Dopamine receptor type 1 of Caenorhabditis elegans expressing in mechanosensory neurons

Until now the results on profiling dopamine receptors in C. elegans have been incomplete and fragmentary. The aim of this study was to investigate the expression profile of dop-1 gene in C. elegans using 3 kb promoter with 3'-end locating before ATG of dop-1gene. Methods. The strain of C. elegans with mutant unc-119 gene was used. To check a pattern of the dop-1 expression, the promoter of this gene was amplified using PCR. The animals were co-bombarded with plasmid pPD95.77 dop-1::GFP and reporter construct containing unc-119 gene. Results. Using GFP as a reporter protein, we built a whole picture of expression of dopamine receptor type 1 in C. elegans and found that this protein could be detected only in mechanosensory neurons such as PLM, PVQR, PVQL, ALNR, ALNL, DVAR, DVC.

Introduction.Dopamine is one of the most essential neurotransmitters for many animals -from nematodes to mammals.In humans it participates in the regulation of locomotor activity, thinking, emotional state, etc [1].The disorder in metabolism of this compound may result in a series of wide-spread human diseases, including schizophrenia, Parkinson disease, and Tourette syn-drome [2].
Plant parasitic nematode C. elegans was used as a model organism for over 20 years [3].Recently its application has been extended to the simulation of neurodegenerative pathologies and design of medical preparations [4].Therefore, a detailed investigation of the nervous system, including the metabolism of neuromediators and their receptors in C. elegans, is rather urgent.
Previous research on the expression profile of dopamine receptor type 1 revealed rather ambiguous results as the works demonstrated different patterns of neurons, where a reporter construction was expressed.This can be explained by uneven length and structure of promoters, used in these works [5][6][7].Therefore, it was decided to conduct a more detailed analysis of expression profile of dop-1 in C. elegans, using 3 kb promoter with 3'-end locating before ATG of the dop-1 gene.The promoter length was selected based on the task of obtaining the most specific expression pattern, independent from other DNA regulating elements, which may be located in longer promoters and cause the expression of the reporter protein in non-target neurons.
Materials and Methods.Strains.The strain C. elegans with a mutant unc-119 gene, leading to almost complete paralysis of animals, was used.The strain was obtained from the Genetic Center of Caenorhabditis (USA).The cultivation was conducted on Petri dishes with NGM-agar.The nutrient medium was Escherichia coli bacteria of OP50 strain (OP-50).
Genetic constructions.To check the expression pattern of dop-1, 3 kb promoter of this gene was amplified using a protocol of single worm polymerase chain reaction (PCR) [8].Then the promoter of dop-1 gene was built into pPD95.77(Add-gene, USA) at restriction sites HindIII and Cfr9I to obtain the construction pPD95.77dop-1:GFP (Fig. 1).

Gene bombardment of C. elegans. Animal cells
were co-bombarded with plasmid pPD95.77dop-1::GFP and reporter construction pRH21Unc119 (Prof.A. Fire's lab, USA) with unc-119 gene, promoting restoration of the wild phenotype.The bombardment was performed using PDS-1000/He system (Biorad, USA) with the pressure of 9.3 MPa and 711 mm Hg in the chamber.
The delivery of DNA was performed using gold nanoparticles, covered with linearized plasmid DNA in 3:1 ratio by a standard method [9].Two days prior to bombardment the C. elegans culture was synchronized, and 1 hour prior to the experiment it was transferred to dishes without bacteria.After bombardment the culture was incubated for 30 min at room temperature and sown to dishes with NGM agar and OP50.
The screening was started 3 days after bombardment and continued for 10 days using light and confocal microscopes.
Results and Discussion.The visualization of GFR expression under the control of the promoter of dop-1 gene revealed that the expression of the target protein is mainly registered in the neurons of the head and tail (Fig. 2, a, b).In the tail of the experimental animals GFR was revealed in neurons PLM, PVQR, PVQL, ALNR, ALNL, DVAR, DVC (Fig. 2, a).In the head of animals the fluorescence of GFR was detected in neurons CEPDL and CEPDR as well as in a numerous group of cells, surrounding the nervous ring (Fig. 2, b), belonging to the mechanosensory neurons, accountable for the response to soft touches.Besides the cranial and caudal compartments, GFP was expressed in mechanosensory neurons PVM and AVM, located in the medium part of C. elegans body (Fig. 2, a, c).
As GFP expression is registered only in the mechanosensory neurons it can be concluded that the dopamine receptor type 1 should determine the response to mechanic stimuli.This is indicated by the previously obtained results of changes in the reactions of animals with the dop-1 gene knockout to mechanic stimuli.However, it is surprising that all the above-76 STADNYK V. V. Conclusions.The expression profile of the 3 kb promoter region of dopamin receptor type 1 of C. elegans nematode was characterized using the expression of reporter gfp gene, which allows employing these results in future elaboration of new approaches to search for the means of treating diseases, related to impairment of the dopaminergic neurotransmission system.