Neurotrophins and their receptors in the primary olfactory neuraxis

The primary olfactory pathway is an elegant and simple system in which to study neurogenesis and neuronal plasticity because of the simple fact that olfactory receptor neurons (ORNs) are continually generated throughout the adult lifetimes of vertebrates. Thus, neuronal birth, differentiation, survival, axon pathfinding, target recognition, synapse formation, and cell death are developmental events that can be examined in the mature olfactory epithelium (OE). Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3, and 4/5) are a family of bioactive peptides that exert their effects by interacting with high- and low-affinity receptors on the surfaces of responsive cells, and have been implicated in several stages of neuronal development throughout the central and peripheral nervous system (CNS and PNS). There has been significant interest within the olfactory community as to how these multifunctional peptides might regulate the cycle of degeneration and regeneration of olfactory receptor neurons. The focus of this review is to highlight what is known about the actions of neurotrophins in the primary olfactory pathway, and to pinpoint future directions that will enable us to further understand their role in olfactory receptor neuron development and turnover.     

Apoptosis in the mature and developing olfactory neuroepithelium

Neuronal apoptosis is important in the developmental sculpting of a normal nervous system and also in the loss of neurons caused by neurodegenerative disease, ischemia or trauma. In a developing embryo, exquisite mechanisms of regulation exist to balance factors that control neuronal birth and death within a given neuronal group, so that sufficient neurons develop and survive to elicit normal function. Postnatally, the only part of the mammalian nervous system where many of these regulatory balance mechanisms are retained is the olfactory epithelium (OE). During the last 30 years, researchers investigating olfactory receptor neuron cellular and developmental biology have focussed on the regeneration of the neuronal population within the olfactory neuroepithelium, following the induced death of the mature neuronal population. This body of work has thus far overshadowed the equally important and intrinsically linked phenomenon of the death of mature olfactory receptor neurons, which is required to initiate regeneration. The purpose of this review is to reveal what has been established about the different forms of cell death that can occur in neurons of the olfactory epithelium, and highlight the identified pro- and anti-apoptotic pathways that control the normal and induced turnover of olfactory receptor neurons.     

Development,growth, and plasticity in the crayfish olfactory system

Decapod crustaceans have a well-defined olfactory system characterised by a set of chemosensitive sensilla grouped together in an array (the olfactory organ) on their antennules. Olfactory receptor neurons in the olfactory organ project exclusively to, and terminate in, cone-shaped olfactory glomeruli in a discrete neuropil in the brain, the olfactory lobe. The olfactory organ appears to be the only afferent input to the olfactory lobe, making the system convenient for the study of its development and growth. The progression of development of the olfactory system is a continuum and can be traced from the first appearance of peripheral receptor cells and central stem cells through to the construction of the tracts and neuropils that constitute the adult system. Cell proliferation leading to the production of peripheral and central olfactory neurons can be observed with mitotic markers in both embryonic stages and in postembryonic growth. Cell proliferation in the olfactory system in crayfish persists throughout the lives of the animals and can be modulated by manipulating the living conditions imposed on growing animals. Large serotonergic neurons that are associated with the olfactory system may play a role in the regulation of cell proliferation.     

Introduction to olfactory neuroepithelium

Among the five senses, the sense of smell (olfaction) is the most sensitive and emotional window on the outside world (Stern and Marx, 1999). The olfactory system recognizes and discriminates myriad odorants of diverse molecular structures. What makes the olfactory system so specific and sensitive? OE harboring the olfactory receptor neurons (ORNs) also has an another unusual characteristic ability that fascinates scientists. Neurogenesis in this tissue continues throughout lifetime. This unique character provides an elegant model to study neurogenesis and neuronal plasticity, since neuronal birth, differentiation, survival, axon pathfinding, target recognition, synapse formation, and cell death can be examined in the mature OE. This special issue of Microscopic Research and Technique presents the recent developments in this exciting field of neuroscience, “structure and function of olfactory neuroepithelium.” Microsc. Res. Tech. 58:133–134, 2002. © 2002 Wiley-Liss, Inc.     

Role of nerve growth factor in the olfactory system

Olfactory neurons are unique in the mammalian nervous system because of their capacity to regenerate in adult animals. It has been shown that olfactory receptor cells located in the olfactory epithelium are replaced on a continuous basis and in response to injury throughout the life span of most species. NGF, which is one of the neurotrophic factors, is present in many areas of the central and peripheral nervous system. It has been shown that NGF in the olfactory bulb plays a role in the survival of cholinergic neurons in the horizontal limb of the diagonal band (HDB). Recent studies of NGF in the olfactory bulb suggest that it is involved in the development, maintenance, and regeneration of olfactory receptor cells. In this study, we review reports examining the relationship between NGF in the olfactory bulb and neuronal regeneration and development in the mammalian olfactory systems. Low- and high-affinity NGF receptor immunoreactivity is markedly expressed during regeneration and at different stages of development in the mouse olfactory system. This level of immunoreactivity is no longer present after completion of regeneration and at maturation. Other findings indicate that NGF injected into the olfactory bulb is transported retrogradely to the olfactory epithelium. It has also been shown that continuous anti-NGF antibody injection into the olfactory bulb causes degeneration and olfactory dysfunction. Administration of NGF directory into nasal cavity results in an increase in the expression of olfactory marker protein within the olfactory epithelium in axotomized rats. These findings suggested that the presence of NGF in the olfactory bulb plays an essential role in regeneration, maintenance, and development in the olfactory system of mammals.     

Immunohistochemical and lectin histochemical studies on the developing olfactory organs of fetal camel

Little is known about the development of the olfactory organs of camel. In this study, prenatal development and neuronal differentiation of the vomeronasal organ (VNO) and the olfactory epithelium (OE) of the one‐humped camel were studied by immunohistochemistry and lectin histochemistry. A neuronal marker, protein gene product (PGP) 9.5, but not a marker of fully differentiated olfactory receptor cells, olfactory marker protein, intensely labeled the olfactory receptor cells of the VNO and OE at 395 mm, 510 mm, and 530 mm fetal ages, indicating that the olfactory receptor cells are differentiated, but not fully matured both in the VNO and the OE. In 187 mm and 190 mm fetuses, PGP 9.5 yielded faint immunoreactive signals in the VNO, but not in the OE, although the presence of olfactory receptor cells were demonstrated in both tissues by intense WGA and LEL stainings. We conclude that the camel VNO and OE bear differentiated, but still immature receptor cells; in addition, the onset of neuronal differentiation seems to be somewhat earlier in the VNO than in the OE till half of the prenatal life. Microsc. Res. Tech. 78:613–619, 2015. © 2015 Wiley Periodicals, Inc.     

Human fetal ductal plate revisited. I. ductal plate expresses NCAM,KIT, MET,PDGFRA, and neuroendocrine antigens (NSE,chromogranin, synaptophysin,and CD56)

Background: The molecular mechanisms of ductal plate (DP) development and differentiation (DD) in human fetal livers (HFLs) are unclear. Materials and Methods: The author immunohistochemically investigated expressions of NCAM, KIT, KIT, PDGFRA, and neuroendocrine antigens in 32 HFLs. Results: The processes of human intrahepatic bile duct (IBD) DD could be categorized into four stages: DP, remodeling DP, remodeled DP, and mature IBD. NCAM was always expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. The biliary elements were positive for cytokeratin (CK)7, 8, 18, and 19. The hepatoblasts were positive for CK8 and CD18, but negative for CK7 and CK19; however, periportal hepatoblasts showed biliary‐type CKs (CK7 and CK19). NCAM was always expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. KIT was occasionally (12/32 cases) expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. NCAM expression was also seen in some hepatoblasts and hematopoietic cells and neurons. KIT was also expressed in some hepatoblasts, hematopoietic cells, and mast cells. MET and PDGFRA were strongly expressed in DP, remodeling DP, remodeled DP, and mature IBD. MET and PDGFRA were also strongly expressed in hepatoblasts and hematopoietic cells. MET and PDGFRA were not expressed in portal mesenchyme, portal veins, sinusoids, and hepatic veins. DP showed immunoreactive chromogranin, synaptophysin, neuron‐specific enolase (NSE), and CD56. Expressions of chromogranin and CD56 were infrequently seen in remodeling DP. No expressions of these four neuroendocrine antigens were seen in remodeled DP and mature IBD. The nerve fibers were consistently positive for chromogranin, synaptophysin, NSE, and CD56 in the portal mesenchyme in the stages of remodeling DP, remodeled DP, and mature IBDs. Conclusions: The data suggest that NCAM, KIT/stem cell factor‐signaling, NSE, hepatocyte growth factor/MET signaling, PDGFα/PDGFRA signaling, chromogranin, synaptophysin, and CD56 play important roles in DD of biliary cells of HFL. They also suggest that the DP cells having neuroendocrine molecules give rise to hepatic stem/progenitor cells. Microsc. Res. Tech. 77:814–824, 2014. © 2014 Wiley Periodicals, Inc.     

Light and transmission electron microscopy study of the peripheral olfactory organ of the guppy, Poecilia reticulata (Teleostei, Poecilidae)

A study of the peripheral olfactory organ, with special attention to the olfactory epithelium, has been carried out in the guppy (Poecilia reticulata). Guppy is well known to have a vision-based sexual behavior. The olfactory chamber caudally opens directly in an accessory nasal sac, which is bent medially and gives rise to two recesses that can be considered secondary accessory nasal sacs, antero-medial and postero-medial, respectively. The sensory epithelium, which lines only the medial wall of the nasal cavity, is basically flat rising in a very low lamella only in the posterior part. The olfactory receptors are not evenly distributed in the olfactory mucosa, but aggregate in shallow folds separated by epithelial cells with evident microridges. Ciliated olfactory sensory neurons and microvillous olfactory sensory neurons are clearly identified by transmission electron microscopy (TEM). Scarce crypt olfactory neurons are found throughout the sensory folds. The nasal sacs indicates the capacity to regulate the flow of odorant molecules over the sensory epithelium, possibly through a pump-like mechanism associated with gill ventilation. The organization of the olfactory organ in guppy is simple and reminds what is found in early posthatching stages of fish which at the adult state have a well developed olfactory organ. This simple organization supports the idea that the guppy rely on olfaction less than other fish species provided with more extended olfactory receptorial surface.     

High-resolution X-ray tomography of the human inner ear: synchrotron radiation-based study of nerve fibre bundles, membranes and ganglion cells

The combination of osmium tetroxide staining and high-resolution tomographic imaging using monochromatic X rays allows visualizing cellular structures of the human inner ear, that is, the organ of Corti, the stria vascularis and further soft tissues of the membranous labyrinth, in three-dimensional space with isotropic micrometre resolution. This approach permits to follow the course of nerve fibre bundles in a major part of the specimen and reveals the detailed three-dimensional arrangement of individual ganglion cells with distinct nuclei by means of X-ray tomography for the first time. The non-destructive neuron cell counting in a selected volume of 125 μm × 800 μm × 600 μm = 0.06 mm³ gives rise to the estimate that 2000 ganglion cells are present along 1 mm organ of Corti.     

Neurotrophins and development of the rod pathway: an elementary deduction

The rodent retina is a particularly attractive model for the study of neuronal developmental processes since considerable neurogenesis, cellular migration, phenotypic differentiation of retinal cell types and synaptogenesis occurs postnatally. In addition, the retina is readily accessible to surgical intervention, pharmacological manipulation, and local suppression of gene expression-tools that can be utilized to study mechanisms underlying the development of retinal neurons and their interconnections that form distinct functional circuits. Here, I review our studies describing the ontogeny of a specific retinal interneuron, the AII amacrine cell, an integral element in the rod (scotopic) pathway. Specifically, we used a number of approaches to examine the potential role of neurotrophic factors on the morphological and neurochemical differentiation of the AII.     

Impulse conduction of olfactory receptor neuron axons

Compound action potentials were recorded from rat olfactory receptor neuron axons at measured distances from the stimulation electrode along the lateral surface of the main olfactory bulb. Distances were plotted as a function of the latencies measured from stimulus onset to the prominent negative trough of the triphasic compound action potential. A straight line was fitted to these data to calculate impulse conduction velocity, 0.42 +/- 0.01 m/s (n = 25). Two procedures were used to investigate whether those axons that project to caudal regions of the bulb had faster conduction velocities than axons projecting to rostral bulb. First, the stimulating electrode was moved to mid-bulb and the recording electrode was placed on the caudal bulb. Alternatively, axons were stimulated antidromically at the caudal bulb. These two procedures stimulate those axons projecting to caudal bulb and bypass olfactory receptor neuron axons that synapse in the rostral bulb. The mean impulse conduction velocities from these caudal and antidromic recordings were 0.58 +/- 0.19 m/s (n = 8) and 0.57 +/- 0.19 m/s (n = 9), respectively. Though both of these means are higher than the impulse conduction velocity calculated for stimulation at the rostral bulb, the differences were not statistically significant.     

Drosophila as a focus in olfactory research: mapping of olfactory sensilla by fine structure, odor specificity, odorant receptor expression, and central connectivity.

This review intends to integrate recent data from the Drosophila olfactory system into an up-to-date account of the neuronal basis of olfaction. It focuses on (1) an electron microscopic study that mapped a large proportion of fruitfly olfactory sensilla, (2) large-scale electrophysiological recordings that allowed the classification of the odor response spectra of a complete set of sensilla, (3) the identification and expression patterns of candidate odorant receptors in the olfactory tissues, (4) central projections of neurons expressing a given odorant receptor, (5) an improved glomerular map of the olfactory center, and (6) attempts to exploit the larval olfactory system as a model of reduced cellular complexity. These studies find surprising parallels between the olfactory systems of flies and mammals, and thus underline the usefulness of the fruitfly as an olfactory model system. Both in Drosophila and in mammals, odorant receptor neurons appear to express only one type of receptor. Neurons expressing a given receptor are scattered in the olfactory tissues but their afferents converge onto a few target glomeruli only. This suggests that in both phyla, the periphery is represented in the brain as a chemotopic map. The major difference between mammals and fruitflies refers to the numbers of receptors, neurons, and glomeruli, which are largely reduced in the latter, and particularly in larvae. Yet, if activated in a combinatorial fashion, even this small set of elements could allow discrimination between a vast array of odorants.     

Presynaptic inhibition of olfactory receptor neurons in crustaceans

Presynaptic inhibition of transmitter release from primary sensory afferents is a common strategy for regulating sensory input to the arthropod central nervous system. In the olfactory system, presynaptic inhibition of olfactory receptor neurons has been long suspected, but until recently could not be demonstrated directly because of the difficulty in recording from the afferent nerve terminals. A preparation using the isolated but intact brain of the spiny lobster in combination with voltage-sensitive dye staining has allowed stimulus-evoked responses of olfactory receptor axons to be recorded selectively with optical imaging methods. This approach has provided the first direct physiological evidence for presynaptic inhibition of olfactory receptor neurons. As in other arthropod sensory systems, the cellular mechanism underlying presynaptic afferent inhibition appears to be a reduction of action potential amplitude in the axon terminal. In the spiny lobster, two inhibitory transmitters, GABA and histamine, can independently mediate presynaptic inhibition. GABA- and histaminergic interneurons in the lobster olfactory lobe (the target of olfactory receptor neurons) constitute dual, functionally distinct inhibitory pathways that are likely to play different roles in regulating primary olfactory input to the CNS. Presynaptic inhibition in the vertebrate olfactory system is also mediated by dual inhibitory pathways, but via a different cellular mechanism. Thus, it is possible that presynaptic inhibition of primary olfactory afferents evolved independently in vertebrates and invertebrates to fill a common, fundamental role in processing olfactory information.     

Strategies for studying microtubule transport in the neuron

Microtubules are prominent cytoskeletal elements within the neuron. They are essential for the differentiation, growth, and maintenance of axons and dendrites. The microtubules within each type of process have a distinct pattern of organization, and these distinct patterns result in many of the morphological and structural features that distinguish axons and dendrites from one another. There are a number of challenges that must be met in order for the neuron to establish the microtubule arrays of axons and dendrites. One attractive model invokes the active transport of microtubules from the cell body of the neuron into and down these processes. In support of this model, specific motor proteins have now been identified within neurons that have the necessary properties to transport microtubules into developing axons and dendrites with the appropriate orientation for each type of process. An important goal is to develop microscopic methods that permit the visualization of microtubule transport within different regions of the neuron. To date, achieving this goal has met with mixed success, probably as a result of the geometry of the neuron and the inherent complexity of the neuronal microtubule arrays. While some approaches have failed to reveal microtubule transport, other more recent approaches have proven successful. These approaches provide strong visual support for a model based on microtubule transport, and provide hope that future approaches can provide even clearer demonstrations of this transport.     

Ciliated and microvillar receptor cells degenerate and then differentiate in the olfactory epithelium of rainbow trout following olfactory nerve section.

We used scanning (SEM) and transmission (TEM) electron microscopy to examine ultrastructural changes in the olfactory epithelium (OE) of rainbow trout following unilateral olfactory nerve section. Both ciliated receptor cells (CRC) and microvillar receptor cells (MRC) degenerated and subsequently differentiated from unidentified precursor cells. The following changes took place in fish that were held at 10 degrees C at the stated period following olfactory nerve section: on day 7, MRC and CRC contained intracellular vacuoles; on day 12, the olfactory knobs appeared disrupted; by day 26, olfactory receptor cells were absent from the OE; on day 42, there were receptor cell bodies and a few CRC with short cilia at the apical surface; and on day 55, a small number of both CRC and MRC had differentiated. By day 76, both CRC and MRC repopulated the OE. Degenerative changes in the cytoplasm of the sustentacular cells (SC) and ciliated nonsensory cells (CNC) were observed in the first 26 days following olfactory nerve section, but these cells remained intact throughout the experiment. The degeneration and subsequent differentiation of CRC and MRC supports and extends previous observations that both cell types are olfactory receptor neurons with axons that extend along the olfactory nerve to the olfactory bulb.     

Ultrastructural neurobiology of the olfactory mucosa of the brown trout, Salmo trutta.

This paper describes four investigations of the olfactory mucosa of the brown trout: 1) the ultrastructure of the olfactory mucosa as revealed by scanning (SEM), conventional transmission (TEM), and high voltage (HVEM) electron microscopy; 2) light and electron-microscopic investigations of retrograde transport of the tracer macromolecule horseradish peroxidase (HRP) when applied to the cut olfactory nerve; 3) SEM and TEM investigations of the effects of olfactory nerve transection on cell populations within the olfactory epithelium; and 4) ultrastructural investigations of reversible degeneration of olfactory receptors caused by elevated copper concentrations. The trout olfactory epithelium contains five cell types: ciliated epithelial cells, ciliated olfactory receptor cells, microvillar olfactory receptor cells, supporting cells, and basal cells. The ciliated and microvillar olfactory receptor cells and a small number of basal cells are backfilled by HRP when the tracer is applied to the cut olfactory nerve. When the olfactory nerve is cut, both ciliated and microvillar olfactory receptor cells degenerate within 2 days and are morphologically intact again within 8 days. When wild trout are taken from their native stream and placed in tanks with elevated copper concentrations, ciliated and microvillar cells degenerate. Replacement of these trout into their stream of origin is followed by morphologic restoration of both types of olfactory receptor cells. Ciliated and microvillar receptor cells are primary sensory bipolar neurons whose dendrites make contact with the environment; their axons travel directly to the brain. Consequently, substances can be transported directly from the environment into the brain via these "naked neurons." Since fish cannot escape from the water in which they swim, and since that water may occasionally contain brain-toxic substances, the ability to close off--and later reopen--this anatomic gateway to the brain would confer a tremendous selective advantage upon animals that evolved the "brain-sparing" capacity to do so. Consequently, the unique regenerative powers of vertebrate olfactory receptor neurons may have their evolutionary origin in fishes.     

Photo‐ and bio‐physical characterization of novel violet and near‐infrared lipophilic fluorophores for neuronal tracing

Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally within nerve cell membranes. Given the hundreds to thousands of connections that a typical neuron makes with its neighbours, a diffusion‐matched set of spectrally distinct dyes is desirable. To extend a set of these dyes to obtain six independent labels, we have characterized the properties of novel violet and near‐infrared candidates. By combining two‐photon and confocal microscopy all of these candidates can be imaged using a single Titanium Sapphire laser. Here we present measurements of the two‐photon action cross‐sections and diffusion properties of the dyes, using either the relative diffusion distance or fluorescence recovery after photobleaching techniques, and demonstrate six‐colour neuronal tracing within the spinal cord and brain tissue.     

Integrated Membrane Proteomic Strategies Revealed Cell Surface Signature during Human Embryonic Stem Cell Differentiation

In stem cell research, cell surface markers are extensively used for stem cell classification, monitoring the differentiation stages as well as purification for their use in regenerative medicine. Quantitative membrane proteomic approaches will provide an in-depth view of the stage- and lineage-specific expression which potentially enhances our understanding on the underlying mechanisms of stem cell differentiation, as well as the opportunity towards isolation of homogenous primary stem cell population. However, the analysis of membrane proteome and its highly heterogeneous glycosylation is experimentally challenging because of their hydrophobic nature and low abundance, which seriously complicate their solubilization, sample handling, separation, and mass spectrometric analysis. In attempt to search for novel stem cell surface markers and differentiation regulators, we have applied a subglobal proteomic approach and glycoproteomic profiling to define a “membrane proteomic profile” of human embryonic stem (hES) cells and 16-day differentiated embryoid body (EB) outgrowth. Using our recently reported gel-assisted digestion and iTRAQ labeling approach, 3842 proteins were identified (p<0.05) and 2783 proteins were quantified with 2 peptides. By labeling strategy with alkynyl sugar derivatives, the preliminary results In glycoproteomic analysis identified 350 glycopeptides (p<0.05) and quantified in 212 glycoproteins. By combining the quantitative information in protein expression and N-glycosylated peptides, the site-specific glycosylation degree of peptides can be confidently determined on a proteome scale. Our study revealed the dramatic change in expression as well as sialylated N-glycosylation on cell surface glycoproteome during stem cell differentiation. Interestingly, the proteomic data revealed that some cell surface markers, previously discovered by gene expression array, have unaltered expression during stem cell differentiation, which may be due to differences in protein turnover and regulation of the abundance of cellular mRNA and proteins. Mapping of these differentially expressed glycoproteins and membrane proteins in multiple cellular pathways related to cell differentiation, proliferation and cell development suggests that not only the protein markers but also the site and degree of glycosylation have distinct pattern or function in the complex process during stem cell differentiation.     

Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: ultrastructure and uptake of tracers.

Mammalian olfactory neurons possess a well-developed system of endocytic vesicles, endosomes, and lysosomes in their dendrites and perikarya. Vomeronasal neurons are similar and also contain much perikaryal agranular endoplasmic reticulum (AER). Olfactory supporting cells contain endocytic vesicles and endosomes associated closely with abundant fenestrated AER, and vesicles and numerous large dense vacuoles are present basally. Vomeronasal supporting cells have little AER, and few dense vacuoles occur in their bases. In olfactory neurons, ultrastructural tracers (0.08% horseradish peroxidase, thorium dioxide, ferritin) are endocytosed by olfactory receptor endings and transported to the cell body, where their movement is halted in lysosomes. Higher concentrations (1%) of horseradish peroxidase penetrate olfactory receptor plasma membranes and intercellular junctions. In olfactory supporting cells, endocytosed tracers pass through endosomes to accumulate in dense basal vacuoles. These observations indicate that olfactory sensory membranes are rapidly cycled and that endocytosed materials are trapped within the epithelium. It is proposed that in the olfactory epithelium, endocytosis presents redundant odorants to the enzymes of the supporting cell AER to prevent their accumulation, whereas in the vomeronasal epithelium the receptor cells carry out this activity.