Abstract
Detailed structural analyses of the mushroom body which plays critical roles in olfactory learning and memory revealed that it is directly connected with multiple primary sensory centers in Drosophila. Connectivity patterns between the mushroom body and primary sensory centers suggest that each mushroom body lobe processes information on different combinations of multiple sensory modalities. This finding provides a novel focus of research by Drosophila genetics for perception of the external world by integrating multisensory signals.
Similar content being viewed by others
Introduction
The mushroom body (MB) is one of the prominent neuropils in the insect brain. Genetic manipulations of the MB intrinsic neurons called Kenyon cells in Drosophila have proved that the MB is an important site for olfactory learning and memory1,2,3. In contrast, the contribution of the Drosophila MB to general learning and memory is under debate. For example, flies in which postembryonic Kenyon cells were ablated by hydroxyurea showed normal visual, spatial, tactile and motor learning, but were defective in olfactory learning4. Instead of the MB, the central complex is suggested to be the site of the memory trace of visual patterns5,6. Conversely, recent works based on the observation of flies in which neural transmission of Kenyon cells were blocked have shown that the MB is required for visual and gustatory learning7,8,9. To resolve these discrepancies, which probably reflect the different fly backgrounds and behavioral paradigms tested, accumulation of anatomical knowledge is indispensable. In Drosophila, it has been reported that only the olfactory antennal lobe (AL)10,11 and visual optic lobe (OL)8 are directly connected with the MB calyx where Kenyon cells receive sensory inputs. On the other hand, the MB calyx in other insects such as the honeybee is connected with not only the OL and AL, but also the gustatory subesophageal zone (SEZ)12,13. We thus analyzed the connectivity patterns between the primary sensory centers and MB calyx by dye injections and genetic labeling methods in Drosophila.
Results
We performed injections of dextran conjugated with fluorescent dye into the primary sensory centers to investigate whether the MB calyx in Drosophila is also connected with multiple primary sensory centers. The MB calyx can be divided into four parts: the main calyx (CA), dorsal, lateral and ventral accessory calyces (d-, l- and vACA)8,14,15,16,17,18,19 (Fig. 1h and Supplementary Movie S1). The dye injections revealed that the MB calyx is connected with not only the AL and OL, but also SEZ via three major antennal lobe tracts (ALTs), three OL-calycal tracts (OLCTs) and one subesophageal-calycal tract (SCT) (Fig. 1a–g). The neurons running through the OLCTs are also called visual projection neuron (VPN)-MBs8. The terminals of ALTs were observed not only in the CA10,11, but also in the dACA and lACA. The OLCTs terminated in both dACA and vACA, whereas the SCT in dACA.
We then screened for the Janelia Farm GAL4 strains14 which visualize neurons running through these tracts and innervating the MB calyx except for the conventional ALT neurons which are known to supply branches into the main calyx10,11,20, subsequently identifying eleven strains, which labeled five OLCTs, two ALTs and one SCT which send projections into the ACAs of the MB (Table 1). The dACA is innervated by two OLCTs, the medial ALT (mALT) and SCT. The lACA is innervated by one OLCT and the mediolateral ALT (mlALT) and the vACA by four OLCTs, respectively (Table 1 and Supplementary Fig. S1). Dye injection and labeling with the GAL4 lines showed that the terminal areas of OLCT and ALT neurons are overlapping in the lACA and posterior area of the dACA, however, are distributed to the vACA and CA, respectively, in a segregated manner (Fig. 1d–g).
We further visualized the morphologies of single cells of these tracts by performing the FLP-out experiments21 using the GAL4 strains (Fig. 2, Supplementary Fig. S2 and Table 1). Most of the OLCTs emanated from the seventh layer of the medulla (ME7, serpentine layer), sixth layer of the lobula (LO6), or accessory medulla (AME). The ME7 and LO6 are the target layers of the chromatic Tm neurons22,23, suggesting that these layers are related to color processing. Only a few neurons innervate the layer entirely, however, most OLCT neurons have specific arborization areas in the OL. For example, one OLCT1 neuron innervates the dorsal three-quarters of the LO6 (Fig. 2a), whereas the innervation of one OLCT2 neuron is restricted to the ventralmost ME7 (Table 1). This result suggests that these neurons transfer information of specific visual fields. It should be noted that none of the OLCT neurons have the same morphologies with recently reported VPNs (VPN-MB1 and VPN-MB2) terminating in the vACA8. For the ALTs, we found two ALT neurons terminating in the ACA. One neuron, which innervates the VP1 glomerulus that receives chemosensory, hygrosensory, or thermosensory inputs from the sensory neurons in the sacculus of the antenna19,24, projects into the dACA through the mALT (Fig. 2d). The other, mlALT neuron, originating from the VP3 glomerulus which receives input from cold-sensing neurons19,25, runs through the mediolateral ALT and then pedunculus and forms a cloud of terminals in the lACA, the structure of which is quite similar with the terminals of the tritocerebral tract in the orthopteran insects26 (Fig. 2e). The morphology of this mlALT neuron appears quite similar with that of the t5ALT neuron14,19, though the neuron we found runs through the mlALT. The SCT neuron originating from the gnathal ganglia (GNG) or flange (FLA) joins the mALT and sends branches into the dACA (Fig. 2f).
Taken together, the dACA is connected with the LO6, ME7, AME, VP1 glomerulus, FLA and GNG. On the other hand, the vACA is linked with the LO6, ME1-7 and AME, whereas the lACA is linked with the AME and VP3 glomerulus. This indicates that each calycal part receives sensory inputs of different combinations of modalities and that the MB in Drosophila may play a role in general learning and memory.
We furthermore investigated how each calycal part is governed by different classes of Kenyon cells. Kenyon cells are classified according to the lobes they terminate in: γ, α/β, or α’/β’ lobe neurons15,16,17,27,28. We analyzed the dendritic patterns of these neurons by using the GAL4 enhancer-trap strains we previously reported28. Since there were no available strains for labeling α’/β’ lobe neurons specifically, we compared staining patterns among the strains labeling Kenyon cells of multiple lobes to estimate the probable innervation areas of α’/β’ lobe neurons. Each population of γ, α/β, or α’/β’ lobe neurons arborizes in the CA, whereas the ACAs are innervated by specific populations (Fig. 3 and Table 2). The dACA is innervated by a subpopulation of α/β lobe (αp/βp) Kenyon cells28, whereas the vACA is innervated by γ lobe neurons8,15,16,17. However, the innervation into the lACA was not observed in those strains that specifically label γ or α/β lobe neurons, but strains which visualize α’/β’ lobe neurons, suggesting that the lACA is innervated by α’/β’ lobe neurons. Since each ACA is connected with different primary sensory centers, these anatomical findings suggest that each lobe may have different functions in multimodal sensory processing (Fig. 4).
Discussion
This study reveals multimodal sensory pathways into the MB calyx in Drosophila as has been reported in such insects as the honeybee, ant, butterfly and cockroach12,13,29,30. Recent work has also revealed that two other OLCT neurons, VPN-MB1 and MB2 are required for visual memories of color and brightness in Drosophila8. In the bumblebee and honeybee, the neurons of the anterior inferior optic tract and the lobula tract which arborize in the serpentine layer of the medulla and in LO5 or LO6 respectively and project to the MB calyx respond to color and motion stimuli31. Due to morphological similarities of the OLCT neurons found in this study to these neurons and VPN-MBs8, the Drosophila MB may also receive similar visual information and combine it with odor, gustatory and temperature information in each lobe. Recent physiological works support this idea: the γ lobe neurons innervating the vACA respond to visual stimuli8, the ALT neuron terminating in the lACA shows calcium responses to temperature shifts19 and taste activity has been observed in the dACA9. Further physiological works revealing the sensory information transferred from the primary sensory centers to the MB calyx by the neurons found in this study need as yet to be determined.
Previous works have shown that each MB lobe contributes to a different temporal phase of memory1,2. This study suggests that each lobe functions in integrating a different combination of multiple sensory modalities. In the cockroach, the MB extrinsic neurons which innervate the MB lobe respond to multimodal sensory inputs in a context-specific manner32. Readily available Drosophila genetics17,18,28 can provide opportunities to reveal novel neural mechanisms by which a rich variety of sensory cues are combined to represent coherent perception of the external world.
Methods
Drosophila strains
To visualize the MB, OK10733, six GAL4 enhancer-trap strains28 (obtained from Kyoto Drosophila Genetic Resource Center) and MB247-DsRed line34 (gifted from Thomas Riemensperger and Andre Fiala) were used. To label the neurons which connect the primary sensory center and the MB calyx, we screened for about 6000 Janelia Farm GAL4 strains14,35 from the expression data of Bloomington Drosophila stock center (BDSC) website (http://flystocks.bio.indiana.edu/Browse/gal4/gal4_Janelia.php) and identified eleven strains. These strains were crossed with a strain carrying UAS-GFP, UAS-mCD8::GFP36 gifted from Aki Ejima, or 20xUAS-IVS-mCD8::GFP37 (#32194, BDSC). To reveal the morphologies of single neurons (see below), the Janelia Farm GAL4 strains were crossed with flies carrying Hs-flp and UAS > CD2, y+ >CD8::GFP21 kindly provided by Gary Struhl. Except for the FLP-out experiments, flies were raised at 25 degrees Celsius with a 12-hour light/12-hour dark cycle. We analyzed female flies between 2 and 8 days after eclosion.
Dextran injection into the primary sensory centers
Flies were anesthetized in a vial on ice for less than a minute and were fixed to plastic chambers with wax and epoxy. The compound eye, top of the head, or proboscis was opened with forceps in Drosophila saline (in mM: NaCl 103, KCl 3, MgCl2 4, CaCl2 1.5, NaHCO3 26, TES 5, trehalose 10, glucose 10, sucrose 7, NaH2PO4 1, adjusted to pH 7.25 with HCl). After fat, air sacs and sheathes around the OL, AL, or SEZ were gently removed with forceps, dextran conjugated with tetramethylrhodamine and biotin (3 kDa, D-3308, Life Technologies) was injected into one of the OL, AL and SEZ with forceps20. Within 20 minutes after dextran injection, the brains were dissected out and fixed with 4% formaldehyde or 4% paraformaldehyde (PFA) for 50 minutes at room temperature. The brains were then washed in phosphate buffered saline (PBS, pH 7.4). When the GFP and tetramethylrhodamine signals needed to be enhanced, the brains were further incubated in the rabbit anti-GFP antibody and Alexa Fluor 568-conjugated streptavidin (S-11226, Life Technologies; 1 mg/l diluted in 0.2% Triton X-100/PBS (PBST)), respectively, followed by the antibody staining procedures (see below). The brains were finally mounted in 50 or 80% glycerol/PBS.
Antibody staining
Brains were dissected out in PBS and fixed with 4% PFA for 50 minutes. After washing with PBS for 5 minutes three times, the brains were then shaken in the blocking solution: 10% goat serum in PBST for an hour. They were incubated in the blocking solution containing primary antibodies overnight at room temperature with shaking. After washing with PBST, the brains were then incubated in the blocking solution containing secondary antibodies overnight at room temperature with shaking. Finally, the brains were washed with PBS and mounted in 50 or 80% glycerol/PBS.
For primary antibodies, we used rabbit anti-GFP antibody (A11122, Life Technologies; diluted at 1:200 except for the FLP-out experiments, see below), mouse anti-GFP antibody (12A6, Developmental Studies Hybridoma Bank at the University of Iowa; diluted at 1:200), or mouse anti-SYNAPSIN antibody38 (3C11, Developmental Studies Hybridoma Bank at the University of Iowa; diluted at 1:10). For secondary antibodies, we used goat anti-rabbit/mouse antibodies conjugated with Alexa Fluor 488 or 568 (A11001, A11004, A11034 and A11036, Life Technologies; diluted at 1:200), except for the FLP-out experiments (see below).
Labeling single neurons with GFP
Janelia Farm GAL4 strains labeled multiple neurons including our targets, which prevented us from analyzing the morphology of single cells. To reduce the number of neurons labeled, we used the FLP-out system21. In this system, we crossed each GAL4 strain with the flies carrying Hs-flp and UAS > CD2, y+ >CD8::GFP and incubated one-day old adult offspring at 37 degrees Celcius for 1 hour to express the flippase gene to induce somatic recombination randomly, which resulted in GFP expression in a fewer numbers of neurons. We then maintained the flies for 6–8 days at 25 degrees Celcius before dissecting the brains out. The brains were then fixed with 4% PFA and immunostained with the rabbit anti-GFP antibody (diluted at 1:1000) and the mouse anti-SYNAPSIN antibody (diluted at 1:20). For secondary antibodies, we used goat anti-rabbit antibody conjugated with Alexa Flour 488 (diluted at 1:1000) and goat anti-mouse antibody conjugated with Alexa Flour 568 (diluted at 1:200). The brains were washed with PBS and finally mounted in 80% glycerol/PBS. The brains expressing CD8::GFP in a single neuron of interest were imaged.
Confocal imaging and three-dimensional reconstructions of optical sections
Confocal serial optical images of the whole-mount brain were taken at 0.7–1 μm z-intervals with a Carl Zeiss confocal microscope LSM 700 equipped with water-immersion C-Apochromat 40×. Three-dimensional reconstruction and surface rendering were performed with Zeiss ZEN 2012 and Avizo 5.1, respectively. The brightness, color and contrast of images were adjusted with Photoshop CS 5.1. For the left panels of Fig. 2d–f, the antibody signal levels of the fibers we analyzed were adjusted to increase the signal/noise ratio by changing the levels manually in Photoshop before the reconstructions. For the right panels of Fig. 2f, antibody signals except for the SCT1, such as the noise backgrounds on the surface of the brain were manually removed to visualize the GAL4 positive neuron more clearly.
Terminology
The spatial definition and nomenclature of each lobe and axonal tract is based on ref. 39. The layer divisions in the medulla and lobula are referred to ref. 40.
Additional Information
How to cite this article: Yagi, R. et al. Convergence of multimodal sensory pathways to the mushroom body calyx in Drosophila melanogaster. Sci. Rep. 6, 29481; doi: 10.1038/srep29481 (2016).
References
Heisenberg, M. Mushroom body memoir: from maps to models. Nat. Rev. Neurosci. 4, 266–275 (2003).
Guven-Ozkan, T. & Davis, R. L. Functional neuroanatomy of Drosophila olfactory memory formation. Learn. Mem. 21, 519–526 (2014).
Stopfer M. Central processing in the mushroom bodies. Curr. Opin. Insect Sci. 6, 99–103 (2014).
Wolf, R. et al. Drosophila mushroom bodies are dispensable for visual, tactile and motor learning. Learn. Mem. 5, 166–178 (1998).
Liu, G. et al. Distinct memory traces for two visual features in the Drosophila brain. Nature 439, 551–556 (2006).
Pan, Y. et al. Differential roles of the fan-shaped body and the ellipsoid body in Drosophila visual pattern memory. Learn. Mem. 16, 289–295 (2009).
Vogt, K. et al. Shared mushroom body circuits underlie visual and olfactory memories in Drosophila. Elife 3, e02395 (2014).
Vogt, K. et al. Direct neural pathways convey distinct visual information to Drosophila mushroom bodies. Elife 5, e14009 (2016).
Kirkhart, C. & Scott, K. Gustatory learning and processing in the Drosophila mushroom bodies. J. Neurosci. 35, 5950–5958 (2015).
Stocker, R. F., Lienhard, M. C., Borst, A. & Fischbach, K. F. Neuronal architecture of the antennal lobe in Drosophila melanogaster. Cell Tissue Res. 262, 9–34 (1990).
Tanaka, N. K., Endo, K. & Ito, K. Organization of antennal lobe-associated neurons in adult Drosophila melanogaster brain. J. Comp. Neurol. 520, 4067–4130 (2012).
Gronenberg, W. Subdivisions of hymenopteran mushroom body calyces by their afferent supply. J. Comp. Neurol. 435, 474–489 (2001).
Schröter, U. & Menzel, R. A new ascending sensory tract to the calyces of the honeybee mushroom body, the subesophageal-calycal tract. J. Comp. Neurol. 465, 168–178 (2003).
Jenett, A. et al. A GAL4-driver line resource for Drosophila neurobiology. Cell Rep. 2, 991–1001 (2012).
Butcher, N. J., Friedrich, A. B., Lu, Z., Tanimoto, H. & Meinertzhagen, I. A. Different classes of input and output neurons reveal new features in microglomeruli of the adult Drosophila mushroom body calyx. J. Comp. Neurol. 520, 2185–2201 (2012).
Aso, Y. et al. The mushroom body of adult Drosophila characterized by GAL4 drivers. J. Neurogenet. 23, 156–172 (2009).
Aso, Y. et al. The neuronal architecture of the mushroom body provides a logic for associative learning. Elife 3, e04577 (2014).
Aso, Y. et al. Mushroom body output neurons encode valence and guide memory-based action selection in Drosophila. Elife 3, e04580 (2014).
Frank, D. D., Jouandet, G. C., Kearney, P. J., Macpherson, L. J. & Gallio, M. Temperature representation in the Drosophila brain. Nature 519, 358–361 (2015).
Tanaka, N. K., Suzuki, E., Ejima, A. & Stopfer, M. Dye fills reveal additional olfactory tracts in the protocerebrum of wild-type Drosophila. J. Comp. Neurol. 520, 4131–4140 (2012).
Wong, A. M., Wang, J. W. & Axel, R. Spatial representation of the glomerular map in the Drosophila protocerebrum. Cell 109, 229–241 (2002).
Gao, S. et al. The neural substrate of spectral preference in Drosophila. Neuron 60, 328–342 (2008).
Lin, T. Y. et al. Mapping chromatic pathways in the Drosophila visual system. J. Comp. Neurol. 524, 213–227 (2016).
Silbering, A. F. et al. Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems. J. Neurosci. 31, 13357–13375 (2011).
Gallio, M., Ofstad, T. A., Macpherson, L. J., Wang, J. W. & Zuker, C. S. The coding of temperature in the Drosophila brain. Cell 144, 614–624 (2011).
Farris, S. M. Tritocerebral tract input to the insect mushroom bodies. Arthropod Struct. Dev. 37, 492–503 (2008).
Crittenden, J. R., Skoulakis, E. M., Han, K. A., Kalderon, D. & Davis, R. L. Tripartite mushroom body architecture revealed by antigenic markers. Learn. Mem. 5, 38–51 (1998).
Tanaka, N. K., Tanimoto, H. & Ito, K. Neuronal assemblies of the Drosophila mushroom body. J. Comp. Neurol. 508, 711–755 (2008).
Nishino, H. et al. Visual and olfactory input segregation in the mushroom body calyces in a basal neopteran, the American cockroach. Arthropod Struct. Dev. 41, 3–16 (2012).
Kinoshita, M., Shimohigashi, M., Tominaga, Y., Arikawa, K. & Homberg, U. Topographically distinct visual and olfactory inputs to the mushroom body in the Swallowtail butterfly, Papilio xuthus. J. Comp. Neurol. 523, 162–182 (2015).
Paulk, A. C. & Gronenberg, W. Higher order visual input to the mushroom bodies in the bee, Bombus impatiens. Arthropod Struct. Dev. 37, 443–458 (2008).
Li, Y. & Strausfeld, N. J. Multimodal efferent and recurrent neurons in the medial lobes of cockroach mushroom bodies. J. Comp. Neurol. 409, 647–663 (1999).
Connolly, J. B. et al. Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies. Science 274, 2104–2107 (1996).
Riemensperger, T., Völler, T., Stock, P., Buchner, E. & Fiala, A. Punishment prediction by dopaminergic neurons in Drosophila. Curr. Biol. 15, 1953–1960 (2005).
Pfeiffer, B. D. et al. Tools for neuroanatomy and neurogenetics in Drosophila. Proc. Natl. Acad. Sci. USA 105, 9715–9720 (2008).
Lee, T. & Luo, L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461 (1999).
Pfeiffer, B. D. et al. Refinement of tools for targeted gene expression in Drosophila. Genetics 186, 735–755 (2010).
Klagges, B. R. et al. Invertebrate synapsins: a single gene codes for several isoforms in Drosophila. J. Neurosci. 16, 3154–3165 (1996).
Ito, K. et al. A systematic nomenclature for the insect brain. Neuron 81, 755–765 (2014).
Fischbach, K. F. & Dittrich, A. P. M. The optic lobe of Drosophila melanogaster. I. A Golgi analysis of wild-type structure. Cell Tissue Res. 258, 441–475 (1989).
Acknowledgements
We thank Kyoto Drosophila Genetic Resource Center, Bloomington Drosophila Stock Center, A. Ejima, T. Riemensperger, A. Fiala and G. Struhl for kindly providing fly strains and Developmental Studies Hybridoma Bank at the University of Iowa for antibodies. We are deeply indebted to S. Takemura, M. Koizumi, Y. Hamanaka and M. Mizunami’s lab members for helpful discussion and to M. Murakami and N. Onoda for maintaining fly stocks. This work was supported by PRESTO, Japan Science and Technology Agency, by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant number: 22770068, 24120509 and 26830026) and by Budget to Promote Executive Office Projects of Hokkaido University to N.K.T.
Author information
Authors and Affiliations
Contributions
R.Y. and N.K.T. designed the project. R.Y., Y.M. and N.K.T. performed the experiments. R.Y., Y.M., M.M. and N.K.T. wrote the manuscript.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Electronic supplementary material
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
Yagi, R., Mabuchi, Y., Mizunami, M. et al. Convergence of multimodal sensory pathways to the mushroom body calyx in Drosophila melanogaster. Sci Rep 6, 29481 (2016). https://doi.org/10.1038/srep29481
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/srep29481
This article is cited by
-
Brain plasticity indicates key cognitive demands in an animal society: caste comparisons in dampwood termites
Insectes Sociaux (2022)
-
Neurofibromin 1 in mushroom body neurons mediates circadian wake drive through activating cAMP–PKA signaling
Nature Communications (2021)
-
Untangling the wires: development of sparse, distributed connectivity in the mushroom body calyx
Cell and Tissue Research (2021)
-
Dopamine modulation of sensory processing and adaptive behavior in flies
Cell and Tissue Research (2021)
-
Structural aspects of plasticity in the nervous system of Drosophila
Neural Development (2018)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.