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The mature cerebellum controls motor skill precision and participates in other sophisticated brain functions that include learning, cognition, and speech. Different types of GABAergic and glutamatergic cerebellar neurons originate in temporal order from two progenitor niches, the ventricular zone and rhombic lip, which express the transcription factors Ptf1a and Atoh1, respectively. However, the molecular machinery required to specify the distinct neuronal types emanating from these progenitor zones is still unclear. Here, we uncover the transcription factor Olig3 as a major determinant in generating the earliest neuronal derivatives emanating from both progenitor zones in mice. In the rhombic lip, Olig3 regulates progenitor cell proliferation. In the ventricular zone, Olig3 safeguards Purkinje cell specification by curtailing the expression of Pax2, a transcription factor that suppresses the Purkinje cell differentiation program. Our work thus defines Olig3 as a key factor in early cerebellar development.

Brain-derived neurotrophic factor (BDNF) is one of the key signaling molecules that supports the viability of neural cells in various brain pathologies, and can be considered a potential therapeutic agent. However, several methodological difficulties, such as overcoming the blood–brain barrier and the short half-life period, challenge the potential use of BDNF in clinical practice. Gene therapy could overcome these limitations. Investigating the influence of viral vectors on the neural network level is of particular interest because viral overexpression affects different aspects of cell metabolism and interactions between neurons. The present work aimed to investigate the influence of the adeno-associated virus (AAV)-Syn-BDNF-EGFP virus construct on neural network activity parameters in an acute hypobaric hypoxia model in vitro. Materials and methods. An adeno-associated virus vector carrying the BDNF gene was constructed using the following plasmids: AAV-Syn-EGFP, pDP5, DJvector, and pHelper. The developed virus vector was then tested on primary hippocampal cultures obtained from C57BL/6 mouse embryos (E18). Acute hypobaric hypoxia was induced on day 21 in vitro. Spontaneous bioelectrical and calcium activity of neural networks in primary cultures and viability tests were analysed during normoxia and during the posthypoxic period. Results. BDNF overexpression by AAV-Syn-BDNF-EGFP does not affect cell viability or the main parameters of spontaneous bioelectrical activity in normoxia. Application of the developed virus construct partially eliminates the negative hypoxic consequences by preserving cell viability and maintaining spontaneous bioelectrical activity in the cultures. Moreover, the internal functional structure, including the activation pattern of network bursts, the number of hubs, and the number of connections within network elements, is also partially preserved. BDNF overexpression prevents a decrease in the number of cells exhibiting calcium activity and maintains the frequency of calcium oscillations. Conclusion. This study revealed the pronounced antihypoxic and neuroprotective effects of AAV-Syn-BDNF-EGFP virus transduction in an acute normobaric hypoxia model.

Glial cell line-derived neurotrophic factor (GDNF) has a pronounced neuroprotective effect in various nervous system pathologies, including ischaemic brain damage and neurodegenerative diseases. In this work, we studied the effect of GDNF on the ultrastructure and functional activity of neuron-glial networks during acute hypoxic exposure, a key damaging factor in numerous brain pathologies. We analysed the molecular mechanisms most likely involved in the positive effects of GDNF. Hypoxia modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). GDNF (1 ng/ml) was added to the culture medium 20 min before oxygen deprivation. Acute hypoxia-induced irreversible changes in the ultrastructure of neurons and astrocytes led to the loss of functional Сa2+ activity and neural network disruption. Destructive changes in the mitochondrial apparatus and its functional activity characterized by an increase in the basal oxygen consumption rate and respiratory chain complex II activity during decreased stimulated respiration intensity were observed 24 hours after hypoxic injury. At a concentration of 1 ng/ml, GDNF maintained the functional metabolic network activity in primary hippocampal cultures and preserved the structure of the synaptic apparatus and number of mature chemical synapses, confirming its neuroprotective effect. GDNF maintained the normal structure of mitochondria in neuronal outgrowth but not in the soma. Analysis of the possible GDNF mechanism revealed that RET kinase, a component of the receptor complex, and the PI3K/Akt pathway are crucial for the neuroprotective effect of GDNF. The current study also revealed the role of GDNF in the regulation of HIF-1α transcription factor expression under hypoxic conditions.

Alzheimer’s disease (AD) is a widespread chronic neurodegenerative pathology characterized by synaptic dysfunction, partial neuronal death, cognitive decline and memory impairments. The major hallmarks of AD are extracellular senile amyloid plaques formed by various types of amyloid proteins (Aβ) and the formation and accumulation of intracellular neurofibrillary tangles. However, there is a lack of relevant experimental models for studying changes in neural network activity, the features of intercellular signaling or the effects of drugs on the functional activity of nervous cells during AD development. In this work, we examined two experimental models of amyloidopathy using primary hippocampal cultures. The first model involves the embryonic brains of 5xFAD mice; the second uses chronic application of amyloid beta 1-42 (Aβ1-42). The model based on primary hippocampal cells obtained from 5xFAD mice demonstrated changes in spontaneous network calcium activity characterized by a decrease in the number of cells exhibiting Ca 2+ activity, a decrease in the number of Ca 2+ oscillations and an increase in the duration of Ca 2+ events from day 21 of culture development in vitro . Chronic application of Aβ1-42 resulted in the rapid establishment of significant neurodegenerative changes in primary hippocampal cultures, leading to marked impairments in neural network calcium activity and increased cell death. Using this model and multielectrode arrays, we studied the influence of amyloidopathy on spontaneous bioelectrical neural network activity in primary hippocampal cultures. It was shown that chronic Aβ application decreased the number of network bursts and spikes in a burst. The spatial structure of neural networks was also disturbed that characterized by reduction in both the number of key network elements (hubs) and connections between network elements. Moreover, application of brain-derived neurotrophic factor (BDNF) recombinant protein and BDNF hyperexpression by an adeno-associated virus vector partially prevented these amyloidopathy-induced neurodegenerative phenomena. BDNF maintained cell viability and spontaneous bioelectrical and calcium network activity in primary hippocampal cultures.

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Table 1. Primer sequences for real-time polymerase chain reaction (RT-PCR). Oaz1Forward 5'-aaggacagttttgcagctctcc-3' Reverse 5'-tctgtcctcacggttcttggg-3' Grik1Forward 5'-ggaggatgaggcggggacc-3' Reverse 5'-gcatgctcttcgggaggcttcaaaac-3' Grik2Forward 5'-ggatgggaaatatggagcccaggatgat-3' Reverse 5'-tcaggggagagaggattcaggaaggag-3' Grin2aForward 5'-tgacggtgagatggaagagctg-3' Reverse 5'-ctgccatgttgtcgatgtccag-3' Grin2bForward 5'-gtcctccaaagacacgagcac-3' Reverse 5'-gccctcctccctctcaatagc-3' Gria1Forward 5'-aggggaatgtggaagcaaggac-3' Reverse 5'-ccaatcccagccctccaatcag-3' Gria2Forward 5'-agccaaggactcgggaagtaagg-3' Reverse 5'-caccagcattgccaaaccaagg-3' Camk2aForward 5'-tctggttactggtggggaactg-3' Reverse 5'-tagcacagcctccaagatctgc-3' Casp3Forward 5'-tcagaggcgactactgccggag-3' Reverse 5'-cgtgagcatggacacaatacacgggt-3' JNK Forward 5'-ggctcaggagctcaaggaatagtgtgt-3' Reverse 5'-cagcagagtgaaggtgcttgattccac-3' Prkca Forward 5'-ccaacgactccacggcgtctc-3' Reverse 5'-tgcttgtgaacattcatgtcgcaggtgt-3' Prkce Forward 5'-tgatcatcgatctctcgggatcatcggg-3' Reverse 5'-gcccacctcgtcaggggtttc-3' Prkcg Forward 5'-tggttcaccgccgatgccac-3' Reverse 5'-ccgcaaagggagggcacg-3' Pik3aForward 5'-ctgagatgggagctgggactgc-3' Reverse 5'-gtgtccacgtgttagacagaacactg-3' Pik3bForward 5'-gaggttatgagtgtgcttccgccctat-3' Reverse 5'-agtcttcgtgtttcgtcttccagttcctc-3' Pik3gForward 5'-gctgcggagttctaccaccgattg-3' Reverse 5'-caggtagtctgggagaggtttggacg-3'

The mature cerebellum controls motor skill precision and participates in other sophisticated brain functions that include learning, cognition and speech. Different types of GABAergic and glutamatergic cerebellar neurons originate in temporal order from two progenitor niches, the ventricular zone and rhombic lip, which express the transcription factors Ptf1a and Atoh1, respectively. However, the molecular machinery required to specify the distinct neuronal types emanating from these progenitor zones is still unclear. Here, we uncover the transcription factor Olig3 as a major determinant in generating the earliest neuronal derivatives emanating from both progenitor zones. In the rhombic lip, Olig3 regulates progenitor cell proliferation. In the ventricular zone, Olig3 safeguards Purkinje cell specification by curtailing the expression of Pax2, a transcription factor that we found to impose an inhibitory interneuron identity. Our work thus defines Olig3 as a key factor in cerebellar development.