Peter C. Brunjes

Maturation and plasticity in the olfactory system of vertebrates

2. Receptor development ................................................................................................................................ 2.1, The olfactory placode ........................................................................................................................... 2.2. Formation of the nasal cavity .................................................................................................................. 2.3. Receptor differentiation ........................................................................................................................ 2.3.1. Olfactory mucosa ........................................................................................................................ 2.3.1.1, Morphological development .............................................................................................. 2.3.1.2. Chemical development ..................................................................................................... 2.3.1.3. Physiological development ................................................................................................ 2.3.2. Vomeronasal organ ..................................................................................................................... 2.3.3. Trigeminal chemoreceptors ........................................................................................................... 2.3.4. Septalorgan .............................................................................................................................. 2.3.5. Nervus terminalis ........................................................................................................................ 2.4. Conclusions .......................................................................................................................................

Unilateral naris closure and olfactory system development.

In most animals there is bilateral access of odorants to the olfactory sensory epithelium. Air enters the nose through two external nares and passes back through the nasal cavity, which is divided down the midline by a cartilaginous nasal septum. The olfactory mucosa, a sheet of ciliated bipolar receptor cells, is found in the caudal two thirds of the nasal cavity. Axons from the sensory cells project to an ipsilateral extension of the telencephalon known as the olfactory bulb. If a single external naris of a rat pup is surgically closed (usually via brief cauterization) on the day after the day of birth (P1) and the subject is examined on P30, the size of the ipsilateral olfactory bulb is reduced by approximately 25%. The large reduction in size, coupled with the clear lamination and other features of the olfactory system, indicates that the manipulation is an ideal preparation for examining the regulation of early growth. We know that both olfactory bulbs are of equal size at the time of occlusion, but that 30 days later there is a large discrepancy. What series of events produces the changes? The present paper outlines what is known about the anatomical, biochemical and physiological changes introduced by naris occlusion in order to lay a framework for further work.

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′‐tetramethylindocarbocyanine 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.

Unilateral odor deprivation: effects on the development of olfactory bulb catecholamines and behavior.

The present studies began an examination of the process by which unilateral odor deprivation results in a 25% reduction in the size of the olfactory bulb. Rat pups had a single naris occluded on the day after the day of birth (Day 1) and were tested at several early postnatal ages. Dopamine (DA) levels were measured to gauge the effects of deprivation on a transmitter system which is intrinsic to the bulb, while norepinephrine (NE) concentrations were assessed to determine how deprivation affects inputs to the bulb from higher brain regions. A significant reduction in DA concentration (pg/mg protein) was observed on Day 8 and persisted until Day 30 although protein concentrations (pg/mg bulb) were not affected. In contrast, deprivation did not significantly alter NE concentration. Deprived and control pups did not differ on a series of behavioral and morphometric measures, suggesting that the surgical procedure did not seriously impair normal growth patterns. The results indicate that unilateral naris occlusion induces rapid and specific changes within the olfactory bulb.

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. J. Comp. Neurol. 431:311–319, 2001.

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.

Comparative Study of Aging in the Mouse Olfactory Bulb

Gene knockout technologies have been used to elevate the mouse as a model species. However, little work has examined age and strain differences in the mouse olfactory system. The present study compared the olfactory bulbs of mature (6 month) and aged (24 month) males of BALB/cBy, C57BL/6J, and DBA/2 strains. Volumes of the glomerular (GLM), external plexiform (EPL), and mitral/granule cell (MIG) layers varied little from strain to strain. Volume measurements increased with age even when corrected for body weight differences. Two nonoverlapping interneuron populations were examined with immunohistochemistry. Staining for the calcium binding protein calretinin varied little between strains, but age‐related increases in staining were seen in EPL of C57BL/6J mice. Typical patterns of tyrosine hydroxylase immunoreactivity were observed in all subjects except for old DBA/2 mice, which evidenced considerable staining in submitral areas. Age‐related increases were observed in BALB/cBy and DBA/2 mice but not in the C57BL/6J strain. Glial fibrillary acidic protein staining was similar in old BALB/cBy and DBA/2 mice, with astrocytes in all layers of the bulb, but more concentrated in the MIG. However, C57BL/6J tissue revealed very large astrocytes relatively evenly distributed in all layers. Cell proliferation dropped dramatically with age. Labeled cells could still be observed along the lateral ventricles, but very few were observed within the rostral migratory stream or subventricular zone. Although TUNEL labeling revealed many apoptotic figures in the granule cell layer of young subjects, almost no staining was seen in aged mice. J. Comp. Neurol. 454:361–372, 2002.

Mitral/tufted cell activity is attenuated and becomes uncoupled from respiration following naris closure

Patterned neural activity helps to establish neuronal connectivity, produce coding of sensory information, and shape synaptic strengths. Here we demonstrate that normal olfactory bulb development might rely on spatial and temporal patterns of afferent neural activity. Neonatal naris occlusion profoundly impacts the development of the ipsilateral olfactory bulb, including reduced bulb volume, decreased protein synthesis, and increased cell death. Relatively few morphologic changes occur if closure is performed postweaning. We examined the immediate electrophysiological consequences of occlusion across this developmentally sensitive period by recording spontaneous and odor-driven mitral/tufted cell responses while the naris was open, closed, and then reopened. In 1-week-old animals, occlusion severely attenuated spontaneous activity, and presentation of the broad-spectrum odorant amyl acetate failed to evoke responses. In 2- and 4-week old rats, spontaneous activity was also reduced by naris closure. However, some cells remained responsive to concentrated odors, even in animals with transected anterior commissures, suggesting passage of odors across the septal window or retronasal pathways. In all age groups, cellular activity became uncoupled from the respiratory cycle. Approximately 47% (18 of 38) of the mitral/tufted cells exhibited activity that was correlated with respiration in the open-naris state, while only 5% (2 of 38) were coupled during naris closure. These data (a) indicate that naris closure reduces both spontaneous and odor-evoked responses, and (b) provide an electrophysiological correlate to a sensitive period in bulb development. The loss of respiration-related synchrony and the reduced activity of mitral/tufted cells may synergistically contribute to the diverse consequences of naris closure on bulb development.

Ontogeny of odorant receptor gene expression in zebrafish, Danio rerio

We cloned three putative odorant receptor (OR) genes from the zebrafish to use as in situ hybridization probes to follow the temporal patterns of neurons expressing OR genes through a developmental progression from embryo (12 h postfertilization) to adult. The identification of these genes is supported by sequence homology to previously reported ORs and by the morphology and location of labeled cells in in situ hybridization experiments. Cells expressing OR mRNA were first observed in the olfactory placodes between 31 and 38 h after fertilization (fish reared at 26 degrees C). Initially, only single cells were observed to hybridize the probe; the number of labeled cells increased throughout the remainder of embryogenesis and through postembryonic growth and morphogenesis of the olfactory organ. At all ages, the positively hybridizing cells were scattered throughout the olfactory epithelium but not in the nonsensory epithelium of the olfactory organ.

Lessons from lesions: the effects of olfactory bulbectomy

Abstract Olfactory bulb removal has been used to examine a wide-ranging number of topics. The present review outlines the categories of studies employing the technique, discusses some problems with the methodology and with previous interpretations of observed results, and suggests some potential avenues of investigation.