. The bottles were removed after 5 minutes and locomotor activity was measured for an additional 15 minutes before animals were returned to home cages. We repeated this procedure for 7 consecutive days. In some experiments, sucrose training was extended to an 8^th day. This brief, non-contingent access to a highly palatable solution allowed us to investigate both acute and cumulative effects of sucrose intake, as animals reliably imbibed sucrose vigorously during the access window within three days of training (Figure 1B). These experimental conditions enabled the comparison of test groups immediately following cessation of ingestion. Our criterion for inclusion in the study was that rats begin to consume at least one gram of sucrose during the access period within three days of training commencement; no animals were excluded from the study on the basis of this criterion.

  

Figure 1

Repeated sucrose ingestion causes transient elevations of spontaneous locomotion.

We observed that within three days of training, Sucrose animals consumed significantly more sucrose solution than Water animals consumed water (Figure 1B). Additionally, while no significant differences in spontaneous locomotion were observed on training days 1–6 (data not shown), we observed significant elevation of total distance traveled in Sucrose animals compared to Water animals in the three minutes after bottle removal on day 7 (Figure 1D), and this difference was also present on day 8 (Figure 1E). No differences in total distance traveled were observed between Water and Sucrose animals in the three minutes prior to bottle introduction on any of the test days (Figure 1C), suggesting elevated locomotion was an acute response to sucrose ingestion specific to the sucrose-trained rat, rather than a conditioned response to the locomotor chamber. In keeping with this possibility, there was a significant positive correlation between the amount of sucrose consumed and the total distance traveled (Figure 1F). There was no difference in animal weights between the Sucrose and Water groups before or after the 7 days of training (data not shown).

Sucrose ingestion induces CPAR incorporation

Sucrose training led to a transient elevation of locomotion on the final training day. To determine whether this consequence of sucrose ingestion was accompanied by electrophysiological changes in the nucleus accumbens, a region that regulates reward behavior, we prepared nucleus accumbens slices immediately following bottle removal on day 7 and recorded from neurons of the accumbens core (Figure 2A). The core subregion has been implicated in locomotor responses to rewarding stimuli (Sesack and Grace, 2010). We found that both the amplitude and the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) were significantly greater in the accumbens core of Sucrose animals compared to Water animals (Figure 2B). This demonstrated that repeated sucrose consumption could positively regulate synaptic transmission in the nucleus accumbens core. To determine if CPAR incorporation played a role in potentiation after sucrose, we determined rectification indices for accumbens core neurons by measuring EPSCs at different membrane potentials (Figures 2C, 2D and 2E). CPARs are inwardly rectifying at depolarized potentials because of endogenous polyamine blockade. We observed significant rectification in recordings from neurons of Sucrose animals, as indicated by nonlinearity in the I/V relationship, compared to Water animals (Figure 2E), in addition to a significant increase in rectification index (Figure 2F).

  

Figure 2

Accumbens core synapses are potentiated after repeated sucrose ingestion.

To confirm elevation of CPAR levels by another method, we recorded from accumbens core neurons after inclusion of the specific CPAR blocker, 1-Naphthylacetyl spermine (Naspm) in the bath. We found that Naspm significantly decreased EPSC amplitude in recordings from neurons from Sucrose but not Water animals (Figures 3A–C). Additionally, after Naspm treatment, the I/V relationship in neurons from Sucrose animals became linear, reflecting the inhibition of CPARs in Sucrose animal synapses, while no significant effect on the I/V relationship was observed after Naspm treatment in neurons from Water animals (Figures 3D). These results demonstrate that repeated sucrose ingestion induces the incorporation of CPARs in synapses of the accumbens core.

  

Figure 3

Sucrose ingestion causes incorporation of Ca2+-permeable AMPA receptors.

Sucrose ingestion induces GluA1 trafficking

CPARs are AMPA receptors that lack the GluA2 AMPA receptor subunit. Thus, synaptic incorporation of CPARs most often involves synaptic activity-dependent trafficking of the GluA1 subunit (He et al., 2009; Isaac et al., 2007; Liu and Zukin, 2007; Plant et al., 2006). To confirm CPAR synaptic incorporation following sucrose training, we investigated whether sucrose ingestion increased synaptic expression of GluA1. Rats were given access to sucrose as described above for 7 consecutive days. On days 1, 3, 5, and 7, we isolated the whole cell, synaptosome and postsynaptic density (PSD) fractions from three brain regions: accumbens core (core), accumbens shell (shell) and somatosensory cortex (cortex). We analyzed the whole cell and PSD fractions by Western blot for expression of GluA1 and GluA2.

We found no changes in GluA1 or GluA2 in the whole cell fractions of accumbens lysates on the test days examined, suggesting repeated sucrose consumption does not regulate the overall levels of these proteins (Figures 4A–C). In the accumbens PSD fractions, however, GluA1 increased significantly on day 7 in the core but not in the shell while GluA2 did not change significantly in either fraction (Figures 4D–4F and data not shown). We observed no significant increase in GluA1 in the accumbens core PSD fractions on the preceding test days (Figures 4D–F) and GluA1 or GluA2 did not change in the cortex PSD fractions on any of the test days (data not shown). Increased GluA1, especially relative to GluA2, in accumbens core PSDs after repeated sucrose ingestion is in keeping with the increased rectification observed in accumbens core neurons, as described above.

  

Figure 4

Postsynaptic density GluA1, but not GluA2, is increased in nucleus accumbens core after sucrose ingestion.

Activity-dependent GluA1 trafficking has been shown to contribute synaptic plasticity in vitro and also in vivo (Lu and Roche, 2011). A rapid, multi-step mechanism for GluA1 trafficking has been demonstrated in vitro (Serulle et al., 2007; Sun et al., 2008; Sun et al., 2005). Thus far, however, the contribution of this multi-step mechanism to GluA1 synaptic accumulation in vivo has not been tested. To determine whether sucrose training induces GluA1 trafficking acutely by the multistep mechanism, we localized GluA1 at nucleus accumbens synapses of sucrose- and water-trained animals by quantitative electron microscopy. Accumbens core tissue was harvested on the seventh day of sucrose training from 3 test groups of rats. These were rats that: 1) consumed water for 7 days (Water), 2) consumed sucrose for 7 days (Sucrose), and 3) consumed sucrose for 6 days and water on day 7 (Sucrose/Water). Rats were sacrificed on the 7^th day, 5 minutes following consumption of sucrose or water. Thus, comparison of two of the test groups, Sucrose/Water and Sucrose animals, to each other and to Water animals revealed the timescale of postsynaptic changes induced by sucrose consumption in sucrose-trained rats. We measured postembed immunogold (PEG)-labeled GluA1 in 5 different postsynaptic compartments: dendritic spine cytosol (intraspinous), extrasynaptic plasma membrane (membrane), PSD, near PSD, and synaptic cleft, with the final three compartments being grouped together as ‘PSD’ (Fig. 5A). To eliminate experimenter bias, electron micrograph test group identities of were triple-blinded.

  

Figure 5

Electron microscopy reveals induction of multi-step GluA1 trafficking by sucrose ingestion.

Both Sucrose and Sucrose/Water animals exhibited significantly elevated intraspinous GluA1 relative to Water animals (Fig. 5B and 5C). This suggests that chronic sucrose consumption increases an intracellular pool of GluA1-containing AMPA receptors adjacent to synaptic sites, receptors that may be readily available for synaptic trafficking, and, importantly, that this intracellular pool can persist for 24 hours following the final consumption of sucrose. We then explored the significant question of whether an acute sucrose stimulus can induce rapid GluA1 trafficking. We observed that extrasynaptic plasma membrane GluA1 was significantly increased in Sucrose animals compared to both Sucrose/Water and Water animals (Figures 5B and 5D). This observation indicates that a natural, orosensory reward provided by a single sucrose stimulus can rapidly (<5 min) but transiently (decay time < 24 h) elevate the extrasynaptic population of GluA1-containing AMPA receptors, creating a labile pool from which the receptors may traffic to the synapse.

Significantly, in vitro studies have suggested that synaptic incorporation of AMPA receptors takes place in 2 steps. In the first, glutamate- or dopamine-dependent Ser 845 phosphorylation elevates the levels of receptors at extrasynaptic sites in the plasma membrane (Esteban et al., 2003; Serulle et al., 2007; Sun et al., 2008; Sun et al., 2005), while in the second, Ser 818 phosphorylation promotes synaptic incorporation (Boehm et al., 2006). Our electron microscopy comparison of Sucrose animals with Sucrose/Water and Water animals demonstrates that the first step of GluA1 trafficking observed in vitro (Makino and Malinow, 2009), rapid trafficking to the extrasynaptic membrane, also takes place in vivo following provision of an orosensory reward.

In keeping with electrophysiology and biochemical results described above, PEG EM demonstrated that sucrose intake also induced the second step in GluA1 trafficking GluA1 receptor entry to the synapse since the level of GluA1-immunoreactivity at the PSD was significantly greater for Sucrose compared to Water rats, and there was a trend toward increased GluA1 in Sucrose/Water compared to Water rats (Figures 5B and 5E). The increase in Sucrose/Water animals is consistent with either rapid incorporation of GluA1 which decays with a synaptic half-life of ~24 hr,, or rapid incorporation of GluA1 and replacement by synaptic GluA1/2 over a similar time period. The percentage of synapses expressing GluA1 in the PSD was also significantly greater in Sucrose rats compared to Water rats (Figure 5F), suggesting that GluA1 trafficked to synapses that previously lacked GluA1. This also suggests that the increase in mEPSC amplitude observed in Sucrose rats results from an increase in synaptic GluA1, and that the increase in mEPSC frequency may result from recruitment of GluA1 to previously silent accumbens synapses, although potentiation of glutamate release cannot be dismissed. We also measured the number of synapses in each of the three test groups to determine whether sucrose ingestion induced synaptogenesis; there were no differences among the three test groups (data not shown). We conclude that repeated sucrose ingestion elevates a stable (>24 hours) intraspinous pool of GluA1, and a single sucrose stimulus to a sucrose trained (6 days) rat is sufficient to rapidly (5 min) elevate GluA1 in the extrasynaptic plasma membrane, potentially drawing receptors from the intraspinous pool. We suggest that a portion of these extrasynaptic receptors is stably incorporated into the PSD, leading to the observed rectification index and PSD GluA1 changes, before the extrasynaptic pool returns to baseline at 24 h following stimulation. These results reveal that a natural reward can acutely induce rapid GluA1 trafficking in a trained animal.

CPAR activity is required for elevated locomotion after sucrose ingestion

Medium spiny neurons receive both dopaminergic and glutamatergic inputs (Calabresi, et al., 1992). In order to assess the involvement of glutamate signaling in the elevated spontaneous locomotion that we observed after sucrose ingestion in sucrose trained rats, we implanted cannulas into the accumbens core of rats, and trained animals in locomotor measurement chambers as described above. On day 8 of sucrose training, we microinjected Naspm into the core prior to placement in the locomotor test chamber. The injection decreased the total distance traveled of the Sucrose animals and eliminated the difference between Sucrose and Water animals seen immediately following bottle removal (Figure 6A). To verify that stress caused by handling of the animals had not affected the sucrose response, we injected saline into the core on the following day (day 9 of sucrose training); again significant hyperactivity was observed in the Sucrose animals immediately after bottle removal (Figure 6B). This demonstrates that Naspm had specifically inhibited sucrose-induced elevation of locomotion. Injection of the NMDAR antagonist, APV, into the core on the following days also eliminated the difference between Sucrose and Water animals (Figure 6C), demonstrating that NMDARs are also required for elevation of spontaneous locomotion after sucrose ingestion. In order to determine whether a conditioned response to the test chamber played a role in hyperactivity induction, animals were placed in the chamber for 35 min without bottle introduction; no differences in distance traveled were observed between Sucrose and Water animals (Figure 6D). Naspm and APV did not affect sucrose consumption (Figure 6E), demonstrating that core CPARs and NMDARs are not required for vigorous intake of sucrose. Animals in which cannulas were not placed in the accumbens core (2 out of 15 animals), as evaluated post-sacrifice (Figure 6F), were not included in the study. In conclusion, these data together demonstrate that consumption of sucrose by a sucrose-trained rat induces synaptic trafficking of GluA1 in 5 minutes, and that blockade of signaling mechanisms that control this trafficking prevents the elevation of spontaneous locomotor activity after sucrose.

  

Figure 6

Elevated spontaneous locomotion after sucrose ingestion requires CPARs and NMDARs.

Two pathways for sucrose signaling may be envisaged. One, a strictly chemosensory or orosensory pathway, is initiated by sucrose binding to the sweet taste receptor, which corresponds to the heteromeric G protein coupled receptor complex, T1R2/T2R3 (Kitagawa et al., 2001; Max et al., 2001; Nelson et al., 2001; Sainz et al., 2001). Calorie-rich nutrients can also regulate brain function by metabolic pathways independent of taste, although the mechanisms are not well understood (de Araujo et al., 2008). To distinguish between these two alternatives for the pathway of GluA1-trafficking induced by sucrose, we repeated the training protocol with three groups of rats (4 rats/group) that were given access for 5 minutes to bottles containing, water, 25% sucrose solution, or 3% saccharin (Sweet and Low). The bottles were removed and the rats remained for 15 minutes longer in the training cage. Training was repeated for 7 days. The volumes of liquid consumed by the sucrose and saccharin test groups were not significantly different from one another and both were greater than consumption by the water group, consistent with reward by both sweet substances (Figure 7A). On the 7th day of drinking, immediately following bottle removal, rats were sacrificed, the accumbens core tissue was harvested and pooled for each test group, the PSD fraction isolated and GluA1 levels probed by Western blot (Figure 7B). As before, animals that consumed sucrose showed elevated GluA1 in the PSD fraction relative to the water group (Figure 7C). Significantly, GluA1 was also elevated in the PSD fraction of animals that consumed saccharin. There was no significant difference in the levels of GluA1 in the whole cell fractions from accumbens core of water, sucrose and saccharin animals, suggesting that the GluA1 increase was specific to the synaptic fraction (Figure 7D). Because saccharin stimulates the same heteromeric G protein coupled taste receptor complex as sucrose (Masuda et al., 2012; Nelson et al., 2001), but lacks caloric value, we conclude that stimulation of the sweet taste receptor is sufficient to initiate signaling that elevates GluA1 levels at nucleus accumbens core synapses.

  

Figure 7

Saccharin Training Induces an Increase in Synaptic GluA1 Similar to Sucrose Training.

We thank members of the Ziff Laboratory, past and present, for technical assistance and helpful discussions, including H. Girma, L. Lee and Drs. B. Fernholz, B. Jordan, W. Lu, G. Rameau, S. Restituito & Y. Serulle. This work was supported by National Institute of Mental Health Predoctoral Fellowship F31MH76617-01 and NIH Training Grant 5T32DC000063 to the New York University Training Program in the Neurosciences (D.S.T.), R01NS061920 from the National Institute of Neurological Disorders and Stroke (E.B.Z.), 1R21MH091445-01 from the National Institute of Mental Health and Office of Research on Women’s Health, Klarman Family Foundation Grants Program in Eating Disorders Research, NYU’s Research Challenge Fund and P30EY13079 (C.A.), National Institute on Drug Abuse grant DA003956 and an Independent Investigator Award from NARSAD (K.D.C.), National Institute on Deafness and other Communications Disorders grant DC009635 to R.C.F, and by a seed grant in the Center of Excellence on Addiction from the New York University Langone Medical Center.

Conflict of interest: The authors declare no competing financial interests.

1. Avena NM, Rada P, Hoebel BG. Evidence for sugar addiction: behavioral and neurochemical effects of intermittent, excessive sugar intake. Neurosci Biobehav Rev. 2008;32:20–39. [PMC free article] [PubMed]
2. Basar K, Sesia T, Groenewegen H, Steinbusch HW, Visser-Vandewalle V, Temel Y. Nucleus accumbens and impulsivity. Prog Neurobiol. 2010;92:533–557. [PubMed]
3. Berridge KC. ‘Liking’ and ‘wanting’ food rewards: brain substrates and roles in eating disorders. Physiology & behavior. 2009;97:537–550. [PMC free article] [PubMed]
4. Boehm J, Kang MG, Johnson RC, Esteban J, Huganir RL, Malinow R. Synaptic incorporation of AMPA receptors during LTP is controlled by a PKC phosphorylation site on GluR1. Neuron. 2006;51:213–225. [PubMed]
5. Boudreau AC, Reimers JM, Milovanovic M, Wolf ME. Cell surface AMPA receptors in the rat nucleus accumbens increase during cocaine withdrawal but internalize after cocaine challenge in association with altered activation of mitogen-activated protein kinases. J Neurosci. 2007;27:10621–10635. [PMC free article] [PubMed]
6. Brebner K, Wong TP, Liu L, Liu Y, Campsall P, Gray S, Phelps L, Phillips AG, Wang YT. Nucleus accumbens long-term depression and the expression of behavioral sensitization. Science. 2005;310:1340–1343. [PubMed]
7. Cacciapaglia F, Saddoris MP, Wightman RM, Carelli RM. Differential dopamine release dynamics in the nucleus accumbens core and shell track distinct aspects of goal-directed behavior for sucrose. Neuropharmacology 2012 [PMC free article] [PubMed]
8. Calabresi P, Maj R, Pisani A, Mercuri NB, Bernardi G. Long-term synaptic depression in the striatum: physiological and pharmacological characterization. J Neurosci. 1992;12:4224–4233. [PubMed]
9. Carr KD, Chau LS, Cabeza de Vaca S, Gustafson K, Stouffer M, Tukey DS, Restituito S, Ziff EB. AMPA receptor subunit GluR1 downstream of D-1 dopamine receptor stimulation in nucleus accumbens shell mediates increased drug reward magnitude in food-restricted rats. Neuroscience. 2010;165:1074–1086. [PMC free article] [PubMed]
10. Conrad KL, Tseng KY, Uejima JL, Reimers JM, Heng LJ, Shaham Y, Marinelli M, Wolf ME. Formation of accumbens GluR2-lacking AMPA receptors mediates incubation of cocaine craving. Nature. 2008;454:118–121. [PMC free article] [PubMed]
11. Day JJ, Carelli RM. The nucleus accumbens and Pavlovian reward learning. Neuroscientist. 2007;13:148–159. [PMC free article] [PubMed]
12. de Araujo IE, Oliveira-Maia AJ, Sotnikova TD, Gainetdinov RR, Caron MG, Nicolelis MA, Simon SA. Food reward in the absence of taste receptor signaling. Neuron. 2008;57:930–941. [PubMed]
13. Ehlers MD, Heine M, Groc L, Lee MC, Choquet D. Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity. Neuron. 2007;54:447–460. [PMC free article] [PubMed]
14. Esteban JA, Shi SH, Wilson C, Nuriya M, Huganir RL, Malinow R. PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity. Nat Neurosci. 2003;6:136–143. [PubMed]
15. Gerfen CR, Surmeier DJ. Modulation of striatal projection systems by dopamine. Annual review of neuroscience. 2011;34:441–466. [PMC free article] [PubMed]
16. Grueter BA, Brasnjo G, Malenka RC. Postsynaptic TRPV1 triggers cell type-specific long-term depression in the nucleus accumbens. Nature neuroscience. 2010;13:1519–1525. [PMC free article] [PubMed]
17. He K, Song L, Cummings LW, Goldman J, Huganir RL, Lee HK. Stabilization of Ca2+-permeable AMPA receptors at perisynaptic sites by GluR1-S845 phosphorylation. Proc Natl Acad Sci U S A. 2009;106:20033–20038. [PMC free article] [PubMed]
18. Hu FB, Malik VS. Sugar-sweetened beverages and risk of obesity and type 2 diabetes: epidemiologic evidence. Physiology & behavior. 2010;100:47–54. [PMC free article] [PubMed]
19. Isaac JT, Ashby MC, McBain CJ. The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity. Neuron. 2007;54:859–871. [PubMed]
20. Jordan BA, Fernholz BD, Boussac M, Xu C, Grigorean G, Ziff EB, Neubert TA. Identification and verification of novel rodent postsynaptic density proteins. Mol Cell Proteomics. 2004;3:857–871. [PubMed]
21. Kalivas PW, Pierce RC, Cornish J, Sorg BA. A role for sensitization in craving and relapse in cocaine addiction. J Pharmacol. 1998;12:49–53. [PubMed]
22. Kitagawa M, Kusakabe Y, Miura H, Ninomiya Y, Hino A. Molecular genetic identification of a candidate receptor gene for sweet taste. Biochemical and biophysical research communications. 2001;283:236–242. [PubMed]
23. Kourrich S, Rothwell PE, Klug JR, Thomas MJ. Cocaine experience controls bidirectional synaptic plasticity in the nucleus accumbens. J Neurosci. 2007;27:7921–7928. [PubMed]
24. Kravitz AV, Freeze BS, Parker PR, Kay K, Thwin MT, Deisseroth K, Kreitzer AC. Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature. 2010;466:622–626. [PMC free article] [PubMed]
25. LaPlant Q, Vialou V, Covington HE, 3rd, Dumitriu D, Feng J, Warren BL, Maze I, Dietz DM, Watts EL, Iniguez SD, et al. Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens. Nat Neurosci. 2010;13:1137–1143. [PMC free article] [PubMed]
26. Liu SJ, Zukin RS. Ca2+-permeable AMPA receptors in synaptic plasticity and neuronal death. Trends in neurosciences. 2007;30:126–134. [PubMed]
27. Lu W, Isozaki K, Roche KW, Nicoll RA. Synaptic targeting of AMPA receptors is regulated by a CaMKII site in the first intracellular loop of GluA1. Proc Natl Acad Sci U S A. 2010;107:22266–22271. [PMC free article] [PubMed]
28. Lu W, Roche KW. Posttranslational regulation of AMPA receptor trafficking and function. Current opinion in neurobiology 2011 [PMC free article] [PubMed]
29. Luscher C, Malenka RC. Drug-evoked synaptic plasticity in addiction: from molecular changes to circuit remodeling. Neuron. 2011;69:650–663. [PMC free article] [PubMed]
30. Makino H, Malinow R. AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis. Neuron. 2009;64:381–390. [PMC free article] [PubMed]
31. Mameli M, Halbout B, Creton C, Engblom D, Parkitna JR, Spanagel R, Luscher C. Cocaine-evoked synaptic plasticity: persistence in the VTA triggers adaptations in the NAc. Nat Neurosci. 2009;12:1036–1041. [PubMed]
32. Masuda K, Koizumi A, Nakajima K, Tanaka T, Abe K, Misaka T, Ishiguro M. Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds. PloS one. 2012;7:e35380. [PMC free article] [PubMed]
33. Matthews K, Wilkinson LS, Robbins TW. Repeated maternal separation of preweanling rats attenuates behavioral responses to primary and conditioned incentives in adulthood. Physiol Behav. 1996;59:99–107. [PubMed]
34. Max M, Shanker YG, Huang L, Rong M, Liu Z, Campagne F, Weinstein H, Damak S, Margolskee RF. Tas1r3, encoding a new candidate taste receptor, is allelic to the sweet responsiveness locus Sac. Nature genetics. 2001;28:58–63. [PubMed]
35. McCutcheon JE, Beeler JA, Roitman MF. Sucrose-predictive cues evoke greater phasic dopamine release than saccharin-predictive cues. Synapse. 2012;66:346–351. [PMC free article] [PubMed]
36. McCutcheon JE, Wang X, Tseng KY, Wolf ME, Marinelli M. Calcium-permeable AMPA receptors are present in nucleus accumbens synapses after prolonged withdrawal from cocaine self-administration but not experimenter-administered cocaine. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2011a;31:5737–5743. [PMC free article] [PubMed]
37. McCutcheon JE, Loweth JA, Ford KA, Marinelli M, Wolf ME, Tseng KY. Group I mGluR activation reverses cocaine-induced accumulation of calcium-permeable AMPA receptors in nucleus accumbenssynapses via a protein kinase C-dependent mechanism. J Neurosci. 2011b;31:14536–14541. [PMC free article] [PubMed]
38. Nedelescu H, Kelso CM, Lazaro-Munoz G, Purpura M, Cain CK, Ledoux JE, Aoki C. Endogenous GluR1-containing AMPA receptors translocate to asymmetric synapses in the lateral amygdala during the early phase of fear memory formation: an electron microscopic immunocytochemical study. The Journal of comparative neurology. 2010;518:4723–4739. [PMC free article] [PubMed]
39. Nelson G, Hoon MA, Chandrashekar J, Zhang Y, Ryba NJ, Zuker CS. Mammalian sweet taste receptors. Cell. 2001;106:381–390. [PubMed]
40. Oh MC, Derkach VA, Guire ES, Soderling TR. Extrasynaptic membrane trafficking regulated by GluR1 serine 845 phosphorylation primes AMPA receptors for long-term potentiation. J Biol Chem. 2006;281:752–758. [PubMed]
41. Pascoli V, Turiault M, Luscher C. Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour. Nature. 2012;481:71–75. [PubMed]
42. Plant K, Pelkey KA, Bortolotto ZA, Morita D, Terashima A, McBain CJ, Collingridge GL, Isaac JT. Transient incorporation of native GluR2-lacking AMPA receptors during hippocampal long-term potentiation. Nat Neurosci. 2006;9:602–604. [PubMed]
43. Rada P, Avena NM, Hoebel BG. Daily bingeing on sugar repeatedly releases dopamine in the accumbens shell. Neuroscience. 2005;134:737–744. [PubMed]
44. Roche KW, O’Brien RJ, Mammen AL, Bernhardt J, Huganir RL. Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit. Neuron. 1996;16:1179–1188. [PubMed]
45. Rumpel S, LeDoux J, Zador A, Malinow R. Postsynaptic receptor trafficking underlying a form of associative learning. Science. 2005;308:83–88. [PubMed]
46. Sainz E, Korley JN, Battey JF, Sullivan SL. Identification of a novel member of the T1R family of putative taste receptors. Journal of neurochemistry. 2001;77:896–903. [PubMed]
47. Serulle Y, Zhang S, Ninan I, Puzzo D, McCarthy M, Khatri L, Arancio O, Ziff EB. A GluR1-cGKII interaction regulates AMPA receptor trafficking. Neuron. 2007;56:670–688. [PMC free article] [PubMed]
48. Sesack SR, Grace AA. Cortico-Basal Ganglia reward network:microcircuitry. Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology. 2010;35:27–47. [PMC free article] [PubMed]
49. Smith GP. Accumbens dopamine mediates the rewarding effect of orosensory stimulation by sucrose. Appetite. 2004;43:11–13. [PubMed]
50. Smith WB, Starck SR, Roberts RW, Schuman EM. Dopaminergic stimulation of local protein synthesis enhances surface expression of GluR1 and synaptic transmission in hippocampal neurons. Neuron. 2005;45:765–779. [PubMed]
51. Sun X, Milovanovic M, Zhao Y, Wolf ME. Acute and chronic dopamine receptor stimulation modulates AMPA receptor trafficking in nucleus accumbens neurons cocultured with prefrontal cortex neurons. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2008;28:4216–4230. [PMC free article] [PubMed]
52. Sun X, Zhao Y, Wolf ME. Dopamine receptor stimulation modulates AMPA receptor synaptic insertion in prefrontal cortex neurons. J Neurosci. 2005;25:7342–7351. [PubMed]
53. Thomas MJ, Beurrier C, Bonci A, Malenka RC. Long-term depression in the nucleus accumbens: a neural correlate of behavioral sensitization to cocaine. Nat Neurosci. 2001;4:1217–1223. [PubMed]
54. Ungless MA, Whistler JL, Malenka RC, Bonci A. Single cocaine exposure in vivo induces long-term potentiation in dopamine neurons. Nature. 2001;411:583–587. [PubMed]
55. Whitlock JR, Heynen AJ, Shuler MG, Bear MF. Learning induces long-term potentiation in the hippocampus. Science. 2006;313:1093–1097. [PubMed]