Long-Read RNA Sequencing Maps Sex Differences in the Rat Hippocampus at Isoform Level

The hippocampus has long been a stage on which scientists watch memory being written, and for decades researchers have reported that male and female animals often perform differently on tasks that depend on this seahorse-shaped structure. What has remained stubbornly opaque is the molecular machinery beneath those differences. A new study published in the journal Biology of Sex Differences takes one of the most detailed looks yet at that machinery, using a cutting-edge sequencing technology to read RNA molecules in the rat hippocampus from end to end, and it finds that the brains of males and females organize their genetic messages in strikingly different ways depending on where inside the neurons those messages reside.

The research, led by J. P. Silva and J. L. Fiedler at the Laboratory of Neuroplasticity and Neurogenetics of the Universidad de Chile, focused on the CA1 region of the dorsal hippocampus, a area essential for forming and retrieving spatial and episodic memories. Rather than treating the tissue as an undifferentiated soup of cells, the team physically separated two functionally distinct compartments: the soma, where neuronal cell bodies house the nucleus and most of the cell’s protein-making infrastructure, and the neuropil, the dense thicket of axons, dendrites and synapses that surrounds those cell bodies and carries out the actual business of neural communication. This spatial split matters because neurons are known to ship specific messenger RNAs out to remote dendritic compartments, where they can be translated into proteins on-site, a process considered essential for synaptic plasticity, the cellular substrate of learning and memory.

To read the RNA in each compartment, the researchers turned to long-read direct RNA sequencing from Oxford Nanopore, a technology that threads intact, full-length RNA molecules through protein nanopores and reads them without the fragmentation and assembly steps required by conventional short-read methods. Where standard RNA sequencing often struggles to distinguish which exon combinations belong to which transcript, the long-read approach resolves individual isoforms, the alternative versions of a gene’s message produced when different exons are stitched together, when transcription starts or stops at different points, or when the poly(A) tail at the message’s end is trimmed or extended. In this study, the technique allowed the team to identify full-length transcripts, quantify isoform usage, and even detect RNA species that had never been annotated in the rat genome’s reference catalog.

The first surprise was a kind of parity. When the researchers compared male rats with females in the estrus phase, a stage of the reproductive cycle characterized by relatively low estrogen levels, they found that the overall numbers of genes and isoforms detected in CA1 were broadly comparable between the sexes. Roughly eighty percent of the transcripts they detected were classified as protein-coding messenger RNAs, and about eight percent corresponded to RNA species absent from existing annotations, a reminder of how much of the transcriptome remains unmapped even in a tissue as intensively studied as the hippocampus. At the coarse level of counting molecules, male and female CA1 looked reassuringly similar.

The differences emerged when the researchers looked at where the molecules were and which versions of them were being used. Sex-associated differences proved more pronounced in the somatic compartment, where males showed a higher variety of RNA isoforms than estrus-phase females, while transcript profiles in the neuropil diverged far less between the sexes. Differential expression analysis told a complementary story: in estrus-phase females, 892 transcripts were enriched in the neuropil and 467 in the soma, whereas in males the corresponding figures were 973 neuropil-enriched and 541 soma-enriched transcripts. Intriguingly, some RNA instructions that were abundant in the neuropil of males were instead more concentrated in the cell bodies of females, suggesting that the two sexes route their molecular cargo to different addresses within the same neurons.

The study also uncovered structural signatures that distinguish the two compartments regardless of sex. Transcripts enriched in both the soma and the neuropil carried longer 5-prime untranslated regions, the stretches of RNA upstream of the protein-coding sequence that regulate how efficiently a message is translated. Somatic transcripts, however, bore longer 3-prime untranslated regions, segments that harbor binding sites for microRNAs and RNA-binding proteins and thus govern message stability and localization, while short poly(A) tails emerged as a hallmark of the neuropil fraction. Because poly(A) tail length can influence both translation efficiency and mRNA decay, this compartmental difference hints at distinct post-transcriptional control regimes operating in cell bodies versus synaptic territories.

One of the most striking sex-specific findings was that female neuropil-enriched transcripts displayed significantly shorter coding sequences than their male counterparts. Functional enrichment analysis added texture to this observation: transcripts enriched in the female neuropil were associated with lipid metabolism and translation-related processes, suggesting a neuropil environment tuned toward local protein synthesis and membrane-related functions. Male-enriched somatic transcripts, by contrast, were linked to memory, the regulation of cellular activity, and the development of neuronal projections, a functional profile consistent with the soma’s role as the command center from which neuronal architecture is built and maintained.

At the level of individual isoforms, the analysis revealed sex- and layer-specific enrichment of RNAs encoding receptors, scaffolding proteins, ribosomal components, and transcriptional regulators. This is a consequential category of molecules. Receptors determine how a neuron responds to its inputs, scaffolding proteins organize the molecular machinery of the synapse, ribosomal components set the capacity for local protein production, and transcriptional regulators feed back to reshape the cell’s entire gene expression program. Sex-dependent selection among isoforms of such genes means that male and female CA1 neurons may not merely express different amounts of the same proteins, but different versions of them, with potentially distinct interaction partners, localization signals, and regulatory properties.

The authors are careful to frame their findings as a baseline map rather than an explanation of behavioral differences. The comparisons were made under basal conditions, and the female cohort was restricted to the estrus phase, one specific hormonal state within the estrous cycle. Since estrogen and other ovarian hormones are known to modulate hippocampal gene expression and spine density, examining other phases of the cycle, or hormonally manipulated animals, would be a natural next step. Nevertheless, the study demonstrates that the molecular landscape of the hippocampus is organized along two axes simultaneously, compartment and sex, and that these axes interact in ways that shorter-read, whole-tissue approaches would have averaged away entirely.

The broader significance lies in the methodological shift the work exemplifies. For years, transcriptomic studies of the brain have catalogued which genes are switched on and by how much, but the layer of biology that determines which isoform of each gene is deployed, where in the neuron it travels, and how its untranslated regions and poly(A) tail are configured has remained largely invisible. By resolving that layer in a memory-critical brain region, and by showing that it differs between the sexes even at rest, the Chilean team has provided a molecular framework for investigating why male and female brains differ in their susceptibility to neurological and psychiatric disorders, from Alzheimer’s disease to depression, conditions in which both hippocampal dysfunction and sex bias are well documented. The map is drawn in rats, but the questions it opens apply to brains of every species, including our own.

Subject of Research: Sex-associated isoform distribution in the soma and neuropil of the rat hippocampal CA1 revealed by long-read direct RNA sequencing

Article Title: Long-read RNA sequencing reveals sex-associated isoform distribution in soma and neuropil of the rat hippocampal CA1

Article References: Silva, J. P., Catalán, J., Olave, F. A., González, P. I., Guarnieri, T., Palacios-Avendaño, N., Corrales, W. A., Alarcón-Mardones, M., Aliaga, E., Munita, R., Maracaja-Coutinho, V., & Fiedler, J. L. (2026). Long-read RNA sequencing reveals sex-associated isoform distribution in soma and neuropil of the rat hippocampal CA1. Biology of Sex Differences. https://doi.org/10.1186/s13293-026-00988-5

Image Credits: AI Generated

DOI: 10.1186/s13293-026-00988-5

Keywords: long-read sequencing, RNA isoforms, hippocampus, CA1, sex differences, neuropil, soma, transcriptome, Oxford Nanopore, poly(A) tail, synaptic plasticity, gene expression