a-comparative-approach-for-quantitative-cell-counting-studies-widely

A Comparative Approach for Quantitative Cell Counting Studies in Widely Different Mammalian Brains

Most neurobiological studies are conducted on laboratory rodents. Despite many similarities across mammalian brains, important differences also exist, ...

Neuroscience Institute Cavalieri Ottolenghi

University of Turin

Brain plasticity is important for translational purposes since most neurological disorders and brain aging problems remain substantially incurable. In the mammalian nervous system, neurons are mostly not renewed throughout life and cannot be replaced. In humans, the increasing life expectancy explains the increase in brain health problems, also producing heavy social and economic burden. An exception to the "static" brain is represented by stem cell niches leading to the production of new neurons. Such adult neurogenesis is dramatically reduced from fish to mammals, and in large-brained mammals with respect to rodents. Some examples of neurogenesis occurring outside the neurogenic niches have been reported, yet these new neurons actually do not integrate in the mature nervous tissue. Non-newly generated, "immature" neurons (nng-INs) are also present: Prenatally generated cells continuing to express molecules of immaturity (mostly shared with the newly born neurons). Of interest, nng-INs seem to show an inverse phylogenetic trend across mammals, being abundant in higher-order brain regions not served by neurogenesis and providing structural plasticity in rather stable areas. Both newly generated and nng-INs represent a potential reservoir of young cells (a "brain reserve") that might be exploited for preventing the damage of aging and/or delay the onset/reduce the impact of neurological disorders.

Newly Generated and Non-Newly Generated "Immature" Neurons in the Mammalian Brain: A Possible Reservoir of Young Cells to Prevent Brain Aging and Disease?

After the discovery of adult neurogenesis (stem cell-driven production of new neuronal elements), it is conceivable to find young, undifferentiated neurons mixed with mature neurons in the neural networks of the adult mammalian brain. This "canonical" neurogenesis is restricted to small stem cell niches persisting from embryonic germinal layers, yet, the genesis of new neurons has also been reported in various parenchymal brain regions. Whichever the process involved, several populations of "young" neurons can be found at different locations of the brain. Across the years, further complexity emerged: (i) molecules of immaturity can also be expressed by non-dividing cells born during embryogenesis, then maintaining immature features later on; (ii) remarkable interspecies differences exist concerning the types, location, amount of undifferentiated neurons; (iii) re-expression of immaturity can occur in aging (dematuration). These twists are introducing a somewhat different definition of neurogenesis than normally assumed, in which our knowledge of the "young" neurons is less sharp. In this emerging complexity, there is a need for complete mapping of the different "types" of young neurons, considering their role in postnatal development, plasticity, functioning, and interspecies differences. Several important aspects are at stake: the possible role(s) that the young neurons may play in maintaining brain efficiency and in prevention/repair of neurological disorders; nonetheless, the correct translation of results obtained from laboratory rodents. Hence, the open question is: how many types of undifferentiated neurons do exist in the brain, and how widespread are they?

How Widespread Are the "Young" Neurons of the Mammalian Brain?

Recently, a population of "immature" neurons generated prenatally, retaining immaturity for long periods and finally integrating in adult circuits has been described in the cerebral cortex. Moreover, comparative studies revealed differences in occurrence/rate of different forms of neurogenic plasticity across mammals, the "immature" neurons prevailing in gyrencephalic species. To extend experimentation from laboratory mice to large-brained mammals, including humans, it is important to detect cell markers of neurogenic plasticity in brain tissues obtained from different procedures (e.g., post-mortem/intraoperative specimens vs. intracardiac perfusion). This variability overlaps with species-specific differences in antigen distribution or antibody species specificity, making it difficult for proper comparison. In this work, we detect the presence of doublecortin and Ki67 antigen, markers for neuronal immaturity and cell division, in six mammals characterized by widely different brain size. We tested seven commercial antibodies in four selected brain regions known to host immature neurons (paleocortex, neocortex) and newly born neurons (hippocampus, subventricular zone). In selected human brains, we confirmed the specificity of DCX antibody by performing co-staining with fluorescent probe for DCX mRNA. Our results indicate that, in spite of various types of fixations, most differences were due to the use of different antibodies and the existence of real interspecies variation.

Consistency and Variation in Doublecortin and Ki67 Antigen Detection in the Brain Tissue of Different Mammals, including Humans.

The recent identification of a population of non-newly born, prenatally generated "immature" neurons in the layer II of the piriform cortex (cortical immature neurons, cINs), raises questions concerning their maintenance or depletion through the lifespan. Most forms of brain structural plasticity progressively decline with age, a feature that is particularly prominent in adult neurogenesis, due to stem cell depletion. By contrast, the entire population of the cINs is produced during embryogenesis. Then these cells simply retain immaturity in postnatal and adult stages, until they "awake" to complete their maturation and ultimately integrate into neural circuits. Hence, the question remains open whether the cINs, which are not dependent on stem cell division, might follow a similar pattern of age-related reduction, or in alternative, might leave a reservoir of young, undifferentiated cells in the adult and aging brain. Here, the number and features of cINs were analyzed in the mouse piriform cortex from postnatal to advanced ages, by using immunocytochemistry for the cytoskeletal marker doublecortin. The abundance and stage of maturation of cINs, along with the expression of other markers of maturity/immaturity were investigated. Despite a marked decrease in this neuronal population during juvenile stages, reminiscent of that observed in hippocampal neurogenesis, a small amount of highly immature cINs persisted up to advanced ages. Overall, albeit reducing in number with increasing age, we report that the cINs are present through the entire animal lifespan.

Age-related changes in layer II immature neurons of the murine piriform cortex.

Neuronal plasticity can vary remarkably in its form and degree across animal species. Adult neurogenesis, namely the capacity to produce new neurons from neural stem cells through adulthood, appears widespread in non-mammalian vertebrates, whereas it is reduced in mammals. A growing body of comparative studies also report variation in the occurrence and activity of neural stem cell niches between mammals, with a general trend of reduction from small-brained to large-brained species. Conversely, recent studies have shown that large-brained mammals host large amounts of neurons expressing typical markers of neurogenesis in the absence of cell division. In layer II of the cerebral cortex, populations of prenatally generated, non-dividing neurons continue to express molecules indicative of immaturity throughout life (cortical immature neurons; cINs). After remaining in a dormant state for a very long time, these cINs retain the potential of differentiating into mature neurons that integrate within the preexisting neural circuits. They are restricted to the paleocortex in small-brained rodents, while extending into the widely expanded neocortex of highly gyrencephalic, large-brained species. The current hypothesis is that these populations of non-newly generated "immature" neurons might represent a reservoir of developmentally plastic cells for mammalian species that are characterized by reduced stem cell-driven adult neurogenesis. This indicates that there may be a trade-off between various forms of plasticity that coexist during brain evolution. This balance may be necessary to maintain a "reservoir of plasticity" in brain regions that have distinct roles in species-specific socioecological adaptations, such as the neocortex and olfactory structures.

Adult neurogenesis and "immature" neurons in mammals: an evolutionary trade-off in plasticity?

From mice to humans: a need for comparable results in mammalian neuroplasticity.

The capacity of the central nervous system for structural plasticity and regeneration is commonly believed to show a decreasing progression from "small and simple" brains to the larger, more complex brains of mammals. However, recent findings revealed that some forms of neural plasticity can show a reverse trend. Although plasticity is a well-preserved, transversal feature across the animal world, a variety of cell populations and mechanisms seem to have evolved to enable structural modifications to take place in widely different brains, likely as adaptations to selective pressures. Increasing evidence now indicates that a trade-off has occurred between regenerative (mostly stem cell-driven) plasticity and developmental (mostly juvenile) remodeling, with the latter primarily aimed not at brain repair but rather at "sculpting" the neural circuits based on experience. In particular, an evolutionary trade-off has occurred between neurogenic processes intended to support the possibility of recruiting new neurons throughout life and the different ways of obtaining new neurons, and between the different brain locations in which plasticity occurs. This review first briefly surveys the different types of plasticity and the complexity of their possible outcomes and then focuses on recent findings showing that the mammalian brain has a stem cell-independent integration of new neurons into pre-existing (mature) neural circuits. This process is still largely unknown but involves neuronal cells that have been blocked in arrested maturation since their embryonic origin (also termed "immature" or "dormant" neurons). These cells can then restart maturation throughout the animal's lifespan to become functional neurons in brain regions, such as the cerebral cortex and amygdala, that are relevant to high-order cognition and emotions. Unlike stem cell-driven postnatal/adult neurogenesis, which significantly decreases from small-brained, short-living species to large-brained ones, immature neurons are particularly abundant in large-brained, long-living mammals, including humans. The immature neural cell populations hosted in these complex brains are an interesting example of an "enlarged road" in the phylogenetic trend of plastic potential decreases commonly observed in the animal world. The topic of dormant neurons that covary with brain size and gyrencephaly represents a prospective turning point in the field of neuroplasticity, with important translational outcomes. These cells can represent a reservoir of undifferentiated neurons, potentially granting plasticity within the high-order circuits subserving the most sophisticated cognitive skills that are important in the growing brains of young, healthy individuals and are frequently affected by debilitating neurodevelopmental and degenerative disorders.

Brain structural plasticity in large-brained mammals: Not only narrowing roads.

Recent research in brain structural plasticity has identified "immature" or "dormant" neurons in layer II of the cerebral cortex (cortical immature neurons; cINs), cells that remain in a prolonged state of arrested development but retain the ability to resume maturation and integrate functionally into mature cortical circuits. These immature cells are far more abundant in large-brained mammals, being restricted to paleocortex (piriform cortex) in small-brained rodents and extending in the widely expanded neocortical mantle of species with large gyrencephalic brains. In a previous systematic analysis, using a comparable method for quantification in eight mammalian species, including mice, chimpanzees, and others of diverse phylogenetic backgrounds and neuroanatomical structure, cIN density showed covariation with brain size. Notably, however, members of the order Carnivora (cats and foxes) displayed the highest cIN densities with respect to sheep and chimpanzees, endowed with larger brains. Here we used the same method to characterize and quantify the cINs in the cerebral cortex of dogs (carnivores) and horses (herbivores with a very large brain) to investigate the position of these two species in the phylogenetic variation. Our results further strengthen the finding of covariance between cIN density and increasing brain size and confirm a relationship with neocortical expansion. These results support the emerging view that immature or dormant neurons may represent a reservoir of undifferentiated (stem cell-independent) neuronal cells for the widely expanded cortices of mammals endowed with high order cognitive functions.

Phylogenetic variation of layer II cortical immature neurons in dog and horse confirms covariance with brain size and neocortical surface.

Structural changes involving new neurons can occur through stem cell-driven neurogenesis, and through incorporation of late-maturing "immature" neurons into networks, namely undifferentiated neuronal precursors frozen in a state of arrested maturation. The latter have been found in the cerebral cortex and are particularly abundant in large-brained mammals, covarying with the size of the brain and cortex. Similar cells have been described in the amygdala of some species, although their features and interspecies variation remain poorly understood. Here, their occurrence, number, morphology, molecular expression, age-related changes, and anatomical distribution in amygdala subdivisions were systematically analyzed in eight diverse mammalian species (including mouse, naked mole rat, rabbit, marmoset, cat, sheep, horse, and chimpanzee) widely differing in neuroanatomy, brain size, life span, and socioecology. We identify converging evidence that these amygdala cells are immature neurons and show marked phylogenetic variation, with a significantly greater prevalence in primates. The immature cells are largely located within the amygdala's basolateral complex, a region that has expanded in primate brain evolution in conjunction with cortical projections. In addition, amygdala immature neurons also appear to stabilize in number through adulthood and old age, unlike other forms of plasticity that undergo marked age-related reduction. These results support the emerging view that large brains performing complex socio-cognitive functions rely on wide reservoirs of immature neurons.

Article	Year
Newly Generated and Non-Newly Generated "Immature" Neurons in the Mammalian Brain: A Possible Reservoir of Young Cells to Prevent Brain Aging and Disease? Journal of clinical medicine\| PubMed ID: 31096632	2019
How Widespread Are the "Young" Neurons of the Mammalian Brain? Frontiers in neuroscience\| PubMed ID: 35733930	2022
Consistency and Variation in Doublecortin and Ki67 Antigen Detection in the Brain Tissue of Different Mammals, including Humans. International journal of molecular sciences\| PubMed ID: 36768845	2023
Age-related changes in layer II immature neurons of the murine piriform cortex. Frontiers in cellular neuroscience\| PubMed ID: 37576566	2023
Adult neurogenesis and "immature" neurons in mammals: an evolutionary trade-off in plasticity? Brain structure & function\| PubMed ID: 37833544	2024
From mice to humans: a need for comparable results in mammalian neuroplasticity. Neural regeneration research\| PubMed ID: 38819049	2025
Brain structural plasticity in large-brained mammals: Not only narrowing roads. Neural regeneration research\| PubMed ID: 40145954	2026
Phylogenetic variation of layer II cortical immature neurons in dog and horse confirms covariance with brain size and neocortical surface. Brain structure & function\| PubMed ID: 40622463	2025
Multispecies characterization of immature neurons in the mammalian amygdala reveals their expansion in primates. PLoS biology\| PubMed ID: 40811712	2025

Marco Ghibaudi

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