Sex Shapes How the Brain’s Inhibitory Scaffolds Build Up During Early Development

Deep inside the brain’s hippocampus, a delicate lattice of extracellular matrix molecules wraps itself around specific inhibitory neurons during development, and new research reveals that this scaffolding does not grow in a simple, steady march toward maturity. Instead, according to a study published in the journal Biology of Sex Differences, the number of these structures, known as perineuronal nets, rises and falls in a distinctive pattern during juvenile development, and the timing of that pattern differs strikingly between males and females. The findings, from researchers at the University of Massachusetts Boston, offer a fresh window into how sex-specific neurodevelopmental trajectories could help explain why conditions such as autism spectrum disorder affect males and females at different rates.

Perineuronal nets, or PNNs, are specialized matrix structures composed of chondroitin sulfate proteoglycans, hyaluronan, and link proteins that encase the cell bodies and proximal dendrites of certain classes of neurons, most prominently fast-spiking parvalbumin interneurons. These nets are far more than passive scaffolding. They are thought to regulate the balance of excitation and inhibition in neural circuits and to govern the closure of critical periods of plasticity, the windows during which the brain is exquisitely malleable. When that balance is disturbed, the consequences can be profound: altered excitation-inhibition ratios and disrupted PNN formation have both been implicated in neurodevelopmental disorders, including autism spectrum disorder, a condition diagnosed substantially more often in biologically male children and adults. Understanding when and how PNNs normally appear, therefore, is not merely an exercise in descriptive neuroanatomy; it could illuminate when the developing brain is most vulnerable to insult.

Yet most of what scientists know about PNNs comes from studies of adolescent and adult animals, long after the nets have consolidated. Very little research has charted the typical, day-by-day emergence of PNNs and the parvalbumin interneurons they envelop during the earlier stretches of development. The research team, led by Abigail W. Hamilton and Susan L. Zup, set out to fill that gap by building a precise timeline of PNN onset in juvenile Sprague Dawley rats across a narrow but crucial developmental span: postnatal days 14, 16, 18, and 22. They focused on the dorsal hippocampus, a region central to learning, memory, and stress regulation, examining five distinct subregions: the subiculum, cornu ammonis 1 (CA1), CA2, CA3, and the dentate gyrus.

The methodology was straightforward but demanding in its precision. Rats at each of the four ages were transcardially perfused, and their brains were sectioned and processed for immunofluorescence staining targeting both perineuronal nets and parvalbumin interneurons. By labeling these two features simultaneously, the researchers could count net-bearing neurons in each hippocampal subregion at each time point and, crucially, compare counts between males and females. This fine-grained sampling at two-day intervals allowed the team to capture developmental dynamics that coarser, week-long sampling intervals might have smoothed away entirely.

The results upended the intuitive assumption that PNN numbers climb steadily as the juvenile brain matures. Instead, the researchers found that PNN counts fluctuated across the p14 to p22 window in a consistent pattern of decline followed by rebound, rather than increasing linearly. Remarkably, male and female rats followed the same general trajectory, but on shifted schedules. Female rats showed a dip in perineuronal net number at postnatal day 16 before the counts rebounded, whereas males exhibited a similarly steep decrease two days later, at postnatal day 18, before their own recovery. In other words, the female developmental program appeared to run ahead of the male one, completing the same sequence of net loss and net gain several days earlier.

Across subregions of the hippocampus, the study also documented a broader sex difference: males generally had fewer perineuronal nets than females. This asymmetry is particularly intriguing in light of the clinical epidemiology of autism spectrum disorder, which is more prevalent in biologically male children and adults. Because PNNs help regulate the excitation-inhibition balance whose disruption is associated with the disorder, the authors suggest that differences in the timing and density of net formation between the sexes could contribute to differential vulnerability. A male brain whose PNN maturation lags behind a female’s may spend a longer stretch of development in a state where its inhibitory circuitry and plasticity controls are still in flux, opening more or longer windows during which environmental, genetic, or biochemical insults could derail typical trajectories.

The fluctuations themselves carry implications for how neuroscientists should think about early brain development. If PNN numbers can drop sharply within a matter of days and then rebound, the developing hippocampus cannot be viewed as a structure steadily locking in its inhibitory architecture. Instead, it appears to pass through alternating phases in which nets are pruned and re-established, potentially reflecting active remodeling of parvalbumin interneuron circuitry during the juvenile period. Each phase of net reduction may correspond to a period of heightened plasticity, when circuits are more open to modification, and each phase of net re-accumulation to a period in which those modifications are being consolidated. Sex differences in the timing of these phases would mean that males and females are not equally susceptible to the same disruption at the same chronological age.

The study’s authors emphasize that these data point to windows of both increased and decreased vulnerability to neurons and neuronal circuitry during early neurodevelopment, windows that are specific to sex. For researchers modeling neurodevelopmental disorders in animals, the message is that the choice of developmental time point is not neutral. A postnatal day 16 female and a postnatal day 18 male may occupy comparable positions in their respective PNN trajectories, while a male and female of the same chronological age may be at entirely different stages. Experimental interventions, environmental manipulations, or pharmacological challenges delivered on identical schedules could therefore produce systematically different outcomes in the two sexes, a confound that precise developmental timelines like this one can now help investigators control for.

More broadly, the work adds to the growing recognition that sex differences are not peripheral curiosities in neuroscience but fundamental features of brain development that deserve systematic study from the earliest ages. By mapping the fluctuating emergence of perineuronal nets and their parvalbumin interneuron partners across the dorsal hippocampus, the University of Massachusetts Boston team has provided a reference framework against which future studies of atypical development can be measured. The research was funded by the University of Massachusetts Boston’s Program Development Grant and College of Liberal Arts Research Funds, and all experiments were approved by the institution’s Institutional Animal Care and Use Committee. As the field continues to probe the developmental roots of conditions like autism spectrum disorder, this sex- and age-resolved atlas of early hippocampal PNN development offers both a caution against assuming steady linear maturation and a promising map of the moments when the juvenile brain may be most, or least, open to lasting change.

Beyond the immediate findings, the study sits within a broader methodological shift in neuroscience. For decades, preclinical research relied disproportionately on male animals, partly on the assumption that cyclic hormonal variation in females would introduce unwanted variability. That assumption has been repeatedly challenged, and funding agencies now routinely require attention to sex as a biological variable. The present work illustrates why such policies matter: had the investigators examined only one sex, or only a single developmental time point, the fluctuating, sex-shifted trajectory of perineuronal net development would have been invisible. A study sampling at postnatal day 16 alone, for instance, would have concluded that females show reduced net density relative to earlier ages, while a day 18 sample would have suggested the same for males, with neither result revealing the underlying shared pattern.

The choice of the dorsal hippocampus also carries significance for interpretation. This region receives extensive attention in research on learning, memory consolidation, and the physiological stress response, and its parvalbumin interneuron population is known to mature gradually across the juvenile period. Parvalbumin cells are fast-spiking inhibitory neurons that depend on precise excitatory input and on metabolic and structural support from the extracellular matrix that surrounds them. Perineuronal nets are believed to stabilize the synapses formed on these cells and to contribute to the maturation of their characteristic rapid firing properties, so fluctuations in net number during the juvenile window may reflect ongoing reorganization of inhibitory microcircuitry rather than simple cell loss or gain.

It is also worth noting what the study does and does not establish. The work documents correlations between age, sex, and net abundance in a typical laboratory strain; it does not demonstrate a causal link between perineuronal net timing and any disorder. Autism spectrum disorder in humans involves many genetic and environmental influences, and rodent hippocampal development, while homologous in broad outline, cannot be mapped directly onto human clinical outcomes. What the data provide is a normative reference: a detailed baseline against which future studies of genetically modified animals, early-life stress models, or environmental exposures can be compared. Such baselines are essential for detecting whether an experimental manipulation accelerates, delays, or flattens the normal developmental curve in one sex but not the other.

The article itself was published as open access under a Creative Commons Attribution license, making the full dataset and methods available to any laboratory without subscription barriers. The version released in early September 2026 is a citable accepted manuscript carrying a permanent digital object identifier, with a final version of record to follow after routine production edits. For a field increasingly attentive to reproducibility, the combination of fine-grained age sampling, dual immunofluorescence labeling, and sex-balanced design offers a template for how descriptive developmental neuroanatomy can be made both rigorous and broadly reusable.

Subject of Research: Sex differences in the developmental timing of perineuronal nets in the rat hippocampus

Article Title: Sex and age impact the developmental trajectory of perineuronal net number in the rat dorsal hippocampus

Article References: Hamilton, A. W., Pritchard, E. K., Hunter III, R. G., & Zup, S. L. (2026). Sex and age impact the developmental trajectory of perineuronal net number in the rat dorsal hippocampus. Biology of Sex Differences. https://doi.org/10.1186/s13293-026-00961-2

Image Credits: AI Generated

DOI: 10.1186/s13293-026-00961-2

Keywords: perineuronal nets, hippocampus, sex differences, neurodevelopment, parvalbumin interneurons, autism spectrum disorder, rats, excitation-inhibition balance, critical periods, postnatal development, extracellular matrix, plasticity