A Ketogenic Diet Improves Cognition and Has Biochemical Effects in Prefrontal Cortex That Are Dissociable From Hippocampus

Abstract

Age-related cognitive decline has been linked to a diverse set of neurobiological mechanisms, including bidirectional changes in proteins critical for neuron function. Importantly, these alterations are not uniform across the brain. For example, the hippocampus (HPC) and prefrontal cortex (PFC) show distinct patterns of dysfunction in advanced age. Because higher cognitive functions require large-scale interactions across prefrontal cortical and hippocampal networks, selectively targeting an alteration within one region may not broadly restore function to improve cognition. One mechanism for decline that the PFC and HPC share, however, is a reduced ability to utilize glucose for energy metabolism. Although this suggests that therapeutic strategies bypassing the need for neuronal glycolysis may be beneficial for treating cognitive aging, this approach has not been empirically tested. Thus, the current study used a ketogenic diet (KD) as a global metabolic strategy for improving brain function in young and aged rats. After 12 weeks, rats were trained to perform a spatial alternation task through an asymmetrical maze, in which one arm was closed and the other was open. Both young and aged KD-fed rats showed resilience against the anxiogenic open arm, training to alternation criterion performance faster than control animals. Following alternation testing, rats were trained to perform a cognitive dual task that required working memory while simultaneously performing a bi-conditional association task (WM/BAT), which requires PFC-HPC interactions. All KD-fed rats also demonstrated improved performance on WM/BAT. At the completion of behavioral testing, tissue punches were collected from the PFC for biochemical analysis. KD-fed rats had biochemical alterations within PFC that were dissociable from previous results in the HPC. Specifically, MCT1 and MCT4, which transport ketone bodies, were significantly increased in KD-fed rats compared to controls. GLUT1, which transports glucose across the blood brain barrier, was decreased in KD-fed rats. Contrary to previous observations within the HPC, the vesicular glutamate transporter (VGLUT1) did not change with age or diet within the PFC. The vesicular GABA transporter (VGAT), however, was increased within PFC similar to HPC. These data suggest that KDs could be optimal for enhancing large-scale network function that is critical for higher cognition.

Keywords: GABA; anxiety; glucose; glutamate; metabolism; monocarboxylate; transporter.

FIGURE 1

Schematic of behavioral tasks utilized and experiment timeline. (A) Elevated figure-8 shaped maze used for all experimental testing in which rats were first trained to alternate between the left and right arms. Note the right arm was enclosed on both sides by white walls (gray for depiction purposes only) and the left arm was open on both sides. (B) Dual task WM/BAT took place using one object pair, but the correct object was contingent on location within the maze. (C) This same maze was used for random arm BAT, with the same objects, but one arm was randomly blocked off each trial by the experimenter. (D) Example of objects used for WM/BAT testing. (E) Timeline of diet implementation and behavioral testing.

FIGURE 2

Confirmation of nutritional ketosis following administration of a ketogenic diet in animals used for the behavioral assay. (A) Glucose levels significantly differed across diet groups, but not across age groups, throughout the duration of the diet. (B) Similarly, β-hydroxybutyrate (BHB) levels were significantly elevated in rats on the KD relative to rats on the CD, but did not differ across age groups, throughout the duration of the diet. All values represent the mean ± SEM, ^∗indicates main effect of diet.

FIGURE 3

Performance by age and diet on ability to alternate within the elevated figure-8 maze. (A) Percent correct alternations during training for young rats within each diet group. (B) Percent correct alternations during training for aged rats within each diet group. (C) Number of incorrect trials before reaching criterion performance (≥80% correct turns) revealed that there was no significant main effect of age (p = 0.37), but there was a significant main effect of diet (p = 0.01). (D) Similarly, there was a main effect of diet (p = 0.02) but not age (p = 0.75) on the turn bias on day 20 of testing, as KD-fed rats were able to overcome their closed side bias by this day while CD-fed rats were not. (E) Performance on the final day of alternation training for all rats indicated all rats were able to perform similarly on alternations prior to moving on to subsequent tasks regardless of age and diet (p > 0.14 for both). All values represent the mean ± SEM; ^∗ indicates main effect of diet.

FIGURE 4

Performance on the working memory/bi-conditional association tasks (WM/BAT). (A) Percent correct object choices (y-axis) on each day of WM/BAT training (x-axis) in young rats within each diet group. (B) Percent correct object choices (y-axis) on each day of WM/BAT training (x-axis) in aged rats within each diet group. The first day on which any group reached criterion performance was day 12, thus performance across days 12–15 were analyzed (blue box). (C) There was a significant main effect of both diet (p < 0.05) and age (p = 0.03) on WM/BAT performance on days 12–15. (D) There was no effect of either age or diet on turn bias during days 12–15 (p ≥ 0.12 for both). (E) Performance on random arm BAT (RA BAT) was not affected by either age or diet (p ≥ 0.28 for both), nor did it differ from performance on regular WM/BAT on the final day of testing (p 0.22). All values represent the mean ± SEM; #indicates main effect of age, ^∗indicates main effect of diet.

FIGURE 5

Metabolic transporter expression within the medial prefrontal cortex, shown as percent of young control rat expression (dotted line), with representative bands from each gel. (A) Schematic of locations of metabolic transporters within the central nervous system. (B–C) Expression of glucose transporters. (D–F) Expression of monocarboxylate transporters. All values are group means expressed as percent of young controls (dotted line) ± SEM, ^∗indicates main effect of diet, #indicates a main effect of age and × indicates significant interaction between age and diet.

FIGURE 6

Vesicular transporter expression within the medial prefrontal cortex, shown as percent of young control rat expression (dotted line), with representative bands from each gel. (A) Vesicular glutamate transporter (VGLUT1) expression did not vary across age (p = 0.11) or diet (p = 0.90) groups. (B) Vesicular GABA transporter (VGAT) expression was significantly higher in rats in the KD-fed group relative to the CD-fed group, regardless of age (p = 0.01). All values are group means expressed as percent of young controls (dotted line) ± SEM, ^∗indicates significant main effect of diet.