Neocortical pyramidal neurons with axons emerging from dendrites are frequent in non-primates, but rare in monkey and human

Abstract

The canonical view of neuronal function is that inputs are received by dendrites and somata, become integrated in the somatodendritic compartment and upon reaching a sufficient threshold, generate axonal output with axons emerging from the cell body. The latter is not necessarily the case. Instead, axons may originate from dendrites. The terms 'axon carrying dendrite' (AcD) and 'AcD neurons' have been coined to describe this feature. In rodent hippocampus, AcD cells are shown to be functionally 'privileged', since inputs here can circumvent somatic integration and lead to immediate action potential initiation in the axon. Here, we report on the diversity of axon origins in neocortical pyramidal cells of rodent, ungulate, carnivore, and primate. Detection methods were Thy-1-EGFP labeling in mouse, retrograde biocytin tracing in rat, cat, ferret, and macaque, SMI-32/βIV-spectrin immunofluorescence in pig, cat, and macaque, and Golgi staining in macaque and human. We found that in non-primate mammals, 10-21% of pyramidal cells of layers II-VI had an AcD. In marked contrast, in macaque and human, this proportion was lower and was particularly low for supragranular neurons. A comparison of six cortical areas (being sensory, association, and limbic in nature) in three macaques yielded percentages of AcD cells which varied by a factor of 2 between the areas and between the individuals. Unexpectedly, pyramidal cells in the white matter of postnatal cat and aged human cortex exhibit AcDs to much higher percentages. In addition, interneurons assessed in developing cat and adult human cortex had AcDs at type-specific proportions and for some types at much higher percentages than pyramidal cells. Our findings expand the current knowledge regarding the distribution and proportion of AcD cells in neocortex of non-primate taxa, which strikingly differ from primates where these cells are mainly found in deeper layers and white matter.

Keywords: axon initial segment; evolution; evolutionary biology; human; inhibitory interneurons; interstitial cells; mouse; neurofilament; neuroscience; rat; rhesus macaque; subplate.

Figure 1.. Confocal tile scan of dorsal neocortex (premotor area) of P60 infant macaque.

(A) Pyramidal cells were stained with SMI-32/βIV-spectrin to label dendrites and the axon initial segment, respectively. Insets depict neurons with an axon emerging from the soma (B), or from an axon carrying dendrite (C), or a shared root (D). Axons indicated by arrows. Scale bars 100 µm for the tile scan and 25 µm for the insets.

Figure 2.. Representative axon carrying dendrite (AcD) neurons.

(A1, A2) From rat visual cortex (biocytin, immunofluorescence); (B1, B2) cat visual cortex (immunofluorescence); (C1, C2) ferret visual cortex (biocytin); (D1, D2) macaque premotor cortex (biocytin, immunofluorescence), the inset shows the axon origin at higher magnification; (E1, E2) human auditory cortex (Golgi method; D2 is a montage of two photos). Apical AcDs (asterisk in C2) were rare, less than 10 were detected among the neurons assessed in adult rat, ferret, and macaque, and none in our human material. In all cases, the axon immediately bent down toward the white matter. Axon origins are marked by large arrows, small arrows indicate the course of biocytin-labeled axons. Scale bars 25 µm.

Figure 2—figure supplement 1.. Variations of axon origins of biocytin-stained pyramidal neurons of rat (A–D) and ferret (E–G) visual cortex, and macaque premotor cortex (H–K), and macaque intraparietal sulcus (L).

The neurons in (L), an AcD cell next to a somatic axon cell, reside millimeters from the injection site, they are long projecting layer III pyramidal neurons. Cells with somatic axons are in B, C-inset, E, F, H, I, L (rightmost cell). Cells with shared root configuration are in C, G. Cells with axon carrying dendrites are D, D-inset, J, K, L. The neurons in K, L (the left one, enlarged in the inset in L) give rise to normal basal dendrites plus a single thick radially descending dendrite which carries the axon. Such cells have been described in macaque cortex (Hendry and Jones, 1983). Note that the axon of the cell in J emerged from the apical dendrite and bent down to the white matter. Axons marked by white arrows, axon collaterals marked by small black arrows. Scales: 15 µm in A–D; 25 µm in E–L.

Figure 2—figure supplement 2.. Variations of axon origins of Golgi-impregnated pyramidal neurons of human temporal cortex, all taken from Individual 2, 56 years of age and sampled from all layers.

Cells with somatic axons are in A, B, C, D, E, F. Cells with shared root configuration are in G, H, I. Cells with axon carrying dendrites are J, K, L, M, N, O. Axons marked by white arrows, axon collaterals marked by small black arrows. Scale: 25 µm.

Figure 3.. Proportion of axon carrying dendrite (AcD) neurons across species.

(A) Shown are mean ± SEM. of the individual percentages listed in Tables 1–4, which also indicate the staining methods. Numbers above the bars are the total number of pyramidal neurons assessed per species/cell class for this graph. Numbers in the bars indicate the number of individuals. (B) Laminar analysis. Non-primate species showed roughly equal proportions of AcD neurons in supra- and infragranular layers. With some individual variability the range was 10–21%. In contrast, in macaque, the cluster was downshifted along the ordinate due to overall much lower proportions. Furthermore, infragranular pyramidal cells displayed much higher proportions of AcD cells compared with supragranular pyramidal cells. A Mann-Whitney rank sum test of ‘all non-primate’ versus ‘all macaque’ percentages of supragranular and infragranular AcD cells, yielded p<0.001 and p<0.001, respectively. Human was not included in the statistical test because only one method was used to detect AcD cells. The legend indicates the number of individuals and the staining methods; IFL, immunofluorescence. Note that we could not do a laminar analysis for all individuals shown in (A) because staining of supragranular layers in some animals delivered too low numbers which might have led to a sampling error.

Figure 4.. Within-species areal comparisons.

(A) Upper left is a photomicrograph of one of the coronal sections stained for immunofluorescence. The regions of interest are color coded. Upper right is the macaque brain (after Paxinos et al., 2009). The dashed boxes and Bregma distances indicate where our assessments were made. The rostral box overlaps the premotor cortex harboring the biocytin injections. Note that the analysis was spanning several millimeters of cortex (see Figure 4—source data 1). The middle box corresponds to the level of the section shown to the left. It is slightly tilted with respect to the stereotaxis coordinates (Paxinos et al., 2009). The posterior box corresponds to a fairly caudal level of the visual cortex. The table summarizes the percentages of AcD neurons obtained in the six areas and three individuals and gives the mean of each area with standard deviation. Abbreviations: arc, arcuate sulcus; cgs, cingulate sulcus; cs, central sulcus; ecal, external calcarine sulcus; ios, inferior occipital sulcus; ips, intraparietal sulcus; lf, lateral fissure; lu, lunate sulcus; prs, principal sulcus; sts, superior temporal sulcus. (B) Upper left is a photomicrograph of one of the coronal sections of cat occipital cortex analyzed for biocytin-stained AcD neurons. The injection site in this case was near the area 17/18 border, some other cats had an additional injection into the suprasylvian gyrus (see Figure 4—source data 1). Area 17 is along the medial flank, areas 18, 19, and 21 are in the lateral sulcus and on the suprasylvian gyrus. Upper right is the cat brain (after Reinoso-Suarez, 1961) with the visual fields indicated. The table summarizes the percentages of AcD neurons obtained in area 17 and the extrastriate areas. The graph pairs the data points of the five cats. To the right, we compared cat (n = 7) to ferret (n = 4) visual cortex (striate and extrastriate). Every point is one individual, the red bar represents the median for each column. The p-values were determined with a Mann-Whitney rank sum test.

Figure 5.. Proportion of axon carrying dendrite (AcD) cells versus shared root cells.

(A) Data from rat (biocytin), ferret (biocytin), macaque (biocytin, immunofluorescence, Golgi), and human (Golgi). The species cluster along the ordinate as already seen in Figure 3B. The Mann-Whitney rank sum test of ‘all non-primate’ versus ‘all macaque’ proportions of AcD cells yielded p <0.001. However, the shared root values scatter considerably along the abscissa. Mann-Whitney rank sum test of ‘all non-primate’ versus ‘all macaque’ proportions of shared root cells yielded p=0.008. (B) The percentages of AcD were graphically compared to the sum of AcD and shared root. For macaque, data were separated by staining methods. Note that the Golgi method in macaque and in human yielded a higher proportion of shared root compared to biocytin and immunofluorescence. Numbers in the bars represent the sample size (individuals and/or cortical areas). (C) Comparison of biocytin and immunofluorescence staining in macaque. (D) Comparison of biocytin and immunofluorescence staining within just one individual macaque. Note in C, D that AcD cells are detected equally well with both methods whereas the biocytin staining yielded higher numbers of shared root cells (SR). In C, D, colors indicate the comparisons, and the p-values were determined with a Mann-Whitney rank sum test. IFL, immunofluorescence.

Figure 6.. Spine density did not systematically differ between regular dendrites (non-axon carrying dendrites [non-AcDs]), dendrites sharing a root with a neighboring axon, and AcDs.

Data from adult rat and ferret biocytin material, values from each cell are connected by a line. For normalization, the average of the non-AcD has been set to 1, and all values were expressed relative to this. Mann-Whitney rank sum test p-values and the sample size are reported above each plot.

Figure 7.. Axon carrying dendrite (AcD) interneurons in human and cat cortex.

(A) Photomicrograph of a representative Golgi-impregnated bitufted neuron with arcade-like initial axon from supragranular layers next to its reconstruction, followed by three further examples of bitufted, Martinotti (2), and basket cells (3). Axons in orange, somata and dendrites in black. Asterisks mark AcD neurons, boxes with arrows show the axon origin at higher magnification. In the table, the percentage of Golgi-impregnated AcD interneurons is reported for Individuals 1, 2, 4, followed by the percentages of Parvalbumin-positive AcD neurons of Individuals 11–13. (B) Photomicrograph of a layer VI neuropeptide Y-positive neuron with somatic axon, and a layer V Somatostatin-positive AcD neuron. Axons marked by white arrows, small black arrows mark collaterals. The graph shows percentages of AcD interneuron subsets at the ages indicated in developing cat occipital cortex (see Figure 7—source data 1 for sample size).

Author response image 1.