Myelination of the developing lateral olfactory tract and anterior commissure.

Both the lateral olfactory tract (LOT) and anterior limb of the anterior commissure (AC) carry olfactory information. The LOT forms the projection from the olfactory bulb to the ipsilateral olfactory cortices, while the AC carries odor information across the midline to the contralateral olfactory cortex and bulb. The LOT and AC differ on a number of dimensions, including early development and functional onset. The present work, examining their myelination in mice, reveals additional important differences. For example, the LOT initiates myelination 3-4 days earlier than the AC, evidenced by both an earlier increase in myelin basic protein staining seen with immunohistochemistry and an earlier appearance of myelinated fibers using electron microscopy. While both exhibit a period of rapid myelination, it occurs 4-5 days earlier in the LOT than the AC. The tracts also respond differently to early sensory restriction. Unilateral naris occlusion from the day after birth to postnatal day 30 had no consistent effects on the AC but resulted in significantly thinner myelin sheaths relative to axon caliber in the LOT. Finally, the two tracts differ structurally (the LOT contains larger, more densely packed axons with significantly thicker myelin sheaths resulting in a conduction velocity that is more than twice as fast as the AC). The findings indicate that these two large, accessible tracts provide an important means for studying brain maturation due to basic differences in both the timing of their maturation and general organization.

Unilateral odor deprivation: early postnatal changes in olfactory bulb cell density and number.

Surgical closure of an external naris of the rat from postnatal day 1 to day 30 results in a 25% decrease in the size of the ipsilateral olfactory bulb. Decreases in size must result from changes in either the number of neurons and/or glia, or their size or both. The present study was designed to quantify cell density (Nv) and number at various early postnatal ages in order to examine both normal patterns of maturation and sequences of change resulting from deprivation. Data from control subjects indicated that numbers of mitral cells remained constant while numbers of external tufted cells increased. Both relay cell populations exhibited increases in nuclear size suggestive of substantial postnatal differentiation. All interneuronal and glial populations increased in number, although differences in maturational patterns were observed between cell species. For example, light and dark subpopulations of granule cells differed in the timing of peak cellular density, and an inside-to-outside gradient of maturation was found for dark granule cells. Growth curves were generally similar in occluded and control pups until approximately day 20, when deprivation resulted in decreased number and nuclear area of external tufted cells and density and number of granule cells and their associated glia. Light granule cells were affected earlier than dark cells, perhaps because of their earlier arrival in the granule cell layer. The affected cell groups represent the last relay and interneuronal populations to be generated, perhaps explaining their particular susceptibility to the effects of experience. Most of the changes emerged late, thus suggesting that they represent the culmination of a series of experience-induced changes within the maturing bulb. The observed effects may result from either altered cellular proliferation or death patterns (or both), alternatives now under investigation.

Development of olfactory glomeruli: temporal and spatial interactions between olfactory receptor axons and mitral cells in opossums and rats.

Mitral cells are the primary output neurons of the vertebrate olfactory bulb and are major recipients of sensory input from the periphery. The morphogenesis of mitral cell dendrites was followed to elucidate their early spatial and temporal interactions with olfactory receptor neurons and glia during the construction of olfactory glomeruli. Monodelphis domestica, a marsupial born at an extremely immature stage, and rats were examined. Mitral cells were retrogradely labeled by application of the lipophilic dye 1,1' dihexadecyl-3,3,3'3'-tetramethylin-docarbocyanine perchlorate (DiI) to the lateral olfactory tract. In double-labeling experiments, olfactory receptor neurons were stained with 3,3' dihexadecyloxacarbocyanine perchlorate (DiO), or olfactory nerve Schwann cells were visualized using S-100 protein immunohistochemistry. Tissue was examined with a confocal laser scanning microscope. Some preparations were subsequently investigated with an electron microscope. In Monodelphis, differentiation of mitral cells starts with an outgrowth of numerous, uniform, and widespread dendrites. As soon as terminals of olfactory receptor axons coalesce into glomerular knots within the presumptive glomerular layer, dendrites of individual mitral cells innervate several adjacent glomeruli where they receive sensory synaptic input. With maturation, supernumerary mitral cell dendrites retract, leaving one primary dendrite bearing a terminal glomerular tuft. Simultaneously, secondary dendrites begin to arise. The formation of glomeruli begins earlier and progresses faster in the rat compared to Monodelphis. Nevertheless, mitral cell differentiation in both species follows a common sequence: overproduction of dendrites, selection of usually one primary apical dendrite, and elimination of supernumerary processes. Since olfactory receptor neurons form synaptic contacts with the widespread mitral cell dendrites, considerable synaptic rearrangement must occur within the olfactory glomeruli during maturation.

Organization of the olfactory system in the adult zebrafish: histological, immunohistochemical, and quantitative analysis.

The zebrafish, Danio rerio, is becoming an important model system for developmental studies. We have used a variety of histological techniques to characterize the adult structure of the olfactory system in this teleost to form a base for future developmental work. The olfactory epithelium in this fish contains ciliated and microvillar sensory neurons, microvillar supporting cells, secretory goblet cells, and basal cells, and the adjacent nonsensory epithelium contains ciliated supporting cells. The olfactory bulb is a diffusely organized structure with four laminae: olfactory nerve, glomerular, mixed mitral cell/plexiform, and granule cell layers. These structures and the synapses observed in the olfactory bulb are typical of what is found in other vertebrates. We also examined the distribution of several neurotransmitter markers (tyrosine hydroxylase, neuropeptide Y, dopamine-beta-hydroxylase, and serotonin) in the olfactory bulb. Antibodies to neuropeptide Y, dopamine-beta-hydroxylase, and serotonin labeled fibers in the olfactory bulb and cell bodies in caudal regions of the brain in distributions comparable to other species. Tyrosine hydroxylase immunoreactivity was observed in a set of intrinsic bulb neurons with extensive processes in the glomerular layer. In addition, the structural proteins glial fibrillary acidic protein and vimentin have distributions similar to those in the olfactory bulbs of other animals. Thus, the adult olfactory structures are analogous to the structures in other vertebrate animals in morphology and chemical neuroanatomy. This similarity, along with its numerous advantages for developmental studies, makes the zebrafish a good model for studies of olfaction and forebrain maturation.

Cell death in the developing and sensory-deprived rat olfactory bulb.

Cell death is ubiquitous in the developing brain and an important regulator of cell number. The olfactory bulb, the first central relay for information from the nose, is a particularly appropriate region for studying cell death. The bulb is constantly infused with new cells, has a strictly organized anatomy, and cell survival is known to depend on levels of afferent activation. The present study examined patterns of cell death in both the normally developing and sensory-deprived rat olfactory bulb terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL). In control pups, TUNEL-labeled profiles were high at postnatal day 5 (P5, day of birth = P0), but then decreased rapidly to constant levels. In contrast, blocking airflow through half of the nasal cavity by surgically closing an external naris on P1 resulted in a gradual increase in TUNEL-positive figures within the ipsilateral olfactory bulb by P20, with the effects being seen in the mitral and granule cell layers until at least P60. The effect was largely age dependent, because subjects occluded from P30 to P60 showed only slight increases in cell death. Furthermore, although interlaminar differences were encountered, the pattern of cell death appeared uniform over much of the bulb. Finally, reopening occluded nares decreased cell death levels to control values, suggesting an inverse relationship between the level of olfactory function and the extent of cell death. Thus, the data indicate that cell death is prevalent in the normal olfactory bulb, and that it is directly regulated by the level of olfactory function.

Early postnatal cellular proliferation and survival in the olfactory bulb and rostral migratory stream of normal and unilaterally odor-deprived rats.

Unilateral naris occlusion in rats on postnatal Day 1 results in dramatic decreases in the size of specific olfactory bulb cell populations when pups are examined 30 days later (Frazier and Brunjes: J. Comp. Neurol. 269: 355-370, '88). The observed reductions must result from alterations in cell proliferation and/or survival, alternatives examined in the present study. During early postnatal development, most cells destined for the bulb are produced in regions caudal to the structure and migrate to the bulb in the massive rostral migratory stream. The dynamics of the stream were examined in both normal rats and pups with a single naris closed on Day 1. 3H-thymidine injections were made on postnatal Days 2, 5, 10, 20, and 30. Groups of pups were killed 2 hours later to assess patterns of proliferation and 24 hours later to gauge initial stages of migration. A gradient of labeled cells was observed in the stream, with higher levels occurring at more caudal locations. The supply of cells to the bulb peaked on Day 5 and was still substantial as late as Day 30. The deprivation procedure did not affect patterns of cell labeling at any stage tested, indicating the procedure does not affect early cellular proliferation. A third group of pups was examined 30 days after thymidine injection to assess both time of cell origin and survival rates. Dark granule cells and glia in the granule cell layer were produced at a consistent rate until Day 20 with cells added during the period evenly spread throughout the layer. Light granule and periglomerular cell production decreased dramatically after P5. Thirty days after injections on P2, fewer labeled dark granule cells and their associated glia were found in deprived bulbs, indicating that enhanced cell death plays a major role in the deprivation-induced decrease in cell number.

Development of the anterior olfactory nucleus in normal and unilaterally odor deprived rats.

The development of a second order structure in the olfactory pathway, the anterior olfactory nucleus, was examined in both normal rat pups and in subjects which underwent unilateral naris closure on postnatal day 1 (P1). Naris occlusion in neonatal rats produces a constellation of changes within the first relay in the pathway, the olfactory bulb, including a 25% reduction in total volume. Such large changes suggest that higher order structures might also be affected. Anterior olfactory nucleus development was quantified in several ways. Laminar volumes were computed by using serial section planimetry. In control animals differential development was observed, with regions extending most rostrally (e.g., pars externa and pars lateralis) exhibiting the least growth. The anterior olfactory nucleus on the "deprived" side of subjects with a single naris occluded was identical in size to that observed in controls, development within the pars lateralis was examined in control animals at P10, P20, P30, and adults. Developmental increases in numbers of both branches per cell and spines were noted, but mean branch length remained relatively constant. Finally, the effects of naris occlusion on histological patterns of succinate dehydrogenase (SDH) staining and 2-deoxyglucose uptake within pars lateralis were examined at P20 to test for more subtle effects of naris occlusion. SDH staining was quite similar in deprived and control rats at P20. However, 3H-2-DG uptake was decreased in rostral areas of the anterior olfactory nucleus ipsilateral to the deprived olfactory bulb, suggesting that naris closure does affect the structure.

Olfactory bulb organization and development in Monodelphis domestica (grey short-tailed opossum).

The olfactory bulbs of adult and developing Monodelphis domestica were examined with a number of techniques. Golgi, Nissl, and Timm stains as well as acetylcholinesterase histochemistry revealed a high degree of order within the adult bulb. All major cell classes characteristic of most mammalian species were observed. Tufted cells appeared to be restricted to the superficial portion of the external plexiform layer. Developing Monodelphis pups were examined with Nissl-stained semithin sections and with immunocytochemistry for tyrosine hydroxylase, microtubule-associated protein 2, vimentin, and glial fibrillary acidic protein. Newborn pups are extremely immature, with few postmitotic cells present in the forebrain. Considerable maturation occurs over the first four postnatal weeks, and by postnatal day 30, the bulb assumes an adult-like organization. The extreme immaturity of the bulb at birth, coupled with its strict organization, suggest that Monodelphis is a particularly appropriate species for experimental examinations of olfactory system development.

Activity-dependent regulation of calcium-binding proteins in the developing rat olfactory bulb.

Intracellular calcium, important in a variety of second messenger cascades, is regulated in part by calcium-binding proteins such as calretinin, parvalbumin, and calbindin. These proteins are highly concentrated in the rat main olfactory bulb and are localized in distinct neuronal populations. In the present study, postnatal expression was characterized immunohistochemically in normal rats and in rats with functional olfactory deprivation caused by unilateral naris closure, a manipulation that attenuates electrical activity in the bulb. Bulbs were examined from rats that had undergone naris closure or sham surgery on either postnatal day 1 (P1) or P30 and were allowed varying subsequent survival times. Each of the calcium-binding proteins showed both distinct patterns of early expression and differential susceptibility to olfactory restriction. For example, at P10, the densest immunoreactivity was observed for calretinin, a protein whose expression was the least affected by naris closure. After occlusion from P1-P30, there was a 30% reduction in the density of calbindin-immunoreactive profiles in the glomerular layer, and parvalbumin-immunoreactive profiles were reduced by 64% in the external plexiform layer. Unlike many other changes induced by deprivation, the effects of olfactory restriction on calbindin and parvalbumin expression were not age dependent: naris closure from P30-P60 caused similar substantial decreases in calbindin and parvalbumin immunoreactivities. These data demonstrate that the expression of calbindin and parvalbumin in rat bulb is regulated, in part, by afferent activity that is associated with full sensory experiences. The reductions of these calcium-binding proteins following olfactory deprivation are likely to be commensurate with altered control of intracellular calcium.

Focal denervation alters cellular phenotypes and survival in the rat olfactory bulb: a developmental analysis.

Our previous work (Couper Leo et al. [2000] J. Comp. Neurol. 417:325-336) introduced a technique for focally denervating the olfactory bulb soon after birth and described the pattern of changes incurred by this procedure by postnatal day (P) 30. The current study extends these findings with a developmental analysis of the effects of focal denervation in P10 and P20 rats. The results suggest that denervation begins to affect bulb architecture and cell survival soon after the procedure is performed, but that alterations within the bulb occur over an extended time period. For example, at P10, bulb and laminar sizes and mitral/tufted cell profile number had begun their decline, and nearly all measurements were significantly reduced by P20. Furthermore, a superficial-to-deep gradient of alterations in bulb architecture and a temporal separation of the effects on mitral/tufted cell dendrites vs. somata were observed. Immunohistochemical analyses of olfactory marker protein (OMP)-, calretinin- calbindin-, parvalbumin-, tyrosine hydroxylase-, and glutamic acid decarboxylase-stained sections indicated that: 1) denervation alters the interaction between olfactory axons and their targets in a developmentally significant manner; 2) the fine structure of denervated cells is altered; 3) cell phenotypes are differentially affected by loss of afferent contact, perhaps due to the age-dependent expression of their defining antigens; and 4) specific cell populations may be lost as a result of denervation.

Focal denervation alters cellular phenotypes and survival in the developing rat olfactory bulb.

Several studies have demonstrated that contact between the olfactory nerve and the forebrain is critical for normal olfactory bulb development. Removal of the embryonic olfactory placode results in a failure of the olfactory bulb to form, as well as causing other forebrain malformations. The current study introduces a technique that permits removal of contact between specific regions of the olfactory nerve and the bulb early in development, without causing damage to other brain regions, and without removing the peripheral olfactory organ. The manipulation, which involves insertion of a small Teflon chip between the cribriform plate and the bulb, prohibits growth of new axons into the "shadow" region behind the implant. Focal denervation of the olfactory bulb causes a decrease in bulb and layer sizes, a reduction in mitral cell number, and changes to bulb architecture. Using a battery of antibodies (OMP, MAP2, TuJ1, calretinin, calbindin, parvalbumin, TH, and GAD), we further demonstrated that 1) focal denervation alters the relationship between the olfactory nerve and the bulb, 2) the fine structure of cells in denervated regions is disrupted, and 3) cellular phenotypes change in response to loss of afferent contact. These results suggest that contact between the olfactory nerve and the bulb is important for maintaining bulb architecture and cell survival, structure, and phenotype. They also point to focal denervation as a useful technique for examining the role of neural contact in olfactory development and maintenance of the central nervous system.

Precocity and plasticity: odor deprivation and brain development in the precocial mouse Acomys cahirinus.

Altering the early olfactory environment of animals can have dramatic consequences on the development of brain regions which subserve olfaction. The present study indicates that early odor deprivation has a more severe effect on a species which is born relatively mature than it does on related species which are not. The results call into question prevailing notions about the developmental continuity between animals born in divergent ontogenetic states.

Activity-dependent regulation of interleukin-1 beta immunoreactivity in the developing rat olfactory bulb.

Interleukin-1beta is a relatively small and abundant polypeptide that plays diverse roles in the central nervous system. In the present study, patterns of interleukin-1beta expression were observed in the olfactory bulbs of rats that had either undergone unilateral closure of the external naris or sham surgery on postnatal day 1 and then survived until postnatal day 30. Interleukin-1beta-immunoreactive fibers occupied distinct layers of the olfactory bulb. Dense immunostaining was found in the periglomerular and granule cell layers. Odor deprivation resulted in a noticeable reduction in interleukin-1beta immunoreactivity only in the periglomerular layer. The data demonstrate that interleukin-1beta is present abundantly in the bulbs, and that it can be regulated in an activity-dependent manner.

Neurogenesis in the olfactory bulb of adult zebrafish.

Cell genesis in the adult brain of zebrafish, with specific reference to the olfactory bulbs, was examined using bromodeoxyuridine immunocytochemistry. Mature fish were exposed to a 1% solution of the thymidine analog 5-bromo-2'-deoxyuridine for 1 h and then killed after short (4-h) or long (3-4-week) survival periods. A monoclonal antibody to bromodeoxyuridine allowed visualization of cells that incorporated the drug during the S phase of mitosis. Four hours after administration of the drug, antibody-labeled cells were found almost exclusively in the proliferative zones around the ventricles and in the cerebellum. Very few labeled nuclei were seen in other locations in the brain, indicating that cell genesis occurs in discrete regions in adults. The few labeled profiles in the olfactory bulbs were located in the olfactory nerve layer; these profiles had the morphology of glial nuclei and did not stain with a neuronal marker, the Hu antibody. After longer survival times, labeled cells were present throughout the layers of the olfactory bulb, and many of the immunoreactive profiles in the internal cell layer were also labeled with the Hu antibody, indicating that they are likely adult-formed interneurons. Thus, neurogenesis continues in the olfactory bulb of adult zebrafish. Understanding the process of the generation of new neurons in the brain of adult animals can lead to important insights into neural regeneration and adult plasticity.

Microglia and the developing olfactory bulb.

The developmental appearance of microglia in the rat olfactory bulb was investigated through the use of selective staining with the B4-isolectin from Griffonia simplicifolia. No changes in the density or distribution of either the spherical, macrophage "ameboid" form or the highly arborized "ramified" variety of microglia were observed in the superficial layers of the bulb between postnatal days 10 and 30. The subependymal zone exhibited the only substantial population of ameboid cells and the only developmental increases in ramified cell density during this time-period. External single naris closure, which enhances cell death in the ipsilateral bulb, did not affect microglia density, presumably due to the unusually high numbers of microglia normally present in the bulb. The olfactory bulb has a dense and relatively uniform population of microglial cells from very early stages of postnatal life, perhaps because of the constant turnover of cells and processes.

Unilateral naris closure and vascular development in the rat olfactory bulb.

The blood supply to the brain has been linked closely to nervous system function and metabolism, thereby possibly playing a direct role in brain maturation. Previously, we demonstrated that closure of an external naris early in life results in large changes within the olfactory bulb, including reductions in laminar volume and cell number and a rapid decline in metabolism and protein synthesis. To understand the role of the blood supply in the dramatic changes following naris closure, the present study examines the development of olfactory bulb vasculature in unilaterally odor-deprived and control rats. On post-partum day 1 (P1; the day after birth), littermate rat pups underwent either unilateral naris occlusion or sham surgery. On P5, P10, P15, P20, P30 and P60, animals were perfused with an india ink-gelatin mixture to assess blood vessel amount and complexity. Densitometric analyses were performed to obtain values of blood vessel area ratios (vessel area/tissue area), branch point number and branch point density. Considerable vessel development in all bulbs occurred over the first two to three weeks post-partum. By P20, large reductions in vessel area ratios were observed in all constituent laminae of deprived bulbs. While similar reductions in number of vessel branch points/tissue area were seen, few changes were noted in the number of branch points/vessel area. The effects were primarily confined to early developmental periods: bulb vasculature in animals deprived at older ages (P40) appeared normal. The results indicate that the vasculature responds to alterations in sensory stimulation early in life, therefore potentially playing an important regulative role in neural development.

Microglial activation in the developing rat olfactory bulb.

The development of the olfactory bulb, the primary central relay of the olfactory system, is characterized by a striking susceptibility to alterations in the amount of afferent input. For example, blocking airflow through one half of the nasal cavity during early life results in a number of dramatic changes in the bulb, including increased cell death. Previous studies reveal high levels of microglia in the olfactory bulb. Microglia function as phagocytes, aid in synaptogenesis, and produce important trophic and cytotoxic factors. In response to a number of tissue perturbations, microglia undergo an activation process that includes, among other changes, the up-regulation of complement receptor 3. Interestingly, a previous study reported that naris closure had no effect on microglia in the bulb; however, the research did not distinguish the functional activation state of microglia. We further examined the role of microglia in the normally developing and olfactory-deprived rat bulb using immunohistochemical detection of complement receptor 3 as a measure of microglial activation. Expression of the receptor in the bulb is relatively high during postnatal development, in particular when compared to levels in cortical regions caudal to the olfactory bulb. In addition, naris closure performed on the day after birth (but not after the first postnatal month) increases levels of the receptor in an age and laminar-dependent fashion. The presence of an inducible pool of activated microglia in the olfactory bulb may be important for normal development and contribute to the plethora of changes seen after early olfactory deprivation.

Oligodendrocyte/myelin-immunoreactivity in the developing olfactory system.

Immunocytochemistry was used to characterize oligodendrocyte maturation in the developing mammalian olfactory system. Postnatal day 10-16, 20, 30 and adult rats were examined, as well as postnatal day 20, 30, 40 and adult Monodelphis domestica (the grey, short-tailed opossum). In rats, oligodendrocyte/myelin-immunoreactivity first appears in the accessory olfactory bulb by day 11, with labeling rapidly increasing throughout the entire bulb over the next five days. An adult pattern of immunoreactivity, characterized by dense labeling in the granule cell layer, sparse immunoreactivity in the external plexiform layer, and staining along the periphery of glomeruli, is attained by day 30. Staining is apparent in both the lateral olfactory tract and anterior commissure by day 11, and becomes heavy by day 20. While patterns of oligodendrocyte/myelin-immunoreactivity in the adult Monodelphis and rat bulb are similar, staining first appears much later in the opossum (around day 30), and maturation occurs more slowly. For example, rostral-caudal gradients in the development of staining in the anterior commissure were noted which were not seen in the rat. These differences emerge because Monodelphis' slower growth allows more resolution into developmental sequences. Finally, in rats, unilateral naris closure on the day after birth, which significantly alters normal patterns of bulb development, has no effect on the pattern and level of immunoreactivity even after long (30 day) survival periods. In both normal and naris occluded rats, oligodendrocyte/myelin-immunoreactivity is found in caudal aspects of the rat bulb on day 11 and subsequently progresses throughout the entire bulb over the next five days. Patterns in the Monodelphis bulb mirror those observed in the rat, however, staining appears later and progresses more slowly, suggesting Monodelphis is a useful animal for examining early myelin formation.

Activity-dependent regulation of dopamine content in the olfactory bulbs of naris-occluded rats.

Several lines of evidence strongly suggest that reduced olfactory nerve activity results in decreased bulb dopamine content. In the present study, high performance liquid chromatography with electrochemical detection was used to assess catecholamine levels in bulbs from postnatal day 60 rats that had undergone either unilateral naris cautery or a sham surgery on day 30. Thirty days of odor deprivation dramatically reduced dopamine and dihydroxyphenylacetic acid levels in functionally-deprived bulbs (ipsilateral to occluded nares) as compared to contralateral controls, while norepinephrine and dihydroxyphenylglycol levels were unchanged. The loss of dopamine was more severe in medial as compared to lateral aspects of experimental bulbs, while the loss of dihydroxyphenylacetic acid was similar on the two sides. To test directly the hypothesis that afferent activity regulates dopamine and dihydroxyphenylacetic acid content, 1 h of high frequency tetanic nerve stimulation was provided to the rostral-medial olfactory nerve layer in deprived olfactory bulbs, and catecholamine levels were assessed from 6 to 192 h later. Partial and temporary recovery of dopamine was observed in medial aspects of the bulb when rats were examined 96 h later, while consistent recovery of dihydroxyphenylacetic acid content was not apparent. These data corroborate evidence that olfactory nerve activity is a potent regulator of bulb dopamine and indicate that continued afferent input is necessary to maintain dopamine levels.

Addition of new cells to the olfactory bulb of adult zebrafish.

We have been examining patterns of cell proliferation in the brain of adult zebrafish. Understanding this process in fish may lead to important insights due to the tremendous regenerative capabilities of these animals. Fish were exposed to a 1% solution of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) for 1 hr before being returned to small aquaria that received frequent water changes. Animals were overanesthetized and perfused with Bouin's fixative solution after two survival periods (4 hr or 3-4 weeks). Paraffin immunohistochemistry using a monoclonal antibody against BrdU was used to visualize the newly generated cells. Quantitative analyses were performed on serial, 10-micron sections from 4 animals for each survival group. Statistical determinations were based on the nonparametric Mann-Whitney U test. The average number of BrdU-labeled nuclear profiles in the bulbs, from analysis of every 5th section, was significantly different between the two groups (22.0 +/- 6.8 [SEM] for the 4-hr group and 136.7 +/- 11.3 for the 4-wk group; p < 0.05). The volumes of the bulbs, however, were not different between the two groups (p > 0.5). These data indicate that either cells divided repeatedly during the longer survival period or cells migrated into the bulb from other brain regions. To examine this phenomenon further, the location of the new cells was analyzed in three mid-bulb sections (20 microns apart) from each animal. Both the area and number of labeled nuclei in each lamina were measured to obtain an average profile density. Comparison of the 4-hr and the 4-wk groups showed that density was significantly greater in all bulb layers in the long survival group (p < 0.05 for all). In the 4-hr survival group, cells were found mainly in the olfactory nerve layer. When examined after 4 wk, proportionately more labeled cells were found in the internal cell layer. This addition of new cells could be a result of neurogenesis, gliogenesis, and/or angiogenesis. We are currently performing double-labeling experiments to determine the types of cells that are added to the adult bulb. In addition, our future plans include investigating the origin of these cells and the signals that direct their formation.