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Intrinsic properties of larval zebrafish neurons in ethanol.

Ikeda H, Delargy AH, Yokogawa T, Urban JM, Burgess HA, Ono F - PLoS ONE (2013)

Bottom Line: The behavioral effects of ethanol have been studied in multiple animal models including zebrafish.The intracellular [Ca(2+)] response in MiD3 neurons decreased in 100 mM ethanol, while Mauthner neurons and vestibulospinal neurons required >300 mM ethanol to elicit similar effects.The ethanol effect in Mauthner neurons was reversible following removal of ethanol.

View Article: PubMed Central - PubMed

Affiliation: Section on Model Synaptic Systems, Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland, United States of America.

ABSTRACT
The behavioral effects of ethanol have been studied in multiple animal models including zebrafish. Locomotion of zebrafish larvae is resistant to high concentrations of ethanol in bath solution. This resistance has been attributed to a lower systemic concentration of ethanol in zebrafish when compared with bath solution, although the mechanism to maintain such a steep gradient is unclear. Here we examined whether the intrinsic properties of neurons play roles in this resistance. In order to minimize the contribution of metabolism and diffusional barriers, larvae were hemisected and the anterior half immersed in a range of ethanol concentrations thereby ensuring the free access of bath ethanol to the brain. The response to vibrational stimuli of three types of reticulospinal neurons: Mauthner neurons, vestibulospinal neurons, and MiD3 neurons were examined using an intracellular calcium indicator. The intracellular [Ca(2+)] response in MiD3 neurons decreased in 100 mM ethanol, while Mauthner neurons and vestibulospinal neurons required >300 mM ethanol to elicit similar effects. The ethanol effect in Mauthner neurons was reversible following removal of ethanol. Interestingly, activities of MiD3 neurons displayed spontaneous recovery in 300 mM ethanol, suggestive of acute tolerance. Finally, we examined with mechanical vibration the startle response of free-swimming larvae in 300 mM ethanol. Ethanol treatment abolished long latency startle responses, suggesting a functional change in neural processing. These data support the hypothesis that individual neurons in larval zebrafish brains have distinct patterns of response to ethanol dictated by specific molecular targets.

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Recovery and adaptation of neuronal activity in 300mM ethanol. Fluorescence intensities of Mauthner neurons and MiD3 neurons were measured in response to sound/vibration stimuli. Sectioned larvae were either washed after 30 min incubation of 300 mM ethanol (A) or bathed continuously in 300 mM ethanol (D, E). Black, red, green and blue traces correspond to 0, 30, 60 and 90 min, respectively. The peaks of ΔF/F normalized to 0 min were plotted against time in B (for A) and F (for D, E) (*P<0.05, ***P<0.001 versus 0 min). Five Mauthner neurons and six MiD3 neurons were measured. Stimuli were applied and calcium transients were elicited at each concentration 2–5 times. The numbers of samplings are shown in parentheses. (C) AO staining in 0 mM (top) and 300 mM (bottom) ethanol are shown. Apoptosis in olfactory organs is marked with arrowheads. Apoptosis of neuromasts, located on the body surface, are shown with arrowheads in insets. Nonspecific signals are observed in the gut (Arrows).
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pone-0063318-g004: Recovery and adaptation of neuronal activity in 300mM ethanol. Fluorescence intensities of Mauthner neurons and MiD3 neurons were measured in response to sound/vibration stimuli. Sectioned larvae were either washed after 30 min incubation of 300 mM ethanol (A) or bathed continuously in 300 mM ethanol (D, E). Black, red, green and blue traces correspond to 0, 30, 60 and 90 min, respectively. The peaks of ΔF/F normalized to 0 min were plotted against time in B (for A) and F (for D, E) (*P<0.05, ***P<0.001 versus 0 min). Five Mauthner neurons and six MiD3 neurons were measured. Stimuli were applied and calcium transients were elicited at each concentration 2–5 times. The numbers of samplings are shown in parentheses. (C) AO staining in 0 mM (top) and 300 mM (bottom) ethanol are shown. Apoptosis in olfactory organs is marked with arrowheads. Apoptosis of neuromasts, located on the body surface, are shown with arrowheads in insets. Nonspecific signals are observed in the gut (Arrows).

Mentions: We examined whether the effects of 300 mM ethanol on zebrafish neurons were reversible. When the sectioned preparation was bathed continuously in 300 mM ethanol, the ΔF/F peak in Mauthner neurons showed a marked decrease between 0 and 30 min, and remained stable after 30 min (Fig. 4D, F). In contrast, when washed after 30 min, Mauthner neurons displayed a robust recovery approaching the level of the pre-ethanol treatment (Fig. 4A, B). This suggests that 300 mM ethanol does not cause an irreversible effect on Mauthner neurons. To further examine the effect of 300 mM on neurons other than Mauthner neurons, AO staining was performed in 4 dpf larval zebrafish after 1 hour exposure of 300 mM ethanol. AO binds to DNA in apoptotic cells and is used widely as a marker of apoptosis in zebrafish [22], [23]. The olfactory organ and neuromasts showed apoptosis in untreated as well as ethanol treated larvae, as was previously reported using TUNEL assays [24]. Unlike chronic exposure of ethanol [19], increase of apoptosis was not observed after 1 hr exposure to 300 mM ethanol, even in larvae with a relatively high level of olfactory organ apoptosis (Fig. 4C).


Intrinsic properties of larval zebrafish neurons in ethanol.

Ikeda H, Delargy AH, Yokogawa T, Urban JM, Burgess HA, Ono F - PLoS ONE (2013)

Recovery and adaptation of neuronal activity in 300mM ethanol. Fluorescence intensities of Mauthner neurons and MiD3 neurons were measured in response to sound/vibration stimuli. Sectioned larvae were either washed after 30 min incubation of 300 mM ethanol (A) or bathed continuously in 300 mM ethanol (D, E). Black, red, green and blue traces correspond to 0, 30, 60 and 90 min, respectively. The peaks of ΔF/F normalized to 0 min were plotted against time in B (for A) and F (for D, E) (*P<0.05, ***P<0.001 versus 0 min). Five Mauthner neurons and six MiD3 neurons were measured. Stimuli were applied and calcium transients were elicited at each concentration 2–5 times. The numbers of samplings are shown in parentheses. (C) AO staining in 0 mM (top) and 300 mM (bottom) ethanol are shown. Apoptosis in olfactory organs is marked with arrowheads. Apoptosis of neuromasts, located on the body surface, are shown with arrowheads in insets. Nonspecific signals are observed in the gut (Arrows).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643919&req=5

pone-0063318-g004: Recovery and adaptation of neuronal activity in 300mM ethanol. Fluorescence intensities of Mauthner neurons and MiD3 neurons were measured in response to sound/vibration stimuli. Sectioned larvae were either washed after 30 min incubation of 300 mM ethanol (A) or bathed continuously in 300 mM ethanol (D, E). Black, red, green and blue traces correspond to 0, 30, 60 and 90 min, respectively. The peaks of ΔF/F normalized to 0 min were plotted against time in B (for A) and F (for D, E) (*P<0.05, ***P<0.001 versus 0 min). Five Mauthner neurons and six MiD3 neurons were measured. Stimuli were applied and calcium transients were elicited at each concentration 2–5 times. The numbers of samplings are shown in parentheses. (C) AO staining in 0 mM (top) and 300 mM (bottom) ethanol are shown. Apoptosis in olfactory organs is marked with arrowheads. Apoptosis of neuromasts, located on the body surface, are shown with arrowheads in insets. Nonspecific signals are observed in the gut (Arrows).
Mentions: We examined whether the effects of 300 mM ethanol on zebrafish neurons were reversible. When the sectioned preparation was bathed continuously in 300 mM ethanol, the ΔF/F peak in Mauthner neurons showed a marked decrease between 0 and 30 min, and remained stable after 30 min (Fig. 4D, F). In contrast, when washed after 30 min, Mauthner neurons displayed a robust recovery approaching the level of the pre-ethanol treatment (Fig. 4A, B). This suggests that 300 mM ethanol does not cause an irreversible effect on Mauthner neurons. To further examine the effect of 300 mM on neurons other than Mauthner neurons, AO staining was performed in 4 dpf larval zebrafish after 1 hour exposure of 300 mM ethanol. AO binds to DNA in apoptotic cells and is used widely as a marker of apoptosis in zebrafish [22], [23]. The olfactory organ and neuromasts showed apoptosis in untreated as well as ethanol treated larvae, as was previously reported using TUNEL assays [24]. Unlike chronic exposure of ethanol [19], increase of apoptosis was not observed after 1 hr exposure to 300 mM ethanol, even in larvae with a relatively high level of olfactory organ apoptosis (Fig. 4C).

Bottom Line: The behavioral effects of ethanol have been studied in multiple animal models including zebrafish.The intracellular [Ca(2+)] response in MiD3 neurons decreased in 100 mM ethanol, while Mauthner neurons and vestibulospinal neurons required >300 mM ethanol to elicit similar effects.The ethanol effect in Mauthner neurons was reversible following removal of ethanol.

View Article: PubMed Central - PubMed

Affiliation: Section on Model Synaptic Systems, Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland, United States of America.

ABSTRACT
The behavioral effects of ethanol have been studied in multiple animal models including zebrafish. Locomotion of zebrafish larvae is resistant to high concentrations of ethanol in bath solution. This resistance has been attributed to a lower systemic concentration of ethanol in zebrafish when compared with bath solution, although the mechanism to maintain such a steep gradient is unclear. Here we examined whether the intrinsic properties of neurons play roles in this resistance. In order to minimize the contribution of metabolism and diffusional barriers, larvae were hemisected and the anterior half immersed in a range of ethanol concentrations thereby ensuring the free access of bath ethanol to the brain. The response to vibrational stimuli of three types of reticulospinal neurons: Mauthner neurons, vestibulospinal neurons, and MiD3 neurons were examined using an intracellular calcium indicator. The intracellular [Ca(2+)] response in MiD3 neurons decreased in 100 mM ethanol, while Mauthner neurons and vestibulospinal neurons required >300 mM ethanol to elicit similar effects. The ethanol effect in Mauthner neurons was reversible following removal of ethanol. Interestingly, activities of MiD3 neurons displayed spontaneous recovery in 300 mM ethanol, suggestive of acute tolerance. Finally, we examined with mechanical vibration the startle response of free-swimming larvae in 300 mM ethanol. Ethanol treatment abolished long latency startle responses, suggesting a functional change in neural processing. These data support the hypothesis that individual neurons in larval zebrafish brains have distinct patterns of response to ethanol dictated by specific molecular targets.

Show MeSH
Related in: MedlinePlus