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.

Neonatal focal denervation of the rat olfactory bulb alters cell structure and survival: a Golgi, Nissl and confocal study.

Contact between sensory axons and their targets is critical for the development and maintenance of normal neural circuits. Previous work indicates that the removal of afferent contact to the olfactory bulb affects bulb organization, neurophenotypic expression, and cell survival. The studies also suggested changes to the structure of individual cell types. The current work examines the effects of denervation on the morphology of mitral/tufted, periglomerular, and granule cells. Focal denervation drastically changed mitral/tufted cell structure but had only subtle effects on periglomerular and granule cells. Denervated mitral/tufted cells lacked apical tufts and, in most cases, a primary dendrite. In addition, the denervated cells had more secondary processes whose orientation with respect to the bulb surface was altered. Our results suggest that contact between olfactory axons and the bulb is necessary for cell maintenance and may be critical for the ability of mitral/tufted cells to achieve adult morphology

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.

NMDA receptor regulation of cell death in the rat olfactory bulb.

Cell death is widespread in the developing nervous system and is under complex regulation by numerous intra- and intercellular mechanisms. Blockade of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor has been shown to promote cell death in the developing brain (Ikonomidou et al., 1999), suggesting that afferent functional activation is an important regulator of cell survival. The olfactory bulb, the first central relay for olfactory information from the nose, is well suited for examining the role of afferent activity in neuronal development. Functional deprivation is easily performed by surgical blockade of airflow to one side of the nasal passage, which results in dramatic alterations in postnatal development of the bulb (Brunjes, 1994), including enhanced neuronal loss (Frazier and Brunjes, 1988; Najbauer and Leon, 1995). The present report examined the specific role of NMDA receptor activation in regulating cell survival within the rat bulb. Pharmacological blockade of receptors with the noncompetitive channel blocker MK-801 (3 x 0.5 mg/kg i.p.) resulted in profound increases in cell death within 24 h. Furthermore, in contrast to other regions, where the effects of receptor blockade were confined to the first 2 postnatal weeks (Ikonomidou et al., 1999), enhancement of cell death was seen in the deeper granule cell-containing regions of the bulb with injections as late as postnatal day 28. In addition, the effects of MK-801 were much more dramatic than those seen after unilateral naris closure, suggesting that NMDA receptor activation may mediate additional survival pathways in the bulb beyond that provided by first nerve input.

Activity modulates neuronal proliferation in the developing olfactory epithelium.

The present experiment demonstrates that environmental stimulation can activate neurogenesis within olfactory mucosae exhibiting depressed proliferation. Rats underwent naris occlusion on P1 with plugs that were removed 20 days later. Normal respiration induced a sharp increase in proliferating neuronal precursors within 24-48 h, and epithelial depth was restored within 5 days.

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.

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.

Sensory deafferentation and olfactory bulb morphology in the zebrafish and related species.

The zebrafish, Danio rerio, has become an important model species for examining olfactory system structure and function, yet little is known about developmental changes in olfactory bulb morphology from embryo to adult. The present study examined both normal growth and the effects of deafferentation on the bulb from hatching to adulthood. In young animals, the bulb is small relative to body size and has a higher percentage of its volume occupied by incoming olfactory nerve fibers. Young animals are also more affected by sensory deafferentation. Olfactory rosette removal resulted in more than 50% reductions in laminar volumes, indicating that sensory input is important during periods of rapid development. In addition, three closely related species were examined to compare how differing bulb morphology might influence the effects of bulb manipulation. The cherry barb, Barbus (=Puntius) titteya, and giant danio, Danio aequipinnatus, have larger bulbs and laminar volumes relative to body size than the zebrafish or scissortail rasbora, Rasbora trilineata. Both are also more affected by deafferentation, with at least a 35% reduction in laminar sizes in many of the bulb layers. The studies are discussed in terms of the importance of the olfactory system to each species and are also compared to the effects of sensory manipulations in other animals.

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.

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.

Trans-neuronal modification of anterior piriform cortical circuitry in the rat.

Long-Evans rats received unilateral naris closure on postnatal day 1 (PN1) or sham surgery. On PN30, brains were processed for anterograde horseradish peroxidase (HRP) labeling of lateral olfactory tract (LOT) fibers in anterior piriform cortex (aPCX) Layer Ia, Timm staining of association/commissural fibers in aPCX Layer Ib, or Golgi staining for reconstruction of aPCX semilunar cell dendrites. The results demonstrate that the width of aPCX Layer Ia was reduced ipsilateral to the sealed naris compared to undeprived controls. No significant change in Layer Ib was detected. Furthermore, semilunar cell dendrites were reduced by unilateral deprivation compared to undeprived controls. The reduction in dendritic tree size was localized to distal dendritic segments, roughly corresponding to Layer Ia. These results suggest an activity-dependent change in both the distribution of cortical afferents and in the dendritic field of their target cells. While these results are similar to those reported for other sensory systems, the relatively simple architecture and laminated organization of bilateral inputs to the aPCX make the olfactory system an ideal model system to examine experience-dependent synaptic reorganization and its functional consequences.

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.

Calcium-binding proteins: differential expression in the rat olfactory cortex after neonatal olfactory bulbectomy.

Calbindin, parvalbumin, and calretinin, members of EF-hand calcium-binding proteins, play important roles in buffering intracellular calcium ions. These proteins are localized in distinct populations of cells in the olfactory bulb (the primary sensory relay in the olfactory system) and its major synaptic target, the primary olfactory cortex (POC). In the present study, the postnatal expression of these calcium-binding proteins in layer III of POC was quantitatively examined 30 days after neonatal bulbectomy, a manipulation known to cause cell death and neurotransmitter changes. The numbers of both calbindin and parvalbumin-immunoreactive profiles showed significant increases (68% and 163%, respectively), while calretinin-immunoreactive profiles exhibited a 46% reduction. The data demonstrate that the expression of these calcium-binding proteins is regulated in part by the afferent input from the olfactory bulb. Furthermore, the resultant increase in calbindin and parvalbumin expression may provide neuroprotective support necessitated by possible alterations in intracellular calcium ions and other neurochemical factors that accompany neonatal bulb removal.

Developmental analysis of the peripheral olfactory organ of the opossum Monodelphis domestica.

The gray, short-tailed opossum, Monodelphis domestica, is born in a very immature state after a brief (14-day) gestation. As a result, the species provides a unique opportunity to examine very early periods of mammalian development. The present study provides the first detailed morphometric analysis of the development of the olfactory mucosa and the nasal cavity in Monodelphis. The extent of the sensory mucosa increases dramatically across development, covering a growing nasal cavity and increasingly elaborate turbinates. Both nasal cavity convolution (a measure of turbinate complexity) and mucosal surface area show extensive growth between birth and adulthood. These measurements are greatest in the central portion of the mucosa (in the caudal portion of the nose) at all ages examined. A developmental BrdU study reveals a robust decrease in cellular proliferation with age; proliferation decreases to near adult-like patterns by postnatal day (P) 40. Results from these studies show that there is dramatic structural and cellular postnatal growth in the opossum peripheral olfactory organ.

Ribosomal RNA expression in the developing rat olfactory bulb.

The rat olfactory bulb undergoes rapid postnatal growth, thus requiring the continuous production of new proteins. The present study examined the development of ribosomes, a key component in protein synthesis, in the bulb using an antibody specific to ribosomal RNA (Y10b). Furthermore, the potential role of afferent activity on patterns of Y10b immunoreactivity, as well as total RNA production (using incorporation of 3H-uridine) was examined by blocking an external naris on postnatal day 1 (P1). In control pups, Y10b was restricted to mitral cells at P2, with labeling expanding to include all layers by P10. Considerable increases in staining density were observed by P30. Although no differences in Y10b immunoreactive patterns were seen in naris-occluded animals 12-48 h after occlusion, or on P10 or P20, by P30, an apparent decrease in numbers of labeled profiles (presumably tufted cells) in the external plexiform layer was found. Furthermore, no changes were seen in levels of 3H-uridine incorporation. Despite the apparent insensitivity to naris closure, the results indicate that ribosome expression, as measured by Y10b immunoreactivity, undergoes rapid postnatal changes that parallel general patterns of growth in the rat olfactory bulb.

The small-eye mutation results in abnormalities in the lateral cortical migratory stream.

Mice with homozygous mutations of the Pax-6 gene exhibit a constellation of developmental problems including the absence of eyes and nasal cavities and problems in the movement of neuroblasts out of the germinal epithelium. In this paper, we demonstrate further disturbances in neuronal migration. Normally, cells produced along the lateral ventricles move laterally across the pallium, ultimately coming to reside in the lateral neocortex and primary olfactory cortex. In mutant animals, these cells continue to migrate to the pial surface of the brain.

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.

The NMDA receptor participates in respiration-related mitral cell synchrony.

The N-methyl-D-aspartate (NMDA)-type glutamate receptor participates in the excitation of olfactory bulb mitral cells and is important in granule-cell-mediated feedback-inhibition. In the present study, extracellular unit recordings were made in vivo to demonstrate that the firing rates of mitral cells are not affected by peripheral administration of the non-competitive NMDA receptor antagonist MK-801. However, while over 50% of odor-driven mitral cell activity is normally correlated with the respiratory cycle, only about 10% of mitral cell activity is correlated with the respiratory cycle 30 min after MK-801 administration. Thus, the NMDA receptor is a participant in normal respiration-related mitral cell activity and may have an important role in the formation of bulb oscillations that encode olfactory information. Furthermore, the NMDA receptor is in a position to mediate activity-dependent changes in the bulb that rely on synchronous activity.

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.

Unilateral naris occlusion and the rat accessory olfactory bulb.

Blocking airflow through half of the nasal cavity during early life results in a 25% reduction in the size of the ipsilateral main olfactory bulb. The present study indicates that the size of the accessory bulb is relatively unaffected by the procedure.