Abstract

Due to the honey bee’s importance as a simple neural model, there is a great need for new functional imaging modalities. Herein we report on the development and new findings of a combined two-photon microscope with a synchronized odor stimulus platform for in-vivo functional and morphological imaging of the honey bee’s olfactory system focusing on its primary centers, the antennal lobes (ALs). Our imaging platform allows for simultaneously obtaining both morphological measurements of the AL’s functional units, the glomeruli, and in-vivo calcium recording of their neural activities. By applying external odor stimuli to the bee’s antennae, we were able to record the characteristic glomerular odor response maps. Compared to previous works where conventional fluorescence microscopy was used, our approach has been demonstrated to offer all the advantages of multi-photon imaging, providing substantial enhancement in both spatial and temporal resolutions while minimizing photo-damages. In addition, compared to previous full-field microscopy calcium recordings, a four-fold improvement in the functional signal has been achieved. Finally, the multi-photon associated extended penetration depth allows for functional imaging of profound glomeruli.

© 2010 OSA

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    [CrossRef]
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    [CrossRef] [PubMed]
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  22. G. Laurent, M. Wehr, K. MacLeod, M. Stopfer, B. Leitch, and H. Davidowitz, “Dynamic encoding of odors with oscillating neuronal assemblies in the locust brain,” Biol. Bull. 191(1), 70–75 (1996).
    [CrossRef]
  23. S. Kirschner, C. J. Kleineidam, C. Zube, J. Rybak, B. Grünewald, and W. Rössler, “Dual olfactory pathway in the honeybee, Apis mellifera,” J. Comp. Neurol. 499(6), 933–952 (2006).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]

2009 (2)

B. Hourcade, E. Perisse, J. M. Devaud, and J. C. Sandoz, “Long-term memory shapes the primary olfactory center of an insect brain,” Learn. Mem. 16(10), 607–615 (2009).
[CrossRef] [PubMed]

N. Yamagata, M. Schmuker, P. Szyszka, M. Mizunami, and R. Menzel, “Differential odor processing in two olfactory pathways in the honeybee,” Front Syst Neurosci 3, 16 (2009).
[CrossRef] [PubMed]

2007 (1)

L. Moreaux and G. Laurent, “Estimating firing rates from calcium signals in locust projection neurons in vivo,” Front Neural Circuits 1, 2 (2007).
[CrossRef] [PubMed]

2006 (2)

P. Peele, M. Ditzen, R. Menzel, and C. G. Galizia, “Appetitive odor learning does not change olfactory coding in a subpopulation of honeybee antennal lobe neurons,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 192(10), 1083–1103 (2006).
[CrossRef] [PubMed]

S. Kirschner, C. J. Kleineidam, C. Zube, J. Rybak, B. Grünewald, and W. Rössler, “Dual olfactory pathway in the honeybee, Apis mellifera,” J. Comp. Neurol. 499(6), 933–952 (2006).
[CrossRef] [PubMed]

2005 (1)

R. Brandt, T. Rohlfing, J. Rybak, S. Krofczik, A. Maye, M. Westerhoff, H.-C. Hege, and R. Menzel, “Three-dimensional average-shape atlas of the honeybee brain and its applications,” J. Comp. Neurol. 492(1), 1–19 (2005).
[CrossRef] [PubMed]

2002 (2)

S. Sachse and C. G. Galizia, “Role of inhibition for temporal and spatial odor representation in olfactory output neurons: a calcium imaging study,” J. Neurophysiol. 87(2), 1106–1117 (2002).
[PubMed]

D. Müller, R. Abel, R. Brandt, M. Zöckler, and R. Menzel, “Differential parallel processing of olfactory information in the honeybee, Apis mellifera L,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 188(5), 359–370 (2002).
[CrossRef] [PubMed]

2001 (3)

C. G. Galizia and R. Menzel, “The role of glomeruli in the neural representation of odours: results from optical recording studies,” J. Insect Physiol. 47(2), 115–130 (2001).
[CrossRef] [PubMed]

R. Menzel and M. Giurfa, “Cognitive architecture of a mini-brain: the honeybee,” Trends Cogn. Sci. (Regul. Ed.) 5(2), 62–71 (2001).
[CrossRef] [PubMed]

T. Faber and R. Menzel, “Visualizing mushroom body response to a conditioned odor in honeybees,” Naturwissenschaften 88(11), 472–476 (2001).
[CrossRef] [PubMed]

1999 (2)

C. G. Galizia, S. L. McIlwrath, and R. Menzel, “A digital three-dimensional atlas of the honeybee antennal lobe based on optical sections acquired by confocal microscopy,” Cell Tissue Res. 295(3), 383–394 (1999), http://neuro.uni-konstanz.de/23bee morph/default.html .
[CrossRef] [PubMed]

C. G. Galizia, S. Sachse, A. Rappert, and R. Menzel, “The glomerular code for odor representation is species specific in the honeybee Apis mellifera,” Nat. Neurosci. 2(5), 473–478 (1999).
[CrossRef] [PubMed]

1997 (2)

A. Gelperin and J. Flores, “Vital staining from dye-coated microprobes identifies new olfactory interneurons for optical and electrical recording,” J. Neurosci. Methods 72(1), 97–108 (1997).
[CrossRef] [PubMed]

K. Svoboda, W. Denk, D. Kleinfeld, and D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature 385(6612), 161–165 (1997).
[CrossRef] [PubMed]

1996 (1)

G. Laurent, M. Wehr, K. MacLeod, M. Stopfer, B. Leitch, and H. Davidowitz, “Dynamic encoding of odors with oscillating neuronal assemblies in the locust brain,” Biol. Bull. 191(1), 70–75 (1996).
[CrossRef]

1993 (1)

E. E. Lieke, “Optical recording of neuronal activity in the insect central nervous system: odorant coding by the antennal lobes of honeybees,” Eur. J. Neurosci. 5(1), 49–55 (1993).
[CrossRef] [PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1989 (1)

D. Flanagan and A. R. Mercer, “An atlas and 3-D reconstruction of the antennal lobes in the worker honey bee, Apis mellifera L. (Hymenoptera: Apidae),” Int. J. Insect Morphol. Embryol. 18(2-3), 145–159 (1989).
[CrossRef]

1985 (1)

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).
[PubMed]

1973 (1)

B. M. Salzberg, H. V. Davila, and L. B. Cohen, “Optical recording of impulses in individual neurones of an invertebrate central nervous system,” Nature 246(5434), 508–509 (1973).
[CrossRef] [PubMed]

1939 (1)

A. L. Hodgkin and A. F. Huxley, “Action potentials recorded from inside a nerve fibre,” Nature 144(3651), 710–711 (1939).
[CrossRef]

Abel, R.

D. Müller, R. Abel, R. Brandt, M. Zöckler, and R. Menzel, “Differential parallel processing of olfactory information in the honeybee, Apis mellifera L,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 188(5), 359–370 (2002).
[CrossRef] [PubMed]

Brandt, R.

R. Brandt, T. Rohlfing, J. Rybak, S. Krofczik, A. Maye, M. Westerhoff, H.-C. Hege, and R. Menzel, “Three-dimensional average-shape atlas of the honeybee brain and its applications,” J. Comp. Neurol. 492(1), 1–19 (2005).
[CrossRef] [PubMed]

D. Müller, R. Abel, R. Brandt, M. Zöckler, and R. Menzel, “Differential parallel processing of olfactory information in the honeybee, Apis mellifera L,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 188(5), 359–370 (2002).
[CrossRef] [PubMed]

Cohen, L. B.

B. M. Salzberg, H. V. Davila, and L. B. Cohen, “Optical recording of impulses in individual neurones of an invertebrate central nervous system,” Nature 246(5434), 508–509 (1973).
[CrossRef] [PubMed]

Davidowitz, H.

G. Laurent, M. Wehr, K. MacLeod, M. Stopfer, B. Leitch, and H. Davidowitz, “Dynamic encoding of odors with oscillating neuronal assemblies in the locust brain,” Biol. Bull. 191(1), 70–75 (1996).
[CrossRef]

Davila, H. V.

B. M. Salzberg, H. V. Davila, and L. B. Cohen, “Optical recording of impulses in individual neurones of an invertebrate central nervous system,” Nature 246(5434), 508–509 (1973).
[CrossRef] [PubMed]

Denk, W.

K. Svoboda, W. Denk, D. Kleinfeld, and D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature 385(6612), 161–165 (1997).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Devaud, J. M.

B. Hourcade, E. Perisse, J. M. Devaud, and J. C. Sandoz, “Long-term memory shapes the primary olfactory center of an insect brain,” Learn. Mem. 16(10), 607–615 (2009).
[CrossRef] [PubMed]

Ditzen, M.

P. Peele, M. Ditzen, R. Menzel, and C. G. Galizia, “Appetitive odor learning does not change olfactory coding in a subpopulation of honeybee antennal lobe neurons,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 192(10), 1083–1103 (2006).
[CrossRef] [PubMed]

Faber, T.

T. Faber and R. Menzel, “Visualizing mushroom body response to a conditioned odor in honeybees,” Naturwissenschaften 88(11), 472–476 (2001).
[CrossRef] [PubMed]

Flanagan, D.

D. Flanagan and A. R. Mercer, “An atlas and 3-D reconstruction of the antennal lobes in the worker honey bee, Apis mellifera L. (Hymenoptera: Apidae),” Int. J. Insect Morphol. Embryol. 18(2-3), 145–159 (1989).
[CrossRef]

Flores, J.

A. Gelperin and J. Flores, “Vital staining from dye-coated microprobes identifies new olfactory interneurons for optical and electrical recording,” J. Neurosci. Methods 72(1), 97–108 (1997).
[CrossRef] [PubMed]

Galizia, C. G.

P. Peele, M. Ditzen, R. Menzel, and C. G. Galizia, “Appetitive odor learning does not change olfactory coding in a subpopulation of honeybee antennal lobe neurons,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 192(10), 1083–1103 (2006).
[CrossRef] [PubMed]

S. Sachse and C. G. Galizia, “Role of inhibition for temporal and spatial odor representation in olfactory output neurons: a calcium imaging study,” J. Neurophysiol. 87(2), 1106–1117 (2002).
[PubMed]

C. G. Galizia and R. Menzel, “The role of glomeruli in the neural representation of odours: results from optical recording studies,” J. Insect Physiol. 47(2), 115–130 (2001).
[CrossRef] [PubMed]

C. G. Galizia, S. L. McIlwrath, and R. Menzel, “A digital three-dimensional atlas of the honeybee antennal lobe based on optical sections acquired by confocal microscopy,” Cell Tissue Res. 295(3), 383–394 (1999), http://neuro.uni-konstanz.de/23bee morph/default.html .
[CrossRef] [PubMed]

C. G. Galizia, S. Sachse, A. Rappert, and R. Menzel, “The glomerular code for odor representation is species specific in the honeybee Apis mellifera,” Nat. Neurosci. 2(5), 473–478 (1999).
[CrossRef] [PubMed]

Gelperin, A.

A. Gelperin and J. Flores, “Vital staining from dye-coated microprobes identifies new olfactory interneurons for optical and electrical recording,” J. Neurosci. Methods 72(1), 97–108 (1997).
[CrossRef] [PubMed]

Giurfa, M.

R. Menzel and M. Giurfa, “Cognitive architecture of a mini-brain: the honeybee,” Trends Cogn. Sci. (Regul. Ed.) 5(2), 62–71 (2001).
[CrossRef] [PubMed]

Grünewald, B.

S. Kirschner, C. J. Kleineidam, C. Zube, J. Rybak, B. Grünewald, and W. Rössler, “Dual olfactory pathway in the honeybee, Apis mellifera,” J. Comp. Neurol. 499(6), 933–952 (2006).
[CrossRef] [PubMed]

Grynkiewicz, G.

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).
[PubMed]

Hege, H.-C.

R. Brandt, T. Rohlfing, J. Rybak, S. Krofczik, A. Maye, M. Westerhoff, H.-C. Hege, and R. Menzel, “Three-dimensional average-shape atlas of the honeybee brain and its applications,” J. Comp. Neurol. 492(1), 1–19 (2005).
[CrossRef] [PubMed]

Hodgkin, A. L.

A. L. Hodgkin and A. F. Huxley, “Action potentials recorded from inside a nerve fibre,” Nature 144(3651), 710–711 (1939).
[CrossRef]

Hourcade, B.

B. Hourcade, E. Perisse, J. M. Devaud, and J. C. Sandoz, “Long-term memory shapes the primary olfactory center of an insect brain,” Learn. Mem. 16(10), 607–615 (2009).
[CrossRef] [PubMed]

Huxley, A. F.

A. L. Hodgkin and A. F. Huxley, “Action potentials recorded from inside a nerve fibre,” Nature 144(3651), 710–711 (1939).
[CrossRef]

Kirschner, S.

S. Kirschner, C. J. Kleineidam, C. Zube, J. Rybak, B. Grünewald, and W. Rössler, “Dual olfactory pathway in the honeybee, Apis mellifera,” J. Comp. Neurol. 499(6), 933–952 (2006).
[CrossRef] [PubMed]

Kleineidam, C. J.

S. Kirschner, C. J. Kleineidam, C. Zube, J. Rybak, B. Grünewald, and W. Rössler, “Dual olfactory pathway in the honeybee, Apis mellifera,” J. Comp. Neurol. 499(6), 933–952 (2006).
[CrossRef] [PubMed]

Kleinfeld, D.

K. Svoboda, W. Denk, D. Kleinfeld, and D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature 385(6612), 161–165 (1997).
[CrossRef] [PubMed]

Krofczik, S.

R. Brandt, T. Rohlfing, J. Rybak, S. Krofczik, A. Maye, M. Westerhoff, H.-C. Hege, and R. Menzel, “Three-dimensional average-shape atlas of the honeybee brain and its applications,” J. Comp. Neurol. 492(1), 1–19 (2005).
[CrossRef] [PubMed]

Laurent, G.

L. Moreaux and G. Laurent, “Estimating firing rates from calcium signals in locust projection neurons in vivo,” Front Neural Circuits 1, 2 (2007).
[CrossRef] [PubMed]

G. Laurent, M. Wehr, K. MacLeod, M. Stopfer, B. Leitch, and H. Davidowitz, “Dynamic encoding of odors with oscillating neuronal assemblies in the locust brain,” Biol. Bull. 191(1), 70–75 (1996).
[CrossRef]

Leitch, B.

G. Laurent, M. Wehr, K. MacLeod, M. Stopfer, B. Leitch, and H. Davidowitz, “Dynamic encoding of odors with oscillating neuronal assemblies in the locust brain,” Biol. Bull. 191(1), 70–75 (1996).
[CrossRef]

Lieke, E. E.

E. E. Lieke, “Optical recording of neuronal activity in the insect central nervous system: odorant coding by the antennal lobes of honeybees,” Eur. J. Neurosci. 5(1), 49–55 (1993).
[CrossRef] [PubMed]

MacLeod, K.

G. Laurent, M. Wehr, K. MacLeod, M. Stopfer, B. Leitch, and H. Davidowitz, “Dynamic encoding of odors with oscillating neuronal assemblies in the locust brain,” Biol. Bull. 191(1), 70–75 (1996).
[CrossRef]

Maye, A.

R. Brandt, T. Rohlfing, J. Rybak, S. Krofczik, A. Maye, M. Westerhoff, H.-C. Hege, and R. Menzel, “Three-dimensional average-shape atlas of the honeybee brain and its applications,” J. Comp. Neurol. 492(1), 1–19 (2005).
[CrossRef] [PubMed]

McIlwrath, S. L.

C. G. Galizia, S. L. McIlwrath, and R. Menzel, “A digital three-dimensional atlas of the honeybee antennal lobe based on optical sections acquired by confocal microscopy,” Cell Tissue Res. 295(3), 383–394 (1999), http://neuro.uni-konstanz.de/23bee morph/default.html .
[CrossRef] [PubMed]

Menzel, R.

N. Yamagata, M. Schmuker, P. Szyszka, M. Mizunami, and R. Menzel, “Differential odor processing in two olfactory pathways in the honeybee,” Front Syst Neurosci 3, 16 (2009).
[CrossRef] [PubMed]

P. Peele, M. Ditzen, R. Menzel, and C. G. Galizia, “Appetitive odor learning does not change olfactory coding in a subpopulation of honeybee antennal lobe neurons,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 192(10), 1083–1103 (2006).
[CrossRef] [PubMed]

R. Brandt, T. Rohlfing, J. Rybak, S. Krofczik, A. Maye, M. Westerhoff, H.-C. Hege, and R. Menzel, “Three-dimensional average-shape atlas of the honeybee brain and its applications,” J. Comp. Neurol. 492(1), 1–19 (2005).
[CrossRef] [PubMed]

D. Müller, R. Abel, R. Brandt, M. Zöckler, and R. Menzel, “Differential parallel processing of olfactory information in the honeybee, Apis mellifera L,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 188(5), 359–370 (2002).
[CrossRef] [PubMed]

C. G. Galizia and R. Menzel, “The role of glomeruli in the neural representation of odours: results from optical recording studies,” J. Insect Physiol. 47(2), 115–130 (2001).
[CrossRef] [PubMed]

R. Menzel and M. Giurfa, “Cognitive architecture of a mini-brain: the honeybee,” Trends Cogn. Sci. (Regul. Ed.) 5(2), 62–71 (2001).
[CrossRef] [PubMed]

T. Faber and R. Menzel, “Visualizing mushroom body response to a conditioned odor in honeybees,” Naturwissenschaften 88(11), 472–476 (2001).
[CrossRef] [PubMed]

C. G. Galizia, S. Sachse, A. Rappert, and R. Menzel, “The glomerular code for odor representation is species specific in the honeybee Apis mellifera,” Nat. Neurosci. 2(5), 473–478 (1999).
[CrossRef] [PubMed]

C. G. Galizia, S. L. McIlwrath, and R. Menzel, “A digital three-dimensional atlas of the honeybee antennal lobe based on optical sections acquired by confocal microscopy,” Cell Tissue Res. 295(3), 383–394 (1999), http://neuro.uni-konstanz.de/23bee morph/default.html .
[CrossRef] [PubMed]

Mercer, A. R.

D. Flanagan and A. R. Mercer, “An atlas and 3-D reconstruction of the antennal lobes in the worker honey bee, Apis mellifera L. (Hymenoptera: Apidae),” Int. J. Insect Morphol. Embryol. 18(2-3), 145–159 (1989).
[CrossRef]

Mizunami, M.

N. Yamagata, M. Schmuker, P. Szyszka, M. Mizunami, and R. Menzel, “Differential odor processing in two olfactory pathways in the honeybee,” Front Syst Neurosci 3, 16 (2009).
[CrossRef] [PubMed]

Moreaux, L.

L. Moreaux and G. Laurent, “Estimating firing rates from calcium signals in locust projection neurons in vivo,” Front Neural Circuits 1, 2 (2007).
[CrossRef] [PubMed]

Müller, D.

D. Müller, R. Abel, R. Brandt, M. Zöckler, and R. Menzel, “Differential parallel processing of olfactory information in the honeybee, Apis mellifera L,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 188(5), 359–370 (2002).
[CrossRef] [PubMed]

Peele, P.

P. Peele, M. Ditzen, R. Menzel, and C. G. Galizia, “Appetitive odor learning does not change olfactory coding in a subpopulation of honeybee antennal lobe neurons,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 192(10), 1083–1103 (2006).
[CrossRef] [PubMed]

Perisse, E.

B. Hourcade, E. Perisse, J. M. Devaud, and J. C. Sandoz, “Long-term memory shapes the primary olfactory center of an insect brain,” Learn. Mem. 16(10), 607–615 (2009).
[CrossRef] [PubMed]

Poenie, M.

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).
[PubMed]

Rappert, A.

C. G. Galizia, S. Sachse, A. Rappert, and R. Menzel, “The glomerular code for odor representation is species specific in the honeybee Apis mellifera,” Nat. Neurosci. 2(5), 473–478 (1999).
[CrossRef] [PubMed]

Rohlfing, T.

R. Brandt, T. Rohlfing, J. Rybak, S. Krofczik, A. Maye, M. Westerhoff, H.-C. Hege, and R. Menzel, “Three-dimensional average-shape atlas of the honeybee brain and its applications,” J. Comp. Neurol. 492(1), 1–19 (2005).
[CrossRef] [PubMed]

Rössler, W.

S. Kirschner, C. J. Kleineidam, C. Zube, J. Rybak, B. Grünewald, and W. Rössler, “Dual olfactory pathway in the honeybee, Apis mellifera,” J. Comp. Neurol. 499(6), 933–952 (2006).
[CrossRef] [PubMed]

Rybak, J.

S. Kirschner, C. J. Kleineidam, C. Zube, J. Rybak, B. Grünewald, and W. Rössler, “Dual olfactory pathway in the honeybee, Apis mellifera,” J. Comp. Neurol. 499(6), 933–952 (2006).
[CrossRef] [PubMed]

R. Brandt, T. Rohlfing, J. Rybak, S. Krofczik, A. Maye, M. Westerhoff, H.-C. Hege, and R. Menzel, “Three-dimensional average-shape atlas of the honeybee brain and its applications,” J. Comp. Neurol. 492(1), 1–19 (2005).
[CrossRef] [PubMed]

Sachse, S.

S. Sachse and C. G. Galizia, “Role of inhibition for temporal and spatial odor representation in olfactory output neurons: a calcium imaging study,” J. Neurophysiol. 87(2), 1106–1117 (2002).
[PubMed]

C. G. Galizia, S. Sachse, A. Rappert, and R. Menzel, “The glomerular code for odor representation is species specific in the honeybee Apis mellifera,” Nat. Neurosci. 2(5), 473–478 (1999).
[CrossRef] [PubMed]

Salzberg, B. M.

B. M. Salzberg, H. V. Davila, and L. B. Cohen, “Optical recording of impulses in individual neurones of an invertebrate central nervous system,” Nature 246(5434), 508–509 (1973).
[CrossRef] [PubMed]

Sandoz, J. C.

B. Hourcade, E. Perisse, J. M. Devaud, and J. C. Sandoz, “Long-term memory shapes the primary olfactory center of an insect brain,” Learn. Mem. 16(10), 607–615 (2009).
[CrossRef] [PubMed]

Schmuker, M.

N. Yamagata, M. Schmuker, P. Szyszka, M. Mizunami, and R. Menzel, “Differential odor processing in two olfactory pathways in the honeybee,” Front Syst Neurosci 3, 16 (2009).
[CrossRef] [PubMed]

Stopfer, M.

G. Laurent, M. Wehr, K. MacLeod, M. Stopfer, B. Leitch, and H. Davidowitz, “Dynamic encoding of odors with oscillating neuronal assemblies in the locust brain,” Biol. Bull. 191(1), 70–75 (1996).
[CrossRef]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Svoboda, K.

K. Svoboda, W. Denk, D. Kleinfeld, and D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature 385(6612), 161–165 (1997).
[CrossRef] [PubMed]

Szyszka, P.

N. Yamagata, M. Schmuker, P. Szyszka, M. Mizunami, and R. Menzel, “Differential odor processing in two olfactory pathways in the honeybee,” Front Syst Neurosci 3, 16 (2009).
[CrossRef] [PubMed]

Tank, D. W.

K. Svoboda, W. Denk, D. Kleinfeld, and D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature 385(6612), 161–165 (1997).
[CrossRef] [PubMed]

Tsien, R. Y.

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).
[PubMed]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Wehr, M.

G. Laurent, M. Wehr, K. MacLeod, M. Stopfer, B. Leitch, and H. Davidowitz, “Dynamic encoding of odors with oscillating neuronal assemblies in the locust brain,” Biol. Bull. 191(1), 70–75 (1996).
[CrossRef]

Westerhoff, M.

R. Brandt, T. Rohlfing, J. Rybak, S. Krofczik, A. Maye, M. Westerhoff, H.-C. Hege, and R. Menzel, “Three-dimensional average-shape atlas of the honeybee brain and its applications,” J. Comp. Neurol. 492(1), 1–19 (2005).
[CrossRef] [PubMed]

Yamagata, N.

N. Yamagata, M. Schmuker, P. Szyszka, M. Mizunami, and R. Menzel, “Differential odor processing in two olfactory pathways in the honeybee,” Front Syst Neurosci 3, 16 (2009).
[CrossRef] [PubMed]

Zöckler, M.

D. Müller, R. Abel, R. Brandt, M. Zöckler, and R. Menzel, “Differential parallel processing of olfactory information in the honeybee, Apis mellifera L,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 188(5), 359–370 (2002).
[CrossRef] [PubMed]

Zube, C.

S. Kirschner, C. J. Kleineidam, C. Zube, J. Rybak, B. Grünewald, and W. Rössler, “Dual olfactory pathway in the honeybee, Apis mellifera,” J. Comp. Neurol. 499(6), 933–952 (2006).
[CrossRef] [PubMed]

Biol. Bull. (1)

G. Laurent, M. Wehr, K. MacLeod, M. Stopfer, B. Leitch, and H. Davidowitz, “Dynamic encoding of odors with oscillating neuronal assemblies in the locust brain,” Biol. Bull. 191(1), 70–75 (1996).
[CrossRef]

Cell Tissue Res. (1)

C. G. Galizia, S. L. McIlwrath, and R. Menzel, “A digital three-dimensional atlas of the honeybee antennal lobe based on optical sections acquired by confocal microscopy,” Cell Tissue Res. 295(3), 383–394 (1999), http://neuro.uni-konstanz.de/23bee morph/default.html .
[CrossRef] [PubMed]

Eur. J. Neurosci. (1)

E. E. Lieke, “Optical recording of neuronal activity in the insect central nervous system: odorant coding by the antennal lobes of honeybees,” Eur. J. Neurosci. 5(1), 49–55 (1993).
[CrossRef] [PubMed]

Front Neural Circuits (1)

L. Moreaux and G. Laurent, “Estimating firing rates from calcium signals in locust projection neurons in vivo,” Front Neural Circuits 1, 2 (2007).
[CrossRef] [PubMed]

Front Syst Neurosci (1)

N. Yamagata, M. Schmuker, P. Szyszka, M. Mizunami, and R. Menzel, “Differential odor processing in two olfactory pathways in the honeybee,” Front Syst Neurosci 3, 16 (2009).
[CrossRef] [PubMed]

Int. J. Insect Morphol. Embryol. (1)

D. Flanagan and A. R. Mercer, “An atlas and 3-D reconstruction of the antennal lobes in the worker honey bee, Apis mellifera L. (Hymenoptera: Apidae),” Int. J. Insect Morphol. Embryol. 18(2-3), 145–159 (1989).
[CrossRef]

J. Biol. Chem. (1)

G. Grynkiewicz, M. Poenie, and R. Y. Tsien, “A new generation of Ca2+ indicators with greatly improved fluorescence properties,” J. Biol. Chem. 260(6), 3440–3450 (1985).
[PubMed]

J. Comp. Neurol. (2)

R. Brandt, T. Rohlfing, J. Rybak, S. Krofczik, A. Maye, M. Westerhoff, H.-C. Hege, and R. Menzel, “Three-dimensional average-shape atlas of the honeybee brain and its applications,” J. Comp. Neurol. 492(1), 1–19 (2005).
[CrossRef] [PubMed]

S. Kirschner, C. J. Kleineidam, C. Zube, J. Rybak, B. Grünewald, and W. Rössler, “Dual olfactory pathway in the honeybee, Apis mellifera,” J. Comp. Neurol. 499(6), 933–952 (2006).
[CrossRef] [PubMed]

J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. (2)

P. Peele, M. Ditzen, R. Menzel, and C. G. Galizia, “Appetitive odor learning does not change olfactory coding in a subpopulation of honeybee antennal lobe neurons,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 192(10), 1083–1103 (2006).
[CrossRef] [PubMed]

D. Müller, R. Abel, R. Brandt, M. Zöckler, and R. Menzel, “Differential parallel processing of olfactory information in the honeybee, Apis mellifera L,” J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 188(5), 359–370 (2002).
[CrossRef] [PubMed]

J. Insect Physiol. (1)

C. G. Galizia and R. Menzel, “The role of glomeruli in the neural representation of odours: results from optical recording studies,” J. Insect Physiol. 47(2), 115–130 (2001).
[CrossRef] [PubMed]

J. Neurophysiol. (1)

S. Sachse and C. G. Galizia, “Role of inhibition for temporal and spatial odor representation in olfactory output neurons: a calcium imaging study,” J. Neurophysiol. 87(2), 1106–1117 (2002).
[PubMed]

J. Neurosci. Methods (1)

A. Gelperin and J. Flores, “Vital staining from dye-coated microprobes identifies new olfactory interneurons for optical and electrical recording,” J. Neurosci. Methods 72(1), 97–108 (1997).
[CrossRef] [PubMed]

Learn. Mem. (1)

B. Hourcade, E. Perisse, J. M. Devaud, and J. C. Sandoz, “Long-term memory shapes the primary olfactory center of an insect brain,” Learn. Mem. 16(10), 607–615 (2009).
[CrossRef] [PubMed]

Nat. Neurosci. (1)

C. G. Galizia, S. Sachse, A. Rappert, and R. Menzel, “The glomerular code for odor representation is species specific in the honeybee Apis mellifera,” Nat. Neurosci. 2(5), 473–478 (1999).
[CrossRef] [PubMed]

Nature (3)

K. Svoboda, W. Denk, D. Kleinfeld, and D. W. Tank, “In vivo dendritic calcium dynamics in neocortical pyramidal neurons,” Nature 385(6612), 161–165 (1997).
[CrossRef] [PubMed]

A. L. Hodgkin and A. F. Huxley, “Action potentials recorded from inside a nerve fibre,” Nature 144(3651), 710–711 (1939).
[CrossRef]

B. M. Salzberg, H. V. Davila, and L. B. Cohen, “Optical recording of impulses in individual neurones of an invertebrate central nervous system,” Nature 246(5434), 508–509 (1973).
[CrossRef] [PubMed]

Naturwissenschaften (1)

T. Faber and R. Menzel, “Visualizing mushroom body response to a conditioned odor in honeybees,” Naturwissenschaften 88(11), 472–476 (2001).
[CrossRef] [PubMed]

Science (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Trends Cogn. Sci. (Regul. Ed.) (1)

R. Menzel and M. Giurfa, “Cognitive architecture of a mini-brain: the honeybee,” Trends Cogn. Sci. (Regul. Ed.) 5(2), 62–71 (2001).
[CrossRef] [PubMed]

Other (2)

T. Franke, “In vivo 2-photon calcium imaging of olfactory interneurons in the honeybee antennal lobe,” Dissertation, FB Biologie, Chemie, Pharmazie, Freie Universit¨at Berlin (2009).

C. G. Galizia, and R. Vetter, “Optical methods for analyzing odor-evoked activity in the insect brain,” in Advances in Insect Sensory Neuroscience, T. A. Christensen, ed. (CRC press, 2004), pp. 349–392.

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Figures (5)

Fig. 1.
Fig. 1.

a) Image of a right antennal lobe at 25µm depth: The line indicates the laser scanning trace, the dots label the measurement’s reference positions corresponding to the vertical lines in Figure 2. (b) Axial projection view of the AL volume image stack, superimposed by the reconstructed surface plots of the involved T1 glomeruli, identified and labeled according to [20].

Fig. 2.
Fig. 2.

Calcium response maps for three different odors (1-Hexanol above, 1-Octanol middle, 1-Nonanol below), recorded along the scanning trace in Figure 1. The stimulus period is enclosed by the horizontal lines, responding glomeruli centers are marked by vertical lines, numbers label the identified glomeruli.

Fig. 3.
Fig. 3.

Odor response maps to 1-Octanol at depths of 25µm and 50µm within the AL. (a) and (d) show the 2D images of the corresponding focal plane together with the line scan traces. In (b) and (e) the activity signal is plotted as a function of position along the line trace (x-axis) and as a function of time (y-axis). The stimulus period is enclosed by horizontal lines, the single responding glomeruli are marked by vertical lines. (c) and (f) show single temporal traces for the strongest responding glomeruli T1-33 and T1-17 at the two corresponding depths.

Fig. 4.
Fig. 4.

a) Image stack examples down to 200µm penetration depth into the right AL. b) Reconstructed glomerular volume images in all projections, glomeruli colored in green are from the T1 tract projecting into the l-ACT, the red glomeruli are from the T3 tract projecting into the m-ACT.

Fig. 5.
Fig. 5.

a) Functional image map of responses to a 1-Hexanol stimulus recorded at 150µm depth from the left AL. b) The imaged focal plane together with the laser scan line. The responding centers are marked by points and labeled by the corresponding glomerular number. Green points correspond to T1, blue ones to T2, and red ones to T3 glomeruli. c) Single time traces of the responses from glomeruli of the T2 and T3 tract.

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