Biogenic amines in microdissected brain regions of Drosophila melanogaster measured with micellar electrokinetic capillary chromatography-electrochemical detection

Abstract

Micellar electrokinetic chromatography with electrochemical detection has been used to quantify biogenic amines in microdissected Drosophila melanogaster brains and brain regions. The effects of pigment from the relatively large fly eyes on the separation have been examined to find that the red pigment from the compound eye masks much of the signal from biogenic amines. The brains of white mutant flies, which have characteristically low pigment in the eyes, have a significantly simplified separation profile in comparison to the red-eyed, wild-type, Canton S fly. Yet, the white mutant flies were found to have significantly less amounts of dopamine, l-3,4-dihydroxyphenylalanine (L-DOPA), salsolinol, and N-acetyltyramine in their dissected brains when compared to dissected brains of Canton S flies. In addition, significant variation has been observed in the dissected brains between individual flies that might be related to changes in neurotransmitter turnover. The transgenic GFP fly line (TH-GFP), for which the overall profile of biogenic amines is not found to be significantly different from Canton S, can be used to visualize the location of dopamine neurons. Biogenic amines were then quantified in three brain regions observed to have dopamine levels, the central brain, optic lobes, and posterior superiormedial protocerebrum (PPM1) region.

Figure 1

(A) Image of two Drosophila heads that are analyzed in B and C. On the left is a wild-type Canton S, and the right is a white mutant. (B) Electropherogram of three homogenized Canton S Drosophila heads with 25 μM catechol internal standard, peak identified with arrow. Many peaks are observed with some being extremely large. At such a low concentration, the internal standard peak can be difficult to find and quantify. (C) Electropherogram of three homogenized white mutant Drosophila heads with 25 μM catechol as internal-standard; peak identified with arrow. The electropherogram is on the same scale as in B and demonstrates that the intense electroactive peaks observed in B are from the pigment in the eye. (Inset) A magnified view of the electropherogram. The absence of eye-pigment in this sample generates a simpler electropherogram with higher detail within the peaks.

Figure 2

Electropherograms of dissected Drosophila brain homogenates for (A) wild-type, (B) white mutant and, (C) TH-GFP mutant flies. Highlighted are peaks corresponding to dopamine (1), salsolinol (2), octopamine (3), N-acetyltyramine (4), N-acetylserotonin (5), N-acetyldopamine (6), L-DOPA (7), and the internal standard catechol (8). Some differences in peak intensities are present between these electropherograms, but intensities can be misleading due to changes in electrode efficiency from fouling which is compensated by the internal standard.

Figure 3

Separation (A) and quantification (B) of neurochemicals from five single fly brains. (A) Highlighted peaks are unidentified but correspond to different changing peak intensities between the five single-fly brain samples. (B) Bars represent the femtomole amounts of each analyte (dopamine, salsolinol, octopamine, N-acetyltyramine, N-acetylserotonin, N-acetyldopamine, and L-DOPA) measured in brains of these five flies. Flies 1–5 have different shades from left to right, and the average of all five is reported as the solid gray bar to the right of each group. Numeric averages and SEM are reported above the bar corresponding to the average amount.

Figure 4

(A) Anterior image of an intact, dissected Drosophila brain. Brackets show the two optic lobes at the sides and the central brain in the middle. (B) False-color fluorescent image of the posterior of a TH-GFP fly. Green areas indicate cells containing tyrosine hydroxylase, the enzyme responsible for catalyzing the conversion of L-tyrosine to L-DOPA and the rate limiting step in the creation of dopamine. White areas are autofluorescence from larger structures within the brain. The white box denotes the location of PPM1 region, which lies ventrally along midline of the central brain that separates it into its two halves. The 500-μm scale bar is applicable for both panels.