CaMKIIα-Positive Interneurons Identified via a microRNA-Based Viral Gene Targeting Strategy

Abstract

Single-cell analysis is revealing increasing diversity in gene expression profiles among brain cells. Traditional promotor-based viral gene expression techniques, however, cannot capture the growing variety among single cells. We demonstrate a novel viral gene expression strategy to target cells with specific miRNA expression using miRNA-guided neuron tags (mAGNET). We designed mAGNET viral vectors containing a CaMKIIα promoter and microRNA-128 (miR-128) binding sites, and labeled CaMKIIα⁺ cells with naturally low expression of miR-128 (Lm128C cells) in male and female mice. Although CaMKIIα has traditionally been considered as an excitatory neuron marker, our single-cell sequencing results reveal that Lm128C cells are CaMKIIα⁺ inhibitory neurons of parvalbumin or somatostatin subtypes. Further evaluation of the physiological properties of Lm128C cell in brain slices showed that Lm128C cells exhibit elevated membrane excitability, with biophysical properties closely resembling those of fast-spiking interneurons, consistent with previous transcriptomic findings of miR-128 in regulating gene networks that govern membrane excitability. To further demonstrate the utility of this new viral expression strategy, we expressed GCaMP6f in Lm128C cells in the superficial layers of the motor cortex and performed in vivo calcium imaging in mice during locomotion. We found that Lm128C cells exhibit elevated calcium event rates and greater intrapopulation correlation than the overall CaMKIIα⁺ cells during movement. In summary, the miRNA-based viral gene targeting strategy described here allows us to label a sparse population of CaMKIIα⁺ interneurons for functional studies, providing new capabilities to investigate the relationship between gene expression and physiological properties in the brain.SIGNIFICANCE STATEMENT We report the discovery of a class of CaMKIIα⁺ cortical interneurons, labeled via a novel miRNA-based viral gene targeting strategy, combinatorial to traditional promoter-based strategies. The fact that we found a small, yet distinct, population of cortical inhibitory neurons that express CaMKIIα demonstrates that CaMKIIα is not as specific for excitatory neurons as commonly believed. As single-cell sequencing tools are providing increasing insights into the gene expression diversity of neurons, including miRNA profile data, we expect that the miRNA-based gene targeting strategy presented here can help delineate many neuron populations whose physiological properties can be readily related to the miRNA gene regulatory networks.

Keywords: GCaMP6; calcium/calmodulin kinase type II; cell type classification; optogenetics.

Figure 1.

Lm128C versus overall CaMKIIα neurons in mouse motor cortex. A, AAV-Lm128C-GCaMP6f-mAGNET and AAV-Lm128C-mRuby-mAGNET vector designs. Lm128C animals (n = 5) were coinjected with these two AAVs for both in vivo calcium activity recordings and static labeling of Lm128C cells. B, AAV-CaMKIIα-GCaMP6f-Control and AAV-Lm128C-mRuby-mAGNET vector designs. CaMKIIα animals (n = 5) were coinjected with these two AAVs for in vivo calcium activity recordings of overall CaMKIIα⁺ cells and static labeling of Lm128C cells. C, Schematic of stereotaxic virus injection site in mouse motor cortex. D, Representative confocal images of GCaMP6f fluorescence from Lm128C cells, immunofluorescence of a CaMKIIα antibody, and a merged overlay. E, Representative low-magnification (top) and high-magnification (bottom) confocal microscopy images of the virus injection site in a CaMKIIα mouse, showing GCaMP6f expression in overall CaMKIIα⁺ neurons, mRuby2 expression in Lm128C neurons, and a merged overlay. F, Same as E, for a Lm128C mouse. G, Quantification of labeled Lm128C cells. Percentage of GCaMP6f⁺ Lm128C cells that are also positive for CaMKIIα immunofluorescence, in Lm128C animals (left, gray); fraction of mRuby2⁺ Lm128C cells over the total GCaMP6f-expressing cells in CaMKIIα animals (middle, blue); percentage of GCaMP6f⁺ Lm128C cells that also express mRuby2 in Lm128C animals (right, red). Scatter plot shows individual animals (circles) and mean (horizontal bar) ± SD across animals (n = 5 mice each for each comparison, and at least 50 neurons were quantified per animal).

Figure 2.

Single-cell sequencing analysis of Lm128C cells. A, Heat map showing gene expression values for individual Lm128C cells in mouse motor cortex slices using the Patch-seq technique (n = 18). Color in top bar indicates the subclass that each cell maps to, and the corresponding dendrogram. Orange, SST; red, PV; green, glutamatergic (Glu). B, Representative electrophysiology traces from cells that map to SST, PV, and glutamatergic subclasses. One-second-long current injections to hyperpolarized potentials show passive properties, while spike train properties are shown with injections for each cell 60 pA above rheobase. C, Box plots of population electrophysiology responses. Top, FI gain, calculated as the change in firing frequency from lowest firing rate to highest firing rate, divided by the current injected. Middle, Rate of spike decay, calculated as the time from peak voltage to the minimum value of the first derivative of voltage. Bottom, Spike half-width, calculated as the time between the two midpoints of the spike waveforms. Individual data points for each cell are overlaid, with color corresponding to the same mapping as in A and B, with gray indicating cells without successful transcriptomic processing.

Figure 3.

Distinct electrophysiological properties of Lm128C and overall CaMKIIα neurons. A, Representative voltage responses (top) of a CaMKIIα neuron (blue) and an Lm128C neuron (orange) to stepped current injections (bottom). Bi, FI for each neuron recorded (n = 5 each). Bii, FI gain (***p = 0.001). Ci, Representative average spike shape of a CaMKIIα neuron (blue) and an Lm128C neuron (orange). Dots indicate spike threshold, and spike waveform midpoint used for calculating spike half-width. Cii, Rate of spike decay (***p = 0.0009). Ciii, Spike half-width (***p = 0.0006). All p values were calculated using two-tailed unpaired Student's t test.

Figure 4.

Electrophysiological properties of Lm128C interneuron subtypes. A, Representative voltage responses (top) of a PV⁺ interneuron (navy), an Lm128C neuron positive for PV (Lm128C/PV⁺, orange), and an Lm128C neuron negative for PV (Lm128C/PV^–, yellow), to stepped current injections (bottom). Bi, FI for each neuron recorded (n = 10 neurons for PV⁺, n = 9 for LM128C/PV⁺, and n = 7 for Lm128C/PV^–). Bii, FI gain for the three neuron groups, calculated as the change in firing frequency from the lowest firing rate to the highest firing rate divided by the amount of current injected. C, Sag for the three neuron groups. D, Adaptation ratio for the three neuron groups (*p = 0.03). Ei, Representative average spike shapes of the three neuron populations. Eii, Spike half-width, calculated as the time between the two midpoints of the spike waveforms. Eiii, Rate of decay, the time from peak voltage to the minimum value of the first derivative of voltage. p Values were calculated using two-tailed unpaired Student's t test, NS, not significant.

Figure 5.

In vivo calcium imaging of overall CaMKIIα and Lm128C neurons in motor cortex. A, Schematic of an imaging window and a head bar implanted on the mouse head. B, In vivo calcium imaging paradigm: animals were awake and head fixed under the imaging microscope during voluntary locomotion on a spherical treadmill. Two USB motion sensors were used for movement tracking. C, Custom GCaMP6f imaging setup with an sCMOS camera. D, Experimental timeline for both the CaMKIIα and Lm128C mice groups. E, Representative wide-field images from in vivo calcium imaging recordings (maximum–mean pixel intensity across all frames) for a CaMKIIα mouse (left) and an Lm128C mouse (right). Top, GCaMP6f fluorescence. Bottom, GCaMP6f fluorescence overlaid with manually identified ROIs corresponding to individual GCaMP6f-expressing neurons. F, Representative calcium traces (top) and waveforms (bottom) from a CaMKIIα neuron (left) and an Lm128C neuron (right). Black traces (bottom) represent the average event waveform, and the red line indicates the average activation threshold. G–J, Calcium event rate (G), event rise time (H), and fall time (I), and intrapopulation pairwise correlation for the overall CaMKIIα population and Lm128C neurons (J). Bar plots represent the mean ± SD across recording sessions (n = 10 recording sessions for each group, 2 recording sessions/mouse; n = 5 mice/group), p values were determined via a two-tailed unpaired Student's t test.

Figure 6.

Differential modulation of calcium activity by movement speed in the overall CaMKIIα population and Lm128C neurons in the motor cortex. A1, A2, Representative examples of mouse movement speed and corresponding calcium traces during a recording session, for a CaMKIIα mouse (A1) and a Lm128C mouse (A2). Periods of high-speed movement are highlighted in red, and periods of low-speed movement are highlighted in green. B–D, Event rate (events per minute; B), event size (area under the curve; C), and intrapopulation pairwise correlation (D) during periods of high-speed and low-speed movement for the overall CaMKIIα population and Lm128C neurons. Bar plots present the mean ± SE (CaMKIIα: n = 1850 neurons for B and D, n = 1659 neurons for C, Lm128C: n = 330 neurons for B and D, n = 322 neurons for C; n = 5 mice for CaMKIIα group, and n = 5 mice for Lm128C group; two-tailed paired Student's t test), NS, not significant.