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Activation of raphe nuclei triggers rapid and distinct effects on parallel olfactory bulb output channels

Nature Neuroscience volume 19, pages 271–282 (2016)Cite this article

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Abstract

The serotonergic raphe nuclei are involved in regulating brain states over timescales of minutes and hours. We examined more rapid effects of raphe activation on two classes of principal neurons in the mouse olfactory bulb, mitral and tufted cells, which send olfactory information to distinct targets. Brief stimulation of the raphe nuclei led to excitation of tufted cells at rest and potentiation of their odor responses. While mitral cells at rest were also excited by raphe activation, their odor responses were bidirectionally modulated, leading to improved pattern separation of odors. In vitro whole-cell recordings revealed that specific optogenetic activation of raphe axons affected bulbar neurons through dual release of serotonin and glutamate. Therefore, the raphe nuclei, in addition to their role in neuromodulation of brain states, are also involved in fast, sub-second top-down modulation similar to cortical feedback. This modulation can selectively and differentially sensitize or decorrelate distinct output channels.

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Figure 1: Raphe stimulation excites mitral and tufted cells at rest.
Figure 2: TC odor responses are sensitized by raphe inputs.
Figure 3: MC odor responses are bidirectionally modulated by raphe inputs.
Figure 4: Optogenetic activation of raphe leads to excitation of principal cells.
Figure 5: Modulation of TCs by optogenetic activation of raphe projections in main olfactory bulb slices.
Figure 6: Modulation of MC activity by optogenetic activation of raphe fibers.
Figure 7: ETCs receive direct excitatory inputs from raphe fibers.
Figure 8: Raphe activation leads to sensitization of TC odor responses and decorrelation of MC odor responses.

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References

  1. Lee, S.-H. & Dan, Y. Neuromodulation of brain states. Neuron 76, 209–222 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Marder, E. Neuromodulation of neuronal circuits: back to the future. Neuron 76, 1–11 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Cools, R., Roberts, A.C. & Robbins, T.W. Serotoninergic regulation of emotional and behavioural control processes. Trends Cogn. Sci. 12, 31–40 (2008).

    PubMed  Google Scholar 

  4. Dayan, P. & Huys, Q.J.M. Serotonin in affective control. Annu. Rev. Neurosci. 32, 95–126 (2009).

    CAS  PubMed  Google Scholar 

  5. Liu, Z. et al. Dorsal raphe neurons signal reward through 5-HT and glutamate. Neuron 81, 1360–1374 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Dacks, A.M., Green, D.S., Root, C.M., Nighorn, A.J. & Wang, J.W. Serotonin modulates olfactory processing in the antennal lobe of Drosophila. J. Neurogenet. 23, 366–377 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Fonseca, M.S., Murakami, M. & Mainen, Z.F. Activation of dorsal raphe serotonergic neurons promotes waiting but is not reinforcing. Curr. Biol. 25, 306–315 (2015).

    CAS  PubMed  Google Scholar 

  8. Hurley, L.M., Devilbiss, D.M. & Waterhouse, B.D. A matter of focus: monoaminergic modulation of stimulus coding in mammalian sensory networks. Curr. Opin. Neurobiol. 14, 488–495 (2004).

    CAS  PubMed  Google Scholar 

  9. Petzold, G.C., Hagiwara, A. & Murthy, V.N. Serotonergic modulation of odor input to the mammalian olfactory bulb. Nat. Neurosci. 12, 784–791 (2009).

    CAS  PubMed  Google Scholar 

  10. Hilaire, G. et al. The role of serotonin in respiratory function and dysfunction. Respir. Physiol. Neurobiol. 174, 76–88 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Jacobs, B.L. & Azmitia, E.C. Structure and function of the brain serotonin system. Physiol. Rev. 72, 165–229 (1992).

    CAS  PubMed  Google Scholar 

  12. Ray, R.S. et al. Impaired respiratory and body temperature control upon acute serotonergic neuron inhibition. Science 333, 637–642 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Hannon, J. & Hoyer, D. Molecular biology of 5-HT receptors. Behav. Brain Res. 195, 198–213 (2008).

    CAS  PubMed  Google Scholar 

  14. Cohen, J.Y., Amoroso, M.W. & Uchida, N. Serotonergic neurons signal reward and punishment on multiple timescales. Elife 4, e06346 (2015).

    PubMed Central  Google Scholar 

  15. Ranade, S.P. & Mainen, Z.F. Transient firing of dorsal raphe neurons encodes diverse and specific sensory, motor, and reward events. J. Neurophysiol. 102, 3026–3037 (2009).

    PubMed  Google Scholar 

  16. Rhoades, R.W., Bennett-Clarke, C.A., Shi, M.Y. & Mooney, R.D. Effects of 5-HT on thalamocortical synaptic transmission in the developing rat. J. Neurophysiol. 72, 2438–2450 (1994).

    CAS  PubMed  Google Scholar 

  17. Suzuki, Y., Kiyokage, E., Sohn, J., Hioki, H. & Toida, K. Structural basis for serotonergic regulation of neural circuits in the mouse olfactory bulb. J. Comp. Neurol. 523, 262–280 (2015).

    CAS  PubMed  Google Scholar 

  18. Varga, V. et al. Fast synaptic subcortical control of hippocampal circuits. Science 326, 449–453 (2009).

    CAS  PubMed  Google Scholar 

  19. Nagayama, S., Homma, R. & Imamura, F. Neuronal organization of olfactory bulb circuits. Front. Neural Circuits 8, 98 (2014).

    PubMed  PubMed Central  Google Scholar 

  20. Haberly, L.B. Parallel-distributed processing in olfactory cortex: new insights from morphological and physiological analysis of neuronal circuitry. Chem. Senses 26, 551–576 (2001).

    CAS  PubMed  Google Scholar 

  21. Fukunaga, I., Berning, M., Kollo, M., Schmaltz, A. & Schaefer, A.T. Two distinct channels of olfactory bulb output. Neuron 75, 320–329 (2012).

    CAS  PubMed  Google Scholar 

  22. Giessel, A.J. & Datta, S.R. Olfactory maps, circuits and computations. Curr. Opin. Neurobiol. 24, 120–132 (2014).

    CAS  PubMed  Google Scholar 

  23. McLean, J.H. & Shipley, M.T. Serotonergic afferents to the rat olfactory bulb: I. Origins and laminar specificity of serotonergic inputs in the adult rat. J. Neurosci. 7, 3016–3028 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Steinfeld, R., Herb, J.T., Sprengel, R., Schaefer, A.T. & Fukunaga, I. Divergent innervation of the olfactory bulb by distinct raphe nuclei. J. Comp. Neurol. 523, 805–813 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Liu, S., Aungst, J.L., Puche, A.C. & Shipley, M.T. Serotonin modulates the population activity profile of olfactory bulb external tufted cells. J. Neurophysiol. 107, 473–483 (2012).

    CAS  PubMed  Google Scholar 

  26. Hardy, A., Palouzier-Paulignan, B., Duchamp, A., Royet, J.-P. & Duchamp-Viret, P. 5-Hydroxytryptamine action in the rat olfactory bulb: in vitro electrophysiological patch-clamp recordings of juxtaglomerular and mitral cells. Neuroscience 131, 717–731 (2005).

    CAS  PubMed  Google Scholar 

  27. Nakamura, K., Matsumoto, M. & Hikosaka, O. Reward-dependent modulation of neuronal activity in the primate dorsal raphe nucleus. J. Neurosci. 28, 5331–5343 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Haddad, R. et al. Olfactory cortical neurons read out a relative time code in the olfactory bulb. Nat. Neurosci. 16, 949–957 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Wachowiak, M. All in a sniff: olfaction as a model for active sensing. Neuron 71, 962–973 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Adam, Y. et al. Functional transformations of odor inputs in the mouse olfactory bulb. Front. Neural Circuits 8, 129 (2014).

    PubMed  PubMed Central  Google Scholar 

  32. Kikuta, S., Fletcher, M.L., Homma, R., Yamasoba, T. & Nagayama, S. Odorant response properties of individual neurons in an olfactory glomerular module. Neuron 77, 1122–1135 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhao, S. et al. Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat. Methods 8, 745–752 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Kapoor, V. & Urban, N.N. Glomerulus-specific, long-latency activity in the olfactory bulb granule cell network. J. Neurosci. 26, 11709–11719 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Gire, D.H. et al. Mitral cells in the olfactory bulb are mainly excited through a multistep signaling path. J. Neurosci. 32, 2964–2975 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Najac, M., De Saint Jan, D., Reguero, L., Grandes, P. & Charpak, S. Monosynaptic and polysynaptic feed-forward inputs to mitral cells from olfactory sensory neurons. J. Neurosci. 31, 8722–8729 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Friedrich, R.W. & Wiechert, M.T. Neuronal circuits and computations: pattern decorrelation in the olfactory bulb. FEBS Lett. 588, 2504–2513 (2014).

    CAS  PubMed  Google Scholar 

  38. Miyazaki, K.W. et al. Optogenetic activation of dorsal raphe serotonin neurons enhances patience for future rewards. Curr. Biol. 24, 2033–2040 (2014).

    CAS  PubMed  Google Scholar 

  39. Gracia-Llanes, F.J. et al. Synaptic connectivity of serotonergic axons in the olfactory glomeruli of the rat olfactory bulb. Neuroscience 169, 770–780 (2010).

    CAS  PubMed  Google Scholar 

  40. Commons, K.G. Locally collateralizing glutamate neurons in the dorsal raphe nucleus responsive to substance P contain vesicular glutamate transporter 3 (VGLUT3). J. Chem. Neuroanat. 38, 273–281 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Fremeau, R.T. Jr., Voglmaier, S., Seal, R.P. & Edwards, R.H. VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate. Trends Neurosci. 27, 98–103 (2004).

    CAS  PubMed  Google Scholar 

  42. Qi, J. et al. A glutamatergic reward input from the dorsal raphe to ventral tegmental area dopamine neurons. Nat. Commun. 5, 5390 (2014).

    CAS  PubMed  Google Scholar 

  43. Okaty, B.W. et al. Multi-scale molecular deconstruction of the serotonin neuron system. Neuron. 88, 774–791 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Otazu, G.H., Chae, H., Davis, M.B. & Albeanu, D.F. Cortical feedback decorrelates olfactory bulb output in awake mice. Neuron 86, 1461–1477 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Burton, S.D. & Urban, N.N. Greater excitability and firing irregularity of tufted cells underlies distinct afferent-evoked activity of olfactory bulb mitral and tufted cells. J. Physiol. (Lond.) 592, 2097–2118 (2014).

    CAS  Google Scholar 

  46. Scott, J.W. Electrophysiological identification of mitral and tufted cells and distributions of their axons in olfactory system of the rat. J. Neurophysiol. 46, 918–931 (1981).

    CAS  PubMed  Google Scholar 

  47. Kiselycznyk, C.L., Zhang, S. & Linster, C. Role of centrifugal projections to the olfactory bulb in olfactory processing. Learn. Mem. 13, 575–579 (2006).

    PubMed  Google Scholar 

  48. Rothermel, M., Carey, R.M., Puche, A., Shipley, M.T. & Wachowiak, M. Cholinergic inputs from Basal forebrain add an excitatory bias to odor coding in the olfactory bulb. J. Neurosci. 34, 4654–4664 (2014).

    PubMed  PubMed Central  Google Scholar 

  49. Shea, S.D., Katz, L.C. & Mooney, R. Noradrenergic induction of odor-specific neural habituation and olfactory memories. J. Neurosci. 28, 10711–10719 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Atasoy, D., Aponte, Y., Su, H.H. & Sternson, S.M. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. J. Neurosci. 28, 7025–7030 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Soucy, E.R., Albeanu, D.F., Fantana, A.L., Murthy, V.N. & Meister, M. Precision and diversity in an odor map on the olfactory bulb. Nat. Neurosci. 12, 210–220 (2009).

    CAS  PubMed  Google Scholar 

  52. Markopoulos, F., Rokni, D., Gire, D.H. & Murthy, V.N. Functional properties of cortical feedback projections to the olfactory bulb. Neuron 76, 1175–1188 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the members of Murthy and Dulac labs for discussions throughout the project, and D. Gire, J. Cohen and M. Thanawala for comments on the manuscript. This work was supported by research grants (DC011291 and DC014453) from the US National Institutes of Health (V.N.M.), a seed grant from the Harvard Brain Initiative (V.N.M.) and fellowships from the US National Science Foundation and the Mortimer and Theresa Sackler Foundation (A.C.P.).

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  1. Vikrant Kapoor and Allison C Provost: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA

    Vikrant Kapoor, Allison C Provost, Prateek Agarwal & Venkatesh N Murthy

  2. Department of Molecular & Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    Vikrant Kapoor, Allison C Provost, Prateek Agarwal & Venkatesh N Murthy

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  1. Vikrant Kapoor
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Contributions

A.C.P., V.K. and V.N.M. designed the project. A.C.P. and V.K. performed in vivo experiments; A.C.P. and P.A. performed and analyzed histological experiments; V.K. perform in vitro experiments. A.C.P. and V.K. analyzed the data with V.N.M.'s guidance. V.N.M. supervised the entire project. A.C.P., V.K. and V.N.M. wrote the paper with input from P.A.

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Correspondence to Vikrant Kapoor or Venkatesh N Murthy.

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Integrated supplementary information

Supplementary Figure 1 Neuronal populations in the Tbx21-Cre (Tbet-Cre) mouse and 5-HT receptors in the OB

a) Tbet Cre mouse olfactory bulb injected with AAV 2.9 flex Gcamp6s virus showing sparse labelling of principal neurons of the OB.

b) 5-HT receptor 2a antibody (green, 1/1000 dilution, abcam ab66049) stains heavily in the external plexiform layer (EPL) and on MC bodies. Labeling performed in Tbet-Cre x Flex tdTomato mouse, so all principal cells are red.

c) Inhibitory 5-HT receptor 1a (red, 1/1000 dilution, abcam ab101914) stains non-GABAergic cells both in the EPL and MC layer (c1) and the glomerular layer (GL, c2). Staining performed in the GAD67-GFP mouse, so inhibitory cells are green.

d) Inhibitory 5-HT receptor 1b (red, 1/1000 dilution, abcam ab102700) stains non-GABAergic cells in the OB. Staining performed in the GAD67-GFP mouse (green). Granule Cell Layer (GCL).

Supplementary Figure 2 Names and structures of odorants used in study

Supplementary Figure 3 Sensitization of TC odor responses by raphe stimulation

a) Scatter plot showing sensitization in TC odor responses in 6 animals. Y axis is the observed response to paired odor and raphe stimulation minus raphe only stimulation response and x axis is odor only response.

b) Bar plot showing mean change in TC responses across animals. Mean response is calculated as the difference between paired odor plus raphe responses and (odor only + raphe only) responses.

c) Scatter plot showing the predicted response (linear model, R2 = 0.7611) vs observed response for 6 animals (1087 odor cell pairs).

d) Scatter plot showing the predicted response (linear model, R2 = 0.8638) vs observed response (1087 odor cell pairs).

Supplementary Figure 4 Odor inputs to the OB are not affected by brief raphe stimulation

a) Resting fluorescence image of glomeruli from an OMP-GCaMP3 mouse.

b) Example traces of odor responses from a single OMP-GCaMP3 glomerulus. Odor duration denoted by green bar at bottom.

c) Odor responses of olfactory sensory neurons measured from multiple glomeruli (left), in an OMP-GCaMP3 mouse, were not altered when the same odor was paired with raphe activation (right).

d) Scatter plot of all glomeruli odor pairs comparing average odor responses (dF/F) with and without raphe activation. Dashes are the unity line (p>0.3, Wilcoxon signed-rank, 2 mice, 26 glomeruli).

Supplementary Figure 5 Raphe projections to the main olfactory bulb

a) Anti-GFP antibody (green, 1/500 dilution; Aves GFP-1010) and anti-serotonin antibody (red, 1/1000 dilution; Sigma S5545) in a TPH2-ChR2 animal stains heavily in the glomerular layer and granule cell layer.

b) Zoomed in version of images shown in a, depicting the heterogeneity of raphe projections in different layers of the olfactory bulb.

c) Bar plots summarizing the density of raphe projections in the olfactory bulb as the mean pixel intensity in the different layers of the olfactory bulb (glomerular layer: 1.0, external plexiform layer: 0.26, mitral cell layer: 0.35, granule cell layer: 0.48, n=4). Data normalized to glomerular layer.

Supplementary Figure 6 Modulation of odor-evoked activity of principal neurons in the olfactory bulb by raphe stimulation in an awake animal

a) Time course of fluorescence changes in 36 TCs in an awake animal in response to ethyl valerate with (right) and without (left) raphe stimulation.

b) Similar plot showing time course responses of 23 MCs in an awake animal in response to ethyl valerate with (right) and without (left) raphe stimulation.

c) Scatter plot showing sensitization of odor responses in TCs with raphe stimulation in awake animals (468 cell-odor pairs, n=2). Mean change in odor response 23.71 ± 1 % (median change of 19.79 %).

d) Scatter plot showing bidirectional modulation of odor responses with raphe stimulation in awake animals (282 cell-odor pairs, n=2). Mean change in odor response 1.94 ± 0.9 % (median change of 1.79 %).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 1119 kb)

Supplementary Methods Checklist (PDF 450 kb)

A 3-dimensional stack showing images of tufted and mitral cells.

Stack is made up of 1 micron optical sections through 360 microns depth, and shows cells imaged in a living mouse with a multiphoton microscope. Green colour represents GCaMP6s fluorescence. Movie starts with the most superficial section with apical tufts visible. Tufted cell somata are visible a bit deeper and past the external plexiform layer (where many lateral dendrites are visible) mitral cell somata become visible. (AVI 6436 kb)

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Kapoor, V., Provost, A., Agarwal, P. et al. Activation of raphe nuclei triggers rapid and distinct effects on parallel olfactory bulb output channels. Nat Neurosci 19, 271–282 (2016). https://doi.org/10.1038/nn.4219

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