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.

Glutamate signaling is essential for the persistent neural activity in prefrontal cortex (PFC) that enables working memory. Metabotropic glutamate receptors (mGluRs) are a diverse class of proteins that modulate excitatory neurotransmission via both presynaptic regulation of extracellular glutamate levels and postsynaptic modulation of ion channels on dendritic spines. This receptor class is of significant therapeutic interest for treatment of cognitive disorders associated with glutamate dysregulation. Working memory impairment and cortical hypoexcitability are both associated with advanced aging. Whether aging modifies PFC mGluR expression, and the extent to which any such alterations are regionally or subtype specific, however, is unknown. Moreover, it is unclear whether specific mGluRs in PFC are critical for working memory, and thus, whether altered mGluR expression in aging or disease is sufficient to play a causative role in working memory decline. Experiments in the current study first evaluated the effects of age on medial PFC (mPFC) mGluR expression using biochemical and molecular approaches in rats. Of the eight mGluRs examined, only mGluR5, mGluR3, and mGluR4 were significantly reduced in the aged PFC. The reductions in mGluR3 and mGluR5 (but not mGluR4) were observed in both mRNA and protein, and were selectively localized to the prelimbic (PrL), but not infralimbic (IL), subregion of PFC. Finally, pharmacological blockade of mGluR5 or mGluR2/3 using selective antagonists directed to PrL significantly impaired working memory without influencing non-mnemonic aspects of task performance. Together, these data implicate attenuated expression of PFC mGluR5 and mGluR3 in the impaired working memory associated with advanced ages. Significance Statement Working memory is impaired in several neuropsychiatric disorders and advanced aging. Glutamate is essential for persistent cellular activity in the prefrontal cortex (PFC) theorized to maintain working memory. Metabotropic glutamate receptors (mGluRs) are well-positioned to coordinate glutamate signaling at PFC synapses; however, studies to date have not yet systematically investigated the contributions of mGluR subtypes to normal working memory and PFC aging. This study shows that aging is accompanied by loss of PFC mGluR2/3 and mGluR5 mRNA and protein, and that pharmacological inhibition of these mGluR subtypes is sufficient to impair working memory. These findings suggest that mGluRs have a normal role in working memory and could serve as a target for treatment of cognitive disorders characterized by PFC dysfunction.

Age-related memory deficits correlate with dysfunction in the CA3 subregion of the hippocampus, which includes both hyperactivity and overly rigid activity patterns. While changes in intrinsic membrane currents and interneuron alterations are involved in this process, it is not known whether alterations in afferent input to CA3 also contribute. Neurons in layer II of the lateral entorhinal cortex (LEC) project directly to CA3 through the perforant path, but no data are available regarding the effects of advanced age on LEC activity and whether these activity patterns update in response to environmental change. Furthermore, it is not known the extent to which age-related deficits in sensory discrimination relate to the inability of aged CA3 neurons to update in response to new environments. Young and aged rats were pre-characterized on a LEGO© object discrimination task, comparable to behavioral tests in humans in which CA3 hyperactivity has been linked to impairments. The cellular compartment analysis of temporal activity with fluorescence in situ hybridization for the immediate-early gene Arc was then used to identify the principal cell populations that were active during two distinct epochs of random foraging in different environments. This approach enabled the extent to which rats could discriminate two similar objects to be related to the ability of CA3 neurons to update across different environments. In both young and aged rats, there were animals that performed poorly on the LEGO object discrimination task. In the aged rats only, however, the poor performers had a higher percent of CA3 neurons that were active during random foraging in a novel environment, but this is not relate to the ability of CA3 neurons to remap when the environment changed. Afferent neurons to CA3 in LEC, as identified with the retrograde tracer choleratoxin B (CTB), also showed a higher percent of cells that were positive for Arc mRNA in aged poor performing rats. This suggests that LEC contributes to the hyperactivity seen in CA3 of aged animals with object discrimination deficits and age-related cognitive decline may be the consequence of dysfunction endemic to the larger network.

Nutritional ketosis is induced by high fat/low carbohydrate dietary regimens, which produce high levels of circulating ketone bodies, shifting metabolism away from glucose utilization. While ketogenic diets (KD) were initially introduced to suppress seizures, they are garnering attention for their potential to treat a myriad of neurodegenerative and metabolic disorders that are associated with advanced age. The feasibility and physiological impact of implementing a long-term KD in old animals, however, has not been systematically examined. In this study, young and aged rats consumed a calorically- and nutritionally-matched KD or control diet for 12 weeks. All KD-fed rats maintained higher levels of BHB and lower levels of glucose relative to controls. However, it took the aged rats longer to reach asymptotic levels of BHB compared to young animals. Moreover, KD-fed rats had significantly less visceral white and brown adipose tissue than controls without a loss of lean mass. Interestingly, the KD led to significant alterations in protein levels of hippocampal transporters for monocarboxylates, glucose, and vesicular glutamate and gamma-aminobutyric acid. Most notably, the age-related decline in vesicular glutamate transporter expression was reversed by the KD. These data demonstrate the feasibility and potential benefits of KDs for treating age-associated neural dysfunction.