Importance of newly generated neurons in the adult olfactory bulb for odor discrimination

Abstract

In adult rodents, neurons are continually generated in the subventricular zone of the forebrain, from where they migrate tangentially toward the olfactory bulb, the only known target for these neuronal precursors. Within the main olfactory bulb, they ascend radially into the granule and periglomerular cell layers, where they differentiate mainly into local interneurons. The functional consequences of this permanent generation and integration of new neurons into existing circuits are unknown. To address this question, we used neural cell adhesion molecule-deficient mice that have documented deficits in the migration of olfactory-bulb neuron precursors, leading to about 40% size reduction of this structure. Our anatomical study reveals that this reduction is restricted to the granule cell layer, a structure that contains exclusively gamma-aminobutyric acid (GABA)ergic interneurons. Furthermore, mutant mice were subjected to experiments designed to examine the behavioral consequences of such anatomical alteration. We found that the specific reduction in the newly generated interneuron population resulted in an impairment of discrimination between odors. In contrast, both the detection thresholds for odors and short-term olfactory memory were unaltered, demonstrating that a critical number of bulbar granule cells is crucial only for odor discrimination but not for general olfactory functions.

Figure 1

Histologic (Nissl) appearance of normal and mutant main olfactory bulbs in adult mice. Comparison of 40-μm thick Vibratome coronal (A) and sagittal (B) sections of olfactory bulbs from wild-type (NCAM^+/+) and knockout (NCAM^−/−) mice, showing the decreased size of the granule cell layer in the mutants. Arrows indicate the mitral cell layer. (C) Details of cresyl violet-stained granule cell layer showing the presence of striated aggregates of cells only in control animals (arrows). (Scale bars = 150 μm in A and B and 200 μm in C.)

Figure 2

Proliferation in the olfactory bulb. BrdUrd immunohistochemistry on sagittal sections revealed the presence of dividing cells in the granule cell layer (gcl) (a–d) and the glomerular layer (e and f) of the olfactory bulb. Labeled cells in the granule cell layer (arrows) were more or less evenly distributed in mutant (a) and control (b). Double labeling and confocal microscopy demonstrated that BrdUrd-positive (red) cells were in all cases embedded in structures expressing GAD67 (green), a marker for GABAergic neurons. In the glomerular layer, which shows the most intense labeling for GAD67, newly generated cells (arrows) were located around glomeruli (gl), suggesting that they represent interneurons of the periglomerular type. (Scale bars = 25 μm in a and b, 10 μm in c and d, and 20 μm in e and f.)

Figure 3

Performance in a habituation task in wild-type and NCAM-deficient mice. Values represent the mean time (±SEM) that wild-type (NCAM^+/+, n = 10) and transgenic (NCAM^−/−, n = 13) mice spent investigating the side of a glass plate scented with an odor (black columns) diluted at 10⁻⁴ (wt/wt) and the other side containing sterilized water (white columns).

Figure 4

Performance in a discrimination task in wild-type and NCAM-deficient mice. Time (in sec) spent investigating a familiar odor (fifth trial) before the novel one (sixth trial) by individual animals in wild-type (filled circles, n = 10) and NCAM-mutant (open circles, n = 13) mice.

Figure 5

Detection threshold in wild-type and NCAM-deficient mice. Normalized values are expressed as the mean ratio (±SEM) between the time spent investigating the odor and the total sniffing time (i.e., odor + water). NCAM-deficient (n = 13) and wild-type (n = 13) mice submitted to a four-step, descending-concentration series (10⁻⁴, 10⁻⁵, 10⁻⁶, and 10⁻⁸) of a given odor (paprika) versus water. Note that only odor at 10⁻⁴ is discriminated from water for both groups.

Figure 6

Performance in a short-term memory task in wild-type and NCAM-deficient mice. Effect of different intertrial intervals on odor recognition in NCAM-deficient (n = 10) and wild-type (n = 10) mice. Each bar represents the mean percentage (±SEM) of time investigating a given odor on the second exposure compared with the time spent investigating the same odor during the first presentation.