Cerebral ("brain") organoids are high-fidelity in vitro cellular models of the developing brain, which makes them one of the go-to methods to study isolated processes of tissue organization and its electrophysiological properties, allowing to collect invaluable data for in silico modeling neurodevelopmental processes. Complex computer models of biological systems supplement in vivo and in vitro experimentation and allow researchers to look at things that no laboratory study has access to, due to either technological or ethical limitations. In this paper, we present the Biological Cellular Neural Network Modeling (BCNNM) framework designed for building dynamic spatial models of neural tissue organization and basic stimulus dynamics. The BCNNM uses a convenient predicate description of sequences of biochemical reactions and can be used to run complex models of multi-layer neural network formation from a single initial stem cell. It involves processes such as proliferation of precursor cells and their differentiation into mature cell types, cell migration, axon and dendritic tree formation, axon pathfinding and synaptogenesis. The experiment described in this article demonstrates a creation of an in silico cerebral organoid-like structure, constituted of up to 1 million cells, which differentiate and self-organize into an interconnected system with four layers, where the spatial arrangement of layers and cells are consistent with the values of analogous parameters obtained from research on living tissues. Our in silico organoid contains axons and millions of synapses within and between the layers, and it comprises neurons with high density of connections (more than 10). In sum, the BCNNM is an easy-to-use and powerful framework for simulations of neural tissue development that provides a convenient way to design a variety of tractable in silico experiments.

Reptiles have long been studied in search of the mechanisms behind neuronal regeneration. This thesis delves into the regenerative areas of two emerging model species to the field of regenerative research: Pogona vitticeps (bearded dragon) and Pantherophis guttatus (corn snake). This fluorescent immunohistochemical study maps out and compares the constitutive proliferative zones in these two species to better define the focus of future comparative neurodegenerative experiments. A BrdU pulse chase experiment in conjunction with PCNA reveals proliferative zones in the lateral ventricular ependyma of both species. Stem cell niches were found in the ependymal lining adjacent to the medial cortex and dorsal ventricular ridge in both species, however, the nucleus sphericus ependyma was an active proliferative zone only in Pantherophis. Imaging of further markers in this study support the findings of the pulse chase experiment. High levels of the stem cell marker Sox2 was found in lateral ventricular ependymal cells in both species. The glial marker GFAP reveals a highly ordered array of radial glia in the cortical areas of Pogona, which is significantly reduced or absent in Pantherophis. And lastly the neuronal marker HU was found in the same cells that were BrdU positive and had migrated a short distance from the proliferative zones, which shows that the proliferative areas in the lateral ventricular lining do indeed produce neurons. The BrdU and PCNA marked cells were quantified in both species, and a brief comparison between the species showed that Pogona had a significantly higher number and concentration of proliferative cells in the proliferative zones than Pantherophis. Scattered BrdU positive cells that were neither neuronal nor positive for any other marker were also found scattered throughout the parenchyma of Pogona, and these cells remain uncharacterized. Differences between these two species are not surprising, as lizards are known to have better regenerative capabilities than snakes, however, more comparative research between these species is needed to gain further insight into the mechanisms behind their contrasting regenerative capabilities.

Hydrocephalus can occur in children alone or in combination with other neurodevelopmental disorders that are often associated with brain overgrowth. Despite the severity of these disorders, the molecular and cellular mechanisms underlying these pathologies and their comorbidity are poorly understood. Here, we studied the consequences of genetically inactivating in mice dual-specificity phosphatase 16 (Dusp16), which is known to negatively regulate mitogen-activated protein kinases (MAPKs) and which has never previously been implicated in brain development and disorders. Mouse mutants lacking a functional Dusp16 gene (Dusp16 =) developed fully-penetrant congenital obstructive hydrocephalus together with brain overgrowth. The midbrain aqueduct in Dusp16 = mutants was obstructed during mid-gestation by an expansion of neural progenitors, and during later gestational stages by neurons resulting in a blockage of cerebrospinal fluid (CSF) outflow. In contrast, the roof plate and ependymal cells developed normally. We identified a delayed cell cycle exit of neural progenitors in Dusp16 = mutants as a cause of progenitor overproliferation during midgestation. At later gestational stages, this expanded neural progenitor pool generated an increased number of neurons associated with enlarged brain volume. Taken together, we found that Dusp16 plays a critical role in neurogenesis by balancing neural progenitor cell proliferation and neural differentiation. Moreover our results suggest that a lack of functional Dusp16 could play a central role in the molecular mechanisms linking brain overgrowth and hydrocephalus.

Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.

The Substantia Nigra pars reticulata (SNpr) is the major information output site of the basal ganglia network and instrumental for the activation and adjustment of movement, regulation of the behavioral state and response to reward. Due to both overlapping and unique input and output connections, the SNpr might also have signal integration capacity and contribute to action selection. How the SNpr regulates these multiple functions remains incompletely understood. The SNpr is located in the ventral midbrain and is composed primarily of inhibitory GABAergic projection neurons that are heterogeneous in their properties. In addition, the SNpr contains smaller populations of other neurons, including glutamatergic neurons. Here, we discuss regionalization of the SNpr, in particular the division of the SNpr neurons to anterior (aSNpr) and posterior (pSNpr) subtypes, which display differences in many of their features. We hypothesize that unique developmental and molecular characteristics of the SNpr neuron subtypes correlate with both region-specific connections and notable functional specializations of the SNpr. Variation in both the genetic control of the SNpr neuron development as well as signals regulating cell migration and axon guidance may contribute to the functional diversity of the SNpr neurons. Therefore, insights into the various aspects of differentiation of the SNpr neurons can increase our understanding of fundamental brain functions and their defects in neurological and psychiatric disorders, including movement and mood disorders, as well as epilepsy.

The aim of this study was to explore the functions of T-type calcium channels, and their possible role in neuronal stem cells migration. The role of T-type calcium channel in mature brain is known to be in producing electroencephalographic oscillations. This action in turn is the key factor in some neuronal physiological and pathophysiological functions, like non-REM sleep, memory, learning and absence epilepsy. In addition, T-type calcium channels have peripheral actions, but this study concerns on its neuronal functions. This low-voltage activated channels functions in neurogenesis is less known than its role in mature brain. It is known to promote neuronal proliferation and differentiation, but what comes to its possible actions in neuronal migration, is poorly studied. This study shows some evidence of T-type calcium channel taking part in neuronal migration in mice embryonic subventricular zones progenitor cells. Selective T-type antagonists, ethosuximide, nickelchloride and a scorpion peptide toxin kurtoxin, decreased the rate of migration in differentiating progenitor cells. This study consists of a literature review and an experimental part. Another aim of this study is to consider an alternative approach to stem cell therapies based on invasive transplantation of the cells. This other attempt is non-invasive manipulating of endogenous stem cells to proliferate and migrate to the injured or depleted area in the brain, differentiate into a desired phenotype and stop their division after they have completed their mission. Non-invasive altering of the stem cells is awaiting pharmacological solutions to resolve the problems being faced in this effort. There are some non-invasive therapies already being used successfully to cure pathological conditions such as spinal cord injury. These methods could be used as well in stem cell based therapies in the treatment of neurodegenerative diseases and brain injuries. These methods are still in the beginning of their way and lacking the full understanding of the key factors that affect neuronal development. These factors include some important endogenous inducing and inhibiting substances. One of the most important inducing substances is calcium ion regulating a variety of events in neurogenesis. T-type calcium channel, as being widely expressed during early brain development, and decaying by neuronal maturation, might have a pivotal role in conducting progenitor cells.