Architecture of a mammalian glomerular domain revealed by novel volume electroporation using nanoengineered microelectrodes

Abstract

Dense microcircuit reconstruction techniques have begun to provide ultrafine insight into the architecture of small-scale networks. However, identifying the totality of cells belonging to such neuronal modules, the "inputs" and "outputs," remains a major challenge. Here, we present the development of nanoengineered electroporation microelectrodes (NEMs) for comprehensive manipulation of a substantial volume of neuronal tissue. Combining finite element modeling and focused ion beam milling, NEMs permit substantially higher stimulation intensities compared to conventional glass capillaries, allowing for larger volumes configurable to the geometry of the target circuit. We apply NEMs to achieve near-complete labeling of the neuronal network associated with a genetically identified olfactory glomerulus. This allows us to detect sparse higher-order features of the wiring architecture that are inaccessible to statistical labeling approaches. Thus, NEM labeling provides crucial complementary information to dense circuit reconstruction techniques. Relying solely on targeting an electrode to the region of interest and passive biophysical properties largely common across cell types, this can easily be employed anywhere in the CNS.

Fig. 1

Effectiveness of standard glass microelectrode electroporation can be predicted by FEM but is restricted in practice by physical limitations. a 3D-FEM model showing the center cut of a standard glass micropipette. The figure illustrates the volume where effective electroporation (transmembrane potential >200 mV) can occur at 1 µA (V_m = transmembrane potential). Inset depicting a twofold magnification of the effective electroporation zone close to the pipette tip. Scale bar = 1 µm. b Plot of corresponding voltage drop along the first micrometer of the central axis of the pipette at 1 µA. Black rings indicate individual elements along the central axis of the pipette (assumed electroporation threshold of 200 mV marked by the dashed red horizontal line; resulting critical distance of 0.285 µm indicated by the dashed red vertical line). c Center cut of the 3D-FEM model employing a standard glass micropipette at 100 µA, illustrating the volume where effective electroporation (transmembrane potential >200 mV) can occur. Scale bar = 5 µm. d Corresponding voltage drop along the first 40 µm of the central axis of the pipette at 100 µA. Black rings indicate individual elements along the central axis of the pipette. Electroporation threshold and critical distance (~22 µm) as in b. e When increasing stimulus intensities beyond 30–40 µA, a jet-like convection movement and gas bubble (black arrow) formation appear, as seen here in an exemplary camera frame under the x20 objective. Scale bar = 20 µm. f Current threshold values (µA) for the jet (red) and gas bubble (blue) phenomenon plotted against tip radius (µm). Dashed lines indicating a linear fit for both (R² = 0.58 for jet, red and R² = 0.74 for bubble, blue)

Fig. 2

Nanoengineered electroporation microelectrodes (NEMs) allow for improved current distribution and electroporation effectiveness by reducing peak potential regions. a Scheme showing the current divider-based electric circuit model with five divisions (“levels”) along a central axis, serving as an equivalent electric circuit model of the newly designed pipette. For simplicity of the scheme, exterior resistances are neglected. b Maximum current density (µA/µm²) of a standard pipette (blue) and an NEM (red) plotted against applied stimulation current (µA). The graph shows that current density of the standard pipette rises twice as quickly when compared to the NEM. c Cross-section of the 3D-FEM model illustrating the total effective electroporation volume and its distribution around the pipette tip at 50 µA employing the NEM (V_m = transmembrane potential). Scale bar = 5 µm. d Cumulated volume of elements for a standard glass pipette (blue) and an NEM (red) beyond a given potential at 50 µA (X-axis, (V) in steps of 0.01 V). e After pulling long-tapered, patch clamp-like pipettes (i), pipettes were successively coated with a thin gold layer (ii) and conductive silver paint (iii). This was necessary to provide efficient grounding of the surface for FIB-assisted milling (iv). f Inside camera view of the vacuum chamber for SEM imaging and FIB-assisted milling. Glass pipettes are mounted and aligned at an angle of 90° relative to the principal axis of the FIB. Gallium ion gun shown in the right upper corner, electron beam mounted vertically in the central portion of the chamber. Scale bar = 5 mm. g Example of an NEM after successful insertion of the five-level hole design, as seen in high-resolution FIB imaging mode. Scale bar = 2 µm

Fig. 3

In vivo electroporation of glomerular neurons using NEMs. a Experimental strategy: In order to probe reliability and comprehensiveness of the method, two differently colored dyes were sequentially and independently electroporated into the same glomerulus. First, electroporation with TMR-dextran. Retraction of first electrode and insertion of the second pipette, after an interval of ~15 min. Second electroporation with fluorescein-dextran. The image panel shows a typical result in the region of interest, as seen by CLSM. b DAPI staining revealing the layered structure of the region of interest. c TMR dye fluorescence. d Fluorescein dye fluorescence, note GFP-marked olfactory sensory neuron axons innervating the MOR174–9 glomerulus. e Overlay of the three channels, note parallel staining of vascular structures (white arrows). Scale bars = 100 µm. f Individual data plot of double-labeling experiments (Expts. A–C, n = 3). For each experiment, the left bar shows the number of TMR-positive cells and the right bar the number of fluorescein-positive cells. Each bar is subdivided into doubly labeled fraction of cells (brown) and singly labeled fraction (red for TMR and green for fluorescein). In g the fraction of doubly labeled, fluorescein-positive cells is shown for different layers. Blue bar and circle indicate mean ± s.d

Fig. 4

Gross cellular analysis of the MOR174–9 domain. a Typical result of the experimental approach. Left image showing the GFP-positive MOR174–9 glomerulus in a horizontal slice of the olfactory bulb. Right image showing TMR-dextran fluorescence after successful electroporation. Scale bars = 100 µm. Blow-up: overlay of the two channels with marked cell bodies (yellow). Scale bar = 100 µm. b In total, a median of 162 (Q1 +25.75; Q3 −26.75) cells (n = 5 animals) projecting to the electroporated MOR174–9 glomerulus is found. Colored triangles indicate the total number of cells for each individual experiment. c Variability of percentile borders (i.e., distance to the center ± s.d. (µm)) across all experiments as a function of decile. d Relative Euclidean distance distribution of the cells associated with the “standard” MOR174–9 glomerulus (red vertical bars, bin size of 10 µm). Red bars refer to the left Y-axis and show the fraction of cells within each bin and the corresponding SDs among the five experiments (dark blue bars). Dashed black line represents the cumulative plot of the histogram and refers to the right Y-axis. e Illustration indicating the four layers of the bulb, which are considered separately: GL (white), outer quarter of the EPL (magenta), inner three quarters of the EPL (yellow), and MCL (red-cyan). For completeness, olfactory nerve layer (ONL), IPL, and main cell types are also shown. PG = periglomerular cell, eTC = external tufted cell, TC = tufted cell, MC = Mitral cell. f Scatter plot of the distances of all cells (Y-axis) to the glomerular center separated by layer identity of the cell soma localization (X-axis). Same color code of the dots as in b. Numbers in the dashed bubbles indicate mean number (Ø) of cells of each layer per glomerulus. g Pie chart illustrating the average relative cellular composition of the MOR174–9 glomerulus, according to layer identity

Fig. 5

Morphological classification of projection neurons in the mitral cell layer. a X–z-plane projection of all mitral cell layer projection neurons (MCLPNs, n = 45). X-axis approximately corresponding to posterior (P) → anterior (A) and Z-axis to dorsal (D) → ventral (V) direction. Triangles represent MCLPN midpoints of the five experiments. b Overlay of preferential MCLPN locations indicated as ellipsoid body around the respective midpoint based on SD of distances (n = 5). Magenta square indicates common MCLPN midpoint and dashed line denotes SD of the distances of all cells. c Based on two principal morphological parameters, MCLPNs were analyzed by fitting a two-component GMM. Probability density function (PDF) shown as discretized mesh (coloring as indicated). Black dots indicate individual cells, red dots their projections onto the 2D plane. Colored PDF isolines according to color bar. Histograms show frequencies of individual dimensions. d Consecutive assignment of cells to one of the two clusters (triangles: Cluster 1, MC or squares: Cluster 2, dTC). Coloring indicates posterior probability of each cell to be part of the MC cluster. Dashed line shows cutoff value of 200 µm² soma area, the assumed lower limit for typical MCs. Cells of the MC group having a smaller soma size are individually marked by green dots. e Cell ranking according to “cluster membership score,” derived from posterior probabilities. f Cell numbers of the two subsets MC and dTC (n = 5, mean ± s.d.). g Comparison of x- and z-spreads per experiment between MCs and dTCs (two-sample t test, *p < 0.05, n.s. not significant). Mean ± s.d. indicated by blue circle and bar. h Ellipsoid body based on SDs of distances to common MC midpoint. The smaller inner ellipsoid (blue) around the common MC midpoint (red circle) is based on SD's of the distances from individual MC midpoints to the common MC midpoint. i Same for dTCs. Green circle illustrates the position of the glomerulus at the origin. Color code for data from different experiments as in Fig. 4b

Fig. 6

Different types of projection neurons form segregated dendritic bands within the EPL. a In the horizontal histogram on the left, average number of neurons per glomerulus of the three separately analyzed TC subregions plotted along the deep-to-superficial axis from the inner edge of the MCL (0) to the GL-EPL-border (1) in bins of 0.025. Bar color represents subgroup of TC (magenta = sTC, yellow = mTC, cyan = dTC). Blue bars indicate ± s.d., dashed blue line adds a line representation. Right, examples of sTCs and mTCs in horizontal partial volume sections of a CLSM image stack. Outline of cellular branching pattern of these cells colored in magenta and yellow. White lines indicate GL-EPL border (top) and MCL (bottom), respectively. Scale bars = 100 µm. Orientation as indicated (A = anterior, P = posterior, L=  lateral, M = medial). b Exemplary whole dendritic reconstruction of all labeled MOR174–9 projection neurons of one experiment. The local dendritic projectome shows a strict laminar pattern when neurons are separated by cellular identity (magenta = sTC, yellow = mTC, cyan = dTC, and red = MC dendrites). Dashed green circle represents the electroporated glomerulus. IPL = internal plexiform layer, GCL = granule cell layer. Scale bar = 100 µm. Orientation as indicated (V = ventral, D = dorsal, L = lateral, M = medial). c–f Histogram plots showing the average distribution of lateral dendrites within the EPL from the four cell types, in bins of 0.05 from the MCL (0) to the GL-EPL border (1) (c = sTC, d = mTC, e = dTC and f = MC). Black vertical bars indicate ± s.d. Overall mean dendritic position for each cell type is shown ± s.d. (number and colored point and horizontal bar above each histogram). g Average relative dendrite contribution from the four cell types (magenta = sTC, yellow = mTC, cyan = dTC, red = MC) per subregion of the normalized EPL width in bins of 0.05. Right Y-axis showing the contributions as absolute distance from MCL within a typical EPL width of 180 µm

Fig. 7

Tufted cell axons project specifically to MCLPN soma locations. a Axonal projections from superficially located TCs traverse the EPL in a column-like arrangement towards the IPL, next to the cluster of the MCLPNs of the same glomerular domain. The same color code is used as in Fig. 6b, indicating different cell types. Red and cyan spheres show the soma positions of MCs and dTCs, respectively. No recurrent axon collaterals from MCs and only minor contributions from dTCs can be found. By far, most axonal branches arise from sTCs. Scale bar = 100 µm. b Example of a 3D-rendered MC (red) and two corresponding sTCs (magenta), which project their axons (“ax”) specifically to the IPL adjacent to the position of the MC, where they support a local collateral mesh. Inset box showing a maximum projection view of the local IPL (depth = 60 µm) next to the MC from a CLSM image stack. Scale bar = 50 µm. c Local axonal IPL mask with positions of the corresponding MCLPNs of the same experiment shown as an overlay (transparent green circles indicating MCs, transparent green triangles marking dTCs). Color bar representing average pixel values ranging from 0 to 1. d Overlay of MCLPN positions form the other two experiments indicated as an intrinsic “shuffled” control (transparent red circles indicating MCs, transparent red triangles marking dTCs). Scale bars = 100 µm. e Normalized pixel counts of all MCLPNs of the three experiments (“matching,” = green lining, MCs = red filled circles, dTCs = cyan filled triangles) are compared to counts of non-matching MCLPN positions from the other two experiments (“control,” = red lining). f, g Same comparison shown for MCs and dTCs individually. Mean values ± s.d. shown in blue. Two-sample t test, **p < 0.01, ***p < 0.001