Cell and Tissue Research

, Volume 376, Issue 1, pp 97–111 | Cite as

Tyrosine hydroxylase-immunoreactive neurons in the mushroom body of the field cricket, Gryllus bimaculatus

  • Yoshitaka HamanakaEmail author
  • Makoto Mizunami
Regular Article


The mushroom body of the insect brain participates in processing and integrating multimodal sensory information and in various forms of learning. In the field cricket, Gryllus bimaculatus, dopamine plays a crucial role in aversive memory formation. However, the morphologies of dopamine neurons projecting to the mushroom body and their potential target neurons, the Kenyon cells, have not been characterized. Golgi impregnations revealed two classes of Kenyon cells (types I and II) and five different types of extrinsic fibers in the mushroom body. Type I cells, which are further divided into two subtypes (types I core and I surface), extend their dendrites into the anterior calyx, whereas type II cells extend many bushy dendritic branches into the posterior calyx. Axons of the two classes bifurcate between the pedunculus and lobes to form the vertical, medial and γ lobes. Immunocytochemistry to tyrosine hydroxylase (TH), a rate-limiting enzyme in dopamine biosynthesis, revealed the following four distinct classes of neurons: (1) TH-SLP projecting to the distal vertical lobe; (2) TH-IP1 extending to the medial and γ lobes; (3) TH-IP2 projecting to the basal vertical lobe; and (4) a multiglomerular projection neuron invading the anterior calyx and the lateral horn (TH-MPN). We previously proposed a model in the field cricket in which the efficiency of synapses from Kenyon cells transmitting a relevant sensory stimulus to output neurons commanding an appropriate behavioral reaction can be modified by dopaminergic neurons mediating aversive signals and here, we provide putative neural substrates for the cricket’s aversive learning. These will be instrumental in understanding the principle of aversive memory formation in this model species.


Insect Dopamine Aversive learning Prediction error Kenyon cell 



We thank Dr. Ian A. Meinertzhagen (Dalhousie University, Canada) for critically reading this manuscript. We are also grateful to Ms. Yoshimi Watanabe for technical support in Golgi impregnation. This work was supported by Grant-in-Aid for Scientific Research (No. 16H04814 and 16K18586) to MM and Grant-in-Aid for Young Scientists (B) No. 26840109 to YH.


  1. Aso Y, Siwanowicz I, Bräcker L, Ito K, Kitamoto T, Tanimoto H (2010) Specific dopaminergic neurons for the formation of labile aversive memory. Curr Biol 20:1445–1451CrossRefGoogle Scholar
  2. Aso Y, Herb A, Ogueta M, Siwanowicz I, Templier T, Friedrich AB, Ito K, Scholz H, Tanimoto H (2012) Three dopamine pathways induce aversive odor memories with different stability. PLoS Genet 8:e1002768CrossRefGoogle Scholar
  3. Aso Y, Hattori D, Yu Y, Johnston RM, Iyer NA, Ngo T-TB, Dionne H, Abbott LF, Axel R, Tanimoto H, Rubin GM (2014) The neuronal architecture of the mushroom body provides a logic for associative learning. eLife 3:e04577CrossRefGoogle Scholar
  4. Awata H, Watanabe T, Hamanaka Y, Mito T, Noji S, Mizunami M (2015) Knockout crickets for the study of learning and memory: dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets. Sci Rep 5:15885CrossRefGoogle Scholar
  5. Awata H, Wakuda R, Ishimaru Y, Matsuoka Y, Terao K, Katata S, Matsumoto Y, Hamanaka Y, Noji S, Mito T, Mizunami M (2016) Roles of OA1 octopamine receptor and Dop1 dopamine receptor in mediating appetitive and aversive reinforcement revealed by RNAi studies. Sci Rep 6:29696CrossRefGoogle Scholar
  6. Balling A, Technau GM, Heisenberg M (1987) Are the structural changes in adult Drosophila mushroom bodies memory traces? Studies on biochemical learning mutants. J Neurogenet 4:65–73CrossRefGoogle Scholar
  7. Blenau W, Baumann A (2001) Molecular and pharmacological properties of insect biogenic amine receptors: lessons from Drosophila melanogaster and Apis mellifera. Arch Insect Biochem Physiol 48:13–38CrossRefGoogle Scholar
  8. Burke CJ, Huetteroth W, Owald D, Perisse E, Krashes MJ, Das G, Gohl D, Silies M, Certel S, Waddell S (2012) Layered reward signalling through octopamine and dopamine in Drosophila. Nature 492:433–437CrossRefGoogle Scholar
  9. Claridge-Chang A, Roorda RD, Vrontou E, Sjulson L, Li H, Hirsh J, Miesenböck G (2009) Writing memories with light-addressable reinforcement circuitry. Cell 139:405–415CrossRefGoogle Scholar
  10. Cognigni P, Felsenberg J, Waddell S (2018) Do the right thing: neural network mechanisms of memory formation, expression and update in Drosophila. Curr Opin Neurobiol 49:51–58CrossRefGoogle Scholar
  11. Denker M, Finke R, Schaupp F, Grün S, Menzel R (2010) Neural correlates of odor learning in the honeybee antennal lobe. Eur J Neurosci 31:119–133CrossRefGoogle Scholar
  12. Frambach I, Schürmann F-W (2004) Separate distribution of deutocerebral projection neurons in the mushroom bodies of the cricket brain. Acta Biol Hung 55:21–29CrossRefGoogle Scholar
  13. Friggi-Grelin F, Coulom H, Meller M, Gomez D, Hirsh J, Birman S (2003) Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase. J Neurobiol 54:618–627CrossRefGoogle Scholar
  14. Giurfa M (2007) Behavioral and neural analysis of associative learning in the honeybee: a taste from the magic well. J Comp Physiol A 193:801–824CrossRefGoogle Scholar
  15. Hamada A, Miyawaki K, Honda-sumi E, Tomioka K, Mito T, Ohuchi H, Noji S (2009) Loss-of-function analyses of the fragile X-related and dopamine receptor genes by RNA interference in the cricket Gryllus bimaculatus. Dev Dyn 238:2025–2033CrossRefGoogle Scholar
  16. Hamanaka Y, Minoura R, Nishino H, Miura T, Mizunami M (2016) Dopamine- and tyrosine hydroxylase-immunoreactive neurons in the brain of the American cockroach, Periplaneta americana. PLoS ONE 11:e0160531CrossRefGoogle Scholar
  17. Heisenberg M (2003) Mushroom body memoir: from maps to models. Nat Rev Neurosci 4:266–275CrossRefGoogle Scholar
  18. Hige T (2018) What can tiny mushrooms in fruit flies tell us about learning and memory? Neurosci Res 129:8–16CrossRefGoogle Scholar
  19. Hinke W (1961) Das relative postembryonale Wachstum der Hirnteile von Culex pipiens, Drosophila melanogaster und Drosophila mutanten. Z Morph Ökol Tiere 50:81–118CrossRefGoogle Scholar
  20. Hörner M, Spörhase-Eichmann U, Helle J, Venus B, Schürmann F-W (1995) The distribution of neurones immunoreactive for ß-tyrosine hydroxylase, dopamine and serotonin in the ventral nerve cord of the cricket, Gryllus bimaculatus. Cell Tissue Res 280:583–604CrossRefGoogle Scholar
  21. Ito K, Shinomiya K, Ito M, Armstrong JD, Boyan G, Hartenstein V, Harzsch S, Heisenberg M, Homberg U, Jenett A, Keshishian H, Restifo LL, Rössler W, Simpson JH, Strausfeld NJ, Strauss R, Vosshall LB (2014) A systematic nomenclature for the insect brain. Neuron 81:755–765CrossRefGoogle Scholar
  22. Liu C, Plaçais PY, Yamagata N, Pfeiffer BD, Aso Y, Friedrich AB, Siwanowicz I, Rubin GM, Preat T, Tanimoto H (2012) A subset of dopamine neurons signals reward for odour memory in Drosophila. Nature 488:512–516CrossRefGoogle Scholar
  23. Malaterre J, Strambi C, Chiang A-S, Aouane A, Strambi A, Cayre M (2002) Development of cricket mushroom bodies. J Comp Neurol 452:215–227CrossRefGoogle Scholar
  24. Mao Z, Davis RL (2009) Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity. Front Neural Circuits 3:5CrossRefGoogle Scholar
  25. Matsumoto H, Tian J, Uchida N, Watabe-Uchida M (2016) Midbrain dopamine neurons signal aversion in a reward-context-dependent manner. eLife 5:e17328CrossRefGoogle Scholar
  26. Menzel R, Leboulle G, Eisenhardt D (2006) Small brains, bright minds. Cell 124:237–239CrossRefGoogle Scholar
  27. Mizunami M, Matsumoto Y (2010) Roles of aminergic neurons in formation and recall of associative memory in crickets. Front Behav Neurosci 4:172Google Scholar
  28. Mizunami M, Iwasaki M, Okada R, Nishikawa M (1998) Topography of modular subunits in the mushroom bodies of the cockroach. J Comp Neurol 399:153–161CrossRefGoogle Scholar
  29. Mizunami M, Yokohari F, Takahata M (1999) Exploration into the adaptive design of the arthropod “microbrain”. Zool Sci 16:703–709CrossRefGoogle Scholar
  30. Mizunami M, Yokohari F, Takahata M (2004) Further exploration into the adaptive design of the arthropod “microbrain”: I. Sensory and memory-processing systems. Zool Sci 21:1141–1151CrossRefGoogle Scholar
  31. Mizunami M, Unoki S, Mori Y, Hirashima D, Hatano A, Matsumoto Y (2009) Roles of octopaminergic and dopaminergic neurons in appetitive and aversive memory recall in an insect. BMC Biol 7:46CrossRefGoogle Scholar
  32. Mizunami M, Matsumoto Y, Watanabe H, Nishino H (2013) Olfactory and visual learning in cockroaches and crickets. In: Menzel R, Benjamin P (eds) Invertebrate learning and memory. Academic Press, San Diego, pp 549–560CrossRefGoogle Scholar
  33. Mizunami M, Hamanaka Y, Nishino H (2015) Toward elucidating diversity of neural mechanisms underlying insect learning. Zool Lett 1:8CrossRefGoogle Scholar
  34. Mobbs PG (1982) The brain of the honeybee Apis mellifera. I. The connections and spatial organization of the mushroom bodies. Philos Trans R Soc Lond B 298:309–354CrossRefGoogle Scholar
  35. Nakatani Y, Matsumoto Y, Mori Y, Hirashima D, Nishino H, Arikawa K, Mizunami M (2009) Why the carrot is more effective than the stick: different dynamics of punishment memory and reward memory and its possible biological basis. Neurobiol Learn Mem 92:370–380CrossRefGoogle Scholar
  36. Nässel DR, Elekes K (1992) Aminergic neurons in the brain of blowflies and Drosophila: dopamine- and tyrosine hydroxylase-immunoreactive neurons and their relationship with putative histaminergic neurons. Cell Tissue Res 267:147–167CrossRefGoogle Scholar
  37. Neder R (1959) Allometrisches Wachstum von Hirnteilen bei drei verschieden großen Schabenarten. Zool Jahrb Anat 4:411–464Google Scholar
  38. Owald D, Waddell S (2015) Olfactory learning skews mushroom body output pathways to steer behavioral choice in Drosophila. Curr Opin Neurobiol 35:178–184CrossRefGoogle Scholar
  39. Rescorla RA, Wagner AR (1972) A theory of Pavlovian conditioning: variations in the effectiveness of reinforcement and nonreinforcement. In: Black A, Prokasy WR (eds) Classical conditioning II. Academic Press, New York, pp 64–99Google Scholar
  40. Roeder T (2002) Biochemistry and molecular biology of receptors for biogenic amines in locusts. Microsc Res Tech 56:237–247CrossRefGoogle Scholar
  41. Schäfer S, Rehder V (1989) Dopamine-like immunoreactivity in the brain and suboesophageal ganglion of the honeybee. J Comp Neurol 280:43–58CrossRefGoogle Scholar
  42. Schildberger K (1984) Multimodal interneurons in the cricket brain: properties of identified extrinsic mushroom body cells. J Comp Physiol A 154:71–79CrossRefGoogle Scholar
  43. Schultz W (2006) Behavioral theories and the neurophysiology of reward. Annu Rev Psychol 57:87–115CrossRefGoogle Scholar
  44. Schultz W (2013) Updating dopamine reward signals. Curr Opin Neurobiol 23:229–238CrossRefGoogle Scholar
  45. Schürmann F-W (1973) Über die Struktur der Pilzkörper des Insektenhirns. I I I. Die Anatomie der Nervenfasern in den Corpora pedunculata bei Acheta domesticus L. (Orthoptera): eine Golgi-Studie. Z Zellforsch 145:247–285CrossRefGoogle Scholar
  46. Schürmann F-W (2016) Fine structure of synaptic sites and circuits in mushroom bodies of insect brains. Arthropod Struct Dev 45:399–421CrossRefGoogle Scholar
  47. Schürmann F-W, Frambach I, Elekes K (2008) GABAergic synaptic connections in mushroom bodies of insect brains. Acta Biol Hung 59:173–181CrossRefGoogle Scholar
  48. Schwaerzel M, Monastirioti M, Scholz H, Friggi-Grelin F, Birman S, Heisenberg M (2003) Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila. J Neurosci 23:10495–10502CrossRefGoogle Scholar
  49. Steinberg EE, Keiflin R, Boivin JR, Witten IB, Deisseroth K, Janak PH (2013) A causal link between prediction errors, dopamine neurons and learning. Nat Neurosci 16:966–973CrossRefGoogle Scholar
  50. Tanaka NK, Endo K, Ito K (2012) Organization of antennal lobe-associated neurons in adult Drosophila melanogaster brain. J Comp Neurol 520:4067–4130CrossRefGoogle Scholar
  51. Tedjakumala SR, Rouquette J, Boizeau M-L, Mesce KA, Hotier L, Massou I, Giurfa M (2017) A tyrosine-hydroxylase characterization of dopaminergic neurons in the honey bee brain. Front Syst Neurosci 11:47CrossRefGoogle Scholar
  52. Terao K, Mizunami M (2017) Roles of dopamine neurons in mediating the prediction error in aversive learning in insects. Sci Rep 7:14694CrossRefGoogle Scholar
  53. Terao K, Matsumoto Y, Mizunami M (2015) Critical evidence for the prediction error theory in associative learning. Sci Rep 5:8929CrossRefGoogle Scholar
  54. Unoki S, Matsumoto Y, Mizunami M (2005) Participation of octopaminergic reward system and dopaminergic punishment system in insect olfactory learning revealed by pharmacological study. Eur J Neurosci 22:1409–1416CrossRefGoogle Scholar
  55. Unoki S, Matsumoto Y, Mizunami M (2006) Roles of octopaminergic and dopaminergic neurons in mediating reward and punishment signals in insect visual learning. Eur J Neurosci 24:2031–2038CrossRefGoogle Scholar
  56. Vergoz V, Roussel E, Sandoz JC, Giurfa M (2007) Aversive learning in honeybees revealed by the olfactory conditioning of the sting extension reflex. PLoS One 2:e288CrossRefGoogle Scholar
  57. Vogt K, Schnaitmann C, Dylla KV, Knapek S, Aso Y, Rubin GM, Tanimoto H (2014) Shared mushroom body circuits underlie visual and olfactory memories in Drosophila. eLife 3:e02395CrossRefGoogle Scholar
  58. Waddell S (2013) Reinforcement signalling in Drosophila; dopamine does it all after all. Curr Opin Neurobiol 23:324–329CrossRefGoogle Scholar
  59. Watanabe T, Sadamoto H, Aonuma H (2013) Molecular basis of the dopaminergic system in the cricket Gryllus bimaculatus. Invertebr Neurosci 13:107–123CrossRefGoogle Scholar
  60. Watanabe T, Noji S, Mito T (2017) Genome editing in the cricket, Gryllus bimaculatus. In: Hatada I (ed) Genome editing in animals: methods and protocols. Humana Press, New York, pp 219–233CrossRefGoogle Scholar
  61. Wendt B, Homberg U (1992) Immunocytochemistry of dopamine in the brain of the locust Schistocerca gregaria. J Comp Neurol 321:387–403CrossRefGoogle Scholar
  62. Yu D, Ponomarev A, Davis RL (2004) Altered representation of the spatial code for odors after olfactory classical conditioning: memory trace formation by synaptic recruitment. Neuron 42:437–449CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of ScienceHokkaido UniversitySapporoJapan
  2. 2.Laboratory of Animal Physiology, Graduate School of ScienceOsaka City UniversitySumiyoshi-kuJapan

Personalised recommendations