Biomedical subjects
Publications and source records attributed to P C Brunjes.
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.
Microtubule-associated protein 2 (MAP2) is a neuron-specific cytoskeletal protein, enriched in dendrites and cell bodies, that helps determine dendritic shape. MAP2 regulates microtubule stability in a phosphorylation-dependent manner. The present study used immunocytochemistry with phosphoepitope-specific and phosphorylation state-independent antibodies to examine experience-dependent changes in MAP2 expression during postnatal development of the olfactory bulb. Our results demonstrate that immunoreactivity reflecting total MAP2 expression reaches a maximal level by postnatal day 20 (P20). The degree of staining for phosphoindependent forms of MAP2 is relatively unaffected by blocking odorant passage to one half the nasal epithelium via unilateral naris closure, a manipulation that attenuates physiological activity in the bulb. However, olfactory restriction from P1 dramatically reduces immunoreactivity for antibody AP18, which recognizes MAP2 only when phosphorylated on Ser136. Quantification of staining in the granule cell layer indicates that the greatest difference (64%) between control and experimental bulbs occurs after occlusion from P1 to P30 compared with animals deprived from P1 to P10 or P1 to P20. The shift in MAP2 phosphorylation occurs even when deprivation is delayed until P30, after the sensitive period for experience-dependent changes in bulb volume. Thus, the degree of the phosphorylation shift depends on the duration but not the time of onset of naris closure. Because staining for phosphorylation-independent forms of MAP2 is unchanged by naris closure, the total amount of the protein per unit area is probably not significantly altered. However, the large reductions of AP18-immunoreactivity in the bulb after olfactory restriction suggest that there is an activity-dependent stimulation of MAP2 phosphorylation.
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.
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.
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.
Unilateral naris closure in young rodents leads to striking alterations in the development of the ipsilateral olfactory system. One of the most pronounced effects is a 25% reduction in the size of the experimental olfactory bulb, a change that stems in part from decreased cell survival. Since naris occlusion in rodents alters the system more during development than in adulthood, we investigated the consequences of olfactory deprivation in a species that is born in a very immature state, Monodelphis domestica. In this pouchless marsupial, offspring are born after a short 14-day gestation. In the present study, the thymidine analogue bromodeoxyuridine was used to examine early postnatal neurogenesis in the olfactory bulb. Unlike rats and mice, neurogenesis of the main output neurons (the mitral cells) continues into postnatal life. Unilateral naris closure was begun on postnatal day 4 (P4) or P5 in Monodelphis and continued for 30 or 60 days. Laminar volume measurements revealed a significant reduction in the size of the experimental bulb following 60, but not 30, days of early olfactory deprivation. Mitral cell number estimates indicated a significant reduction after both 30 and 60 days of naris closure. The immaturity of Monodelphis offspring may render the population of mitral cells susceptible to the effects of olfactory deprivation. These findings suggest that afferent activity plays a role in the survival of all bulb neurons, irrespective of cell class.
Dramatic alterations occur in the developing olfactory bulb when air flow is reduced through one-half of the nasal cavity. Naris closure on the day after the day of birth (P1) in rats, for example, results in reduced cell survival in the ipsilateral bulb by P20 and a substantial (25%) decrease in bulb size by P30. Almost immediate changes in protein synthesis and cell metabolism are also observed, and one prevalent theory suggests that these changes may be important in specifying which cells are subsequently eliminated. In the present study we used a reversible technique for unilateral naris closure to examine the sensitive period for the effects of olfactory deprivation on bulb size and cell survival. This technique involves the insertion of removable plugs into a rat pup's external naris. We occluded the naris for increasing periods of time (P1-P10, P1-P15, or P1-P20), reared all animals to P30, and measured volumes of bulb laminae. In addition, we examined the duration of naris closure needed to affect cell survival by injecting animals with the thymidine analogue bromodeoxyuridine to label cells born soon after the onset of olfactory deprivation. Results indicate that relatively long periods of naris occlusion (P1-P15 or longer) are required to produce a substantial reduction in experimental bulb size. Cell survival was decreased following olfactory deprivation from P1 to P10, but not after deprivation from P1 to P3. These data support the hypothesis that changes that occur within 48 h of naris closure are not sufficient to affect cell survival.
While the anterior commissure has been shown to be an important route of information transfer in the forebrain, relatively little is known about its anatomical development. Glial substrates and extracellular spaces have been associated with the maturation of other large-fiber tracts, such as the corpus callosum and retinofugal pathway. The present study examined early stages in the maturation of the commissure in the gray short-tailed opossum, Monodelphis domestica. Monodelphis offspring are born after a short 14-day gestation, and, unlike in rats and mice, the anterior commissure develops entirely during the postnatal period. A number of techniques were employed: the carbocyanine dye Dil was used to label early axons in the region, semithin plastic sections were used to examine the extracellular environment of the developing commissure, and immunocytochemistry for glial fibrillary acidic protein (GFAP) was used to characterize glial components. Results suggest that the first commissural fibers that cross the midline pass through a region of large extracellular spaces and may use GFAP-immunoreactive cells and processes as guides during their midline decussation.
The anterior olfactory nucleus (AON) is located caudal to the olfactory bulb in the olfactory peduncle. Although this important structure is involved in the bilateral coordination of olfactory information, relatively little is known about its development, structure, or function. The present report details results from an immunohistochemical examination of specific neuronal (microtubule-associated protein-2: MAP2, calbindin D28-k, neuropeptide-Y: NPY) and glial (astrocytes: glial fibrillary-associated protein, or GFAP, oligodendrocytes; RIP) populations in postnatal Days 10, 20, and 30 rats. MAP2-immunoreactivity (-ir), was present throughout the AON, although most dense in the outer plexiform layer. Increases in labeling occurred from Day 10 to Day 30, reflecting the maturation of dendritic processes. Both temporal and regional differences in expression were found with the two neuronal markers. For example, although substantial numbers of calbindin-ir cells were observed as early as Day 10, relatively few cells exhibited NPY-ir. An apparent decline in the number of stained figures was observed from Days 20-30 with both markers. Most cells exhibiting calbindin- or NPY-ir were found in the inner half of the cellular zone of the AON. GFAP-ir was localized mainly to the subependymal zone and the lateral olfactory tract (LOT) at Day 10, with successive increases in staining in the cellular and plexiform layers at Days 20 and 30. Oligodendrocyte-ir was restricted to the anterior commissure and the LOT at Day 10, with dramatic increases in labeling of the cellular and plexiform layers observed by Days 20 and 30. These results represent some of the first analyses of the maturation of specific cellular phenotypes within this large neural region.
The rostral migratory stream consists of a large number of cells migrating from the lateral ventricles to the rostral telencephalon, primarily the olfactory bulb. The pathway continually provides neuro- and glioblasts throughout life. The present paper indicates that a considerable number of cells undergo apoptotic cell death en route, even in young (day 3) rats when presumably many vacant sites are still available in the developing brain.
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.
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.
The distribution of the adenosine-producing ecto-enzyme 5'-nucleotidase was investigated histochemically in the developing rat olfactory bulb. Rat pups underwent either unilateral surgical occlusion of the right external naris or sham surgery on postnatal day 1. At 10, 20, or 30 days postpartum, horizontal sections of the olfactory bulb were reacted histochemically to reveal the locus and intensity of 5'-nucleotidase activity. Relative staining levels were determined by optical densitometry in standardized bulb regions. A marked, age-related increase in staining density was observed. Reaction product was found primarily in neuropil areas. The P10 and P20 control animals did not exhibit right/left differences in bulb staining; however, some laterality was observed in P30 animals. Inter-glomerular and regional variations were observed throughout the developmental period, including (1) differences between neighboring glomeruli; (2) a gradient in the dorsal-ventral axis of the bulb; and (3) a higher staining density in the medial-caudal portion of the bulb. In subjects with occluded nares, asymmetries in right/left bulb 5'-nucleotidase staining patterns were detected throughout development. Bulbs ipsilateral to the blocked nares exhibited increased staining density, suggesting that the procedure enhanced enzymatic activity. Understanding these variations in 5'-nucleotidase staining may be important for a complete understanding of the mechanisms of olfactory bulb maturation and may give insight into the possible role of this enzyme in synaptic malleability during nervous system development and regeneration.
Luteinizing hormone-releasing hormone (LHRH) containing neurons arise in the region of the medial olfactory placode and migrate into the developing olfactory bulbs and basal forebrain along branches of the terminal and vomeronasal nerves. The neurons ultimately come to reside in olfactory and septo-preoptic areas and project extensively to several brain regions, including the preoptic area and median eminence. The present study examined the expression of a glial-associated guidance molecule, S100, as a possible substrate for this migration. Monodelphis domestica (the Brazilian grey, short-tailed opossum) was studied since this species gives birth to very immature, young, allowing access to early periods of mammalian forebrain development. Immunoreactivity for both S100 and LHRH-containing neurons and fibers were observed to be closely associated along the entire LHRH migratory route from the vomeronasal organ to the septo-preoptic areas as early as the day of birth (PO). By P10, S100-immunoreactivity was also seen in areas containing LHRH-immunoreactive fibers such as the preoptic area and median eminence. We suggest that S100, a protein with neurotrophic properties in vitro, acts as a guidance molecule for migrating LHRH-immunoreactive neurons and elongating processes.
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.
Immediate early genes such as c-fos may be a route through which extracellular events affect genomic expression. Expression of immediate early genes is important in the transcriptional regulation necessary for the normal development of the nervous system. Developmental patterns of Fos protein (the product of c-fos immediate early gene expression) were studied in the main olfactory bulb of the rat using immunocytochemistry. Embryonic Day 21 (E21, the last prenatal day), as well as Postnatal Day 0 (P0), P1, P5, P10, P15, P20 and P30 subjects were examined. Although staining was absent in the E21 bulb, there was a rapid onset of Fos synthesis within hours after birth. Distribution of Fos-immunoreactive (Fos-ir) nuclei corresponded to the sequence of bulb maturation: numerous mitral/tufted and granule cells were labeled on P0, followed by the appearance of Fos-ir in the nuclei of periglomerular cells and an increase in the number of stained granule cells with development. Surgical closure of an external naris on P1 resulted in a 70% reduction in the number of Fos-ir granule cell nuclei as early as 2 h after the manipulation. During the next 30 days, levels of Fos staining further diminished in experimental bulbs when compared to their contralateral controls. Nevertheless, electrical stimulation of the contralateral bulb in P20 pups resulted in a robust increase of Fos labeling in most main and accessory olfactory bulb mitral cells and in many granule and periglomerular neurons, suggesting that the experimental bulbs remain competent to express Fos protein.
We report that intracellular injections of Lucifer Yellow into lightly fixed mitral cells revealed dye-coupling between mitral cells and between mitral and granule cells in the form of discrete, radially oriented cell clusters. Dye-coupling was observed in animals as early as postnatal day 10 (P10) and at least until P30. In P10 rats, a mean of 2.5 dye-coupled mitral cells and 27 granule cells were observed per column. Mean column depth and width were 169 microns and 86 microns respectively. Most of the dye-filled granule cells were found within 150 microns of the mitral cell layer. No significant changes were found at P20. By P30, the mean number of granule cells per column increased to 42 and the addition of granule cells occurred in areas proximal to the mitral cell layer. Immunocytochemical results indicate that the developing bulb contains a large concentration of the gap junction protein Connexin 43 (Cx43). Cx43-like immunoreactivity was found at all ages examined, with the most intense staining in the nerve and glomerular layers. Less intense Cx43-like immunoreactivity was found in both the mitral and granule cell layers, with Cx43-like immunoreactive puncta observed between and around cell body profiles. Freeze-fracture analysis revealed the presence of gap junction-like plaques on mitral cells, further suggesting that the dye-coupling occurred across interneuronal gap junctions. Neuronal coupling during development could provide an inter-cellular pathway for the passage of relevant developmental signals which could influence the formation and/or strengthening of synaptic contacts. The coupling could also be involved in the synchronization of neuronal activity, which may be important for olfactory coding.
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.