Article Figures & Data
Figures
- Figure 1.
Laminar distribution patterns of GFP+ interneurons. GFP expression was visualized by anti-GFP immunocytochemistry in 40-μm-thick coronal sections from brains of 2- to 3-month-old animals. A–C, Low-power images. D–F, High-power images from different sections of the same brains. Histograms at the left margin of A–C show the laminar distribution of GFP+ neurons, counted in 50 μm bins in vertical strips through the barrel cortex. Bin heights in the three panels are to the same scale; the highest bin in A represents 30 counts. Note the nearly complementary distribution patterns of the three lines. The laminar boundaries indicated in C apply also to A and B; the dotted lines indicate the white matter border. D–F are not aligned by layers. Scale bar: A–C, 250 μm; D–F, 100 μm.
- Figure 2.
Neurochemical identity of GFP+ interneurons. Confocal images of parasagittal sections from X94 (A, C, E, G) and X98 (B, D, F, H) barrel cortex are shown. GFP fluorescence is pseudocolored green; anti-SOM (A, B), anti-PV (C, D), anti-CB (E, F), and anti-NPY (G, H) immunoreactivity is pseudocolored red. Note that in both lines, all GFP+ neurons were SOM+ and PV−, but only X98 cells were CB+ (all) and NPY+ (some). The yellow seen in C represents overlap in the z-dimension between red-labeled SOM+ cell bodies and green-labeled GFP+ processes, not colocalization of the two labels.
- Figure 3.
Comparison of axonal projections to layer 1. A–C, Digitally inverted confocal image stacks showing GFP-containing cell bodies and processes in the upper cortical layers of each line. Note the dense band of fluorescent fibers in layer 1 of X98 and GIN, but not X94, cortex.
- Figure 4.
Morphological reconstructions of representative GFP+ neurons. A–L, Neurons were reconstructed in three dimensions using Neurolucida; cell bodies and dendrites are shown in green, axons in red. For ease of comparison, individual drawings were normalized to the same width of layer 4; average width of layer 4 was 240 ± 7.5 μm (mean ± SEM). The arrowheads in B–D point to a turning point of the axon, from the upper layers back to layer 4.
- Figure 5.
Suprathreshold and subthreshold responses of GFP+ neurons. A, Spike trains in response to current steps of increasing amplitudes. Aa–Ac, Ad–Af, and Ag–Ai are responses to high, medium, and low current levels, respectively; the three current steps for each neuron are shown superimposed in Aj–Al. The asterisks in Ad denote interruptions in firing characteristic of stuttering X94 neurons; some GIN neurons also stuttered, but X98 neurons never did. The insets in Ag–Ai are the first action potential from the corresponding trace, shown at half the vertical scale and at a 100-fold expanded horizontal scale; note the pronounced difference in spike widths between lines. The arrowheads in Ah and Ai point to the characteristic triphasic AHP in X98 and GIN neurons. Calibration: 40 mV (Aa–Ai), 1000 pA (Aj–Al), 200 ms. B, Superimposed voltage responses (Ba–Bc) to the hyperpolarizing and small depolarizing current steps shown in Bd–Bf, in three other neurons. Note the very low input resistance of the X94 neuron compared with that of the X98 and GIN neurons (much larger current steps required to elicit similar voltage changes). The asterisks in Ba–Bc indicate the sag attributable to the hyperpolarization-activated cationic current Ih. The open arrowheads in Ba and Bc denote a depolarizing rebound, also attributable to Ih. The filled arrowheads in Bb point to bursts of action potentials riding on low-threshold Ca2+ spikes. Calibration: 20 mV (Ba–Bc), 300 pA (Bd–Bf), 200 ms.
- Figure 6.
The ionic basis of the rebound burst. In all panels, control traces are drawn as thinner lines. CsCl (3 mm), which blocks the hyperpolarization-activated cationic current Ih, blocked both the sag and the rebound depolarization in X94 and GIN cells (A, B, open arrowheads). In bursting X98 cells, CsCl blocked the sag (C, open arrowhead) but not the burst (filled arrowhead). In contrast, the IT channel blocker mibefradil (10 μm) did not block the sag (D, open arrowhead), but blocked the burst despite the large depolarizing rebound (filled arrowhead) evoked by a stronger hyperpolarization (−120 pA in mibefradil, compared with −40 pA in control). Note that in A–C, the superimposed responses in each panel were evoked by the same current step. Calibration: 40 mV, 200 ms.
- Figure 7.
Multivariate analysis of electrophysiological parameters. A, Grouping of parameters according to their CV and their η2. The nine numbered data points correspond, respectively, to the numbered parameters in B. The half-box (near the origin of axes) encloses parameters with η2 < 0.1 and CV < 0.35. B, Density plots (computed with a Gaussian kernel) of the nine parameters with η2 > 0.1, separated by transgenic line. Only the two extreme x-values are indicated for each plot. Parameters 1–3 are plotted in a logarithmic scale. C, Total-sample correlation matrix of parameters 1–9; the absolute values of the pairwise Pearson correlation coefficients are coded by color. D, Pooled within-group correlation matrix of the nine parameters. Note the separate clusters of passive (1–3) and active (4–9) parameters; parameters are correlated within, but not between, each cluster. E, Scatterplot of the electrophysiological parameters of the three neuronal subtypes in the principal component plane. Each principal component is a linear combination of the original 9 parameters; PC1 correlates strongly with active parameters, PC2 with passive parameters. X94 data points are separated into layer 4 and layer 5B cells. F, Scatterplot of the electrophysiological parameters of the three neuronal subsets in the canonical discriminant function plane. Each CDF is a linear combination of 11 electrophysiological parameters. X94 neurons are separated into layer 4 cells and layer 5B cells. The two intersecting lines separate the plane into three regions, with good segregation of the three groups into separate regions.
Tables
- Table 1.
Overlap between GFP and SOM, CB, and NPY expression, by line and by laminar position
Line Layer SOM+ GFP+ CB+ GFP+ NPY+ GFP+ n % DL n % DL n % DL n % DL n % DL n % DL X94 2–3 382 5.9 23 100.0 4 408 40.9 174 96.2 48 0 78 0 48 0 30 0 5–6 1079 13.0 151 92.7 425 0 88 0 177 0 65 0 All 1869 17.6 348 94.9 473 0 166 0 225 0 95 0 X98 2–3 271 12.9 35 100.0 173 20.2 38 92.1 126 1.6 13 15.4 4 188 1.7 3 100 5–6 694 20.3 148 95.2 558 21.3 124 96.0 456 12.3 137 40.9 All 1153 15.6 186 96.2 530 20.8 115 95.7 582 10.0 150 38.7 GIN 2–3 299 34.8 110 95.9 172 14.0 76 31.6 291 6.2 80 26.9 4 171 26.5 45 100 40 0 35 0 114 0 71 0 5–6 492 10.8 56 97.4 417 11.3 96 49.0 159 0.6 71 1.4 All 96 21.2 211 97.1 629 11.3 207 34.3 564 3.4 222 8.6 -
For each marker, both the percentage of marker-immunopositive cells expressing GFP and the percentage of GFP-expressing cells immunopositive for the marker are indicated. DL, Double-labeled cells.
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All (n = 150) X94 (n = 58) X98 (n = 59) GIN (n = 33) Vrest(mV) η2 0.05 p value 0.0230 10% −72.5 −73.2 −72.3 −69.8 90% −63.9 −64.6 −63.7 −64.7 Mean −68.3 −69.1 −68.2 −67.1 CV 0.05 0.05 0.05 0.04 Threshold (mV) η2 0.02 pvalue 0.2420 10% −47.5 −47.4 −48.4 −47.2 90% −38.3 −37.9 −37.4 −40.5 Mean −43.0 −42.8 −42.6 −44.0 CV 0.10 0.11 0.10 0.06 Spike height (mV) η2 0.06 pvalue 0.0120 10% 51.7 51.0 53.0 48.1 90% 71.8 70.1 73.5 69.3 Mean 60.9 60.3 63.0 58.1 CV 0.13 0.12 0.12 0.13 Sag–V slope η2 0.01 pvalue 0.5240 10% −0.41 −0.42 −0.40 −0.38 90% −0.19 −0.19 −0.19 −0.22 Mean −0.30 −0.31 −0.29 −0.30 CV 0.30 0.33 0.28 0.27 Adaptation ratio η2 0.04 pvalue 0.0620 10% 0.39 0.41 0.36 0.41 90% 0.71 0.77 0.68 0.63 Mean 0.54 0.57 0.51 0.54 CV 0.23 0.25 0.23 0.18 F–I slope (Hz/pA) η2 0.06 pvalue 0.0071 10% 0.47 0.53 0.40 0.55 90% 1.12 1.26 0.98 0.81 Mean 0.74 0.82 0.68 0.71 CV 0.35 0.34 0.39 0.25 Rin (MΩ) η2 0.55 p value 0.0000 10% 92 73 304 149 90% 602 218 796 403 Mean 316 132 514 282 CV 0.73 0.43 0.44 0.39 τm (ms) η2 0.66 pvalue 0.0000 10% 7.2 6.0 24.1 12.5 90% 41.4 17.3 47.2 33.9 Mean 22.2 9.9 35.0 20.9 CV 0.62 0.42 0.29 0.41 Rheobase (pA) η2 0.63 p value 0.0000 10% 11 77 7 13 90% 266 331 57 90 Mean 100 202 27 51 CV 1.03 0.47 0.78 0.59 Rate of rise (V/s) η2 0.38 pvalue 0.0000 10% 161 217 137 181 90% 293 307 241 295 Mean 227 261 191 232 CV 0.22 0.14 0.22 0.19 Rate of fall (V/s) η2 0.59 pvalue 0.0000 10% −183 −201 −122 −164 90% −89 −130 −79 −103 Mean −133 −166 −101 −132 CV 0.28 0.17 0.20 0.17 Spike width (ms) η2 0.72 pvalue 0.0000 10% 0.42 0.38 0.62 0.48 90% 0.79 0.50 0.86 0.63 Mean 0.58 0.45 0.74 0.55 CV 0.27 0.13 0.16 0.10 Fmax, steady-state (Hz) η2 0.57 p value 0.0000 10% 49 106 41 80 90% 175 201 103 148 Mean 113 152 69 120 CV 0.44 0.26 0.33 0.26 Fmax, initial (Hz) η2 0.67 pvalue 0.0000 10% 106 201 86 180 90% 304 343 188 265 Mean 206 270 135 221 CV 0.36 0.19 0.27 0.18 AHP (mV) η2 0.16 pvalue 0.0000 10% 13.7 13.2 15.8 13.9 90% 23.3 23.0 24.1 19.8 Mean 18.3 17.5 20.2 16.5 CV 0.21 0.23 0.18 0.15 -
The 10th and 90th percentiles, mean, and CV are indicated for each parameter. Also indicated for each parameter are its η 2 and the p value of η 2. The nine parameters in boldface had η 2 > 0.1 and p values lower than our computational limit of p = 0.0001 (probably by several orders of magnitude). All parameters are defined in Materials and Methods.
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Phenotypic property X94 X98 Cell body position Layers 4 and 5B Layers 5B and 6 Layer 4 axonal arborizations Dense None or sparse Layer 1 axonal arborizations None or sparse Dense Somatostatin All All Calbindin None All Neuropeptide Y None ∼40% Input resistance <250 MΩ >300 MΩ Time constant <20 ms >20 ms Spike width ≤0.5 ms ≥0.6 ms Fmax, initial >200 Hz <200 Hz Fmax, steady-state >100 Hz <100 Hz Robust stuttering ∼60% ∼3% Low-threshold spikes ∼2% ∼40% -
Phenotypic properties are arranged in five groups: laminar position, axonal distributions, neurochemical content, electrophysiological parameters, and firing properties. Inequalities in the electrophysiological parameters group apply to at least 90% of each population.
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