Critical period for sensory experience-dependent survival of newly generated granule cells in the adult mouse olfactory bulb

Abstract

Granule cells in the olfactory bulb (OB) are continually produced and added into the neuronal circuit in the adult brain. Sensory input to the OB plays a key role in the survival of newly generated granule cells. Here, we examined in the adult mice whether there is a time window after the generation of new granule cells when their survival is strongly influenced by sensory input. New granule cells were labeled by BrdUrd injection, and the mice were deprived of sensory input unilaterally by naris cauterization. During the initial 14 days after BrdUrd labeling, the number of BrdUrd-positive granule cells was similar for deprived and undeprived OBs. At 28 days or later, the BrdUrd-positive cell number remarkably decreased in the deprived OB. Cauterization at days 14-28 effectively reduced the number of BrdUrd-positive granule cells, whereas 2-week cauterization before or after this period produced little effect. Administration of diazepam, a GABAA receptor modulator, decreased the number of BrdUrd-positive granule cells. The diazepam administration was most effective at days 14-28. Histochemical examination showed that activation of caspase-3 was accompanied by apoptotic cell death of granule cells that was induced by sensory deprivation or diazepam administration. Double labeling with activated caspase-3 and BrdUrd indicated that granule cells at days 14-20 were most susceptible to cell death. These results indicate that there is a critical period when the survival of new granule cells is determined in a sensory experience-dependent manner and that the pharmacological manipulation can mimic the effect of sensory deprivation.

Fig. 1.

Olfactory sensory deprivation reduces the number of BrdUrd-labeled cells. (A) Shown is the timetable for sensory deprivation. Newly generated cells were labeled by i.p. BrdUrd injection at day 0. On the same day, one nostril was cauterized. At days 7–56 after the injection, mice were perfusion-fixed and analyzed. (B) Coronal sections through the OBs were examined at day 28. (Upper) BrdUrd-labeled cells in the OB. (Lower) The layer organization using anti-NeuN antibody staining (green). BrdUrd-labeled cells (red) in the GCL, IPL, and MCL were counted. (Scale bar: 100 μm.) (C) The number of BrdUrd-labeled cells in the OB at various periods is shown. ▴, control OB; ○, undeprived OB of animals that received unilateral sensory deprivation; •, sensory-deprived OB. Values indicate mean ± SD from six OBs for control, three OBs for undeprived side, and three OBs for deprived side. Statistical analysis was conducted by paired or unpaired Student's t test (**, P < 0.01).

Fig. 2.

A critical period for the sensory experience-dependent survival of new granule cells. (A) A diagram for the protocol of sensory deprivation. Newly generated cells were labeled by BrdUrd injection at day 0. One nostril was cauterized at various periods after the BrdUrd labeling. (B and C) The effect of sensory deprivation for 28 (B) and 14 (C) days. Hatched columns show the number of labeled cells in the undeprived OB, and filled columns show the number of labeled cells in the deprived OB. Values indicate mean ± SD from three OBs. Statistical analysis was conducted by paired Student's t test (**, P < 0.01).

Fig. 3.

Diazepam administration mimics the effect of sensory deprivation on the survival of new granule cells. (A) A diagram for the protocol of diazepam administration. Newly generated cells were labeled by BrdUrd at day 0. Diazepam was i.p. administered once per day for 14 consecutive days at various periods after the BrdUrd labeling. (B) The effect of diazepam on the survival of new granule cells. Hatched columns indicate the number of BrdUrd-labeled cells of vehicle-administered animals. Filled columns show the number of BrdUrd-labeled cells of diazepam-administered animals. Values indicate mean ± SD from six OBs. (C) Simultaneous manipulation of sensory deprivation and diazepam administration during days 14–28 or days 42–56. Hatched and filled columns represent undeprived and deprived OBs, respectively. Veh, vehicle-injected animals; DZ, diazepam-injected animals. Values indicate mean ± SD from four OBs. Statistical analysis was conducted by paired or unpaired Student's t test (**, P < 0.01).

Fig. 4.

Caspase-3 activation and apoptotic cell death in granule cells induced by sensory deprivation and diazepam administration. Immunoreactivity for activated caspase-3 is shown in green (A–E) or red (F and H). (A–C) Coronal section of the control OB (A), OB after 28 days of sensory deprivation (B), and OB after 8 h of diazepam administration (C). (D and E) Morphology of dendrites of caspase-3-activated granule cells in the OB after 28 days of sensory deprivation (D) and after 8 h of diazepam administration (E). Some radially elongating dendrites extended into EPL and made branches in the layer. The layer structure was shown by fluorescent Nissl staining (blue). Arrows indicate EPL. The pictures are the stack of confocal images within 15-μm thickness. (F and G) Confocal images of double labeling for activated caspase-3 (F) and TUNEL (G) after 28 days of sensory deprivation. (H) Merged view of F and G. (Scale bars: 100 μm, A–C; 50 μm, D and E; and 20 μm, F.)

Fig. 5.

Determination of the age of caspase-3-activated cells. (A) A diagram showing the experimental procedure. Newly generated cells were labeled by BrdUrd injection for 7 consecutive days. The last day of BrdUrd injection was defined as day 0. At either day 7, 14, 21, or 28, diazepam was administered, and the animals were perfusion-fixed 8 h after the diazepam administration. (B and C) Confocal images of a cell that was positive for both activated caspase-3 (B) and BrdUrd (C) in the animal that received diazepam administration at day 14. (D) Merged view of B and C. (E) Morphology of caspase-3-activated cells at days 14–20. An OB slice was obtained from an animal that received diazepam administration at day 14. Activated caspase-3 is shown in green, BrdUrd in red, and fluorescent Nissl staining in blue. Cells indicated by arrowheads are positive for both activated caspase-3 and BrdUrd. The picture is the stack of confocal images within 15-μm thickness. The magnified views of single confocal images of cell somata are indicated. (Scale bars: 20 μm, B;50 μm, E.) (F) The age of caspase-3-activated cells determined by the codetection of BrdUrd. Each column indicates the number of double-positive cells for activated caspase-3 and BrdUrd in the GCL, IPL, and MCL of the OB. Open columns, untreated OBs; filled columns, OBs 8 h after diazepam administration; hatched columns, subtracted values between filled columns and open columns showing the net increase caused by diazepam administration. Values indicate mean ± SD from six OBs.