Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

Abstract

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population.The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing "Golgi-like" complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills.Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.

Morphological heterogeneity among corticogeniculate neurons in ferrets: quantification and comparison with a previous report in macaque monkeys

“…All the experimental methods involving the use of animals were described in detail in Section 2 and Supporting Information Appendix: Materials and Methods of Hasse and Briggs () and were approved by the Institutional Animal Care and Use Committee at Dartmouth. Detailed descriptions of the methods for stereotaxic injection of SAD‐B19 rabies virus into the LGN, tissue harvesting, sectioning, staining, and reconstruction of labeled neurons were also described in detail previously (Bragg & Briggs, ; Bragg, Fairless, Liu, & Briggs, ; Briggs et al, ).…”

“…Neurons infected with virus express fluorescent proteins throughout their dendritic arbors, enabling complete reconstruction of dendritic morphology (see Figure ). Virus‐mediated delivery of fluorescent markers causes widespread labeling of individual neurons, making it possible to reconstruct large numbers of neurons including rare neuronal types (Bragg & Briggs, ; Bragg et al, ; Briggs et al, ; Hasse & Briggs, ). This high‐throughput approach facilitates rigorous statistical analyses of distinct morphological subtypes within select neuronal populations.…”

“…We applied an independent cluster analysis algorithm using all of the independent morphological metrics exported from each of the 98 CG reconstructions as the input. Because the cluster analysis algorithm assumes each parameter is independent (Bragg & Briggs, ; Bragg et al, ; Cauli et al, ; Thorndike, ), we excluded number of endings because this metric is not independent of number of nodes. The 13 parameters included in the cluster analysis were cell body size, cell body roundness, number of nodes, proportion of basal dendrite in Layers 5, 6, and the white matter, proportion of apical dendrite in Layers 2/3, 4, 5, and 6, location of the cell body in Layer 6, height of the apical dendrite (tallest point on stellate neurons), and the angle of the apical dendrite.…”

“…We used the PCA scores as the input to the “fitgmdist” Matlab function to test each GMM using three different evaluations: negative log likelihood, Akaike information criterion, and Bayes information criterion. The GMM with the lowest criteria across the three evaluations indicates the optimal number of clusters (Bragg & Briggs, ).…”

“…Robust infection and expression of fluorescent markers cause labeling of thousands of neurons per animal and enables resolution of unusual and/or rare neuronal types, which may be missed with conventional retrograde tracing methods (Callaway & Luo, ). Additionally, fluorescent markers are expressed throughout the complete dendritic arborization of infected neurons, enabling accurate, complete, and high‐throughput reconstructions of dendritic morphology followed by rigorous statistical analysis of morphological variation (Bragg & Briggs, ; Briggs et al, ).…”