Xiaoqing Yuan

Postnatal NG2 proteoglycan–expressing progenitor cells are intrinsically multipotent and generate functional neurons

Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan–positive progenitor cells that express the 2′,3′-cyclic nucleotide 3′-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

Expression of the green fluorescent protein in the oligodendrocyte lineage: a transgenic mouse for developmental and physiological studies.

We generated a transgenic mouse expressing the enhanced green fluorescent protein (EGFP) under the control of the 2′‐3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) promoter. EGFP+ cells were visualized in live tissue throughout embryonic and postnatal development. Immunohistochemical analysis in brain tissue and in sciatic nerve demonstrated that EGFP expression was restricted to cells of the oligodendrocyte and Schwann cell lineages. EGFP was also strongly expressed in “adult” oligodendrocyte progenitors (OPs) and in gray matter oligodendrocytes. Fluorescence‐activated cell sorting allowed high‐yield purification of EGFP+ oligodendrocyte‐lineage cells from transgenic brains. Electrophysiological patch clamp recordings of EGFP+ cells in situ demonstrated that OP cells displayed large outward tetraethylammonium (TEA)‐sensitive K+ currents and very small inward currents, whereas mature oligodendrocytes were characterized by expression of large inward currents and small outward K+ currents. The proliferation rate of EGFP+ cells in developing white matter decreased with the age of the animals and was strongly inhibited by TEA. Oligodendrocyte development and physiology can be studied in live tissue of CNP‐EGFP transgenic mice, which represent a source of pure EGFP+ oligodendrocyte‐lineage cells throughout development.

A Blueprint for the Spatiotemporal Origins of Mouse Hippocampal Interneuron Diversity

Although vastly outnumbered, inhibitory interneurons critically pace and synchronize excitatory principal cell populations to coordinate cortical information processing. Precision in this control relies upon a remarkable diversity of interneurons primarily determined during embryogenesis by genetic restriction of neuronal potential at the progenitor stage. Like their neocortical counterparts, hippocampal interneurons arise from medial and caudal ganglionic eminence (MGE and CGE) precursors. However, while studies of the early specification of neocortical interneurons are rapidly advancing, similar lineage analyses of hippocampal interneurons have lagged. A “hippocampocentric” investigation is necessary as several hippocampal interneuron subtypes remain poorly represented in the neocortical literature. Thus, we investigated the spatiotemporal origins of hippocampal interneurons using transgenic mice that specifically report MGE- and CGE-derived interneurons either constitutively or inducibly. We found that hippocampal interneurons are produced in two neurogenic waves between E9–E12 and E12–E16 from MGE and CGE, respectively, and invade the hippocampus by E14. In the mature hippocampus, CGE-derived interneurons primarily localize to superficial layers in strata lacunosum moleculare and deep radiatum, while MGE-derived interneurons readily populate all layers with preference for strata pyramidale and oriens. Combined molecular, anatomical, and electrophysiological interrogation of MGE/CGE-derived interneurons revealed that MGE produces parvalbumin-, somatostatin-, and nitric oxide synthase-expressing interneurons including fast-spiking basket, bistratified, axo-axonic, oriens-lacunosum moleculare, neurogliaform, and ivy cells. In contrast, CGE-derived interneurons contain cholecystokinin, calretinin, vasoactive intestinal peptide, and reelin including non-fast-spiking basket, Schaffer collateral-associated, mossy fiber-associated, trilaminar, and additional neurogliaform cells. Our findings provide a basic blueprint of the developmental origins of hippocampal interneuron diversity.

Voltage-activated K+ channels and membrane depolarization regulate accumulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(CIP1) in glial progenitor cells.

Neural cell development is regulated by membrane ion channel activity. We have previously demonstrated that cell membrane depolarization with veratridine or blockage of K+channels with tetraethylammonium (TEA) inhibit oligodendrocyte progenitor (OP) proliferation and differentiation (Knutson et al., 1997); however the molecular events involved are largely unknown. Here we show that forskolin (FSK) and its derivative dideoxyforskolin (DFSK) block K+ channels in OPs and inhibit cell proliferation. The antiproliferative effects of TEA, FSK, DFSK, and veratridine were attributable to OP cell cycle arrest in G1 phase. In fact, (1) cyclin D accumulation in synchronized OP cells was not affected by K+ channel blockers or veratridine; (2) these agents prevented OP cell proliferation only if present during G1 phase; and (3) G1 blockers, such as rapamycin and deferoxamine, mimicked the anti-proliferative effects of K+channel blockers. DFSK also prevented OP differentiation, whereas FSK had no effect. Blockage of K+ channels and membrane depolarization also caused accumulation of the cyclin-dependent kinase inhibitors p27Kip1 and p21CIP1 in OP cells. The antiproliferative effects of K+channel blockers and veratridine were still present in OP cells isolated from INK4a−/− mice, lacking the cyclin-dependent kinase inhibitors p16INK4a and p19ARF. Our results demonstrate that blockage of K+ channels and cell depolarization induce G1 arrest in the OP cell cycle through a mechanism that may involve p27Kip1 and p21CIP1 and further support the conclusion that OP cell cycle arrest and differentiation are two uncoupled events.

mGluR7 undergoes rapid internalization in response to activation by the allosteric agonist AMN082

The G-protein coupled receptor (GPCR) metabotropic glutamate receptor 7 (mGluR7) is widely expressed throughout the nervous system and is implicated in diverse physiological processes ranging from synaptic plasticity to neuroprotection. To date, unequivocally assigning specific functions to mGluR7 has been hampered by a lack of specific pharmacological tools, however, an mGluR7 specific allosteric agonist, AMN082, was recently discovered. Accumulating evidence indicates that in addition to G-protein activation, GPCRs trigger critical intracellular signalling cascades during agonist-induced internalization. Thus, to determine if AMN082 will be useful for evaluating signalling events related to mGluR7 internalization as well as receptor activation we have examined whether AMN082 induces mGluR7 endocytosis. Using an immunofluorescence assay we demonstrate that AMN082 induces robust internalization of mGluR7 overexpressed in dissociated hippocampal neurons. AMN082-induced mGluR7 internalization was resistant to inhibition by a competitive antagonist consistent with the distinct binding site of the allosteric agonist from the glutamate-binding pocket utilized by conventional orthosteric ligands. Finally, as an independent assay of receptor internalization we overexpressed N-terminal pHluorin-tagged mGluR7 in neurons, allowing live imaging of surface receptors in real time. AMN082 treatment produced a rapid loss of surface mGluR7 as indicated by decreased fluorescence confirming the ability of allosteric receptor activation to trigger mGluR7 endocytosis. Thus, AMN082 will be effective for investigating physiological processes related to both mGluR7 activation and internalization such as control of bidirectional plasticity at mossy fiber-st. lucidum interneuron synapses.

Pentraxins Coordinate Excitatory Synapse Maturation and Circuit Integration of Parvalbumin Interneurons.

Circuit computation requires precision in the timing, extent, and synchrony of principal cell (PC) firing that is largely enforced by parvalbumin-expressing, fast-spiking interneurons (PVFSIs). To reliably coordinate network activity, PVFSIs exhibit specialized synaptic and membrane properties that promote efficient afferent recruitment such as expression of high-conductance, rapidly gating, GluA4-containing AMPA receptors (AMPARs). We found that PVFSIs upregulate GluA4 during the second postnatal week coincident with increases in the AMPAR clustering proteins NPTX2 and NPTXR. Moreover, GluA4 is dramatically reduced in NPTX2(-/-)/NPTXR(-/-) mice with consequent reductions in PVFSI AMPAR function. Early postnatal NPTX2(-/-)/NPTXR(-/-) mice exhibit delayed circuit maturation with a prolonged critical period permissive for giant depolarizing potentials. Juvenile NPTX2(-/-)/NPTXR(-/-) mice display reduced feedforward inhibition yielding a circuit deficient in rhythmogenesis and prone to epileptiform discharges. Our findings demonstrate an essential role for NPTXs in controlling network dynamics highlighting potential therapeutic targets for disorders with inhibition/excitation imbalances such as schizophrenia.

Dual origins of functionally distinct O-LM interneurons revealed by differential 5-HT 3A R expression

Forebrain circuits rely upon a relatively small but remarkably diverse population of GABAergic interneurons to bind and entrain large principal cell assemblies for network synchronization and rhythmogenesis. Despite the high degree of heterogeneity across cortical interneurons, members of a given subtype typically exhibit homogeneous developmental origins, neuromodulatory response profiles, morphological characteristics, neurochemical signatures and electrical features. Here we report a surprising divergence among hippocampal oriens-lacunosum moleculare (O-LM) projecting interneurons that have hitherto been considered a homogeneous cell population. Combined immunocytochemical, anatomical and electrophysiological interrogation of Htr3a-GFP and Nkx2-1-cre:RCE mice revealed that O-LM cells parse into a caudal ganglionic eminence–derived subpopulation expressing 5-HT3A receptors (5-HT3ARs) and a medial ganglionic eminence–derived subpopulation lacking 5-HT3ARs. These two cohorts differentially participate in network oscillations, with 5-HT3AR-containing O-LM cell recruitment dictated by serotonergic tone. Thus, members of a seemingly uniform interneuron population can exhibit unique circuit functions and neuromodulatory properties dictated by disparate developmental origins.

State-Dependent cAMP Sensitivity of Presynaptic Function Underlies Metaplasticity in a Hippocampal Feedforward Inhibitory Circuit

At hippocampal mossy fiber (MF)-st. lucidum interneuron (SLIN) synapses, mGluR7 serves as a metaplastic switch controlling bidirectional plasticity. mGluR7 activation during high-frequency stimulation (HFS) triggers presynaptic LTD due to persistent P/Q-type Ca(2+) channel inhibition. However, following mGluR7 internalization HFS produces presynaptic LTP. Surprisingly, LTP is not a simple molecular reversal of Ca(2+) channel depression. Rather, mGluR7 activation/internalization controls plasticity polarity by gating cAMP sensitivity of release. While naive surface mGluR7 expressing MF-SLIN synapses are insensitive to cAMP elevation, synapses that have internalized mGluR7 robustly potentiate following cAMP increases. Moreover, MF-SLIN LTP requires adenylate cyclase (AC) and protein kinase A (PKA) activities. We also discovered an association between mGluR7 and RIM1alpha, an active zone molecule required for AC/PKA-dependent presynaptic LTP. Importantly, the mGluR7-RIM1alpha interaction is regulated by mGluR7 activation, and mice lacking RIM1alpha are deficient in MF-SLIN LTP. We conclude that state-dependent cAMP sensitivity controlled by mGluR7-RIM1alpha interactions underlies MF-SLIN metaplasticity.

Unraveling Oligodendrocyte Origin and Function by Cell-Specific Transgenesis

Besides the role of mature oligodendrocytes in myelin synthesis during the development of the central nervous system (CNS), the oligodendrocyte lineage also encompasses the largest pool of postnatal proliferating progenitors whose behavior in vivo remains broadly elusive in health and disease. We describe here transgenic models that allow us to track the functions and origins of such cells by using proteolipid protein and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) gene promoters to direct oligodendroglial expression of different reporters, in particular the green fluorescent protein (GFP). We emphasize that the CNP-GFP mouse, which targets the entire oligodendroglial lineage from embryonic life to adulthood, provides an outstanding tool to study the in vivo properties of oligodendrocyte progenitor cells in normal and damaged CNS.

Expression and Functional Analysis of Glutamate Receptors in Glial Cells

The brain consists of a complex network in which neurones and glial cells are structurally and functionally interwoven. Astrocytes, the most numerous member of the glial family, were originally considered, along with the whole glial population, to be only of structural importance (Virchow, 1846). For example, during development the radial glia, the precursors of astrocytes, serve as a scaffold at which neurones migrate to form the layered structure of different brain regions such as the cortex, the hippocampus or the cerebellum. During the last two decades, considerable knowledge about astrocytes has accumulated regarding their physiological function. One exciting function is their contribution to the regulation of the extracellular space and, thereby, also of brain excitability (Walz, 1989). Qualities such as their capacity for uptake and metabolism of transmitters, buffering capacity of ions and ability to convey external signals via surface receptors to biological responses within the cells indicate an intimate crosstalk between glial cells and neurones. The other major glial population in the brain are the oligodendrocytes. As small cells with few processes they form the myelin sheath, a highly lipid enriched stack of cell membranes enwrapping 50 to 300αm long axonal segments to enhance the conduction of electrical signals and to inhibit electrical crosstalk between individual axons. Oligodendrocytes are capable of myelinating up to 50 axonal segments simultaneously. Mature oligodendrocytes develop from progenitors originating from the subventricular zone as the germinative layer (Miller, 1996). In vertebrates, progenitors start to migrate to their final destination regions, the presumptive white matter, during the first postnatal week.