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The neuronal cell-specific proteomeThe neuronal cell transcriptomeExpression specificity within neuronsInhibitory neuronsExcitatory neuronsSpecialized neurotransmitter neuronsNeurosecretoryOther neuronsBackground
Single nuclei brain methods
The neuronal cell-specific proteomeNeurons are the primary signaling cells of the brain, forming complex networks that coordinate sensory processing, movement, cognition, and other essential functions. The human brain contains an exceptionally diverse range of neuronal populations, which can be distinguished by their neurotransmitter identity, developmental origin, anatomical location, connectivity, and molecular expression profiles. To explore this diversity, neuronal cell clusters identified by single-nucleus RNA sequencing have been annotated and organized into 34 neuronal cell types, further divided into five main groups. These include inhibitory neurons, primarily associated with GABAergic and glycinergic signaling, and excitatory neurons, predominantly using glutamate as a neurotransmitter. Additional groups comprise specialized neurotransmitter neurons, including dopaminergic, serotonergic, noradrenergic and cholinergic populations, as well as neurosecretory neurons involved in hormonal regulation and a small group of other neurons that do not fit into the main categories. Within these groups, neuronal cell types are further distinguished based on developmental origin, regional distribution, and characteristic gene expression markers. The tables and examples below provide an overview of the annotated neuronal populations and highlight genes with elevated expression in specific neuronal cell types, illustrating the molecular diversity of the human brain. Transcriptome analysis shows that 68% (n=13658) of all human proteins (n=20162) are detected in neuronal cells and 2478 of these genes show an elevated expression in any neuronal cells compared to other brain cell types. The neuronal cell transcriptomeThe snRNA-seq-based neuronal cell transcriptome can be analyzed with regard to specificity, illustrating the number of genes with elevated expression in neuronal cell type compared to other brain cell types (Table 1). Genes with an elevated expression are divided into three subcategories:
Table 1. Number of genes in the subdivided specificity categories of elevated expression in the analyzed neuronal cell types compared to other grouped cell types.
General neuronal markersNeuronal cells are highly variable, for example with regard to the signaling properties, what neurotransmitter they use as a pre-synaptic neuron, as well as the receptors and ability to receive different types of input as a post-synaptic neuron. Additionally, the regional variation and circuitry (connectivity) plays a role in the identity of a neuron. However, certain markers are pan-neuronal, such as RBFOX3, also called NeuN, which was utilized for neuronal enrichment when generating the data, by Siletti K et al. (2023).
Inhibitory or excitatory neuronsInterneurons, which typically have local connections, are often inhibitory, meaning they reduce the membrane potential and decrease the likelihood of action potential propagation in the postsynaptic neuron. Common neurotransmitters released by inhibitory neurons include GABA and glycine. As a result, proteins associated with these molecules, such as GAD1 and GAD2, both essential for GABA synthesis, and used as markers for inhibitory neurons. In contrast, long-range projecting neurons are often excitatory, increasing the membrane potential in the postsynaptic neuron. Glutamate is a common excitatory neurotransmitter, and markers for these neurons include various glutamate transporters like VGLUT1(SLC17A7) and VGLUT2(SLC17A6). The separation of inhibitory and excitatory neurons is one of the main features used for naming the different cell type clusters. Expression specificity within neuronsTable 2. Number of genes in the subdivided specificity categories of elevated expression within the analyzed neuronal cell types, and their brain region of origin.
Inhibitory neuronsAs shown in Table 1, 1431 genes show elevated expression in inhibitory neurons as a group, compared to non-neuronal cells and other grouped neuronal populations. Inhibitory neurons primarily use GABA or glycine as neurotransmitters and play an essential role in regulating neuronal activity and maintaining the balance between excitation and inhibition in the brain. Although many inhibitory neurons function as local interneurons, inhibitory identity does not necessarily imply an interneuron, as some inhibitory populations also form long-range projections to other brain regions. MGE derived interneuronInhibitory interneurons originating from the medial ganglionic eminence (MGE), commonly characterized by expression of GAD1, GAD2, LHX6, and subtype-specific markers such as PVALB, NPY and SST. As shown in Table 2, 75 genes show elevated expression in MGE interneuron compared to other neuronal cell types. Somatostatin (SST) is a well-studied peptide hormone, expressed by MGE derived interneurons, although is also expressed by many other cells through out the brain, and therefore chategorized as elevated in MGE derived interneurons.
CGE derived InterneuronsInhibitory interneurons originating from the caudal ganglionic eminence (CGE), comprising diverse populations characterized by markers such as VIP, RELN, LAMP5, and ADARB2. As shown in Table 2, 99 genes show elevated expression in the CGE interneuron cluster compared to other neuronal cell types. Among the genes with elevated expression we can find Vasoactive intestinal peptide, VIP, highly expressed and specific for the CGE interneuron cluster. VIP can act as neurotransmitter and neuromodulator, it is a key marker to separate different types of cortical interneurons. The ligand-gated ion channel and serotonin transporter HTR3A is also classified as enriched among CGE interneurons compared to other cell clusters in the brain. The cells with overlapping expression of VIP and HTR3A could be bipolar cells, a cortical interneuronal subtype, based on suggested dependency of serotonergic inputs (Wong FK et al. (2022)). Cholecystokinin CCK, often mentioned in interneuronal subclassification, is also found among the genes with elevated expression in the CGE interneuron cluster.
Medium Spiny Neurons (MSN)Medium spiny neurons (MSNs) are the principal inhibitory projection neurons of the striatum, characterized by expression of PPP1R1B and distinct populations expressing dopamine receptors DRD1 or DRD2, often associated with TAC1, PDYN, or PENK. As shown in Table 2, 175 genes show elevated expression in medium spiny neurons compared to other neuronal cell types. Dopamine receptor D2 (DRD2), a well-established marker of a major MSN subpopulation in the basal ganglia, is classified as cell type enriched, while the serotonin receptor 5-hydroxytryptamine receptor 4 (HTR4) shows its highest expression in this cluster, despite variable expression across other neuronal populations. Additional examples of elevated expression include PTPN5, a protein tyrosine phosphatase involved in neuronal signaling and synaptic plasticity, and PENK, encoding the neuropeptide precursor proenkephalin, associated particularly with DRD2-expressing MSNs. Protein expression of PTPN5 and PENK is also illustrated by immunohistochemistry (IHC).
MSN-like neuronsInhibitory neurons sharing molecular characteristics with striatal medium spiny neurons, including expression of DRD1, DRD2, PENK, or TAC1, but distributed across additional forebrain regions. As shown in Table 2, 59 genes show elevated expression in MSN-like neurons compared to other neuronal cell types. Other InhibitoryA heterogeneous group of inhibitory neurons expressing GAD1 and GAD2 that do not clearly belong to the more specialized developmental or molecular categories. As shown in Table 2, 21 genes show elevated expression in other Inhibitory neurons compared to other neuronal cell types. Midbrain derived inhibitory neuronsInhibitory neuronal populations associated with midbrain developmental programs, characterized by GAD1 and GAD2 expression and additional region- or subtype-specific molecular markers. As shown in Table 2, 139 genes show elevated expression in Midbrain-derived inhibitory neurons compared to other brain cell clusters. This cluster of neurons has several genes classified as enriched, indicating a distinct expression profile compared to other neurons. Prolactin (PRL) is highly enriched in this cluster, and so is the transcription factor SOX14. Additionally, a serotonin receptors with variable expression across the neuronal cell population, 5-hydroxytryptamine receptor 2A (HTR2A) show high expression in this cluster. Glutamate receptor interacting protein 2 (GRIP2) and Glutamate ionotropic receptor kainate type subunit 1 (GRIK1) both show highest expression in this cluster. TFAP2B+ InhibitoryA molecularly distinct population of inhibitory neurons characterized by expression of the transcription factor TFAP2B. These neurons are found in the cerebellum, medulla oblongata, midbrain, and pons, with a particularly characteristic population represented by the molecular layer interneurons of the cerebellar cortex, including stellate and basket cells. As shown in Table 2, 137 genes show elevated expression in TFAP2B+ inhibitory neurons compared to other neuronal cell types. One example is SCGN, encoding secretagogin, a calcium-binding protein expressed in subsets of inhibitory interneurons, including molecular layer interneurons of the cerebellum. SCGN is also expressed outside the brain, particularly in pancreatic islet cells, illustrating how genes associated with specific neuronal populations may have additional functions in peripheral tissues. Another example is TFAP2B itself, a transcription factor associated with the molecular identity of these inhibitory neurons.
Midbrain-hindbrain inhibitory neuronsInhibitory neuronal populations distributed across the midbrain and hindbrain, characterized by GAD1 and GAD2 expression and molecular identities associated with their regional developmental origin. As shown in Table 2, 28 genes show elevated expression in Midbrain-hindbrain inhibitory neurosn compared to other neuronal cell types. One example of enriched expression in midbrain–hindbrain inhibitory neurons is PAX7, encoding a transcription factor involved in the development and regional specification of neuronal populations in the dorsal midbrain and hindbrain. Its elevated expression reflects the distinct developmental identity of this inhibitory neuronal population.
LAMP5+ glycinergic inhibitoryA specialized population of inhibitory neurons present in medulla oblongata, pons, spinal cord, characterized by expression of LAMP5 and the glycine transporter SLC6A5, indicating glycinergic neurotransmission. As shown in Table 2, 40 genes show elevated expression in LAMP5+ glycinergic inhibitory neurons compared to other neuronal cell types.
Cerebellar Purkinje cellsLarge inhibitory projection neurons of the cerebellar cortex that integrate synaptic input and provide the principal output of the cerebellar cortex, characterized by markers such as CALB1 and PCP2. As shown in Table 2, 781 genes show elevated expression in Cerebellar Purkinje cells compared to other neuronal cell types. Examples of genes with elevated expression in cerebellar Purkinje cells include HOMER3, encoding a postsynaptic scaffolding protein involved in neuronal signaling, SKOR1, encoding a transcriptional corepressor associated with Purkinje cell differentiation and identity, and PRKCG, encoding protein kinase C gamma, an enzyme involved in intracellular signaling, synaptic plasticity, and regulation of Purkinje cell dendritic development. All three are established Purkinje cell-associated proteins, representing different aspects of neuronal function.
Excitatory neuronsAs shown in Table 1, 1374 genes show elevated expression in excitatory neurons as a group, compared to non-neuronal cells and other grouped neuronal populations. Excitatory neurons primarily use glutamate as a neurotransmitter and play an essential role in transmitting signals and facilitating communication across neuronal networks. Many excitatory neurons function as long-range projection neurons, connecting different brain regions, while others form local circuits involved in processing and integrating information. These populations exhibit considerable molecular diversity, reflecting their distinct anatomical locations, connectivity, and functional roles. Forebrain ExcitatoryA heterogeneous group of glutamatergic neurons distributed across forebrain structures, characterized by excitatory neurotransmitter markers such as SLC17A7 and region-specific molecular expression profiles. As shown in Table 2, 74 genes show elevated expression in Forebrain Excitatory neurons compared to other neuronal cell types. Forebrain excitatory neurons show enriched expression of SMYD1, encoding a protein involved in epigenetic regulation through histone methylation. Although SMYD1 is primarily known for its role in cardiac and skeletal muscle development, its function in neuronal populations remains poorly characterized. Another example is, EMX1 encoding a homeobox transcription factor involved in forebrain development and the specification of cortical excitatory neurons. EMX1 shows group-enriched expression shared with forebrain projection neurons, reflecting the developmental and molecular similarities between these excitatory neuronal populations.
Forebrain projection neuronsExcitatory projection neurons primarily associated with the cerebral cortex and other forebrain structures, including populations with distinct laminar identities and projection patterns. This group includes upper- and deep-layer intratelencephalic neurons, as well as deep-layer corticothalamic and layer 6b neurons. As shown in Table 2, 70 genes show elevated expression in Forebrain projection neurons compared to other neuronal cell types. One characteristic example is SATB2, a transcription factor involved in the development and identity of cortical projection neurons, particularly those forming connections within and between the cerebral hemispheres. SATB2 shows elevated expression across several forebrain projection neuron clusters, reflecting their shared molecular characteristics.
Another example is NEUROD6, a transcription factor involved in neuronal differentiation and the development of excitatory neurons. Within the forebrain projection neuron group, NEUROD6 shows its highest expression in neurons sampled from the hippocampus, illustrating how a broadly defined neuronal population can exhibit distinct regional expression patterns.
CA1-3 ExcitatoryHippocampal pyramidal neurons located in the CA1, CA2, and CA3 subfields, involved in hippocampal information processing and memory-related neuronal circuits. As shown in Table 2, 127 genes show elevated expression in Hippocampal CA1-3 neurons compared to other brain cell clusters. CA4 ExcitatoryExcitatory neurons associated with the CA4 region of the hippocampus, exhibiting distinct molecular profiles and contributing to local hippocampal circuitry. As shown in Table 2, 127 genes show elevated expression in CA4 Excitatory neurons compared to other neuronal cell types. Dentate gyrus excitatoryExcitatory granule neurons of the hippocampal dentate gyrus, characterized by markers such as PROX1 and involved in the initial processing of information entering the hippocampal circuit. As shown in Table 2, 190 genes show elevated expression in Dentate gyrus excitatory neurons compared to other neuronal cell types. Hypothalamic VGLUT2+ neuronsExcitatory hypothalamic neurons characterized by expression of SLC17A6 (VGLUT2), contributing to neuronal circuits regulating homeostasis, endocrine function, and behavior. As shown in Table 2, 90 genes show elevated expression in Hypothalamic VGLUT2+ neurons compared to other neuronal cell types. Subcortical VGLUT2+ excitatoryExcitatory neurons distributed across subcortical structures, characterized by SLC17A6 expression and diverse region-specific functions. As shown in Table 2, 32 genes show elevated expression in Subcortical VGLUT2+ excitatory neurons compared to other neuronal cell types. Thalamic projection neuronsExcitatory neurons of the thalamus, primarily expressing SLC17A6, that transmit and integrate information between subcortical structures and the cerebral cortex. As shown in Table 2, 121 genes show elevated expression in Thalamic projection neurons compared to other neuronal cell types. PVALB+ excitatory neuronsA distinct population of excitatory neurons characterized by expression of PVALB, encoding parvalbumin, a calcium-binding protein involved in regulating intracellular calcium signaling. These neurons are found in the cerebellum, medulla oblongata, midbrain, pons, and thalamus. Although PVALB is commonly associated with inhibitory interneurons, it is also expressed in excitatory neuronal populations. As shown in Table 2, 75 genes show elevated expression in PVALB+ excitatory neurons compared to other neuronal cell types. In several of these brain regions, PVALB expression is also observed in inhibitory neurons, including the TFAP2B+ inhibitory group, highlighting its expression across functionally distinct neuronal populations.
Upper rhombic lip-derived neuronsExcitatory neurons originating from the upper rhombic lip during development, including cerebellar-associated populations with specialized molecular and anatomical identities. As shown in Table 1, 245 genes show elevated expression in Upper rhombic lip neurons compared to other brain cell clusters. BarH like homeobox 1 (BARHL1) is an example of a gene with shared enriched expression in the lower and upper rhombic lip clusters. EOMES is a transcription factor, that plays an important role in neurodevelopment Lv X et al. (2019), with enriched expression in this cluster, combined with leukocytes. Glutamate metabotropic receptor 4 (GRM4) and transcription factor UNC homeobox (UNCX) are more examples of genes with elevated expression in this cluster. While, EN2 and CRTAM both are examples of enriched genes shared between this cluster and cerebellar interneurons. Lower rhombic lip-derived neuronsExcitatory neurons originating from the lower rhombic lip, primarily associated with hindbrain structures and characterized by developmental markers such as ETV1 and ZIC1 in selected populations. As shown in Table 1, 0 genes show elevated expression in Lower rhombic lip neurons compared to other brain cell clusters. Neurofilament light (NEFL), medium (NEFM) and heavy (NEFH) chain, all show elevated expression levels in this cluster. Tubulointerstitial nephritis antigen (TINAG) is a basement membrane glycoprotein with selective expression in this cluster. BarH like homeobox 1 (BARHL1) is an example of a gene with shared enriched expression in the lower and upper rhombic lip clusters. Calretinin (CALB2), a calcium-binding protein expressed by subtypes of neurons, especially interneurons in cerebral cortex and hippocampus, show elevated expression in the CGE interneuron cluster as well as the lower rhombic lip cluster and thalamic excitatory cluster cells.
Cerebellar granular cellsSmall, highly abundant excitatory neurons of the cerebellar granular layer that receive mossy fiber input and transmit signals to Purkinje cells through their parallel fibers. As shown in Table 2, 245 genes show elevated expression in Cerebellar granular cells compared to other neuronal cell types. Brainstem VGLUT2+ ExcitatoryExcitatory neuronal populations of the brainstem characterized by SLC17A6 expression, contributing to diverse sensory, motor, and autonomic circuits. As shown in Table 2, 27 genes show elevated expression in Brainstem VGLUT2+ Excitatory neurons compared to other neuronal cell types. Spinal peptidergic extitatory neuronsExcitatory neurons of the spinal cord characterized by glutamatergic signaling and expression of neuropeptides involved in sensory processing and local spinal circuitry. As shown in Table 2, 0 genes show elevated expression in Spinal peptidergic extitatory neurons compared to other neuronal cell types. Other Excitatory neuronsA heterogeneous group of glutamatergic neurons characterized by SLC17A7, SLC17A6, or SLC17A8 expression that do not fit into the more anatomically or developmentally defined excitatory populations. As shown in Table 2, 56 genes show elevated expression in Other Excitatory neurons compared to other neuronal cell types. Specialized neurotransmitter neuronsAs shown in Table 1, 741 genes show elevated expression in specialized neurotransmitter neurons as a group, compared to non-neuronal cells and other grouped neuronal populations. This group includes dopaminergic, noradrenergic, serotonergic, cholinergic, monoaminergic, and orexinergic neurons, characterized by the production and release of specific neurotransmitters or neuropeptides. These populations play important roles in modulating neuronal activity and regulating functions such as movement, arousal, sleep, attention, and mood. Although relatively small in number, many of these neurons form widespread projections that influence activity across multiple brain regions. CholinergicNeurons characterized by expression of CHAT and SLC5A7, responsible for acetylcholine synthesis and transport and involved in attention, motor control, and autonomic regulation. As shown in Table 2, 150 genes show elevated expression in Cholinergic neurons compared to other neuronal cell types. Choline acetyltransferase (CHAT) is expressed by cholinergic neurons, sparesly detected, in selected clusters and brain regions. On protein level, we can observe CHAT in a subset of large neurons in caudate nucleus (basal ganglia).
DopaminergicNeurons characterized by dopamine synthesis machinery, including TH, and involved in motor control, reward processing, motivation, and neuroendocrine regulation. As shown in Table 2, 77 genes show elevated expression in Dopaminergic neurons compared to other neuronal cell types.
NoradrenergicNeurons characterized by expression of DBH, TH, and SLC6A2, particularly associated with the locus coeruleus and involved in arousal, attention, and stress responses. As shown in Table 2, 113 genes show elevated expression in Noradrenergic neurons compared to other neuronal cell types.
SerotonergicNeurons characterized by expression of TPH2 and SLC6A4, primarily associated with brainstem raphe nuclei and involved in mood, sleep, and behavioral regulation. As shown in Table 2, 76 genes show elevated expression in Serotonergic neurons compared to other neuronal cell types.
MonoaminergicA group of neurons sharing molecular characteristics associated with monoamine synthesis and signaling, representing populations that cannot be clearly assigned to one specific monoaminergic subtype. As shown in Table 2, 181 genes show elevated expression in Monoaminergic neurons compared to other neuronal cell types. OrexinergicSpecialized hypothalamic neurons characterized by expression of HCRT, encoding orexin/hypocretin, which plays an essential role in maintaining wakefulness and regulating arousal and energy balance. As shown in Table 2, 119 genes show elevated expression in Orexinergic neurons compared to other neuronal cell types.
NeurosecretoryAs shown in Table 1, 233 genes show elevated expression in neurosecretory neurons as a group, compared to non-neuronal cells and other grouped neuronal populations. Neurosecretory neurons are specialized neurons that produce and release neurohormones, providing an important functional link between the nervous and endocrine systems. They are primarily associated with the hypothalamus, where populations producing hormones such as oxytocin (OXT), vasopressin (AVP), and corticotropin-releasing hormone (CRH) contribute to the regulation of water balance, stress responses, reproduction, and other physiological processes. These neurons exhibit distinct molecular expression profiles reflecting their specialized roles in hormonal regulation. AVP
Other neuronsVGLUT3 neuronsA distinct neuronal population characterized by expression of SLC17A8 (VGLUT3), often associated with PENK expression, exhibiting molecular characteristics that distinguish them from conventional excitatory and inhibitory populations. As shown in Table 2, 0 genes show elevated expression in VGLUT3 neurons compared to other neuronal cell types. Other neuronsA heterogeneous group of neuronal populations with distinct molecular identities that cannot be confidently assigned to the main excitatory, inhibitory, or specialized neurotransmitter categories. As shown in Table 2, 0 genes show elevated expression in Other neurons compared to other neuronal cell types. BackgroundSingle nuclei RNAseq dataSiletti K et al. (2023) published single nuclei RNA sequencing result, based on over 3 million cells from multiple brain regions, in Science magazine and created an interactive portal (The Human Brain Cell Atlas v1.0 ) available for single cell exploration across human gene expression in healthy brain cells. The Human Protein Atlas aims to generate a comprehensive resource representing the human body and its complexity, and with a need for better representation of the different cell types of the human brain, we imported the expression profiles re-processed the data by reclustering, naming and grouping cell clusters into main cell types. This way providing bar charts of pooled data for each cell type cluster and calculating the average normalized protein-coding transcripts per million). Some of the supercluster names were kept thanks to good correlation and overlap with the new cell cluster annotation. The expression profile of the different clusters is shown for each of the 11 different brain regions. More details, related to number of M reads and number of cells per brain region/UMAP can be found here. The published cerebral cortex data is represented by a larger number of cells, and we only included a random selection of 500 thousand cells. In total, expression data for 2526725 brain cells is displayed in the Brain single nuclei dataset, for browsing the gene expression and provide easy comparison to cell type expression in peripheral tissues. Exploring the original data inIn the Human Brain Cell Atlas v1.0 you can explore the specific clusters of cells and their expression in single cell level based on the original annotation and cell type grouping.
Relevant publications Uhlén M et al., Tissue-based map of the human proteome. Science (2015) |
The Human Protein Atlas project is funded
