In vivo visualization of olfactory pathophysiology induced by intranasal cadmium instillation in mice

Abstract

Intranasal exposure to cadmium has been related to olfactory dysfunction in humans and to nasal epithelial damage and altered odorant-guided behavior in rodent models. The pathophysiology underlying these deficits has not been fully elucidated. Here we use optical imaging techniques to visualize odorant-evoked neurotransmitter release from the olfactory nerve into the brain's olfactory bulbs in vivo in mice. Intranasal cadmium chloride instillations reduced this sensory activity by up to 91% in a dose-dependent manner. In the olfactory bulbs, afferents from the olfactory epithelium could be quantified by their expression of a genetically encoded fluorescent marker for olfactory marker protein. At the highest dose tested, cadmium exposure reduced the density of these projections by 20%. In a behavioral psychophysical task, mice were trained to sample from an odor port and make a response when they detected an odorant against a background of room air. After intranasal cadmium exposure, mice were unable to detect the target odor. These experiments serve as proof of concept for a new approach to the study of the neural effects of inhaled toxicants. The use of in vivo functional imaging of the neuronal populations exposed to the toxicant permits the direct observation of primary pathophysiology. In this study optical imaging revealed significant reductions in odorant-evoked release from the olfactory nerve at a cadmium chloride dose two orders of magnitude less than that required to induce morphological changes in the nerve in the same animals, demonstrating that it is a more sensitive technique for assessing the consequences of intranasal neurotoxicant exposure. This approach is potentially useful in exploring the effects of any putative neurotoxicant that can be delivered intranasally.

Figure 1. Intranasal cadmium instillation reduces neurotransmitter release from the olfactory nerve into olfactory bulb glomeruli in a dose dependent manner

(Upper) Baseline fluorescence images of the dorsal olfactory bulbs, viewed through a cranial window in vivo. (Lower) Pseudocolored response maps showing the increase in fluorescence during odorant presentation relative to the pre-odorant baseline. Each spot corresponds to a single olfactory bulb glomerulus. Callouts indicate individual traces showing the change in fluorescence in the corresponding glomerulus during the baseline and odorant presentation part of each trial.

Figure 2. Intranasal cadmium instillation reduces the magnitude of odorant-evoked synaptic input to the olfactory bulb glomeruli and the number of glomeruli receiving sensory input

(A) Ratio of the number of glomeruli receiving synaptic input from the olfactory nerve in the olfactory bulb on the cadmium-exposed side to the corresponding number on the vehicle-exposed side, shown as a function of cadmium chloride dose. The dashed line at 1 denotes no difference between the olfactory bulbs. (B) Frequency-distribution of glomerular response amplitudes in vehicle-exposed (above the x-axis) and cadmium-exposed (below the x-axis) olfactory bulbs. Data are shown for the 20µg (top), 2 µg (middle), and 0.2 µg (bottom) cadmium chloride doses. (C) Plot of the Kolmogorov-Smirnov z-scores for the paired distributions shown in part B for each cadmium dose. * denotes p < 0.05, ** denotes p < 0.001.

Figure 3. Axonal projection density from the olfactory epithelium to the olfactory bulb is moderately reduced at the highest cadmium dose

(A) Representative sections of cadmium- and vehicle-exposed olfactory bulbs across decreasing cadmium chloride doses. The DAPI nuclear stain marks rings of periglomerular interneurons, while the GFP is selectively expressed in the afferents from the olfactory epithelium into olfactory bulb glomeruli. (B) The ratio of GFP optical density in glomeruli from cadmium-exposed olfactory bulbs to that from vehicle-exposed olfactory bulbs, plotted relative to cadmium chloride dose. Dashed line at 1 represents no difference in GFP expression between bulbs. ** denotes p < 0.01.

Figure 4. Performance on an olfactory detection task approaches zero in mice receiving intranasal cadmium instillations

(A) Performance cadmium- and vehicle-exposed mice on an odorant detection task before and after cadmium chloride instillations. (B) Average number of odorant detection trials initiated during the test session two days after instillations.