Alterations in biogenic amines levels associated with age-related muscular tissue impairment in Drosophila melanogaster

Abstract

While holding on youth may be a universal wish, aging is a natural process associated with physical and physiological impairment in living organisms. Drosophila provides useful insights into aging-related events. Hence, this study was conducted to investigate the age-related changes in muscle function and architecture in relation to the biogenic amine titers. To achieve this aim, visceral and skeletal muscles performance was tested in newly-eclosed, sexually mature and old adult flies using climbing and gut motility assays. In addition, age-related ultrastructural alterations of muscular tissue were observed using transmission electron microscopy (TEM). The titer of selected biogenic amines was measured using high-performance liquid chromatography (HPLC). The results demonstrated that old flies were dramatically slower in upward movement than either newly-eclosed or sexually mature flies. Similarly, gut contraction rate was significantly lower in old flies than the sexually mature, although it was markedly higher than that in the newly-eclosed flies. In TEM examination, there were several ultrastructural changes in the midgut epithelium, legs and thorax muscles of old flies. Regarding biogenic amine titers, the old flies had significantly lower concentrations of octopamine, dopamine and serotonin than the sexually mature. We concluded that aging has adverse effects on muscular system function and ultrastructure, synchronized with biogenic amine titers changes. Our results highlighted the need for more researches on therapeutics that may balance the levels of age-related alterations in biogenic amines.

Keywords: Aging; Biogenic amines; Drosophila; Muscles; TEM.

Graphical abstract

Fig. 1

Schematic diagram representing the time scale of the entire experimental design components. The repeated tasks were: to evaluate male climbing ability (see text), to measure males and females' gut peristalsis, to examine ultra-structural alterations in thoracic, abdominal and gut muscles of both males and females and finally, to measure the titer of biogenic amines level in both males and females. This full scheme was repeated at least three times unless other replicate numbers were mentioned in the text.

Fig. 2

Climbing speed (Median, 25%, and 75% percentiles) of newly-eclosed, sexually mature and old adult Drosophila melanogaster. n = 27 replicates. The bars are significantly different when P < 0.05 (Kruskal-Wallis test). *** refers to a significant difference between means when P < 0.0002 (Bonferroni correction on multiple comparisons). ns refers to a non– significant difference between means when P ≥ 0.033.

Fig. 3

Number of gut contractions (Mean ± SD) of newly-eclosed, sexually mature and old adult Drosophila melanogaster. n = 15 replicates. ** and *** refers to significant differences when P < 0.002 and P < 0.0002, respectively (Bonferroni correction on multiple comparisons).

Fig. 4

Transmission electron micrograph of part of Drosophila’s midgut showing: A. Normal surface epithelium of midgut with continuous long microvilli (arrowhead), intact junctional complexes (curved arrow), rounded nucleus (N) surrounded by homogenous cytoplasm (asteric), mitochondria (arrow) and other normal cellular organelles in the newly-eclosed group (Bar = 5 µm). B. Normal surface epithelium of midgut with the normal architecture of epithelium such as microvilli (arrowhead), junctional complex (curved arrow), mitochondria, cytoplasm and rough endoplasmic reticulum. Note, massive condensation of chromatin in the nucleus (N) in the sexually mature group (Bar = 5 µm). C. Cytoplasmic vacuolation (V) of the epithelium surface, irregular nucleus (N) and a few swollen degenerated mitochondria (arrow) can be observed in the old group (Bar = 10 µm). D. Focal loss of microvilli of the epithelium (arrowhead), disrupted rough endoplasmic reticulum (circle), dilated smooth endoplasmic reticulum (arrow), widening of intracellular space between the cells (curved arrow), cristolysis of some mitochondria (thin arrow) and cytoplasmic vacuolation (V) can be observed in the old group (Bar = 5 µm).

Fig. 5

Transmission electron micrograph of part of Drosophila’s thorax muscle showing: A. More or less normal oval nucleus (N), sarcoplasm (arrow), mitochondria (circle) with abundant cristae are distributed between sarcofibrils (S) in an organized sarcomeric structure. Note, slightly widening with a decrease of electron density of connective tissue between sarcomeres (curved arrow) in the newly-eclosed group (Bar = 2 µm). B. Normal architecture of sarcomere in an organized form of fibrils (S), oval regular nucleus (N) surrounded by a mild dilated perinuclear membrane (curved arrow), mitochondria (circle), sarcoplasm (arrow) and rough sarcoplasmic reticulum (arrowhead) in the sexually mature group (Bar = 2 µm). C. Vacuolated sarcoplasm (V) surrounded irregular nucleus (N) with fragmented chromatin, Oblique section of fibrils showing Z line and M line, mitochondria (circle) in the old group (Bar = 2 µm). D. Disorganized sarcomeric structure with lysis of most of the sarcofibrils (arrow), massive rarification of sarcoplasm (asteric), a decrease of electron density of nucleus (N), partial lysis of cristea of mitochondria (circle), dilated SER (arrowhead) and disrupted RER (curved arrow) in the old group (Bar = 2 µm).

Fig. 6

Transmission electron micrograph of part of Drosophila’s legs muscle showing: A. mild indented nucleus (arrow) with dispersed heterochromatin (N). Sarcoplasm between fibrils rich with vessels (arrowhead) and the fibrils showing alternates dark bands (A) which bisected by (H) zone & light bands (I) which bisected by Z line. Mitochondria (M) with abundant cristae are distributed between fibrils in the newly-eclosed group (Bar = 10 µm). B. More or less normal oval nucleus with regular outline (N). Most probably normal architecture of sarcomere showing alternate dark bands (A) which are bisected by (H) zone (arrowhead) & light bands (I) which are bisected by Z line. Normal mitochondria (M) can be observed between myofibrils in the sexually mature group (Bar = 10 µm). C. Detached junction between sarcomeres (asteric), mild rarified sarcoplasm (arrowhead), dilated SER (arrow) and focal lysis area of fibril (curved arrow) in the old group (Bar = 5 µm). D. Pyknotic nucleus (N), vacuolated sarcoplasm (V), dilated SER (arrow), slightly lysis of fibril (curved arrow), dilated sarcolemma (arrowhead), rarified sarcoplasm (asteric), dense mitochondria (M) with different size in the old group (Bar = 10 µm).

Fig. 7

Concentration (Mean ± SD) of Acetylcholine (A), dopamine (B), histamine (C), octopamine (D), tyramine (E), and serotonin (F) estimated by HPLC in newly-eclosed, sexually- mature and old adult Drosophila melanogaster. n = 3 replicates. The bars in dopamine, octopamine, and serotonin are significantly different when P < 0.05 (One-way ANOVA). *, **, and *** refer to significant differences between means when P < 0.033, P < 0.002 and P < 0.0001, respectively (Bonferroni correction on multiple comparisons). ns refers to a non-significant difference between means when P ≥ 0.033.