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. 2026 Jul 8;16:31371. doi: 10.1038/s41598-026-61283-2

Cellular and subcellular localization of the copper transporter CTR1 in human postmortem hippocampus and striatum

Cheyenne F Griffin 1, Charlene B Farmer 1, Alexis Henry 1,2, Christine Nguyen 1,3, Kirsten E Schoonover 1,✉
PMCID: PMC13645664  PMID: 42420436

Abstract

Copper is essential for neurological function, and its transport across the blood-brain barrier (BBB) is mediated by the copper transporter CTR1. Via CTR1, copper enters endothelial cells at the BBB and is transported into astrocytes and neurons. Although critical for homeostasis, copper distribution varies across brain regions and within cells. Knockdown of CTR1 causes developmental defects, highlighting its significance. This study examined CTR1 localization in human postmortem hippocampus and striatum. Postmortem brain samples from unaffected individuals were analyzed using immunohistochemistry, in situ hybridization, and ultrastructural electron microscopy (EM). Immunolabeling was observed in putative endothelial-like, astrocytic-like and oligodendroglial-likeprofiles, pyramidal neurons, and bipolar-appearing interneuronal profiles. Multiplex fluorescent in situ hybridization demonstrated SLC31A1 mRNA signal in neuronal, vascular-associated, and glial-associated cellular populations corresponding to those identified by immunohistochemistry. Using ultrastructural EM, CTR1-associated labeling was observed in axon terminals forming asymmetric synapses in the hippocampus and striatum, including labeling adjacent to vesicular structures. Additionally, CTR1-associated labeling was observed adjacent to mitochondrial profiles in both regions. These findings provide descriptive baseline data on CTR1 localization in human brain and may inform future studies of copper transport in health and disease. disorders.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1038/s41598-026-61283-2.

Keywords: Copper, CTR1, Ultrastructure, Striatum, Hippocampus, Entorhinal cortex, Electron microscopy, Fluorescent in situ hybridization

Subject terms: Cell biology, Neuroscience

Introduction

Copper plays an essential role in maintaining critical brain functions, but is not endogenously synthesized. Instead, copper is transported across the blood-brain barrier from the bloodstream into the brain by copper transporter CTR11–3, encoded by the SLC31A1 gene3. CTR1 knockdown results in developmental defects and knockout results in embryonic lethality4, exemplifying the incredible importance of copper in early development. However, copper is also crucial across the lifespan, serving as the catalytic core for enzymes involved in catecholamine synthesis, energy metabolism5, and oxidative stress6–8. These pathways have long been implicated in a myriad of psychiatric disorders9–15, but only recently has copper been implicated as a potential underlying factor in serious mental illness16–23. Because baseline cellular localization of CTR1 in human brain remains incompletely described, defining its regional and subcellular distribution in postmortem tissue is an important prerequisite for interpreting future disease-focused studies.

The hippocampus and striatum are critical brain regions implicated in psychiatric disorders due to their distinct yet interconnected roles in cognition, emotion, and perception24–28. The hippocampus is essential for learning, memory, and emotional regulation29–31. Dysfunction in this region is strongly linked to psychiatric disorders such as schizophrenia32,33, major depressive disorder34–36, and bipolar disorder37,38. Copper dysregulation in the hippocampus of various pathological models has been implicated in oxidative stress39,40, synaptic dysfunction41, and apoptosis42, making it a relevant target for studying CTR1 localization. Understanding what cellular types express CTR1 within the hippocampus could provide insights into its role in maintaining copper homeostasis and synaptic integrity. Additionally, the striatum is a key component of the basal ganglia, playing a central role in motor control43,44, habit formation45,46, and reward processing47. It is heavily implicated in psychiatric conditions such as obsessive-compulsive disorder48,49, schizophrenia50–53, and addiction54–56. Given the involvement of the striatum in dopamine regulation and synaptic plasticity, disruptions in copper transport mechanisms could influence neuronal excitability and neurotransmitter balance, as copper plays a critical role in enzymes involved in energy production and neurotransmitter synthesis57,58.

Thus, we selected the hippocampus and striatum as initial investigational sites based on their involvement and susceptibility to dysregulation in various serious mental illnesses. The purpose of the present study was to characterize CTR1 localization in unaffected human hippocampus and striatum using immunohistochemistry, multiplex fluorescent in situ hybridization (FISH), and electron microscopy, with the goal of providing descriptive reference data for future studies of copper transport in human brain.

Methods

Postmortem brains

This study used postmortem brains from 16 cases obtained from the Maryland (Baltimore, MD) and Alabama (Birmingham, AL) Brain Collections. Brains were collected and used in compliance with the University of Alabama at Birmingham Institutional Review Board-approved protocol (IRB-F080306003). Informed consent was obtained for brain tissue, next-of-kin phone interviews, and donor medical records, all of which were used for diagnostic purposes. Two psychiatrists established an unaffected diagnosis according to the DSM (DSM-III-R through DSM-IV-TR) at the time of diagnosis. Exclusionary criteria for this cohort have been previously reported22. Demographic information is listed in Table 1 and Supplementary Table 1.

Table 1.

A, B. Demographics and tissue quality measures for the cases in the hippocampus (Hipp) and entorhinal cortex (ENT), and striatum, respectively. ARS, age, race, sex; PMI, postmortem interval; AA, African American; C, Caucasian; M, male; F, female; Values shown are mean ± standard deviation.

Hipp/ENT ARS PMI (hrs) pH
A
A039 32CF 8 6.3
A163 65CM 17 7.2
554 32CF 7 7.37
874 40AAM 7 6.68
n = 4 42.3 ± 15.6 9.8 ± 4.9 6.9 ± 0.5
3 C/1AA
2 M/2F
B
Striatum
A008 61CM 4 6.5
A010 88CM 7 6.2
A063 30CM 15 NA
A163 65CM 17 7.2
481 50AAM 5 6.45
553 30AAF 10 6.85
570 43AAF 17 NA
n = 7 52.4 ± 20.8 10.7 ± 5.6 6.6 ± 0.4
4 C/3AA
5 M/2F
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All experimental protocols and methods were performed in accordance with the relevant guidelines and regulations. De-identified human tissue samples for immunohistochemistry and electron microscopy from hippocampus and adjacent entorhinal cortex (Table 1A) and striatum (Table 1B) were stored in 4% paraformaldehyde with 1% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4, until use. Tissue was collected with postmortem intervals ranging from 4 to 17 h, as detailed in Table 1. After fixation, tissue was sectioned with a vibratome into six series of 40-µm thick sections. For striatal analyses, tissue included caudate nucleus and putamen; where relevant, observations are described separately for ventricular/superficial caudate regions, deeper caudate neuropil, and putamen.

Tissue samples for FISH from hippocampus and striatum were flash-frozen using cold isopentane and stored at − 80 °C until sectioning. The tissue was sectioned with a cryostat into 20-µm thick sections and mounted onto charged glass slides and stored at − 20 °C until FISH was performed.

Immunohistochemistry

Immunohistochemical methods are described previously in full22. The sections of striatum were processed identically as the sections of the hippocampus, except for the immunohistochemical antibody dilution (1:2000 for striatum and 1:4000 in hippocampus and entorhinal cortex). Briefly, sections from one series per brain region were prepared for the immunohistochemical localization of CTR1 using a polyclonal rabbit anti-CTR1 (Novus Biologicals NB100-402). Solutions were made in 1x phosphate-buffered saline (PBS), and after each step, sections were rinsed multiple times in 1x PBS; incubations occurred at room temperature on an agitator unless specified otherwise. The protocol began with incubation in 1% sodium borohydride for 15 minutes, incubation in 5% hydrogen peroxide for 30 minutes, and incubation in 10% normal goat serum for one hour. Sections were incubated for 48 hours at 4°C in the primary antibody in a solution of 10% normal goat serum, then incubated for 45 minutes in a solution of biotinylated goat anti-rabbit serum. The sections were then incubated in an avidin-biotin complex solution for 45 minutes. To visualize the reaction product, sections were incubated for 4 minutes in a 3, 3’-diaminobenzidine solution (10 mg diaminobenzidine, 15 ml PBS, 12µL 0.03% hydrogen peroxide; Vector Laboratories, SK-4100).

Antibody validation

Antibody controls were previously conducted and reported22 and included omission of the primary antibody and preadsorption with blocking peptide, both of which eliminated specific staining. In the present study, multiplex FISH was used as an orthogonal approach to assess whether the SLC31A1 transcript signal was present in cellular populations corresponding to those identified by immunohistochemistry. Although background staining was present in some sections, negative controls did not produce comparable signal, and consistent labeling patterns across regions and modalities support the specificity of the observed signal. However, these approaches do not fully resolve all questions of antibody specificity.

Light microscopy

The sections used for light microscopy were mounted on slides and, when dry, placed in increasing concentrations of ethanol and then in xylene for dehydration. Eukitt was used to coverslip the slides. A Nikon DS-Fi1 color digital camera connected to a Nikon Eclipse 50i microscope was used to take photomicrographs of the sections. All photomicrographs of CTR1 immunolabeled sections were taken with a 20× lens, aperture of 0.4, quality capture (2560 × 1920 pixels), exposure length of six milliseconds, medium contrast, and a gain of 1.2. Each photomicrograph was 0.283mm2, yielding a total of 1.698mm2 analyzed per area (6 micrographs × 0.283mm2)22.

All photomicrographs were taken by author CBF from the following 19 areas: entorhinal cortex (superficial layers, pyramidal cell layer, subcortical white matter), subiculum (superficial layers, pyramidal cell layer, subcortical white matter), CA1 (stratum oriens, pyramidale, radiatum, and lacunosum/moleculare), CA3 (stratum oriens, pyramidale, and radiatum/ lacunosum/moleculare), dentate gyrus (granule cell layer, molecular layer and polymorphic layer), the alveus over CA1 and over CA3, and the fornix. Superficial layers of the subiculum and entorhinal cortex refer to the distal, apical dendritic region. Two series of sections, yielding a total of six sections, were analyzed.

Fluorescent in situ hybridization and immunofluorescence

To validate the expression of CTR1 in differential cell types and regions, FISH was conducted on hippocampal and striatal fresh-frozen sections. FISH was processed according to manufacturer’s specifications (Advanced Cell Diagnostics, Inc RNAscope 2.0 Assay) and as described in59. FISH probes were used on human hippocampus and striatum tissue to detect mRNAs encoding GAD1 (Cat.# 404031), SLC31A1 (Cat. # 1333501-C2), GJA1 (Cat. # 444281-C3), and PECAM1 (Cat. # 487381-C4). Fluorophores (Akoya Biosciences) were assigned to each target mRNA in a stepwise manner: (GAD1 in Opal 520 (1:750; Cat. # OP-001001), SLC31A1 in Opal 570 (1:300; Cat. # OP-001003), GJA1 in TSA DIG (1:1500; Cat. # OP-001007)/Opal 780 (1:500; Cat. # OP-001008), and PECAM1 in TSA Cy5 (1:750; Cat. # TS-000203). Sections were stored in 1x PBS overnight awaiting immunofluorescent NeuN labeling.

Immunofluorescence labeling began the following day and all steps were carried out at room temperature. First, slides were incubated in immunocytochemistry (ICC) blocking buffer59 for 60 min. Blocking buffer incubation was followed by NeuN antibody (1:100; Synaptic Systems; Cat. # 266004) incubation for 30 min. The slide was then washed twice in ICC blocking buffer for 3 min and followed by biotinylated anti-guinea pig (1:200; Biosynth; Cat. # 43R-ID014BT) secondary antibody incubation for 30 min. Slides were washed twice in ICC blocking buffer for 3 min each, and Strep 405 (1:100; Invitrogen; Cat. # S32351) for NeuN labeling and Opal 780 for FISH GJA1 labeling were then added to slides for 15 min. Slides were then washed twice in 1x PBS for 1 min each, and diluted DAPI (1:100 in 1x PBS; ACDbio; Cat. # 323108) was added for 30 s for nuclear labeling. Slides were then washed in 1x PBS, coverslipped with Prolong Diamond Antifade Mountant (Invitrogen; Cat. # P36970), and cured at 4 °C until imaging on Olympus VS200 slide scanner.

Fluorescent microscopy

Images were collected on an Olympus VS200 SlideView slide scanner (Center Valley, PA, USA) equipped with a 9-channel X-CITE NOVEM light engine and a Hamamatsu ORCA-flash 4.0 camera. All equipment was controlled using the Olympus VS200 software suite. Using the slide scanning microscope, the entirety of the section was captured using a low-magnification (20x, 0.80 N.A., UPlanXApo extended apochromat objective, Olympus Lifesciences). From the 20x overview image, high-magnification (60x, 1.42 N.A. UPlanXApo extended apochromat oil immersion objective, Olympus Lifesciences) images were collected of three separate 1.0 mm wide traverses of striatum and hippocampus where the section was cut perpendicular to the pial surface. Images were captured as 5 successive 2D images with an imaging depth of 0.6 μm in the z-dimension (equivalent to the depth of field for this objective). Five fluorescent channels were captured for DAPI/NeuN, GAD1, SLC31A1, GJA1, and PECAM1 channels, and lipofuscin autofluorescence as previously described59. These channels were captured at set exposure times that were optimized to prevent saturation of fluorescent signal intensity and were held constant throughout the project. The high-magnification images were automatically stitched together within the VS200 slide scanning software and exported for offline analysis.

Electron microscopy

Following immunohistochemical staining, sections of all regions from a subset of each case were processed for electron microscopy using standard techniques60–62. Briefly, tissue was incubated in a series of steps starting with a 1 h incubation in a solution of 1% osmium tetroxide in PB, followed by PBS washes. The tissue was then dehydrated by sequential washes in 50% and 70% ethanol. The tissue was then incubated in a solution of 1% uranyl acetate in 70% ethanol in the dark for 1 h, followed by washes in 70% ethanol. After that, the tissue was stored overnight in 70% ethanol, and then underwent sequential dehydrations in 95% and 100% ethanol, followed by washes in 100% propylene oxide. To finish the procedure, the tissue was incubated in a 1:1 epon resin: propylene oxide solution for 1 h, a 2:1 epon resin: propylene oxide solution for 1 h and then a 100% epon resin solution for 3 h. The tissue was then mounted on slides and polymerized for 72 h at 60 °C. After semi-thin sections were obtained to verify location and immunolabeling, 90 nm thick sections were cut with a diamond knife on a Leica EM UC6 ultramicrotome and collected on slot grids. These grids were viewed on a Hitachi transmission electron microscope.

It is important to note that the current study is wholly descriptive and does not include any formal quantification. Future work is needed to determine the overall proportions of CTR1 expression within cellular types and organelles.

Results

Hippocampus and entorhinal cortex

Cellular Identification

We have previously reported regional CTR1 immunolabeling in hippocampus22. Here, we extend those observations by focusing on additional cellular and ultrastructural features in hippocampus, entorhinal cortex, and stratum. To further validate the CTR1 antibody and prior to undertaking the extensive process of electron microscopy in human postmortem tissue, we conducted multiplex FISH on the hippocampus proper to ensure the immunolabeled cell types also expressed the gene responsible for CTR1 protein production.

We observed CTR1 immunolabeling of pyramidal neurons across the CA1, CA2, and CA3 subregions of the hippocampus (Fig. 1A-D). Within the entorhinal cortex adjacent to the hippocampus, we observed CTR1 expression in pyramidal neurons in both the superficial and deep layers (Fig. 1E-G). Corresponding SLC31A1 mRNA signal in hippocampal pyramidal neurons was confirmed by FISH (Fig. 2A-D). Pyramidal neurons were identified by their classic morphological shape and presence of an apical dendrite, as labeled by NeUN immunohistochemistry.

Fig. 1.

Fig. 1

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The pyramidal neurons across hippocampal subfields exhibit immunolabeling of copper transporter CTR1. Panels A–D show CTR1 immunolabeling in the CA1 (A), CA2 (B), CA3 (C), and CA4 (D) subregions, respectively. Panel E displays a broader view of the entorhinal cortex (ENC), and distribution of CTR1 immunolabeling across layers. Panels F and G present a higher-magnification image of the superficial and deeper layers of ENC, respectively. Scale bars: A-D) 50 μm; E) 200 μm; F-G) 50 μm. These images are intended to illustrate regional distribution and representative labeling patterns; fine cellular identification is supported by FISH and ultrastructural analyses.

Fig. 2.

Fig. 2

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Pseudo-colored fluorescent in situ hybridization and immunohistochemical labeling of a pyramidal neuron in the CA1 region of the hippocampus. A) DAPI (white) and NeUN (gray) co-label showing the nucleus and soma of the pyramidal neuron, respectively. B) Lipofuscin (yellow) shows autofluorescence within the cell. C) SLC31A1 (red) mRNA expression for cognate protein CTR1; D) Merged image. Scale bar: 20 μm.

Within the subiculum, bipolar-appearing interneuronal profiles were immunolabeled near the gray/white matter transition zone (Fig. 3). Because these cells were identified on the basis of morphology and location, neurotransmitter phenotype cannot be assigned definitively from immunohistochemistry alone. However, corresponding FISH experiments indicated SLC31A1 mRNA presence in GAD1-positive cellular populations within the hippocampus and striatum (Fig. 4). Immunolabeling did not differ within the white matter of the entorhinal cortex (data not shown).

Fig. 3.

Fig. 3

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Immunolabeling of bipolar-appearing interneuronal profiles in the human subiculum. Panels A and B show representative immunolabeled putative bipolar interneurons and their processes. Panel C displays the transition between grey and white matter (dotted line), illustrating the varied spatial distribution and morphology of immunolabeled interneurons within the subiculum region. Scale bars: A-C) 50 μm. These images are intended to illustrate regional distribution and representative labeling patterns; fine cellular identification is supported by FISH and ultrastructural analyses.

Fig. 4.

Fig. 4

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Pseudo-colored fluorescent in situ hybridization and immunohistochemical labeling of GAD1 + interneurons in the hippocampus (HIPP) and the striatum (STR), respectively. A, F) DAPI (white) and NeUN (gray) co-label showing the nucleus and soma of the interneuron, respectively. B, G) Lipofuscin (yellow) shows autofluorescence within the cell. C, H) GAD1 (cyan) mRNA expression for cognate protein GAD67 allows identification of GABAergic interneurons; D, I) SLC31A1 (red) mRNA expression for cognate protein CTR1; E, J) Merged image. Scale bar: 10 μm.

Lastly, robustly immunolabeled glial-like cells and their processes were present along the ventricular surface of the hippocampus (Fig. 5A). The alveus of the hippocampus proper exhibited CTR1 immunolabeling in glial-appearing profiles and vasculature (Fig. 5B, C). These findings were supported by the presence of SLC31A1 mRNA in vascular-associated (labeled by PECAM1) and glial-associated (labeled by GJA1) cellular profiles (Fig. 6A-E), as shown by FISH.

Fig. 5.

Fig. 5

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CTR1 immunolabeling of glial-appearing cells in the human hippocampus. Panel A shows a putative glial profile labeling along the ventricular surface. Panels B and C depict immunolabeled fibrous astrocyte-like and oligodendrocyte-like profiles within the alveus region of the hippocampus. Scale bars: A-C) 20 μm. These images are intended to illustrate regional distribution and representative labeling patterns; fine cellular identification is supported by FISH and ultrastructural analyses.

Fig. 6.

Fig. 6

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Pseudo-colored fluorescent in situ hybridization labeling of endothelial (PECAM1) astrocytic (GJA1) cells in the hippocampus (HIPP) and the striatum (STR), respectively. A, F) Lipofuscin (yellow) shows autofluorescence within the cell. B, G) PECAM1 (pink) mRNA expression allows the identification of endothelial cells. C, H) GJA1 (blue) mRNA expression allows identification of astrocytic glial cells. D, I) SLC31A1 (red) mRNA expression for cognate protein CTR1; E, J) Merged image. Scale bar: HIPP: 60 μm. STR: 20 μm.

Electron microscopy

Following validation of the antibody labeling via FISH in the hippocampus, we sought to investigate CTR1 and its ultrastructural localization at the electron microscopic level. Original micrographs without pseudo-coloring are available in the supplement.

CTR1 immunolabeling was present in several types of cells in the hippocampus. Within the entorhinal cortex, we observed immunolabeling within the cytoplasm of a cell morphologically consistent with an oligodendrocyte, such as its size and features of densely packed heterochromatin within the nucleus (Fig. 7A). We also observed immunolabeling within the nucleus and cytoplasm of a cell morphologically consistent with an astrocyte (Fig. 7B) and a granule cell (Fig. 7C). Within the cytoplasm of the granule cell, we also observed immunolabeling adjacent to a mitochondrion and endoplasmic reticulum (Fig. 7C).

Fig. 7.

Fig. 7

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Electron microscopic localization of CTR1 in various types of cells in entorhinal cortex. A) A putative oligodendrocyte has immunolabeling in the cytoplasm (circles). It is identified by a cellular profile morphologically consistent with an oligodendrocyte, such as size and features, including a round nucleus filled with heterochromatin. B) A putative astrocyte has immunolabeling in the cytoplasm and nucleus (circles). It is identified by characteristics morphologically consistent with an astrocyte, such as an irregularly shaped nucleus with sparse heterochromatin and fibrils in the cytoplasm (orange). C) A portion of a putative granule cell shows immunolabeling in the cytoplasm and the nucleus (circles). A mitochondrion (m), vesicular structures adjacent to mitochondria (pink), and endoplasmic reticulum (blue) are shown adjacent to the labeling. D) A portion of a neuronal soma with immunolabeling in the cytoplasm (circles), with vesicular structures adjacent to mitochondria (pink), also exhibiting label both adjacent to and far from a mitochondrion (m). E) A higher magnification of the cytoplasm of a neuronal somata showing immunolabeling (circle) between endoplasmic reticulum (blue) and mitochondria (m), with vesicular structures adjacent to mitochondria (pink). F) In a dendritic process (red), CTR1 immunolabeling (circles) is observed adjacent to vesicular structures near mitochondria (pink). Scale bars: A-D) 2 μm; E-F): 500 nm. Subcellular localization is based on DAB reaction product and should be interpreted cautiously.

Within the superficial layers of the entorhinal cortex, CTR1 immunolabeling was observed within the cytoplasm of a neuronal soma morphologically consistent with a pyramidal neuron, with labeling adjacent to a mitochondrial profile and nearby vesicular structures (Fig. 7D). At higher magnification, immunolabeling was observed within neuronal cytoplasm adjacent to vesicular structures and endoplasmic reticulum (Fig. 7E). Additionally, CTR1 immunolabeling was observed adjacent to a mitochondrial profile and nearby vesicular structures within a dendritic process (Fig. 7F).

Ultrastructural examination of the neuropil within the entorhinal cortex revealed CTR1 immunolabeling within axonal processes, with labeling observed adjacent to mitochondrial profiles and nearby vesicular structures, including regions adjacent to asymmetric axospinous synapses (Fig. 8A). Multiple examples of immunolabeling were observed near asymmetric synapses (Fig. 8B–E), including labeling adjacent to vesicular structures in axon terminals (Fig. 8B, C) and within dendritic processes (Fig. 8D). Immunolabeling was also observed within dendritic processes adjacent to mitochondrial profiles and nearby vesicular structures in proximity to asymmetric synapses (Fig. 8E). Labeled axon terminals varied in size and formed asymmetric synapses with dendritic spines or shafts.

Fig. 8.

Fig. 8

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Electron microscopic localization of CTR1 in axon terminals in entorhinal cortex. A) An axon terminal (blue) emerges from a varicose axonal process (blue). Immunolabeling (circles) of this axon is observed away from the asymmetric axospinous synapse (open arrow), and adjacent to a mitochondrial profile (m) and nearby vesicular structures (pink) within the varicosity. B) Another axon terminal (blue) emerges from a thin preterminal axon and forms an asymmetric synapse (open arrow) with a spine (yellow). In this axon terminal, immunolabeling (circle) is associated with vesicular structures near the synapse. C) A small axon terminal (blue) with immunolabeling (circle) near the synapse (open arrow; spine shown in yellow). D) A small axon terminal (blue) filled with immunolabeling (circle) forms an asymmetric axodendritic synapse (open arrow; dendrite shown in red). E) A dendrite (red) with an adjacent axospinous synapse (open arrow) contains immunolabeling (circles). An unlabeled axon terminal (blue) is shown in apposition to the spine (yellow). Immunolabeling is present adjacent to the mitochondrial outer membrane and nearby vesicular structures (pink). Scale bars: A-E) 500 nm. Subcellular localization is based on DAB reaction product and should be interpreted cautiously.

Within the alveus, CTR1 immunolabeling was observed within the nucleus and cytoplasm of a cellular profile morphologically consistent with a fibrous astrocyte (Fig. 9A), with labeling adjacent to mitochondrial profiles and nearby vesicular structures. Additionally, immunolabeling was observed associated with vascular elements, including endothelial cells, pericyte profiles, and surrounding putative astrocytic endfeet (Fig. 9B). Finally, asymmetric synapses within the white matter alveus showed immunolabeling within axon terminals, with labeling observed adjacent to vesicular structures (Fig. 9C). An adjacent myelinated axonal process also exhibited immunolabeling (Fig. 9C).

Fig. 9.

Fig. 9

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Electron microscopic localization of CTR1 immunolabeling in the subhippocampal white matter. A) A fibrous astrocyte-like profile shows immunolabeling (circles) within the cytoplasm, with labeling observed adjacent to a mitochondrial profile (m) and nearby vesicular structures (pink). B) A capillary containing red blood cells (red) shows immunolabeling (circles)associated with the endothelial cell and surrounding perivascular elements, including a pericyte (pink) and surrounding putative astrocytic endfeet (orange). C) Immunolabeling (circles) within an axospinous synapse composed of an axon terminal (blue) onto a dendritic spine (yellow), adjacent to an immunolabeled myelinated process. Scale bars: A) 2 μm; B-C) 1 μm. Subcellular localization is based on DAB reaction product and should be interpreted cautiously.

Striatum

Cellular Identification

At the light microscopic level, the caudate and putamen exhibited broadly similar patterns of CTR1 immunolabeling. Representative findings are shown from the caudate nucleus, including ventricular surface/subependymal and deeper neuropil regions; similar patterns were observed in the putamen where assessed. At the ventricular surface of the caudate nucleus, immunoreactive ependymal cells and adjacent processes were prominent and extended approximately 50–100 μm into the subependymal zone (Fig. 10A). A region of relatively lighter immunolabeling was present between the ependymal layer and deeper portions of the caudate (Fig. 10A, B). Immunoreactive processes traversed the subependymal zone and extended into the caudate with moderate density (Fig. 10B, C).

Fig. 10.

Fig. 10

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Light microscopic distribution of CTR1 immunolabeling in the caudate nucleus of the striatum. A) Immunoreactive ependymal cells and adjacent immunolabeled processes are observed at the ventricular surface of the caudate nucleus. B) A region of relatively lighter immunolabeling is present between the ependymal layer and deeper regions of the caudate, with many labeled processes extending through this zone. C) A higher magnification view of the area immediately adjacent to the ventricular surface of the caudate nucleus. An immunoreactive cell body and processes are visible. D) Deeper within the caudate nucleus, immunolabeling is very light in the neuropil and minimal within fiber bundles. E) Within the rostrum of the corpus callosum, white matter adjacent to the caudate, immunolabeling is observed in glial-appearing cellular profiles. F) An immunolabeled capillary and adjacent glial-appearing profile within the rostrum of the corpus callosum. Scale bars: A-E) 50 μm; F) 20 μm.

In deeper regions of the caudate nucleus (Fig. 10D), and similarly in the putamen (not shown), immunolabeling was diffuse within the neuropil and minimal within fiber bundles. Distinct neuronal somata or glial cell bodies were not readily resolved by light microscopy in the gray matter. Within white matter adjacent to the caudate (rostrum of the corpus callosum), immunolabeling was observed in glial-appearing cellular profiles and vascular elements (Fig. 10E, F).

To assess regional generalizability, multiplex FISH was conducted in fresh-frozen human striatum. Consistent with findings in the hippocampus, SLC31A1 mRNA signal was observed in cellular populations expressing GAD1, GJA1, and PECAM1 (Figs. 4F-J and 6F-J) in both caudate and putamen. Cellular profiles were more clearly resolved by FISH than by DAB-based light microscopy, indicating that transcript detection and protein immunolabeling did not have equivalent cellular sensitivity.

Electron microscopy

At the ultrastructural level, immunolabeling was observed within the cytoplasm of a cellular profile morphologically consistent with an astrocyte in the caudate (Fig. 11A). Similar to observations in the hippocampus, immunolabeling was associated with vascular elements, including endothelial cells, circulating blood cells, and surrounding putative astrocytic endfeet (Fig. 11B). Adjacent to this vasculature, an axon terminal forming an asymmetric synapse showed immunolabeling, with labeling observed adjacent to a mitochondrial profile and nearby vesicular structures (Fig. 11B). Another capillary containing red blood cells showed immunolabeling associated with the endothelial cell and surrounding putative astrocytic endfeet (Fig. 11C).

Fig. 11.

Fig. 11

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CTR1 immunolabeling in the caudate nucleus of the striatum at the electron microscopic level. A) A cellular profile morphologically consistent with an astrocyte shows immunolabeling (circles) within the cytoplasm. B) A capillary containing red blood cells (red) shows immunolabeling (circles) associated with a circulating blood cell and surrounding perivascular elements, including putative astrocytic endfeet (orange). The basement membrane is indicated by arrows. An adjacent axon terminal (blue) forms an asymmetric synapse (open arrow) with a dendritic spine (yellow) and contains immunolabeling (circles) observed adjacent to a mitochondrial profile (m) and nearby vesicular structures (pink). C) Another capillary containing red blood cells (red) shows immunolabeling (circles) associated with the endothelial cell (pink) and surrounding putative astrocytic endfeet (orange). The basement membrane is indicated by arrows. D) Axon terminals (blue) form asymmetric synapses (open arrows) with dendritic spines (yellow) near the ventricular surface. One spine contains a small amount of immunolabeling (circle). E) A perforated asymmetric synapse (open arrows) near the ventricular surface shows an axon terminal containing a mitochondrial profile (m), while the apposing spine (yellow) shows a small amount of immunolabeling (circle) distal from the synapse. F) An axon terminal (blue) forming an axospinous synapse (open arrow) with a dendritic spine (yellow) contains immunolabeling (circles). Scale bars: A) 2 μm; B-F) 500 nm. Subcellular localization is based on DAB reaction product and should be interpreted cautiously.

Within the neuropil of the caudate, multiple axon terminals forming asymmetric synapses with dendritic spines showed immunolabeling (Fig. 11E, F). At the ventricular surface, labeled axon terminals contacting lightly labeled spines were observed. In a perforated asymmetric synapse, the presynaptic terminal contained a mitochondrial profile, and the apposing spine showed a small amount of immunolabeling distal to the synapse.

Discussion

The present findings indicate that CTR1 immunolabeling varies across hippocampal, entorhinal, and striatal compartments and is detectable in neuronal, vascular-associated, and glial-associated profiles. FISH demonstrated SLC31A1 transcript signal in neuronal and non-neuronal cellular populations corresponding broadly to those identified by immunohistochemistry. At the ultrastructural level, labeling was observed in association with barrier-associated cellular elements, excitatory synapses, and organelle-adjacent cytoplasmic profiles. Light microscopic interpretation of CTR1 immunolabeling was limited by section thickness and DAB-based detection, which can reduce contrast and obscure fine cellular boundaries; therefore, conclusions regarding cellular identity rely primarily on FISH. Furthermore, because the ultrastructural reaction product was DAB-based, subcellular assignments should be interpreted cautiously.

Based on neuronal morphology and laminar distribution, CTR1 was localized primarily in excitatory neurons such as pyramidal and granule cells. In axons and axon terminals, CTR1-associated labeling was observed adjacent to vesicular structures within axon terminals forming asymmetric synapses, consistent with prior observations of copper release following depolarization63,64. However, it was previously believed that the copper transporter ATP7A inserts copper into synaptic vesicles65. Taken together, these observations suggest that CTR1-associated labeling can occur near synaptic vesicle pools at asymmetric synapses in human brain tissue, consistent with a speculative presynaptic role for CTR1, although the current data does not demonstrate definitive localization to synaptic vesicles nor direct involvement in neurotransmitter release. Importantly, functional implications cannot be determined from the present data.

White matter labeling of CTR1 protein differed substantially between regions. CTR1 was abundant in the hippocampal and entorhinal white matter, particularly within putative fibrous astrocytic and oligodendrocytic profiles, but sparse in the striatum. The function of CTR1 within the bulbous varicosities of fibrous astrocytes remains unclear, but the presence of CTR1 in presumed astrocytes and oligodendrocytes aligns with prior work showing that copper deficiency leads to demyelination and decreased oligodendrocyte marker expression21,66–69. Together, these findings underscore high copper demand in putative glial populations, particularly those that support excitatory cortical networks.

CTR1-associated labeling was repeatedly observed adjacent to mitochondrial profiles, which is consistent with, but does not establish, a role in mitochondrial copper handling. However, copper serves as a cofactor for cytochrome c oxidase, the terminal enzyme of the electron transport chain, required for ATP production57. Deficiency in mitochondrial copper reduces ATP synthesis and downregulates regional energy metabolism70. Labeling adjacent to mitochondrial and endoplasmic reticulum profiles raises the possibility of association with organelle contact regions, although this interpretation remains speculative.

The present findings provide descriptive data on CTR1 immunolabeling in human postmortem hippocampus, entorhinal cortex, and striatum across cellular and ultrastructural compartments. The data support further study of CTR1 in neuronal, glial, and barrier-associated elements, while also highlighting the need for additional validation of subcellular localization using complementary approaches. These observations may serve as a reference framework for future studies of copper transport in neuropsychiatric and neurodegenerative disease.

Limitations

In addition to the inherent constraints of postmortem human tissue, several methodological limitations should be noted. First, DAB-based immunolabeling in 40-µm vibratome sections limits the precision of cellular identification by light microscopy. Second, subcellular interpretation of electron-dense reaction product is necessarily inferential, particularly for vesicular and mitochondrial-associated profiles. Third, some cellular identities were assigned on the basis of morphology and anatomical location rather than direct double-label confirmation. Finally, variation in tissue quality measures, including PMI and tissue pH, may have influenced labeling intensity. Furthermore, the current study is wholly descriptive and does not include any formal quantification. Future work is needed to determine the overall proportions of CTR1 expression within cellular types and organelles. Thus, the data presented here should be interpreted with caution and not used to infer CTR1 localization in all cells or in a particular organelle type.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (8.9MB, docx)

Acknowledgements

The authors would like to thank the Alabama and Maryland Brain Collections staff for the samples used in this study. We also want to thank the brain donors and their families, as this research is only made possible through their generous donations during an extremely difficult time.

Author contributions

K.E.S.: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft and revisions.G.N.: Investigation (immunohistochemistry, electron microscopy).G.G.: Investigation (FISH), Writing – review & editing.H.H.: Investigation (FISH).C.B.F.: Conceptualization, Methodology, Writing – review & editing.All authors: Writing – review & editing, Approval of final manuscript.

Funding

We would like to acknowledge and thank our funding sources: Brain & Behavior Research Foundation NARSAD Young Investigator Grant (31567), the NIH FIRST Scholars Program (U54CA267746), the NIMH (K00 MH122943), and UAB Psychiatry and Behavioral Neurobiology Startup Funding.

Data availability

All data generated during this study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Materials

Supplementary Material 1 (8.9MB, docx)

Data Availability Statement

All data generated during this study are available from the corresponding author on reasonable request.

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