Sucrose ingestion induces rapid AMPA receptor trafficking

Abstract

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 (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs 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 Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep 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.

Figure 1.

Repeated sucrose ingestion causes transient elevations of spontaneous locomotion. A, Adult male rats (n = 8, water; n = 10, sucrose) were habituated to a test room for 3 d before being placed in a locomotor measurement chamber for 7 consecutive days; spontaneous locomotor activity was measured 15 min before and 15 min after 5 min access to a bottle containing water or 25% sucrose. B, Sucrose rats consumed significantly more liquid than Water rats starting on day 3. C–E, Total distance traveled (centimeters) was significantly greater in Sucrose than Water rats in the 3 min time bin immediately after Sucrose on days 7 and 8 while pre-sucrose activity did not change. F, Distance traveled in the 3 min time bin immediately after bottle removal was positively correlated with the amount of sucrose consumed. B–E, Data are expressed as mean + SEM and were analyzed using one-way ANOVA on each training day followed by Fisher's post hoc tests. F, Data were analyzed using a Kendall tau rank correlation test (τ = 0.285, Z = 3.156).

Figure 2.

Accumbens core synapses are potentiated after repeated sucrose ingestion. A–C, Spontaneous EPSCs in accumbens core neurons (n = 10 cells from 3 rats, Water; n = 10 cells from 3 rats, Sucrose) after 7 d sucrose or water training. Slices were prepared immediately after bottle removal. D, E, Evoked EPSCs in accumbens core neurons (n = 10 cells from 4 rats, Water; n = 11 cells from 5 rats, Sucrose) measured at −70, −50, −30, 0, +20, +40, and +60 mV. In F, evoked EPSCs from individual records were normalized to the current at −70 mV and averaged for water and sucrose animals as indicated. Rectification index (F) calculated as EPSC₋₇₀/EPSC₊₄₀. B–G, Data are represented as mean ± SEM and analyzed using unpaired, two-tailed t tests.

Figure 3.

Sucrose ingestion causes incorporation of Ca²⁺-permeable AMPARs. A, B, Evoked EPSCs before and after bath Naspm (200 μm) wash-in. B, Mean evoked EPSCs before and after bath Naspm wash-in (n = 9 cells from 4 rats, water; n = 10 cells from 5 rats, sucrose). C, Mean EPSCs_{Naspm/baseline}. D, I/V relationship before and after wash-in of Naspm into the bath. C, D, Data are represented as mean ± SEM and analyzed with an unpaired, two-tailed t test (C).

Figure 4.

PSD GluA1, but not GluA2, is increased in nucleus accumbens core after sucrose ingestion. A, Western blots of accumbens core whole-cell lysates harvested from Water or Sucrose rats on days 1, 3, 5, and 7 of training (n = 3 rats for Water and Sucrose on each training day). B, C, GluA1:tubulin or GluA2:tubulin integrated density of Sucrose rats normalized to Water rats. D, Western blots of accumbens core PSD fractions harvested from Water or Sucrose rats on days 1, 3, 5, and 7 of training (n = 3 rats for Water and Sucrose on each training day). E, F, GluA1 or GluA2 integrated density of Sucrose rats normalized to Water rats. B, C, E, F, Data are represented as mean + SEM and were analyzed using one-way ANOVA followed by Fisher's post hoc tests on each test day.

Figure 5.

Electron microscopy reveals induction of multistep GluA1 trafficking by sucrose ingestion. A, GluA1 was PEG labeled and particles were classified into five postsynaptic regions: (1) intraspinous, (2) extrasynaptic membrane, or (3) PSD (cleft + at PSD + near PSD). B, Electron micrographs were prepared from Water, Sucrose/Water, and Sucrose animals (n = 3 animals/test group) and the number of GluA1 PEG particles was quantified for each individual synapse (n = 279 synapses/condition). Scale bar, 500 nm. C–F, Repeated sucrose ingestion elevates intraspinous and PSD GluA1 while acute sucrose ingestion induces rapid trafficking of GluA1 to the extrasynaptic membrane. Data are represented as mean + SEM and were analyzed using one-way ANOVA followed by Fisher's post hoc tests. C–E, Data are presented as averages of the number of particles per spine.

Figure 6.

Elevated spontaneous locomotion after sucrose ingestion requires CPARs and NMDARs. A–D, Total distance traveled in 3 min time bin after sucrose or water bottle removal (n = 6 Water rats, 7 Sucrose rats). Bilateral microinjections of Naspm (40 μg/side) or APV (4 μg/side) were performed before placement of rats in locomotor measurement chambers. E, Sucrose or water consumed after microinjections. F, Placement of cannulae in the accumbens core. Data are represented as mean + SEM and were analyzed using one-way ANOVA followed by Fisher post hoc tests.

Figure 7.

Saccharin training induces an increase in synaptic GluA1 similar to sucrose training. Rats were given access to water, 3% saccharin, or 25% sucrose daily (4 rats per group), in a training cage for 5 min per day, whereupon the feeding bottle was removed and the rats were maintained in the cage for 15 additional minutes. This training was performed for 7 consecutive days. A, The mass of Saccharin and Sucrose solution consumed were similar, and both were greater than the mass of Water consumed. B, Western blot analysis of GluA1 and actin (control) in the PSD and whole-cell fractions of the nucleus accumbens core isolated from Water, Saccharin, and Sucrose rats. Relative to Water training, Saccharin and Sucrose training significantly elevated GluA1 levels similarly in the PSD (F_c = 10.6; *p = 0.009, **p < 0.001) (C) but not the whole-cell fractions (D) of the accumbens core. Western blot quantitation of GluA1 was normalized to actin levels.