Article Figures & Data
Figures
- Figure 1.
OB projection neurons in the ML include differently sized and shaped cells. A, An example image of the OB outer layers. Excitatory projection neurons labeled with antibodies against neurofilament marker protein (SMI-32, white) span the EPL and the ML. Dopaminergic interneurons stained with antibodies against TH (cyan) indicate the location of the GL, adjacent to which are the SMI-32 positive somas of the excitatory interneurons external TCs (ETCs). The soma of three representative projection neurons across EPL and ML has been manually traced in dashed magenta to calculate the maximum diameter and area. B, Correlation of the soma area and maximum diameter for OB projection neurons located in the lower EPL (green, n = 137) or ML (cyan, n = 1,671). Blue circles represent confirmed MCs from the Lbhd2-CreERT2 transgenic mouse line, meta-analyzed from Koldaeva et al. 2021 (n = 23). The canonical diameter and area dividers between MCs and TCs are indicated on the axes. C, Frequency distribution of maximum diameters for OB projection neurons located in the lower EPL (green, n = 137), ML (cyan, n = 1,671), and confirmed MCs from the Lbhd2-CreERT2 transgenic mouse line. D, Soma roundness [(4 * area / (π * major_axis2)] of EPL TCs (green) and confirmed MCs (blue) compared with ML cells (cyan) when split by the 20 µm max diameter classifier proposed in the literature. Circles are individual cells; lines are mean ± SEM; ***p < 0.001; ns, not significant.
- Figure 2.
The soma size and passive electrical properties differ between OB projection neurons. A, Schematic representation of the location of OB projection neurons targeted for whole-cell patch–clamp recordings in acute horizontal mouse brain slices. B, Two cells in the ML, a putative tufted (pTC) and a putative mitral cell (pMC) patched with biocytin-supplemented intracellular solution and postfixed for morphological analysis. C, Unbiased k-means analysis of the soma size of patched cells in the ML returns two clusters separable by a 45 pF capacitance classifier. D–E, Membrane capacitance (Cm) and input resistance (Ri) in eplTC (n = 21) and ML's pTC (n = 28) and pMC (n = 42) classed using the 45 pF divider. Circles are individual cells; lines are mean ± SEM; *p < 0.05; ***p < 0.001. GL, glomerular layer; EPL, external plexiform layer; GRL, granule cell layer.
- Figure 3.
The depolarization response to hyperpolarization (sag potential) is extremely variable among OB projection neurons. A, Schematic visualization of the voltage sag analysis parameters and formulas used to calculate sag amplitude and index. B, Example traces of the voltage sag response to hyperpolarizing current injections in eplTC (green, n = 10), pTCs (cyan, n = 13), and pMC (blue, n = 28). Note the variability in pTCs. C–F, Peak amplitude, steady-state voltage, sag amplitude, and sag index in the three classes of OB projection neurons. Circles are individual cells; lines are mean ± SEM; further quantification and statistical analysis in Table 1.
- Figure 4.
AP threshold and waveforms differ between OB projection neurons. A, Example traces of the membrane voltage response to the minimum depolarizing 10-ms-long current injection needed to evoke an AP in eplTCs (green, n = 13), pTCs (cyan, n = 13), and pMCs (blue, n = 27). Waveform parameters for the three OB projection neurons subtypes include (B) injected current density needed to evoke an AP, (C) membrane potential at which the AP was evoked, (D) maximum voltage reached by the AP, (E) AP peak amplitude, (F) AP width at half the maximum height, (G) minimum voltage reached by the AP, and (H) peak amplitude of the AP AHP. Circles are individual cells, lines are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; further quantification and statistical analysis in Table 1.
- Figure 5.
EPL cells have similarly long but more distal axon initial segments than ML neurons. A, Example maximum intensity projection images of eplTC, pTC, and pMC neurons visualized via SMI-32 immunolabel (white) with an identified AnkG-positive AIS (magenta, arrows). The solid line indicates the emergence of the axonal process from the soma (asterisk). EPL, external plexiform layer; ML, mitral layer. B, Mean AIS (magenta) start and end position for each group. C, D, AIS distance from soma and length in eplTCs (green, n = 23); pTCs (cyan, n = 21); and pMCs (blue, n = 34). Circles represent individual cells, different color shades represent different mice; orange border indicates AIS originating from dendrite; lines are mean ± SEM; **p < 0.01; ***p < 0.001.
- Figure 6.
Comparable repetitive AP firing among OB projection neurons. A, Example traces of the membrane voltage response to 500 ms depolarizing current injections at 300 pA, rheobase, and max AP firing in eplTCs (green, n = 15); pTCs (cyan, n = 11); and pMCs (blue, n = 27). B–D, Raw input–output plots showing the number of APs fired at each current injection in individual neurons. E, Mean number of APs and SEM at each current density (i.e., injected current normalized for cell capacitance) in eplTCs (green), pTCs (cyan), and pMCs (blue). Repetitive firing parameters include (F) rheobase, (G) slope of the number of APs versus current density input–output curves, (H) maximum AP firing frequency, (I) number of APs at threshold (light shades) and at maximum firing (dark shades), (J) latency of the first AP at the current injection level where the max AP number was fired, (I, J) CV of the ISIs (CV) and of adjacent ISIs (CV2; see inset for graphical description) at the current injection level where the max AP number was fired. M, Input–output curve with the 500 ms current injection divided into five 100 ms bins (see inset). N–O, Correlation of sag amplitude with firing properties. Circles and thin lines are individual cells; thick black lines are mean ± SEM; *p < 0.05; **p < 0.01; further quantification and statistical analysis in Table 1.
- Figure 7.
PCA of firing properties fails to reveal clear clustering of OB projection neurons. A, PC score plot for eplTC (green, n = 9) and ML's pTC (cyan, n = 8) and pMC (blue, n = 20) based on passive properties and all measurements obtained from AP firing recordings (Figs. 2, 4, 6; Table 1). Each circle represents a cell plotted against its primary and secondary PC scores. B, Individual (circles) and cumulative (bars) proportion of variance explained by each PC. C, Loading scores for all variables showing their respective contributions to PC1 (light gray) and PC2 (dark gray). See also Extended Data Figure 7-1.
Tables
Intrinsic electrophysiological properties eplTC mean ±
SEM, (n)pTC mean ±
SEM, (n)pMC mean ±
SEM, (n)ANOVA/Kruskal–
Wallis p valueTukey's/Dunn's multiple comparisons p value eplTC
versus
pTCeplTC
versus
pMCpTC
versus
pMCSag potentials properties Peak amplitude (mV) 71.97 ± 7.24 [10] 88.7 ± 15.82 [13] 67.27 ± 5.63 [28] A, 0.24 (F = 1.46) ns ns ns Steady state (mV) −121.3 ± 6.5 [10] −109 ± 8.39 [13] −115.5 ± 4.89 [28] KW, 0.52 ns ns ns Sag amplitude (mV) 11.20 ± 3.43 [10] 39.28 ± 17.80 [13] 11.53 ± 3.80 [28] KW, 0.63 ns ns ns Sag Index 0.86 ± 0.05 [10] 0.74 ± 0.09 [13] 0.86 ± 0.03 [28] KW, 0.74 ns ns ns APS properties Current threshold (pA/pF) 9.63 ± 1.64 [13] 5.46 ± 0.87 [13] 3.06 ± 0.37 [27] KW, <0.001 ns *** * Voltage threshold (mV) −30.22 ± 2.18 [13] −37.26 ± 2.76 [13] −43.14 ± 1.14 [27] A, <0.001 (F = 13.14) ns *** ns Maximum voltage (mV) 10.48 ± 2.01 [13] 14.89 ± 2.24 [13] 14.10 ± 1.76 [27] KW, 0.40 ns ns ns Peak amplitude (mV) 40.17 ± 1.83 [13] 58.56 ± 4.60 [13] 58.29 ± 2.06 [27] KW, <0.001 ** *** ns WHH (ms) 0.80 ± 0.08 [13] 0.93 ± 0.06 [13] 0.83 ± 0.03 [27] KW, 0.25 ns ns ns Minimum voltage (mV) −46.22 ± 2.05 [15] −54.03 ± 2.30 [11] −56.26 ± 1.87 [27] A, 0.003 (F = 6.41) ns ** ns AHP on amplitude (mV) 17.13 ± 1.57[15] 12.61 ± 1.18 [11] 16.59 ± 1.10 [27] A, 0.08 (F = 2.58) ns ns ns Repetitive firing properties Rheobase (pA/pF) 3.03 ± 0.69 [14] 1.59 ± 0.29 [11] 1.82 ± 0.31 [28] A, 0.08 (F = 2.58) ns ns ns Slope I/O curve (Hz/pA/pF) 7.98 ± 1.38 [13] 6.33 ± 1.24 [11] 7.21 ± 1.01 [26] A, 0.72 (F = 0.34) ns ns ns First AP delay (ms) 109.8 ± 20.6 [15] 169.5 ± 42.1 [11] 161.1 ± 29.1 [27] KW, 0.78 ns ns ns n APs at rheobase 8.14 ± 2.95 [14] 5.36 ± 1.22 [11] 8.64 ± 2.56 [28] KW, 0.86 ns ns ns Max number of APs 29.87 ± 4.76 [15] 22.27 ± 3.65 [11] 25.321 ± 2.67 [28] KW, 0.67 ns ns ns Max frequency (Hz) 110.9 ± 28.1 [15] 131.2 ± 25.4 [11] 80.1 ± 7.3 [28] KW, 0.19 ns ns ns ISI CV at max firing 0.19 ± 0.03 [14] 0.47 ± 0.16 [11] 0.19 ± 0.02 [27] KW, 0.20 ns ns ns ISI CV2 at max firing 0.10 ± 0.13 [14] 0.27 ± 0.10 [11] 0.11 ± 0.01 [27] KW, 0.33 ns ns ns Mean values ± SEM and number of cells (n) of sag currents, AP properties, and repetitive firing properties for eplTCs and ML's pTCs and pMC. Statistical differences between cell types were calculated independently with a one-way ANOVA with Tukey's post hoc correction for normally distributed data (A) or with a Kruskal–Wallis test with Dunn's post hoc correction for non-normally distributed data (KW). Individual data points and example traces are presented in Figures 3, 4, and 6.
Fig 7-1
Animal age impact on PCA of firing properties. As in figure 7A, principal component (PC) score plot for principal projection neurons based on passive properties and all measurements obtained from cells with stable passive properties and AP firing recordings (Figures 2-4-6, Table 1). Each circle represents a cell plotted against its primary and secondary PC scores and it has been colour-coded to indicate the animal age, ranging from P20 (grey) to P68 (purple). Download Fig 7-1, TIF file.
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