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

Lindsley TA, Mazurkiewicz JE - Brain Sci (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College (MC-136), 47 New Scotland Ave., Albany, NY 12208, USA. lindslt@mail.amc.edu.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

(a) Spontaneous Ca2+ waves in axonal growth cones of control and acute ethanol-exposed hippocampal pyramidal neurons. Time-series confocal images of cells loaded with Fluo-3 demonstrate a Ca2+ wave in a control neuron (top panels), and in a neuron exposed to 87 mM ethanol (bottom panels) added 15 min before starting the recording. Images are pseudocolored with peak elevations of [Ca2+]i indicated in red. (b) Plots of relative fluorescence over time in representative growth cones illustrate variation in temporal pattern of waves. Traces A–C are typical Ca2+ waves recorded in the growth cones of neurons in control medium without ethanol. Trace D shows a Ca2+ wave observed when ethanol is added to the medium 15 min before imaging. Trace A is the growth cone on top in (a). Trace D EtOH is the growth cone on the bottom in (a).
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brainsci-03-00615-f002: (a) Spontaneous Ca2+ waves in axonal growth cones of control and acute ethanol-exposed hippocampal pyramidal neurons. Time-series confocal images of cells loaded with Fluo-3 demonstrate a Ca2+ wave in a control neuron (top panels), and in a neuron exposed to 87 mM ethanol (bottom panels) added 15 min before starting the recording. Images are pseudocolored with peak elevations of [Ca2+]i indicated in red. (b) Plots of relative fluorescence over time in representative growth cones illustrate variation in temporal pattern of waves. Traces A–C are typical Ca2+ waves recorded in the growth cones of neurons in control medium without ethanol. Trace D shows a Ca2+ wave observed when ethanol is added to the medium 15 min before imaging. Trace A is the growth cone on top in (a). Trace D EtOH is the growth cone on the bottom in (a).

Mentions: Calcium waves were defined according to the criteria of Gomez et al. [15], which includes localization to the growth cone, fluorescence increases >150% of baseline, slow rise time (>3 s to peak), and return to baseline. Rarely, Ca2+ transients propagated to the growth cone from the cell body or axon shaft, and these were excluded from the analyses. These characteristics distinguish Ca2+ waves from Ca2+ spikes and from morphological movements and noise. To determine whether spontaneous Ca2+ waves also occur in neurons exposed to ethanol, ethanol was added to the medium (final concentration of 43 or 87 mM), either acutely or chronically. For acute exposure, ethanol was added 24 h after plating in normal medium, just before imaging. For chronic exposure, ethanol was added shortly after plating and was present in the medium continuously until imaging 24 h later. In both conditions, the concentration of ethanol in the medium was also present in buffers during imaging. None of these experimental conditions resulted in any observable effect on baseline calcium levels, neuron survival or growth cone morphology, either during 10 min recordings or 60 min afterwards. Calcium waves with varying temporal patterns were observed in axonal growth cones in all treatment groups. Figure 2 shows spontaneous Ca2+ waves in growth cones of control and ethanol-treated neurons.


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

Lindsley TA, Mazurkiewicz JE - Brain Sci (2013)

(a) Spontaneous Ca2+ waves in axonal growth cones of control and acute ethanol-exposed hippocampal pyramidal neurons. Time-series confocal images of cells loaded with Fluo-3 demonstrate a Ca2+ wave in a control neuron (top panels), and in a neuron exposed to 87 mM ethanol (bottom panels) added 15 min before starting the recording. Images are pseudocolored with peak elevations of [Ca2+]i indicated in red. (b) Plots of relative fluorescence over time in representative growth cones illustrate variation in temporal pattern of waves. Traces A–C are typical Ca2+ waves recorded in the growth cones of neurons in control medium without ethanol. Trace D shows a Ca2+ wave observed when ethanol is added to the medium 15 min before imaging. Trace A is the growth cone on top in (a). Trace D EtOH is the growth cone on the bottom in (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061854&req=5

brainsci-03-00615-f002: (a) Spontaneous Ca2+ waves in axonal growth cones of control and acute ethanol-exposed hippocampal pyramidal neurons. Time-series confocal images of cells loaded with Fluo-3 demonstrate a Ca2+ wave in a control neuron (top panels), and in a neuron exposed to 87 mM ethanol (bottom panels) added 15 min before starting the recording. Images are pseudocolored with peak elevations of [Ca2+]i indicated in red. (b) Plots of relative fluorescence over time in representative growth cones illustrate variation in temporal pattern of waves. Traces A–C are typical Ca2+ waves recorded in the growth cones of neurons in control medium without ethanol. Trace D shows a Ca2+ wave observed when ethanol is added to the medium 15 min before imaging. Trace A is the growth cone on top in (a). Trace D EtOH is the growth cone on the bottom in (a).
Mentions: Calcium waves were defined according to the criteria of Gomez et al. [15], which includes localization to the growth cone, fluorescence increases >150% of baseline, slow rise time (>3 s to peak), and return to baseline. Rarely, Ca2+ transients propagated to the growth cone from the cell body or axon shaft, and these were excluded from the analyses. These characteristics distinguish Ca2+ waves from Ca2+ spikes and from morphological movements and noise. To determine whether spontaneous Ca2+ waves also occur in neurons exposed to ethanol, ethanol was added to the medium (final concentration of 43 or 87 mM), either acutely or chronically. For acute exposure, ethanol was added 24 h after plating in normal medium, just before imaging. For chronic exposure, ethanol was added shortly after plating and was present in the medium continuously until imaging 24 h later. In both conditions, the concentration of ethanol in the medium was also present in buffers during imaging. None of these experimental conditions resulted in any observable effect on baseline calcium levels, neuron survival or growth cone morphology, either during 10 min recordings or 60 min afterwards. Calcium waves with varying temporal patterns were observed in axonal growth cones in all treatment groups. Figure 2 shows spontaneous Ca2+ waves in growth cones of control and ethanol-treated neurons.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College (MC-136), 47 New Scotland Ave., Albany, NY 12208, USA. lindslt@mail.amc.edu.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus