Laminar organization of pyramidal neuron cell types defines distinct CA1 hippocampal subregions

Pachicano, Maricarmen; Mehta, Shrey; Hurtado, Angela; Ard, Tyler; Stanis, Jim; Breningstall, Bayla; Bienkowski, Michael S.

doi:10.1038/s41467-025-66613-y

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Published: 03 December 2025

Laminar organization of pyramidal neuron cell types defines distinct CA1 hippocampal subregions

Nature Communications volume 16, Article number: 10604 (2025) Cite this article

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Abstract

Investigating the cell type organization of hippocampal CA1 is essential for understanding its role in memory and cognition and its susceptibility to neurological disorders like Alzheimer’s disease and epilepsy. Multiple studies have identified different organizational principles for gene expression and how it reflects cell types within the CA1 pyramidal layer including gradients or mosaic. Here, we identify sublaminar gene expression patterns within the mouse CA1 pyramidal layer that span across the entire hippocampal axis. Our findings reveal that CA1 subregions (CA1d, CA1i, CA1v, CA1vv) contain differentially distributed layers of constituent cell types and can be identified by regional gene expression signatures. This work offers a new perspective on the organization of CA1 cell types that can be used to further explore hippocampal cell types across species.

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Introduction

CA1 is a laminar cortical region in the hippocampus containing a pyramidal cell layer, with pyramidal neurons whose dendrites extend into superficial and deep input layers. CA1 pyramidal neurons are notable for their unique cell type-specific functional properties (e.g. ‘place’ cells)¹ and these cell types can be characterized by a distinct combination of factors, including anatomical location, connectivity, morphology, molecular expression, electrophysiological properties, and functional attributes². Previous studies of the cellular and molecular differences of CA1 pyramidal cells have demonstrated differential gene expression within the pyramidal layer^{3,4,5,6,7,8,9}. However, how differential gene expression reflects the cell type organization across the entire CA1 is still debated^10,11.

Early mouse studies investigating the molecular organization of CA1 proposed that CA1 is made up of discrete molecular domains across its longitudinal axis, delineating dorsal, intermediate, and ventral subregions (CA1d, CA1i, and CA1v)^3,6,7,12. More recently, RNA-seq single-cell transcriptomics studies of the mouse CA1 characterized gene expression heterogeneity at the single-cell level with analysis at dorsal, intermediate, and ventral parts that suggested smooth gradients of continuously distributed cell types^9,13. In contrast, a spatial transcriptomic study of the CA1 pyramidal layer identified spatially discrete cell types organized as a mosaic-like pattern that changes within distinct CA1 subregions across the dorsal-ventral axis¹⁴. Finally, a laminar organization of gene expression has been observed specifically in CA1d^3,15 and extensive research investigating electrophysiological differences in the CA1 pyramidal layer supports that CA1d cell types form distinct ‘superficial’ and ‘deep’ sublayers within the pyramidal layer^{16,17,18,19,20,21,22,23}. Deep and superficial CA1d pyramidal neurons innervate distinct functional circuits in the hippocampus^24,25, but how this laminar organization extends beyond CA1d is unknown.

Our previous work creating the mouse Hippocampus Gene Expression Atlas (HGEA) utilized Allen Brain Atlas in situ hybridization (ISH) data (mouse.brain-map.org) to define 20 distinct genetic subdivisions of the hippocampus and subiculum based on mapped gene expression patterns²⁶. The HGEA defines hippocampal cell types based on connectivity and gene expression, where we found that the CA1 was divided into four distinct molecular subregions with differential brain-wide connectivity. We hypothesized that CA1 gene expression patterns reflected a hidden sublaminar organization across the entire CA1, similar to what we observed in the subiculum (SUB)²⁷, but these layers were difficult to distinguish due to the CA1’s more mixed heterogeneity. Here, we tested the hypothesis that complementary CA1 gene expression patterns are spatially distinct and demarcate sublayers of differentially distributed CA1 pyramidal cell types across the entire hippocampal axis reflecting the CA1 subregions previously identified in the HGEA (CA1d, CA1i, CA1v, CA1vv; Fig. 1 and Supplementary Fig. 1).

**Fig. 1: Refined CA1 Hippocampus Gene Expression Atlas.**

Our study uses RNAscope single molecule fluorescent in situ hybridization (smFISH)²⁸ to label 4 marker genes (Supplementary Fig. 2) at high resolution to investigate CA1 spatial organization. 20μm thick coronal C57BL/6 mouse brain sections were hybridized with probes targeting CA1 gene expression markers using RNAscope HiPlex V2 kit (Advanced Cell Diagnostics) (Supplementary Fig. 3a; see Methods). To analyze CA1 across all levels of the hippocampus, we have curated an analysis pipeline using QuPath cell segmentation (Supplementary Fig. 3b-h)²⁹ and SCAMPR analysis software³⁰ to quantify 332,938 marker gene transcripts within 58,065 detected pyramidal layer cells in coronal tissue sections spanning all of CA1 (Fig. 2) which corresponds to HGEA Levels 69-91. Using this approach, we show that the CA1 pyramidal layer comprises four transcriptionally distinct cell type sublayers; that each HGEA subregion is defined by characteristic laminar gene expression signatures with clear sublayer transitions; and that in the ventral hippocampus, CA1 and SUB layers form a complex transition where superficial CA1 sublayers align with deep SUB sublayers. Together, these findings provide a comprehensive view of CA1 cell type organization.

**Fig. 2: CA1 laminar expression patterns across all rostrocaudal levels.**

Results

General structure of the CA1 within the hippocampus

CA1 pyramidal neurons are organized as a discrete layer across the hippocampal axis. In all species, the CA1 pyramidal layer is continuous with the pyramidal layers of the CA3, CA2 and SUB hippocampal subregions, however, the thickness of the CA1 pyramidal layer changes as it approaches these adjacent structures^15,31. Whereas the proximal CA1 pyramidal layer is only a few cells deep, the distal CA1 is nearly as thick as the SUB, making it difficult to determine the distal CA1/SUB boundary based solely on cytoarchitecture. As recent studies have characterized deep vs. superficial pyramidal neurons in the distal CA1^{16,17,18,21,22}, the gradual thickening of the CA1 pyramidal layer along the hippocampal axis suggests that either these two cell type sublayers become thicker or there are additional sublayers within other parts of CA1.

Distinct subregions of CA1 are composed of different combinations of layered cell types

Using Allen Institute in situ hybridization data, we previously defined the four CA1 subregions – CA1d, CA1i, CA1v, and CA1vv – based on four gene expression patterns that were spatially located at different positions along the dorso-ventral axis and their differential input/output connectivity (Fig. 1, left)²⁶. Although it was clear these gene expression patterns appeared laminar, the thin size of the layers made it difficult to segregate and determine the full extent of the laminar organization across the CA1.

For RNAscope smFISH experiments, we chose the HGEA marker genes Lrmp, Ndst4, Trib2, and Peg10 as examples for each of these spatially distinct patterns (Supplementary Fig. 2). Rather than each gene being restricted to a specific CA1 subregion, we found that each gene expression pattern defined complementary sublayers of the CA1 pyramidal layer that varied along the longitudinal axis (Fig. 1, right). Notably, the previous HGEA subregion boundaries demarcate shifts or transitions in the size, shape, and position of each of these newly-defined sublayers (high Lrmp expression defines CA1 Layer 1 cell types, high Ndst4 expression defines CA1 Layer 2 cell types, high Trib2 expression defines CA1 Layer 3 cell types, high Peg10 expression defines CA1 Layer 4 cell types). In the following sections, we will provide quantitative data across multiple hippocampal sections (Fig. 2) to support delineation of each of the CA1 subregions and their constituent cell type sublayers. In total, 38,484 Lrmp transcripts, 137,075 Ndst4 transcripts, 80,837 Trib2 transcripts, and 76,542 Peg10 transcripts were detected across 58,065 CA1 pyramidal layer cells. We demonstrate that the HGEA CA1 subregions (CA1d, CA1i, CA1v, and CA1vv) can be defined by unique gene expression signatures characterized by distinct combinations of layer-specific patterns across all CA1 levels (Fig. 3a). In contrast to the previous HGEA delineation, this analysis identifies the caudal CA1 as a potentially independent subregion, supported by its distinct gene expression profile (Fig. 3b) and trilaminar organization. Quantification of marker gene expression across superficial to deep parts of the pyramidal layer within each CA1 subregion show clear enrichment patterns consistent with our laminar boundaries (Supplementary Fig. 4). Additional supporting evidence from coexpression analyses (Supplementary Fig. 5) showed limited overlap among neighboring layer-specific markers, as visualized by LOESS fits of normalized single-cell expression values, supporting that adjacent layers represent distinct CA1 cell types.

**Fig. 3: CA1 subregional gene expression signatures across all sections.**

CA1d is defined by Layer 1 and Layer 2 cell types

In the dorsal region of CA1 (Fig. 4a, b), pyramidal neurons appear to be organized into a superficial Layer 1, marked by the gene Lrmp (Fig. 4c, e) and a deep Layer 2, marked by the gene Ndst4 (Fig. 4c, f). Trib2 expression is moderate (Fig. 4g), but spatially spread in cells across both layers, whereas Peg10 expression is almost entirely absent except for a few scattered cells (Fig. 4h). At rostral levels of CA1d (Supplementary Fig. 6a, b), Layers 1 and 2 each occupy roughly half the thickness of the pyramidal layer (Supplementary Fig. 6e, f) whereas Layer 2 is thicker at caudal levels of CA1d with Layer 1 tapering off as it approaches the CA1i boundary, as illustrated in the normalized gene expression topographic heatmap (Fig.4f; right). Quantification of normalized gene expression across all levels of CA1d shows similar gene expression signature (Fig. 3a, Fig. 4d and Supplementary Fig. 6d) and display similar spatial laminar organization in topographic heatmaps (Fig. 4e-h and Supplementary Fig. 6e-h; right).

**Fig. 4: CA1d Gene Expression Signature: Layer 1 and Layer 2.**

CA1i is defined by Layer 2 and Layer 3 cell types

Intermediate CA1 (Fig. 5a, b) consists of a continued and widened Layer 2 cell type (gene marker Ndst4), joined by the deep emerging Layer 3 cell type (gene marker Trib2) (Fig. 5c, f, g). Notably, Layer 1 ends at the CA1d/CAi boundary (Fig. 5e) and Layer 2 now becomes the most superficial part of the pyramidal layer (Fig. 5g; topographic heatmap). Starting at level 82, a thin Layer 2 transitions superficial to the caudal part of CA2 becoming thicker as it moves ventrally and caudally (Fig. 5f; right-to-left). Moreover, the Layer 3 cell type, delineated by Trib2, shows a marked widening particularly toward the ventral axis (Fig. 5g; right-to-left), as it occupies the deep pyramidal cell layer in this subregion. In contrast to CA1d, CA1i gene expression signature is identified by higher Trib2 gene expression, moderate Ndst4 expression, and minimal expression of Lrmp and Peg10 (Figs. 3a and 5d). CA1i gene expression profile is also apparent in data from the sagittal CA1 sections (Supplementary Fig. 8c, j). Although Trib2 is also expressed in CA2, Trib2 expression is more robust across the full pyramidal layer compared to CA1 and this region was anatomically outlined and excluded from CA1 quantifications at all levels. To clarify this boundary distinction, we examined comparisons with Allen ISH data for Grp (CA1 Layer 3) and Amigo2 (CA2) that validate the placement of the CA1i-CA2 border (Supplementary Fig. 7). In addition to the original HGEA, which identified gene markers for CA2 in the context of other hippocampal subregions²⁶, our recent investigations refining CA2 boundaries provide further explanation of this delineation³¹.

**Fig. 5: CA1i Gene Expression Signature: Layer 2 and Layer 3.**

CA1v is defined by Layer 2, Layer 3, and Layer 4 cell types

As we approach the ventral CA1 (Fig. 6a, b), the Layer 3 cell type, marked by Trib2, evidently dominates the majority of the pyramidal cell layer and is flanked by a thinner superficial Layer 2 and deeper Layer 4 (Fig. 6c, g; topographic heatmap). Although superficial Layer 2 cell types, identified by the gene marker Ndst4, persist in this subregion (Fig. 6c, f), there is a marked decrease in their abundance near the ventral axis (Fig. 6f; topographic heatmap), which ultimately leads to its gradual tapering towards the center of CA1v. Concurrently, where the Layer 2 cells fade out, we observe the emergence of Layer 4 cell types, characterized by the gene marker Peg10, in the deep pyramidal cell layer (Fig. 6h). The appearance of three cell type layers at HGEA Level 82 suggests a more complex organization in the CA1v (Fig. 6d). This tri-laminar organization can also be clearly observed across all other CA1v levels as well as in sagittal sections (Fig. 3a, Supplementary Fig. 8d,e-h (bottom),k). This aligns with previous reports highlighting the intricate organization and functional complexity of the hippocampal circuitry as it transitions toward CA1v^3,4,32,33.

**Fig. 6: CA1v Gene Expression Signature: Layer 2, Layer 3, and Layer 4.**

CA1vv is defined by a monolayer of the Layer 4 cell type

The ventral tip of CA1 (Fig. 7a, b) is defined by the strong expression of the gene Peg10 across the whole pyramidal layer suggesting this region is a monolayer comprising Layer 4 cell types (Fig. 3a, Fig. 7c, h). Some low/moderate expression of Trib2 is also present in these cells (Fig. 7g), but Lrmp and Ndst4 expression are minimal (Fig. 7e-f). In contrast to the original HGEA²⁶ which describes the end of CA1vv at HGEA Level 82, we found that Layer 4 continues further caudal where the layers of the ventral subiculum also begin to appear (Supplementary Fig. 1 and Supplementary Fig. 9).

**Fig. 7: CA1vv Gene Expression Signature: Layer 4.**

Identifying the boundaries between ventral CA1 and SUB

Identifying the boundaries between ventral CA1 and SUB is challenging based on cytoarchitecture and the extension of CA1 Layer 4 caudally into the SUB suggested a revised interpretation of the original HGEA boundary was needed. To further investigate the boundaries of the ventral CA1 and SUB, we performed RNAscope HiPlex smFISH Assay for 7 CA1(Lrmp, Ndst4,Trib2, and Peg10) and SUB (SUB_2 gene marker Dlk1, SUB_3 marked by Teddm3, and SUB_4 labeled by Tle4) probes on the same section (see Methods). We performed probe hybridization, amplification, and imaging for our four CA1 gene markers using caudal hippocampal sections. Subsequently, we cleaved the fluorophores, stripped the CA1 gene probes, and re-hybridized the tissue with probes for ventral subiculum gene markers (more details in Methods). By employing the RNAscope HiPlex Image Registration software, we overlaid the two images by co-registering the DAPI staining, allowing us to examine the border between CA1vv and SUBv.

We found that throughout HGEA Levels 84-86, the ventral hippocampus undergoes a transitional shift where CA1vv becomes gradually displaced by the SUBv cell type layers (SUB_2, SUB_3, SUB_4; Supplementary Fig. 9). Starting at level 84 (Supplementary Fig. 9a), SUB_2 emerges underneath CA1vv, followed by SUB_3 and SUB_4 at Level 85 (Supplementary Fig. 9b), which continue to advance superficially more caudally until the complete disappearance of CA1vv at HGEA Level 86 (Supplementary Fig. 9c). These data suggest that the ventral tip of the hippocampus contains both layers of CA1 and SUB cell types as these two structures transition rostrocaudally.

Caudal CA1 – a potentially distinct subregion

The gene expression profiles of the caudal CA1 are distinct from the CA1 subregions rostral to HGEA level 85 (Fig. 3b). Whereas the HGEA had the CA1d, CA1i, and CA1v continue through the caudal extent of the CA1, our new data suggests that the caudal CA1 could be considered a distinct subregion based on its laminar organization. Our results indicate that CA1 Layer 1, which is confined to CA1d up to HGEA level 85, extends ventrally through the entire CA1, creating a tri-laminar organization in the caudal CA1 (Supplementary Fig. 11). Consequently, the gene expression signatures for HGEA levels 86-91 can be redefined such that the previously distinct HGEA CA1d, CA1i, and CA1v are characterized by the same laminar organization: CA1 Layer 1 as the most superficial layer, followed by layer 2, with layer 3 forming the deep pyramidal layer.

The caudal continuation of Layer 1 is critical to this interpretation, and we have found that some genes are organized as rostrocaudal gradients within Layer 1. The marker gene Lrmp we have used in our RNAscope experiments is stronger within rostral Layer 1 and weaker in caudal Layer 1. To confirm that Layer 1 does extend caudally, we examined ABA data to observe other Layer 1-specific genes and verified caudal CA1_1 gene expression does extend caudally. Gene markers Htr1a, Lamp5, and Fibcd1 (Supplementary Fig. 12c-e) all together show heightened gene expression in the superficial layer at the caudal levels of CA1, further supporting that CA1_1 cell types occupy the superficial layer along the dorso-ventral axis in caudal CA1 (Supplementary Fig. 12f-h). Htr1a and Lamp5 are expressed in Layer 1 across the whole rostrocaudal axis. Fibcd1 is expressed in all CA1 layers except Layer 2, so its expression provides a clear example of the tri-laminar organization in caudal CA1. To further confirm the laminar identity of deep CA1, we examined Trib2 expression in caudal sections. While Trib2 is also expressed in SUB_1, comparison with the SUB_1 marker Nts (Supplementary Fig. 13) demonstrates that Trib2 is additionally localized within the deep CA1 layer where Nts is absent. This complementary labeling confirms that Trib2 marks CA1_3 at caudal levels, consistent with our delineations of caudal CA1 lamination. Importantly, the change in gene expression signature (Fig. 3b) suggests caudal CA1 (HGEA Levels 86-91) may be an independent subregion of CA1 compared to the subregions in the more rostral levels of CA1 (HGEA Level 69-85) – but further investigation based on functional connectivity is needed.

Discussion

Our findings demonstrate that a sublaminar organization of pyramidal cell types exists across the entire CA1 longitudinal axis. Similar to the cortex and SUB²⁷, the CA1 contains both laminar and regional organization; where each subregion (CA1d, CA1i, CA1v, CA1vv) is distinguished by the number and representation of four specific cell type layers (CA1_1, CA1_2, CA1_3, CA1_4) characterized by a unique gene expression signature (Fig. 3, Fig. 8a-e; see Supplementary Movie 1 or use Schol-AR app). The RNAscope smFISH data presented here examined 4 exemplar marker genes in coronal tissue sections across the entire CA1 (Figs. 2 and 3). These patterns are substantiated by Allen Brain Atlas in situ hybridization data which demonstrates that other genes are expressed with similar combinatorial laminar patterns (Supplementary Fig. 14b). Whereas previous studies have identified and investigated differences between deep and superficial cells within CA1d^16,21,34, we found that the cell type laminar organization extends beyond CA1d (Fig. 8f; left-to-right). Notably, the deep CA1d cell layer (CA1 Layer 2) shifts to displace CA1 Layer 1 as the superficial sublayer of CA1i. The CA1d/CA1i boundary also marks the emergence of CA1 Layer 3 in the deepest part of the CA1i pyramidal layer. The CA1v is similar to the CA1i, except for a tapering of the superficial CA1 Layer 2 and the addition of a thin and deep CA1 Layer 4; whereas the CA1vv is a monolayer of CA1 Layer 4 cell types.

**Fig. 8: Defining CA1 subregions (CA1d, CA1i, CA1v, CA1vv) with distinct cell type layers.**

CA1 cell types: Gradients, Mosaic, and Laminar organization

In recent years, anatomical and molecular studies have shed light on the unique organization of the CA1 pyramidal cell types^3,9,14,27. Identifying the cellular architecture of the CA1 pyramidal layer is critical for understanding the functional role that CA1 plays in learning and memory. Our results find that pyramidal cell types in CA1 comprise distinct cell type layers across its longitudinal axes, which exist throughout the entire CA1 rostral-caudal axis (Supplementary Fig. 1). Our findings agree with multiple electrophysiological studies that reveal distinct functional sublayering between superficial and deep CA1 pyramidal cells, including those where neurons were anatomically identified by depth or labeling^18,21. However, previously reported superficial (Calb1, Zbtb20, Satb2) and deep (Sox5, Col11a1, Ndst4) CA1d markers^3,35, with both superficial and deep genes largely aligning with the layers we define as Layers 1 and 2 but some are also combinatorially expressed in multiple layers (ex. Calb1 in Layers 1 and 3; Supplementary Fig. 14a). Notably, our finding that CA1 Layer 2, marked by Ndst4, shifts from a deep to a superficial position at intermediate and ventral subregions further challenges the assumption of a fixed deep/superficial identity across the longitudinal axis, a spatial variation that likely carries important consequences for CA1 function along the longitudinal axis.

In line with this, comparison with the Allen Brain Cell (ABC) Atlas MERFISH dataset³⁶ demonstrates that the same four gene markers analyzed here (Lrmp, Ndst4, Trib2, Peg10) display stratified expression within the CA1 pyramidal layer across rostro-caudal levels (Supplementary Fig. 15a). The ABC atlas recapitulates a superficial layer marked by Lrmp, deeper layers labeled by Ndst4 and Trib2, and the ventral tip enriched for Peg10. In addition, ABC atlas-defined CA1-ProS glutamatergic neurons organized into six CA1 ‘supertypes’ and Glut1/Glut2 ‘cluster’ populations also reveal a stratified laminar organization, further corroborating the layered cell type organization we see in CA1 pyramidal cells. Similarly in the ABC 10x scRNAseq UMAP data, CA1 gene markers appear differentially expressed, supporting their identification as distinct CA1 cell types (Supplementary Fig. 15b). While large-scale approaches such as MERFISH and RNAseq capture similar overall patterns, our complementary high-resolution approach using RNAscope quantification of curated marker genes within restricted brain areas can help to sharpen cell type boundaries that may otherwise be difficult to visualize.

Previous single-cell transcriptomics mouse studies have reported that CA1 pyramidal cells are organized along dorso-ventral gradients^9,37, while others described the CA1 as a mosaic of cell types constituting distinct subregions across the longitudinal axis¹⁴. It’s possible that all three organizational principles can exist within the CA1’s transcriptomic landscape but at different scales. For example, we find that Lrmp expression has a strong rostrocaudal gradient within CA1 Layer 1 and the CA1 gene expression laminar patterns are thinner and more heterogeneous compared to the more discrete SUB layers. In addition, mosaicism of cell types within the layers could exist due to the high degree of heterogeneity at the single-cell level. Ultimately, we believe the CA1 layers are a tissue-level organizational principle that is strongly supported by the accumulating evidence of publicly available resources as well as multiple electrophysiological studies in CA1d, but additional studies in other CA1 subregions are needed to investigate the functional differences among cell type laminae.

The elusive ventral border between CA1 and subiculum

While the compact arrangement of superficial CA1 cells and the sparser packing of deep CA1 cells provide useful landmarks for approximating the CA1/SUB boundary, similar thickness in the pyramidal layer leads many studies to use the more general term ‘ventral hippocampus’. Progressing from rostral to caudal (Supplementary Fig. 9; left-to-right), our data show a transitional shift of the CA1 layers where CA1v layer 3 and 4 cell types are displaced by SUBv layer 2, 3, and 4 cell types in the deepest parts of the pyramidal layer. Across HGEA Levels 84-86 (Supplementary Fig. 9a, b), both CA1 and SUB layers are present creating a multi-layered appearance that has been previously attributed as part of the SUB organization³⁸. Our data showing the progressive transition across multiple coronal sections of CA1 and SUB gene expression patterns demonstrates that the multi-layered appearance occurs as a result of both CA1 and SUB layers being present at these coronal levels, culminating the eventual absence of CA1 in the ventral hippocampus at HGEA Level 86 (Supplementary Fig. 9c). Notably, these findings are also consistent with the morphological studies of Lorente de Nó, who noted a superficial layer of ‘Ammonshorn’ pyramidal neurons in his original observations of the ProSUB as well as a layer of ‘deeply displaced subicular pyramids’ within the CA1³⁹. Overall, these data suggest that CA1 and SUB layers can extend beyond their regional boundaries creating the presence of mixed-layered cell types at the CA1/SUB border in coronal tissue sections.

Furthermore, the area of the CA1/SUB transitional shift (HGEA Levels 84-86) corresponds to the Hippocampo-amygdalar transition area (HATA) region delineated in the ABA Mouse Reference Atlas CCFv3 (ABA Levels 82-94). Our data suggests that the area currently designated as HATA in the CCFv3 may partially represent a transitional zone between CA1 and SUB layers. Because the Allen ABC Atlas³⁶ reports high Peg10 (a CA1_4 marker in this study) expression in the posterior amygdala (PA), we evaluated whether the CA1/SUB shift might instead correspond to HATA/PA. However, ABA ISH data at HGEA Levels 84-86 showed absence of PA maker Slc17a6 (Supplementary Fig. 10a) and complementary labeling of CA1 (Peg10, St18) and SUB_2 (Dlk) (Supplementary Fig. 10b, c), supporting a CA1/SUB boundary at these levels. Consistent with this, the SUB_1 marker Nts demonstrates that what we delineate as CA1_3, marked by Trib2, is distinct from SUB (Supplementary Fig. 13). Additional CA1_3 and CA1_4 markers further reinforce that the trilaminar organization represents CA1 rather than SUB, although we note that this boundary remains challenging to define (Supplementary Fig. 14b). While our data show an intersection between CA1 and SUB, it’s also possible that cell types from adjacent amygdala and entorhinal cortex regions are present as well.

Functional implications of CA1 cell type laminar architecture

The laminar organization of pyramidal cell types, in conjunction with the overall laminar organization of inputs to the CA1 (stratum oriens, stratum radiatum, stratum moleculare), suggests that CA1 cell types are organized into functional circuits similar to the cerebral cortex and SUB. Numerous electrophysiological studies have reported on functional differences between ‘deep’ and ‘superficial’ parts of the pyramidal layer, identifying sublayers. Although these studies have been limited to the CA1d region, the evidence suggests deep vs superficial laminar differences in CA1 pyramidal cell type firing rates^{16,18,19,24,25,40,41,42,43}, functional circuit connectivity^22,24,42,44, oscillatory patterns^21,23,34, and behavioral role in spatial navigation^17,20,45,46. Our observation that CA1 Layer 2 changes position across the subregions suggests that these functional differences may not be uniform along the longitudinal axis. This spatial variation has important implications, particularly in light of physiological studies showing that anatomically identified deep and superficial pyramidal cells differ in connectivity and network oscillations such as sharp-wave ripples and theta rhythms^16,18,22. Such variation may help reconcile conflicting physiological reports of deep vs. superficial activity and suggests that region-specific laminar identity contributes to function diversity in CA1.

A laminar functional organization in the CA1 pyramidal layer has also been reported^16,21,34,40. Our findings are consistent with Abbaspoor et al. who show a strata-specific organization to CA1 pyramidal cell type assemblies which also display strata-specific firing patterns¹⁶. Other reports have shown that grouped CA1 pyramidal cells with like-oscillation patterns form functionally distinct layers^21,34. Moreover, our identification of the 4 CA1 pyramidal cell layers (CA1_1, CA1_2, CA1_3, CA1_4) suggests additional functional roles in other CA1 layers and may provide an atlas/blueprint to target areas of interest. Also, electrophysiology studies in CA1v demonstrate that laminar identity also maps onto distinct functional roles beyond CA1d. For example, Sánchez-Bellot et al. (2022) showed that superficial and deep CA1v neurons form opposite pathways to the prefrontal cortex that differentially control approach and avoidance behavior⁴⁷. Pi et al. (2019) further demonstrated that basolateral amygdala inputs target superficial versus deep CA1v pyramidal cells to drive distinct behavioral outcomes⁴⁸, while Jimenez et al. (2018) identified functionally distinct ‘anxiety cells’ in CA1v projecting to hypothalamus⁴⁹. Together, these findings underscore that laminar distinctions in CA1 extend across the longitudinal axis and have critical implications for anatomy and function.

Leveraging CA1 lamination in translational research applications

Multiple studies have found evolutionarily-conserved anatomical and functional roles of the hippocampus across the mammalian species^{4,7,33,50,51,52}. Previously, we found evidence for homologous laminar organization in the mouse and human SUB²⁷, suggesting that a similar homology likely exists for CA1 cell types. A recent study investigating the CA1 pyramidal cell layer in macaque primates¹⁶ reports consistent functional roles in deep and superficial cells with rodent studies^21,34 and describes a laminar-specific organization within CA1. Notably, the increasing thickness of the CA1 pyramidal layer along the proximo-distal axis is found across numerous species; including opossums, cats, and primates³¹. A conserved laminar architecture of the CA1 pyramidal layer in nonhuman mammals and humans provides a critical framework for translational research.

Laminar organization represents a fundamental principle of cortical architecture with direct implications for disease. Our findings of a new CA1 laminar organization may reveal cell type-specific susceptibility in hippocampal-affected diseases. Recent studies demonstrate that deep and superficial CA1d pyramidal neurons are differentially affected in Alzheimer’s disease models and epilepsy^53,54, but it’s unclear how the laminar organization may be affected across the entire hippocampus. These findings highlight that this layered structure is not only essential for understanding basic circuit organization but also central to interpreting selective cell type susceptibility in neurological disorders and for identifying therapeutic targets.

Methods

8-week-old (3 male, 2 female) C57BL/6 mice (Jackson Labs strain # 000664) were pair-housed in a room controlled for temperature (21–22 °C), humidity (51%), and light (12 h light:12 h dark cycle with lights on at 6:00 a.m. and off at 6:00 p.m.) with ad libitum access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Southern California. Sex was considered in the study design, and sample composition (sex, age, and n per experiment) is detailed in Supplementary Fig. 16 and the Source Data file.

Probe Design and Manufacturing

Marker genes were carefully selected based on the annotated gene expression dataset from the Hippocampus Gene Expression Atlas (HGEA): Lrmp, Ndst4, Trib2, and Peg10 (Bienkowski et al, 2018). HGEA CA1 gene markers for CA1 subregions were selected based on their restricted expression in adjacent hippocampal subregions, allowing for clear differentiation of CA1. Probes Lrmp (catalog # 559391-T8), Ndst4 (catalog # 313631-T6), Trib2 (catalog # 514021-T7, and Peg10 (catalog # 512921-T5) were designed using the Advanced Cell Diagnostics RNAscope HiPlex12 Reagent Kit (488, 550, 650, 750) v2 Standard Assay (catalog #324409). Each target probe is tagged with a distinct tail for cleavable fluorophores AF488 (T1), Dylight 550 (T2), Dylight 650 (T3), AF750 (T4). RNAscope HiPlex12 Positive Control Probe (RTU) (catalog # 324433) and RNAscope HiPlex12 Negative Control Probe (RTU) (catalog # 324341) reagents containing probes targeting housekeeping genes for each color channel were hybridized onto tissue sections on separate slides (Supplementary Fig. 17).

Brain Extraction and Tissue Sectioning

Mice were euthanized by injection of Euthasol followed by trans-cardial perfusion of fresh 0.9% saline solution and 4% PFA fixatives (PFA; pH 9.5). Brains were extracted from the skull and post-fixed in 4% PFA for 24-48 hours at 4 °C. For sectioning, fixed brains were embedded in 3% Type1-B agarose (Sigma-Aldrich) and sectioned into four series of 20 m-thick coronal or sagittal sections using a VF-510-0Z Compresstome (Precisionary Instruments). Sections were mounted onto SuperFrost® Plus Slides (Fisher Scientific catalog # 12-550-15).

Sample Pretreatment and Hybridization

Tissue sections were processed for in situ hybridization following the RNAscope protocol. Immediately following tissue mounting, sections were baked using a HybEZ II Oven (catalog # 321710) at 60 °C and dehydrated with a series of 50%, 75%, and 100% EtOH dilutions for 5 minutes. Samples were permeabilized with RNAscope Target Retrieval Reagent (catalog # 322000) for 5 minutes and RNAscope Protease III (catalog # 322340) for 30 minutes. After pretreatment, samples undergo probe hybridization with 50X RNAscope HiPlex Target Probes diluted in RNAscope HiPlex Probe Diluent at 40 °C for 2 hours, followed by three 30-minute amplification steps using RNAscope HiPlex Amp 1-3. Sections were hybridized with RNAscope Fluoro T1–T4 v2 (AF488, Dylight 550, Dylight 650, AF750) for 15 minutes and counterstained with RNAscope DAPI. Each microscope slide was coverslipped using ProLong Gold antifade mountant solution (Fisher Scientific catalog # P36930).

For the caudal CA1/SUBv analysis (Supplementary Fig. 9), we utilized the RNAscope HiPlex v2 Assay’s de-coverslipping and cleaving fluorophore protocol. After hybridizing and imaging Round 1 target probes, slides de-coverslipped by soaking in 4X SSC buffer (Fisher Scientific catalog # BP1325) in room temperature for approximately 30 minutes. Once de-coverslipped, slides were incubated in 10% RNAscope HiPlex Cleaving Stock Solution v2 (diluted in 4X SSC buffer) for 15 minutes at room temperature in order to cleave Round 1 fluorophores from the tissue. Sections were washed with PBST (10% Tween) twice, before repeating the cleaving incubation. Once cleaved, sections were re-hybridized with Round 2 probes targeting Dlk1 (catalog # 405971-T5), Tle4 (catalog # 417301-T6), and Teddm3 (catalog # 1040001-T7) gene markers using the previously described RNAscope probe and fluorophore hybridization.

Microscopy Imaging and Image Post-Processing

Sections were imaged using an Andor Dragonfly spinning disk confocal microscope with 40X Nikon silicon oil objective. Images were captured tile by tile using appropriately matched fluorescent filters through the entire thickness of the tissue section (15–20 µm) at 1 µm Z steps and then deconvolved and stitched together as whole-section 3D volumes. Range of microscopy exposure times were between 100–200 ms and 30–90% laser intensity. Whole section volumes were imported into Fiji software at full resolution and converted to Max Intensity Projection (MIP) for quantification analysis.

Animals and Sectioning

A total of five mice (3 male, 2 female; 57 CA1 sections) were processed (Supplementary Fig. 16). Coronal sections (20 μm) spanning HGEA Levels 69-91 were obtained, with one representative section per level selected from male cases for analysis; each section was analyzed independently as its own dataset. To account for potential dorsoventral variability introduced by coronal sectioning, we examined 52 coronal sections across 4 animals and observed consistent CA1 delineation, including the CA1/SUB transition zone in ventral hippocampus. Complementary analysis of sagittal CA1 sections, where dorsoventral gradients are minimized, further support the accuracy of laminar delineations. Normalized gene expression values from all five cases were aggregated to assess expression consistency across animals, as shown in Supplementary Fig. 16.

Cell Segmentation and Probe Detection

MIP images containing RNAscope fluorescence were analyzed using QuPath open-source software for bioimaging analysis (Supplementary Fig. 3) (Bankhead et al, 2017). Annotation ROIs of the CA1 pyramidal layer were manually drawn using QuPath’s ‘magic wand’ tool. The QuPath cell detection algorithm was used to detect DAPI-labeled nuclei and define non-overlapping cellular ROI’s with DAPI nuclei and surrounding 5 area to estimate for cell body cytoplasm with a pixel intensity threshold of 5. Following cell detection, QuPath’s subcellular detection algorithm was used to detect and identify fluorescent ‘dots’ in the individual gene expression channels. Subcellular detection threshold parameters were determined visually and maintained consistently across all sections (note that the analysis shown in Supplementary Fig. 4, subcellular detection thresholds in QuPath were slightly adjusted on a per-probe basis to better capture true transcript counts across CA1 layers). Detection measurements were exported as cell-by-gene matrix CSV files and cellular ROI’s were exported as zip files.

Gene Expression and Cell Type Analysis

Exported QuPath CSV files were converted into cell-by-gene matrices using the ‘num spots estimated’ metric as a measurement of gene expression. The ‘num spots estimated’ value includes both the number of individual fluorescent ‘dots’ as well as estimated ‘dots’ based on the size of detected fluorescent clusters. The cell-by-gene matrix file and cellular ROI zip file was imported into the R environment using the SCAMPR pipeline (https://github.com/ramin-ali-marandi-ghoddousi/SCAMPR) (Ghoddousi et al, 2022). Gene expression values for each cell were normalized by converting area fraction measurements to absolute expression (area fraction × ROI area / 100) and then applying a log transformation to reduce skewness. These normalized values were used for all visualizations and quantifications. SCAMPR scripts were used to generate violin plots of log-normalized gene expression and spatial topographic heat maps of the log-normalized CA1 gene expression. To generate maps and quantify cell types, we adapted the SCAMPR script for topographic heat maps to assign a color to the cell ROI based on ‘majority rule’ of the highest expressing marker gene. For quantification of mean expression across superficial and deep sublayers (Supplementary Fig. 4), boundaries of the pyramidal cell layer were manually annotated in QuPath at HGEA Level 82. Segmented cells were assigned to superficial, middle (CA1v only), or deep categories. Transcript counts were normalized and log-transformed as described above, and zero-expressing cells were excluded from the mean expression plots. For co-expression analysis (Supplementary Fig. 5), pairs of adjacent-layer markers were evaluated in segmented cells from each subregion at HGEA Level 82. SCAMPR Loess Coexpression analysis (Ghoddousi et al., 2022) was applied to visualize residuals at the single-cell level and assess overlap between markers. Only layer-enriched markers were included; CA1vv was excluded because only Peg10 was enriched in this region.

In total, 58,065 CA1 pyramidal neurons were analyzed across all sections corresponding to HGEA Levels 69-91 (and sagittal HGEA Level 3). From these cells, we quantified 38,484 Lrmp, 137,075 Ndst4, 80,837 Trib2, and 76,542 Peg10 single-molecule transcripts, which were used to refine the CA1 HGEA.

All raw detection data and ROI annotation files underlying these analyses are available in the accompanying Source Data file.

3D Models of HGEA

In order to create 3D models from image data, we first aligned the manually labeled sequential images using an optimization-based registration method that applied Gaussian blur, edge detection, and differential evolution to optimize rotation and translation parameters, ensuring spatial coherence across the stack. The aligned images were then upsampled across the Z-dimension to match the physical dimensions of the brain volume. Surface models were generated using marching cubes on integer-labeled volumes created from the processed images, followed by mesh repair and quadric decimation to reduce polygon counts without sacrificing anatomical detail. Final GLB-format models retained region-specific coloring, watertight geometry, and oriented normals for optimal visualization. Image processing and mesh generation were done with Python. Image and video renders of the resulting models were created with 3D studio max⁵⁵. Schol-AR was used to generate the QR code and augment associated images with 3D visualizations in Figs. 1 and 8⁵⁶.

Allen Brain Atlas ISH and ABC Data

The Allen Institute offers an online open-access resource featuring single-gene mouse in situ hybridization and ABC Atlas images (https://www.mouse.brain-map.org). Individual gene names were searched and images at the desired anatomical level were downloaded (Supplementary Figs. 2, 7, 10, 12-15). All ABA images are available at the Allen Brain Atlas website.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information. Source data underlying all figures are provided in the accompanying Source Data file. Analyses were performed using the publicly available SCAMPR pipeline (https://github.com/ramin-ali-marandi-ghoddousi/SCAMPR). External reference datasets were obtained from the Hippocampus Gene Expression Atlas (https://cic.ini.usc.edu) and the Allen Brain Atlas (https://mouse.brain-map.org/). Source data are provided with this paper.

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Acknowledgements

The authors would like to thank Arthur Toga and the entire USC Mark and Mary Stevens Institute of Neuroimaging and Informatics for their guidance and support of the Center for Integrative Connectomics (CIC). We also acknowledge Jared Simmons and other members of the CIC for their advice and technical assistance. The authors are also thankful to Allen Institute and Ramin Ali Marandi Ghoddousi for their open-source data and code that helped in the analysis of this study. This work was financially supported by NIH/NIA K01AG066847 (M.S.B), NSF Grant 2121164 (M.S.B.), NIH/NIA R36AG087310-01 (M.P), NIH/NIA supplement P30-AG066530-03S1 (M.P), and funding from the USC Center for Neuronal Longevity. Research data reported in this publication was supported by the Office of the Director, National Institutes of Health under award number S10OD032285.

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Stevens Neuroimaging and Informatics Institute, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, USA
Maricarmen Pachicano, Shrey Mehta, Angela Hurtado, Tyler Ard, Jim Stanis, Bayla Breningstall & Michael S. Bienkowski
Center for Integrative Connectomics, Stevens Neuroimaging and Informatics Institute, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, USA
Maricarmen Pachicano, Shrey Mehta, Angela Hurtado, Bayla Breningstall & Michael S. Bienkowski
Zilkha Neurogenetic Institute, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, USA
Michael S. Bienkowski
Department of Physiology and Neuroscience, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, USA
Michael S. Bienkowski
Alfred E. Mann Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, USA
Michael S. Bienkowski

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Maricarmen Pachicano
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Contributions

M.P. and M.S.B. designed the experiments, M.P. performed the experiments with the help of A.H., M.P. and S.M. analyzed the data with the help of B.B., T.A. and J.S. generated 3D rendering of HGEA and movie. M.P. wrote the manuscript draft, M.P. and M.S.B. edited and revised the final publication.

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Correspondence to Michael S. Bienkowski.

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Pachicano, M., Mehta, S., Hurtado, A. et al. Laminar organization of pyramidal neuron cell types defines distinct CA1 hippocampal subregions. Nat Commun 16, 10604 (2025). https://doi.org/10.1038/s41467-025-66613-y

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Received: 14 May 2025
Accepted: 11 November 2025
Published: 03 December 2025
Version of record: 03 December 2025
DOI: https://doi.org/10.1038/s41467-025-66613-y