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. 2011 Mar;216(1):1-15.
doi: 10.1007/s00429-010-0291-8. Epub 2010 Dec 3.

Axonal Morphometry of Hippocampal Pyramidal Neurons Semi-Automatically Reconstructed After in Vivo Labeling in Different CA3 Locations

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Free PMC article

Axonal Morphometry of Hippocampal Pyramidal Neurons Semi-Automatically Reconstructed After in Vivo Labeling in Different CA3 Locations

Deepak Ropireddy et al. Brain Struct Funct. .
Free PMC article

Abstract

Axonal arbors of principal neurons form the backbone of neuronal networks in the mammalian cortex. Three-dimensional reconstructions of complete axonal trees are invaluable for quantitative analysis and modeling. However, digital data are still sparse due to labor intensity of reconstructing these complex structures. We augmented conventional tracing techniques with computational approaches to reconstruct fully labeled axonal morphologies. We digitized the axons of three rat hippocampal pyramidal cells intracellularly filled in vivo from different CA3 sub-regions: two from areas CA3b and CA3c, respectively, toward the septal pole, and one from the posterior/ventral area (CA3pv) near the temporal pole. The reconstruction system was validated by comparing the morphology of the CA3c neuron with that traced from the same cell by a different operator on a standard commercial setup. Morphometric analysis revealed substantial differences among neurons. Total length ranged from 200 (CA3b) to 500 mm (CA3c), and axonal branching complexity peaked between 1 (CA3b and CA3pv) and 2 mm (CA3c) of Euclidean distance from the soma. Length distribution was analyzed among sub-regions (CA3a,b,c and CA1a,b,c), cytoarchitectonic layers, and longitudinal extent within a three-dimensional template of the rat hippocampus. The CA3b axon extended thrice more collaterals within CA3 than into CA1. On the contrary, the CA3c projection was double into CA1 than within CA3. Moreover, the CA3b axon extension was equal between strata oriens and radiatum, while the CA3c axon displayed an oriens/radiatum ratio of 1:6. The axonal distribution of the CA3pv neuron was intermediate between those of the CA3b and CA3c neurons both relative to sub-regions and layers, with uniform collateral presence across CA3/CA1 and moderate preponderance of radiatum over oriens. In contrast with the dramatic sub-region and layer differences, the axon longitudinal spread around the soma was similar for the three neurons. To fully characterize the axonal diversity of CA3 principal neurons will require higher-throughput reconstruction systems beyond the threefold speed-up of the method adopted here.

Figures

Fig. 1
Fig. 1. Axonal reconstruction process: from micrographs to digital trees
a) Representative micrographs of dorsal hippocampus captured by lenses with different magnification power: 4× (top) with captioned sub-regions (Sub, Supra, and Infra indicate subiculum and the two granular blades of the dentate gyrus, respectively) and asterisk marking the somatic position within CA3c; 8× (bottom) with visible dendritic tree further enlarged at 20× and 40× (boxed insets indicated by black arrows) to highlight branches and spines (white ‘s’ arrows), as well as two axonal stretches enlarged at 40×. b) Illustration of a single labeled slice tracing, assembled from dozens of US letter-sized sheets of paper tiled together, each with manually pencil-traced neurites. One sheet is enlarged on the left, showing representative line traces. c) After high-resolution serial scanning, tracing images (here represented for one sheet only) are digitized into pixel format and vectorized. Dashed lines represent untraced segments joined by nearest-neighborhood. d) In the 3D arbor-stitching step, all planar vector sets, each corresponding to a histological section, are first aligned by translation and rotation. Then, they are joined together in 3D using information of the apparent terminations marked to be in focus at the top or bottom of the slice
Fig. 2
Fig. 2. Digital rendering of reconstructed CA3 pyramidal cell axons
a) Axonal arbors of neurons with somatic locations (black dots) in different CA3 sub-regions (CA3b, red; CA3pv, blue; and CA3c, green) 3D-embedded within the rat hippocampal template, with arrow captions indicating dorso-ventral (D/V), medio-lateral (M/L) and rostro-caudal (R/C) orientations. b) Individual renderings of the four CA3 axonal reconstructions (including CA3cNL), with fiduciary boundaries showing strata oriens (OR) and radiatum (RAD) and the pyramidal layer marked in light blue color.
Fig. 3
Fig. 3. Intrinsic morphometry of CA3 pyramidal cell axons
a) Cumulative axonal length as a function of Euclidean distance from the soma for each of the four neurons (CA3b, red; CA3pv, blue; CA3c, light green; and CA3cNL: dark green). b) Distribution of the proportion of arbor bifurcations with respect to Euclidean distance from the soma, overlaid with Gaussian curve fits.
Fig. 4
Fig. 4. Axonal length distribution along main hippocampus axes and canonical brain orientations
a) Differential distribution of axonal projections in the septo-temporal axis for the reconstructed neurons (CA3b, red; CA3pv, blue; CA3c, light green; and CA3cNL: dark green). The captions ‘S’ and ‘T’ on the abscissa indicate septal and temporal directions, respectively and arrows point to corresponding soma positions. The inset illustrates the position of the hippocampus (light blue) within an outline of the rat brain, relating the canonical hippocampus and brain axes. b) Cumulative axonal length distributions in the medio-lateral (left panel) and dorso-ventral (right panel) orientations. Solid circles, stars, and triangles represent the 5th, 50th, and 95th percentiles of the axonal length for every neuron. The captions ‘M’, ‘L’, ‘V’, and ‘D’ on the abscissa indicate medial, lateral, ventral, and dorsal directions, respectively.
Fig. 5
Fig. 5. Axonal length distributions across hippocampal sub-regions and layers
a) Axonal collaterals for the four reconstructed neurons within each of six CA sub-regions (CA3c, CA3b, CA3a, CA1c, CA1b and CA1a). Bar lengths are proportional to absolute measures, while the numerical labels in the bars indicate percentages within every neuron. b) Axonal collaterals for the four reconstructed neurons within each of four individual layers (oriens, OR; pyramidal cell, PC; radiatum, RAD; and lacunosum-moleculare, LM). Bar heights are proportional to absolute measures, while the numerical labels in the bars indicate percentages within every neuron.
Fig. 6
Fig. 6. Absolute and relative axonal length distributions within individual sub-regional layers
For each of the three neurons (CA3b, top; CA3pv, middle; CA3c, bottom), the size of the pie charts is scaled according to total axonal length. The surface area of each pie slice is proportional to the axonal distributions in the respective three CA3 (white) and three CA1 (grey) sub-regions, and further divided based on the arbor length in each cytoarchitectonic layer (OR, PC, RAD and LM). These pie charts were generated by measuring sector surface areas with ImageJ (v.1.38, http://rsb.info.nih.gov/ij), and marking the appropriate slice boundary positions with Paint Shop Pro (v.8.0, http://corel.com).

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