Qi-Ying Liu

Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

Increasing evidence has shown that some neurotransmitters act as growth‐regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell‐derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF‐deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth‐stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2‐bis‐(O‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA‐AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G‐proteins, the protein kinase C inhibitor H7 and the mitogen‐activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth‐regulatory signal during neurogenesis via pathways involving pertussis toxin‐sensitive G‐proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepithelial cells.

Ionotropic glutamate receptors mediate fast forms of excitatory synaptic transmission in mature neurons and may play critical roles in neuronal development. However, the developmental stage at which neuronal cells begin to express functional receptors and their roles in lineage progression remain unclear. In the present study, neural precursor cells were isolated from the cortical neuroepithelium of embryonic day 13 rats, and rapidly expanded in serum‐free medium in response to basic fibroblast growth factor. RT‐PCR revealed the presence of mRNAs encoding AMPAA, AMPAC, KA1, KA2, NMDA1, and NMDA2D subunits after 3 days in culture. The functional expression of AMPA/kainate and NMDA receptors was investigated using Ca2+ imaging and whole‐cell patch‐clamp recording techniques in cells pulse‐labeled with bromodeoxyuridine (BrdU) for 1–4 hr. The recorded cells were then double‐immunostained for BrdU incorporation and neuron‐specific β‐tubulin (TuJ1). The results show that AMPA/kainate and NMDA induced increases in cytosolic Ca2+ and inward currents only in differentiating neurons. In contrast, proliferating (BrdU+TuJ1‐) cells failed to respond to any ionotropic glutamate receptor agonists. Interestingly, Ca2+ imaging revealed that a subpopulation of BrdU+TuJ1+ cells also responded to AMPA, indicating the emergence of functional ionotropic AMPA/kainate receptors during terminal cell division and the earliest commitment to neuronal cell lineage. These in vitro results were supported by flow cytometric sorting of AMPA‐responsive cells pulse‐labeled with BrdU for 1 hr in vivo, which revealed that functional AMPA receptors appear in BrdU+TuJ1+ cells under physiological conditions and may play a role in terminal cell division. J. Neurosci. Res. 61:652–662, 2000.

CENTRAL NEURONAL SYNAPSE FORMATION ON MICROPATTERNED SURFACES

Controlling synapse formation is a key to patterning of neurons into functional circuits and networks in vitro. However, the process of synapse formation among neurons grown on artificial surfaces is relatively unstudied. We cultured embryonic hippocampal cells on trimethoxysilylpropyl-diethylenetriamine (DETA) and tridecafluoro-1, 1,2,2-tetrahydrooctyl-1-dimethylchlorosilane (13F), and on patterns composed of DETA lines separated by 13F spaces. For comparison, neurons were concurrently plated on surfaces coated with uniform poly-d-lysine (PDL). Pre- and postsynaptic specializations were identified by immunostaining for synapsin I and microtubule-associated protein-2 (MAP-2). Spontaneous (SPCs) and evoked (EPCs) postsynaptic currents were recorded using dual patch-clamp techniques. We found that DETA promoted synapse formation, whereas evidence for synapse formation on 13F was barely detected. MAP-2+ neuronal soma and rapidly growing dendrites were co-localized with synapsin I puncta faithfully along DETA lines. The expression of synapsin I puncta, and MAP-2+ soma and dendrites correlated well with the appearance of SPCs. Synapsin I, MAP-2 and SPCs emerged together at days 3-4 and increased at day 7, when EPCs appeared. Synaptic signals occurring during 4-7 days in culture were all GABAergic. These results indicate that fully functional synapses are formed on silane surfaces, demonstrating the suitability of patterned silane surfaces for organizing synapse formation in vitro.

Upregulation of GABAA Current by Astrocytes in Cultured Embryonic Rat Hippocampal Neurons

Embryonic rat hippocampal neurons were cultured on poly-d-lysine (PDL) or a monolayer of postnatal cortical astrocytes to reveal putative changes in neuronal physiology that involve astrocyte-derived signals during the first 4 d of culture. GABA-induced Cl− current (IGABA) was quantified using outside-out and whole-cell patch-clamp recordings beginning at 30 min, when cells had become adherent. The amplitude and density (current normalized to membrane capacitance) of IGABA in neurons grown on astrocytes became statistically greater than that recorded in neurons grown on PDL after 2 hr in culture (HIC). Although the current density remained unchanged in neurons on astrocytes, that in neurons on PDL decreased and became statistically lower beginning after 2 HIC. The differences in amplitude and density of IGABA in the two groups of neurons were maintained during the 4 d experiment. The upregulation effect of astrocytes on neuronal IGABA required intimate contact between the neuronal cell body and underlying astrocytes. Suppression of spontaneous Cac2+ elevations in astrocytes by bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid that was loaded intracellularly decreased their modulatory effects on IGABA. IGABA in all cells was blocked completely by bicuculline and exhibited virtually identical affinity constants, Hill coefficients, and potentiation by diazepam in the two groups. Outside-out patch recordings revealed identical unitary properties of IGABA in the two groups. More channels per unit of membrane area could explain the astrocyte enhancement of IGABA. The results reveal that cortical astrocytes potentiate IGABA in hippocampal neurons in a contact-dependent manner via a mechanism involving astrocyte Cac2+elevation.

Astrocytes Regulate Amino Acid Receptor Current Densities in Embryonic Rat Hippocampal Neurons

Embryonic rat hippocampal neurons were cultured in a serum-free defined medium (MEM/N3) either directly on poly-D-lysine (PDL) or on a confluent monolayer of postnatal cortical astrocytes, C6 glioma cells, or Rat2 fibroblasts. Neurons on PDL were grown in MEM/N3 or in MEM/N3 conditioned for 24 h by astrocytes or C6 cells. Membrane capacitance (Cm) and gamma-aminobutyric acid (GABA)-, glycine-, kainate-, and N-methyl-D-aspartate (NMDA)-induced currents were quantified using whole-cell patch-clamp recordings. Cm as well as the amplitude and the density of these currents in neurons cultured on astrocytes were significantly greater than those in neurons grown on PDL after 24 and 48 h. C6 cells mimicked astrocytes in promoting Cm and GABA-, glycine-, and NMDA-evoked, but not kainate-evoked, currents. Cm and currents in neurons grown on Rat2 cells were comparable to those in neurons on PDL. Astrocytes maintained in culture for 3 months were noticeably less effective than freshly prepared ones just grown to confluence. Suppression of spontaneous cytoplasmic Ca2+ (Ca[c]2+) elevations in astrocytes by 1,2-bis(2-aminophenoxy) ehane-N, N, N, N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) loaded intracellularly blocked the observed modulatory effects. Medium conditioned by either astrocytes or C6 cells mimicked the effects of direct coculture of neurons on these cells in promoting Cm and amino acid-evoked currents. Inclusion of antagonists at GABA and glutamate receptors in coculture experiments blocked the observed effects. Thus, diffusible substances synthesized and/ or secreted by astrocytes in a Ca(c)2+-dependent manner can regulate neuronal growth and aminoacid receptor function, and these effects may involve neuronal GABA and glutamate receptors.

A role for inwardly rectifying K+ channels in differentiation of NG108‐15 neuroblastoma × glioma cells

The whole-cell patch-clamp technique was used to assess the current carried by inwardly rectifying K+ channels (K(ir)) and the resting membrane potential (RMP) during long-term culture of NG108-15 cells. Culture of this cell line in serum-free medium triggers differentiation of a type I, neuron-like cell type followed by an eventual predominance of a type II, proliferative cell type. NG108-15 K(ir) currents, which strongly resemble currents carried by human ether-a-go-go related gene (HERG) K+ channels, exhibited significantly smaller current density for the more depolarized undifferentiated cells in growth media (GM) and type II cells compared to the neuron-like type I cells. Detailed examination of the transition from undifferentiated GM cells to type I cells revealed a shift in the voltage dependence of K(ir) activation which paralleled the more hyperpolarized RMP, neurite outgrowth, and biochemical differentiation characteristic of type I cells. Reverse-transcription polymerase chain reaction experiments using primers for the rat variant of HERG, RERG, revealed a a nearly twofold increase in RERG mRNA as cells differentiate from GM to type I, a finding entirely consistent with the increased K(ir) current density derived from patch-clamp recordings. Administration of CsCl(5 mM) blocked K(ir) currents and depolarized the RMP of type I cells. Furthermore, culture of NG108-15 cells in serum-free medium but with CsCl added significantly prevented neurite extension, an effect which was entirely reversible upon subsequent removal of CsCl. In contrast, other K+ channel inhibitors (4-aminopyridine and tetraethylammonium), at concentrations without marked effects on K(ir), failed to affect neurite extension. These results suggest an important role of the K(ir) channels in determining the RMP and triggering morphological differentiation of the cell line.

Kainate induces an intracellular Na+-activated K+ current in cultured embryonic rat hippocampal neurones

1 In embryonic rat hippocampal neurones cultured for < 3 days, kainate induced an inward current at negative potentials that recovered to baseline levels immediately upon termination of agonist application. However, in neurones cultured for longer, the kainate‐induced current was often followed by a long‐lasting inward current that slowly recovered to baseline levels. The amplitude of the delayed current (Idelay) triggered by kainate was positively related both to the duration of application at constant agonist concentration and to concentration at constant application duration. 2 I delay could last for several minutes and was accompanied by a conductance increase, which closely paralleled current amplitude. Depression of the kainate‐induced current response at receptor level with CNQX or at ionic level with Na+‐free solution eliminated Idelay. However, when applied during Idelay neither CNQX nor Na+‐free solution had any effect on Idelay. Li+ effected the same response as Na+ in mediating kainate‐induced Idelay. 3 GABA‐activated Cl− current, which was associated with the same amount of inwardly directed charge flow at the same potential as that induced by kainate, did not trigger a long‐lasting delayed current. 4 I delay depended on the existence of extracellular K+ and its amplitude increased with the increase in K+ concentration. Neither applying Cl−‐ or Ca2+‐free solutions nor increasing intracellular Ca2+ buffering speed and capacity altered Idelay. Exposure to the specific KCa channel blockers apamin and charybdotoxin also failed to alter Idelay. However, Idelay could be blocked by Cs+, Ba2+ and high concentrations of 4‐aminopyridine (4‐AP) and TEA. 5 Inside‐out excised patch‐clamp recordings revealed that low density or highly clustered Na+‐activated K+ channels were expressed in the cell bodies of cultured embryonic rat hippocampal neurones. These could be the elementary channels underlying Idelay.

Exogenous GABA persistently opens Cl− channels in cultured embryonic rat thalamic neurons

We recorded whole-cell Cl− currents in cultured embryonic rat thalamic neurons by brief applications of GABA or the structural analogue muscimol. In 17 of 141 neurons (12%) the Cl− current persisted for a minute or more after the pipette was removed from the bath. Cl− current never persisted after muscimol exposure even in those cells exhibiting persistent GABA-activated currents (PGC). The half decay times (T50) of PGCs were exponentially and asymptotically related to the duration of GABA exposure and could be interrupted or completely aborted by low-pressure application of saline. PGCs were insensitive to membrane potential, to Tiagabine, a nipecotic acid analogue known to block GABA uptake, and persisted in Cao2+-free medium. Fluctuation analysis revealed that PGCs exhibited inferred Cl− channel properties whose kinetic components and estimated average elementary conductance showed no significant difference from those estimated during GABA exposure. The relative contribution of low frequency components was consistently reduced and that of high frequency components modestly increased during PGC compared to those recorded during GABA exposure. Taken together, the results suggest the existence of a superficial compartment in these embryonic neurons that can momentarily accumulate and release exogenous GABA.

Astrocyte-conditioned saline supports embryonic rat hippocampal neuron differentiation in short-term cultures

Embryonic rat hippocampal neurons were cultured for 1-2 days in serum-free, HEPES-buffered Tyrodes solution. The effects of cortical astrocytes and astrocyte-conditioned saline on neuron survival, membrane surface area and the expression of functional amino acid neurotransmitter receptors were studied. Neurons grown in Tyrodes solution alone survived well for 1 day but deteriorated thereafter both in terms of percent neurons surviving and the amplitudes and densities of GABA-, glycine-, kainate-and NMDA-induced currents. Neurons grown in Tyrodes previously conditioned by astrocytes for 24 h had significantly larger apparent plasma membrane surface area, as indexed by whole-cell membrane capacitance, and larger amplitudes and densities of the amino acid-induced currents after both 1 and 2 days. The survival rate and neurite outgrowth were also greater in the astrocyte-conditioned saline group after 2 days in culture. Similarly, neurons cultured on glass cover-slips facing a confluent monolayer of astrocyte were larger in apparent plasma membrane area and amino acid-induced currents than neurons cultured in Tyrodes alone. Neurons cultured in saline conditioned by astrocytes provide a strategy to study the physiological basis of astrocyte-directed neuronal differentiation in the absence of ambiguities arising from the inclusion of sera and other additives often used in vitro.

GABA induces GABAergic MSCs in cultured embryonic rat thalamic neurons

Application of 0.1–10 μM GABA in the vicinity of cultured embryonic rat thalamic neurons recorded with patch pipettes in the presence of 2 μM TTX induced or increased the frequency of miniature synaptic currents (MSCs) that reversed polarity at the Cl− equilibrium potential. These MSCs were blocked by the GABAA receptor antagonist bicuculline and exhibited exponential decay kinetics that closely paralleled those estimated from fluctuation analysis of Cl− channels activated pharmacologically by applying 1–10 μM GABA to the same cells. We conclude that the MSCs are mediated by GABA. Application of the GABAA receptor agonist muscimol activated Cl− current but failed to induce GABAergic MSCs while submicromolar concentrations of GABA evoked GABAergic MSCs but did not activate Cl− channels. The GABAB receptor agonist (‐)baclofen did not mimic GABA in inducing MSCs. Induction of GABAergic MSCs by GABA required extracellular Ca2+. Verapamil and Co2+, which block voltage‐dependent calcium channels, completely blocked GABA‐induced MSCs independent of their effects on the direct activation of a Cl− current response. The results indicate that GABA can trigger GABAergic Cl−‐dependent MSCs in a Cao2+‐dependent manner. The mechanism may involve a novel receptor and/or signal transduction pathway. Synapse 25:15–23, 1997.