PhD in Veterinary Sciences for Animal Health and Food Safety

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Supervisor

Curriculum vitae

Phd thesis

TITLE: Features and possible modulation of “immature” neurons in gyrencephalic mammals

• Scientific background

“Immature” neurons (INs) are a recently discovered form of brain structural plasticity involving cells that are generated prenatally, then remaining undifferentiated for long time, eventually maturing and integrating into preexisting circuits at different life stages^1,2. The INs have been described in cortical layer II and in amygdala³ by using immaturity markers (e.g., the cytoskeletal protein doublecortin - DCX) that allow their identification by immunocytochemistry. A recent comparative analysis conducted by our lab in the cerebral cortex of 10 mammals spanning from small-brained, lissencephalic (rodents) to large-brained, gyrencephalic species (sheep, chimpanzee), showed far higher densities of INs in the latter⁴. The remarkable variation of IN location (restricted to paleocortex in rodents, extended to whole neocortex in gyrencephalic species), and amount (one order of magnitude difference in density) observed across mammals underlines the need for systematic comparative studies involving non-rodent species, to avoid misleading interpretation of result translation from laboratory rodents^5,6.

• Aims, Methods, Results

In the first year, I performed a systematic study of INs in cortical layer II of a domestic animal species (Beagle dog, n=4; 1 year old, raised at Marshall Bio, Lyon) as part of a wide phylogenetic study in mammals started in prior PhD projects. INs were identified by using anti-DCX antibody and quantified in the whole cerebral cortex as linear density (DCX+ cells/mm of layer II perimeter). Results indicate that canine IN density is high, close to that of other carnivores (cat and fox, being the most enriched in INs to date)⁴.

In the first half of my second year, I performed a similar analysis in the cortical layer II of young-adult horses (age: 3-7 years), as an example of very large-brained mammals. Also in this case, high densities were found, confirming the importance of brain size. Dog and horse results suggest that IN evolutionary maintenance might be linked to both brain size and phylogenetic groups.

In February, I moved to the Sherwood lab to study the possible IN inter-individual variation in two groups of postmortem-fixed primate brains (4 to 5 brain hemispheres/group; middle aged). My initial focus was on evaluating alternative anti-DCX primary antibody options on different primates (chimpanzee, macaque, marmoset) to overcome the lack of Santa Cruz anti-DCX polyclonal antibody (the best tool to identify INs, no longer produced since 2021) and ensure consistent staining across samples. Based on the results (none of the new DCX products work on chimpanzee tissue), we decided to shift on macaques (Rhesus monkey), a nonhuman primate used in translational neuroscience because of its similarities to human brain anatomy, phylogenetics, and cognitive functions^7,8. We also added this species to the IN phylogenetic study, and I am currently analyzing 4 young-adult macaques (age: 7-10 years) by quantifying DCX+ cells in cortical layer II and amygdala.

All analyses are carried out by using the same procedure established in previous PhD projects, to allow comparable results⁶: for cortical INs, qualitative and quantitative analyses are conducted in each brain hemisphere on 12 coronal sections belonging to 4 anterior-posterior levels established using corresponding neuroanatomical structures (results expressed as linear density); the amygdala INs are quantified on serial coronal sections of the entire structure, spaced each other 480µm (results expressed as DCX+ cells/mm²).

• Future developments

In the coming year, I will complete the IN analysis on young-adult macaques, and I will study the possible modulation of these cells as potential inter-group variation associated with the animal rearing history (mother Vs nursery reared, a condition that has been shown to impact brain structure) in two groups of post-mortem fixed macaque brains (4-5 brain hemispheres/group; age: 23 years).

• Bibliography

1. Rotheneichner P, Belles M, Benedetti B, König R, Dannehl D, Kreutzer C, Zaunmair P, Engelhardt M, Aigner L, Nacher J, Couillard-Despres S (2018) Cellular plasticity in the adult murine piriform cortex: continuous maturation of dormant precursors into excitatory neurons. Cereb Cortex 28(7):2610-2621.
2. Benedetti B, Dannehl D, König R, Coviello S, Kreutzer C, Zaunmair P, Jakubecova D, Weiger TM, Aigner L, Nacher J, Engelhardt M, Couillard-Després S (2020) Functional integration of neuronal precursors in the adult murine piriform cortex. Cereb Cortex 30(3): 1499-1515.
3. Piumatti M, Palazzo O, La Rosa C, Crociara P, Parolisi R, Luzzati F, Lévy F, Bonfanti L (2018) Non-newly generated, “immature” neurons in the sheep brain are not restricted to cerebral cortex. J Neurosci 38:826-842.
4. La Rosa C, Cavallo F, Pecora A, Chincarini M, Ala U, Faulkes CG, Nacher J, Cozzi B, Sherwood CC, Amrein I, Bonfanti L (2020a) Phylogenetic variation in cortical layer II immature neuron reservoir of mammals. eLife 9:e55456.
5. Bonfanti L, La Rosa C, Ghibaudi M, Sherwood CC (2023) Adult neurogenesis and “immature” neurons in mammals: An evolutionary trade-off in plasticity? Brain Struct Funct (epub).
6. Ghibaudi M, Boda E, Bonfanti L (2024) From mice to humans: a need for comparable results in mammalian neuroplasticity. Neural Regen Res 20(2):464-466.
7. Alldritt S, et al. (2024) Brain charts for the Rhesus macaque lifespan. bioRxiv [Preprint]. doi: 10.1101/2024.08.28.610193.
8. Phillips KA, Bales KL, Capitanio JP, Conley A, Czoty PW, 't Hart BA, Hopkins WD, Hu SL, Miller LA, Nader MA, Nathanielsz PW, Rogers J, Shively CA, Voytko ML. (2014) Why primate models matter. Am J Primatol 76(9):801-27.

Research activities

Co supervisor

Chet Sherwood

Attended Congresses

• National Congress of the Italian Society for Neuroscience (SINS): 14th to 17th September 2023
• Gruppo Italiano per lo Studio della Neuromorfologia (GISN): 24th to 25th November 2023 
• Greater Baltimore Society for Neuroscience (GBSfN): 8th November 2024