Extracellular dopamine and alterations on dopamine transporter are related to reserpine toxicity in Caenorhabditis elegans

Abstract

Reserpine is used as an animal model of parkinsonism. We hypothesized that the involuntary movements induced by reserpine in rodents are induced by dopaminergic toxicity caused by extracellular dopamine accumulation. The present study tested the effects of reserpine on the dopaminergic system in Caenorhabditis elegans. Reserpine was toxic to worms (decreased the survival, food intake, development and changed egg laying and defecation cycles). In addition, reserpine increased the worms' locomotor rate on food and decreased dopamine levels. Morphological evaluations of dopaminergic CEP neurons confirmed neurodegeneration characterized by decreased fluorescence intensity and the number of worms with intact CEP neurons, and increased number of shrunken somas per worm. These effects were unrelated to reserpine's effect on decreased expression of the dopamine transporter, dat-1. Interestingly, the locomotor rate on food and the neurodegenerative parameters fully recovered to basal conditions upon reserpine withdrawal. Furthermore, reserpine decreased survival in vesicular monoamine transporter and dat-1 loss-of-function mutant worms. In addition, worms pre-exposed to dopamine followed by exposure to reserpine had decreased survival. Reserpine activated gst-4, which controls a phase II detoxification enzymes downstream of nuclear factor (erythroid-derived-2)-like 2. Our findings establish that the dopamine transporter, dat-1, plays an important role in reserpine toxicity, likely by increasing extracellular dopamine concentrations.

Glial loss of the metallo β-lactamase domain containing protein, SWIP-10, induces age- and glutamate-signaling dependent, dopamine neuron degeneration

“…We examined the DA neurons of multiple mutant swip-10 alleles crossed to BY250, a strain that stably expresses the integrated transcriptional fusion p dat-1 :: GFP ( vtIs7) ( Fig 1A ) [ 46 ]. We focused our evaluations on CEP DA neurons, and quantitatively evaluated degeneration by three distinct morphological assessments: 1) neurite truncations and breaks in GFP-labeled dendrites ( Fig 1B and 1C ), 2) shrunken cell soma ( Fig 1D ) and 3) missing cell soma ( Fig 1E ), as previously described [ 47 , 48 ]. From these categories, we also calculated an overall degeneration score where the appearance of any of the components qualifies an animal as displaying CEP degeneration [ 48 ].…”

“…We focused our evaluations on CEP DA neurons, and quantitatively evaluated degeneration by three distinct morphological assessments: 1) neurite truncations and breaks in GFP-labeled dendrites ( Fig 1B and 1C ), 2) shrunken cell soma ( Fig 1D ) and 3) missing cell soma ( Fig 1E ), as previously described [ 47 , 48 ]. From these categories, we also calculated an overall degeneration score where the appearance of any of the components qualifies an animal as displaying CEP degeneration [ 48 ]. We found that all three available swip-10 alleles ( vt29 and vt33 from our forward genetic screen, and the larger deletion allele, tm5915 ) exhibited elevations in the degeneration index, relative to wildtype animals ( Fig 1F–1I ).…”

“…Thus, elevations in cytosolic DA that arise with pharmacological blockade of the vesicular monoamine transporter (VMAT, cat-1 in C . elegans) by reserpine results in DA neuron degeneration [ 48 ], and a genetic reduction of VMAT2 expression causes progressive DA neuron degeneration in mammals [ 56 ]. The degeneration of DA neurons in swip-10 animals does not appear to arise as a consequence of elevations of intracellular DA as disruption of DA synthesis capacity arising from a loss of function mutation in cat-2 , the C .…”

“…The neurodegeneration assay was adapted from a previously described method [ 145 ]. In our case, we transferred 20 worms to normal NGM/OP50 plates as L4s and incubated these plates for 48hrs at 19°C until animals reached the gravid adult stage, unless otherwise noted.…”

Highly sensitive isotope-dilution liquid-chromatography–electrospray ionization–tandem-mass spectrometry approach to study the drug-mediated modulation of dopamine and serotonin levels in Caenorhabditis elegans

“…Only a few publications measuring neurotransmitters in brain microdialysates exist, including Suominen et al presenting LODs for DA and SRT analyzed by LC-ESI-MS/MS of 0.20 nM (3 fmol injected onto the column) and an LOQ of 0.5 nM for DA and 1 nM for SRT [16, 17, 43]. Due to the high sensitivity of the developed method (LOQ DA: 0.37 nM; LOQ SRT: 0.14 nM; summarized in Table 2), the number of requisite worms (L1 stage) could be reduced to about 2,000 per sample compared to other HPLC methods requiring hundreds of thousands worms (200,000 worms) per sample [34, 44, 45]. While several methods extracting neurotransmitters from brain tissue are often laborious due to pretreatment or derivatization steps [13, 18, 35, 46], we developed a fast and simple extraction protocol allowing high-throughput analyses.…”