Saturated free fatty acids and association with memory formation

Learning and memory formation are complex processes involving various proteins and genes. While phospholipids and their metabolites are abundant in the brain and play roles in neural signaling, their contribution to learning and memory is understudied. Phospholipids are essential components of neuronal membranes and are involved in neurotransmission and synaptic plasticity. Phospholipases modify the phospholipid landscape, generating metabolites like free fatty acids (FFAs), which influence membrane dynamics and act as signaling molecules. Saturated FFAs, such as myristic and palmitic acids, are involved in protein lipidation (myristoylation and palmitoylation), crucial for synaptic plasticity. Previous studies focused mainly on unsaturated FFAs, particularly arachidonic acid, while the role of saturated FFAs remains unclear. This study aimed to investigate the distribution of FFAs and phospholipids in the rat brain and how these are altered during learning, using auditory fear conditioning (AFC) as a model. The researchers hypothesized that changes in saturated FFAs, along with unsaturated FFAs, contribute to memory acquisition.

Decades of research have established the involvement of proteins and genes in learning and memory. However, the role of phospholipids and their metabolites, despite their abundance in the brain, remains largely unexplored. Phospholipids are crucial for membrane structure and function in neurons, impacting neurotransmission and synaptic plasticity. Phospholipase activity releases FFAs, which affect membrane dynamics and can act as signaling molecules. Unsaturated FFAs like arachidonic acid have been extensively studied and linked to neurotransmitter release, membrane fluidity, and long-term potentiation (LTP). However, saturated FFAs are less understood despite their prevalence in the brain and their role in protein acylation, which is essential for synaptic plasticity. The relatively low abundance of arachidonic acid compared to other FFAs raises questions about the roles of saturated FFAs in these processes. Prior in vitro studies by the authors revealed the predominant generation of saturated FFAs during neural stimulation, indicating their potential involvement in neurotransmission, learning, and memory.

The study employed two complementary lipidomic techniques: Free Fatty Acid Stable Isotope Tagging (FFAST) and liquid chromatography-mass spectrometry (LCMS). FFAST enabled quantitative analysis of 18 FFA species across six rat brain regions (amygdala, prefrontal cortex, hippocampus, cerebellum). LCMS quantified 135 phospholipid species across five classes (phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine). Male Sprague-Dawley rats underwent auditory fear conditioning (AFC), where a neutral auditory tone was paired with a mild electric footshock in the paired group, and unpaired in the control group. A group received NMDA receptor antagonist CPP to block learning. Brains were rapidly extracted and analyzed using the lipidomic methods after perfusion with ice-cold ACSF to minimize post-mortem lipid changes. The FFA and phospholipid concentrations were measured in each brain region for each group, and statistical analysis including Student's t-test and multivariate analysis (isomap) were used to compare groups and identify significant changes in lipid profiles. Statistical significance was determined using two-tailed t-tests (p<0.05). Freezing behavior (a measure of fear memory) was also assessed.

The study revealed a non-homogeneous distribution of FFAs and phospholipids across brain regions, with the highest concentrations in the amygdala and prefrontal cortex. Auditory fear conditioning (AFC) induced significant changes in lipid profiles. Specifically, paired AFC in saline-treated rats led to increased saturated FFAs (especially myristic and palmitic acids) and, to a lesser extent, unsaturated FFAs (arachidonic acid) in the amygdala and prefrontal cortex. These FFA increases were largely absent when AFC was blocked by the NMDA receptor antagonist CPP. Multivariate analysis (isomap) confirmed the clear separation of FFA profiles based on CPP treatment and AFC pairing, strongly correlating with long-term memory consolidation. Phospholipid levels generally decreased following paired AFC, although some increases in PG were observed. The amygdala showed the most dramatic changes in FFA levels, especially saturated FFAs. The changes were most pronounced in the central amygdala, with myristic acid exhibiting the strongest response. While short-term memory acquisition showed no difference between saline and CPP treated rats, long-term memory consolidation was significantly impaired in the CPP group.

The study's findings demonstrate a significant link between FFA changes and memory consolidation. The increase in saturated FFAs (myristic and palmitic acids) during AFC, particularly in the amygdala, suggests their crucial role in memory formation. These FFAs might serve as substrates for protein acylation (myristoylation and palmitoylation), modifying protein-membrane interactions and impacting synaptic plasticity. The dependence of these changes on NMDA receptor activation highlights the connection between synaptic plasticity and lipid metabolism. The amygdala's prominent response in saturated and unsaturated FFAs aligns with its known role in fear memory. The prefrontal cortex's substantial response suggests its involvement in cognitive processes during fear learning. The lack of significant changes in the cerebellum, primarily involved in motor learning, supports the hypothesis of a specific role for these lipid changes in fear memory.

This study provides the first comprehensive survey of phospholipid metabolite changes during memory acquisition. The findings strongly support a role for saturated FFAs, particularly myristic acid, in long-term memory consolidation, a process dependent on NMDA receptor activation. Further research is needed to elucidate the exact mechanisms by which FFAs contribute to memory. Investigating the relationship between FFA generation and synaptic protein acylation is a promising avenue. Combining advanced lipidomics techniques with animal models of learning and memory will be crucial to unravel the complex interplay between lipids and cognition.

The study used only male rats, limiting the generalizability of findings to females. The time point for lipid analysis (2h post-AFC) might not fully capture all the dynamic changes in lipid profiles during memory consolidation. The study focused on a specific type of learning (fear conditioning) and might not be directly generalizable to other types of learning and memory.