Developmental Formation of the GABAergic and Glycinergic Networks in the Mouse Spinal Cord

Gamma-aminobutyric acid (GABA) and glycine act as inhibitory neurotransmitters. Three types of inhibitory neurons and terminals, GABAergic, GABA/glycine coreleasing, and glycinergic, are orchestrated in the spinal cord neural circuits and play critical roles in regulating pain, locomotive movement, and respiratory rhythms. In this study, we first describe GABAergic and glycinergic transmission and inhibitory networks, consisting of three types of terminals in the mature mouse spinal cord. Second, we describe the developmental formation of GABAergic and glycinergic networks, with a specific focus on the differentiation of neurons, formation of synapses, maturation of removal systems, and changes in their action. GABAergic and glycinergic neurons are derived from the same domains of the ventricular zone. Initially, GABAergic neurons are differentiated, and their axons form synapses. Some of these neurons remain GABAergic in lamina I and II. Many GABAergic neurons convert to a coreleasing state. The coreleasing neurons and terminals remain in the dorsal horn, whereas many ultimately become glycinergic in the ventral horn. During the development of terminals and the transformation from radial glia to astrocytes, GABA and glycine receptor subunit compositions markedly change, removal systems mature, and GABAergic and glycinergic action shifts from excitatory to inhibitory.

FASN-dependent de novo lipogenesis is required for brain development

Fate and behavior of neural progenitor cells are tightly regulated during mammalian brain development. Metabolic pathways, such as glycolysis and oxidative phosphorylation, that are required for supplying energy and providing molecular building blocks to generate cells govern progenitor function. However, the role of de novo lipogenesis, which is the conversion of glucose into fatty acids through the multienzyme protein fatty acid synthase (FASN), for brain development remains unknown. Using Emx1Cre-mediated, tissue-specific deletion of Fasn in the mouse embryonic telencephalon, we show that loss of FASN causes severe microcephaly, largely due to altered polarity of apical, radial glia progenitors and reduced progenitor proliferation. Furthermore, genetic deletion and pharmacological inhibition of FASN in human embryonic stem cell–derived forebrain organoids identifies a conserved role of FASN-dependent lipogenesis for radial glia cell polarity in human brain organoids. Thus, our data establish a role of de novo lipogenesis for mouse and human brain development and identify a link between progenitor-cell polarity and lipid metabolism.

Study of the glial cytoarchitecture of the developing olfactory bulb of a shark using immunochemical markers of radial glia

During development of the olfactory bulb (OB), glial cells play key roles in axonal guiding/targeting, glomerular formation and synaptic plasticity. Studies in mammals have shown that radial glial cells and peripheral olfactory glia (olfactory ensheathing cells, OECs) are involved in the development of the OB. Most studies about the OB glia were carried out in mammals, but data are lacking in most non-mammalian vertebrates. In the present work, we studied the development of the OB glial system in the cartilaginous fish Scyliorhinus canicula (catshark) using antibodies against glial markers, such as glial fibrillary acidic protein (GFAP), brain lipid-binding protein (BLBP), and glutamine synthase (GS). These glial markers were expressed in cells with radial morphology lining the OB ventricle of embryos and this expression continues in ependymal cells (tanycytes) in early juveniles. Astrocyte-like cells were also observed in the granular layer and surrounding glomeruli. Numerous GS-positive cells were present in the primary olfactory pathway of embryos. In the developmental stages analysed, the olfactory nerve layer and the glomerular layer were the regions with higher GFAP, BLBP and GS immuno-reactivity. In addition, numerous BLBP-expressing cells (a marker of mammalian OECs) showing proliferative activity were present in the olfactory nerve layer. Our findings suggest that glial cells of peripheral and central origin coexist in the OB of catshark embryos and early juveniles. These results open the path for future studies about the differential roles of glial cells in the catshark OB during embryonic development and in adulthood.

Developmental Formation of the GABAergic and Glycinergic Networks in the Mouse Spinal Cord

Gamma-aminobutyric acid (GABA) and glycine act as inhibitory neurotransmitters. Three types of inhibitory neurons and terminals, GABAergic, GABA/glycine co-releasing, and glycinergic, are orchestrated in the spinal cord neural circuits and play key roles in the regulation of pain, locomotive movement, and respiratory rhythms. Herein, we first describe GABAergic and glycinergic transmission and inhibitory networks, which consist of three types of terminals, in the mature mouse spinal cord. Second, we describe the developmental formation of GABAergic and glycinergic networks, with specific focus on the differentiation of neurons, formation of synapses, maturation of removal systems, and changes in their action. GABAergic and glycinergic neurons are derived from the same domains of the ventricular zone. Initially, GABAergic neurons are differentiated and their axons form synapses. Some of these neurons remain GABAergic in lamina I and II. Many of GABAergic neurons convert to co-releasing state. The co-releasing neurons and terminals remain in the dorsal horn, whereas many of co-releasing ones ultimately become glycinergic in the ventral horn. During the development of terminals and the transformation from radial glia to astrocytes, GABA and glycine receptor subunit compositions markedly change, removal systems mature, and GABAergic and glycinergic action shifts from excitatory to inhibitory.

Excitatory neurotransmission activates compartmentalized calcium transients in Müller glia without affecting lateral process motility

Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here we explore this phenomenon in Müller glia, which are specialized radial astroglia that are the predominant glial type of the vertebrate retina. Müller glia extend fine filopodia-like processes into retinal synaptic layers, in similar fashion to brain astrocytes and radial glia which exhibit perisynaptic processes. Using two-photon volumetric imaging, we found that during the second postnatal week, Müller glial processes were highly dynamic, with rapid extensions and retractions that were mediated by cytoskeletal rearrangements. During this same stage of development, retinal waves led to increases in cytosolic calcium within Müller glial lateral processes and stalks. These comprised distinct calcium compartments, distinguished by variable participation in waves, timing, and sensitivity to an M1 muscarinic acetylcholine receptor antagonist. However, we found that motility of lateral processes was unaffected by the presence of pharmacological agents that enhanced or blocked wave-associated calcium transients. Finally, we found that mice lacking normal cholinergic waves in the first postnatal week also exhibited normal Müller glial process morphology. Hence, outgrowth of Müller glial lateral processes into synaptic layers is determined by factors that are independent of neuronal activity.

Calcium Signaling in the Cerebellar Radial Glia and Its Association with Morphological Changes during Zebrafish Development

Radial glial cells are a distinct non-neuronal cell type that, during development, span the entire width of the brain walls of the ventricular system. They play a central role in the origin and placement of neurons, since their processes form structural scaffolds that guide and facilitate neuronal migration. Furthermore, glutamatergic signaling in the radial glia of the adult cerebellum (i.e., Bergmann glia), is crucial for precise motor coordination. Radial glial cells exhibit spontaneous calcium activity and functional coupling spread calcium waves. However, the origin of calcium activity in relation to the ontogeny of cerebellar radial glia has not been widely explored, and many questions remain unanswered regarding the role of radial glia in brain development in health and disease. In this study we used a combination of whole mount immunofluorescence and calcium imaging in transgenic (gfap-GCaMP6s) zebrafish to determine how development of calcium activity is related to morphological changes of the cerebellum. We found that the morphological changes in cerebellar radial glia are quite dynamic; the cells are remarkably larger and more elaborate in their soma size, process length and numbers after 7 days post fertilization. Spontaneous calcium events were scarce during the first 3 days of development and calcium waves appeared on day 5, which is associated with the onset of more complex morphologies of radial glia. Blockage of gap junction coupling inhibited the propagation of calcium waves, but not basal local calcium activity. This work establishes crucial clues in radial glia organization, morphology and calcium signaling during development and provides insight into its role in complex behavioral paradigms.

Overexpression of Lin28A in neural progenitor cells in vivo does not lead to brain tumor formation but results in reduced spine density

LIN28A overexpression has been identified in malignant brain tumors called embryonal tumors with multilayered rosettes (ETMR) but its specific role during brain development remains largely unknown. Radial glia cells of the ventricular zone (VZ) are proposed as a cell of origin for ETMR. We asked whether an overexpression of LIN28A in such cells might affect brain development or result in the formation of brain tumors.Constitutive overexpression of LIN28A in hGFAP-cre::lsl-Lin28A (GL) mice led to a transient increase of proliferation in the cortical VZ at embryonic stages but no postnatal brain tumor formation. Postnatally, GL mice displayed a pyramidal cell layer dispersion of the hippocampus and altered spine and dendrite morphology, including reduced dendritic spine densities in the hippocampus and cortex. GL mice displayed hyperkinetic activity and differential quantitative MS-based proteomics revealed altered time dependent molecular functions regarding mRNA processing and spine morphogenesis. Phosphoproteomic analyses indicated a downregulation of mTOR pathway modulated proteins such as Map1b being involved in microtubule dynamics.In conclusion, we show that Lin28A overexpression transiently increases proliferation of neural precursor cells but it is not sufficient to drive brain tumors in vivo. In contrast, Lin28A impacts on protein abundancy patterns related to spine morphogenesis and phosphorylation levels of proteins involved in microtubule dynamics, resulting in decreased spine densities of neurons in the hippocampus and cortex as well as in altered behavior. Our work provides new insights into the role of LIN28A for neuronal morphogenesis and development and may reveal future targets for treatment of ETMR patients.

The Effect of Opiates on the Developing Cerebral Cortex

<p>Opiate drugs, such as codeine, morphine and heroin are powerful analgesics and drugs of abuse. The unborn child is invariably exposed to opiate drugs as a consequence of maternal use. Studies that have investigated the impact of opiate drugs demonstrated opioid system expression in proliferating regions of the developing brain, as well as on proliferative astroglia taken from the developing central nervous system. The effects of opiates on astroglial proliferation (largely mediated by the mu opioid receptor) are predominantly inhibitory, but are extremely context dependent. This context dependency exists because of the complexity resident within the opioid signalling system. However, since this previous research was conducted, there has been impressive progress made in the field of developmental neurobiology with the demonstration that cells of astrocytic lineage are responsible for the generation of the central nervous system. It was therefore the aim of the current research project to investigate the developmental impact of opiate exposure in the context of the foetal mouse cerebral cortex. This aim was divided into 3 separate aims that comprised of; determining the cellular localisation of the mu opioid receptor, the effects of opiate exposure on cortical progenitor cells, and to determine the effect of opiate exposure on the development of the cerebral cortex itself. The mu opioid receptor was expressed on proliferative radial glia of both the embryonic day 15.5 (neurogenic) and embryonic day 18.5 (gliogenic) ventricular zone of the dorsal forebrain. Interestingly and significantly, the mu opioid receptor-positive glia observed in the embryonic day 18.5 mouse forebrain were also observed at a comparable developmental stage in the foetal human forebrain. Morphine exposure slowed down G2 phase of the cell cycle at embryonic day 15.5 in the neurogenic murine cortical ventricular zone. This opiate-induced slowing in cell cycle progression was shown not to impact on proliferation in the ventricular zone, although future research should address whether this perturbation altered differentiation or developmental maturation of the radial glia. Morphine exposure throughout corticogenesis decreased levels of doublecortin expression (a migratory neuronal marker) at the end of gestation. Postnatally, mice exposed to morphine during corticogenesis also showed decreased numbers of neurons in layer V of the cerebral cortex. Collectively, this thesis presents the first evidence that shows morphine affects cortical progenitor cells in vivo. This research supports the possibility that the opioid system plays an endogenous role in corticogenesis. The clinical significance is morphine has the potential to perturb normal development of the cerebral cortex.</p>

The Effect of Opiates on the Developing Cerebral Cortex

<p>Opiate drugs, such as codeine, morphine and heroin are powerful analgesics and drugs of abuse. The unborn child is invariably exposed to opiate drugs as a consequence of maternal use. Studies that have investigated the impact of opiate drugs demonstrated opioid system expression in proliferating regions of the developing brain, as well as on proliferative astroglia taken from the developing central nervous system. The effects of opiates on astroglial proliferation (largely mediated by the mu opioid receptor) are predominantly inhibitory, but are extremely context dependent. This context dependency exists because of the complexity resident within the opioid signalling system. However, since this previous research was conducted, there has been impressive progress made in the field of developmental neurobiology with the demonstration that cells of astrocytic lineage are responsible for the generation of the central nervous system. It was therefore the aim of the current research project to investigate the developmental impact of opiate exposure in the context of the foetal mouse cerebral cortex. This aim was divided into 3 separate aims that comprised of; determining the cellular localisation of the mu opioid receptor, the effects of opiate exposure on cortical progenitor cells, and to determine the effect of opiate exposure on the development of the cerebral cortex itself. The mu opioid receptor was expressed on proliferative radial glia of both the embryonic day 15.5 (neurogenic) and embryonic day 18.5 (gliogenic) ventricular zone of the dorsal forebrain. Interestingly and significantly, the mu opioid receptor-positive glia observed in the embryonic day 18.5 mouse forebrain were also observed at a comparable developmental stage in the foetal human forebrain. Morphine exposure slowed down G2 phase of the cell cycle at embryonic day 15.5 in the neurogenic murine cortical ventricular zone. This opiate-induced slowing in cell cycle progression was shown not to impact on proliferation in the ventricular zone, although future research should address whether this perturbation altered differentiation or developmental maturation of the radial glia. Morphine exposure throughout corticogenesis decreased levels of doublecortin expression (a migratory neuronal marker) at the end of gestation. Postnatally, mice exposed to morphine during corticogenesis also showed decreased numbers of neurons in layer V of the cerebral cortex. Collectively, this thesis presents the first evidence that shows morphine affects cortical progenitor cells in vivo. This research supports the possibility that the opioid system plays an endogenous role in corticogenesis. The clinical significance is morphine has the potential to perturb normal development of the cerebral cortex.</p>