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The participation of cortical amygdala in innate, odour-driven behaviour

Nature volume 515, pages 269–273 (2014)Cite this article

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Abstract

Innate behaviours are observed in naive animals without prior learning or experience, suggesting that the neural circuits that mediate these behaviours are genetically determined and stereotyped. The neural circuits that convey olfactory information from the sense organ to the cortical and subcortical olfactory centres have been anatomically defined1,2,3, but the specific pathways responsible for innate responses to volatile odours have not been identified. Here we devise genetic strategies that demonstrate that a stereotyped neural circuit that transmits information from the olfactory bulb to cortical amygdala is necessary for innate aversive and appetitive behaviours. Moreover, we use the promoter of the activity-dependent gene arc to express the photosensitive ion channel, channelrhodopsin, in neurons of the cortical amygdala activated by odours that elicit innate behaviours. Optical activation of these neurons leads to appropriate behaviours that recapitulate the responses to innate odours. These data indicate that the cortical amygdala plays a critical role in generating innate odour-driven behaviours but do not preclude its participation in learned olfactory behaviours.

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Figure 1: Behavioural assay for innate responses to odour.
Figure 2: The projections from the olfactory bulb to cortical amygdala are required for innate aversion and attraction to odours.
Figure 3: Activation of odour-responsive neurons within cortical amygdala is sufficient to recapitulate behavioural responses.
Figure 4: The spatial distribution of neurons responsive to different odours within the cortical amygdala.

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Acknowledgements

We thank L. Abbott, T. Jessell and D. Costantini for comments and reading the manuscript; B. Bader for assistance with experiments; M. Mendelsohn and N. Zabello for help with mice; P. Kisloff for assistance in preparation of the manuscript; and A. Nemes and M. Gutierrez for laboratory support. This work was supported by the Howard Hughes Medical Institute and the Mathers Foundation.

Author information

Authors and Affiliations

  1. Department of Neuroscience and the Howard Hughes Medical Institute, College of Physicians and Surgeons, Columbia University, New York, 10032, New York, USA

    Cory M. Root & Richard Axel

  2. Department of Biological Sciences, New York State Psychiatric Institute, New York, 10032, New York, USA

    Christine A. Denny

  3. Department of Neuroscience and Psychiatry, New York State Psychiatric Institute, New York, 10032, New York, USA

    Christine A. Denny & René Hen

  4. Division of Integrative Neuroscience, New York State Psychiatric Institute, New York, 10032, New York, USA

    Christine A. Denny & René Hen

  5. Department of Pharmacology, Columbia University, New York State Psychiatric Institute, New York, 10032, New York, USA

    René Hen

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  1. Cory M. Root
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  2. Christine A. Denny
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  4. Richard Axel
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Contributions

C.M.R. and R.A. conceived the project, participated in its development, wrote the manuscript and analysed data. C.M.R. performed all experiments. C.A.D. and R.H. conceived and generated the ArcCreERT2 transgenic mouse.

Corresponding author

Correspondence to Richard Axel.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Quantification of the four-quadrant behaviour assay.

a–c, Images of the four-field behaviour chamber. b, An image, taken from the camera that tracked the position of the animal, showing a mouse tethered to optic fibres that entered the chamber through a port on the left side. c, A motorized fibre retraction system adjusted the length of the fibre as the mouse traversed the arena. The trajectory of a representative mouse is plotted for a 10 minute period in the absence of odour (left), or after the addition of odour to the lower right quadrant (right). The raster plots below the trajectory graphs represent quadrant occupancy over time. e, The velocity over time in the absence of odour (left) or in the presence of TMT (right) reveals bouts of inactivity associated with freezing behaviour in the presence of TMT. f, The average amount of time spent in each quadrant either in the absence of odour, or the presence of TMT, 2-phenylethanol or isoamyl acetate. g, This quantification is reduced when plotted as the performance index (n = 5). h, Pauses in locomoter activity are quantified as the percentage time immobile in the presence and absence of TMT, 2-phenylethanol or isoamyl acetate. Immobility is defined as velocity less than 1 cm s−1 for at least 1 s. f–h, **P < 0.01, ***P < 0.001, paired t-test comparing performance index with and without odour for each odour group; error bars, s.e.m.

Extended Data Figure 2 Location of optical fibres implanted in cortical amygdala for photoactivation of halorhodopsin.

Schematics show coronal sections throughout most of the region containing cortical amygdala. The posterolateral cortical amygdala is highlighted in grey and the location of bilaterally implanted fibres is indicated.

Extended Data Figure 3 Locomotor activity of mice with optical suppression in cortical amygdala.

a, b, Mice with halorhodopsin in the olfactory bulb and optical fibres in cortical amygdala were optically coupled to a yellow laser and tested in the behavioural assay for the response to TMT (a) or 2-phenylethanol (b) with and without laser stimulation. The position of a representative mouse during a 10 minute period in the presence of TMT (a) or 2-phenylethanol (b) either in the absence (left) or presence (right) of photoactivation during the 10 minute behavioural testing is shown. Raster plots show quadrant occupancy over time for each animal (a, n = 11; b, n = 6). c, d, The percentage time immobile in the absence and presence of photoactivation. Immobility is defined as velocity less than 1 cm s−1 for at least 1 s. c, Response to TMT in mice receiving photostimulation of halorhodopsin in different experimental animals. Bulb halo and COA halo describe mice with halorhodopsin expression in the olfactory bulb and cortical amygdala, respectively. Optical fibres were placed above cortical amygdala (COA, n = 11), olfactory tubercle (OT, n = 7) or in piriform cortex (Pir, n = 8) as denoted below the site of injection. Control animals received no viral injection, and fibres implanted into cortical amygdala (n = 4). d, The percentage immobility for mice exposed to 2-phenylethanol in the absence and presence of photoactivation of bulbar axons in cortical amygdala (n = 6). c, d, *P < 0.05, ***P < 0.001, paired t-test comparing with and without laser; error bars, s.e.m.

Extended Data Figure 4 Optical suppression of bulbar input to cortical amygdala selectively reduces odour-evoked activity in this region.

Mice were unilaterally injected with AAV5-eNpHR3.0–eYFP into the olfactory bulb and exposed to TMT while photoactivating the mitral-tufted cell axon terminals in cortical amygdala. a, Coronal section of the olfactory bulbs (top) and of the brain region with cortical amygdala (bottom) reveal halorhodopsin expression in the bulb and the lateral olfactory tract. b, Magnified and cropped image showing the cortical amygdala of both sides of the posterolateral cortical amygdala. The region in the bottom image received photoactivation of halorhodopsin in bulbar axon terminals during odour exposure, whereas the contralateral side (top) did not. Scale bars, 200 μm (a) and 100 μm (b). c, The number of neurons expressing c-fos was counted for each side of the brain (n = 4) as well as in control animals that did not receive any stimuli (n = 2) across multiple regions of cortical amygdala. Numbers are normalized by area (square millimetres) of 100 μm thick sections for comparison between brain areas. *P < 0.05, paired t-test comparing with and without laser; error bars, s.e.m. d–f, Coronal sections showing the olfactory tubercle (d), anterior piriform (e) and posterior piriform (f); the region in the bottom images received photoactivation of bulbar axon terminals in the cortical amygdala. Scale bars, 200 μm. Bar graphs show the number of neurons expressing c-fos counted for each side of the brain (n = 4) as well as in control animals that did not receive any stimuli (n = 2). Error bars, s.e.m. a–f, Images are taken from sections at the following anterior–posterior distances from bregma: −1.7 (b), 1.2 (d, e), −1.6 (f).

Extended Data Figure 5 Halorhodopsin expression of neurons within cortical amygdala.

Injection of AAV into cortical amygdala leads to broad expression of halorhodopsin within cortical amygdala and neighbouring areas; however, optical silencing is restricted to the cortical amygdala. a, Schematic of a coronal section showing cortical amygdala (COApl) in relation to other ventral brain regions. b–d, Mice were unilaterally injected with AAV5-eNpHR3.0–eYFP into the cortical amygdala and exposed to TMT while photoactivating halorhodopsin in cortical amygdala neurons. b, Coronal section reveals broad expression of halorhodopsin in the cortical amygdala region. This expression is broad, covering at least 90% of posterolateral cortical amygdala throughout the anterior–posterior axis. c, d, Magnified and cropped image showing c-fos expression in the cortical amygdala. The right side (d) received photoactivation during odour exposure, whereas the contralateral side (c) did not. Scale bar, 200 μm. e, The number of neurons expressing c-fos was counted for each side of the brain (n = 3) across multiple regions of the cortical amygdala (top) as well as the medial amygdala (MEPV, bottom). The mean cell number for each animal is shown in the bar graph. *P < 0.05, paired t-test comparing with and without laser stimulation; error bars, s.e.m.

Extended Data Figure 6 The arc promoter can be used to drive channelrhodopsin expression faithfully in odour-specific neurons.

Arc-Cre-ERT2 mice were administered tamoxifen and exposed to odour to induce ChR2–eYFP expression, and subsequently exposed to either the same odour or a different odour and then sampled for endogenous arc expression. a, b, The timeline for odour exposure is indicated at the top, and images from two different regions of cortical amygdala are shown from representative animals for each experiment. a, TMT exposure induced ChR2–eYFP expression in the cortical amygdala and re-exposure to the same odour-induced arc expression detected by immunocytochemistry. b, 2-Phenylethanol induced expression of ChR2–eYFP followed by re-exposure to TMT. Scale bar, 100 μm. c, The number of channelrhodopsin-expressing neurons that also expressed the endogenous arc protein. Neurons were counted across randomly chosen sections throughout the cortical amygdala (n = 4).

Extended Data Figure 7 Location of optical fibres implanted in cortical amygdala for photoactivation of odour-responsive neurons.

a, Schematics show unilateral coronal sections throughout most of the region containing cortical amygdala. The posterolateral cortical amygdala is highlighted in grey and the location of unilaterally implanted fibres is indicated. Fibres were not preferentially targeted to one side of the brain, but the fibre positions are collapsed onto unilateral schematics. b, The extent of light-induced activation of channelrhodopsin-expressing neurons as a function of distance from the fibre tip using c-fos expression. ArcCreERT2 mice were injected with AAV5-ef1α-DIO-ChR2–eYFP into cortical amygdala, administered tamoxifen and exposed to TMT to induce channelrhodopsin expression. Three weeks later, the cortical amygdala was photoactivated for 10 min with cycles of 30 s of pulsed light (10 Hz, 50% duty cycle) and 30 s off. Mice were sampled for c-fos immunoreactivity as a function of distance from the fibre tip in 200 μm bins (n = 3). P < 0.001, one-way ANOVA; error bars, s.e.m.

Extended Data Figure 8 Locomotor activity of mice during activation of odour-responsive neurons within cortical amygdala.

Mice with odour-driven channelrhodopsin expression were tested in the open field assay where they received pulsed photoactivation upon entrance into the lower right quadrant. a–c, The trajectory graphs (top) show the position of representative animals with ChR2–eYFP in neurons activated by TMT (a), 2-phenylethanol (b) or isoamyl acetate (c). The raster plots (bottom) show quadrant occupancy over time. d, The percentage time immobile in the absence and presence of photoactivation. Immobility is defined as velocity less than 1 cm s−1 for at least 1 s. a–c, TMT (n = 6), 2-phenylethanol (n = 4) and isoamyl acetate (n = 6); ***P < 0.001, paired t-test comparing with and without laser; error bars, s.e.m.

Extended Data Figure 9 The spatial distribution of neurons responsive to different odours within cortical amgydala.

a, ArcCreERT2 mice were administered tamoxifen and exposed to one of five different odours and then consecutive, serial, coronal sections were collected throughout the cortical amygdala region. Images reveal odour-driven ChR2–eYFP expression in serial sections across the cortical amygdala of representative mice for each odour. Images of the ventral brain region are magnified and cropped to show only the posterolateral cortical amygdala, and sections are displayed at 200 μm intervals across 1.2 mm of the anterior–posterior axis. Scale bar, 100 μm. b, Representative images showing the expression of odour-induced ChR2–eYFP with magnified images that reveal identifiable cell bodies for counting. The top image shows a z-projection of 40 μm through cortical amygdala (left), and the bottom images (i–iv) show magnified single z-plane images of small areas revealing neuronal cell bodies, indicated by the white arrows. Scale bar, 60 μm. c, The average number of neurons counted per 100 μm coronal section throughout the posterolateral cortical amygdala for different odours. Error bars, s.e.m. d, A one-way ANOVA was performed for each point along the anterior–posterior axis, comparing the means between different odours (excluding the no-odour condition). The no-odour condition was compared with each odour at −2.3 from bregma using an unpaired t-test; P < 0.001 for each odour.

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Root, C., Denny, C., Hen, R. et al. The participation of cortical amygdala in innate, odour-driven behaviour. Nature 515, 269–273 (2014). https://doi.org/10.1038/nature13897

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