High-fructose corn syrup consumption in adolescent rats causes bipolar-like behavioural phenotype with hyperexcitability in hippocampal CA3-CA1 synapses

Abstract

Background and purpose: Children and adolescents are the top consumers of high-fructose corn syrup (HFCS) sweetened beverages. Even though the cardiometabolic consequences of HFCS consumption in adolescents are well known, the neuropsychiatric consequences have yet to be determined.

Experimental approach: Adolescent rats were fed for a month with 11% weight/volume carbohydrate containing HFCS solution, which is similar to the sugar-sweetened beverages of human consumption. The metabolic, behavioural and electrophysiological characteristics of HFCS-fed rats were determined. Furthermore, the effects of TDZD-8, a highly specific GSK-3B inhibitor, on the HFCS-induced alterations were further explored.

Key results: HFCS-fed adolescent rats displayed bipolar-like behavioural phenotype with hyperexcitability in hippocampal CA3-CA1 synapses. This hyperexcitability was associated with increased presynaptic release probability and increased readily available pool of AMPA receptors to be incorporated into the postsynaptic membrane, due to decreased expression of the neuron-specific α3-subunit of Na⁺ /K⁺ -ATPase and an increased ser⁸⁴⁵ -phosphorylation of GluA1 subunits (AMPA receptor subunit) respectively. TDZD-8 treatment was found to restore behavioural and electrophysiological disturbances associated with HFCS consumption by inhibition of GSK-3B, the most probable mechanism of action of lithium for its mood-stabilizing effects.

Conclusion and implications: This study shows that HFCS consumption in adolescent rats led to a bipolar-like behavioural phenotype with neuronal hyperexcitability, which is known to be one of the earliest endophenotypic manifestations of bipolar disorder. Inhibition of GSK-3B with TDZD-8 attenuated hyperexcitability and restored HFCS-induced behavioural alterations.

Figure 1

Tracking of the consumption of corresponding diets and the metabolic characterization of the rats. (A) Rats received their corresponding diets for six consecutive weeks, beginning from the postnatal day 21. After a month of feeding with their corresponding diets, rats were randomized for either vehicle or TDZD‐8 treatments for additional 2 weeks. The last week of the 2 weeks of treatment period consisted of a battery of behavioural tests. At the end of the 6th week, rats were undergone in vivo electrophysiology experiments and killed afterward for tissue collection. OGTT was performed in a different set of rats in order to avoid any interference due to fasting. (B) Weekly liquid consumption was significantly greater in HFCS group compared to the control group, starting from the second week (n = 6 cage per group, each cage houses three rats). (C) HFCS consumption caused a significant reduction in weekly chow consumption throughout the tracking period of a month (n = 6 cage per group, each cage houses three rats). (D) The mean body weight of HFCS group was slightly less than that of control group, starting from day 17. The means of final weights were significantly different between control and HFCS groups (n = 9 per group, P < 0.05). (E) HFCS consumption caused glucose intolerance as evident in OGTT, and TDZD‐8 reversed HFCS‐induced glucose intolerance (n = 10 per group for control and HFCS, n = 5 per group for TDZD‐8). (F) HFCS group displayed elevated blood glucose levels after 6 h of fasting, and TDZD‐8 partially reversed this elevation (F _(2,22) = 56.5, P < 0.05). Two hours after the oral glucose load, the blood glucose levels of HFCS remained high compared to those of control and TDZD‐8 rats (F _(2,22) = 24.9, P < 0.05). (G) Area under the OGTT curve was higher in HFCS group compared to control group, and TDZD‐8 treatment restored this towards control levels (F _(2,22) = 8.46, P < 0.05). (H) Total weight of extracted fat pads normalized to total body weight was higher in HFCS group, suggesting a greater fatty body percentage, which was not reversed by TDZD‐8 treatment (F _(2,26) = 7.0, P < 0.05).

Figure 2

HFCS consumption caused spontaneous hyperlocomotion, decreased anxiety, increased risk‐taking behaviour, hyperhedonia and susceptibility to behavioural despair with significant impairments in hippocampal learning in adolescent rats. (A) Total distance travelled in the OFA (n = 8 per group, ANOVA F _(2,21) = 7.39, P < 0.05). (B) Representative track plots of OFA. (C) Time spent in open arms of the EPM (n = 9 per group, ANOVA F _(2,24) = 6.21, P < 0.05). (D) Averaged group heat maps of EPM. (E) Distance travelled in the open arms of the EPM (n = 9 per group, ANOVA F _(2,24) = 8.01, P < 0.05). (F) FUST. All three groups had significantly increased sniffing durations when presented with female urine after distilled water (n = 8 per group, repeated measures two‐way ANOVA, stage effect F _(1,21) = 21.8, P < 0.05). When groups were compared to each other, the HFCS group had significantly higher sniffing durations of female urine compared to the other groups (n = 8 per group, repeated measures two‐way ANOVA, Group effect F _(2,21) = 6.24, P < 0.05). (G) Immobility duration in the forced swim test (n = 9 per group, ANOVA F _(2,24) = 4.84, P < 0.05). (H) Latency to find the hidden platform in the acquisition stage of the MWM and mean swim speeds (inlet). The mean swim speeds of groups were not significantly different from each other (n = 9 per group, ANOVA F _(2,456) = 0.474, P > 0.05). Repeated measures two‐way ANOVA revealed a significant group effect in the acquisition stage of MWM (Acquisition Days × Groups F _(6,420) = 1.70, P > 0.05; Acquisition Days F _(3,420) = 118, P < 0.05; Groups F _(2,420) = 6.90, P < 0.05). In addition, Tukey's multiple comparisons test detected a difference between HFCS and TDZD‐8 groups compared to control group on the first acquisition day. (I) Time spent in the target quadrant in the probe trial of the MWM was not statistically different between groups (n = 9 per group but one outlier data point was removed from control group as identified by ROUT test, F _(2,23) = 0.08, P > 0.05).

Figure 3

HFCS consumption caused neuronal hyperexcitability without altering GABAergic inhibitory activity that was restored by TDZD‐8 in rat hippocampal CA3‐CA1 synapses. (n = 9 rats per group, maximum of one rat per group was excluded because of either low‐quality recording, severe bleeding or death.) (A) Input–output curve of stratum radiatum. HFCS group exhibited hyperexcitability compared to control and TDZD‐8 groups (repeated measures two‐way ANOVA, Group F _(2,23) = 4.66, P < 0.05). (B) Paired‐pulse paradigm in stratum radiatum. HFCS and TDZD‐8 groups showed significantly less facilitation compared to control group when interpulse interval was 20 ms (Tukey's multiple comparisons test, control vs. HFCS and control vs. TDZD‐8, P < 0.05). (C) Representative recordings from stratum radiatum. I/O traces were selected from the responses to stimuli of 7 V. Paired‐pulse traces were given for interpulse intervals of 20 and 1000 ms. Traces from control group are yellow, whereas traces from HFCS and TDZD‐8 groups are red and blue, respectively, in their corresponding columns. (D) Input–output curve of stratum pyramidale. (E) Paired‐pulse paradigm of stratum pyramidale. (F) Representative recordings from stratum pyramidale. I/O traces were selected from the responses to stimuli of 7 V. Paired‐pulse traces were given for interpulse intervals of 20 and 1000 ms. Traces from control group are yellow, whereas traces from HFCS and TDZD‐8 groups are red and blue, respectively, in their corresponding columns.

Figure 4

HFCS consumption in adolescent rats caused increased ser⁸⁴⁵‐phosphorylation of GluA1 subunits and decreased transcription of neuron‐specific α3‐subunit of NKA in the hippocampus (n = 4 per group for immunoblot and n = 7 per group for qRT‐PCR, maximum of one per group was excluded from the qRT‐PCR experiments as the isolation yielded samples of low purity). (A) Relative protein levels of ser⁴⁷³‐phosphorylated Akt to Akt normalized to β‐actin compared to control group. (B) Relative protein levels of thr308‐phosphorylated Akt to Akt normalized to β‐actin compared to control group. (C) Relative protein levels of ser⁹‐phosphorylated GSK‐3B to GSK‐3B normalized to β‐actin compared to control group. (D) Relative protein levels of ser⁸⁴⁵‐phosphorylated GluA1 subunits to GluA1 subunits normalized to β‐actin compared to control group (statistical analysis was not performed as n < 5 per group). (E) Raw immunoblot images of bands of ser⁸⁴⁵‐phosphorylated GluA1 and GluA1 subunits, and β‐actin. (F) Relative protein levels of GluN2A subunits normalized to β‐actin compared to control group. (G) Relative protein levels of GluN2B subunits normalized to β‐actin compared to control group. (H) Relative expression of α1‐subunit (ATP1A1) compared to control group. (I) Relative expression of α2‐subunit (ATP1A2) compared to control group. (J) Relative expression of α3‐subunit (ATP1A3) compared to control group.

Figure 5

A systemic inflammatory response was evoked by HFCS consumption, but not locally in the hippocampi (n = 9 per group). (A) IL‐1β levels in serum. (B) IL‐6 levels in serum. (C) TNF‐α levels in serum. (D) IL‐1β levels normalized to total protein in hippocampus. (E) IL‐6 levels normalized to total protein in hippocampus. (F) TNF‐α levels normalized to total protein in hippocampus.