Angélique Bordey

The astrocyte odyssey.

Neurons have long held the spotlight as the central players of the nervous system, but we must remember that we have equal numbers of astrocytes and neurons in the brain. Are these cells only filling up the space and passively nurturing the neurons, or do they also contribute to information transfer and processing? After several years of intense research since the pioneer discovery of astrocytic calcium waves and glutamate release onto neurons in vitro, the neuronal-glial studies have answered many questions thanks to technological advances. However, the definitive in vivo role of astrocytes remains to be addressed. In addition, it is becoming clear that diverse populations of astrocytes coexist with different molecular identities and specialized functions adjusted to their microenvironment, but do they all belong to the umbrella family of astrocytes? One population of astrocytes takes on a new function by displaying both support cell and stem cell characteristics in the neurogenic niches. Here, we define characteristics that classify a cell as an astrocyte under physiological conditions. We will also discuss the well-established and emerging functions of astrocytes with an emphasis on their roles on neuronal activity and as neural stem cells in adult neurogenic zones.

Nonsynaptic GABA signaling in postnatal subventricular zone controls proliferation of GFAP-expressing progenitors

In the postnatal subventricular zone (SVZ), local cues or signaling molecules released from neuroblasts limit the proliferation of glial fibrillary acidic protein (GFAP)-expressing progenitors thought to be stem cells. However, signals between SVZ cells have not been identified. We show that depolarization of neuroblasts induces nonsynaptic SNARE-independent GABAA receptor currents in GFAP-expressing cells, the time course of which depends on GABA uptake in acute mouse slices. We found that GABAA receptors are tonically activated in GFAP-expressing cells, consistent with the presence of spontaneous depolarizations in neuroblasts that are sufficient to induce GABA release. These data demonstrate the existence of nonsynaptic GABAergic signaling between neuroblasts and GFAP-expressing cells. Furthermore, we show that GABAA receptor activation in GFAP-expressing cells limits their progression through the cell cycle. Thus, as GFAP-expressing cells generate neuroblasts, GABA released from neuroblasts provides a feedback mechanism to control the proliferation of GFAP-expressing progenitors by activating GABAA receptors.

GABA Release and Uptake Regulate Neuronal Precursor Migration in the Postnatal Subventricular Zone

In the postnatal subventricular zone (SVZ), astrocyte-like cells tightly encapsulate chains of migrating neuronal precursors, although an influence of the astrocyte-like cells on precursor migration has not yet been demonstrated. Cell migration was studied in acute sagittal brain slices to determine whether GABA signaling between astrocyte-like cells and neuronal precursors controls the speed of neuronal precursor migration in the anterior SVZ and rostral migratory stream of juvenile and adult mice. Application of GABA at 10 μm, a nondesensitizing concentration for GABAA receptors (GABAARs), reduced the rate (mean of ∼50 μm/hr) of cell migration by 21% via GABAAR activation. Application of the GABAAR antagonist bicuculline enhanced the migration rate by 30%, suggesting that endogenous GABA tonically reduces the speed of cell migration via GABAAR activation. Using immunohistochemistry, we found that astrocyte-like cells express the high-affinity GABA transporter subtype GAT4 on processes ensheathing neuronal precursors that contain GABA. Inhibition of GABA uptake into astrocyte-like cells or enhancement of GABA release from neuronal precursors during high K+ application further reduced the migration rate by increasing ambient GABA levels. GABA altered the migration speed by interfering with intracellular Ca2+ signaling independently of cell depolarization, because high K+ application did not alter the speed of cell migration in the presence of bicuculline. These data indicate that astrocyte-like cells create a microenvironment in which their uniquely positioned GABA transporters control the degree of GABAAR activation and the migration of neuronal precursors.

MicroRNA miR-137 regulates neuronal maturation by targeting ubiquitin ligase Mind Bomb-1

The maturation of young neurons is regulated by complex mechanisms and dysregulation of this process is frequently found in neurodevepmental disorders. MicroRNAs have been implicated in several steps of neuronal maturation including dendritic and axonal growth, spine development, and synaptogenesis. We demonstrate that one brain‐enriched microRNA, miR‐137, has a significant role in regulating neuronal maturation. Overexpression of miR‐137 inhibits dendritic morphogenesis, phenotypic maturation, and spine development both in brain and cultured primary neurons. On the other hand, a reduction in miR‐137 had opposite effects. We further show that miR‐137 targets the Mind bomb one (Mib1) protein through the conserved target site located in the 3′ untranslated region of Mib1 messenger RNA. Mib1 is an ubiquitin ligase known to be important for neurodevelopment. We show that exogenously expressed Mib1 could partially rescue the phenotypes associated with miR‐137 overexpression. These results demonstrate a novel miRNA‐mediated mechanism involving miR‐137 and Mib1 that function to regulate neuronal maturation and dendritic morphogenesis during development. STEM Cells 2010;28:1060–1070

NMDA Receptors Activated by Subventricular Zone Astrocytic Glutamate Are Critical for Neuroblast Survival Prior to Entering a Synaptic Network

Even before integrating into existing circuitry, adult-born neurons express receptors for neurotransmitters, but the intercellular mechanisms and their impact on neurogenesis remain largely unexplored. Here, we show that neuroblasts born in the postnatal subventricular zone (SVZ) acquire NMDA receptors (NMDARs) during their migration to the olfactory bulb. Along their route, neuroblasts are ensheathed by astrocyte-like cells expressing vesicular glutamate release machinery. Increasing calcium in these specialized astrocytes induced NMDAR activity in neuroblasts, and blocking astrocytic vesicular release eliminated spontaneous NMDAR activity. Single-cell knockout of NMDARs using neonatal electroporation resulted in neuroblast apoptosis at the time of NMDAR acquisition. This cumulated in a 40% loss of neuroblasts along their migratory route, demonstrating that NMDAR acquisition is critical for neuroblast survival prior to entering a synaptic network. In addition, our findings suggest an unexpected mechanism wherein SVZ astrocytes use glutamate signaling through NMDARs to control the number of adult-born neurons reaching their final destination.

GABA Depolarizes Neuronal Progenitors of the Postnatal Subventricular Zone Via GABAA Receptor Activation

Previous studies have reported the presence of migrating and dividing neuronal progenitors in the subventricular zone (SVZ) and rostral migratory stream (RMS) of the postnatal mammalian brain. Although the behaviour of these progenitors is thought to be influenced by local signals, the nature and mode of action of the local signals are largely unknown. One of the signalling molecules known to affect the behaviour of embryonic neurons is the neurotransmitter GABA. In order to determine whether GABA affects neuronal progenitors via the activation of specific receptors, we performed cell‐attached, whole‐cell and gramicidin perforated patch‐clamp recordings of progenitors in postnatal mouse brain slices containing either the SVZ or the RMS. Recorded cells displayed a morphology typical of migrating neuronal progenitors had depolarized zero‐current resting potentials, and lacked action potentials. A subset of progenitors contained GABA and stained positive for glutamic acid decarboxylase 67 (GAD‐67) as shown by immunohistochemistry. In addition, every neuronal progenitor responded to GABA via picrotoxin‐sensitive GABAA receptor (GABAAR) activation. GABAARs displayed an ATP‐dependent rundown and a low sensitivity to Zn2+. GABA responses were sensitive to benzodiazepine agonists, an inverse agonist, as well as a barbiturate agonist. While GABA was hyperpolarizing at the zero‐current resting potentials, it was depolarizing at the cell resting potentials estimated from the reversal potential of K+ currents through a cell‐attached patch. Thus, our study demonstrates that neuronal progenitors of the SVZ/RMS contain GABA and are depolarized by GABA, which may constitute the basis for a paracrine signal among neuronal progenitors to dynamically regulate their proliferation and/or migration.

GFAP‐expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes

Neural stem cells in the adult subventricular zone (SVZ) derive from radial glia and express the astroglial marker glial fibrillary acidic protein (GFAP). Thus, they have been termed astrocytes. However, it remains unknown whether these GFAP‐expressing cells express the functional features common to astrocytes. Using immunostaining and patch clamp recordings in acute slices from transgenic mice expressing green fluorescent protein (GFP) driven by the promoter of human GFAP, we show that GFAP‐expressing cells in the postnatal SVZ display typical glial properties shared by astrocytes and prenatal radial glia such as lack of action potentials, hyperpolarized resting potentials, gap junction coupling, connexin 43 expression, hemichannels, a passive current profile, and functional glutamate transporters. GFAP‐expressing cells express both GLAST and GLT‐1 glutamate transporters but lack AMPA‐type glutamate receptors as reported for dye‐coupled astrocytes. However, they lack 100 μM Ba2+‐sensitive inwardly rectifying K+ (KIR) currents expressed by astrocytes, but display delayed rectifying K+ currents and 1 mM Ba2+‐sensitive K+ currents. These currents contribute to K+ transport at rest and maintain hyperpolarized resting potentials. GFAP‐expressing cells stained positive for both KIR2.1 and KIR4.1 channels, two major KIR channels in astrocytes. Ependymal cells, which also derive from radial glia and express GFAP, display typical glial properties and KIR currents consistent with their postmitotic nature. Our results suggest that GFAP‐expressing cells in concert with ependymal cells can perform typical astrocytic functions such as K+ and glutamate buffering in the postnatal SVZ but display a unique set of functional characteristics intermediate between astrocytes and radial glia.

Refuting the challenges of the developmental shift of polarity of GABA actions: GABA more exciting than ever!

During brain development, there is a progressive reduction of intracellular chloride associated with a shift in GABA polarity: GABA depolarizes and occasionally excites immature neurons, subsequently hyperpolarizing them at later stages of development. This sequence, which has been observed in a wide range of animal species, brain structures and preparations, is thought to play an important role in activity-dependent formation and modulation of functional circuits. This sequence has also been considerably reinforced recently with new data pointing to an evolutionary preserved rule. In a recent “Hypothesis and Theory Article,” the excitatory action of GABA in early brain development is suggested to be “an experimental artefact” (Bregestovski and Bernard, 2012). The authors suggest that the excitatory action of GABA is due to an inadequate/insufficient energy supply in glucose-perfused slices and/or to the damage produced by the slicing procedure. However, these observations have been repeatedly contradicted by many groups and are inconsistent with a large body of evidence including the fact that the developmental shift is neither restricted to slices nor to rodents. We summarize the overwhelming evidence in support of both excitatory GABA during development, and the implications this has in developmental neurobiology.

GFAP-GFP neural progenitors are antigenically homogeneous and anchored in their enclosed mosaic niche

Study of the different stages of postnatal neurogenesis relies on using antigenic markers and transgenic mice. In particular, neural stem cells that express GFAP are studied using mice expressing GFP under the human GFAP promoter (GFAP‐GFP). However, it remains unclear whether GFP and the commonly used progenitor markers label different cell populations in the neurogenic subventricular zone (SVZ) and its rostral extension into the olfactory bulb (i.e. rostral migratory stream, RMS). Here, we found that all GFP‐fluorescent cells express GFAP, the radial glia marker brain lipid‐binding protein (BLBP), Lewis X (LeX), and the astrocytic marker GLAST. Faint GFP fluorescence could be detected in a few cells expressing EGF receptors (EGFRs), Olig2, or S100, suggesting that GFAP‐GFP cells generate these diverse cell types. GFP‐fluorescent cells were slowly cycling, as shown by their long‐term retention of BrdU, and less than 10% expressed the proliferative markers Ki67 and Mcm2. The majority of EGFR‐expressing cells and Olig2‐expressing cells were cycling. NG2 and EGFR identified distinct progenitor populations while Olig2 labeled a subset of EGFR‐expressing cells. The entire neurogenic zone contained a mosaic of different cell types and was ensheathed by processes of GFAP‐expressing cells and NG2 cells. Finally, using time‐lapse imaging in acute slices, we show that GFP‐fluorescent cells are stationary within the SVZ. Our findings collectively highlight the cellular mosaic of the neurogenic niche, show that the slowly‐cycling GFAP‐expressing cells are stationary and generate distinct intermediate progenitors.

Single-cell Tsc1 knockout during corticogenesis generates tuber-like lesions and reduces seizure threshold in mice

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by mutations in Tsc1 or Tsc2 that lead to mammalian target of rapamycin (mTOR) hyperactivity. Patients with TSC suffer from intractable seizures resulting from cortical malformations known as tubers, but research into how these tubers form has been limited because of the lack of an animal model. To address this limitation, we used in utero electroporation to knock out Tsc1 in selected neuronal populations in mice heterozygous for a mutant Tsc1 allele that eliminates the Tsc1 gene product at a precise developmental time point. Knockout of Tsc1 in single cells led to increased mTOR activity and soma size in the affected neurons. The mice exhibited white matter heterotopic nodules and discrete cortical tuber-like lesions containing cytomegalic and multinucleated neurons with abnormal dendritic trees resembling giant cells. Cortical tubers in the mutant mice did not exhibit signs of gliosis. Furthermore, phospho-S6 immunoreactivity was not upregulated in Tsc1-null astrocytes despite a lower seizure threshold. Collectively, these data suggest that a double-hit strategy to eliminate Tsc1 in discrete neuronal populations generates TSC-associated cortical lesions, providing a model to uncover the mechanisms of lesion formation and cortical hyperexcitability. In addition, the absence of glial reactivity argues against a contribution of astrocytes to lesion-associated hyperexcitability.