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Neurochemical measurements in the zebrafish brain.

Jones LJ, McCutcheon JE, Young AM, Norton WH - Front Behav Neurosci (2015)

Bottom Line: In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon.We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain.This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Psychology and Behaviour, University of Leicester Leicester, UK.

ABSTRACT
The zebrafish is an ideal model organism for behavioral genetics and neuroscience. The high conservation of genes and neurotransmitter pathways between zebrafish and other vertebrates permits the translation of research between species. Zebrafish behavior can be studied at both larval and adult stages and recent research has begun to establish zebrafish models for human disease. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the detection of neurotransmitter release and reuptake. In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon. We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain. This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain.

No MeSH data available.


Dopamine reuptake inhibition with cocaine modifies analyte release profile in the telencephalon. (A–D) Color plots showing the time course (x-axis) of changes in current as a function of the applied waveform (y-axis) after a control stimulation (A) and 10 (B), 20 (C), and 40 (D) min after 10 μM cocaine administration. (E–H) Current vs. time plots showing the time course of changes in current after a control stimulation (E) and 10 (F), 20 (G), and 40 (H) min after 10 μM cocaine administration. (I–L) Cyclic voltammograms extracted immediately following onset of release with oxidation and reduction peaks becoming larger at ~ +0.65 V and ~ −0.25 V over time; control stimulation (I) and 10 (J), 20 (K), and 40 (L) min after 10 μM cocaine administration. (M–P) Individual data points showing peak height (M), peak area (N), half width (O), and T half (P) for control stimulations (C1, C2, C3) or at the time points indicated.
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Figure 9: Dopamine reuptake inhibition with cocaine modifies analyte release profile in the telencephalon. (A–D) Color plots showing the time course (x-axis) of changes in current as a function of the applied waveform (y-axis) after a control stimulation (A) and 10 (B), 20 (C), and 40 (D) min after 10 μM cocaine administration. (E–H) Current vs. time plots showing the time course of changes in current after a control stimulation (E) and 10 (F), 20 (G), and 40 (H) min after 10 μM cocaine administration. (I–L) Cyclic voltammograms extracted immediately following onset of release with oxidation and reduction peaks becoming larger at ~ +0.65 V and ~ −0.25 V over time; control stimulation (I) and 10 (J), 20 (K), and 40 (L) min after 10 μM cocaine administration. (M–P) Individual data points showing peak height (M), peak area (N), half width (O), and T half (P) for control stimulations (C1, C2, C3) or at the time points indicated.

Mentions: The identity of analytes released during FSCV can be further confirmed by pharmacological validation (Dankoski and Wightman, 2013). The results of the CV match and PCA analyses suggest that dopamine is likely to be a major contributor to the changes in current that we measured. We further investigated this prediction by manipulating dopamine pharmacologically. We treated sagittal slices of the adult zebrafish brain with either cocaine, a non-selective monoamine reuptake inhibitor that has been shown to increase dopamine reuptake within the rodent nucleus accumbens, caudate putamen and substantia nigra (Jones et al., 1995a,b; Davidson et al., 2000; John and Jones, 2007a,b; España et al., 2008; Yorgason et al., 2011) or the selective long-acting dopamine reuptake inhibitor GBR 12909 (España et al., 2008; Esposti et al., 2013). Treatment with 10 μM cocaine produced an increase in current at ~ +0.6 V that appeared to be prolonged (Figures 9B–D) compared to controls (Figure 9A). Current vs. time plots showed peaks that become broader over time (Figures 9E–H) indicating a slowing down of reuptake kinetics. Comparison of cyclic voltammograms from these experiments further demonstrated that oxidation- and reduction peaks became more prominent at ~ +0.65 and ~ −0.25 V, respectively (Figures 9I–L) following cocaine application, with a shape that was more similar to the dopamine voltammogram obtained in the flow cell (Figure 2B). Current vs. time plots further illustrated this, as peaks became broader over time (Figures 9I–L) indicating a slowing down of reuptake kinetics. Moreover, there was a slight increase in the amplitude of peaks (Figures 9M,N), suggesting that cocaine may also affect dopamine release in the zebrafish telencephalon. We used One-Way repeated measures ANOVA tests followed by Dunnett's multiple comparisons tests with p-value adjustment to compare the average value of three control stimulations with four time-points following cocaine perfusion (n = 6 fish in each case). Non-parametric tests were used when the data were not normally distributed. There was a significant effect of cocaine on peak height [nA; F(4, 20) = 4.91, p < 0.01]. Post-hoc Dunnett's tests revealed that peak height was significantly larger than control at 10 min (p < 0.005), 20 min (p < 0.005), and 30 min (p < 0.05) after cocaine perfusion, but not after 40 min (p > 0.05). Cocaine also had a significant effect on half width [s; F(4, 20) = 23.14, p < 0.0001]. Half width was significantly increased 10 min (p < 0.01), 20 min (p < 0.0001), 30 min (p < 0.0001), and 40 min (p < 0.0001) after perfusion. There was also a significant effect on T Half [s; F(4, 20) = 23.23, p < 0.0001]. T Half was significantly increased at 10 min (p = 0.005), 20 min, (p < 0.0001), 30 min (p < 0.0001), and 40 min (p < 0.0001). Cocaine also significantly influenced peak area [nA*s; F(4, 20) = 19.17, p < 0.0001]. Dunnett's tests revealed peak area was significantly larger 10 min, (p < 0.005), 20 min (p < 0.0001), 30 min (p < 0.0001), and 40 min (p < 0.0001) following cocaine perfusion. Tau decay (s) was also significantly altered [, p = 0.001 Friedman test]. Post-hoc tests showed that tau decay was significantly larger 20 min (p = 0.001), 30 min (p < 0.05), and 40 min (p < 0.005) following cocaine application (Figures 9M–P). Treatment with 10 μM of the more selective dopamine reuptake inhibitor GBR 12909 led to an increase in the amplitude and time course of current at ~ +0.6 V following electrical stimulation (Figures 10A–D). The related current vs. time plots show that peaks become larger and somewhat broader over time indicating an increase in release and possibly a slowing of reuptake kinetics as well (Figures 10E–H). Comparison of cyclic voltammograms from these experiments provided further evidence for an increase in the amplitude of release, as the peak around the oxidation potential for dopamine (~ +0.65 V) became considerably larger (Figures 10I–L). The effects of 10 μM GBR 12909 application were analyzed using non-parametric tests to account for significant deviation from normality. Friedman tests followed by post-hoc Dunn's multiple comparisons with p-value adjustment were used to compare the average value of three control stimulations with four time-points post GBR 12909 perfusion (n = 6 fish in each case). GBR 12909 had a significant effect on peak height [nA; = 16.93, p < 0.005]. Peak height was significantly increased after 30 min, (p < 0.05) and 40 min (p < 0.0001) drug application. GBR 12909 did not significantly alter half width (s; p = 0.0504), however there was a significant effect on T half [s; , p < 0.05]. Dunn's tests revealed that T half was significantly increased after 20 min, (p < 0.05), 30 min (p < 0.05), and 40 min (p < 0.05). There was also a significant effect of GBR 12909 on peak area [nA*s; = 14.67, p < 0.01]. Peak area was significant increased after 20 min, (p < 0.05), 30 min, (p < 0.05), and 40 min, (p < 0.005) GBR 12909 perfusion (Figures 10M–P). GBR 12909 did not significantly alter tau decay (p > 0.05). Taken together, the combination of statistical analysis and pharmacological studies demonstrates that stimulation of the telencephalon evokes the release of dopamine, with possible release of histamine and a concomitant acidic change in pH as well.


Neurochemical measurements in the zebrafish brain.

Jones LJ, McCutcheon JE, Young AM, Norton WH - Front Behav Neurosci (2015)

Dopamine reuptake inhibition with cocaine modifies analyte release profile in the telencephalon. (A–D) Color plots showing the time course (x-axis) of changes in current as a function of the applied waveform (y-axis) after a control stimulation (A) and 10 (B), 20 (C), and 40 (D) min after 10 μM cocaine administration. (E–H) Current vs. time plots showing the time course of changes in current after a control stimulation (E) and 10 (F), 20 (G), and 40 (H) min after 10 μM cocaine administration. (I–L) Cyclic voltammograms extracted immediately following onset of release with oxidation and reduction peaks becoming larger at ~ +0.65 V and ~ −0.25 V over time; control stimulation (I) and 10 (J), 20 (K), and 40 (L) min after 10 μM cocaine administration. (M–P) Individual data points showing peak height (M), peak area (N), half width (O), and T half (P) for control stimulations (C1, C2, C3) or at the time points indicated.
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Related In: Results  -  Collection

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Figure 9: Dopamine reuptake inhibition with cocaine modifies analyte release profile in the telencephalon. (A–D) Color plots showing the time course (x-axis) of changes in current as a function of the applied waveform (y-axis) after a control stimulation (A) and 10 (B), 20 (C), and 40 (D) min after 10 μM cocaine administration. (E–H) Current vs. time plots showing the time course of changes in current after a control stimulation (E) and 10 (F), 20 (G), and 40 (H) min after 10 μM cocaine administration. (I–L) Cyclic voltammograms extracted immediately following onset of release with oxidation and reduction peaks becoming larger at ~ +0.65 V and ~ −0.25 V over time; control stimulation (I) and 10 (J), 20 (K), and 40 (L) min after 10 μM cocaine administration. (M–P) Individual data points showing peak height (M), peak area (N), half width (O), and T half (P) for control stimulations (C1, C2, C3) or at the time points indicated.
Mentions: The identity of analytes released during FSCV can be further confirmed by pharmacological validation (Dankoski and Wightman, 2013). The results of the CV match and PCA analyses suggest that dopamine is likely to be a major contributor to the changes in current that we measured. We further investigated this prediction by manipulating dopamine pharmacologically. We treated sagittal slices of the adult zebrafish brain with either cocaine, a non-selective monoamine reuptake inhibitor that has been shown to increase dopamine reuptake within the rodent nucleus accumbens, caudate putamen and substantia nigra (Jones et al., 1995a,b; Davidson et al., 2000; John and Jones, 2007a,b; España et al., 2008; Yorgason et al., 2011) or the selective long-acting dopamine reuptake inhibitor GBR 12909 (España et al., 2008; Esposti et al., 2013). Treatment with 10 μM cocaine produced an increase in current at ~ +0.6 V that appeared to be prolonged (Figures 9B–D) compared to controls (Figure 9A). Current vs. time plots showed peaks that become broader over time (Figures 9E–H) indicating a slowing down of reuptake kinetics. Comparison of cyclic voltammograms from these experiments further demonstrated that oxidation- and reduction peaks became more prominent at ~ +0.65 and ~ −0.25 V, respectively (Figures 9I–L) following cocaine application, with a shape that was more similar to the dopamine voltammogram obtained in the flow cell (Figure 2B). Current vs. time plots further illustrated this, as peaks became broader over time (Figures 9I–L) indicating a slowing down of reuptake kinetics. Moreover, there was a slight increase in the amplitude of peaks (Figures 9M,N), suggesting that cocaine may also affect dopamine release in the zebrafish telencephalon. We used One-Way repeated measures ANOVA tests followed by Dunnett's multiple comparisons tests with p-value adjustment to compare the average value of three control stimulations with four time-points following cocaine perfusion (n = 6 fish in each case). Non-parametric tests were used when the data were not normally distributed. There was a significant effect of cocaine on peak height [nA; F(4, 20) = 4.91, p < 0.01]. Post-hoc Dunnett's tests revealed that peak height was significantly larger than control at 10 min (p < 0.005), 20 min (p < 0.005), and 30 min (p < 0.05) after cocaine perfusion, but not after 40 min (p > 0.05). Cocaine also had a significant effect on half width [s; F(4, 20) = 23.14, p < 0.0001]. Half width was significantly increased 10 min (p < 0.01), 20 min (p < 0.0001), 30 min (p < 0.0001), and 40 min (p < 0.0001) after perfusion. There was also a significant effect on T Half [s; F(4, 20) = 23.23, p < 0.0001]. T Half was significantly increased at 10 min (p = 0.005), 20 min, (p < 0.0001), 30 min (p < 0.0001), and 40 min (p < 0.0001). Cocaine also significantly influenced peak area [nA*s; F(4, 20) = 19.17, p < 0.0001]. Dunnett's tests revealed peak area was significantly larger 10 min, (p < 0.005), 20 min (p < 0.0001), 30 min (p < 0.0001), and 40 min (p < 0.0001) following cocaine perfusion. Tau decay (s) was also significantly altered [, p = 0.001 Friedman test]. Post-hoc tests showed that tau decay was significantly larger 20 min (p = 0.001), 30 min (p < 0.05), and 40 min (p < 0.005) following cocaine application (Figures 9M–P). Treatment with 10 μM of the more selective dopamine reuptake inhibitor GBR 12909 led to an increase in the amplitude and time course of current at ~ +0.6 V following electrical stimulation (Figures 10A–D). The related current vs. time plots show that peaks become larger and somewhat broader over time indicating an increase in release and possibly a slowing of reuptake kinetics as well (Figures 10E–H). Comparison of cyclic voltammograms from these experiments provided further evidence for an increase in the amplitude of release, as the peak around the oxidation potential for dopamine (~ +0.65 V) became considerably larger (Figures 10I–L). The effects of 10 μM GBR 12909 application were analyzed using non-parametric tests to account for significant deviation from normality. Friedman tests followed by post-hoc Dunn's multiple comparisons with p-value adjustment were used to compare the average value of three control stimulations with four time-points post GBR 12909 perfusion (n = 6 fish in each case). GBR 12909 had a significant effect on peak height [nA; = 16.93, p < 0.005]. Peak height was significantly increased after 30 min, (p < 0.05) and 40 min (p < 0.0001) drug application. GBR 12909 did not significantly alter half width (s; p = 0.0504), however there was a significant effect on T half [s; , p < 0.05]. Dunn's tests revealed that T half was significantly increased after 20 min, (p < 0.05), 30 min (p < 0.05), and 40 min (p < 0.05). There was also a significant effect of GBR 12909 on peak area [nA*s; = 14.67, p < 0.01]. Peak area was significant increased after 20 min, (p < 0.05), 30 min, (p < 0.05), and 40 min, (p < 0.005) GBR 12909 perfusion (Figures 10M–P). GBR 12909 did not significantly alter tau decay (p > 0.05). Taken together, the combination of statistical analysis and pharmacological studies demonstrates that stimulation of the telencephalon evokes the release of dopamine, with possible release of histamine and a concomitant acidic change in pH as well.

Bottom Line: In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon.We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain.This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Psychology and Behaviour, University of Leicester Leicester, UK.

ABSTRACT
The zebrafish is an ideal model organism for behavioral genetics and neuroscience. The high conservation of genes and neurotransmitter pathways between zebrafish and other vertebrates permits the translation of research between species. Zebrafish behavior can be studied at both larval and adult stages and recent research has begun to establish zebrafish models for human disease. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the detection of neurotransmitter release and reuptake. In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon. We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain. This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain.

No MeSH data available.