GSK-3β has occupied a central place in Alzheimer’s research for decades. The enzyme is a serine/threonine kinase that phosphorylates a wide array of substrates, including the tau protein whose hyperphosphorylated tangles are a hallmark of the disease. In a mouse model engineered to overexpress GSK-3β, previous work had already documented a decrease in dendritic spine density in the dentate gyrus, the gateway through which cortical information enters the hippocampus. What remained unknown was how the rest of the trisynaptic circuit—the entorhinal cortex, CA3, and CA1—responds to elevated levels of this kinase. The new study, led by María Llorens-Martín and colleagues at the Centro de Biología Molecular Severo Ochoa in Madrid, set out to fill that gap.

To capture the fine structure of neurons, the researchers turned to a classical but technically demanding technique: intracellular injection of Lucifer Yellow, a fluorescent dye that fills individual neurons in fixed tissue and reveals their dendritic trees and spines in exquisite detail. Using this method, the team analyzed dendritic spine morphology and density on both apical and basal dendrites of pyramidal neurons in the entorhinal cortex, CA3, and CA1, comparing wild-type mice with mice overexpressing GSK-3β. Spines—the tiny protrusions studding dendrites where most excitatory synapses reside—come in distinct morphological classes, including mushroom, thin, and stubby forms, each thought to reflect different stages of synaptic maturity and stability. The head diameter and length of each spine type were quantified, alongside the overall density of spines.

The first striking result came from the wild-type animals themselves. Even in healthy brains, the study found intrinsic differences in spine density and in the distribution of spine types between apical and basal dendrites across the different regions of the trisynaptic circuit. This baseline regional heterogeneity matters, because it establishes that apical and basal dendrites— which receive different complements of afferent inputs—are not interchangeable substrates. Any pathological process acting on the circuit will therefore be superimposed on a landscape already patterned by region- and compartment-specific wiring.

When the researchers compared the GSK-3β-overexpressing mice to their wild-type littermates, a clear hierarchy of vulnerability emerged. The entorhinal cortex, despite being the region typically hit earliest by Alzheimer’s pathology in humans, showed only subtle morphological changes in this model, a finding the authors interpret as a form of partial structural resilience. The CA3 region, likewise, presented minimal alterations overall—although here the basal dendrites were more affected than their apical counterparts, a pattern the authors attribute to the differential inputs these two dendritic compartments receive. In sharp contrast, CA1 stood out as the most affected region of the entire circuit, a result consistent with the well-documented vulnerability of CA1 to Alzheimer’s disease pathology in both human patients and animal models.

Beyond spine shape, the study probed the functional architecture of the circuit by examining excitatory innervation. The researchers stained for vesicular glutamate transporter 1, or VGlut1, a marker of excitatory presynaptic boutons, and measured the percentage of area occupied by VGlut1-positive terminals around the dendritic compartments. In both CA1 and the basal dendrites of CA3, the GSK-3β-overexpressing mice showed a reduced percentage of area occupied by VGlut1-positive boutons, indicating a loss of excitatory innervation. Because excitatory drive is the currency of synaptic plasticity—the cellular substrate of learning and memory—this loss suggests that GSK-3β overexpression does not merely remodel the shape of spines but actively disrupts the connectivity needed for plasticity to occur.

The regional selectivity of these alterations carries significant implications for how scientists think about the progression of Alzheimer’s disease. Rather than a diffuse, homogeneous degeneration of the hippocampal formation, the data support a model in which specific subregions and even specific dendritic compartments are differentially susceptible to a single molecular insult. The vulnerability of CA1 is particularly noteworthy: this region serves as the major output hub of the hippocampus, relaying processed information back to the cortex. Structural compromise at this node, combined with lost excitatory innervation, could plausibly undermine the coherence of the entire memory circuit even while upstream regions such as the entorhinal cortex and CA3 appear comparatively preserved.

The study also underscores the value of apical–basal comparisons. In CA3, basal dendrites were more affected than apical dendrites, likely reflecting the different sources of input they receive—apical dendrites in CA3 are heavily targeted by perforant path fibers from the entorhinal cortex and associational inputs, whereas basal dendrites sample a different mix of local and long-range afferents. That GSK-3β overexpression left a compartment-specific fingerprint on the circuit suggests that the enzyme’s effects are mediated not only by intrinsic neuronal properties but also by the identity of the inputs each dendrite integrates. This level of resolution would have been impossible with bulk tissue measurements and highlights why single-cell anatomy remains indispensable in neuroscience.

Methodologically, the work relied on carefully characterized mouse cohorts, with the GSK-3β-overexpressing model validated by β-galactosidase and NeuN staining to confirm transgene expression and neuronal counts across the entorhinal cortex, CA3, and CA1. Excitatory and inhibitory balance was further assessed by quantifying GAD65-positive inhibitory boutons alongside VGlut1-positive excitatory terminals, allowing the team to compute ratios that capture the excitatory–inhibitory landscape around each dendritic compartment. Statistical comparisons were performed across regions, genotypes, and dendritic compartments, with sample sizes of four to five animals per group for the various analyses, lending statistical weight to the observed regional differences.

Taken together, the study paints a nuanced picture of GSK-3β as a driver of regionally selective synaptic deterioration within the memory circuit. By demonstrating that the entorhinal cortex retains partial structural resilience, that CA3 is largely spared except for its basal compartment, and that CA1 is the most affected node, the researchers provide a cellular framework for understanding why certain hippocampal areas fail early in Alzheimer’s disease while others endure. The loss of excitatory innervation in CA1 and basal CA3 further implicates disrupted synaptic plasticity as a downstream consequence of kinase overexpression. As the field continues to pursue GSK-3β as a therapeutic target, these findings caution that interventions will need to account for the striking regional and compartmental heterogeneity of the hippocampal circuit—and they offer a detailed anatomical benchmark against which future protective strategies can be measured.

Subject of Research: Regional dendritic spine and innervation changes caused by GSK-3β overexpression in the hippocampal trisynaptic circuit of a mouse model relevant to Alzheimer's disease.

Article Title: Regional differences in dendritic spine morphology and innervation of the trisynaptic circuit of a mouse model of GSK-3β overexpression

Article References: Alonso-Moreno, M. C., Terreros-Roncal, J., Rodríguez-Moreno, C. B., Ávila, J., & Llorens-Martín, M. (2026). Regional differences in dendritic spine morphology and innervation of the trisynaptic circuit of a mouse model of GSK-3β overexpression. Cellular and Molecular Life Sciences. https://doi.org/10.1007/s00018-026-06304-z

Image Credits: AI Generated

DOI: 10.1007/s00018-026-06304-z

Keywords: Alzheimer's disease, GSK-3β, dendritic spines, hippocampus, trisynaptic circuit, entorhinal cortex, CA1, CA3, pyramidal neurons, VGlut1, synaptic plasticity, mouse model