Skip to main content
SpringerLink
Log in

Analysis of various physiological salines for heart rate, CNS function, and synaptic transmission at neuromuscular junctions in Drosophila melanogaster larvae

  • Original Paper
  • Published:
Journal of Comparative Physiology A Aims and scope Submit manuscript

Abstract

Drosophila serves as a playground for examining the effects of genetic mutations on development, physiological function and behavior. Many physiological measures that address the effects of mutations require semi-intact or cultured preparations. To obtain consistent physiological recordings, cellular function needs to remain viable. Numerous physiological salines have been developed for fly preparations, with emphasis on nervous system viability. The commonly used saline drifts in pH and will cause an alteration in the heart rate. We identify a saline that maintains a stable pH and physiological function in the larval heart, skeletal neuromuscular junction, and ventral nerve cord preparations. Using these common assays, we screened various pH buffers of differing concentrations to identify optimum conditions. Buffers at 25 mM produce a stable heart rate with minimal variation in pH. Excitatory junction potentials evoked directly on larval muscles or through sensory-CNS-motor circuits were unaffected by at buffers at 25 mM. The salines examined did not impede the modulatory effect of serotonin on heart rate or neural activity. Together, our results indicate that the higher buffer concentrations needed to stabilize pH in HL3 hemolymph-like saline do not interfere with the acute function of neurons or cardiac myocytes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

BES:

5 N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid

HEPES:

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

TRIZMA:

Tris(hydroxymethyl)aminomethane

HR:

Heart rate

CNS:

Central nervous system

EJP:

Excitatory junction potential

NMJ:

Neuromuscular junction

HL3:

Hemolymph-like saline

References

  • Akasaka T, Ocorr K (2009) Drug discovery through functional screening in the Drosophila heart. Meth Mol Biol 577:235–249. doi:10.1007/978-1-60761-232-2_18

    Article  CAS  Google Scholar 

  • Badre NH, Cooper RL (2008) Reduced calcium channel function in Drosophila disrupts associative learning in larva, and behavior in adults. Int J Zool Res 4(3):152–164

    Article  Google Scholar 

  • Badre NH, Martin ME, Cooper RL (2005) The physiological and behavioral effects of carbon dioxide on Drosophila larvae. Comp Biochem Physiol A 140:363–376

    Article  Google Scholar 

  • Ball R, Xing B, Bonner P, Shearer J, Cooper RL (2003) Long-term maintenance of neuromuscular junction activity in cultured Drosophila larvae. Comp Biochem Physiol A 134:247–255

    Article  Google Scholar 

  • Becnel J, Johnson O, Majeed ZR, Tran V, Yu B, Roth BL, Cooper RL, Nichols CD (2013) A pharmacogenetic approach for robustly controlling behavior, neuronal signaling, and physiology in Drosophila. Cell Rep 4(5):1049–1059

    Article  CAS  PubMed  Google Scholar 

  • Bhatt D, Cooper RL (2005) The pharmacological and physiological profile of glutamate receptors at the Drosophila larval neuromuscular junction. Physiol Entomol 30:205–210

    Article  CAS  Google Scholar 

  • Bier E, Bodmer R (2004) Drosophila, an emerging model for cardiac disease. Gene 342(1):1–11

    Article  CAS  PubMed  Google Scholar 

  • Bodmer W (1995) Where will genome analysis lead us forty years on? Ann N Y Acad Sci 758:414–426

    Article  CAS  PubMed  Google Scholar 

  • Cooper AS, Rymond KE, Ward MA, Bocook EL, Cooper RL (2009) Monitoring heart function in larval Drosophila melanogaster for physiological studies. J Vis Exp (JoVE). 32: http://www.jove.com/index/details.stp?id=1596

  • Cripps RM, Olson EN (2002) Control of cardiac development by an evolutionarily conserved transcriptional network. Dev Biol 246(1):14–28

    Article  CAS  PubMed  Google Scholar 

  • Dasari S, Cooper RL (2004) Modulation of sensory to motor circuits by serotonin, octopamine, and dopamine in semi-intact Drosophila larva. Neurosci Res 48:221–227

    Article  CAS  PubMed  Google Scholar 

  • Dasari S, Cooper RL (2006) Direct influence of serotonin on the larval heart of Drosophila melanogaster. J Comp Physiol B 176:349–357

    Article  CAS  PubMed  Google Scholar 

  • Dasari S, Viele K, Turner AC, Cooper RL (2007) Influence of p-CPA and MDMA on physiology, development and behavior in Drosophila melanogaster. Eur J Neurosci 26:424–438

    Article  PubMed  Google Scholar 

  • Dasari S, Wang L, Harrison DA, Cooper RL (2009) Reduced and misexpression of 5-HT2 receptors alters development, behavior and CNS activity in Drosophila melanogaster. Int J Zool Res 5(3):1–14

    Google Scholar 

  • Desai-Shah M, Papoy AR, Ward M, Cooper RL (2010) Roles of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, plasma membrane Ca2+-ATPase and Na+/Ca2+ exchanger in regulation of heart rate in larval Drosophila. Open Physiol J 3:16–36

    Article  CAS  Google Scholar 

  • Durst RA, Staples BR (1972) Tris/Tris HCl: a standard buffer for use in the physiologic pH range. Clin Chem 18:206

    CAS  PubMed  Google Scholar 

  • Echalier G (1976) In vitro culture of Drosophila cells and applications in physiological genetics. In: Kurstak E, Maramorosch K (eds) Invertebrate tissue culture. Academic Press, New York, pp 131–150

    Chapter  Google Scholar 

  • Feng Y, Ueda A, Wu CF (2004) A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae. J Neurogenet 18:377–402

    Article  CAS  PubMed  Google Scholar 

  • Fink M, Callol-Massot C, Chu A, Ruiz-Lozano P, Belmonte JC, Giles W, Bodmer R, Ocorr K (2009) A new method for detection and quantification of heartbeat parameters in Drosophila, zebrafish, and embryonic mouse hearts. Biotechniques 46(2):101–113

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Frank CA (2013) Homeostatic plasticity at the Drosophila neuromuscular junction. Neuropharmacology. doi:10.1016/j.neuropharm.2013.06.015 (in press)

  • Ganetzky B (2000) Genetic analysis of ion channel dysfunction in Drosophila. Kidney Int 57(3):766–771

    Article  CAS  PubMed  Google Scholar 

  • Gu GG, Singh S (1995) Pharmacological analysis of heartbeat in Drosophila. J Neurobiol 28:269–280

    Article  CAS  PubMed  Google Scholar 

  • Guerrero G, Reiff DF, Agarwal G, Ball RW, Borst A, Goodman CS, Isacoff EY (2005) Heterogeneity in synaptic transmission along a Drosophila larval motor axon. Nat Neurosci 8:1188–1196

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Illingworth JA (1981) A common source of error in pH measurements. Biochem J 195:259–262

    CAS  PubMed  Google Scholar 

  • Jan LY, Jan YN (1976) Properties of the larval neuromuscular junction in Drosophila melanogaster. J Physiol 262:189–214

    CAS  PubMed  Google Scholar 

  • Johansen J, Halpern ME, Keshishian H (1989) Axonal guidance and the development of muscle fiber-specific innervation in Drosophila embryos. J Neurosci 9:4318–4332

    CAS  PubMed  Google Scholar 

  • Johnson E, Ringo J, Dowse H (1997) Modulation of Drosophila heartbeat by neurotransmitters. J Comp Physiol B 167(2):89–97

    Article  CAS  PubMed  Google Scholar 

  • Kapur S, Wasserstrom JA, Kelly JE, Kadish AH, Aistrup GL (2009) Acidosis and ischemia increase cellular Ca2+ transient alternans and repolarization alternans susceptibility in the intact rat heart. Am J Physiol Heart Circ Physiol 296:H1491–H1512

    Article  CAS  PubMed  Google Scholar 

  • Lalevee N, Monier B, Senatore S, Perrin L, Semeriva M (2006) Control of cardiac rhythm by ORK1, a Drosophila two-pore domain potassium channel. Curr Biol 16:1502–1508

    Article  CAS  PubMed  Google Scholar 

  • Lee J-Y, Bhatt D, Bhatt D, Chung W-Y, Cooper RL (2009) Furthering pharmacological and physiological assessment of the glutamatergic receptors at the Drosophila neuromuscular junction. Comp Biochem Physiol C 150:546–557

    Google Scholar 

  • Lehmacher C, Abeln B, Paululat A (2012) The ultrastructure of Drosophila heart cells. Arthropod Struct Dev 41(5):459–474. doi:10.1016/j.asd.2012.02.002

    Article  PubMed  Google Scholar 

  • Li H, Peng X, Cooper RL (2002) Development of Drosophila larval neuromuscular junctions: maintaining synaptic strength. Neuroscience 115:505–513

    Article  CAS  PubMed  Google Scholar 

  • Macleod GT, Hegström-Wojtowicz M, Charlton MP, Atwood HL (2002) Fast calcium signals in Drosophila motor neuron terminals. J Neurophysiol 88(5):2659–2663

    Article  CAS  PubMed  Google Scholar 

  • Marrus SB, Portman SL, Allen MJ, Moffat KG, DiAntonio A (2004) Differential localization of glutamate receptor subunits at the Drosophila neuromuscular junction. J Neurosci 24:1406–1415

    Article  CAS  PubMed  Google Scholar 

  • Marsh JL, Thompson LM (2006) Drosophila in the study of neurodegenerative disease. Neuron 52:169–178

    Article  CAS  PubMed  Google Scholar 

  • Mitsuhashi J (1982) Media for insect cell cultures. In: Maramorosch K (ed) Advances in cell culture, vol 2. Academic Press, New York, pp 133–196

    Google Scholar 

  • Muller KJ, Nicholls JG, Stent GS (1981) Neurobiology of the Leech. Cold Spring Harbor Laboratory. Cold spring Harbor, New York p254

    Google Scholar 

  • Na J, Musselman LP, Pendse J, Baranski TJ, Bodmer R, Ocorr K, Cagan R (2013) A Drosophila model of high sugar diet-induced cardiomyopathy. PLoS Genet 9(1):e1003175. doi:10.1371/journal.pgen.1003175

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Neckameyer WS, Argue KJ (2012) Comparative approaches to the study of physiology: Drosophila as a physiological tool. Am J Physiol Reg Integr Comp Physiol 304(3):R177–R188. doi:10.1152/ajpregu.00084.2012

    Article  Google Scholar 

  • Nishimura M, Ocorr K, Bodmer R, Cartry J (2011) Drosophila as a model to study cardiac aging. Exp Gerontol 46(5):326–330. doi:10.1016/j.exger.2010.11.035

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ocorr K, Perrin L, Lim H-Y, Qian L, Wu X, Bodmer R (2007) Genetic control of heart function and aging in Drosophila. Trends Cardiovasc Med 17(5):177–182. doi:10.1016/j.tcm.2007.04.001

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pandey UB, Nichols CD (2011) Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery. Pharmacol Rev 63(2):411–436. doi:10.1124/pr.110.003293

    Article  CAS  PubMed  Google Scholar 

  • Park S-I, Jun SB, Park S, Kim HC, Kim SJ (2003) Application of a new Cl-plasma-treated Ag/AgCl reference electrode to micro machined glucose sensor. IEEE Sens J 3(3):267–272

    Article  CAS  Google Scholar 

  • Piyankarage SC, Augustin H, Grosjean Y, Featherstone DE, Shippy SA (2008) Hemolymph amino acid analysis of individual Drosophila larvae. Anal Chem 80(4):1201–1207. doi:10.1021/ac701785z

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Qin G, Schwarz T, Kittel RJ, Schmid A, Rasse TM, Kappei D, Ponimaskin E, Heckmann M, Sigrist SJ (2005) Four different subunits are essential for expressing the synaptic glutamate receptor at neuromuscular junctions of Drosophila. J Neurosci 25:3209–3218

    Article  CAS  PubMed  Google Scholar 

  • Ruffner ME, Cromarty SI, Cooper RL (1999) Depression of synaptic efficacy in Drosophila neuromuscular junctions by the molting hormone (20-Hydroxyecdysone). J Neurophysiol 81:788–794

    CAS  PubMed  Google Scholar 

  • Ryan MF (1969) Unreliable results. Science 165:851

    Article  CAS  PubMed  Google Scholar 

  • Schneider I, Blumenthal A (1978) Drosophila cell and tissue culture. In: Ashburner M, Wright TRF (eds) Biology and genetics of Drosophila, vol 2B. Academic Press, New York, pp 266–315

    Google Scholar 

  • Shields G, Sang JH (1977) Improved medium for culture of Drosophila embryonic cells. Drosophila Inf Serv 52:161

    Google Scholar 

  • Sigma (1996) Technical Bulletin No. 106B (9/96) Tris(hydroxymethyl)aminomethane; Tris http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/1/106bbul.Par.0001.File.tmp/106bbul.pdf

  • Stewart BA, Atwood HL, Renger JJ, Wang J, Wu CF (1994) Improved stability of Drosophila larval neuromuscular preparation in haemolymph-like physiological solutions. J Comp Physiol A 175:179–191

    Article  CAS  PubMed  Google Scholar 

  • Stewart BA, Schuster CM, Goodman CS, Atwood HL (1996) Homeostasis of synaptic transmission in Drosophila with genetically altered nerve terminal morphology. J Neurosci 16(12):3877–3886

    CAS  PubMed  Google Scholar 

  • Stone GC, Koopowitz H (1974) Mechanisms of action of CO2 on the visual response of Galleria mellonella. J Insect Physiol 20:485–496

    Article  CAS  PubMed  Google Scholar 

  • Strausfeld NJ, Hirth F (2013) Deep homology of arthropod central complex and vertebrate basal ganglia. Science 340:157–161

    Article  CAS  PubMed  Google Scholar 

  • Suzuki H, Hirakawa T, Sasaki S, Karube I (1998) Micro machined liquid-junction Ag/AgCl reference electrode. Sens Actuators B 46:146–154

    Article  CAS  Google Scholar 

  • Titlow JS, Rufer J, King K, Cooper RL (2013) Pharmacological analysis of dopamine modulation in the Drosophila melanogaster larval heart. Physiol Rep 1(2):e00020. doi:10.1002/phy2.20

    Article  PubMed Central  PubMed  Google Scholar 

  • Tsai MT, Lee CK, Chang FY, Wu JT, Wu CP, Chi TT, Yang CC (2012) Noninvasive imaging of heart chamber in Drosophila with dual-beam optical coherence tomography. J Biophotonics. doi:10.1002/jbio.201200164

    Google Scholar 

  • Venken KJ, Simpson JH, Bellen HJ (2011) Genetic manipulation of genes and cells in the nervous system of the fruit fly. Neuron 72:202–230

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wang JW, Wong AM, Flores J, Vosshall LB, Axel R (2003) Two-photon calcium imaging reveals an odorevoked map of activity in the fly brain. Cell 112(2):271–282

    Article  CAS  PubMed  Google Scholar 

  • Wessells RJ, Bodmer R (2004) Screening assays for heart function mutants in Drosophila. Biotechniques 37(1):58–60

    CAS  PubMed  Google Scholar 

  • Xing B, Long AA, Harrison DA, Cooper RL (2005) Developmental consequences of NMJs with reduced presynaptic calcium channel function. Synapse 57:132–147

    Article  CAS  PubMed  Google Scholar 

  • Zhou Q, Bett GC (2010) Regulation of the voltage-insensitive step of HERG activation by extracellular pH. Am J Physiol Heart Circ Physiol 298:H1710–H1718

    Article  CAS  PubMed  Google Scholar 

  • Zornik E, Paisley K, Nichols R (1999) Neural transmitters and a peptide modulate Drosophila heart rate. Peptides 20:45–51

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was funded by the Higher Committee for Education Development (HCED) in Iraq (ZRM) and personal funds (RLC). We thank the former high school students from Lexington, Kentucky Ms. Kylah Rymond and Ms. Valarie Sarge for preliminary studies in modifying the concentrations in amino acids and Ca2+ in the HL3 saline which lead to this current project.

Author information

Authors and Affiliations

  1. Sayre School, Upper School, 194 North Limestone, Lexington, KY, 40507, USA

    Clara de Castro

  2. Department of Biology, University of Kentucky, 675 Rose Street, Lexington, KY, 40506-0225, USA

    Clara de Castro, Josh Titlow, Zana R. Majeed & Robin L. Cooper

  3. Center for Muscle Biology, University of Kentucky, Lexington, KY, 40506-0225, USA

    Clara de Castro, Josh Titlow, Zana R. Majeed & Robin L. Cooper

  4. Department of Biology, College of Science, University of Salahaddin, Erbil, Iraq

    Zana R. Majeed

Authors
  1. Clara de Castro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Josh Titlow
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zana R. Majeed
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robin L. Cooper
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robin L. Cooper.

Additional information

The authors C. de Castro, J. Titlow, Z. R. Majeed, and R. L. Cooper contributed equally.

Rights and permissions

Reprints and permissions

About this article

Cite this article

de Castro, C., Titlow, J., Majeed, Z.R. et al. Analysis of various physiological salines for heart rate, CNS function, and synaptic transmission at neuromuscular junctions in Drosophila melanogaster larvae. J Comp Physiol A 200, 83–92 (2014). https://doi.org/10.1007/s00359-013-0864-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00359-013-0864-0

Keywords

Search

Navigation