The Müller (glial) cell in normal and diseased retina: a case for single-cell electrophysiology

In the retina of most vertebrates there exists only one type of macroglia, the Müller cell. Müller cells express voltage-gated ion channels, neurotransmitter receptors and various uptake carrier systems. These properties enable the Müller cells to control the activity of retinal neurons by regulating the extracellular concentration of neuroactive substances such as K+, GABA and glutamate. We show here how electrophysiological recordings from enzymatically dissociated mammalian Müller cells can be used to study these mechanisms. Müller cells from various species have Na(+)-dependent GABA uptake carriers, but only cells from primates have additional GABA receptors that activate Cl- channels. Application of glutamate analogues causes enhanced membrane currents recorded from Müller cells in situ but not from isolated cells. We show that mammalian Müller cells have no ionotropic glutamate receptors but respond to increased K+ release from glutamate-stimulated retinal neurons. This response is involved in extracellular K+ clearance and is mediated by voltage-gated (inwardly rectifying) K+ channels which are abundantly expressed by healthy Müller cells. In various cases of human retinal pathology, currents through these channels are strongly reduced or even extinguished. Another type of voltage-gated ion channels, observed in Müller cells from many mammalian species, are Na+ channels. In Müller cells from diseased human retinae, voltage-dependent Na+ currents were significantly increased in comparison to cells from control donors. Thus, the expression of glial ion channels seems to be controlled by neuronal signals. This interaction may be involved in the pathogenesis of retinal gliosis which inevitably accompanies any degeneration of retinal neurons. In particular, Müller cell proliferation may be triggered by mechanisms requiring the activation of Ca(2+)-dependent K+ channels. Ca(2+)-dependent K+ currents are easily elicitable in Müller cells from degenerating retinae and can be blocked by 1 mM TEA (tetraethylammonium). In purified Müller cell cultures, the application of 1 mM TEA greatly reduces the proliferative activity of the cells. These data clearly show that Müller cells are altered in cases of neuronal degeneration and may be crucially involved in pathogenetic mechanisms of the retina.     

Rapid sprouting of filopodia in nerve terminals of chromaffin cells, PC12 cells, and dorsal root neurons induced by electrical stimulation

Rapid morphological changes induced by direct electrical stimulation of nerve terminals were studied by using video-enhanced differential interference contrast microscopy at a very high magnification (12,000x). We used mainly cultured bovine chromaffin cells, which developed neurite-like processes, and PC12 cells, which showed neuronal differentiation upon NGF treatment. In a few cases, primary neurons of the rat dorsal root ganglion were also examined. Brief pulse stimulation of the terminals and varicosities induced exocytosis accompanied by rapid formation of filopodia. These filopodia, 0.1-0.2 micron in diameter and up to 10 microns in length, formed within a few hundreds of milliseconds and then retracted within tens of seconds. They could also be induced by K depolarization. This rapid filopodial sprouting strongly depended on the presence of extracellular Ca2+ and could be abolished in a medium containing a Ca chelator (EGTA) or La2+. Anti-cytoskeletal agents colchicine and cytochalasin B failed to block this response completely but lidocaine fully suppressed it. Quantitative analysis of exocytosis and filopodial sprouting showed that they were independent events, not directly linked to each other, having different thresholds usually higher for filopodial formation. In PC12 cells, the extent of filopodial sprouting varied with the state of differentiation of the cells, suggesting a functional role of rapid sprouting during a particular phase of their differentiation. Filopodia could be induced with greater ease by repetitive stimulation. The same responses may occur at growth cones approaching the target cells or even at mature synapses particularly after repetitive electrical activity, possibly playing a role in use-dependent synapse formation or plasticity.     

Take Five, a nutrition education intervention to increase fruit and vegetable intakes: impact on consumer choice and nutrient intakes

Employing clonal cell lines derived from rat embryonic hippocampal cells, we detected neuropeptide Y (NPY) mRNA in three progenitor subcloned cell lines. These cell lines upon differentiation express markers indicative of commitment to either neuronal (H19-7; NF +, GFAP -), glial (H19-5; GFAP +, NF -), or bipotential (H583-5; NF +, GFAP + ) lineages. Induction of differentiation was associated with the persistence of the NPY mRNA, however, in the differentiated H19-7 cells a 20-fold increase in NPY mRNA levels was observed (P<0.05). NPY immunoreactivity was observed only in cells with a differentiated neuronal phenotype. The cellular radioimmunoassayable NPY peptide levels increased twelve-fold without a change in extracellular NPY peptide levels by multi-factorially induced neuronal or glial cell differentiation. The differentiated H19-5 cells expressed lower levels of NPY that could not be immunocytochemically detected. The peripheral sympathetic PC-12 neuronal cells examined in the undifferentiated and nerve growth factor-driven differentiated states expressed NPY only upon differentiation. We conclude that NPY is expressed by the cultured undifferentiated and differentiated rat hippocampal clonal cell lines, while the peripheral sympathetic PC-12 neuronal cell line only expresses the NPY gene upon differentiation. These immortalized embryonic neural cell line(s) will provide a hippocampal cell line(s) to conduct future in-vitro investigations targeted at determining the cellular and molecular mechanisms governing NPY gene expression.     

Postnatal development of type I and type II hair cells in the mouse utricle: acquisition of voltage-gated conductances and differentiated morphology

The type I and type II hair cells of mature amniote vestibular organs have been classified according to their afferent nerve terminals: calyx and bouton, respectively. Mature type I and type II cells also have different complements of voltage-gated channels. Type I cells alone express a delayed rectifier, gK,L, that is activated at resting potential. We report that in mouse utricles this electrophysiological differentiation occurs during the first postnatal week. Whole-cell currents were recorded from hair cells in denervated organotypic cultures and in acutely excised epithelia. From postnatal day 1 (P1) to P3, most hair cells expressed a delayed rectifier that activated positive to resting potential and a fast inward rectifier, gK1. Between P4 and P8, many cells acquired the type I-specific conductance gK,L and/or a slow inward rectifier, gh. By P8, the percentages of cells expressing gK,L and gh were at mature levels. To investigate whether the electrophysiological differentiation correlated with morphological changes, we fixed utricles at different times between P0 and P28. Ultrastructural criteria were developed to classify cells when calyces were not present, as in cultures and neonatal organs. The morphological and electrophysiological differentiation followed different time courses, converging by P28. At P0, when no hair cells expressed gK,L, 33% were classified as type I by ultrastructural criteria. By P28, approximately 60% of hair cells in acute preparations received calyx terminals and expressed gK,L. Data from the denervated cultures showed that neither electrophysiological nor morphological differentiation depended on ongoing innervation.     

G-Protein-dependent facilitation of neuronal alpha1A, alpha1B, and alpha1E Ca channels

Modulation of neuronal voltage-gated Ca channels has important implications for synaptic function. To investigate the mechanisms of Ca channel modulation, we compared the G-protein-dependent facilitation of three neuronal Ca channels. alpha1A, alpha1B, or alpha1E subunits were transiently coexpressed with alpha2-deltab and beta3 subunits in HEK293 cells, and whole-cell currents were recorded. After intracellular dialysis with GTPgammaS, strongly depolarized conditioning pulses facilitated currents mediated by each Ca channel type. The magnitude of facilitation depended on current density, with low-density currents being most strongly facilitated and high-density currents often lacking facilitation. Facilitating depolarizations speeded channel activation approximately 1.7-fold for alpha1A and alpha1B and increased current amplitudes by the same proportion, demonstrating equivalent facilitation of G-protein-inhibited alpha1A and alpha1B channels. Inactivation typically obscured facilitation of alpha1E current amplitudes, but the activation kinetics of alpha1E currents showed consistent and pronounced G-protein-dependent facilitation. The onset and decay of facilitation had the same kinetics for alpha1A, alpha1B, and alpha1E, suggesting that Gbeta gamma dimers dissociate from and reassociate with these Ca channels at very similar rates. To investigate the structural basis for N-type Ca channel modulation, we expressed a mutant of alpha1B missing large segments of the II-III loop and C terminus. This deletion mutant exhibited undiminished G-protein-dependent facilitation, demonstrating that a Gbeta gamma interaction site recently identified within the C terminus of alpha1E is not required for modulation of alpha1B.     

Properties of ectopic neurons induced by Xenopus neurogenin1 misexpression

We have examined cells cultured from ectoderm-misexpressing Neurogenin1 (Ngn1) to describe better the extent to which this gene can control aspects of neuronal phenotype including motility, morphology, excitability, and synaptic properties. Like primary spinal neurons which normally express Ngn1, cells in Ngn1-misexpressing cultures exhibit a motility-correlated behavior called circus movements prior to neuritogenesis. Misexpression of NeuroD also causes circus movements and later neuronal differentiation. GSK3beta, which inhibits NeuroD function in vivo, blocks both Ngn1-induced and NeuroD-induced neuronal differentiation, while Notch signaling inhibits only Ngn1-induced neuronal differentiation, confirming that NeuroD is downstream of Ngn1 and insensitive to Notch inhibition. While interfering with NeuroD function in ventral ectoderm inhibits both circus movements and neuronal differentiation, such inhibition in the neural plate inhibits only neuronal differentiation, suggesting that additional factors regulate circus movements in the neural ectoderm. Ngn1-misexpressing cells extend N-tubulin-positive neurites and exhibit tetrodotoxin-sensitive action potentials. Unlike the majority of cultured spinal neurons, however, Ngn1-misexpressing cells do not respond to glutamate and do not form functional synapses with myocytes, suggesting that these cells are either like Rohon-Beard sensory neurons or are not fully differentiated.     

Lidocaine toxicity in primary afferent neurons from the rat

Evidence from both clinical studies and animal models suggests that the local anesthetic, lidocaine, is neurotoxic. However, the mechanism of lidocaine-induced toxicity is unknown. To test the hypothesis that toxicity results from a direct action of lidocaine on sensory neurons we performed in vitro histological, electrophysiological and fluorometrical experiments on isolated dorsal root ganglion (DRG) neurons from the adult rat. We observed lidocaine-induced neuronal death after a 4-min exposure of DRG neurons to lidocaine concentrations as low as 30 mM. Consistent with an excitotoxic mechanism of neurotoxicity, lidocaine depolarized DRG neurons at concentrations that induced cell death (EC50 = 14 mM). This depolarization occurred even though voltage-gated sodium currents and action potentials were blocked effectively at much lower concentrations. (EC50 values for lidocaine-induced block of tetrodotoxin-sensitive and -resistant voltage-gated sodium currents were 41 and 101 microM, respectively.) At concentrations similar to those that induced neurotoxicity and depolarization, lidocaine also induced an increase in the concentration of intracellular Ca++ ions ([Ca++]i; EC50 = 21 mM) via Ca++ influx through the plasma membrane as well as release of Ca++ from intracellular stores. Finally, lidocaine-induced neurotoxicity was attenuated significantly when lidocaine was applied in the presence of nominally Ca(++)-free bath solution to DRG neurons preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Our results indicate: 1) that lidocaine is neurotoxic to sensory neurons; 2) that toxicity results from a direct action on sensory neurons; and 3) that a lidocaine-induced increase in intracellular Ca++ is a mechanism of lidocaine-induced neuronal toxicity.     

Production of sweat gland cholinergic differentiation factor depends on innervation

Sympathetic neurons innervating sweat glands undergo a target-directed developmental switch in neurotransmitter properties. Using cultured sympathetic neurons as a bioassay for cholinergic differentiation factors, we and others found that extracts containing soluble proteins from developing and adult footpads caused the same changes in transmitter properties in sympathetic neurons in vitro that the target does in vivo. In the present studies, using footpads from Tabby mutant mice that lack sweat glands, we found that the presence of sweat glands is correlated with the presence of cholinergic differentiation activity in footpad extracts. We examined the conditions necessary for secretion of differentiation activity from primary cultures of sweat gland cells. Surprisingly, sweat gland cells cultured alone do not produce or secrete cholinergic differentiation activity. When grown in the presence of sympathetic neurons, however, gland cells induce cholinergic function, increase vasoactive intestinal peptide content, and reduce catecholamine production in the neurons. Medium conditioned by sweat gland/neuron cocultures has a similar effect on the transmitter properties of cultured sympathetic neurons, indicating that the target influence on phenotype is mediated by a secreted factor(s). The innervation-dependence of cholinergic differentiation factor production provides evidence that reciprocal interactions between neurons and sweat glands are necessary for acquisition of the mature transmitter phenotype.     

Heterodimeric neurotrophins induce phosphorylation of Trk receptors and promote neuronal differentiation in PC12 cells

Neurotrophins are a family of highly conserved proteins that affect the development and maintenance of distinct neuronal populations. Neurotrophins exist in vivo as homodimers, but we show that neurotrophins can exist as heterodimers in vitro and are pluripotent, being able to bind and to activate different Trk tyrosine kinase receptors as well as promote neuronal differentiation in PC12 cells as effectively as wild type homodimers. These asymmetric neurotrophin dimers allow unique characterization of neurotrophin structure-function relationships with Trk receptors. The chimeric Trk activities of these heterodimers suggest an alternative model of neurotrophin-Trk receptor activation in which the critical Trk-interacting elements may be attributed to a single protomer.     

Basal ganglia precursors found in aggregates following embryonic transplantation adopt a striatal phenotype in heterotopic locations

Transplantation of immature CNS-derived cells into the developing brain is a powerful approach to investigate the factors that regulate neuronal position and phenotype. CNS progenitor cells dissociated from the embryonic striatum and implanted into the brain of embryos of the same species generate cells that reaggregate to form easily recognizable structures that we previously called clusters and cells that disperse and integrate as single cells into the host brain. We sought to determine if the neurons in the clusters differentiate according to their final location or acquire a striatal phenotype in heterotopic positions. We transplanted dissociated cells from the E14 rat medial and lateral ganglionic eminences, either combined or in isolation, into the E16 embryonic rat brain. At all time points, we found clusters of BrdU- and DiI-labelled donor cells located in the forebrain and hindbrain, without any apparent preference for striatum. Immunocytochemical analyses revealed that cells in the clusters expressed DARPP-32 and ARPP-21, two antigens typically co-expressed in striatal medium-sized spiny neurons. In agreement with observations previously noted by several groups, isolated cells integrated into heterologous host areas do not express basal ganglia phenotypes. These data imply that immature striatal neuronal progenitors exert a community effect on each other that is permissive and/or instructive for development of a striatal phenotype in heterotopic locations.     

Induction of neuronal morphology in adrenal chromaffin cells cocultured with denervated Schwann cells

We have studied the interactions of adrenal chromaffin and Schwann cells in a coculture system to observe whether denervated Schwann cells induce and support chromaffin cell differentiation in a manner analogous to nerve growth factor (NGF). Schwann cells induce both the accumulation of intense clumps of cocultured chromaffin cells on their surfaces and intense neurite outgrowth. This interaction is not blocked by antibodies to NGF or laminin. Conditioned medium from Schwann cell cultures fosters neurite outgrowth in chromaffin cells in a fashion qualitatively similar to NGF. Our data indicate that denervated Schwann cells exert a profound aggregating and differentiating effect upon chromaffin cells, inducing the expression of a neuronal phenotype via a predominantly NGF-independent mechanism.     

Neurotrophin effects on survival and expression of cholinergic properties in cultured rat septal neurons under normal and stress conditions

These studies tested the hypothesis that survival-promoting effects of neurotrophins on basal forebrain cholinergic neurons are enhanced under stress. Septal neurons from embryonic day 14-15 rats exposed for 10-14 d to neurotrophin [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4 (NT-4), each at 100 ng/ml] showed a two- to threefold increase in choline acetyltransferase (ChAT) activity, with little evidence of synergistic interactions. Neurotrophins produced no significant increase in the survival of total or acetylcholinesterase (AChE)-positive neurons at moderate plating density (1200-1600 cells/mm2). However, with very low plating densities (2-28 cells/mm2) BDNF, NT-3, and NT-4 (but not NGF) increased total neuronal survival, and BDNF increased survival of AChE-positive neurons. NGF and BDNF enhanced ChAT activity and survival of cholinergic neurons after a 24 hr hypoglycemic stress, even when added 1 hr after stress onset. All four tested neurotrophins increased total neuronal survival after hypoglycemic stress. These results suggest that neurotrophins are important for preservation of central cholinergic function under stress conditions, with different neurotrophins protecting against different stresses. The stress-associated survival-promoting effects of neurotrophins were not limited to the cholinergic subpopulation.     

Induction of NG108-15 cells differentiation by human bone marrow stromal cells

The survival and differentiation of neuronal cells is dependent on factors such as neurotrophins, cytokines and components of extracellular matrix. Bone marrow stromal cells have been shown to support the growth and differentiation of neuroblastoma cells. In an attempt to study the effects of bone marrow stromal cells on neuronal differentiation, we have co-cultured neuroblastoma x glioma hybrid NG108-15 cells with human bone marrow stromal cells. After co-culturing, clones exhibiting morphological differentiated phenotype and high level of neurofilament expression were isolated. Interestingly, these clones maintain their ability to proliferate in contrast to differentiated NG108-15 cells induced by dibutyryladenosine 3',5'-cyclic monophosphate. These results suggested that bone marrow stromal cells can induce partial differentiation of NG108-15 cells.     

Altered ganglioside expression by SH-SY5Y cells upon retinoic acid-induced neuronal differentiation

SH-SY5Y Neuroblastoma cells were used to study the effect of retinoic acid (RA)-induced differentiation on the expression of gangliosides and neuronal markers. In the presence of 10 microM RA, more than 70% of the cells differentiate to a neuronal phenotype within 8 days. They extend long neuritic processes and show an enhanced immuno-expression of neurone-specific enolase (NSE), neurofilament protein (NF-M), and polysialic acid (PSA). SH-SY5Y cells were found to express at least 12 different gangliosides. RA-induced neuronal differentiation led to a decrease in the content of GM2, GD3, and GD2 and to a 3-7 fold increased concentration of the ganglio-tetraosyl gangliosides GM1, GD1a, GT1a, GD1b, and GT1b. Thus, RA-induced neuronal differentiation of SH-SY5Y cells is accompanied by ganglioside changes similar to those observed during embryonic neuronal differentiation.     

Docosahexaenoic acid block of neuronal voltage-gated K+ channels: subunit selective antagonism by zinc

The omega-3 polyunsaturated fatty acid docosahexaenoic acid is highly enriched in neuronal membranes, and several studies suggest that DHA is critical for neuronal development. We have investigated the effects of exogenously applied DHA on voltage-gated K+ channels using patch-clamp techniques. DHA produced a concentration-dependent inhibition of the sustained outward current in isolated neocortical neurons. This blocking action was examined in more detail with two cloned neuronal K+ channels (Kv1.2 and Kv3.1a) expressed in mammalian fibroblasts. DHA produced a potent inhibition of depolarization-activated K+ currents from cells expressing these channels (Kd values, 1.8 +/- 0.1 muM and 690 +/- 60 nM, for Kv1.2 and Kv3.1a, respectively, at +40 mV). The DHA block of both channel types was rapidly reversed (approximately 2 sec) by bovine serum albumin, which binds the fatty acid. Micromolar concentrations of extracellular Zn2+ non-competitively antagonized DHA inhibition of Kv1.2 channels, whereas there was little effect on DHA block of Kv3.1a channels. Experiments with membrane patches from Kv1.2 transfected cells demonstrated that the DHA block occurred from the outside, suggesting that the fatty acid interacts directly with an external domain of the ion channel. DHA may serve as a local messenger molecule that selectively modulates the activity of certain voltage-gated K+ channels in a Zn2(+)-dependent fashion.     

Differential effects of physostigmine and organophosphates on nicotinic receptors in neuronal cells of different species

The effects of the carbamate physostigmine and of the organophosphates (OPs) parathion, paraoxon and phenyl saligenin cyclic phosphate (PSP) were examined on different subtypes of neuronal nicotinic acetylcholine receptors (nAChR). Stimulation with 1 mM ACh induced transient nicotinic inward currents in mouse N1E-115 and human SH-SY5Y neuroblastoma and in locust thoracic ganglion cells. All four acetylcholinesterase (AChE) inhibitors reduced the nicotinic currents in a concentration-dependent manner. Parathion is about 50 times more potent in blocking nAChR, compared to its active AChE inhibiting metabolite paraoxon. The relative blocking potency of the different AChE inhibitors was the same in all cell types, and followed the order parathion > physostigmine > PSP > paraoxon. In N1E-115 cells the IC50 values of block amounted to 2 microM, 30 microM, 39 microM and 96 microM for parathion, physostigmine, PSP and paraoxon, respectively. In all cell types, the nicotinic currents were equally blocked by parathion. Human nAChR in SH-SY5Y cells appeared more sensitive to block by physostigmine, PSP and paraoxon, while these AChE inhibitors similarly inhibited nicotinic currents in insect cells and in mouse neuroblastoma cells. The observation that the concentration-dependence of block is different from that of AChE inhibition, indicates a distinct interaction of AChE inhibitors with nAChR. Only in locust cells physostigmine induced a non-desensitizing inward current, that appeared to originate from nAChR activation. Occasionally, the OPs were able to activate slow ionic currents in mouse, but not in human and locust cells. As the OP-induced agonistic activity in mouse cells was not associated with the blocking action, the target site appeared to be distinct from nAChR. These results show that AChE inhibitors block nAChR with different potencies, dependent on the compound and the receptor subtype, and may activate distinct ion currents in neuronal cells of different species origin.     

Role of neurotrophins and their receptors in human neuroblastomas: a primary culture study

Expression of trk family genes are prognostic indicators of neuroblastoma. However, the functional role of neurotrophins and their receptors in neuroblastomas in vivo is still unclear. We studied the expression of neurotrophin receptors (trk-A, trk-B, trk-C) and their responsiveness to neurotrophins (NGF, BDNF, NT-3) in 25 human neuroblastomas using a primary culture system. The tumours in early stages and stage 4s responded to both NGF and NT-3, but not to BDNF, by surviving and differentiating terminally and the responsiveness was correlated with high levels of trk-A, especially the neuronal isoform. However, in many advanced stage tumours, the expression of trk-A was down-regulated and the response pattern to neurotrophins was diverse, without showing terminal differentiation. Interestingly, a stage 4 tumour with MYCN amplification which expressed high level of neuronal trk-A was dependent on nerve growth factor (NGF) for both survival and differentiation in primary culture. The results suggest that the NGF/trk-A signalling may be the main regulatory pathway for differentiation and survival of neuroblastoma in vivo and that trk-A overexpression may overcome aggressiveness, even of the tumour with MYCN amplification.