Summary

约束和自由飞行,蜜蜂的神经药理学操纵,<em>蜜蜂</em

Published: November 26, 2016
doi:

Summary

这份手稿介绍了管理药剂对蜜蜂,包括自由飞行的蜜蜂简单的无创方法,以及更具侵入性的变异,让内敛的蜜蜂精确的局部治疗多种协议。

Abstract

蜜蜂展示惊人的学习能力和先进的社会行为和沟通。此外,他们的大脑体积小,便于可视化和学习。因此,蜜蜂也早已之间神经生物学家和neuroethologists为研究社会和自然行为的神经基础青睐的机型。是很重要的,但是,用于研究蜜蜂不与行为干扰的实验技术正在研究中。由于这个原因,一直需要开发出一系列的为蜜蜂的药理学处理的技术。在本文中,我们演示了具有广泛的药理剂治疗内敛或自由飞行的蜜蜂的方法。这些包括非侵入性的方法,如口服和局部治疗,以及更侵入性的方法,其允许在任一全身或局部的方式精确药物递送。最后,我们讨论每种方法的优点和缺点,并描述常见的障碍以及如何最好地克服它们。我们的结论与适应实验方法的生物学问题,而不是周围的其他方式的重要性进行了讨论。

Introduction

由于卡尔·冯·弗里希阐明他们的舞蹈语言1,蜜蜂仍然是一个热门的研究物种的动物行为和神经生物学的研究人员。近年来新学科无数已经出现在这两个领域,和其他几个学科(如分子生物学,基因组学和计算机科学)已经出现和他们一起的交集。这导致了新的理论和模式的快速发展,为理解的行为从活动结果如何神经系统之内。由于独特的生活方式,丰富的行为库,并且易于实验和药理操纵的,蜜蜂一直维持在这个革命的最前沿。

蜜蜂被用来研究基本神经生物学问题那些潜在学习和记忆2,3,决策4,嗅觉5,或可视处理6如。在最近几年,议员EY蜂甚至被用作研究主题通常保留用于医学研究,例如成瘾药物7的影响的模型 11,睡眠12,老化13,或机制基础麻醉14。

对于不同的经典遗传模式生物( 例如果蝇线虫 ,M.家鼠 ),也有极少数可用于蜜蜂操纵的神经功能的遗传工具,虽然这是目前改变15。相反,蜜蜂的研究主要依靠药理操作。这是非常成功;然而,蜂研究的多样性是使得所需的药理给药方法的范围内。研究与蜜蜂满足高度多样化的问题,是由来自不同学科和背景的研究人员研究,并采用了多种实验方法。许多RESE弓问题需要蜜蜂要么是自由飞行,在他们的殖民地,或两者相互作用的自由。这可以使它很难跟踪个别实验动物,并使得限制或插管不可行的。

容纳蜜蜂研究的多样性,需要多种药物递送方法,允许强大和灵活的管理,同时确保药物动力学和药效型材,该方法的侵袭,其可靠性,适合所讨论的范例。由于这些多样化的需求,大部分的研究小组已经开发出了自己独特的给药方法。到目前为止,这一直是蜜蜂研究界的力量;它导致了允许在不同的情况下的同种药物的给药方法阵列的发展。我们的目标不是要开发蜜蜂药理操纵一个标准化的方法,而是突出的方法已被证明是特别成功的,并帮助研究人员采用这些。我们讨论的是如何工作的基本原则,以及它们的优点和缺点。

Protocol

1.药品监督管理局铁甲蜜蜂 口服药物治疗 通过混合257克蔗糖用​​500毫升水制备1.5M的蔗糖溶液(它是比较容易溶解于沸水蔗糖的这个量)。储存在4℃直至使用蔗糖溶液。 注:蔗糖溶液中某些微生物提供了一个非常好客的环境,因此很容易被污染和令人不快的蜜蜂。散装蔗糖溶液可以等分并储存在-20℃直到使用。 决定适当的药物剂量(如何实现的,在下面?…

Representative Results

用于上述方法的代表性结果的选择示,主要是为了证明方法允许药剂到达脑部,并影响蜜蜂行为。 对大脑处理具体影响如下胸部注射容易获得。 因为药剂注入通过胸廓可作用于在主体多个目标,并在到达大脑之前得到稀释到体内,这种技术可以提高可能特异性的担忧。尽管如此,它…

Discussion

上面提到的方法允许任何自由飞行或利用,蜜蜂的简单,有效和有力的治疗方法。这些方法有许多实验范式和生物学问题( 表1)兼容。所有的自由飞行的方法可以很容易地应用到利用,蜜蜂。相反的是不太成功的,但是,由于暂时克制和侵入性治疗方法往往能危及蜜蜂的飞行能力。

该方法已经从大脑为中心的立体呈现。这不是由于技术固有的局限性,但作者的?…

Declarações

The authors have nothing to disclose.

Acknowledgements

This project was funded by ARC grant DP0986021 and NHMRC grant 585442. ABB is supported by an ARC Future Fellowship (FT140100452). JAP is supported by an iMQRES scholarship awarded by Macquarie University and by a DAAD-Doktorandenstipendium awarded by the German Academic Exchange Service. JMD is supported by CNRS and University Paul Sabatier.

Materials

Sucrose Sigma-Aldrich S8501 Any supplier will do
Sodium Chloride Sigma-Aldrich S7653
Potassium Chloride Sigma-Aldrich P9333 
Magnesium Chloride hexahydrate Sigma-Aldrich M2670
Calcium Chloride dihydrate Sigma-Aldrich C8106
Dextrose monohydrate Sigma-Aldrich 49159
Phosphate Buffer Saline (PBS) Sigma-Aldrich P4417
Protection Wax Dentaurum 124-305-00
HEPES Sigma-Aldrich H3375
dimethylformamide Sigma-Aldrich D4551
95% Ethanol Sigma-Aldrich 493511
Glass capillary WPI 1B100F-3
23 G NanoFil needle WPI NF33BV-2
Very fine forsceps Dumont 0208-55-PO
Electrode puller SRI 2001
FemtoJet Microinjector Eppendorf 5247 000.01
Eicosane Sigma-Aldrich 219274
manual micromanipulator Brinkmann Instrumentenbau MM-33
electronic micromanipulator Luigs & Neumann Feinmechanik + Elektortechnik Junior unit XYZ
stereomicroscope Leica M80
soldering iron Weller WESD51
Dextran, Alexa Fluor 546, 10000 MW ThermoFisher Scientific D-22911
Dextran, Alexa Fluor 568, 10000 MW ThermoFisher Scientific D-22912
small Petri dish Sigma-Aldrich P5481
mineral oil Sigma-Aldrich M5904
50 mL Centrifuge tube ThermoFisher Scientific 339652
forceps Australian Entomological Supplies
Blade holder and breaker Australian Entomological Supplies E130
Feather double edged razor blade ThermoFisher Scientific 50-949-135
Nichrome wire Any supplier will do
Electrical wires Any supplier will do
Model paint Tamiya USA Depends on colour
Repeating dispenser Hamilton company PB-600-1
Glass syringe WPI NANOFIL
flourescence viewing system Nightsea SFR-GR
graticule ProSciTech S8014-24
microcapillary with holder Drummond 1-000-0010
Liquid silicone Any supplier will do
Thermocouple Digitech QM-1324
Micropipette Eppendorf

Referências

  1. Frisch, K. .. . v. o. n. . B. e. e. s. . Their Vision, Chemical Senses, and Language. , (1971).
  2. Giurfa, M. The amazing mini-brain: lessons from a honey bee. Bee World. 84 (1), 5-18 (2003).
  3. Giurfa, M. Behavioral and neural analysis of associative learning in the honeybee: a taste from the magic well. J. Comp. Physiol. 193 (8), 801-824 (2007).
  4. Perry, C. J., Barron, A. B. Honey bees selectively avoid difficult choices. Proc. Natl. Acad. Sci. U. S. A. 110 (47), 19155-19159 (2013).
  5. Giurfa, M., Sandoz, J. -. C. Invertebrate learning and memory: Fifty years of olfactory conditioning of the proboscis extension response in honeybees. Learn. Mem. 19 (2), 54-66 (2012).
  6. Srinivasan, M. V. Honey bees as a model for vision, perception, and cognition. Annu. Rev. Entomol. 55, 267-284 (2010).
  7. Søvik, E., Cornish, J. L., Barron, A. B. Cocaine tolerance in honey bees. PLoS One. 8 (5), e64920 (2013).
  8. Søvik, E., Barron, A. B. Invertebrate models in addiction research. Brain. Behav. Evol. 82 (3), 153-165 (2013).
  9. Søvik, E. . Reward processing and responses to drugs of abuse in the honey bee, Apis mellifera. , (2013).
  10. Søvik, E., Even, N., Radford, C. W., Barron, A. B. Cocaine affects foraging behaviour and biogenic amine modulated behavioural reflexes in honey bees. Peer J. 2, e662 (2014).
  11. Abramson, C. I., Stone, S. M., et al. The development of an ethanol model using social insects I: behavior studies of the honey bee (Apis mellifera L.). Alcohol. Clin. Exp. Res. 24, 1153-1166 (2000).
  12. Sauer, S., Kinkelin, M., Herrmann, E., Kaiser, W. The dynamics of sleep-like behaviour in honey bees. J. Comp. Physiol. A Neuroethol. Sensory, Neural, Behav. Physiol. 189 (8), 599-607 (2003).
  13. Münch, D., Kreibich, C. D., Amdam, G. V. Aging and its modulation in a long-lived worker caste of the honey bee. J. Exp. Biol. 216 (Pt 9), 1638-1649 (2013).
  14. Cheeseman, J. F., Winnebeck, E. C., et al. General anesthesia alters time perception by phase shifting the circadian clock. Proc. Natl. Acad. Sci. , (2012).
  15. Schulte, C., Theilenberg, E., Müller-Borg, M., Gempe, T., Beye, M. Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee (Apis mellifera). Proc. Natl. Acad. Sci. U. S. A. 111 (24), 9003-9008 (2014).
  16. Felsenberg, J., Gehring, K. B., Antemann, V., Eisenhardt, D. Behavioural pharmacology in classical conditioning of the proboscis extension response in honeybees (Apis mellifera). J. Vis. Exp. (47), e2282 (2011).
  17. Burger, H., Ayasse, M., Dötterl, S., Kreissl, S., Galizia, C. G. Perception of floral volatiles involved in host-plant finding behaviour: Comparison of a bee specialist and generalist. J. Comp. Physiol. A Neuroethol. Sensory, Neural, Behav. Physiol. 199 (9), 751-761 (2013).
  18. Pan, K. C., Goodman, L. J. Ocellar projections within the central nervous system of the worker honey bee, Apis mellifera. Cell Tissue Res. 176 (4), 505-527 (1977).
  19. Ito, K., Shinomiya, K., et al. A systematic nomenclature for the insect brain. Neuron. 81, 755-765 (2014).
  20. Bitterman, M. E., Menzel, R., Fietz, A., Schäfer, S. Classical conditioning of proboscis extension in honeybees (Apis mellifera). J. Comp. Psychol. 97 (2), 107-119 (1983).
  21. Barron, A. B., Robinson, G. E. Selective modulation of task performance by octopamine in honey bee (Apis mellifera) division of labour. J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 191 (7), 659-668 (2005).
  22. Schulz, D. J., Sullivan, J. P., Robinson, G. E. Juvenile Hormone and Octopamine in the Regulation of Division of Labor in Honey Bee Colonies. Horm. Behav. 42 (2), 222-231 (2002).
  23. Schulz, D. J., Elekonich, M. M., Robinson, G. E. Biogenic amines in the antennal lobes and the initiation and maintenance of foraging behavior in honey bees. J. Neurobiol. 54 (2), 406-416 (2003).
  24. Barron, A. B., Vander Meer, R. K., Maleszka, J., Robinson, G. E., Maleszka, R. Comparing injection, feeding and topical application methods for treatment of honeybees with octopamine. J. Insect Physiol. 53 (2), 187-194 (2007).
  25. McClung, C., Hirsh, J. Stereotypic behavioral responses to free-base cocaine and the development of behavioral sensitization in Drosophila. Curr. Biol. 8 (2), 109-112 (1998).
  26. Martin, B. R., Lue, L. P., Boni, J. P. Pyrolysis and volatilization of cocaine. J. Anal. Toxicol. 13 (3), 158-162 (1989).
  27. Lefer, D., Perisse, E., Hourcade, B., Sandoz, J. -. C., Devaud, J. -. M. Two waves of transcription are required for long-term memory in the honeybee. Learn. Mem. 20 (1), 29-33 (2012).
  28. Urlacher, E., Soustelle, L., et al. Honey Bee Allatostatins Target Galanin/Somatostatin-Like Receptors and Modulate Learning: A Conserved Function?. PLoS One. 11 (1), e0146248 (2016).
  29. Stollhoff, N., Menzel, R., Eisenhardt, D. Spontaneous recovery from extinction depends on the reconsolidation of the acquisition memory in an appetitive learning paradigm in the honeybee (Apis mellifera). J. Neurosci. 25 (18), 4485-4492 (2005).
  30. Barron, A. B., Maleszka, R., Helliwell, P. G., Robinson, G. E. Effects of cocaine on honey bee dance behaviour. J. Exp. Biol. 212 (2), 163-168 (2009).
  31. Devaud, J. -. M., Papouin, T., Carcaud, J., Sandoz, J. -. C., Grünewald, B., Giurfa, M. Neural substrate for higher-order learning in an insect: Mushroom bodies are necessary for configural discriminations. Proc. Natl. Acad. Sci. , 1-9 (2015).
  32. Vergoz, V., Roussel, E., Sandoz, J. -. C., Giurfa, M. Aversive learning in honeybees revealed by the olfactory conditioning of the sting extension reflex. PLoS One. 2 (3), e288 (2007).
  33. Henry, M., Béguin, M., et al. A common pesticide decreases foraging success and survival in honey bees. Science. 336 (6079), 348-350 (2012).
  34. Søvik, E., Perry, C. J., LaMora, A., Barron, A. B., Ben-Shahar, Y. Negative impact of manganese on honeybee foraging. Biol. Lett. 11 (3), 20140989 (2015).
  35. Farooqui, T., Vaessin, H., Smith, B. H. Octopamine receptors in the honeybee (Apis mellifera) brain and their disruption by RNA-mediated interference. J. Insect Physiol. 50 (8), 701-713 (2004).
  36. Guo, X., Su, S., et al. Recipe for a Busy Bee: MicroRNAs in Honey Bee Caste Determination. PLoS One. 8 (12), e81661 (2013).
  37. Cristino, A. S., Barchuk, A. R., et al. Neuroligin-associated microRNA-932 targets actin and regulates memory in the honeybee. Nat. Commun. 5, 5529 (2014).
  38. Vargaftig, B. B., Coignet, J. L., de Vos, C. J., Grijsen, H., Bonta, I. L. Mianserin hydrochloride: Peripheral and central effects in relation to antagonism against 5-hydroxytryptamine and tryptamine. Eur. J. Pharmacol. 16 (3), 336-346 (1971).
  39. Beggs, K. T., Tyndall, J. D. A., Mercer, A. R. Honey bee dopamine and octopamine receptors linked to intracellular calcium signaling have a close phylogenetic and pharmacological relationship. PLoS One. 6 (11), (2011).
  40. Matsumoto, Y., Menzel, R., Sandoz, J. -. C., Giurfa, M. Revisiting olfactory classical conditioning of the proboscis extension response in honey bees: a step toward standardized procedures. J. Neurosci. Methods. 211 (1), 159-167 (2012).
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Søvik, E., Plath, J. A., Devaud, J., Barron, A. B. Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera. J. Vis. Exp. (117), e54695, doi:10.3791/54695 (2016).

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