PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Luca Bonfanti

Curriculum vitae

Curriculum Vitae

Phd thesis

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

• Scientific background/state of the art

Brain structural plasticity, namely the ability of neural elements to perform adaptive changes, remarkably differ in animal species, anatomical regions, and ages. It occurs in different forms, e.g., synaptic plasticity, stem cell-driven adult neurogenesis (AN), and the recently demonstrated concept of “immature” neurons (INs). INs are generated prenatally, then remaining in an undifferentiated state for long time, eventually maturing and integrating in pre-existing circuits (Rotheneichner et al., 2018). While AN is restricted to neurogenic niches (subventricular zone, subgranular zone), the INs are present in cortical and subcortical regions. A recent comparative analysis performed by our lab on IN density in ten mammals, spanning from small-brained, lissencephalic to large-brained, gyrencephalic species, showed that it is higher in the latter, the INs extending from paleocortex in laboratory rodents to neocortex in gyrencephalic species. The remarkable variation of IN occurrence, location and amount across mammals can lead to misleading translation of results obtained from laboratory rodents, thus interspecies studies addressing such differences are needed. In this project, I will pursue the mapping of INs in mammals (here considering domestic dogs), and I will investigate their possible modulation in the cerebral cortex of chimpanzees (in both cases by using a previously established method; La Rosa et al., 2020) as a translational model, to identify and quantify the cortical INs in a comparable way.

• Aims

The goal of the project is twofold: the investigation of IN anatomical distribution, morphology, and density in cortical layer II of dogs, and the possible correlates of inter-individual variation in chimpanzees.

Experiments will include:

1. A systematic study of INs in the cerebral cortex of a domestic animal species (Canis lupus) to expand the current knowledge on this neuronal population in gyrencephalic species, and compare its density to the other mammals. During the first year, this analysis will allow me to learn the skills needed to correctly identify/count the IN cell types, in view of my stay abroad to perform the chimpanzee brain analysis.
2. An investigation of IN variation in chimpanzee (Pan troglodytes) cortex, to be conducted during the period abroad (2^nd year) on two groups of postmortem fixed brains collected at the National Chimpanzee Brain Resource (USA), associated with the animal's rearing history. One cohort of chimpanzees was raised by their mothers while the other was raised in a nursery setting with care provided by human handlers until three years of age, then placed in enclosures among peers (the early life social experiences have been shown to impact brain structure and behavior). The animals had participated during their life in a Primate Cognition Test Battery (PCTB), used to measure general intelligence by comparing cognitive and social skills.

   Depending on time, an additional subproject might be carried out:

3. The extension of the dog IN analysis to subcortical areas (amygdala, claustrum).

• Materials and methods

For both dog and chimpanzee analyses, postmortem, formalin-fixed brains will be used. Tissues will be serially cut on a cryostat (40 µm thick), then, immunohistochemistry for doublecortin (DCX; a cytoskeletal marker for immaturity) will be performed to visualize the IN cell population (using an anti-DCX Abcam antibody - ab18723). The methodology adopted in two previous PhD projects (La Rosa, Ghibaudi) will be maintained for IN mapping. Qualitative and quantitative analyses will be conducted on 4 anterior-posterior brain levels, established using correspondent neuroanatomical structures. Cell counting will be performed using Neurolucida-Stereoinvestigator software. Since DCX+ INs are arranged in a monolayer (cortical layer II), counting will be expressed as linear density (number of DCX+ cells/layer II perimeter length in mm). Finally, GraphPad Prism Software will be used for statistical analyses, using parametric or nonparametric tests based on data distribution.

Animals: 4 Beagle dog breed (2 males and 2 females; age 1 year; same number as in previous studies, to obtain comparable results) will be considered, raised in the same environmental conditions at MarshallBio (Lyon, France; authorization: Art. R.214-89 of Décret 2013-118), euthanized, and their brain extracted within 30 minutes. 8 to 12 brain hemispheres of adult chimpanzees (both sexes; age range 30-46 years) will be considered, belonging to two groups (4 to 6 each) that responded similarly to the PCTB but have different rearing histories.

• Expected results

The expected results of the present project are based on current knowledge in brain plasticity and INs [ i) plasticity is usually modulated by experience (e.g., rearing history); ii) IN amount remarkably vary among mammals; iii) INs appear more abundant in animal species endowed with gyrencephalic brains and high computational capabilities], hence:

1. in the analysis of the chimpanzee model, we expect to find a possible difference in IN density between the chimpanzee groups considered (i.e., an aspect that has not yet been addressed until now);
2. in the canine model study, we expect to uncover the placement of dog IN density with respect to other mammals, in the mapping of IN phylogenetic variation.

Finally, comparing the canine model, involving animals maintained in the same, standardized environmental conditions since birth (similarly to laboratory rodents), and the chimpanzee model, based on different animal groups, will help to understand the importance of interindividual variation.

• Bibliography

La Rosa, C., Cavallo, F., Pecora, A., Chincarini, M., Ala, U., Faulkes, C. G., Nacher, J., Cozzi, B., Sherwood, C. C., Amrein, I., & Bonfanti, L. (2020). Phylogenetic variation in cortical layer II immature neuron reservoir of mammals. ELife, 9, 1–21. https://doi.org/10.7554/eLife.55456

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. Cerebral Cortex, 28(7), 2610–2621. https://doi.org/10.1093/cercor/bhy087

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