The research, conducted by a Bulgarian team, set out to test whether newly synthesized creatine derivatives could enhance learning and memory, increase physical endurance, and influence depressive-like behavior. The work builds on a long scientific record suggesting that creatine is far more than a sports supplement. The molecule, produced endogenously at roughly one gram per day in the liver, kidneys, and pancreas from the amino acids glycine, methionine, and arginine, also enters the body through meat, fish, and dietary supplements. Its central role is energetic: phosphocreatine acts as an instant reservoir of high-energy phosphate bonds that can rapidly regenerate adenosine triphosphate, the cellular fuel currency, in tissues with intense energy demands such as skeletal muscle and the brain.

That energy-supply role is precisely why neuroscientists have long suspected creatine could matter for cognition. Neurons carry out many energy-hungry processes, and genetic disorders of creatine metabolism, including deficiencies of the enzymes AGAT and GAMT or the creatine transporter SLC6A8, are characterized by global developmental delay and intellectual disability. Oral creatine supplementation largely reversed these dysfunctions in AGAT and GAMT deficiency, though not in transporter deficiency. Mental training has been shown to elevate brain creatine levels, and higher resting creatine concentrations have been associated with better performance on recognition memory tasks. In humans, supplementation has been reported to improve attention under short-term hypoxic conditions, enhance mood and psychomotor function in sleep-deprived subjects, raise intelligence test scores and working memory, and reduce mental fatigue, with brain imaging confirming that such doses increase brain creatine pools and the phosphocreatine-to-ATP ratio.

To probe these possibilities experimentally, the researchers worked with male albino mice of the H line, weighing 28 to 32 grams, divided into nine groups of six animals each. One group served as controls and received only drinking water. A second received conventional creatine monohydrate at 1.5 grams per kilogram per day. Five further groups received newly synthesized creatine derivatives, each conjugated with a different amino acid, at 5 grams per kilogram per day: creatine lysinate, creatine leucinate, creatine arginate, creatine isoleucinate, and creatine valinate. The derivatives were synthesized and provided by Professor Lyubomir Vezenkov of the University of Chemical Technology and Metallurgy in Sofia. Two final groups received established antidepressants as comparators, citalopram hydrobromide and fluoxetine hydrochloride, each at 20 milligrams per kilogram. The creatine compounds were delivered through drinking water for two weeks, while the antidepressants were given by oral gavage. Behavioral testing was performed on days 1, 7, and 14 of the experiment, which was approved by the Bulgarian Food Safety Agency and conducted under European Directive 2010/63/EU on animal protection.

The team used three complementary behavioral assays. Learning and memory were assessed with the passive avoidance test, a fear-motivated, one-trial task that exploits rodents’ natural preference for dark spaces. Each mouse was placed in a lit compartment of a two-chamber apparatus; when it entered the dark chamber, it received a mild 0.5 milliampere electric shock lasting three seconds. On later trials, the latency to re-enter the dark compartment served as the memory measure, with longer latencies indicating better retention. Motor function and fatigue were evaluated with an accelerating rotarod, on which mice were placed on a rotating rod speeding from 4 to 40 revolutions per minute over 30 seconds, with the latency to fall recorded across three consecutive trials. Depressive-like behavior was measured with the tail suspension test, in which mice suspended by their tails were observed for six minutes, with the final four minutes scored for immobility time, a standard proxy for antidepressant-like effects, alongside the latency to first immobility.

The memory results were striking. On day 14, the group treated with creatine leucinate showed the most significant increase in latency compared with controls, with a p-value at or below 0.0001, and fluoxetine-treated animals also showed significantly prolonged latency at p at or below 0.001. Creatine leucinate significantly outperformed not only creatine monohydrate, creatine arginate, and creatine valinate, but also creatine isoleucinate and citalopram. Creatine lysinate produced a smaller but statistically significant improvement in latency compared with controls on day 14. In other words, the leucine-conjugated derivative appeared to enhance memory retention more effectively than the parent compound creatine monohydrate itself, and even more than one of the two reference antidepressants in this particular test.

The rotarod results told a different story, with a different compound in the lead. Creatine isoleucinate produced a significant increase in latency to fall between days 1 and 14 within its own group, and by the final week it showed significantly better motor coordination than creatine leucinate, creatine valinate, creatine lysinate, fluoxetine, and the control group. The authors suggest this effect may relate to the direct positive effects of leucine and isoleucine on muscle strength and recovery. Branched-chain amino acids are essential substrates and regulators of muscle protein synthesis, and leucine in particular is known to enhance protein synthesis through the mTOR signaling pathway and to stimulate insulin secretion. Previous work has shown that twelve weeks of leucine-rich amino acid supplementation can counteract the strength losses induced by eccentric exercise, lending plausibility to the idea that attaching these amino acids to creatine could amplify benefits for motor performance.

The antidepressant question yielded the most cautious findings. In the tail suspension test, there was no statistically significant difference in immobility time among the groups, meaning none of the creatine derivatives demonstrated a clear antidepressant-like effect by this primary measure. A downward trend in immobility time, especially on days 7 and 14, was observed in the groups treated with creatine monohydrate, creatine lysinate, and fluoxetine, consistent with a previous study by the same team that found a slight decrease in immobility with creatine monohydrate and creatine lysinate. On the latency measure, a statistically significant increase in time to first immobility was observed only on day 1, with creatine monohydrate, creatine leucinate, creatine isoleucinate, and citalopram all significantly prolonging this latency compared with controls. Interestingly, the two reference antidepressants diverged: fluoxetine produced a gradual decrease in immobility over the two weeks, while citalopram-treated animals showed a gradual increase in immobility from the first to the second week, even though citalopram significantly prolonged time to first immobility on day 1.

The authors place these mixed antidepressant findings in context by noting that the effects of fluoxetine and citalopram vary across studies depending on the test used, the mouse strain, and the dose applied. In prior work, fluoxetine weakly and dose-dependently decreased immobility duration in the forced swim test in NMRI mice and increased latency to first immobility, while in C57BL6/J mice it did not significantly affect immobility duration but did increase latency at 32 milligrams per kilogram. The serotonergic system, densely represented in the amygdala, hippocampus, and prefrontal cortex, is deeply involved in fear learning and emotional memory, which may explain why fluoxetine also improved memory retention in the passive avoidance task over the two-week course of treatment in the present study.

The broader significance of the work lies in its demonstration that the molecular company creatine keeps may shape its effects on the brain and body. By conjugating creatine with different amino acids, the researchers produced compounds with distinct behavioral profiles: creatine leucinate stood out for memory enhancement, creatine isoleucinate for motor coordination, and creatine lysinate showed modest benefits on both memory and immobility trends. These are early findings from a small study, with six animals per group and a two-week treatment window, conducted in a single mouse strain and sex, so the results cannot be extrapolated directly to humans or to clinical use. Nor do they establish mechanisms; the study measured behavior, not brain creatine levels or signaling pathways. Still, the data add to a growing body of evidence that creatine metabolism influences brain physiology and function, and they suggest that rational chemical modification of this well-known molecule could open new avenues for supporting cognitive and motor performance, even as the promise of creatine-based antidepressant effects remains, for now, unproven.

Subject of Research: Effects of newly synthesized creatine derivatives on memory, motor coordination, and depressive-like behavior in mice

Article Title: Effects of newly synthesized creatine derivatives on memory, motor coordination, and depressive-like behavior in mice

Article References: Valchinkova, I., & Kostadinova, I. (2026). Effects of newly synthesized creatine derivatives on memory, motor coordination, and depressive-like behavior in mice. Pharmacia, 73, Article e201921. https://doi.org/10.3897/pharmacia.73.e201921

Image Credits: AI Generated

DOI: 10.3897/pharmacia.73.e201921

Keywords: creatine, creatine derivatives, mice, memory, motor coordination, passive avoidance test, rotarod test, tail suspension test, antidepressant, branched-chain amino acids, fluoxetine, citalopram