Single-cell RNA-Seq characterization of anatomically identified OLM interneurons in different transgenic mouse lines

Abstract

Inhibitory GABAergic interneurons create different brain activity patterns that correlate with behavioural states. In this characterizing study, we used single-cell RNA-Seq to analyse anatomically- and electrophysiologically identified hippocampal oriens-lacunosum moleculare (OLM) interneurons. OLMs express somatostatin (Sst), generate feedback inhibition and play important roles in theta oscillations and fear encoding. Although an anatomically- and biophysically homogenous population, OLMs presumably comprise of two functionally distinct types with different developmental origins, inferred from the expression pattern of serotonin type-3a (5-HT3a, or Htr3a) receptor subunit and 5-HT excitability in a set of OLMs. To broadly characterize OLM cells, we used the Sst-Cre and the BAC transgenic Htr3a-Cre mouse lines and separately analysed SstCre-OLM and Htr3aCre-OLM types. We found a surprisingly consistent expression of Npy in OLMs, which was previously not associated with the identity of this type. Our analyses furthermore revealed uniform expression of developmental origin-related genes, including transcription factors and neurexin isoforms, without providing support for the current view that OLMs may originate from multiple neurogenic zones. Together, we found that OLMs constitute a highly homogenous transcriptomic population. Finally, our results revealed surprisingly infrequent expression of Htr3a in only ~10% of OLMs and an apparently specific expression of the 5-HT3b subunit-coding gene Htr3b in Htr3aCre-OLMs, but not in SstCre-OLMs. However, additional in situ hybridization experiments suggested that the differential expression of Htr3b may represent an unexpected consequence arising from the design of the Htr3a-Cre BAC transgenic line.

Keywords: Htr3a; OLM; interneuron; neurexin; single-cell RNA-Seq.

Figure 1

Combined anatomical, physiological and transcriptomic analysis of OLM interneurons. (a) In acute hippocampal brain slices, we identified OLM neurons based on tdTomato expression using the Htr3a‐Cre::Ai14 and Sst‐Cre::Ai14 transgenic lines. Single cells were electrophysiologically characterized, and their cytosolic mRNA was subsequently aspirated. Single‐cell RNA sequencing was performed after confirming OLM identity by post hoc visualization of axons and dendrites. (b₁‐b₂) Representative examples of Htr3aCre‐OLM (cell ID ‘H52’) and SstCre‐OLM neurons (‘Som138’), respectively

Figure 2

Anatomical characterization of OLM interneurons. (a,b) Neurolucida reconstruction of all analysed Htr3aCre‐ (in a) and SstCre‐OLM neurons (in b). The cell ID of each neuron is displayed next to the cell (Htr3aCre‐OLM neurons are labelled as H#, and SstCre‐OLM neurons are labelled as Som#). Each cell's dendrites and soma are shown in black, whereas the axon is shown in red. Scale bar is shown in panel a (bottom, right) and is applicable to every cell in panels a and b. In each drawing, cell layers are shown by dashed grey lines. The key for cell layers is shown in panel a (bottom, right) and is applicable to every drawing in panels a and b. (c) Sholl analysis of OLM dendritic segments. Two‐dimensional projections of cells H53 and Som15 are shown as examples with overlaid concentric spheres (seen as circles). The radius of each sphere is shown in the right. (d) Dendritic length (Length, measured in three dimensions, d₁), number of dendrites intersecting a sphere (Intersections, d₂), number of dendrites ending in a spheric shell (Endings, d₃) and the number of dendritic branching points (Nodes, d₄) are shown. In each plot and spheric shell, data points represent single cells, and statistical data are overlaid as box plots

Figure 3

Electrophysiological characterization of OLM interneurons. (a) Electrophysiological parameters measured from patch‐clamp recordings of OLM cells. (a₁) Schematic drawing shows a current transient in response to a voltage step that was used to quantify input resistance (applied voltage step divided by the measured steady‐state current) and capacitance (measured charge divided by the applied voltage step) of cells, and series resistance of patch pipettes (applied voltage step divided by the measured peak capacitive current). (a₂) Example trace shows a cell's voltage response to hyperpolarizing and depolarizing current pulses that were used to quantify resting membrane potential (RMP), firing rate, attenuation and amplitude of the sag potential. (a₃) Analysis of action potential (AP) trains elicited by depolarizing current pulses. For each cell, AP firing frequencies in response to defined current injection pulses were quantified and fitted with a sigmoid curve. The amplitude of the sigmoid was used to extrapolate maximal firing rate, whereas a linear fit on the sigmoid curve's 20–80% segment was used to extrapolate firing threshold. (a₄) Time‐averaged APs were used to determine peak AP amplitude, symmetry, base‐ and half‐widths. (a₅) Drawing depicts linear fits that were used to determine peak AP amplitude, symmetry, base‐ and half‐widths. First, the pre‐AP baseline was fit with a linear function, which intersected the AP trace at inflection point ‘a’. Then, a horizontal line (‘A’) was drawn through point ‘a’ to determine point ‘b’. Second, the 20–80% between peak AP amplitude (determined as maximal value throughout the AP trace) and intersections ‘a’ and ‘b’ were separately fit with one‐one linear function, characterizing the ascending and descending AP phase, respectively. The intersection point of these two linear fits was used to determine peak AP time. Third, a horizontal line (‘B’) was drawn through 50% of peak AP was used to determine points ‘c’ and ‘d’, which defined AP half‐width. Finally, a vertical line (‘C’) though the peak AP time was used to determine point ‘e’ and its relative distance between points ‘a’ and ‘b’ that was used to define a value for AP symmetry. (b) Box plots depict measured electrophysiological parameters for Htr3aCre‐OLM and SstCre‐OLM interneurons. Each plot is labelled on the left, and data points represent single cells. Statistical significance (p values) using t test comparison is shown on top. None of the statistical comparisons were below 0.05

Figure 4

Single‐cell RNA‐Seq profiling of OLM interneurons. (a) Single‐cell RNA sequencing quality parameters for SstCre‐OLM and Htr3aCre‐OLM neurons. Violin plots show sequencing and alignment parameters, including counts for reads, aligned reads, mapped reads, alignment rate, mapping rates and gene count. Each plot is labelled on the left, and data points represent single cells. None of the sequencing parameters were statistically different between SstCre‐OLM and Htr3aCre‐OLM neurons (the lowest adjusted p‐value was for the alignment rate, p = .187). (b) Heatmap shows single‐cell (left) and averaged (right) expression of key marker genes in SstCre‐OLM and Htr3aCre‐OLM types. Each column represents a single cell, for which cell ID is shown in the top. Averaged values are shown on the right. Circle's colour represents normalized gene expression levels (left scale bar), and size represents the proportion of cells in which the gene was detected within the cell type (applicable only to averaged values on the right, right scale bar). (c) Violin plots show Npy (neuropeptide Y) expression in SstCre‐OLM and Htr3aCre‐OLM cells. (d) Violin plots show 5‐HT3 subunit‐coding gene Htr3a, Htr3b and Chrna4 expression in Htr3aCre‐OLM and SstCre‐OLM cells. (e) Averaged expression level of MGE‐associated developmental markers was significantly higher than expression of CGE‐associated markers in both Htr3aCre‐OLM and SstCre‐OLM cells. (f) Regression analysis of CGE‐associated Htr3a, Prox1 and Sp8 and MGE‐associated Lhx6, Satb1 and Sox6 developmental markers reveals strong correlation between Htr3aCre‐OLM and SstCre‐OLM cells

Figure 5

Meta‐analysis of multiple transcriptomic cell populations. Violin plots show normalized gene expression levels for Htr3aCre‐OLMs, SstCre‐OLMs, cell populations of a recent transcriptional survey (Harris et al., 2018) as well as a control group of extracellular aspirations. Each column represents a single Sst, Pvalb, Cck, Vip or control group, and each row represents a single gene, grouped as interneuron markers, 5‐HT3 receptor subunits, developmental, astrocytic and glial marker genes. The number of single cells included for each cell population is indicated in parentheses

Figure 6

NPY peptide expression in OLM interneurons. (a–e) Electrophysiologically recorded and morphologically identified OLM neurons. During patch‐clamp recordings, tdTomato+ cells were filled with biocytin, and after recordings, brain slices containing the recorded cells were fixed and re‐sectioned into 60‐μm‐thick slices for immunochemical staining for biocytin and NPY. Left panels show morphological reconstructions after DAB conversion (scale bar: 100 μm), and inserts show the cells response to hyperpolarizing and depolarizing current pulses (horizontal scale: 0.5 s; vertical scale: 25 mV). Right panels show confocal images for biocytin, tdTomato, NPY and their merge (scale bar: 20 μm). OLM neurons in panels a, c and d were NPY+, whereas NPY content in OLMs in panels b and e could not be confirmed

Figure 7

In situ hybridization of Htr3b and Chrna4 in stratum oriens of hippocampal CA1. (a,b) Two confocal images show expression pattern of Htr3a/tdTomato (red), Htr3b (cyan) and Chrna4 (green) in the BAC transgenic Htr3a‐Cre::Ai14 mouse line (scale bar: 100 μm). Cells with high Htr3b but without tdTomato expression (‘Htr3b+ and tdTomato‐’) are labelled with asterisks. Inserts a₁‐a₂ and b₁‐b₂ show higher magnification images of presumed OLM cells in a and b, respectively (scale bar: 25 μm). In each insert, left image shows Htr3b and right image shows Chrna4 staining, overlaid on tdTomato. (c,d) Two confocal images show expression pattern of Sst/tdTomato (red), Htr3b (cyan) and Chrna4 (green) in the Sst‐Cre::Ai14 mouse line (scale bar: 100 μm). Inserts c₁‐c₂ and d₁‐d₂ show higher magnification images of presumed OLM cells in c and d, respectively (scale bar: 25 μm). In each insert, left image shows Htr3b and right image shows Chrna4 staining, overlaid on tdTomato. (e) Bar graph shows the percentage of presumed OLM cells co‐expressing Htr3b with tdTomato (grey for low expressing, L, and dark grey for high expressing, H) and Chrna4 (black) in Htr3a‐Cre::Ai14 and Sst‐Cre:Ai14 mice. (f) Left bar graph shows the percentage of all hippocampal cells co‐expressing Htr3b (H) with tdTomato in Htr3a‐Cre::Ai14 and Sst‐Cre::Ai14 mice. Right bar graph shows the total number of high Htr3b‐expressing, but tdTomato‐negative hippocampal cells

Figure 8

Neurexin isoform expression in OLM interneurons. (a) Exon structure of the Nrxn1 gene. Introns are shortened for clarity, and exons (in purple, numbered in the bottom) are shown proportional to their length. Alternatively spliced exon–exon junctions are shown in black; red lines represent alternative splice donor or acceptor sites. Alternative splice sites 1–6 (ASS1‐ASS6) are labelled on top. (b) Bar plots show side‐by‐side comparison of Nrxn1 alternative splicing between Htr3aCre‐OLM and SstCre‐OLM cells. Upward bars represent exon inclusion (‘spliced‐in’), and downward bars represent exon exclusion (‘spliced‐out’) in the final mRNA product. In case, for example ASS1, multiple alternatively spliced exons are present, bar plots represent total read counts. None of the paired comparisons revealed statistically significant difference (Welch's t test, p > .05). (c) Exon structure of the Nrxn3 gene. (d) Bar plots show side‐by‐side comparison of Nrxn3 alternative splicing between Htr3aCre‐OLM and SstCre‐OLM cells. None of the paired comparisons revealed statistically significant difference (Welch's t test, p > .05). (e) Regression analysis of Nrxn1 and Nrxn3 alternative splicing levels reveals strong correlation between OLM cells (where SstCre‐OLM and Htr3aCre‐OLM types are pooled together) and the mean MGE neurexin profiles as described in Lukacsovich et al. (2019). Insert shows regression analysis of neurexin isoforms between OLM and the mean CGE neurexin profiles

Figure 9

Transcriptional differences and similarities between Htr3aCre‐OLM and SstCre‐OLM neurons. (a) Volcano plot shows comparison of gene expression between Htr3aCre‐OLM and SstCre‐OLM cells. Yellow dots represent genes with at least fourfold difference expression and p less than .05. Genes that are enriched in Htr3a‐OLM cells appear in the left, whereas genes that are enriched in Sst‐OLM cells appear in the right. Labelled genes include 5‐HT3 subunits, transcription‐related genes, developmental origin‐associated genes, as well as genes with ligase, acetase, kinase activity and unknown function (Tmem229b). (b) Venn diagram showing the overlap of gene expression between Htr3aCre‐OLM and SstCre‐OLM cells. Less than 2% of the considered genes that belong to just one population were from SstCre‐OLMs. (c) Heatmap generated by a 2D Kernel density estimation (KDE), showing the expression rates of genes across the two cell types. White line: unity line; dashed line: LOWESS fit of distribution, showing a shift towards more consistent expression in Htr3aCre‐OLM cells. (d) We used a bootleg method to separate our data set into a train and test set 1,000 times, and evaluated the accuracy of a linear svm on classifying OLM cells into Htr3a‐OLM and Sst‐OLM types using between 2 and 500 genes. For each gene number, we show the average accuracy (blue line) plus or minus the standard deviation (pink shaded region). An average accuracy of above 50% indicates the existence of differences between the two types in a higher dimensional space. (e) PCA plot based on HVG (see Methods) in Htr3aCre‐OLM and SstCre‐OLM cells, showing that the two populations do not separate