Dopamine directly modulates GABAA receptors.

Dopamine is a critical neuromodulator that activates GPCRs in mammals or ligand-gated ion channels in invertebrates. The present study demonstrates that dopamine (0.1-10 mm) exerts novel, opposing effects on different populations of mammalian (rat) GABAA receptors. Using whole-cell patch-clamp electrophysiology, we observed direct dopamine-mediated inhibition of tonic-level (1 µm) GABA-evoked currents in untransfected striatal neurons that could be recapitulated in HEK293 cells containing α1ß3 or α1ß2γ2 subunits. Surprisingly, direct activation by dopamine was seen in the absence of GABA with α1ß2γ2, α5ß3γ2, or α1ß3γ2 transfections. This activity was also present in α1ß3γ2 receptors containing a mutant ß3 subunit (H267A [(Z)ß3]) insensitive to trace levels of inhibitory Zn(2+). Dopamine activation required ß and γ subunits but not α subunits ((Z)ß3γ2 EC50 value, 660 µm). Dopamine activity was fully blocked by picrotoxin but not GABAA competitive antagonists, and was strongly correlated with spontaneous receptor activity. We also report opposing effects of bicuculline and gabazine, such that bicuculline surprisingly activated non-α-containing (ß3γ2) GABAA receptors, whereas gabazine suppressed spontaneous activity in these receptors. Our results suggest that dopamine may directly inhibit GABAA receptors that are both immediately adjacent to dopamine release sites in the striatum and activated by tonic GABA. Furthermore, synaptic/phasic release of dopamine may directly enhance signaling at some spontaneously active noncanonical GABAA receptors that lack α subunits.

Native serotonin 5-HT2C receptors are expressed as homodimers on the apical surface of choroid plexus epithelial cells.

G protein-coupled receptors (GPCRs) are a prominent class of plasma membrane proteins that regulate physiologic responses to a wide variety of stimuli and therapeutic agents. Although GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain whether native receptors expressed in their natural cellular environment are monomers, dimers, or oligomers. The goal of this study was to determine the monomer/oligomer status of a native GPCR endogenously expressed in its natural cellular environment. Native 5-HT2C receptors in choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH). An anti-5-HT2C fragment antigen binding protein was used to label native 5-HT2C receptors. A known monomeric receptor (CD-86) served as a control for decoding the oligomer status of native 5-HT2C receptors by molecular brightness analysis. FCS with PCH revealed molecular brightness values for native 5-HT2C receptors equivalent to the molecular brightness of a homodimer. 5-HT2C receptors displayed a diffusion coefficient of 5 × 10(-9) cm(2)/s and were expressed at 32 receptors/µm(2) on the apical surface of choroid plexus epithelial cells. The functional significance and signaling capabilities of the homodimer were investigated in human embryonic kidney 293 cells using agonists that bind in a wash-resistant manner to one or both protomers of the homodimer. Whereas agonist binding to one protomer resulted in G protein activation, maximal stimulation required occupancy of both protomers. This study is the first to demonstrate the homodimeric structure of 5-HT2C receptors endogenously expressed in their native cellular environment, and identifies the homodimer as a functional signaling unit.

Ethanol modulates spontaneous calcium waves in axonal growth cones in vitro.

In developing neurons the frequency of long duration, spontaneous, transient calcium (Ca2+) elevations localized to the growth cone, is inversely related to the rate of axon elongation and increases several fold when axons pause. Here we report that these spontaneous Ca2+ transients with slow kinetics, called Ca2+ waves, are modulated by conditions of ethanol exposure that alter axonal growth dynamics. Using time-series fluorescence calcium imaging we found that acute treatment of fetal rat hippocampal neurons with 43 or 87 mM ethanol at an early stage of development in culture decreased the percent of axon growth cones showing at least one Ca2+ wave during 10 min of recording, from 18% in controls to 5% in cultures exposed to ethanol. Chronic exposure to 43 mM ethanol also reduced the incidence of Ca2+ waves to 8%, but exposure to 87 mM ethanol increased their incidence to 31%. Neither chronic nor acute ethanol affected the peak amplitude, time to peak or total duration of Ca2+ waves. In some experiments, we determined the temporal correlation between Ca2+ waves and growth and non-growth phases of axonal growth dynamics. As expected, waves were most prevalent in stationary or retracting growth cones in all treatment groups, except in cultures exposed chronically to 87 mM ethanol. Thus, the relationship between growth cone Ca2+ waves and axon growth dynamics is disrupted by ethanol.

Oligomer size of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor revealed by fluorescence correlation spectroscopy with photon counting histogram analysis: evidence for homodimers without monomers or tetramers.

Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) are techniques with single molecule sensitivity that are well suited for examining the biophysical properties of protein complexes in living cells. In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT(2C)) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 µm(2)/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescence-tagged 5-HT(2C) receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT(2C) receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT(2C) receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT(2C) receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. Therefore, it is concluded that 5-HT(2C) receptors freely diffusing within the plasma membrane are dimeric.

Degraded mitochondrial DNA is a newly identified subtype of the damage associated molecular pattern (DAMP) family and possible trigger of neurodegeneration.

We previously showed a preferential degradation and down-regulation of mitochondrial DNA and RNA in hamster fibroblasts in response to hydrogen peroxide. Subsequent studies by others demonstrated that mitochondrial DNA can stimulate immune cells as a DAMP (damage associated molecular patterns) family member. However, the actual physical structure of this mitochondrial DNA DAMP and its importance in non-immune cell types are poorly understood. Here we report that transfected oxidant-initiated degraded mitochondrial polynucleotides, which we term "DeMPs", strongly induce the proinflammatory cytokines interleukin 6, monocyte chemotactic protein-1, and tumor necrosis factor α in mouse primary astrocytes. Additionally, proinflammatory IL1ß was induced, implicating DeMPs in inflammasome activation. Furthermore, human cerebrospinal fluid (CSF) and plasma were found to contain detectable DeMP signal. Finally, significant degradation of mitochondrial DNA was observed in response to either a bolus or steady state hydrogen peroxide. Combined, these studies demonstrate, all for the first time, that a pathophysiologically relevant form of mitochondrial DNA (degraded) can elicit a proinflammatory cytokine induction; that a brain cell type (astrocytes) elicits a proinflammatory cytokine induction in response to these DeMPs; that this induction includes the inflammasome; that astrocytes are capable of inflammasome activation by DeMPs; that DeMPs are detectable in CSF and plasma; and that hydrogen peroxide can stimulate an early stage cellular degradation of mitochondrial DNA. These results provide new insights and are supportive of our hypothesis that DeMPs are a newly identified trigger of neurodegenerative diseases such as Alzheimer's disease, which are known to be associated with early stage inflammation and oxidation.

Ethanol alters BDNF-induced Rho GTPase activation in axonal growth cones.

BACKGROUND: The effects of ethanol on development of postmitotic neurons include altered neurite outgrowth and differentiation, which may contribute to neuropathology associated with fetal alcohol spectrum disorders. We previously reported that ethanol exposure alters axon growth dynamics in dissociated cultures of rat hippocampal pyramidal neurons. Given the important regulatory role of small Rho guanosine triphosphatases (GTPases) in cytoskeletal reorganization associated with axon growth, and reports that ethanol alters whole cell Rho GTPase activity in other cell types, this study explored the hypothesis that ethanol alters Rho GTPase activity specifically in axonal growth cones. METHODS: Fetal rat hippocampal pyramidal neurons were maintained in dissociated cultures for 1 day in control medium or medium containing 11 to 43 mM ethanol. Some cultures were also treated with brain-derived neurotrophic factor (BDNF), an activator of Rac1 and Cdc42 GTPases that promotes axon extension. Levels of active Rho GTPases in growth cones were measured using in situ binding assays for GTP-bound Rac1, Cdc42, and RhoA. Axon length, growth cone area, and growth cone surface expression of tyrosine kinase B (TrkB), the receptor for BDNF, were assessed by digital morphometry and immunocytochemistry. RESULTS: Although ethanol increased the surface area of growth cones, the levels of active Rho GTPases in axonal growth cones were not affected in the absence of exogenous BDNF. In contrast, ethanol exposure inhibited BDNF-induced Rac1/Cdc42 activation in a dose-dependent manner and increased RhoA activation at the highest concentration tested. Similar TrkB expression was observed on the surface of axonal growth cones of control and ethanol-treated neurons. CONCLUSIONS: These results reveal an inhibitory effect of ethanol on growth cone signaling via small Rho GTPases during early stages of hippocampal development in vitro, and suggest a mechanism whereby ethanol may disrupt neurotrophic factor regulation of axon growth and guidance.

Mathematical modeling of axonal formation. Part I: Geometry.

A stochastic model is proposed for the position of the tip of an axon. Parameters in the model are determined from laboratory data. The first step is the reduction of inherent error in the laboratory data, followed by estimating parameters and fitting a mathematical model to this data. Several axonogenesis aspects have been investigated, particularly how positive axon elongation and growth cone kinematics are coupled processes but require very different theoretical descriptions. Preliminary results have been obtained through a series of experiments aimed at isolating the response of axons to controlled gradient exposures to guidance cues and the effects of ethanol and similar substances. We show results based on the following tasks; (A) development of a novel filtering strategy to obtain data sets truly representative of the axon trail formation; (B) creation of a coarse graining method which establishes (C) an optimal parameter estimation technique, and (D) derivation of a mathematical model which is stochastic in nature, parameterized by arc length. The framework and the resulting model allow for the comparison of experimental and theoretical mean square displacement (MSD) of the developing axon. Current results are focused on uncovering the geometric characteristics of the axons and MSD through analytical solutions and numerical simulations parameterized by arc length, thus ignoring the temporal growth processes. Future developments will capture the dynamic growth cone and how it behaves as a function of time. Qualitative and quantitative predictions of the model at specific length scales capture the experimental behavior well.

NeuroRhythmics: software for analyzing time-series measurements of saltatory movements in neuronal processes.

Time-lapse imaging of living neurons both in vivo and in vitro has revealed that the growth of axons and dendrites is highly dynamic and characterized by alternating periods of extension and retraction. These growth dynamics are associated with important features of neuronal development and are differentially affected by experimental treatments, but the underlying cellular mechanisms are poorly understood. NeuroRhythmics was developed to semi-automate specific quantitative tasks involved in analysis of two-dimensional time-series images of processes that exhibit saltatory elongation. This software provides detailed information on periods of growth and nongrowth that it identifies by transitions in elongation (i.e. initiation time, average rate, duration) and information regarding the overall pattern of saltatory growth (i.e. time of pattern onset, frequency of transitions, relative time spent in a state of growth vs. nongrowth). Plots and numeric output are readily imported into other applications. The user has the option to specify criteria for identifying transitions in growth behavior, which extends the potential application of the software to neurons of different types or developmental stage and to other time-series phenomena that exhibit saltatory dynamics. NeuroRhythmics will facilitate mechanistic studies of periodic axonal and dendritic growth in neurons.

Signaling pathways regulating cell motility: a role in ethanol teratogenicity?

This article summarizes the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism in Santa Barbara, California. The organizer and chair was Tara A. Lindsley. The presentations were (1) Ethanol and Neuron Migration in the CNS, by Michael W. Miller; (2) Ethanol and L1-mediated Neurite Outgrowth, by Yoav Littner and Cynthia F. Bearer; and (3) Ethanol and Axon Guidance, by Tara A. Lindsley.

Ethanol withdrawal influences survival and morphology of developing rat hippocampal neurons in vitro.

BACKGROUND: Previous studies in this laboratory have shown that, like their counterparts in vivo, fetal rat hippocampal pyramidal neurons in culture develop abnormally small dendritic arbors when exposed to ethanol. This study asked whether ethanol's inhibitory effects on dendritic development differ when the duration of ethanol exposure and timing of withdrawal are varied to correspond with early versus later stages of development and whether ethanol withdrawal influences survival of these neurons. METHODS: We compared neurons exposed continuously for 6 or 14 days to ethanol (70 mM) with neurons transferred from ethanol-containing medium to control medium either 1 day after adding ethanol (before dendrites elongated) or 6 days after adding ethanol (after dendrites began elongating). We then performed morphometric and cell density analyses at 6 and 14 days using digital images of neurons immunostained with microtubule-associated protein 2 (MAP2) to visualize dendrites. RESULTS: Continuous exposure to ethanol decreased the length and number of dendrites formed but had no effect on neuron survival compared with controls without ethanol. Dendritic length was less inhibited when ethanol was withdrawn after 1 day, but the number of dendrites per cell was unchanged compared with neurons continuously exposed to ethanol. Withdrawal from ethanol at 1 day slightly enhanced the survival of neurons assessed at 14 days compared with neurons in control medium and with neurons exposed continuously to ethanol. In contrast, withdrawal from ethanol at 6 days severely decreased the number of neurons at 14 days. CONCLUSIONS: These results suggest that dendrites can achieve normal length when ethanol exposure is limited to only 1 day and withdrawal occurs before dendrites begin elongating. However, a persistent reduction in dendrite number results in smaller overall dendritic arbor size. Although continuous exposure to ethanol has little effect on neuron survival in these cultures, and exposure limited to 1 day followed by withdrawal can be neuroprotective against cell death associated with increased time in culture, longer exposure before withdrawal can trigger cell death.

Time-lapse analysis of ethanol's effects on axon growth in vitro.

The cortical abnormalities found in animal models of fetal alcohol syndrome (FAS) suggest a disruption of axon growth. After emerging from the cell body, axons exhibit saltatory growth, cycling between periods of extension and periods of retraction. The timing of neuronal process outgrowth an the balance between extension and retraction together determine the net rate of axon elongation, and may be independently regulated. In this study, we used time-lapse digital microscopy and custom-designed analytic software to assess the effects of ethanol on the growth of axons from embryonic rat hippocampal pyramidal neurons in culture during 24 h of development, beginning approximately 7 h after plating. We recorded the amount of time elapsed before axons emerged, the relative amount of time spent in periods of growth and nongrowth, and the rate and direction of change in axon length during both periods of growth and nongrowth. The initiation of axonal outgrowth was significantly delayed by ethanol in a dose-dependent fashion at concentrations in the medium at or above 100 mg/dl. However, once established, axons exhibited accelerated growth in the presence of ethanol. This increase in overall growth rate was primarily due to a significant decrease in axon retraction during nongrowth periods. Ethanol did not affect the duration or frequency of growth and nongrowth periods. We propose, therefore, that mechanisms underlying ethanol-mediated changes in axon growth are linked to signaling events that differentially regulate outgrowth and retraction.

Astrocyte-derived factors modulate the inhibitory effect of ethanol on dendritic development.

Numerous studies in vivo and in vitro have demonstrated that ethanol disrupts neuromorphogenesis. However, it has not been determined what role, if any, is played by non-neuronal cells in mediating this effect. We recently reported that ethanol inhibits dendritic development in low-density cultures of fetal rat hippocampal pyramidal neurons (Yanni and Lindsley, 2000: Dev Brain Res 120:233-243). In this culture system, cortical astrocytes precondition neuronal culture media for 2 days before the addition of neurons, which then develop on a separate substrate in coculture with the astrocytes. To determine whether astrocyte response to ethanol mediates the effects of ethanol on neurons, the present study compared dendritic development of neurons after 6 days in medium containing 400 mg/dl ethanol in coculture with live astrocytes and in conditioned medium from astrocytes that were never exposed to ethanol. The same experiment was also performed with and without ethanol present during astrocyte preconditioning of the medium. The effects of ethanol differed depending on when it was added to the cultures relative to addition of newly dissociated neurons. However, the effects of ethanol were not related to whether neurons were cocultured with live astrocytes. When astrocytes preconditioned the medium normally, ethanol added at plating inhibited dendritic development of neurons regardless of whether they were maintained in coculture with live astrocytes or in conditioned medium. In surprising contrast, the presence of ethanol during astrocyte preconditioning of the media had a growth promoting effect on subsequent dendrite development despite the continued presence of ethanol in the medium. Thus, astrocytes release soluble factors in response to ethanol that can protect neurons from the inhibitory effects of ethanol on dendritic growth, but the timing of neuronal exposure to these factors, or their concentration, may influence their activity.

Morphologic and neurotoxic effects of ethanol vary with timing of exposure in vitro.

Results of investigations with animal models of fetal alcohol syndrome (FAS) seem to indicate that neuronal vulnerability to ethanol-induced cell death may be correlated with specific developmental events. In the present study, we sought to test this observation in a cell culture model of neuronal development in which morphogenesis as well as survival could be assessed. Using embryonic rat hippocampal pyramidal neurons in primary cultures, we compared the sensitivity of neurons to ethanol added, at 400 mg/dl, to the medium at different times relative to the development of axons and dendrites. Quantitative morphometric analysis was performed by using phase contrast at 12 h (0.5 day) and 24 h (1 day), or fluorescence microscopy after microtubule-associated protein-2 (MAP2) immunostaining at 6 and 14 days. Survival was assessed by counting the number of neurons per unit area of the substrate at 14 days. Addition of ethanol 1 day after plating, when most neurons had developed an axon, had no effect on survival up to 14 days in vitro, but resulted in significantly shorter, less branched dendrites than observed when ethanol was added 2 h after plating. Despite the shorter duration of ethanol exposure, the addition of ethanol on day 6, after rapid growth of dendrites and synapses had begun, resulted in loss of all but about one third of the neurons by 14 days. This supports the suggestion that increased neuronal vulnerability to the morphoregulatory effects of ethanol is correlated with the establishment of polarity, but that the sensitivity of neurons to the cytotoxic effects of ethanol occurs later, when dendrites and synapses are rapidly forming.