Olfactory bulb recovery after early sensory deprivation

Abstract

Olfactory bulbs retain the ability to acquire new neurons throughout life. Unilateral olfactory deprivation during the first postnatal month in rats results in a dramatic reduction in the size of the experimental olfactory bulb. Part of this reduction is attributable to the death of neurons and glia. To examine the regenerative capacity of the juvenile olfactory bulb, we developed a technique for reversible olfactory deprivation. Reversible blockade from postnatal day 1 (P1) to P20 or P30 results in reduced bulb volume and tyrosine hydroxylase immunostaining, and decreased depth in the olfactory mucosa. In another experiment, normal stimulation was restored for varying periods of time, and experimental and control bulb volumes were measured. Recovery of bulb size occurs after 40 d of normal stimulation. Rats injected with a thymidine analog to label dividing cells during the recovery period revealed that rescue results at least in part from the addition of new neurons and glia. Thus, cells born after the return of normal levels of environmental stimulation can replace some of the neurons and glia that are lost during olfactory deprivation. This system can be used to study mechanisms that underlie neuronal regeneration in the maturing mammalian brain.

Fig. 1.

Diagram indicating how plugs were constructed. (1) A length of suture was threaded through a piece of tubing. (2) A piece of hair (or strands of dental floss) was tied around the thread, and (3) the thread was tied into a knot around the hair. (4) The knot was pulled into the lumen of the tubing, and (5) the ends of thread and hair were trimmed until only 2 mm of hair remained extending from the tubing. The opposite end of tubing was beveled for ease of insertion.

Fig. 2.

Photomicrograph: a Nissl-stained section depicting the layers of the olfactory bulb. Laminar volume measurements of the glomerular (GLM), external plexiform (EPL), and granule cell (GCL) layers of experimental and control bulbs were taken and expressed as percentage differences in the graph. The mitral cell layer (asterisk) was included in the measurement of the GCL.Graph, Filled bars represent data from animals that had a naris permanently closed with cautery from P1 to P30 (Brunjes and Borror, 1983). Open bars represent animals that had a naris reversibly occluded from P1 to P30. NL, Nerve layer. Scale bar, 100 μm.

Fig. 4.

Top, Photomicrographs of sections through the olfactory epithelium on the experimental (E) and control (C) side of the septum. Sections of the mucosa were stained with hematoxylin and eosin. Bottom, Graph of percentage differences in epithelial thickness in animals that had a naris closed from P1 to P30 (filled bar), from P1 to P20 (open bar), or from P1 to P20 and then reopened for 10 d (hatched bar). Scale bar, 20 μm.

Fig. 3.

Photomicrograph of a coronal section through the bulbs of an animal that had a naris reversibly closed from P1 to P30 depicts immunoreactivity (ir) for tyrosine hydroxylase (TH). TH-ir is substantially reduced in the experimental bulb (E) compared with the contralateral control bulb (C). Scale bar, 400 μm.

Fig. 5.

Graph of mean percentage differences of laminar volume measurements between experimental and control bulbs. A baseline group of animals had a naris reversibly closed from P1 to P20 (filled bars). Recovery groups had a naris closed from P1 to P20 and then opened for 10 (open bars), 20 (hatched bars), or 40 (striped bars) d.

Fig. 6.

Photomicrographs depicting BrdU-ir along the ventromedial aspects of the bulbs of an animal that was injected with BrdU on P21, 24 hr after the naris was reopened, and survived for 30 d. Many more BrdU-containing profiles are visible among periglomerular cells of the GLM of the experimental bulb (A) compared with the contralateral control bulb (B). Graph, Mean percentage differences in numbers of BrdU-containing profiles between experimental and control bulbs. Subjects had a naris closed from P1 to P20, were injected with BrdU on P21 or P50, and survived 30 d after the BrdU injections. GLM, Glomerular layer; GCL, granule cell layer. C, Photomicrograph of a section adjacent to the one shown in B that was processed through immunocytochemistry for BrdU (brown) and calretinin (blue). A double-labeled cell (arrow) is visible along with single-labeled cells that were immunoreactive for either BrdU (filled arrowheads) or calretinin (open arrowhead). A process from the double-labeled cell can be seen extending in the direction of a single-labeled BrdU-ir cell. GLM, Glomerular layer; NL, nerve layer. Scale bar (shown inC): A, B, 40 μm; C, 10 μm.