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, 21 (9), 1185-1195

Transcriptomic and Morphophysiological Evidence for a Specialized Human Cortical GABAergic Cell Type


Transcriptomic and Morphophysiological Evidence for a Specialized Human Cortical GABAergic Cell Type

Eszter Boldog et al. Nat Neurosci.


We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1-SST-CALB2-PVALB-) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.

Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interest.


Figure 1
Figure 1. Identification of transcriptomic cell types in layer 1 of human temporal cortex.
A, Isolation of single nuclei from post-mortem adult human cortex for RNA-sequencing. Scale bars, left 1 cm, right, 20 µm. B, Left: Nuclei were grouped based on similar gene expression profiles using an automated iterative clustering procedure. Clustering was repeated 100 times on random subsets of 80% of nuclei. Right: Hierarchical clustering of nuclei that were consistently co-clustered across iterations identified 24 clusters. 16 clusters remained after removal of clusters associated with quality control metrics and merging of clusters that lacked at least one binary marker gene. C, 4 non-neuronal, 1 excitatory and 11 inhibitory neuron clusters were identified, although the excitatory cluster and one inhibitory cluster were likely in Layer 2 due to incidental capture superficial layer 2 with Layer 1 dissection. For each cluster, the constellation diagram shows the cell type class (based on canonical marker gene expression), relative frequency (disc area), and discreteness (line thickness proportional to the number of nuclei with ambiguous cluster membership) of clusters. D, Clusters arranged by transcriptomic similarity based on hierarchical clustering, with the expression distributions of selective marker genes shown across clusters as violin plots. Expression is on a linear scale and dots indicate median expression. Cluster sample sizes: i2 (n=77); i1 (n=90); i5 (n=47); i3 (n=56); i4 (n=54); i7 (n=31); i10 (n=16); i6 (n=44); i8 (n=27); i9 (n=22); i11 (n=6); e1 (n=299); g2 (n=27); g1 (n=48); g3 (n=18); g4 (n=9). E, ISH of select marker genes in human temporal cortex at low magnification (left columns with near adjacent Nissl stain for cytoarchitectonic laminar identification) and high magnification in layers 1-3 (right column). Red arrows highlight cells expressing genes in layer 1. Note that LHX6 marks a single cluster (i2) that is not expressed in layer 1 and therefore nuclei in this cluster were likely sampled from upper layer 2. Other clusters are restricted to layer 1 (e.g. NDNF+) or may be distributed across layers 1 and 2. Scale bars=250 µm (low mag), 100 µm (high mag). ISH experiments were conducted on multiple tissue donors as follows: SLC17A7, LHX6, CNR1, SEMA3C (n=3); CXCL14 (n=5); GAD1, CCK, RELN, NDNF (n=6); SST (n=7); PVALB (n=8); VIP (n=10).
Figure 2
Figure 2. Morphological phenotype of rosehip cells in layer 1 of the human cerebral cortex.
A1, A2, Anatomical reconstructions of RCs biocytin filled during whole cell recordings (somata and dendrites, burgundy; axons, red). B, Anatomical reconstructions of layer 2/3 BCs in the human cerebral cortex (somata and dendrites, black; axons, gray). C, Anatomical reconstructions of NGFCs in layer 1 of the human cerebral cortex (somata and dendrites, dark blue; axons, light blue). D, Left, Light micrographs of RCs (n=130) showing somata and proximal dendrites with stub-like spines (arrows). Right, Axons of RCs arborized densely with large, round boutons (top). Tortuous neurogliaform axons (n=16) posess very small boutons (middle). Axons of BCs (n=5) form longer segments with less convoluted branches with longer interbouton intervals (bottom). Scale bars: 10 µm. E, Quantitative comparison of axonal and dendritic parameters of rosehip (red, n=6), neurogliaform (blue, n=5) and basket (gray, n=5) cells. Top, bouton densities determined by Sholl analysis in 10 µm thick spherical shells were lower in BCs 30-50 µm and higher in NGFCs 70-220 µm and in BCs 130-220 µm from the soma compared to that of RCs. Bottom, Bouton volume (p<0.001)and the number of primary dendrites (p<0.04) of RCs were significantly different from that of NGFCs. Maximal vertical extent of axon (p<0.005), total dendritic length (p<0.031) and dendritic node frequency (/100 µm, p<0.009) of RCs differed significantly from that of BCs. Axonal tortuosity of RCs was similar in the two other cell types, however, the frequency of axonal branch points in RCs was 2.5 and 2.95 times that of neurogliaform (p<0.005) and BCs (p<0.005), respectively. Furthermore, interbouton interval, total axon length and maximal horizontal extent of the axon were also significantly different (two-sided Mann-Whitney U Test, * p ≤ 0.05; ** p ≤ 0.01; columns and error bars represent mean and standard deviation, respectively).
Figure 3
Figure 3. Molecular phenotype of rosehip cells in layer 1 of the human cerebral cortex.
A, Whole cell recorded and biocytin (red) filled RCs shows CCK (green; n=10) immunopositivity. All biocytin (red) labeled RCs tested for CB1 cannabinoid receptors (CNR1; n=11), somatostatin (SST; n=9), and calretinin (CALB2; n=2) were immuno-negative in spite of having labeled cells in the vicinity. Scale bars, 10 µm. B, Violin plots of gene expression for broad cell type and putative rosehip specific markers. Expression is on a linear scale and dots indicate median expression. Cluster sample sizes: i2 (n=77); i1 (n=90); i5 (n=47); i3 (n=56); i4 (n=54); i7 (n=31); i10 (n=16); i6 (n=44); i8 (n=27); i9 (n=22); i11 (n=6); Smad3 (n=12); Ndnf Car4 (n=24); Ndnf Cxcl14 (n=30); Igtp (n=10). Expression validated for select genes by immunohistochemistry (red stars), colorimetric ISH (black), multiplex FISH (orange), and single cell digital PCR (blue) in morphologically identified RCs. C, ISH of select marker genes in human temporal cortex (left) and mouse cortex (right). Red arrows highlight cells expressing genes in layer 1. Scale bars=250 µm (low mag), 100 um (high mag). ISH experiments were repeated on multiple human donors as follows: LAMP5 (n=2); EYA4, CPLX3 (n=3); SV2C (n=5). For mouse, ISH experiments were repeated on multiple specimens as follows: Lamp5, Sv2c, Cplx3 (n=2); Eya4 (n=3). D, Multiplex FISH validation of rosehip marker co-expression. Arrowheads and arrows show examples of RCs that are triple- and double-positive (i.e. CNR1-), respectively, for marker genes based on RNA-Seq expression data. Scale bar=25 µm. Multiplex FISH experiments were repeated on n=2 tissue donors. E, RCs comprise 10-15% of layer 1 interneurons based on multiplex FISH quantification of 408 GAD1+ cells in 2 subjects. 15% (+/- 3) of GAD1+ cells express the rosehip specific marker PDGFRA, although a small fraction of these cells may be oligodendrocyte precursor cells (see Suppl. Fig.5). 10% (+/-1) of GAD1+ cells express PDGFRA and a second rosehip marker TRPC3, although some RCs may lack TRPC3 expression based on RNA-seq. Error bars represent standard deviation. Cell counts were conducted on n=3 tissue sections from n=2 tissue donors. F, Expression of rosehip cluster markers in cytoplasm of whole cell recorded RCs. Quantified by single cell digital PCR and reported as a percentage of housekeeping gene (TBP) expression in n=9 cells (CNR1) or n=4 cells (CCK, CPLX3, NDNF, SV2C, TRPC3) per gene. Note that NDNF expression was not detected in any of the cells tested. Columns and error bars represent mean and standard deviation.
Figure 4
Figure 4. Intrinsic electrophysiological properties of rosehip cells.
A, Examples of different firing patterns induced by current injections in layer 1 interneurons. Firing pattern of a RC (top), a NGFC (middle) and an unidentified layer 1 interneuron (bottom). B, Support vector machine (SVM) based wrapper feature selection of electrophysiological parameters for the identification of RCs. Anatomically identified RCs (red dots) and other types of interneurons with known morphology (black dots) are mapped to the distribution of electrophysiological features ranked as the two best delineators by SVM. Black lines show the best hyperplane separating RCs from other interneuron types. C-D, RCs exhibit distinct impedance profile relative to neurogliaform and other human interneurons in layer 1. C, Individual responses of anatomically identified rosehip (red), neurogliaform (blue) and other (black) interneurons to current injections with an exponential chirp (0.2-200 Hz, top). Traces were normalized to the amplitude of the rosehip response at 200 Hz. D, Left, Normalized impedance (Z) profiles of distinct groups of interneurons. RCs (n=5) had higher impedance in the range of 0.9 - 12.4 Hz compared to neurogliaform (n=5) and other (n=5) interneurons. Shaded regions represent standard deviation. Right, Impedances were similar at the lowest frequency (Z0.2 Hz, left), but resonance magnitude (Q) calculated as maximal impedance value divided by the impedance at lowest frequency (middle) and frequencies of maximal impedance (fmax, right) showed significant differences (p<0.05, ANOVA with and Bonferroni post hoc correction). E, Automatized selection of recording periods for the assessment of subthreshold membrane potential oscillations (boxed segments) and detection of bursts (bars) for measuring intraburst spiking frequency demonstrated on a RC response to near rheobasic stimulation showing stuttering firing behavior. F, Averaged fast Fourier transforms (FFT) of membrane potential oscillations had higher power between 3.8 and 80 Hz in RCs compared to neurogliaform and other interneurons. G, Intraburst frequency of RCs peaked in the gamma range.
Figure 5
Figure 5. Connections of rosehip cells in the local microcircuit.
A, Distribution of local connections mapped in layers 1-3 between RCs (rh, red), pyramidal cells (pyr, green), NGFCs (ngf, blue) and other types of layer 1 interneuron (int, black) based on unbiased targeting of postsynaptic cells. RCs predominantly innervate pyramidal cells, receive monosynaptic EPSPs from layer 2-3 pyramidal cells, monosynaptic IPSPs from neurogliaform and other types of interneurons, however, IPSPs arriving from RCs were not encountered. In addition, RCs are interconnected by homologous electrical synapses (gap junctions). B, Example of a NGFC to RC connection. Left, Firing patterns of the presynaptic NGFC (blue) and the postsynaptic RC (red). Right, Anatomical reconstruction of the recorded NGFC (soma, dark blue; axon, light blue) and RC (soma and dendrites, burgundy; axon: red). Action potentials in the NGFC (blue) elicited slow IPSPs in the RC (red). C, Example of a pyramidal cell to RC connection. Left, Anatomical reconstruction and firing pattern of the presynaptic pyramidal cell (firing, soma and dendrites, green; axon, black) and the postsynaptic RC (firing, soma and dendrites, burgundy; axon, red). Right, action potentials in the pyramidal cell (green) elicited EPSPs in the RC (burgundy). D, Spatial distribution of coupled and uncoupled neurons tested as postsynaptic targets of RCs. Note the relative dominance of layer 2-3 pyramidal cells among neurons receiving input from RCs. E, The only RC to NGFC connection successfully tested for synaptic coupling. Left, Firing patterns of the presynaptic RC (burgundy) and the postsynaptic NGFC (blue). Middle, Anatomical reconstruction of the RC (soma and dendrites, burgundy; axon, red) and the NGFC (soma and dendrites, blue; axon not shown). Right, Action potentials in the RC (red) elicited slow IPSPs in the NGFC (blue). F, Example of RC to layer 3 pyramidal cell connections (n=16). Left, Firing patterns of the presynaptic RC (red) and the postsynaptic pyramidal cell (green). Action potentials in the RC (burgundy) elicited IPSPs in the pyramidal cell (green). Right, Confocal fluorescence image showing the recorded RC (rh) forming its axonal cloud in the tuft of the apical dendrite of the layer 2-3 pyramidal cell (pyr). G, Pharmacological characterization of a rosehip-to-pyramidal cell connection. Presynaptic spikes in the RC (red) elicited IPSPs in the layer 2-3 pyramidal cell (green) which could be blocked by application of gabazine (n=4, 10 µM). H, Functional test of presynaptic CNR1expression in RCs show the absence of modulation by the CNR1antagonist AM251 (n=4). Presynaptic spikes in the RC 1 (red, top) elicited IPSPs in the RC 2 (red, bottom). Application of AM251 (5 µM) had no effect on IPSPs (black). I, Representative electron microscopic images (left) and three-dimensional reconstructions (right, n=31) showing axon terminals (b, red) of biocytin filled RCs (n=3) targeting exclusively dendritic shafts (d, green) (100%, n=31). Synaptic clefts are indicated between arrowheads. Scale bars: 200 nm. J, Representative electron microscopic image (left) and three-dimensional reconstruction (right) of a biocytin filled RC bouton (b, red) targeting a pyramidal dendritic shaft (d, green) identified based on emerging dendritic spines (s, arrows). Scale bars: 500 nm. K, RCs form a network of electrical synapses. Top left, firing patterns of three RCs (red, rh1; orange, rh2; burgundy, rh3). Bottom left, Hyperpolarization of RC rh1 was reciprocally transmitted to RCs rh2 and rh3 confirming electrical coupling. Right, Route of the hyperpolarizing signals through putative dendro-dendritic gap junctions (arrows) between RCs rh1, rh2 and rh3 is shown by corresponding colors in the dendritic network of the three cells (gray).
Figure 6
Figure 6. Human rosehip interneurons perform segment specific regulation of action potential backpropagation to apical dendritic tufts of pyramidal cells.
A, Top, Firing patterns of a presynaptic RC (burgundy) and a postsynaptic pyramidal cell (green). Bottom, Action potentials in the RC (burgundy) elicited IPSPs in the pyramidal cell (green). B, Anatomical reconstruction of the RC (soma and dendrites: burgundy; axon, red) and the layer 2-3 pyramidal cell (soma and dendrites, green; axon not shown). Presynaptic axonal boutons of the RC formed close appositions (a, b, and c) with three separate branches on the tuft of the pyramidal apical dendrite. C, Repetitive burst firing was triggered to initiate backpropagating Ca2+ signals in the pyramidal cell (green) while the output of the RC (red) was switched on and off timed prior and during every second pyramidal burst. Simultaneously, Ca2+ dynamics of the pyramidal apical dendritic tuft was measured at several locations and signals detected at location no.1 shown on panels E and F are shown in black. D, The area boxed in panel B shows the dendritic branch of the apical tuft of the pyramidal cell (green) with a putative synaptic contact (a) arriving from the RC. E, Confocal Z-stack image of the same area shown on panel D taken during paired whole cell recordings. The soma of the RC (rh, red), the dendrite of the pyramidal cell (pyr, green), the putative synaptic contact (a) arriving from the RC to the pyramidal cell and sites of line scans performed across the dendrite (1, 2 and 3) are indicated. Cytoplasmic lipofuscin autofluorescence characteristic to human tissue is seen as green patches. The experiment was repeated independently with similar results in n=4 cell pairs. F, Superimposition of the anatomical reconstruction of panel D and the confocal image of panel E. G, Normalized amplitudes of Ca2+ signals during pyramidal cell firing with and without coactivation of the RC detected at the three sites of line scans (1, 2 and 3) on the pyramidal dendrite. Rosehip input simultaneous with the backpropagating pyramidal action potentials was significant (p=0.02) in suppressing Ca2+ signals only at site 1 which was closest (8 µm) to the putative synapse between the two cells, no effect (n=10 trials, p=1 and p=0.27, respectively, two-sided Wilcoxon-test) of the RC was detected at sites 2 and 3 located at distances of 21 and 28 µm, respectively from the putative synaptic contact.

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