Visualizing odor representation in the brain: a review of imaging techniques for the mapping of sensory activity in the olfactory glomeruli

Abstract

The brain transforms clues from the external world, the sensory stimuli, into activities in neuroglial networks. These circuits are activated in specialized sensory cortices where specific functional modules are responsible for the spatiotemporal coding of the stimulus. A major challenge in the neuroscience field has been to image the spatial distribution and follow the temporal dynamics of the activation of such large populations in vivo. Functional imaging techniques developed in the last 30 years have enabled researchers to solve this critical issue, and are reviewed here. These techniques utilize sources of contrast of radioisotopic, magnetic and optical origins and exploit two major families of signals to image sensory activity: the first class uses sources linked to cellular energy metabolism and hemodynamics, while the second involves exogenous indicators of neuronal activity. The whole panel of imaging techniques has fostered the functional exploration of the olfactory bulb which is one of the most studied sensory structures. We summarize the major results obtained using these techniques that describe the spatial and temporal activity patterns in the olfactory glomeruli, the first relay of olfactory information processing in the main olfactory bulb. We conclude this review by describing promising technical developments in optical imaging and future directions in the study of olfactory spatiotemporal coding.

Fig. 1

The olfactory bulb, first structure for olfactory information processing in the brain. a Top: Sagittal view of a rodent head showing in green the location of the MOE at the very end of the nasal cavity and the MOB at the anterior position in the brain. Bottom: OSNs, primary receptor cells located in the MOE, which express the same odorant receptor, converge onto the same glomeruli in the MOB. b Olfactory glomeruli, the round-shaped neuropils located at the surface of the olfactory bulb, about 100 μm deep just under the olfactory nerve layer (ONL), are constituted by incoming convergent OSN axon fibers. In each glomerulus, projections of OSNs ramify and constitute synapses (purple arrows) with the apical dendritic tufts of principal excitatory cells (M/TCs) and inhibitory interneurons (periglomerular cells, PGCs). M/TCs and PGCs interact via dendrodendritic synapses: PGC dendrites project inhibitory GABAergic synapses onto M/TC dendrites (lower black circle), whereas M/TC dendrites make glutamatergic contacts onto PGC dendrites (purple arrow). PGCs also make GABAergic synapses onto OSN axonal terminals (upper black circle). This interneuronal population includes short axon cells which could allow lateral interactions between distant glomeruli (black arrow to the left). Numerous astrocytic projections are also part of this neuropil that they compartmentalize: they enwrap both synapses and blood vessels (green star-like branches) cleaning up glutamate and regulating local hemodynamics. Note that a very dense and complex vascular network is present at the glomerular level (arteriole, capillaries, venule). The dendrodendritic interactions in the external plexiform layer (EPL) allowing lateral inhibition between M/TCs are due to the activation of local inhibitory interneurons, the granule cells (GCs). Each M/TC is specific to one glomerulus and sends information via the lateral olfactory tract (LOT) mainly to the anterior piriform cortex and to the limbic system without a thalamic relay. These structures send back centrifugal fibers (CF) to the MOB for feedback regulation. c Coronal section of a mouse MOB (top dorsal, bottom ventral, left medial, right lateral). Inset lower magnification of image d. Scale bar 500 μm. d Anatomical view of MOB layers. Note the olfactory glomeruli aligned close to the MOB surface and the very thin layer of M/TCs. Scale bar 100 μm. GL glomerular layer, GCL granule cell layer, MCL mitral cell layer, ON olfactory nerve. (a Adapted from reference [1] with permission from Macmillan Publishers; copyright 2004)

Fig. 2

Mapping of 2DG uptake during sustained MOB activation. Top left: Normalized z-score flattened patterns of the whole bulb in a ventral-centered representation for three odorants in groups of 3-week-old rats (n = 10). Top right: Topographic explanation of the flattened map (details of the method in reference [23]). Bottom: Reconstructed relative glucose uptake maps. GL/SEZ glomerular layer/subependymal zone (area with no specific uptake). (Adapted from reference [30] with permission from Oxford University Press)

Fig. 3

Mapping of MOB activation using BOLD-fMRI. Left: Coronal BOLD image through a rat MOB. Significant odor-activated zones are represented in red. Right: Time-course of odor-induced BOLD signal changes showing delayed activation of the ventral region. (Reprinted from reference [42] with permission from Elsevier; copyright 2007))

Fig. 4

Wide-field optical imaging set-up for mapping odor-evoked activities in rodents. 1 Anesthetized animal with either full craniotomy or thinned bone above the MOB. Optical window is made of dental cement walls, agarose well and a microscope cover slip. 2 Wide-field microscopy optics with C-mount stereomicroscope or dual lens macroscope. 3 Cooled CCD camera, typically of 12-bit dynamics, with a frame rate of ten images per second or more at full frame. 4 Stabilized white light source with associated filter wheel. Light is shone onto the exposed brain tissue using a goose-neck optical light guide, using an annular fiber ring attached to the optics lens A, or through the epi-illumination port of the microscope B. Green light is used to visualize the blood vessel architecture. Bandpass interference filters are used to select the light wavelength for reflectance imaging or excitation of VSD or calcium dyes. For fluorescence studies the emission wavelength is selected using bandpass or long-pass filters placed in the detection path 5. Odors at a given concentration are deposited onto a filter paper and put into sealed vials (here only one odored paper is shown in the left vial in the figure). An olfactometer allows olfactory stimulation control and poststimulation air clean-up. In particular, the use of an air compressor with a manometer and computer-controlled electrovanes allows the mixing of several pure odorants if needed and stimulation for a given intensity and duration. 6 A computer allows the synchronization of illumination, olfactory stimulation and image acquisition. Activation maps are generally processed after acquisition but can also be displayed in video rate mode

Fig. 5

VSD imaging resolves glomerular activation with high temporal and spatial resolution. a Blood vessel pattern visualized in VSD-loaded dorsal MOB tissues illuminated at 540 nm. b VSD map evoked by presentation of 1% methylbenzoate. Frames were averaged during the period of the shaded square shown in e. The grayscale (clipping range) 0.08–0.18% is inverted for comparison with the intrinsic map shown in d. c Blood vessel pattern before intrinsic imaging. d IOS map evoked by the same odor. Frames were averaged during the period of the shaded square shown in e, grayscale 0.12–0%. Maps obtained using the two techniques are very similar although some of the caudal glomeruli did not appear in the VSD map presumably due to poor dye staining. e Reflectance and fluorescence changes plotted as a function of time for the red and the blue regions shown in inset and in b and d. The VSD signal is significantly faster and locked to inspirations after odor application. IOS peaks after a long delay and does not resolve the respiration-coupled activity. The decline in the VSD signal parallels the beginning of IOS during odor stimulation and may partly reflect contamination of the dye signal by intrinsic optical changes. The trace labeled “Dye-Pharma” shows the time-course for the region with the biggest remaining response after local application of the glutamate receptor antagonists (average of eight repetitions). (Reprinted from reference [72] with permission from Elsevier, copyright 2002)

Fig. 6

Comparison of calcium fluorescence maps at the presynaptic level and IOS maps acquired sequentially in the same mice. Left: Time-course and amplitude for each optical signal in a region of interest activated under odorant presentation. Right: Activation maps obtained sequentially in the same mice using calcium and intrinsic imaging for increasing stimulus intensities. (Adapted from reference [80], used with permission, copyright 2003))

Fig. 7

Presynaptic imaging of SpH for olfactory mapping. a, b Confocal microscopy images of the surface of MOB slices for comparison of fluorescence resulting from anterograde calcium labeling and SpH. Labeling of axon bundles in the ONL and GL are seen with Alexa Fluor dextran, whereas only glomeruli are labeled in SpH mice. For both labelings, no fluorescence is seen in the periglomerular area. c Comparison of the amplitude and time-course of calcium (high-affinity rhod tracer) and SpH signals recorded alternately in the same glomerulus. Both traces are from a single trial. The onsets of the signals are the same following stimulation, but the rise time of the rhod signal is much shorter than that of SpH (amplitude bar ∆F/F: SpH 1%, rhod 0.2%). d Resting fluorescence in SpH mice. e Activation maps induced by a 2-s presentation of 1% hexanone shows bilaterally symmetrical maps. (a–c Adapted from reference [88] used with permission; d, e reprinted from reference [87] with permission from Elsevier, copyright 2003)

Fig. 8

Odorant-evoked GCaMP2 calcium responses in the MOB. a Left: Resting fluorescence image of the MOB surface observed through the thinned skull of a GCaMP2 mouse. Right: A single-trial odor response map induced by butyraldehyde (0.25% of saturated vapor). Arrows indicate the locations where the odor-evoked response traces shown in b were obtained. b Top: Raw fluorescence traces without photobleaching correction. The red and gray traces are the GCaMP2 signals measured, respectively, in odor-activated (trace 2) and nonactivated regions (trace 1) as indicated in a. The black horizontal bar indicates a 2-s odor delivery. Bottom: Same odor responses with photobleaching subtraction using a no-odor imaging trial. c Comparison of the averaged ΔF/F response spatial profile in odor-evoked hot spots in the GCaMP2 mice (blue) and from SpH-labeled olfactory glomeruli in the OMP-SpH mice (red). d Quantitative comparison of the mean widths measured at half-maximum in the GCaMP2 and OMP-SpH mice. e Odor-evoked GCaMP2 response maps are bilaterally symmetrical (adapted from reference [98], used with permission)