Journal of Molecular Histology

, Volume 43, Issue 4, pp 405–419

Locust primary neuronal culture for the study of synaptic transmission

  • Stefan Weigel
  • Petra Schulte
  • Simone Meffert
  • Peter Bräunig
  • Andreas Offenhäusser
Original Paper

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.

Keywords

Locust Cell culture Chemical synapse Electrical synapse Patch-clamp 

References

  1. 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 PubMedCrossRefGoogle Scholar
  2. 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 PubMedCrossRefGoogle Scholar
  3. 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):8410PubMedCrossRefGoogle Scholar
  4. 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 PubMedCrossRefGoogle Scholar
  5. 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 PubMedCrossRefGoogle Scholar
  6. 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):1939PubMedGoogle Scholar
  7. Benson JA (1992) Electrophysiological pharmacology of the nicotinic and muscarinic cholinergic responses of isolated neuronal somata from locust thoracic ganglia. J Exp Biol 170:31Google Scholar
  8. Bräunig P, Pflüger HJ (2001) The unpaired median neurons of insects. Adv Insect Physiol 28Google Scholar
  9. 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):171PubMedCrossRefGoogle Scholar
  10. Burrows M (1980) The control of sets of motoneurones by local interneurones in the locust. J Physiol 298:213–233PubMedGoogle Scholar
  11. Burrows M (1996) The neurobiology of an insect brain. Oxford University Press, OxfordCrossRefGoogle Scholar
  12. Burrows M, Siegler MV (1978) Graded synaptic transmission between local interneurones and motor neurones in the metathoracic ganglion of the locust. J Physiol 285:231–255PubMedGoogle Scholar
  13. Comer CM, Robertson RM (2001) Identified nerve cells and insect behavior. Prog Neurobiol 63(4):409–439PubMedCrossRefGoogle Scholar
  14. 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 PubMedCrossRefGoogle Scholar
  15. 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 PubMedCrossRefGoogle Scholar
  16. 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 PubMedGoogle Scholar
  17. Gauglitz S, Pflüger HJ (2001) Cholinergic transmission via central synapses in the locust nervous system. J Comp Physiol A 187(10):825–836PubMedCrossRefGoogle Scholar
  18. 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):231–236CrossRefGoogle Scholar
  19. 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):53CrossRefGoogle Scholar
  20. 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 PubMedCrossRefGoogle Scholar
  21. 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 PubMedCrossRefGoogle Scholar
  22. 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 83:95PubMedGoogle Scholar
  23. 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 PubMedCrossRefGoogle Scholar
  24. Grünewald B, Levine RB (1998) Ecdysteroid control of ionic current development in Manduca sexta motoneurons. J Neurobiol 37(2):211–223PubMedCrossRefGoogle Scholar
  25. Hancox JC, Pitman RM (1995) Spontaneous bursting induced by convulsant agents in an identified insect neurone. Gen Pharmacol 26(1):195–204PubMedCrossRefGoogle Scholar
  26. 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 PubMedCrossRefGoogle Scholar
  27. 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 PubMedCrossRefGoogle Scholar
  28. 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):3Google Scholar
  29. 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 PubMedCrossRefGoogle Scholar
  30. 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 PubMedCrossRefGoogle Scholar
  31. Kirchhof B, Bicker G (1992) Growth properties of larval and adult locust neurons in primary cell culture. J Comput Neurol 323(3):411CrossRefGoogle Scholar
  32. Kloppenburg P, Hörner M (1998) Voltage-activated currents in identified giant interneurons isolated from adult crickets Gryllus bimaculatus. J Exp Biol 201(17):2529PubMedGoogle Scholar
  33. 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):459PubMedCrossRefGoogle Scholar
  34. 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):893CrossRefGoogle Scholar
  35. Laurent G (1990) Voltage-dependent nonlinearities in the membrane of locust nonspiking local interneurons, and their significance for synaptic integration. J Neurosci 10(7):2268PubMedGoogle Scholar
  36. Laurent G (1991) Evidence for voltage-activated outward currents in the neuropilar membrane of locust nonspiking local interneurons. J Neurosci 11(6):1713PubMedGoogle Scholar
  37. 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):1484–1498PubMedGoogle Scholar
  38. Lee D, O’Dowd DK (1999) Fast excitatory synaptic transmission mediated by nicotinic acetylcholine receptors in Drosophila neurons. J Neurosci 19(13):5311PubMedGoogle Scholar
  39. 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):4625PubMedGoogle Scholar
  40. Leibovitz A (1963) The growth and maintainance of tissue-cell cultures in free gas exchange with the atmosphere. Am J Hyg 78:173PubMedGoogle Scholar
  41. 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 CrossRefGoogle Scholar
  42. 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 PubMedCrossRefGoogle Scholar
  43. Lutz EM, Tyrer NM (1987) Immunohistochemical localization of choline acetyltransferase in the central nervous system of the locust. Brain Res 407(1):173–179PubMedCrossRefGoogle Scholar
  44. 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):117PubMedCrossRefGoogle Scholar
  45. 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):323–329Google Scholar
  46. 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 PubMedCrossRefGoogle Scholar
  47. 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):289–308PubMedGoogle Scholar
  48. Parker D, Newland PL (1995) Cholinergic synaptic transmission between proprioceptive afferents and a hind leg motor neuron in the locust. J Neurophysiol 73(2):586–594PubMedGoogle Scholar
  49. Pfahlert C, Lakes-Harlan R (1997) Responses of insect neurones to neurotrophic factors in vitro. Die Naturwissenschaften (84):163–165Google Scholar
  50. Pfahlert C, Lakes-Harlan R (2008) Interneurons, motoneurons and sensory neurons of Locusta migratoria (Insecta: Orthoptera) in primary cell culture. Open J Entomol (2):6–13Google Scholar
  51. 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 PubMedCrossRefGoogle Scholar
  52. 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):2695–2701PubMedCrossRefGoogle Scholar
  53. 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):2751–2755Google Scholar
  54. Rossler W, Bickmeyer U (1993) Locust medial neurosecretory cells in vitro: morphology, electrophysiological properties and effects of temperature. J Exp Biol 183(1):323Google Scholar
  55. 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):429–438CrossRefGoogle Scholar
  56. 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):021905Google Scholar
  57. 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 PubMedCrossRefGoogle Scholar
  58. Smith PJ, Howes EA (1996) Long-term culture of fully differentiated adult insect neurons. J Neurosci Methods 69(1):113PubMedCrossRefGoogle Scholar
  59. 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):781–802PubMedGoogle Scholar
  60. 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):9246PubMedGoogle Scholar
  61. 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):221–229CrossRefGoogle Scholar
  62. Thomas MV (1984) Voltage-clamp analysis of a calcium-mediated potassium conductance in cockroach (Periplaneta americana) central neurones. J Physiol 350:159–178PubMedGoogle Scholar
  63. 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 PubMedCrossRefGoogle Scholar
  64. 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):665–667PubMedCrossRefGoogle Scholar
  65. Trimarchi JR, Murphey RK (1997) The shaking-B2 mutation disrupts electrical synapses in a flight circuit in adult Drosophila. J Neurosci 17(12):4700–4710PubMedGoogle Scholar
  66. Vanhems E, Delbos M, Girardie J (1990) Insulin and Neuroparsin promote neurite outgrowth in cultured locust CNS. Eur J Neurosci 2(9):776PubMedCrossRefGoogle Scholar
  67. 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):1562PubMedCrossRefGoogle Scholar
  68. Wafford KA, Bai D, Sattelle DB (1992) A novel kainate receptor in the insect nervous system. Neurosci Lett 141(2):273–276PubMedCrossRefGoogle Scholar
  69. Watson AH (1988) Antibodies against GABA and glutamate label neurons with morphologically distinct synaptic vesicles in the locust central nervous system. Neuroscience 26(1):33–44PubMedCrossRefGoogle Scholar
  70. 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 CrossRefGoogle Scholar
  71. 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):210PubMedCrossRefGoogle Scholar
  72. 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):547–557PubMedCrossRefGoogle Scholar
  73. 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 PubMedCrossRefGoogle Scholar
  74. Yaksi E, Wilson RI (2010) Electrical coupling between olfactory glomeruli. Neuron 67(6):1034–1047. doi:10.1016/j.neuron.2010.08.041 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Stefan Weigel
    • 1
    • 3
  • Petra Schulte
    • 1
    • 4
  • Simone Meffert
    • 1
  • Peter Bräunig
    • 2
  • Andreas Offenhäusser
    • 1
  1. 1.Peter Grünberg InstituteInstitute of Complex Systems, BioelectronicsJülichGermany
  2. 2.Unit for Developmental Biology and Morphology, Institute of Biology IIRWTH Aachen UniversityAachenGermany
  3. 3.Technische Universität MünchenFreising-WeihenstephanGermany
  4. 4.Projectmanagement Group Jülich, Biotechnology, EU and InternationalJülichGermany

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