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, 35 (20), 7892-902

Integrating Temperature With Odor Processing in the Olfactory Bulb

Affiliations

Integrating Temperature With Odor Processing in the Olfactory Bulb

Eugen Kludt et al. J Neurosci.

Abstract

Temperature perception has long been classified as a somesthetic function solely. However, in recent years several studies brought evidence that temperature perception also takes place in the olfactory system of rodents. Temperature has been described as an effective stimulus for sensory neurons of the Grueneberg ganglion located at the entrance of the nose. Here, we investigate whether a neuronal trace of temperature stimulation can be observed in the glomeruli and mitral cells of the olfactory bulb, using calcium imaging and fast line-scanning microscopy. We show in the Xenopus tadpole system that the γ-glomerulus, which receives input from olfactory neurons, is highly sensitive to temperature drops at the olfactory epithelium. We observed that thermo-induced activity in the γ-glomerulus is conveyed to the mitral cells innervating this specific neuropil. Surprisingly, a substantial number of thermosensitive mitral cells were also chemosensitive. Moreover, we report another unique feature of the γ-glomerulus: it receives ipsilateral and contralateral afferents. The latter fibers pass through the contralateral bulb, cross the anterior commissure, and then run to the ipsilateral olfactory bulb, where they target the γ-glomerulus. Temperature drops at the contralateral olfactory epithelium also induced responses in the γ-glomerulus and in mitral cells. Temperature thus appears to be a relevant physiological input to the Xenopus olfactory system. Each olfactory bulb integrates and codes temperature signals originating from receptor neurons of the ipsilateral and contralateral nasal cavities. Finally, temperature and chemical information is processed in shared cellular networks.

Keywords: activity correlation imaging; chemosensitivity; integration; mitral cells; olfactory bulb; thermosensitivity.

Figures

Figure 1.
Figure 1.
Responses of the γ-glomerulus upon stimulation. A, Overview of the Xenopus olfactory bulb with the different layers and cell types indicated. The positions of the lateral, medial, and small glomerular clusters are labeled. The γ-glomerulus is situated close to the posterior and ventral border of the glomerular layer within the small cluster and is marked in blue. The top right side shows an image of a tadpole. The red rectangle indicates the area used for sample preparation. Scale bar, 2 mm. On the bottom right side, a scheme of the experiment including the glomerular afferents of the right ventral olfactory bulb is provided. Olfactory sensory neurons electroporated with Fluo-4 dextran are shown in green. B, Raw Fluo-4 fluorescence signal before stimulation. C, Top, Responses of primary olfactory projections in the ventral olfactory bulb upon stimulation with different temperature changes (I–IV) or a mixture of amino acids (VI). The images show the spatial map of the ΔF/F. Bottom, Time courses of responses of the γ-glomerulus (Glγ) and glomeruli in the lateral cluster (lat), along with the underlying temperature stimuli (ΔT), plotted as deviations from ambient temperature. D, Summary of normalized temperature response curves of the γ-glomerulus at two ambient temperatures of 22–24°C (green, n = 17 animals) and 18–20°C (blue, n = 9 animals). The points show the responses of individual animals, and the rectangles indicate the average responses with SD. The sigmoid curves represent the average fit for each ambient temperature. E, Representative response of the γ-glomerulus upon prolonged stimulation (temperature step, ΔT = −3.5°C). Dashed line, exponential fit. F, Odorant responses of the glomeruli in the lateral cluster as a function of temperature (n = 5 animals). γ, Glγ, γ-glomerulus; ob, olfactory bulb; oe, olfactory epithelium; on, olfactory nerve; lat, lateral cluster; C, caudal; L, lateral; M, medial; R, rostral. Scale bar, 20 μm.
Figure 2.
Figure 2.
Temperature sensitivity and chemosensitivity in the olfactory bulb. A, Temperature response of the postsynaptic neuropil of a γ-glomerulus and two mitral cells. Dashed curves, Boltzmann fit. B, Integration of temperature and odor sensitivity in individual mitral cells. Left, Maximum projection of a 30-μm-thick volume of the olfactory bulb stained with Fluo-4 AM. Arrowheads highlight six mitral cells. Scale bar, 50 μm. Right, Ca2+ responses of the six mitral cells to a mixture of amino acids, a temperature drop (ΔT = −1.1°C), or the control with ambient temperature Ringer's solution. The red and blue bars under the traces indicate the application of amino acids (100 μm) and cold Ringer's solution (T = 0°C), respectively. C, Response patterns of the small glomerular cluster and surrounding mitral cells to temperature and chemical stimulation. Presynaptic Alexa Fluor staining is shown in green (arrow, γ-glomerulus; I). Postsynaptic Fluo-8 AM staining was performed through bolus loading. Stimulus sequence: cold Ringer's solution, cold Ringer's solution, l-histidine, cold Ringer's solution. II, Activity correlation map for the postsynaptic Ca2+ responses. Red, correlation to a pure His response; cyan, correlation to pure temperature drop responses (reference traces as inset). III, Regions responding to both stimuli (yellow). All images are maximum projections of the same 36-μm-thick volume. Scale bar, 20 μm. D, ΔF/F traces representing different mitral cells from the measurement in C. The relative response strength ranges from pure thermosensitivity to exclusive histidine sensitivity over various degrees of dual sensitivity. The bars under the traces depict the stimulus application in the same manner as in B.
Figure 3.
Figure 3.
Bilateral projections of OSN axons. Maximum intensity projection along the dorsoventral axis of a 128-μm-thick volume within the ventral olfactory bulb. A–C, Primary olfactory projections (A) from the left (green, B) and right (red, C) olfactory epithelium, overlaid with a widefield image of the same preparation (gray). ac, Anterior commissure; e, extrabulbar olfactory fibers; on, olfactory nerve; med, medial cluster; int, intermediate cluster; lat, lateral cluster; C, caudal; L, lateral; M, medial; R, rostral. Scale bar, 100 μm. D–F, Innervation of the γ-glomerulus by contralateral OSN axons. D, The imaged volume is presented in three maximum intensity projections (dorsoventral, I; mediolateral, II; rostrocaudal, III). E, OSN axons emerging from the left, ipsilateral olfactory epithelium are presented in green; and OSN axons from the right, contralateral olfactory epithelium in red. F, Overlay of the fluorescence images in D and E. β, β-Glomeruli; γ, γ-glomeruli; δ, δ-glomeruli; e, extrabulbar olfactory fibers; med, medial cluster; int, intermediate cluster; lat, lateral cluster; C, caudal; L, lateral; M, medial; R, rostral; V, ventral; D, dorsal. Scale bar, 50 μm.
Figure 4.
Figure 4.
Temperature sensitivity of the contralateral OSN glomerular afferents. A, Primary ipsilateral olfactory projections of the medioventral olfactory bulb, traced with Alexa Fluor 568. B, Peak response (ΔF/F) of a single stimulation (ΔT = −2.5°C). C, Pixel correlation map of a single response upon a stimulation (ΔT = −2.5°C) at the contralateral olfactory epithelium. D, Overlay of A and C. The ROI used to compute response traces plotted in EG is marked by a dotted line. E–G, Temperature responses (ΔF/F) obtained from the γ-glomerulus encircled in D. Applied stimuli: E, ΔT = −2.2 ± 0.5°C; F, ΔT = −0.2°C; G, ΔT= +2.3°C. The black bar under the trace shows the length of the stimulus application. Scale bar, 50 μm.
Figure 5.
Figure 5.
Postsynaptic response of a γ-glomerulus upon temperature stimulation in the olfactory epithelium. A, Maximum intensity projections (dorsoventral, I; mediolateral, II; rostrocaudal, III) of the ventral olfactory bulb after bolus injection of Fluo-4 AM (green). OSN axons originating from the contralateral olfactory epithelium (red, Alexa Fluor 568) enter the imaged volume at the ventrocaudal rim, protrude rostrodorsally (II) and form a glomerular tuft in the region of the γ-glomerulus. B, Stimulation (ΔT = −1.3°C) at the ipsilateral olfactory epithelium. C, The contralaterally projecting fibers indicate the region of the γ-glomerulus. D, ΔF/F peak response of postsynaptic elements upon cold stimulus occurs in the same region. Arrow, Neuropil; arrowheads, mitral cells. E–G, Stimulation (ΔT = −3.3°C) at the contralateral olfactory epithelium of the same brain preparation. The ipsilateral nerve was cut (E). F, The contralaterally projecting fibers indicate the region of the γ-glomerulus. G, ΔF/F peak response of postsynaptic elements upon cold stimulus occurs in the same region. Arrow, Neuropil; arrowhead, mitral cell; γ, γ-glomerulus. Scale bars, 20 μm.

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