Skip to main content
SpringerLink
Log in

Locust primary neuronal culture for the study of synaptic transmission

  • Original Paper
  • Published:
Journal of Molecular Histology Aims and scope Submit manuscript

Abstract

We have designed a cell culture system for thoracic neurons of adult Locusta migratoria that enables the establishment of functional synapses in vitro. Patch-clamp recordings revealed three different neuron classes. About half of the neurons (47%) had unexcitable somata with outward and no inward conductance. The other half generated either single (37%) or multiple action potentials (18%) and differed mainly in lower outward conductance. Selectively stained motor neurons were analyzed to demonstrate varied physiological properties due to culture conditions. Using paired patch clamp recordings we demonstrate directly synaptic transmission in morphologically connected neurons in vitro. Presynaptic stimulation resulted in postsynaptic potentials in 42 pairs of neurons tested, independent of the type of neuron. According to pharmacological experiments most of these synapses were either glutamatergic or GABAergic. In addition to these chemical synapses, electrical synapses were found. With the demonstration of synapse formation in cell culture of adult locust neurons, this study provides the basis for the future analysis of more defined insect neuronal circuits in culture.

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
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Anava S, Greenbaum A, Ben Jacob E, Hanein Y, Ayali A (2009a) The regulative role of neurite mechanical tension in network development. Biophys J 96(4):1661–1670. doi:10.1016/j.bpj.2008.10.058

    Article  PubMed  CAS  Google Scholar 

  • Anava S, Rand D, Zilberstein Y, Ayali A (2009b) Innexin genes and gap junction proteins in the locust frontal ganglion. Insect Biochem Mol Biol 39(3):224–233. doi:10.1016/j.ibmb.2008.12.002

    Article  PubMed  CAS  Google Scholar 

  • Barrio LC, Suchyna T, Bargiello T, Xu LX, Roginski RS, Bennett MV, Nicholson BJ (1991) Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage. Proc Natl Acad Sci USA 88(19):8410

    Article  PubMed  CAS  Google Scholar 

  • Bar-Yehuda D, Korngreen A (2008) Space-clamp problems when voltage clamping neurons expressing voltage-gated conductances. J Neurophysiol 99(3):1127–1136. doi:10.1152/jn.01232.2007

    Article  PubMed  Google Scholar 

  • Beadle DJ (2006) Insect neuronal cultures: an experimental vehicle for studies of physiology, pharmacology and cell interactions. Invert Neurosci 6(3):95–103. doi:10.1007/s10158-006-0024-0

    Article  PubMed  CAS  Google Scholar 

  • Benkenstein C, Schmidt M, Gewecke M (1999) Voltage-activated whole-cell K + currents in lamina cells of the desert locust schistocerca gregaria. J Exp Biol 202(Pt 14):1939

    PubMed  CAS  Google Scholar 

  • Benson JA (1992) Electrophysiological pharmacology of the nicotinic and muscarinic cholinergic responses of isolated neuronal somata from locust thoracic ganglia. J Exp Biol 170:31

    Google Scholar 

  • Bräunig P, Pflüger HJ (2001) The unpaired median neurons of insects. Adv Insect Physiol 28

  • Brone B, Tytgat J, Wang DC, Van KE (2003) Characterization of Na(+) currents in isolated dorsal unpaired median neurons of Locusta migratoria and effect of the alpha-like scorpion toxin BmK M1. J Insect Physiol 49(2):171

    Article  PubMed  CAS  Google Scholar 

  • Burrows M (1980) The control of sets of motoneurones by local interneurones in the locust. J Physiol 298:213–233

    PubMed  CAS  Google Scholar 

  • Burrows M (1996) The neurobiology of an insect brain. Oxford University Press, Oxford

    Book  Google Scholar 

  • Burrows M, Siegler MV (1978) Graded synaptic transmission between local interneurones and motor neurones in the metathoracic ganglion of the locust. J Physiol 285:231–255

    PubMed  CAS  Google Scholar 

  • Comer CM, Robertson RM (2001) Identified nerve cells and insect behavior. Prog Neurobiol 63(4):409–439

    Article  PubMed  CAS  Google Scholar 

  • Duch C, Vonhoff F, Ryglewski S (2008) Dendrite elongation and dendritic branching are affected separately by different forms of intrinsic motoneuron excitability. J Neurophysiol 100(5):2525–2536. doi:10.1152/jn.90758.2008

    Article  PubMed  Google Scholar 

  • Fuchs E, Ayali A, Robinson A, Hulata E, Ben-Jacob E (2007) Coemergence of regularity and complexity during neural network development. Dev Neurobiol 67(13):1802–1814. doi:10.1002/dneu.20557

    Article  PubMed  CAS  Google Scholar 

  • Ganfornina MD, Sanchez D, Herrera M, Bastiani MJ (1999) Developmental expression and molecular characterization of two gap junction channel proteins expressed during embryogenesis in the grasshopper Schistocerca americana. Dev Genet 24(1–2):137–150. doi:10.1002/(SICI)1520-6408(1999)24:1/2<137:AID-DVG13>3.0.CO;2-7

    PubMed  CAS  Google Scholar 

  • Gauglitz S, Pflüger HJ (2001) Cholinergic transmission via central synapses in the locust nervous system. J Comp Physiol A 187(10):825–836

    Article  PubMed  CAS  Google Scholar 

  • Giles D, Usherwood PN (1985a) The effects of putative amino acid neurotransmitters on somata isolated from neurons of the locust central nervous system. Comp Biochem Physiol, C: Comp Pharmacol Toxicol 80(2):231–236

    Article  CAS  Google Scholar 

  • Giles DP, Usherwood PN (1985b) Locust nymphal neurones in culture: a new technique for studying the physiology and pharmacology of insect central neurones. Comput Biochem Physiol C 80(1):53

    Article  CAS  Google Scholar 

  • Göbbels K, Thiebes AL, van Ooyen A, Schnakenberg U, Bräunig P (2010) Low density cell culture of locust neurons in closed-channel microfluidic devices. J Insect Physiol 56(8):1003–1009. doi:10.1016/j.jinsphys.2010.05.017

    Article  PubMed  Google Scholar 

  • Gocht D, Wagner S, Heinrich R (2009) Recognition, presence, and survival of locust central nervous glia in situ and in vitro. Microsc Res Tech 72(5):385–397. doi:10.1002/jemt.20683

    Article  PubMed  Google Scholar 

  • Goodman CS, Heitler WJ (1979) Electrical properties of insect neurones with spiking and non-spiking somata: normal, axotomized, and colchicine-treated neurones. J Exp Biol 83:95

    PubMed  CAS  Google Scholar 

  • Greenbaum A, Anava S, Ayali A, Shein M, David-Pur M, Ben-Jacob E, Hanein Y (2009) One-to-one neuron-electrode interfacing. J Neurosci Methods 182(2):219–224. doi:10.1016/j.jneumeth.2009.06.012

    Article  PubMed  Google Scholar 

  • Grünewald B, Levine RB (1998) Ecdysteroid control of ionic current development in Manduca sexta motoneurons. J Neurobiol 37(2):211–223

    Article  PubMed  Google Scholar 

  • Hancox JC, Pitman RM (1995) Spontaneous bursting induced by convulsant agents in an identified insect neurone. Gen Pharmacol 26(1):195–204

    Article  PubMed  CAS  Google Scholar 

  • Heck C, Kunst M, Härtel K, Hülsmann S, Heinrich R (2009) In vivo labeling and in vitro characterisation of central complex neurons involved in the control of sound production. J Neurosci Methods 183(2):202–212. doi:10.1016/j.jneumeth.2009.06.032

    Article  PubMed  CAS  Google Scholar 

  • Heidel E, Pflüger HJ (2006) Ion currents and spiking properties of identified subtypes of locust octopaminergic dorsal unpaired median neurons. Eur J Neurosci 23(5):1189–1206. doi:10.1111/j.1460-9568.2006.04655.x

    Article  PubMed  CAS  Google Scholar 

  • Hoyle G, Burrows M (1973) Neural mechanisms underlying behavior in the locust Schistocerca gregaria. I. Physiology of identified motorneurons in the metathoracic ganglion. J Neurobiol 4(1):3

    Google Scholar 

  • Jackson C, Bermudez I, Beadle DJ (2002) Pharmacological properties of nicotinic acetylcholine receptors in isolated Locusta migratoria neurones. Microsc Res Tech 56(4):249–255. doi:10.1002/jemt.10028

    Article  PubMed  CAS  Google Scholar 

  • Janssen D, Derst C, Buckinx R, Van den Eynden J, Rigo JM, Van Kerkhove E (2007) Dorsal unpaired median neurons of Locusta migratoria express ivermectin- and fipronil-sensitive glutamate-gated chloride channels. J Neurophysiol 97(4):2642–2650. doi:10.1152/jn.01234.2006

    Article  PubMed  CAS  Google Scholar 

  • Kirchhof B, Bicker G (1992) Growth properties of larval and adult locust neurons in primary cell culture. J Comput Neurol 323(3):411

    Article  CAS  Google Scholar 

  • Kloppenburg P, Hörner M (1998) Voltage-activated currents in identified giant interneurons isolated from adult crickets Gryllus bimaculatus. J Exp Biol 201(17):2529

    PubMed  CAS  Google Scholar 

  • Küppers-Munther B, Letzkus JJ, Lüer K, Technau G, Schmidt H, Prokop A (2004) A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses. Dev Biol 269(2):459

    Article  PubMed  Google Scholar 

  • Lapied B, Tribut F, Sinakevitch I, Hue B, Beadle DJ (1993) Neurite regeneration of long-term cultured adult insect neurosecretory cells identified as DUM neurons. Tissue Cell 6(25):893

    Article  Google Scholar 

  • Laurent G (1990) Voltage-dependent nonlinearities in the membrane of locust nonspiking local interneurons, and their significance for synaptic integration. J Neurosci 10(7):2268

    PubMed  CAS  Google Scholar 

  • Laurent G (1991) Evidence for voltage-activated outward currents in the neuropilar membrane of locust nonspiking local interneurons. J Neurosci 11(6):1713

    PubMed  CAS  Google Scholar 

  • Laurent G, Seymour-Laurent KJ, Johnson K (1993) Dendritic excitability and a voltage-gated calcium current in locust nonspiking local interneurons. J Neurophysiol 69(5):1484–1498

    PubMed  CAS  Google Scholar 

  • Lee D, O’Dowd DK (1999) Fast excitatory synaptic transmission mediated by nicotinic acetylcholine receptors in Drosophila neurons. J Neurosci 19(13):5311

    PubMed  CAS  Google Scholar 

  • Lee D, Su H, O’Dowd DK (2003) GABA receptors containing Rdl subunits mediate fast inhibitory synaptic transmission in Drosophila neurons. J Neurosci 23(11):4625

    PubMed  CAS  Google Scholar 

  • Leibovitz A (1963) The growth and maintainance of tissue-cell cultures in free gas exchange with the atmosphere. Am J Hyg 78:173

    PubMed  CAS  Google Scholar 

  • Leitch B, Watkins BL, Burrows M (1993) Distribution of acetylcholine receptors in the central nervous system of adult locusts. J Comput Neurol 334(1):47–58. doi:10.1002/cne.903340104

    Article  CAS  Google Scholar 

  • Loesel R, Weigel S, Bräunig P (2006) A simple fluorescent double staining method for distinguishing neuronal from non-neuronal cells in the insect central nervous system. J Neurosci Methods 155(2):202–206. doi:10.1016/j.jneumeth.2006.01.006

    Article  PubMed  CAS  Google Scholar 

  • Lutz EM, Tyrer NM (1987) Immunohistochemical localization of choline acetyltransferase in the central nervous system of the locust. Brain Res 407(1):173–179

    Article  PubMed  CAS  Google Scholar 

  • Melville JM, Hoffman KL, Jarrard HE, Weeks JC (2003) Cell culture of mechanoreceptor neurons innervating proleg sensory hairs in Manduca sexta larvae, and co-culture with target motoneurons. Cell Tissue Res 311(1):117

    Article  PubMed  CAS  Google Scholar 

  • Newland PL, Smith PJS, Howes EA (1993) Regenerating adult cockroach dorsal unpaired median neurons in vitro retain their in vivo membrane characteristics. J Exp Biol 179(1):323–329

    Google Scholar 

  • Oh HW, Campusano JM, Hilgenberg LG, Sun X, Smith MA, O’Dowd DK (2008) Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture. Dev Neurobiol 68(3):281–294. doi:10.1002/dneu.20575

    Article  PubMed  Google Scholar 

  • O’Shea M, Rowell CH (1976) The neuronal basis of a sensory analyser, the acridid movement detector system. II. response decrement, convergence, and the nature of the excitatory afferents to the fan-like dendrites of the LGMD. J Exp Biol 65(2):289–308

    PubMed  Google Scholar 

  • Parker D, Newland PL (1995) Cholinergic synaptic transmission between proprioceptive afferents and a hind leg motor neuron in the locust. J Neurophysiol 73(2):586–594

    PubMed  CAS  Google Scholar 

  • Pfahlert C, Lakes-Harlan R (1997) Responses of insect neurones to neurotrophic factors in vitro. Die Naturwissenschaften (84):163–165

  • Pfahlert C, Lakes-Harlan R (2008) Interneurons, motoneurons and sensory neurons of Locusta migratoria (Insecta: Orthoptera) in primary cell culture. Open J Entomol (2):6–13

  • Phelan P, Goulding LA, Tam JL, Allen MJ, Dawber RJ, Davies JA, Bacon JP (2008) Molecular mechanism of rectification at identified electrical synapses in the Drosophila giant fiber system. Curr Biol 18(24):1955–1960. doi:10.1016/j.cub.2008.10.067

    Article  PubMed  CAS  Google Scholar 

  • Raymond V, Sattelle DB, Lapied B (2000) Co-existence in DUM neurones of two GluCl channels that differ in their picrotoxin sensitivity. NeuroReport 11(12):2695–2701

    Article  PubMed  CAS  Google Scholar 

  • Reska A, Kuenzel T, Gasteier P, Schulte P, Moeller M, Offenhäusser A, Groll J (2008) Ultrathin coatings with change of reactivity over time enable functional in vitro networks of insect neurons. Adv Mater 20(14):2751–2755

    Google Scholar 

  • Rossler W, Bickmeyer U (1993) Locust medial neurosecretory cells in vitro: morphology, electrophysiological properties and effects of temperature. J Exp Biol 183(1):323

    Google Scholar 

  • Sattelle DB (1992) Receptors for L-glutamate and GABA in the nervous system of an insect (Periplaneta americana). Comp Biochem Physiol, C: Comp Pharmacol Toxicol 103(3):429–438

    Article  CAS  Google Scholar 

  • Shefi O, Golding I, Segev R, Ben-Jacob E, Ayali A (2002) Morphological characterization of in vitro neuronal networks. Phys Rev E Stat Nonlin Soft Matter Phys 66(2 Pt 1):021905

    Google Scholar 

  • Shefi O, Golebowicz S, Ben-Jacob E, Ayali A (2005) A two-phase growth strategy in cultured neuronal networks as reflected by the distribution of neurite branching angles. J Neurobiol 62(3):361–368. doi:10.1002/neu.20108

    Article  PubMed  Google Scholar 

  • Smith PJ, Howes EA (1996) Long-term culture of fully differentiated adult insect neurons. J Neurosci Methods 69(1):113

    Article  PubMed  CAS  Google Scholar 

  • Spruston N, Jaffe DB, Williams SH, Johnston D (1993) Voltage- and space-clamp errors associated with the measurement of electrotonically remote synaptic events. J Neurophysiol 70(2):781–802

    PubMed  CAS  Google Scholar 

  • Su H, O’Dowd DK (2003) Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors and picrotoxin-sensitive GABA receptors. J Neurosci 23(27):9246

    PubMed  CAS  Google Scholar 

  • Suter C, Usherwood PN (1985) Action of acetylcholine and antagonists on somata isolated from locust central neurons. Comp Biochem Physiol, C: Comp Pharmacol Toxicol 80(2):221–229

    Article  CAS  Google Scholar 

  • Thomas MV (1984) Voltage-clamp analysis of a calcium-mediated potassium conductance in cockroach (Periplaneta americana) central neurones. J Physiol 350:159–178

    PubMed  CAS  Google Scholar 

  • Torkkeli PH, Widmer A, Meisner S (2005) Expression of muscarinic acetylcholine receptors and choline acetyltransferase enzyme in cultured antennal sensory neurons and non-neural cells of the developing moth Manduca sexta. J Neurobiol 62(3):316–329. doi:10.1002/neu.20097

    Article  PubMed  CAS  Google Scholar 

  • Tribut F, Lapied B, Duval A, Pelhate M (1991) A neosynthesis of sodium channels is involved in the evolution of the sodium current in isolated adult DUM neurons. Pflugers Arch 419(6):665–667

    Article  PubMed  CAS  Google Scholar 

  • Trimarchi JR, Murphey RK (1997) The shaking-B2 mutation disrupts electrical synapses in a flight circuit in adult Drosophila. J Neurosci 17(12):4700–4710

    PubMed  CAS  Google Scholar 

  • Vanhems E, Delbos M, Girardie J (1990) Insulin and Neuroparsin promote neurite outgrowth in cultured locust CNS. Eur J Neurosci 2(9):776

    Article  PubMed  Google Scholar 

  • Vogt AK, Lauer L, Knoll W, Offenhäusser A (2003) Micropatterned substrates for the growth of functional neuronal networks of defined geometry. Biotechnol Prog 19(5):1562

    Article  PubMed  CAS  Google Scholar 

  • Wafford KA, Bai D, Sattelle DB (1992) A novel kainate receptor in the insect nervous system. Neurosci Lett 141(2):273–276

    Article  PubMed  CAS  Google Scholar 

  • Watson AH (1988) Antibodies against GABA and glutamate label neurons with morphologically distinct synaptic vesicles in the locust central nervous system. Neuroscience 26(1):33–44

    Article  PubMed  CAS  Google Scholar 

  • Watson AH, Laurent G (1990) GABA-like immunoreactivity in a population of locust intersegmental interneurones and their inputs. J Comput Neurol 302(4):761–767. doi:10.1002/cne.903020408

    Article  CAS  Google Scholar 

  • Watson AH, Schürmann FW (2002) Synaptic structure, distribution, and circuitry in the central nervous system of the locust and related insects. Microsc Res Tech 56(3):210

    Article  PubMed  Google Scholar 

  • Watson AH, Burrows M, Leitch B (1993) GABA-immunoreactivity in processes presynaptic to the terminals of afferents from a locust leg proprioceptor. J Neurocytol 22(7):547–557

    Article  PubMed  CAS  Google Scholar 

  • Wu CL, Shih MF, Lai JS, Yang HT, Turner GC, Chen L, Chiang AS (2011) Heterotypic gap junctions between two neurons in the drosophila brain are critical for memory. Curr Biol 21(10):848–854. doi:10.1016/j.cub.2011.02.041

    Article  PubMed  CAS  Google Scholar 

  • Yaksi E, Wilson RI (2010) Electrical coupling between olfactory glomeruli. Neuron 67(6):1034–1047. doi:10.1016/j.neuron.2010.08.041

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was in part supported by the EC project CICADA of the IST Programme FET Key Action Life Like Perception funded in FP 5. We are very grateful to M. Knierim-Grenzebach for her help and advice with neuronal cell culture. We thank A. Reska for her initial work in the early phase of this project and M. Gebhardt and K. Göbbels for critical comments on the manuscript.

Author information

Author notes

  1. Stefan Weigel & Petra Schulte

    Present address: Technische Universität München, 85354, Freising-Weihenstephan, Germany

Authors and Affiliations

  1. Peter Grünberg Institute, Institute of Complex Systems, Bioelectronics, Forschungszentrum Jülich, 52425, Jülich, Germany

    Stefan Weigel, Petra Schulte, Simone Meffert & Andreas Offenhäusser

  2. Unit for Developmental Biology and Morphology, Institute of Biology II, RWTH Aachen University, 52074, Aachen, Germany

    Peter Bräunig

  3. Projectmanagement Group Jülich, Biotechnology, EU and International, 52425, Jülich, Germany

    Petra Schulte

Authors
  1. Stefan Weigel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Petra Schulte
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Simone Meffert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter Bräunig
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andreas Offenhäusser
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stefan Weigel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Weigel, S., Schulte, P., Meffert, S. et al. Locust primary neuronal culture for the study of synaptic transmission. J Mol Hist 43, 405–419 (2012). https://doi.org/10.1007/s10735-012-9395-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10735-012-9395-1

Keywords

Search

Navigation