Non-allometric expansion and enhanced compartmentalization of Purkinje cell dendrites in the human cerebellum

Reviewer #1 (Public review):

Summary:

Busch and Hansel present a morphological and histological comparison between mouse and human Purkinje cells (PCs) in the cerebellum. The study reveals species-specific differences that have not previously been reported despite numerous observations of these species. While mouse PCs show morphological heterogeneity and occasional multi-innervation by climbing fibers (CFs), human PCs exhibit a widespread, multi-dendritic structure that exceeds expectations based on allometric scaling. Specifically, human PCs are significantly larger, and exhibit increased spine density, with a unique cluster-like morphology not found in mice.

Strengths:

The manuscript provides an exceptionally detailed analysis of PC morphology across species, surpassing any prior publication. Major strengths include a systematic and thorough methodology, rigorous data analysis, and clear presentation of results. This work is likely to become the go-to resource for quantitation in this field. The authors have largely achieved their aims, with the results effectively supporting their conclusions.

Weaknesses:

There are a few concerns that need to be addressed, specifically related to details of the methodolology as well as data interpretation based on the limits of some experimental approaches. Overall, these weaknesses are minor.

Reviewer #2 (Public review):

Summary:

This manuscript aims to follow up on a previously published paper (Busch and Hansel 2023) which proposed that the morphological variation of dendritic bifurcation in Purkinje cells in mice and humans is indicative of the number of climbing fiber inputs, with dendritic bifurcation at the level of the soma resulting in a proportion of these neurons being multi-innervated. The functional and anatomical climbing fiber data was obtained solely from mice since all human tissue was embalmed and fixed, and the extension of these findings to human Purkinje cells was indirect. The current comparative anatomy study aims to resolve this question in human tissue more directly and to further analyse in detail the properties of adult human Purkinje cell dendritic morphology.

Strengths:

The authors have carried out a meticulous anatomical quantification of human Purkinje cell dendrites, in tissue preparations with a better signal-to-noise ratio than their previous study, comparing them with those from mice. Importantly, they now present immunolabelling results that trace climbing fiber axons innervating human PCs. As well as providing detailed analyses of spine properties and interesting new findings of human PC dendritic length and spine types, the work confirms that human PCs that have two clearly distinct dendritic branches have an approximately x% chance of receiving more than one CF input, segregated across the two branches. Albeit entirely observational, the data will be of widespread interest to the cerebellar field, in particular, those building computational models of Purkinje cells.

Weaknesses:

The work is, by necessity, purely anatomical. It remains to be seen whether there are any functional differences in ion channel expression or functional mapping of granule inputs to human PCs compared with the mouse that might mitigate the major differences in electronic properties suggested.

Author response:

We plan to submit a revised version of our manuscript eLife-RP-RA-2024-105013, in which we address all comments raised by the two expert reviewers.

Below we describe what we like to address in this revision. We understand that the provisional response is not meant to be a point-by-point reply. Therefore, our revision plan more generally summarizes the comments of the reviewers and how we plan to address them.

Reviewer #1:

This reviewer is overall very positive and states that our ‘work is likely to become the go-to resource for quantification in this field’. This reviewer raises few weaknesses of the manuscript that are explicitly described as minor.

Microscopic resolution sufficient to support quantitative spine assessments?

In the detailed revision, we will provide quantification of microscopic resolution and will relate this to the spine comparisons offered. Where needed, we will add caveats discussing measurement limits.

Age of the human tissue.

Most analysis is based on the study of three brains from elderly individuals. For the analysis of dendritic spines, we added measures from a younger brain (37 years-old). We will make it more clear, which datasets contained these measures and what the results of our comparative analysis have been.

Genetic diversity contributing to species differences?

We provide an updated discussion on this interesting topic.

Reviewer #2:

This reviewer also expresses a largely positive view of the manuscript, noting that ‘..the data will be of widespread interest to the cerebellar field…’.

Microscopic resolution:

see above.

Figure panels / Fig. 3:

We will make sure that the figures are readable and will provide a clarification of gray scales used in Fig. 3.

Vertical vs horizontal dendrite orientation:

This is a point that requires clarification. Per our definition, all dendrites fall either into the vertical or horizontal category. We will make sure that this is defined sufficiently well.