Fig. 2: Techniques used for investigating microRNAs and executive functions in human and animal models.

The methods used to assess the role of microRNA in executive functions (EFs) differ in various aspects. The most common were: (a) Regarding the selection or induction of the underlying pathology or age group being studied: a.1) In animal models: no genetic manipulation (examples are natural aging by using older animals or other behaviorally induced models, such as early life stress or social defeat); use of transgenic animals (with manipulation of genes such as DGCR8 or DICER that code for relevant enzymes related to the biosynthesis process); and administration of microRNAs inhibitors or mimics peripherally, e.g., by intranasal injection, or directly into the brain by stereotactic injections into the interventricular area or hippocampus; and a.2) In human studies: observational cohort, case-control, or cross-sectional studies without an intervention and experimental designs such as randomized controlled trials. b Regarding the methods used for assessing the phenotype, i.e., executive functioning: b.1) In animal models: behavioral assessments of working memory (other domains are still barely established) with the eight-arm water maze, Morris water maze, Novel Object Recognition test, Y water maze, or T water maze (these evaluations may require correction for visual and motor abilities); and b.2) In human studies: the domains of executive functions are broader, for instance the Trail Making Test can measure set shifting, the Card Sorting Test evaluates inhibitory control, and the digit symbol test assesses working memory. 3) Regarding the assessment of the genotype or the microRNA changes: 3a) sample collection, with examples of biomaterial ranging from saliva to brain-specific areas collected post mortem; 3b) measurement of microRNA expression either with tests for specific microRNAs that use polymerase chain reaction methods or with broader analysis by sequencing techniques; and (c) differential microRNA expression analysis by using pipelines in programming or statistical software, with possible further bioinformatics analysis, e.g., network analysis or gene set enrichment analysis. And (d) Regarding additional methods that can complement the research, for example: d.1) studies of cellular electrical or morphological changes after microRNA manipulation (with inhibitors or mimics) in cellular cultures derived from primary or human-derived induced pluripotent stem cells or organoids; and d.2) other omics techniques, such as proteome analysis or correlations with changes in structural or functional imaging markers. Created in BioRender. Navarro Flores, A. (2026) https://BioRender.com/ztbg5cd. Partially adapted from Faber, S. [164].